^ t

DRUG RESISTANCE IN
MICRO-O RGANISMS

Mechanisms of Development

Ciba Foundation Symposia

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^ . • 1

CIBA FOUNDATION SYMPOSIUM

ON

DRUG RESISTANCE IN
MICRO-ORGANISMS

Mechanisms of Development

Editors for the Ciba Foundation

G. E. W. WOLSTENHOLME, O.B.E., M.A., M.B., B.Ch.

and

CECILIA M. O'CONNOR, B.Sc.

With 62 Illustrations

LITTLE, BROWN AND COMPANY

BOSTON

THE CIBA FOUNDATION

for the Promotion of International Co-operation in Medical and Chemical Research
41 Portland Place, London, W.l.

Trustees :

The Right Hon. Lord Adrian, O.M., F.R.S.

The RiCxHT Hon. Lord Beveridge, K.C.B., F.B.A.

Sir Russell Brain, Bt.

The Hon. Sir George Lloyd-Jacob

Sir Raymond Needham, Q.C.

Director, and Secretary to the Executive Council:
Dr. G. E. W. Wolstenholme, O.B.E.

Assistant to the Director:
Dr. H. N. H. Genese

Assistant Secretary :
Miss N. Bland

Librarian :
Miss Joan Etherington

Editorial Assistants :

Miss Cecilia M. OTonnor, B.Sc.

Miss Maeve O'Connor, B.A.

All Rights Reserved

l^his book may not be reproduced by
any means, in whole or in part, with-
out the permission of the Publishers

Published in London by

J. & A. Churchill Ltd.

104 Gloucester Place, W.l

First Published 1957
Printed in Great Britain

PREFACE

It was Sir Charles Harington, Director of the National
Institute for Medical Research, and also Chairman of the
Medical Research Council's Committee on Chemotherapy,
who put forward to the Director of the Ciba Foundation the
proposal of a symposium on drug resistance. It was his
belief that the more fundamental problems at the basis of
chemotherapy were not attracting as much attention in re-
search as was desirable, and his hope that out of a thorough
discussion of the question some suggestions might come which
would stimulate fresh investigations, more particularly perhaps
on the part of chemists.

The Director of the Foundation thought it would be most
profitable, and more in keeping with the facilities of the
Foundation, if the subject to be considered were narrowed
down to "Mechanisms of Development of Drug Resistance in
Micro-Organisms." With the expert advice and ready
assistance of Sir Charles, and also of Dr. M. R. Pollock, such
a meeting was realized in March 1957, Sir Charles himself
acting as its Chairman. The Trustees and the Director of the
Foundation remain much indebted to both of them, and to
the Members who contributed so freely and informatively in
the papers and discussions.

The group was a small one, as usual at the Ciba Foundation,
partly because the Foundation's accommodation is severely
limited, but mainly because experience has shown that useful
discussions can best be conducted when members can get to
know each other quickly and well, and can be seated in a
convenient and comfortable manner for conversation.

This record of the papers presented and the discussions
they aroused is prepared for the many people who could not
be invited on this occasion, and the Editors hope it will prove
an acceptable substitute for personal participation.

vi Preface

To some readers this book may form an introduction to the
work of the Ciba Foundation, and it may be helpful to add a
few words about its interests.

Under its eminent Trustees, the Foundation is engaged in a
number of activities with the purpose of improving co-
operation in medical and chemical research between workers
in different countries and different disciplines. At its house in
London the Foundation provides accommodation for scientists,
organizes conferences, conducts a medical post-graduate ex-
change scheme between Great Britain and France, arranges
a variety of informal discussions, awards two annual lecture-
ships, and is building up a library service in special fields.
The Foundation assists international congresses and scientific
institutions, and it is hoped that in its hospitality, its meetings,
and in such a volume as this, it is also usefully helping the
individual scientist.

CONTENTS

Chairman's opening remarks

Sir Charles Harixgtox ...... 1

Aspects of the problem of drug resistance in bacteria

by A. C. R. Deax and Sir Cyril Hinshelwood . . 4

Discussion: Dean, Eagle, Hinshelwood, Lederberg,

Pollock, Pontecorvo ....... 24

Indirect selection and origin of resistance

by L. L. Cavalli-Sforza ...... 30

Discussio7i : Cavalli-Sforza, Davis, Dean, Gyorffy,
Hinshelwood, Hotchkiss, Kunicki-Goldfinger,
Lederberg, Pontecorvo, Yudkin .... 40

Genetic aspects of drug resistance

by M. Demerec ....... 47

Discussion: Alexander, Davis, Demerec, Hinshelwood,
Hotchkiss, Lederberg, Pollock, Pontecorvo, Slonimski,
Walker, Yudkin . . . . . . .58

Inheritance in single bacterial cells

by W. Howard Hughes ...... 64

Discussion: Cavalli-Sforza, Davis, Demerec, Eagle,
Hayes, Hughes, Ierusalimsky, Lederberg, Pontecorvo,
Stocker, Yudkin ....... 71

Penicillin-induced resistance to penicillin in cultures of

Bacillus cereus

by M. R. Pollock 78

Discussion : Alexander, Barber, Davis, Hayes, Hotchkiss,
Knox, Lederberg, Pollock, Pontecorvo, Slonimski,
Stocker ......... 96

Directed hereditary changes of fermentative properties of
yeast by a specific substrate

by K. V. KossiKOV ....... 102

Discuss^ion : Harington, Ierusalimsky .... 136

vii

73503

viii Contents

PAGE

Multiple mechanisms of acquired drug resistance

by Margaret J. Thornley, Jehudith Sinai and John

YUDKIN ........ 141

Discussion: Davis, Dean, Fredericq, Fulton, Hotchkiss,

Hughes, Pollock, Rose, Slonimski, Walker, Yudkin . 161

Physiological (phenotypic) mechanisms responsible for
drug resistance

by B. D. Davis 165

Discussion: Davis, Eagle, Knox, Pollock, Pontecorvo . 180

Genetic and metabolic mechanisms underlying multiple
levels of sulphonamide resistance in pneumococci

by RoLLiN D. Hotchkiss and Audrey H. Evans . 183

Discussion: Cavalli-Sforza, Davis, Demerec, Hotchkiss,

Lederberg, Pontecorvo, Stocker .... 193

The phenotypic expression of genes determining various
types of drug resistance following their inheritance by
sensitive bacteria

by W. Hayes . . 197

Discussion: Barber, Cavalli-Sforza, Davis, Fredericq,
Hayes, Hotchkiss, Lederberg, Pollock, Pontecorvo,
Stocker ......... 205

Specific polyhydroxy compounds as cofactors of enzymic
adaptation and its inheritance

by P. P. Slonimski and H. de Robichon-Szulmajster 210

Discussion: Davis, Fulton, Pollock, Slonimski, Wester-

GAARD ......... 230

Development of resistance to streptomycin in Serratia
marcescens

by B. Gyorffy and I. Kallay ..... 233

Discussion: Cavalli-Sforza, Davis, Dean, Eagle, Fulton,
Gyorffy, Hayes, Hotchkiss, Knox, Lederberg, Stocker,
Yudkin ......... 237

Distribution of drug -resistant individuals in cultures of

Mycobacterium tuberculosis

by R. Knox 241

Discussion : Davis, Dean, Eagle, Hayes, Knox, Lederberg,

Pollock, Pontecorvo, Stocker, Westergaard . . 246

Contents ix

PAGE

Physiological adaptation of bacteria to antibiotics

by W. KUNICKI-GOLDFINGER ..... 251

Discussion: Alexander, Davis, Gyorffy, Knox, Kunicki-

GoLDFiNGER, Lederberg, Slonimski, Stocker, Yudkin . 259

Drug resistance of staphylococci with special reference to
penicillinase production

by Mary Barber ....... 262

Discussion: Barber, Bishop, Cavalli-Sforza, Davis,
Eagle, Hayes, Hughes, Knox, Lederberg, Pollock,
Pontecorvo, Westergaard ..... 274

On the identification of genetic and non- genetic variation
in bacteria

by M. Westergaard ....... 280

Discussion: Alexander, Davis, Demerec, Hayes, Hughes,

Knox, Lederberg, Pontecorvo, Rose, Westergaard . 290

The reactions of the mutagenic alkylating agents with
proteins and nucleic acids

by P. Alexander, Sheila F. Cousens and K. A. Stagey 294

Discussion: Alexander, Davis, Hayes, Lederberg, Ponte-
corvo, Rose, Walker, Westergaard . . . .318

Genetics of two different mechanisms of resistance to
colicins: resistance by loss of specific receptors and im-
munity by transfer of colicinogenic factors

by P. Fredericq ....... 323

Discussion : Cavalli-Sforza, Fredericq, Hayes, Lederberg,

Pollock, Pontecorvo, Stocker ..... 335

General Discussion: Davis, Dean, Hayes, Hotchkiss,

Lederberg, Pollock, Slonimski .... 339

Chairman's closing remarks

Sir Charles Harington ...... 344

List of those participating in or attending the Symposium on

Drug Resistance in Micro-Organisms

26th-28th March, 1957

P. Alexander .
Mary Barber .

Ann Bishop

L. L. Cavalli-Sforza
B. D. Davis

A. C. R. Dean .
M. Demerec

H. Eagle .

P. Fredericq

J. D. Fulton

B. Gyorffy

Sir Charles Harington

P. Hartman

W. Hayes .

Sir Cyril Hinshelwood
r. d. hotchkiss

W. Howard Hughes

N. D. Ierusalimski

R. Knox .

K. V. KossiKOv .

Chester Beatty Research Inst., London
Dept. of Bacteriology, St. Thomas's Hospital

Medical School, London
Molteno Inst, of Biolojjy and Parasitology,

University of Cambridfje
Istituto Sieroterapico Milanese, Milan
Dept. of Bacteriology and Immunology,

Harvard Medical School, Boston
Physical Chemistry Laboratory, Oxford
Dept. of Genetics, Carnegie Inst, of Washing-
ton, Cold Spring Harbor, Long Island, New

York
Dept. of Experimental Therapeutics, National

Inst, of Allergy and Infectious Diseases,

Bethesda, Maryland
Dept. of Microbiology and Hygiene, L^niversity

of Liege
National Inst, for ^Medical Research, Mill Hill,

London
Inst, of Genetics, Hungarian Academy of

Sciences, Budapest
National Inst, for Medical Research, Mill Hill,

London
Dept. of Bacteriology and Immunology,

Harvard Medical School, Boston; and

Faculte des Sciences, Brussels
Dept. of Bacteriology, Postgraduate Medical

School, London
Physical Chemistry Laboratory, Oxford
Rockefeller Inst, for Medical Research, New

York
Wright-Fleming Inst, for Microbiology, St.

Mary's Hospital Medical School, London
Inst, of Genetics, Academy of Sciences of the

U.S.S.R., Moscow
Dept. of Bacteriology, Guy's Hospital Medical

School, London
Inst, of Genetics, Academy of Sciences of the

U.S.S.R., Moscow

Xll

List of Participants

W. KUNICKI-GOLDFINGER

J. Lederberg
Esther M. Lederberg
M. R. Pollock .

G. PONTECORVO .

F. L. Rose

P. Sloximski

B. A. D. Stocker
J. Walker

M. Westergaard

J. YUDKIN

Dept. of Microbiology, The University,

Warsaw
Tlie Medical School, Dept. of Medical Genetics,

University of Wisconsin
The Medical School, Dept. of Medical Genetics,

University of Wisconsin
National Inst, for Medical Research, Mill Hill,

London
Dept. of Genetics, The University, Glasgow
Research Dept. Chemical Group, Pharma-
ceuticals Division, Imperial Chemical
Industries Ltd., Manchester
Laboratoire de Genetique Physiologique du

C.N.R.S., Universite de Paris
Lister Inst, of Preventive Medicine, London
National Inst, for Medical Research, Mill Hill,

London
Dept. of Genetics, The University, Copen-
hagen
Dept. of Nutrition, Queen Elizabeth College,
University of London

OPENING REMARKS

Sir Charles Harington

In spite of the great advances that have been made in
recent years in the chemotherapeutic treatment of infectious
diseases — advances that have brought under some measure
of control the majority of protozoal and bacterial infections
and some helminthic infections — the subject of chemotherapy
remains distressingly empirical. The relationship between
chemical structure and biological action in this field is still
so ill-defined that we have only one significant guiding prin-
ciple, based on biological theory, to help us in the search for
new synthetic drugs for specific chemotherapeutic purposes.
As for antibiotics the attempt to discover new substances of
therapeutic value is admittedly based on no scientific prin-
ciple at all, but is an operation such as oil prospecting would
be with no adequate background of geological information.

Even when a new synthetic drug is discovered that proves
to be of value in the treatment of a particular infection, it is
usually impossible to explain the nature of the action of the
drug; indeed the type of activity found is not infrequently
quite different from that which is being sought, as is shown
for instance by the discovery of a valuable antimalarial, pyri-
methamine, in the course of a search for folic acid antagonists.
Sometimes the drug proves not to have a direct action on the
infecting organism at all, although it can suppress or cure the
disease which the micro-organism causes ; thus the anti-malarial
drug proguanil has no direct lethal effect on plasmodia,
but it is metabolized in the body of the host to a substance
that has such an effect. Again hetrazan, which is the most
effective remedy so far known for filariasis, does not kill the
microfilariae directly but so alters them that they become
susceptible to attack by the natural defence mechanisms of the
host. The most striking example of such indirect chemothera-
peutic action is afforded by the high-molecular surface-active

DRUa RES. 1

2 Sir Charles Harington

compounds that are curative of experimental tuberculosis
and leprosy; in this instance there is clear evidence that the
drugs, which are quite innocuous to the infecting organisms,
confer on the monocytes of the host the power of inhibiting
the growth of these organisms, thus exercising their effect by
reinforcing the natural defence mechanisms of the host. The
discovery of these drugs again was a totally unexpected out-
come of the line of research that was being pursued.

All this means that the life of a chemist working in chemo-
therapy is apt to consist of long periods of unexciting work,
punctuated if he is fortunate by occasional successes; even
these successes however, whilst practically satisfying, may
well be intellectually disappointing, since they will very likely
bear little or no relation to the thought that he has put into
his research.

By emphasizing as I have done the uncertainties and lack
of fundamental knowledge that bedevil chemotherapy I must
appear to have painted a very gloomy picture of the subject.
If this is so, it is certainly not because I wish to say anything
in disparagement of its importance. On the contrary, my
object is to analyse the difficulties that we face, and which are
particularly discouraging to chemists, in the attempt to see
how they may be overcome.

It might be argued that in spite of all that I have said the
situation is not unsatisfactory. New and effective chemothera-
peutic agents continue to be discovered and the range of
diseases brought under control increases. But so long as we
cannot explain the reason for our successes we must remain
scientifically dissatisfied, and there is one biological pheno-
menon, namely drug resistance, which makes the situation
much less favourable than it appears even from a strictly
utilitarian point of view. We can hardly be easy about a state
of affairs in which it is reported that in many hospitals over
50 per cent of the strains of staphylococci causing infections
have become resistant to penicillin, even though we now have
other antibiotics with which they can be controlled; nor is
the encouraging emptying of our tuberculosis sanatoria cause

Opening Remarks 3

for complacency, when we reflect that this would not be
occurring had not the discovery of streptomycin been oppor-
tunely followed by those of the antituberculous effects of
^^-aminosalicylic acid and isoniazid. We cannot be sure that the
searchers for new drugs and antibiotics will always win the race.

However hard and successfully we may work in the search
for new drugs we shall therefore continue to labour under
discouragement so long as we are faced with the bugbear of
drug resistance. The problem is one of microbial biochemistry,
physiology and genetics, and can only be solved by work in
these fields. Until we understand the problem we shall have
no hope of overcoming it, and until we overcome it we shall
have no real sense of security in our chemotherapy. The sub-
ject of this symposium therefore is not only of the greatest
scientific interest and importance; it has also a background
of practical medical urgency, and I think we should do well to
keep this thought in our minds.

There are, I am sure, plenty of chemists who would be eager
to devote their abilities to research in chemotherapy if they
could see it as a less empirical subject than it still is. This is
obvious indeed from the mass of work that has resulted from
the Woods-Fildes hypothesis of metabolic interference, a
theory which, born by biochemistry out of microbiology, has
been the most encouraging lead that the chemists have yet
received from the biologist; it has systematized thought in
the search for new drugs, and if its practical yield, apart from
the folic acid antagonists, has so far been small, this is in my
view because full fructification of the idea cannot be ex-
pected until microbiology is further advanced.

Now a further lead is needed, which can only come from
the biochemists and the microbiologists. The greatest en-
couragement to chemical research would be the achievement
of clearer insight into the development of drug resistance
together with even a glimmering of an indication that this
phenomenon may ultimately be subject to control. If our
discussions bring nearer the day when a confident lead in this
direction can be given our time will not have been wasted.

ASPECTS OF THE PROBLEM OF DRUG
RESISTANCE IN BACTERIA

A. C. R. Dean and Sir Cyril Hinshelwood

Physical Chemistry Laboratory, Oxford

General Observations

In presenting this brief review of our present ideas on the
subject of drug resistance it may be well to begin by mention-
ing views which have at one time or another been attributed
to us, but which we have never held and of which no expres-
sion could be quoted from any of our publications.

We have never doubted that the essential characters of a
cell are inherent in the structure of certain fundamental units
including (though not necessarily exclusively) the deoxy-
ribonucleic acid (DNA). We do not suppose these basic
structures to be easily susceptible to change, and indeed in
our experience easily provoked changes are normally destruc-
tive. The maintenance of species characters is of course a
matter of the copying of the genetic patterns, and if and when
these have been changed the heredity will be changed. We
have never denied that structural mutations leading to
increased drug resistance or improved utilization of nutrient
sources can and do occur, or the obvious consequence that the
mutants so arising would be rapidly selected in the appro-
priate environment.

On the other hand, we have contested the assumption that
random mutation and selection is the sole mechanism (or
perhaps even the major mechanism) for adaptation to new
media or for the development of drug resistance. We have
proposed more direct mechanisms, and quoted what appears
to us to be good experimental evidence that in various specific
examples these mechanisms operate.

4

Drug Resistance in Bacteria 5

Before outlining the proposed mechanisms and sum-
marizing this evidence another point should be made clear.
The primary concern of the work has been to explore the
problem of the way in which cell reactions are co-ordinated,
and the manner in which adjustments in the cell economy can
take place, not to assert the relative importance of this or
that evolutionary mechanism. A sound judgement on this
latter question will probably be reached only when the
number of examples studied is considerably greater than it
is at present.

In quoting the kinds of evidence on which our current
views are based we shall group together examples of adapta-
tion to drugs and certain examples of adaptation to new sub-
strates. The mechanisms will be often, though not always,
the same. The addition of a drug to the medium often im-
pedes certain essential enzyme reactions and so imposes a
new reaction pattern on the cell. This is not unlike what
happens when an unfamiliar substrate has to be used. On the
other hand, drug resistance could arise, as in some cases phage
resistance seems to, by a mutation leading to a deficiency
whereby receptors for the drug in the cell are extirpated. In
such a case the analogy with enzymic adaptation would be
absent.

In its essentials the mechanism of adaptive change which
we believe to operate in certain examples is the following.
Although the major characters are determined by the basic
gene structures, their quantitative expression is a function
not merely of what structures are present but of the propor-
tions in which they occur in the cell. When the medium is
changed so that some parts of the reaction sequence are im-
peded relatively to others, corresponding changes in the rela-
tive proportions of the major cell constituents must occur.
If division is governed even approximately by the attainment
of a threshold amount of some key substance (and DNA seems
to be roughly an invariant in this respect), then it is easily
shown that the cell composition adjusts itself automatically
to give an optimum growth rate.

6 A. C. R. Dean and Sir Cyril Hinshelwood

The details need not be repeated here, but a very crude
analogy may be cited in illustration. Suppose we have a tank
with an inflow and an outflow of water. A certain level is
established. If now the outflow is restricted the head of water
rises until eventually outflov/ equals inflow once more. The
kinetic theory of the automatic adjustments in cells is less
crude than this analogy but still, no doubt, far cruder than
reality. Nevertheless, it is very general, and one point which
is worth emphasizing is that if certain quite general and
extremely likely conditions are fulfilled the development of
drug resistance becomes a predictable phenomenon. If the
mechanism just mentioned does not operate in nature there
should be a positive explanation of why it does not.

In principle, such adjustments are reversible when the
original conditions are restored, and a major clash of opinion
has occurred in this connexion. Drug resistance and proper-
ties such as ready utilization of substrates are often rather
persistent, and are often cited as manifestations of "stable
heredity". We differ from this view in two ways. In the first
place, we believe these phenomena in fact to be essentially
reversible, on experimental grounds which will be sum-
marized later. The reversion can be slow, for reasons not
wholly unlike those which account for the delay of many
other chemical transformations. In the second place, we con-
sider that the term "heredity" is a rather ambiguous one to
use of organisms which multiply by binary fission. With such
organisms new individuals do not develop from special cells
which are a minute fraction onh^ of the total somatic make-up
of the progenitors. When the two new cells are formed by
division there is no question of parent and offspring. Each is
roughly half of the original cell and retains its cytoplasmic
make-up. If there is an inertial lag to adjustments in this
(and reasons can be imagined why there should be) the exist-
ing organization will persist. But the persistence might just
as well be called the stability of the physiology of an individual
as a hereditary quality.

In fact this stability is not absolute, and in some examples

Drug Resistance in Bacteria 7

it is very low. Since the phenomenon is neither one of com-
pletely stable changes, nor one to which the term hereditary
has a clear meaning, we consider arguments invoking the name
of Lamarck to be largely meaningless or irrelevant. The way
is thus opened for the consideration on its merit of experi-
mental evidence (which, it should be repeated, applies only
to the particular examples with which it is found — except in
so far as it illustrates what can happen as distinct from what
must happen).

Some aspects of the mutation-adaptation controversy offer
an interesting analogy with the history of the phlogiston
theory in chemistry. This doctrine, it has been said, was
never formally abandoned by its supporters. But under the
pressure of facts they changed it, added to it, and buttressed
it with auxiliary hypotheses until it became indistinguishable
from its rival, except for some superfluous nomenclature
which was presently forgotten.

Is something of the sort in process of happening with the
uncompromising version of the mutation theory? At first
mutations w^ere catastrophic, one-step events, occurring in a
purely random manner to an excessively minute proportion
of the population, and essentially during the hazards of
nuclear division. Gradually the lines of the picture have
softened. Mutations may occur in the absence of nuclear
division and may affect almost the whole population (Ryan,
1955; Szybalski, 1954-55); they may be induced by a drug
to which the mutant subsequently shows resistance, a marked
departure from randomness (Akiba, 1955; Szybalski, 1954-55) ;
they are sufficiently dependent on cytoplasmic events to be
associated with a considerable "phenotypic delay" (New-
combe, 1948, 1953); and as to their discreteness, so elaborate
a polygenic system is frequently assumed that the results of
recombination experiments become indistinguishable from
those which would be given by quantitative cytoplasmic
changes.

This last fact would be even more commonly realized but for
the practice of creating an illusory impression of discreteness

8 A. C. R. Dean and Sir Cyril Hinshelwood

in the phenomena by dividing continuous ranges of quan-
titative variation into two arbitrarily defined ranges such as
"fast" and "slow" or + at 24 hours and — at 24 hours. Add
to this the fact that the resulting changes are not nearly as
stably heritable as is sometimes implied (Dean and Hinshel-
wood, 1954a), and the theory of rather sluggishly reversible
cytoplasmic adaptations no longer looks quite so much like
belonging to a different ideological world.

Stability and Reversibility

In our experience partial reversion often occurs rather
quickly, slow subsequent reversion following an erratic course.
Sometimes almost complete reversion occurs rather quickly,
as wdth Bacterium coli trained to utilize D-arabinose (Cross
and Hinshelwood, 1956), and with some yeast strains made
drug-resistant (Wild and Hinshelwood, 1956). Sometimes
reversion is hastened by growth in media to which fresh
adaptation is needed, the disturbing effect of the new adjust-
ment relieving the metastability of the old. For example,
growth in the presence of phenols resulted in a loss of the
adaptation of Bacterium lactis aerogenes (Aerohacter aerogenes)
to proflavine (Davies, Hinshelwood and Pryce, 1945) and
adaptation to proflavine of a sulphanilamide-trained strain
of the same organism led to the loss of the sulphanilamide
adaptation (James and Hinshelwood, 1947). With Aero-
hacter aerogenes, adaptation to D-arabinose is gradually
removed in this way. That the phenomenon is not due to the
re-selection of a few reverse mutants has been shown by the
fact that deliberately added cells of the original untrained
strain are in fact not preferentially supported by the media
used (Cross and Hinshelwood, 1956; Baskett and Hinshel-
wood, 1951).

In general, the more thoroughly the training to drugs has
been impressed on the bacterial cells the less readily is it lost.
For example, cells which have just acquired the ability to
grow in the presence of the drug readily lose it on subculture

Drug Resistance in Bacteria 9

in a drug-free medium. As training proceeds reversion takes
place less readily until eventually the adaptation appears to
be relatively stable. This pattern of behaviour is seen in the
adaptation of Aerohacter aerogenes to proflavine (Davies,
Hinshelwood and Pryce, 1944, 1945; Pryce and Hinshelwood,
1947), and to sulphanilamide (Davies and Hinshelwood, 1943).
Reversion when it does take place need not be complete but
a lower level of immunity, the "equilibrium state", may be
reached and held for a considerable time.

The stability, however, is never absolute. For example.
Dean and Hinshelwood (1954a) have trained Aerohacter
aerogenes to moderately high concentrations of proflavine,
propamidine and chloramphenicol and have subcultured these
trained strains for a very long time (about 1,000 generations)
in the drug medium. The adaptations, although of consider-
able stability, were eventually lost on long-continued sub-
culture in a drug-free medium, thus emphasizing the fact that
arguments from stable heredity cannot by themselves be used
to disprove the theory of environmental response. Moreover,
the entire pattern of events in these and in the earlier drug ex-
periments was more easily explained by an adaptive hypothesis
than by a theory involving mutations and reverse mutations.

Similar results have been obtained with Aerohacter aerogenes
and acetate and with Bact. coli mutahile and lactose as sole
carbon sources. In the latter example although it has been
relatively easy to detrain the Lac^ strains partially (Dean
and Hinshelwood, 1954c) complete reversion to the Lac~ state
has proved more difficult. It has, however, been achieved in
one or two cases (Dean and Hinshelwood, unpublished).

Mass -Number Relations

If the only cells to develop and multiply in a given medium
are pre-existent mutants, the bulk of the population remaining
inert, then there can be no increase in the mass of the culture
as a whole without a corresponding increase in the number of
cells. If the average size of a mutant is nearly equal to that

10 A. C. R. Dean and Sir Cyril Hinshelwood

of a non-mutant (and it will never differ from it by more than
a small factor) then an increase of mass of x per cent will only
occur as a result of an increase in number of approximately
the same amount.

The proportion of mutants initially present is usually
assumed to be about 10"^ (to account for observed delays in
growth), and the approximate doubling of the mass of each
mutant, which would precede its division, would make an
unobservably minute contribution to the change in the total
mass of the culture.

If, on the other hand, most of the cells in the culture de-
velop after a suitable lag period, there can be a substantial
increase in mass before any detectable increase in number
occurs. In the simplest case, where the lags are all equal and
each cell doubles in size before dividing, there will be a
100 per cent increase in mass before the numbers increase.
This limiting case would, however, not be observed since
some of the cells divide before others have completed their lag.

Nevertheless, in several examples of adaptation to new
carbohydrate sources increases in mass of about 40 per cent
have been observed without any observable multiplication.
Since no cell is likely to grow to much more than about
double its original size, this result indicates that at least a
considerable proportion of the population is concerned in the
adaptive process (Baskett and Hinshelwood, 1951; Kilkenny
and Hinshelwood, 1951; Minis and Hinshelwood, 1953).

Tests for the Presence of Mutant Forms in Massive

Inocula

A mutation rate of about 1 in 10^ is not infrequently
assumed for bacteria. This would mean a very small pro-
bability of any mutants at all in an inoculum of 10* and near
certainty of the presence of several in an inoculum of 10^.
Frequently the plating of 10 to 100 cells on a medium to
which adaptation is required leads to the formation of
colonies in 100 per cent yield, but only after a long lag. In

Drug Resistance in Bacteria 11

such cases the further assumption is sometimes made that an
initial proUferation of the inoculated cells occurs through the
intervention of impurities in the plate-medium, and that
during the process mutations occur so that each of the micro-
colonies formed in this preliminary growth contains at least
one mutant. This mutant can then eventually multiply to
give the final colony. Objections to the universal application
of the underlying assumptions have already been mentioned.
However, when they are accepted they are usually coupled
w^ith the analogous one that if a large enough inoculum is
plated those few cells which form colonies not later than a
given relatively short time, regarded as the normal develop-
ment time, represent the mutants initially present.

To test the likelihood of this interpretation experiments
have been made on the rate of development of colonies of
Bad. coli rnutabile on lactose-agar and of Bad. ladis aerogenes
on D-arabinose-agar. Inocula were varied from 10 to 10^, and
distributions of sizes and numbers of colonies at various times
were recorded (Dean and Hinshelwood, 1956; McCarthy and
Hinshelwood, 1957).

The conclusion reached was that in these examples the
colonies which appeared the earliest need not be ascribed to
any special mutant type but represented nothing more than
the tail of the nearly Gaussian distribution which the develop-
ment times (for a given colony size), or the sizes at a given
time, were found to follow.

Time -Number Relations

When resistance can be developed by training, the assump-
tion commonly made is that drug-resistant mutants are
present in minute proportion in the culture before it has ever
been exposed to the drug. Some cultures may, of course, be
heterogeneous, containing cells with a higher natural resist-
ance than others, and such cells would be enriched by selection.
This, however, is quite a different proposition from the denial
of direct adaptive processes as possible in themselves.

12 A. C. R. Dean and Sir Cyril Hinshelwood

That in certain examples the resistant forms, which
emerge from the process of "training" in the presence of the
drug, are not present at all in the original culture is strongly
supported by a study of the development as a function of
time of colonies on drug plates. Suppose a given number of
cells to be plated. Let aQ^ be the fraction which ever form
colonies and a^ the fraction which has done so at time t.
With a trained strain, a^^ will approach unity, while with an
untrained strain it may vary from near unity to a vanish-
ingly small value according to the drug concentration. Even
when aoo is very small countable numbers of colonies can never-
theless be obtained by the use of large enough inocula. If
these inocula contain preformed resistant mutants similar
to those in the trained strain, then clJol^, no matter how small
aQo may be, will be a function of time similar to that which
would be found in experiments with the trained strain itself.
In a number of examples tested, however, this consequence
was not verified. The time required for a given fraction of the
final number of colonies to appear was much longer for the
untrained strain. Thus it would seem that the nature as well
as the number of the resistant cells in the trained culture
differs from that of anything present in the original culture
(Dean and Hinshelwood, 1955).

The only way of reconciling these observations with the
uncompromising mutation-selection theory is to postulate an
almost continuous series of minute mutational steps, and to
assume that the chance of a considerable number of successive
mutations is negligible unless cells that have already taken a
few of the steps are first selected and then given further
opportunities for taking subsequent steps. But in some ex-
amples aQQ may be not far short of unity (i.e., nearly all the
population consists of mutants) and yet the time of colony
formation is longer than for a trained strain. So it seems
(a) that there would have to be a very high proportion of
early step mutants and (b) that even these are not fully adapted
at first. If (a) is true then the complete absence of the more
profoundly mutated cells is strange, and if {h) has to be

Drug Resistance in Bacteria 13

assumed, the usefulness of the basic assumption about pre-
existing mutants loses most of its point.

Adaptation of Bacterial Cultures during the Lag Phase
in Media containing New Substrates or Antibacterial
Agents

There is a long lag when cultures of Bad. lactis aerogenes
are introduced for the first time into media containing
D-arabinose as the sole carbon source. Baskett and Hinshel-
wood (1951) showed that if samples are withdrawn at intervals
during this lag phase and are plated on D-arabinose agar the
time taken by the colonies to reach the A+ size progressively
diminishes as the time of sojourn in the liquid medium
increases. Similar results have been reported for Bad. coli
mutabile and lactose (Dean and Hinshelwood, 19546). Since
the majority of the cells in the culture took part in the
adaptive response and since the response preceded the growth
of the culture these results were interpreted as showing that
the substrate induces the adaptation. An explanation based on
the selection of spontaneously-arising pre-adapted cells is not
compatible with the experimental findings.

More recently Dean (unpublished) has investigated this
topic in greater detail. He used Bad. coli mutabile with
lactose, Escherichia coli Kl2 with D-arabinose and with dul-
citol and Bad. ladis aerogenes with D-arabinose. His experi-
ments confirmed the earlier interpretation but in addition he
found that the response of the cells to the environment was
of three types. Usually there was a considerable increase in
cell mass towards the end of the lag phase and this preceded
any division. Less frequently, however, division preceded
any swelling of the cells. The third type of behaviour was
characterized by swelling and division taking place almost
simultaneously.

The experiments in which the first two types of behaviour
were observed showed the progressive reduction in plate lag
reported earlier. Those in which the third type of behaviour

14 A. C. R. Dean and Sir Cyril Hinshelwood

was in evidence also showed the reduction in lag but since
the increase in mass and the onset of cell division took place
almost at the same time as the plate lag began to fall it was
not possible to draw any definite conclusions from them. Of
a series of eighteen experiments nine were of the first two types
and nine were of the third type.

A technique somewhat similar to that of Baskett has been
used by Akiba (1955) and by Szybalski (1954-55). They have
shown in certain cases that cells exposed to streptomycin
were, at the end of a definite period, if they had survived at
all, fully resistant to the drug. Akiba and Szybalski, however,
used a medium lacking the materials essential for division
while Baskett and Hinshelwood used a medium which would
support growth and division.

Dean (unpublished) has carried out experiments of the
Akiba-Szybalski type with Bad. coli mutabile and lactose and
with Bad. ladis aerogenes and D-arabinose by omitting a
nitrogen source from the medium. Out of four experiments
two gave definite positive results as regards the plate lag
whilst in the other two very little adaptation took place. In
another experiment which involved Bad. ladis aerogenes and
both D-arabinose and streptomycin, the plate lag on D-arabi-
nose and D-arabinose-streptomycin agar dropped progressively
in the usual manner while the survival on streptomycin plates
containing either glucose or D-arabinose as sole carbon sources
gradually increased to 100 per cent. In a final experiment
involving Bad. coli mutabile and chloramphenicol the survival
on chloramphenicol-agar gradually increased to 100 per cent.

Although this experiment was continued until the viable
population had fallen from 10^/ml. to 400 cells/ml. there was
no evidence of lysis, a fact which excludes the possibility of a
multiplication of mutants on the debris from other cells.

Graded Response

Sometimes the degree of resistance of bacteria to a drug is
continuously graded to conform to the exact concentration

Drug Resistance in Bacteria 15

at which "training" has been carried out. A good example
of this type of behaviour is found with Bad. lactis aerogenes
and proflavine and it is easily explained on an adaptive
theory involving an automatic adjustment of the enzyme
systems in the cells in response to the environment (Davies,
Hinshelwood and Pryce, 1945; Dean, 1955). The alternative
is to assume a complex polygenic system — a theory which
encounters difficulties when the resistance of colonies picked
from proflavine plates is re-tested. It is found that resistance
or non-resistance on re-test depends on the buffering capacity
of the agar medium in the primary plating. Since proflavine
is antagonized by the acids produced by growing cells, a simple
explanation of a non-genetic nature can be given. It is that on
the lightly buffered plates cells which have just begun to
adapt to proflavine will de-adapt when the acid antagonizes
the drug and hence on re-test would be expected to be no more
resistant than in the primary test. On the well buffered plates,
however, the acids produced by the growing cells will not be
present in sufficient amount to change the pH of the medium
and hence no considerable antagonism of the drug or de-
adaptation will take place (Dean and Hinshelwood, 1955).

Accelerated Adaptation to Drugs

It has been shown that if proflavine is added gradually to
an actively growing culture of Bad. ladis aerogenes the cells
can be rapidly adapted to grow in concentrations of drug
which if added directly to the culture would cause long lags
or even cessation of growth. Using this method Baskett
(1952) was able to adapt cells of Bad. ladis aerogenes to
110 mg./l. of proflavine in 220 minutes, a time interval too
short for an appreciable selection of pre-existing proflavine-
resistant mutants in the culture.

Dean (unpublished) has carried out similar experiments
with Bad. ladis aerogenes and proflavine and sodium azide.
Any pH changes in the medium were carefully followed since
both proflavine and azide would be expected to be less active

16 A. C. R. Dean and Sir Cyril Hinshelwood

at lower pH values. In the proflavine experiments concen-
tration levels of 42 and 63 mg./l. were reached in 98 and
185 minutes respectively. In the first case the pH was un-
changed at the end of the experiment and in the second case
it had dropped from 7-0 to 6-7. Controls in which the cells
were inoculated directly into media adjusted to the pH
reached at the end of the respective experiments and con-
taining 42 and 63 mg./l. of drug respectively had lags of
380 and 1,300 minutes respectively.

In experiments with sodium azide, concentrations of 263
and 430 mg./l. were reached in two experiments lasting for
130 and 245 minutes respectively. Controls put up as in the
proflavine experiments had lags of 3,000 minutes and infinity
respectively.

There can be little doubt that these experiments involve
the adjustment of the cells to the adverse environment since
the time intervals are too short for any extensive selection of
pre-existing resistant mutants. This "physiological" adapta-
tion, is however, unstable on subculture. Continued subcul-
ture in the drug media stabilizes it. In this process there
would be time for selection. It would, however, be rather
surprising if there were two quite distinct mechanisms
involved in the development of a resistance and the gradual
stabilization of that same degree of resistance to the same
drug by the same organism. This matter is under further
investigation.

Binding or Adsorption of Drugs by Resistant and Non-
Resistant Cells

Proflavine-resistant cells of Bad. lactis aerogenes take up
from solution not less but more proflavine than those of the
non-resistant strain from which they have been derived
(Peacocke and Hinshelwood, 1948). On the other hand,
certain phage-resistant forms of Esch. coli B take up no phage,
in contrast with the corresponding sensitive forms for which
Brenner (1955) determined the adsorption isotherms. Eagle

Drug Resistance in Bacteria 17

(1954) found that some penicillin-resistant forms of bacteria
take up more and some take up less of the drug than the
sensitive forms.

These facts support the idea that there may be more than
one mechanism of resistance. If the cell has suffered damage
(e.g. by exposure to radiation) so that it has lost receptors
which bind the drug, then it may thereby acquire a certain
passive kind of resistance which would contrast with a more
active type of resistance to be suspected in those examples
where the drug is actually taken up more readily by the
adapted form.

The passive type due to loss of a function would be that for
which a spontaneous origin could be most readily explained.

Among drug resistances, as distinct from resistance to
phage, that to streptomycin seems more likely than many to
have, on occasion though not necessarily always, a spon-
taneous origin. In this connexion the adsorption isotherms
are of interest. Dean (unpublished) found that the sensitive
form of Bad. lactis aerogenes took up streptomycin according
to a conventional type of adsorption isotherm. A resistant
strain took up little or none in most experiments, but occasion-
ally showed a positive adsorption. If the resistant bacteria
were grown anaerobically, however, the adsorption was once
more considerable. The phenomena are still under investiga-
tion but they seem, on the whole, to provide evidence that loss
of receptors may play some part in one type of streptomycin
resistance of at least some bacteria.

The complexity of the situation is, however, illustrated by
the fact that Neumark and Pasynskii (1954) found about
equal adsorptions of streptomycin for resistant and sensitive
varieties of the same strain of Staphylococcus aureus.

Papilla Formation

The papillae or secondary colonies which, in certain con-
ditions, form on the edge or on the surface of primary colonies
have generally been supposed to owe their origin to mutant

18 A. C. R. Dean and Sir Cyril Hinshelwood

cells. We have set forth elsewhere evidence in support of our
view that this is an oversimplified, and sometimes incorrect
interpretation of the phenomenon (Dean and Hinshelwood,
1957). Our present views, and the arguments for them, may
be summarized as follows.

(1) Secondary colony formation is essentially a pheno-
menon shown by the ageing primary colony. In this we are
in complete agreement with the work and views of Haddow
(1937).

(2) Observations on the very early stages of colony
growth show that cells align themselves into more or less
regular arrays giving the colony a characteristic internal
structure. This is generally very close-packed.

(3) Growth of the colony stops when nutrient is exhausted
or toxic products accumulate. Renewed growth is only pos-
sible when one of several things has occurred. The cells have
thrown off mutants or have adapted themselves to utilize
a substrate not utilized at first, or they have become resistant
to something which has hitherto impeded their growth, or
regions of lysis occur permitting cannibalism in parts of the
existing colony, or cracks and channels may develop in
the mass of the colony allowing nutrient to diffuse from the
medium below to its surface so that fresh growth can take
place there.

(4) The renewed growth can result in papilla formation,
when for any of a number of reasons it is localized. If, for
example, capillary channels to feed nutrient to the aerated
surface of the colony are required, the papillae will occur at
the points where such "craters" exist. The surface may be
preferred to the periphery because the concentration of toxic
products is lower. Particular points on the periphery may be
preferred for such reasons as that the highly heterogeneous
microstructure of the agar gel there provides adsorption sites
which remove inhibitors, or concentrate growth factors.

(5) If renewed growth depends in this way on fortuitous
structural factors, the new array of cells will not conform
to the former one in orientation and packing, and a visibly

Drug Resistance in Bacteria 19

distinct secondary colony will result. Heterogeneity of colony
form is sometimes manifested in ways other than papilla
formation — colonies with crinkled edges, "rough" colonies
and so on. In the course of our work we have often observed
in ageing colonies the development of "lenticular" areas of
changed internal colony texture, possibly connected with
local lysis and re-growth. The papilla is one of quite a series
of departures from regular monotonous colony development.
(6) Several lines of experimental evidence show that
papillae need not arise from mutants.

(a) When drugs, such as phenol or thymol, are added to the
solid medium, papillae occur in numbers which do not increase
with the total number of cells grown (i.e. with the chance of
mutation) but simply with the age and diameter of the colonies.
The same total growth distributed among a large number of
smaller colonies may be associated with no papilla formation
at all, even though the chance of mutation is as great, and the
opportunity for mutants to develop is probably greater.

(b) Re-spreading of inocula derived from the secondary
colonies sometimes give colonies still showing papillae — even
to the sixth re-spreading. In general, re-spreading may lead
to fresh papilla formation or not, according to circumstances.
Where it does not, the disappearance is usually attributed to
the selection of the mutants. This conclusion is, however,
ambiguous. When any form of adaptive response, whether
by mutation or otherwise, has once occurred (as it can do
during the slow growth of a primary colony on an initially
unsuitable medium) the times of colony formation in the
"re-spreading" experiment are less than in the original test.
Thus the period elapsing between the initiation of growth
and the final complete exhaustion of the medium is reduced.
By the time any parts of the colony have aged enough for
papilla formation to occur, all nutrients may have been
removed and no secondary growths can develop. In the first
plating the utilization is so slow that some parts of the colony
age sufficiently while there is still unexhausted material for
growth.

20 A. C. R. Dean and Sir Cyril Hinshelwood

(c) When colonies are formed in such examples as the growth
of Bact coli mutabile on lactose or Bact. lactis aerogenes on
D-arabinose, the primary colony has often become so large
before any of the secondaries appear that to attribute all the
utilization of the adaptation-requiring substrate to these is
impossible. Moreover, re-spreading of inocula taken from
primaries and papillae on lactose-endo-agar plates may result
in nearly as many Lac"*" colonies from the former as from the
latter. In general, however, the papillae should yield more
thoroughly adapted cells since they have utilized lactose after
the lag required for the adaptation, whereas parts of the
primary grew on peptone, and had no opportunity for using
lactose (Dean and Hinshelwood, 1957).

Fluctuation Tests

The belief that the Luria and Delbriick fluctuation test is a
reliable proof of the spontaneous origin of drug-resistant
mutants or of mutants capable of utilizing new substrates is
now less widely held than formerly. Factors other than
mutation, which are not usually controlled rigidly in the test,
have been shown to be capable of producing the observed
variation between samples from the same culture and samples
from different cultures. It has also been questioned whether
Newcombe's spreading technique provides unambiguous
evidence for the existence of spontaneous mutants and
whether the results of the fluctuation test are necessarily
strengthened by the inclusion of a test for correlation between
relatives or by invoking the Lea-Coulson distribution (Dean
and Hinshelwood, 1952a and b; Hinshelwood, 1953; Dean,
1955).

The Technique of Replica Plating and Related
Methods

The natural level of resistance of bacteria to drugs differs
among varieties of the same strain and among closely related

Drug Resistance in Bacteria 21

strains. Bad. lactis aerogenes, for example, will grow without
previous adaptation in presence of proflavine at concentra-
tions several times greater than those which completely
inhibit the growth of most strains of Esch. coli.

The biochemical history of the strain also affects the
natural resistance level. A strain of Esch. coli transferred from
a broth medium to a minimal glucose-ammonium sulphate
medium acquired substantially increased resistance to pro-
flavine (in the minimal medium) as it became thoroughly
adapted to the medium itself. The maximum concentration
of proflavine tolerated rose about threefold (McConnell,
unpublished). Adaptation of Esch. coli to various nutrient
sources caused changes in the resistance level to various drugs
within a range of 50 to 150 per cent.

These relatively minor diff'erences may be of structural
(genetic) origin, or may reflect changes in the enzymic
organization of the cell as the case may be. Cavalli-Sforza
and Lederberg (1956), by a selection technique with liquid
media, obtained strains which showed an increase from about
10 to about 35 mg./l. in the maximum concentration of
chloramphenicol which they would tolerate. We have, as
stated, observed variations of this order in the proflavine
resistance of mass cultures not subjected to selective tech-
niques, and the increase from 10 to 35 mg./l. in the chloram-
phenicol, even if it is due to the selection of mutants is very
small compared with the increase to many hundreds which is
readily achieved by culture in presence of the drug itself. A
strain of Esch. coli has been trained to resist 1,100 mg./l. of
chloramphenicol.

With streptomycin, as with phage, much more drastic
increases in resistance have been reported (Lederberg and
Lederberg, 1952; Cavalli-Sforza and Lederberg, 1956). One
of the forms of streptomycin resistance is probably due to loss
of receptors for the drug, just as some phage resistance is due
to inability of the cell to take up the phage. This passive type
of resistance is, in our view, much more likely than any other
to arise spontaneously, since most controlled mutations seem

22 A. C. R. Dean and Sir Cyril Hinshelwood

to be destructive, and in the course of their normal hfe the
cells encounter destructive agencies such as radiations in an
unpredictable manner.

Nevertheless, in the absence of more detailed knowledge of
the structural changes which determine difference in bio-
chemical properties, it would be unwise to neglect the possi-
bility that spontaneous mutations giving rise to positive new
capacities in the cell may sometimes occur, and if they do
they could be selected. The fact that sometimes such mutants
can be enriched by selective methods (though we are rather
doubtful about the evidence of successful application of these
methods except in the case where the passive type of resist-
ance is at least possible) does not in any way show that direct
adaptation is not also a common method by which drug
resistance appears.

The positive evidence for this direct adaptation is strong,
and the arguments have already been summarized. One final
comment may be added: by methods of accelerated training
high degrees of unstable resistance can be produced under
conditions where selection could not possibly have operated.
We do not believe that the gradual and progressive stabiliza-
tion of this represents the complete substitution of one
phenomenon by another having no connexion with it.

In conclusion, perhaps we should make clear once more
that we have never contested the part that selection of
mutants may play {cf. Hinshelwood, 1946). If that denial has
sometimes been gratuitously made for us by others, it is per-
haps because we have been more interested in investigating
and following up the nature of the adaptation process itself.
This, indeed, is a matter of very great biochemical and bio-
physical interest, and should need no apology, though it may
not seem the aspect of major interest to those whose approach
is through classical genetics. Similar efforts to form a picture
of how spontaneous mutations occur, and how they lead to
resistance, is another problem well worthy of attention.

Drug Resistance in Bacteria 23

REFERENCES

Akiba, T. (1955). In Origins of Resistance to Toxic Agents, p. 82. Ed.,

Sevag, M. G., Reid, R. D., and Reynolds, O. E. New York:

Academic Press.
Baskett, a. C. (1952). Proc. roy. Soc. B, 139, 251.
Baskett, a. C, and Hinsiielwood, Sir Cyril (1951). Proc. roy. Soc. B,

139, 58.
Brenner, S. (1955). Proc. roy. Soc. B, 144, 93.

Cavalli-Sforza, L. L., and Lederberg, J. (1956). Genetics, 41, 365.
Cross, J. R., and Hinshelwood, Sir Cyril (1956). Proc. roy. Soc. B,

145, 516.
Davies, D. S., and Hinshelwood, C. N. (1943). Trans. Faraday Soc.y

39, 431.
Davies, D. S,, Hinshelwood, C. N., and Pryce, J. M. G. (1944).

Trans. Faraday Soc, 40, 397.
Davies, D. S., Hinshelwood, C. N., and Pryce, J. M. G. (1945).

Trans. Faraday Soc, 41, 778.
Dean, A. C. R. (1955). In Origins of Resistance to Toxic Agents, p. 42.

Ed., Sevag, M. G., Reid, R. D., and Reynolds, O. E. New York:

Academic Press.
Dean, A. C. R., and Hinshelwood, Sir Cyril (1952a). Proc. roy. Soc B,

139, 236.

Dean, A. C. R., and Hinshelwood, Sir Cyril (1952&). Proc. roy. Soc. B,

140, 339.

Dean, A. C. R., and Hinshelwood, Sir Cyril (1954«). Proc roy. Soc,

B, 142, 45.
Dean, A. C. R., and Hinshelwood, Sir Cyril (1954&). Proc roy. Soc,

B, 142, 225.
Dean, A. C. R., and Hinshelwood, Sir Cyril (1954c). Proc. roy. Soc,

B, 142, 471.
Dean, A. C. R., and Hinshelwood, Sir Cyril (1955). Proc. roy. Soc. B,

144, 297.
Dean, A. C. R., and Hinshelwood, Sir Cyril (1956). Proc. roy. Soc.

B, 146, 109.
Dean, A. C. R., and Hinshelwood, Sir Cyril (1957). Proc. roy. Soc.

B, in press.
Eagle, H. (1954). J. exp. Med., 99, 207; 100, 103; 100, 117.
Haddow, a. (1937). Acta Un. int. Cancr., 2, 376.
Hinshelwood, C. N. (1946). The Chemical Kinetics of the Bacterial

Cell. Oxford: Clarendon Press.
Hinshelwood, Sir Cyril (1953). Symp. Soc. gen. Microbiol., 3, 18.
James, A.M., andHiNSHELwooD,C.N.(1947).rm/i5.Fararfa?/ 5*00., 43, 274.
Kilkenny, B. C, and Hinshelwood, Sir Cyril (1951). Proc roy. Soc.

B, 139, 575.
Lederberg, J., and Lederberg, E. M. (1952). J. Bact., 63, 399.
McCarthy, B. J., and Hinshelwood, Sir Cyril (1957). Proc roy. Soc.

B, in press.
MiMS, N. M., and Hinshelwood, Sir Cyril (1953). J. chem. Soc, p. 663.

24 A. C. R. Dean and Sir Cyril Hinshelwood

Neumark a, M., and Pasynskii, A. G. (1954). Dokl. Akad. Nauk.,

USSR, 95, 329.
Newcombe, H. B. (1948). Genetics, 33, 447.
Newcombe, H. B. (1953). Genetics, 38, 134.
Peacocke, a. R., and Hinshelwood, Sir Cyril (1948). J. chem. Soc,

p. 2290.
Pryce, J. M. G., and Hinshelwood, C. N. (1947). Trans. Faraday

Soc, 43, 1.
Ryan, F. J. (1955). Genetics, 40, 726.
SzYBALSKi, W. (1954-55). Antibiotics Annual, p. 174.
Wild, D. G., and Hinshelwood, Sir Cyril (1956). Proc. roy. Soc. B,

145, 32.

DISCUSSION

Lederberg: In 1954, Sir Cyril, you sent us a culture of your Bad. lactis
aerogenes ; we have been experimenting with it from time to time, and I
regret to say that we do not get the observational results that you have
indicated. According to your published results (Baskett and Hinshel-
wood, 1951, loc. cit.), the inoculation of this organism into tubes of
minimal medium containing D-arabinose as a sole carbon source is
followed by an interval in which there is an increase in total mass;
and during this interval there may be a homogeneous response during
which each of the cells in the suspension has become gradually (but
all of the cells uniformly) better adapted to this environment, so that
when they are plated they show a uniform decrease in the lag for the
time of colony development. Our own findings are that when samples
are taken from a tube inoculated — say with 10'' or 2 x 10' cells per ml.
— in such a medium, and these samples are plated out at half-daily or
daily intervals, there is a variable time of onset of visibly turbid growth.
That is invariably preceded by the appearance of a large colony-forming
type, and during the interval this increase in turbidity gives a very
clearcut distinction between small and large colonies, and the large
colonies simply increase. I have seen nothing in this system which is not
most readily explainable by the sporadic occurrence of a better adapted
mutant which will form large colonies. However, there are several
features in this system which make it less precise to work with than some
other selectable systems, and which may perhaps account for some of the
questions of increase in mass. It is quite clear that the original type is
capable of utilizing arabinose, but I think at a much slower rate than
the wild type. Even unadapted cells will form colonies, so that as these
colonies are replated they continue to form visible colonies ; it may take
a week before they reach a size of 1 mm. or so in diameter. Every once in
a while one finds a colony — one of a number of hundreds of colonies on a
plate — that has become quite large over a period of a day or two, and
this on replating always gives a large colony. In addition, there is a very
large stimulation of colonies by products from the adapted types, and I
suspect that is one of the reasons for the conclusion that there is a uni-
form response. If the colonies are plated too densely there is a marked

Discussion 25

stimulation from the plus to the minus colonies so that there appears to
be a uniformity of colony type which in fact does not exist, as can be
shown by replating. To summarize: my own experience has been that
there occurs from time to time, in these cultures held in D-arabinose
medium, a new cell type which increases very rapidly over a period of
about 1-1 • 5 days, and that this overtakes the bulk of the population ;
but one can see these two colonies side by side with one another in these
platings, provided they are not plated too densely.

Hinshelwood: We never get this division into two sharply defined types
unless actual growth and multiplication of numbers in the culture is
already well under way. With the arabinose-negative t>T)e and the
lactose-negative type we get at any given time a distribution of colony
size, or for any given size a distribution of times, which is practically a
Gaussian distribution.

The essential point of our argument is that before any growth or multi-
plication in the liquid medium begins all or most of the cells show a
reduction in the actual time required to form a colony of standard size
on the plate. I should attribute Prof. Lederberg's large colonies to cells
taken from a culture in which growth in the liquid has already begun to
make some appreciable progress. A very careful watch on total and
viable counts is necessary. There are two arguments : increase in mass
precedes increase in number, and decrease in plate lag may precede either.
Plating after measurable increase in turbidity may give colonies from
cells which have reached a still more advanced stage of adaptation.

Lederberg: We have done a variety of experiments. If we put e.g. 100
cells into a plate then we get a fairly uniform development which gives
colonies of quite good size over a period of a week. When these colonies
are replated they almost invariably give exactly the same picture,
though it may take another week before they come out. Occasionally
one finds a colony that has an actual burgeoning-out from it, and when
such a colony is replated it gives the plus-type which immediately grows
up (it takes about a day and a half to reach the same colony type). This
experiment can be done, for example, by making rather dense platings
of about 10* or 10^ cells into a plate; this gives a sort of ground glass
appearance, and with a binocular microscope one can see the individual
colonies. Undoubtedly the minus-type is capable of utilizing at a fair
rate either D-arabinose (which I suspect is the case) or some contaminant
in the preparation. I think some of the imprecision that may creep in
here is due to the fact that we are not jumping from a zero state to a
100 state, but from a 5 state to a 75 state, so to speak, as compared with
the rate of utilization of glucose. In such densely inoculated plates there
appear from time to time, over a period of a week or ten days, occasional
large colonies which are surrounded by a very dense halo of satellites of
colonies from the background. Now, if we replate the large ones, which
are quite rare, these give new platings which consist mostly of large
colonies. If we replate from the satellites immediately around them, or
from the background growth, we generally get a small colony develop-
ment. So here are cells, side by side in the same environment, which have
quite diverse histories; and there must occur a sudden "catastrophe"

26 Discussion

which strikes an occasional part of the plate and gives rise to wide
divergences in subsequent behaviour.

Hinshelwood: Do you keep complete time-size logs of all the colonies
on the plate? We have always thought it necessary to do this, i.e. to
measure or photograph the plate day by day or every half-day. Other-
wise, if you inspect things at arbitrary intervals and arbitrary sizes you
do tend to create sharp distinctions which may not exist. These are
especially marked if the liquid culture has already begun to multiply.

Lederberg: No, we have not done this. Nor are we acquainted with such
a detailed presentation in the literature.

Pollock: We have done some work on the original strain of Bad. coli
mutabile used by Sir Cyril (see Dean and Hinshelwood, 1954&, loc. cit.)
for studying the lactose training phenomenon which he maintains is
specifically induced in most of the cells by lactose. Most of our findings
correspond to what Lederberg has described for D-arabinose training.
Cells untrained to lactose (Lac~) plated into lactose-agar produce
small colonies (Fig. la) compared to cells from a lactose-trained culture
(Lac+) which form much larger, denser colonies (Fig. lb) in the same
period (5-7 days). If you leave the Lac~ colonies for longer, most of
them eventually grow into large colonies (Fig. Ic) comparable in size
to the Lac+ colonies (as described by Dean and Hinshelwood) but
mottled and irregular in appearance. A series of stages in the develop-
ment of Lac~ colonies is illustrated in Fig. 2a to / showing clearly
the emergence of papillae. Subcultures from the papillae yield nearly
100 per cent colonies of the Lac+ type (Fig. lb) in lactose agar. It
might be quite easy to miss these papillae in colonies growing deep
in agar and they were only easily visible by the use of a binocular
plate microscope. With surface-inoculations, the papillae were quite
obvious to the naked eye and it was much easier to distinguish between
colonies of the Lac+ and Lac~ type. Fig. 3 is a re-analysis of the Hin-
shelwood and Dean curve of increase in cell numbers of a glucose-grown
culture inoculated into a liquid lactose medium. As well as plating
samples out into glucose agar (for total viable count) they were also
plated into lactose agar and Lac+ and Lac~ colonies counted. The total
viable count corresponds exactly with Dean and Hinshelwood's curve,
but it can be seen that the final rise in numbers is due entirely to growth
of Lac''' cells (which have, indeed, been increasing logarithmically during
most of the experiment). With surface inoculation on lactose agar there
is absolutely no difficulty, after 4 days of incubation, in distinguishing
the two types of colony. The Lac"'" colonies are opaque and dome-shaped
and nearly twice the diameter of the much thinner and flatter Lac~
colonies. Fig. 4a shows the appearance of colonies, after 8 days of incu-
bation, from a Lac~ culture inoculated on the surface of lactose agar.
Fig. 4<b shows 3 Lac"^ and 3 Lac~ colonies from an artificial 50 : 50 mixture
of Lac+ and Lac~ cells inoculated in the same way and incubated for
the same period. The Lac~ colonies in Fig. 46 are about twice the dia-
meter of those in Fig. 4a; their growth has obviously been greatly
stimulated by a substance produced by the Lac+ cells. It might be
something as simple as glucose. It seems possible that this effect might

•

Fig. 1 (Pollock). Single deep colonies of Bad.
coli mutabile orowing in lactose-aoar. X 80.
a. From Lac " inoculum. 7 days" incubation.
h. From Lac+ inoculum, .'j days" incubation.
c. From Lac~ inoculum. 9 days" incul)ati()n.

[fitcing page 26

#

f

Fig. 2 (Pollock). Single deep colonies (chosen at random) from Lac"
moculum of Bact. coU mutabile growing in lactose-agar. 7-9 days' incubation

X80.

Total

Fig. 3 (Pollock), Numbers of Lac + and Lac ~ colonies found on plating out on

the surface of lactose-agar samples of a culture of Bact. coli tnutabUe (from a

glucose-grown inoculum) during "• training** in a liquid lactose medium.

m

• id^

^^P-

Fig 4 (Pollock). Colonies of Bad. coli mntahile orowing on the surface
of lactose-agar. 8 days' incubation. X 8.

a. From Lac - inoculum (all Lac ' colonies). ^f t «p - and T ac +

h From an inoculum containing a 50 : oO mixture of Lac and Lac
cells (showing 3 Lac + and 3 Lac - colonies).

Discussion 27

explain why Dean and Hinshelwood failed to observe the two distinct
types of colony in their experiments during the course of training to
lactose. All these findings obviously do not explain the origin of papillae ;
nor do they exclude the possibility of some direct induced change in
individual cells, as has been claimed. I only suggest that it is much
simpler to assume that you have got some kind of mutation in a very
few cells, followed by selection.

Hinshelwood: We don't agree with the interpretation of that curve.
We don't agree that if you plate out and then observe the relation be-
tween the time and the size of the colony you find this distinction
between the two types. Moreover, we are very well aware of the mutual
stimulation, and indeed have published a paper on it. But if you plate
a culture that has been lagging in a medium, and you get perhaps 30-40
colonies all in a short space of time, it is hardly possible for those to have
stimulated one another. If thej^ have been lagging in the medium for
24 hours, and you plate out enough cells to give a total count of 50 and
these all come up, e.g. within 30 hours of one another and not at widely
different intervals, then they do not have much chance to stimulate one
another.

Pollock: I am thinking of the half- trained culture.

Hinshelwood: Yes, that is exactly what I am talking about.

Pollock: Would that not be 2 or 3 or 4 days, rather than 24 hours?

Hinshelwood: It depends on how advanced the stage of training is.

Pollock: We did not pursue the problem any further, and we did not
prove anything, but mutation followed by selection seems a likely
explanation.

Hinshelwood: I do not see how this matter of having two juxtaposed
colonies, one affecting the other, is relevant to this particular question.

Pollock: My point is that it might be very misleading when you were
scoring your large colonies and your small colonies.

Dean : The colonies are too far apart on the Petri dish to stimulate one
another.

Pollock: We find that you get stimulation over a very long distance.

Hinshelwood: That is when a big colony has been formed and the small
colony is near it.

Lederberg: I have seen this develop in the course of two days, e.g. with
D-arabinose.

Pontecorvo: Prof. Hinshelwood has clearly stated that, of course, he
does not deny that there are changes in cell structures which segregate ;
on the other hand, no geneticist will deny that there are permanent
heritable changes definitely not due to changes in structural elements like
chromosomes. If we denied this of course we would deny somatic differ-
entiation in higher organisms! Now what seems to me an important
point to discuss is that of the relations between kinetic changes and
changes in cell structures during a process of phylogenetic adaptation.
This has been attempted at the level of higher organisms : Waddington,
for instance, has developed theories to show how responses which are
at first directly elicited by an external stimulus may be taken over and
made permanent by changes in structures later on.

28

Discussion

Eagle: Although I agree with Dr. Pontecorvo in general, I think the
question that Prof. Hinshelwood and Dr. Dean have raised is specifically
whether the development of resistance to antibiotics is adaptational or
mutational. We have some data, very similar to those presented by
Dr. Pollock, in relation to this point. We made a serious effort to show
that the development of resistance was indeed the result of adaptation,
but were reluctantly forced to the conclusion that all our data were

10^

10=

10" -

O I03

I02

NUMBER RESISTANT
TO 0.036 fig./cc.

6 8 10

TIME IN HOURS
Fig. 1 (Eagle). Development of resistance to penicillin in broth
cultures of Micrococcus pyogenes (concentration of penicillin in
medium, 0-036 (jig./ml.).

consistent with a rare mutational event, followed by selection. There
was no conclusive evidence for adaptation.

With staphylococci and penicillin we get precisely the curves which
Dr. Pollock showed. As indicated in Fig. 1, at an appropriate concentra-
tion of penicillin in a fluid medium, the total number of viable organisms
at first falls steeply, after which the survivors apparently begin to
remultiply. Actually, however, throughout the entire period, resistant
organisms present ab initio have been multiplying, and eventually take
over the population.

Discussion 29

In this instance, precisely as with the Lac~ and Lac+, we are deahng
with the selective multiplication, in a large population, of the rare
resistant organism.

A more interesting experiment was that with chloramphenicol and
Strep, faecalis in a sealed agar plate. If the plate was poured with no
more than 50-100 organisms, then at threshold concentrations of chloram-
phenicol, after periods of as long as 50 days, every organism inoculated
ultimately grew out to form a visible colony. At this point we thought
we had demonstrated adaptation, because each colony which appeared
proved to be relatively resistant to chloramphenicol. However, if during
this period of supposed adaptation, and before visible colonies had ap-
peared, the agar were cut into e.g. 100 sectors, and the number of
organisms in each sector determined, we found that all through this
period there had been an extremely slow but progressive multiplication
of the organisins. Initially, these organisms were not resistant. It was
only after the population of the microcolony reached fairly large pro-
portions that resistant organisms began to appear; and those resistant
organisms then multiplied at a relatively rapid rate. These results are
again consistent with mutation and selection, and do not prove adapta-
tion. At least with penicillin and chloramphenicol, we have therefore
found no need to invoke physiological adaptation as the basis for the
development of increased resistance. All our data are consistent with
the rare appearance of mutation, followed by selection.

Hinshehvood: Dr. Eagle, what is the criterion of a resistant organism?

Eagle: An organism which is capable of growing rapidly to form a
colony at a concentration of penicillin or chloramphenicol at which
normally no colony appears.

Hinshelwood: Under what conditions?

Eagle: In the case of penicillin, plating out in agar and watching the
plates for about 5 or 6 days. The proportion of organisms which develop
colonies is of course a function of the concentration used. With our
particular strain of staphylococcus, for instance, at 0-02 [jig./ml. 100
per cent will grow up in time ; at • 03 (ig. the number begins to fall off
very sharply ; at • 04 \Lg. one gets down to a very small fraction of viable
organisms.

Hinshelwood: In the curv^e you showed, the cells are dying off in one
part, obviously. There is a race between dying and adaptation. At a
given stage you plate, and the surviving cells will partially adapt. Sup-
pose that slow adaptation is going on, in competition with the dying off;
now you plate your samples at intervals, and naturally a larger and
larger number of cells can survive on the plate as the adaptation in the
liquid approaches completion. The number that can survive is steadily
increasing as the adaptation approaches more nearly to perfection. But
if you have too small an inoculum all the cells will be dead before any-
thing like adaptation has set in, so on the adaptive hypothesis you would
expect precisely the result found. I would suggest that the experiment
is not unambiguous and is made uncertain by your very criterion of what
is a resistant form. It is a form which has achieved some measure of
adaptation by the time the sample was taken.

INDIRECT SELECTION AND ORIGIN OF
RESISTANCE

L. L. Cavalli-Sforza

Istituto Sieroterapico, Milan

Adaptation of individuals and of populations

Adaptation to environmental changes in living organisms
can take place both at an individual and at a populational
level. Mechanisms of the first type are often efficient enough
to cope with the altered situation, but there will be some
variation in the individual responses to the changed environ-
ment. If the following two conditions are fulfilled: (i) that
this variation is at least in part heritable, (ii) that there is
differential reproduction of individuals showing different
degrees of adaptation, then the population is also bound to
change, in the sense that the frequencies with which the
variously adaptable types are represented will be modified.

This mechanism is of course nothing but natural selection
and its consequence is evolution. How much evolution takes
place in any given population in a given time is dependent
on how much heritable variation is available, and this is a
property of the population; and on how much variation in
the reproduction of the various types is created by the change
in living conditions. The use of antibacterial drugs in con-
centrations at which they exert their specific effect con-
stitutes a fairly drastic change in conditions, which is bound
to affect deeply the rates of reproduction of individual cells.
One should therefore expect drugs to effect major changes
in the composition of a bacterial population whenever this
contains — either because of original heterogeneity or because
of new hereditary change — types which have different
adaptability.

30

Indirect Selection and Drug Resistance 31

Heritable changes

There would probably be full agreement so far but for the
question of the heritability of individual changes in drug
adaptation. In order to avoid irreconcilable disagreement at
a later stage, we must agree on operational definitions of what
is heritable and what is not; only the former type of change is
of direct interest to the geneticists. Considering the necessity
of distinguishing individual from populational adaptation, the
heritability of an observed change in a population must be
examined as much as possible at the level of single individuals,
and not limited to that of the population as a whole. As
this is often technically impossible, compromises should be
attempted and their results analysed with care. For most
ordinary work the following definition, which corresponds to
what is routinely done in many laboratories, was found to
be useful. A population is considered to contain resistant
individuals, if single cells isolated (usually by plating) from
it and allowed to grow into colonies in the absence of the
selection medium, are found to be resistant, the test being
carried out on samples from such colonies, or sub-cultures
from them.

These "minimum" requirements for heritability have
obvious shortcomings, but unless we accept an unequivocal
definition, confusion is inevitable, as has indeed happened.
Further definitions may be elaborated: this one has the ad-
vantage of corresponding to the usual procedure, and of being
simple. It will fail on exceptional occasions, for highly mutable
and poorly growing mutants. But if any sluggishly reversible,
directly induced adaptation is taking place, it should be picked
up with this procedure.

Pre- or postadaptation

Since it has been ascertained that heritable changes in drug
resistance, or any other trait, do in fact occur after treatment
of the population, the question has repeatedly arisen: is the
change induced by the drug itself or does, this simply select

32 L. L. Cavalli-Sforza

pre-existing, or independently forming, variants? These two
mechanisms have also been called post- and preadaptation,
respectively (Cavalli-Sforza and Lederberg, 1953).

The available data may be considered. In higher organisms
the distinction between phenotype and genotype is an absolute
necessity if confusion is to be avoided. The effects of environ-
mental conditions, bringing about individual adaptation,
affect the phenotype but not the genotype, i.e. the sum of the
hereditary potentialities from which successive generations are
moulded. Therefore, selection can be effective only when it
picks up spontaneous variation, in other words, variation
which occurred at a level where the selective conditions
cannot act: that of the hereditary determinants. To clear the
issue we have, it is true, to simplify and forget about a few
systems, essentially cytoplasmic inheritance; but the cost
does not seem to be too high, at least at present.

Higher organisms, on which these conclusions were de-
veloped, are probably best defined in this connexion as those
in which the ratio of sizes between the adult soma and the
gamete is high. In fact, some w^orkers have developed the
view that in unicellular organisms (where this ratio is low,
soma and germ being of nearer orders of magnitude in size)
the distinction between genotype and phenotype is an arti-
ficial one. However, experiments like those reported by
Hayes (this symposium, p. 197) will be helpful in showing, if
necessary, which differences exist between what is hereditarily
determinant, and what is determined, even in a unicellular
organism. Also, the fact that observation of simple indi-
viduals usually takes place on colonies produced from them
helps to eliminate, though perhaps only partially, the effects
of the " phenotype " of the individual giving rise to the colony,
by the "dilution" to which it is subject when a large clone
is built from it.

Adaptation of bacterial populations

The facts on "lower" organisms are simply summarized.
Early attempts were made (early at least in the short history

Indirect Selection and Drug Resistance 33

of bacterial genetics) to see if the model which was so fruitful
for the study of evolution in higher organisms, namely the
selection of spontaneous genotypic changes, could be applied
to explain changes in resistance, in particular to viruses and
drugs. The elegant methods of Luria and Delbriick, and of
Newcombe, were used successfully and gave practically unequi-
vocal answers in favour of the preadaptation theory.

Both the strength of these methods and the validity of the
conclusions which rely on the statistical properties of clones
have been questioned. While the present author would agree
as to their insufficient strength, which leaves the door ajar to
equivocal results or interpretations, the evidence collected
later has fully confirmed the validity of the early conclusions.
In view of the existence of such stronger evidence, methods of
analysis such as the so-called "fluctuation test", the test of
" average clones " and the " correlation between relatives " will
not be considered here. They have been reviewed elsewhere
(Cavalli-Sforza, 1952; Cavalli-Sforza and Lederberg, 1953).
A method will be considered instead, the strength of which
nobody would question, namely that of indirect selection.

Indirect selection

Indirect selection was first introduced by Lederberg and
Lederberg (1952) in streptomycin- and Tl phage-resistance.
It uses sib-selection (or, in general, selection by tests on
relatives) to obtain strains which are resistant to some agent,
without using this agent for sorting out the resistant mutants.
By substituting genetic testing for direct isolation with the
drug, it can be proved, and has been proved unequivocally
that the resistant cells are present in the population spon-
taneously, as they can be isolated from it without exposure
of the population to the drug. To express the principle in
simple terms, suppose we can isolate the two descendants of
one cell for a great number of cells, and test for resistance to
a drug one of the daughter cells, keeping the other for repro-
duction in the absence of the drug: if the mother cell was
sensitive, apart from rare mutational events both cells should

DRUG RES. — 2

34 L. L. Cavalli-Sforza

also be sensitive. Therefore, the test on one of the two
daughters will permit us to formulate with good probability
the prediction that also the other daughter is sensitive. If
the mother cell was resistant, both daughters should be
resistant; this situation being revealed by the test carried out
on one of the two daughter cells, it will be possible to verify
the prediction that the untested sister should give rise to a
pure colony of resistant cells.

This method has been made technically possible, for the
first time, by the use of replica plating on solid media; and
it has permitted the indirect isolation of streptomycin- and
phage-resistant mutants. Later (Cavalli-Sforza and Leder-
berg, 1956), a method was developed for carrying out indirect
selection in liquid cultures — which has the advantage over
replica plating of leading more easily to quantitative analysis
— with a view to answering the question: Have all resistant
cells arisen by spontaneous mutations, or have some arisen
by other mechanisms, such as mutation induced by the drug,
or adaptation not controlled by nuclear determinants?

Concentration by limiting dilution

The principle of this method (Cavalli-Sforza and Leder-
berg, 1956) is to concentrate spontaneous mutants to resist-
ance by using a sample which contains few resistant cells,
possibly only one, and subdividing it further. If there is in
the sample before subdivision just one resistant mutant and
(N— 1) normal sensitives, i.e. if the relative frequency of

mutant cells is ^j^, after subdivision in n tubes the frequency

n ,
of mutants will be :j^ in the tube which happened to receive

the single mutant, and zero in the other tubes. Each tube will,
in fact, receive 'Njn bacteria but only one of them contains
the resistant mutant. This tube will therefore show an
enrichment of mutants of n times. It will be possible to
identify it by incubating all tubes after addition of fresh
broth, and testing samples from them for drug resistance.

Indirect Selection and Drug Resistance 35

When statistical fluctuations are considered, as shown by
Dr. J. Pfanzagl of Vienna (personal communication), the
enrichment expected is :

l_e-'» _A

l-e-""" /o

where E is the ratio between the relative frequency of mutant
cells in tubes which have received at least one of them (/j),
and that one in the original suspension (/o); while m is the
expected number of mutants in the sample which has been
subdivided into n tubes.

One such experiment, e.g. on 10 tubes, will give at best a
tenfold enrichment; but on repeating the indirect selection
experiment it is possible to isolate in a predictable number of
cycles a pure culture of resistant mutants, which will never
have experienced direct contact with the drug.

This experiment was successfully carried out for high
degree resistance to streptomycin and for low degree resist-
ance to chloramphenicol in Escherichia coli. The speed of
selection observed was comparable to that predicted, as is
shown in greater detail in the paper by Cavalli-Sforza and
Lederberg (1956).

A short report is given here of data in which this experi-
ment was amplified, considering that every experiment of
indirect selection tests just one culture, and that the most
informative stage is the first cycle (or, occasionally, the first
two cycles) of indirect selection. This usually permits the
counting of spontaneous mutants, and the comparison of
their number with that of the resistant cells counted by direct
selection, i.e. by plating in presence of the drug. In view of
the greater simplicity of the system, the experiment was
made on streptomycin resistance.

Samples from a number of independent saturated cultures
were tested for streptomycin resistance and concentrations
of resistant cells per ml. ranging from to 105 resistants
were found. The tests on two cultures will be considered in
detail.

36 L. L. Cavalli-Sforza

Table I shows the results obtained with a saturated culture
of Esch. coli Kl2 no. 176 which was expected, from the
assay, to contain zero streptomycin-resistant mutants. Three
samples from it (of 0-5 ml., 0-25 ml. and 0-125 ml., respec-
tively) were diluted each to 20 ml. with fresh broth. Each
20-ml. quantity was distributed into 10 tubes, in quantities
of 2 ml. per tube, and the 30 cultures thus obtained were
incubated to saturation. Eventually the total number of
resistants per culture was counted on streptomycin agar

Table I

A Protocol of Quantitative Indirect Selection

From a culture which has been assayed for streptomycin resistants and
found to contain zero resistants per ml. , further samples have given :

Sample
Multiplication factor

0-5 ml.
40 X

0-25 ml.

80 X

125 ml.
160 X

Resistants

5

2

6

1

12

10

4

10

10

6

3

6

13

6

17

5

5

1

1

11

6

14

5

3

14

Average per subculture 4-9 6-4

6-3

(500 (jLg./ml.). Some resistant cells were found (Table I), but
only a few, and often there were none per culture ; the average
number of mutants expected in such conditions from more
extensive tests is 4-8 ±2-0 per culture, and the results from
the three series agree within the limits of error with this
number.

Table II shows what happened instead when a culture
known to contain 39-5 resistant cells (from the assay of a
sample from it plated in streptomycin agar) was treated in
the same way. Here the sample of 0-125 ml. was expected
to contain 5 mutants and if, on subdivision into 10 tubes,
each of these fell into a separate tube, 5 out of these would

Indirect Selection and Drug Resistance 37

be expected to contain a mutant at the beginning of growth.
As the sample of 0-125 ml. was made up to 20 ml. with fresh
broth, every cell inoculated was allowed to grow into a clone
of 160 cells on average. There should then be about 160
resistant cells in 5 out of 10 tubes of the 0-125-ml. series.
This was found to be true of 4 tubes instead of 5. In other
samples, of 0-5 and 0-25 ml., there is evidence that, as would

Table II

A Protocol of Quantitative Indirect Selection

From a culture which has been assayed for streptomycin resistants and
found to contain 39-5 resistants per ml., further samples, after addition
of fresh broth to a total of 20 ml. and incubation, have given:

Sample

0-5 ml.

• 25 ml.

125 ml.

Multiplication factor

40 X

SOX

160 X

Resistants

160

19

44

59

7

65

89

132

31

59

184

53

102

156

49

105

159

164

3

82

Spontaneous mutants :

Expected

19-8

9-9

50

Found

11

8

4

Found (corr.)

13-2

9-1

4-5

be expected, more than one mutant fell into some tubes (e.g.
the first and ninth tube of the 0-25-ml. sample, etc.).

One can in this way count the number of spontaneous
mutants in a culture and compare it with the expected one,
if the hypothesis is made that all resistants observed on drug
plates are the consequence of spontaneous mutation. These
two values are given for each sample in the second-last and
third-last lines of Table II.

Corrections have to be made, however, to account for
the statistical distribution of mutants and for the possible

38 L. L. Cavalli-Sforza

differences in growth rate between mutant and parent in drug-
free medium. Corrected figures for the numbers of spon-
taneous mutants are given in the last Hne of Table II. When
this was done for 18 cultures, no significant deviation was
found from the hypothesis that all resistants are the con-
sequence of spontaneous mutations.

Some precautions

The following precautions should be taken in applying this
test:

(1) The growth rate of mutants is usually lower than that
of the normal sensitive. In 18 independent mutants, the dis-
tribution of relative growth rates k given in Table III was

Table III

Distribution of the Growth Rates of Streptomycin-resistant Mutants
k = Growth rate of resistant relative to growth rate of sensi tive

k Number of strains

less than

0-70

1

from 0-71 to

0-75

4

„ 0-76 „

0-80

2

„ 0-81 „

0-85

6

„ 0-86 „

0-90

1

„ 0-91 „

0-95

„ 0-96 „

100

4

Total 18

obtained, where k is the ratio between the growth rate of the
resistant and that of the sensitive parent in mixed culture.

If A^T^l, the expected numbers of resistants per culture
differ from those expected on the basis of the ratio of increase
in total cell numbers (the "multiplication factors" in Tables I,
II and IV). Table IV gives the expected numbers of resistants
for some k values. When k is small the "enriched" mutants
are not easily sorted out from the background of new mutants.

(2) In some circumstances, e.g. chloramphenicol resistance,
first-step mutation does not determine a high level of resist-
ance, and resistants may be incompletely recovered in tests

Indirect Selection and Drug Resistance 39

Table IV

Number of Resistants expected from the Multiplication of a Single

Cell
/^ = Growth rate of resistant relative to growtli rate of sensitive

MuUiplication factor
k 40 X 80 X 160 X

0-0

9-2

13-8

21

0-7

13-2

21-4

34-8

0-8

19 1

33-3

58-0

0-9

27-6

51-6

960

0-95

33-2

64-0

124

10

40

80-0

160

with the drug concentrations necessary to ehminate all or
most of the sensitives. There may also be interactions
between sensitives and resistants in mixed populations,
increasing (protection: Cavalli-Sforza and Lederberg, 1956)
or decreasing (co-killing or suppression: Eagle, 1955) the
count of the resistant type. Such situations can usually be
revealed, and their consequences evaluated, by appropriate
reconstruction experiments.

(3) Cells do not always divide regularly. For example,
chains may be formed with the strain used here if static
unaerated, but not aerated, cultures are employed. Where
static unaerated cultures are used, situations of the type
shown in Table V may be encountered. The irregularity

Table V

An Unaerated Culture containing 102 Resistants per ml. (Strepto-
mycin Agar Assay) tested by Indirect Selection

Sample • 25 ml. ■ 125 ml. • 0625 ml.

MuUiplication factor 20 x 40 x 80 X

Resistants

8

83

47

10

24

17

350

15

24

42

11

15

42

132

24

32

14

43

5

77

49

8

27

610

40 L. L. Cavalli-Sforza

in the distribution of the number of resistants per tube is
immediately apparent. Thus the last tube of the 0-0625-ml.
sample must have contained 8 or 9 mutants at least ; the same
is true of the third tube of the 0-125-ml. sample, and so on.
This distribution could hardly be random. Presumably,
resistant cells tend to form short chains which are not split
on dilution in broth, while they are more easily broken up in
agar, perhaps as a consequence of the joint action of tempera-
ture of the agar plus its chelating power. In such cases one
would tend to underestimate the number of resistant mutants
if the frequency of tubes showing enrichment were used for its
assessment, while if enrichment ratios were used one would
tend to overestimate it.

Conclusions

Tests of adaptation in bacterial and other populations are
available that permit the assessment of the relative import-
ance of genetic and non-genetic adaptation, defining the
former as the selection of spontaneous mutants. In the cases
tested so far — essentially streptomycin and chloramphenicol
resistance — evidence was found for the adequacy of the
hypothesis of genetic adaptation, and no need arose for addi-
tional alternative explanations.

REFERENCES

Cavalli-Sforza, L. L. (1952). Bull. Wld. Hlth. Org., 6, 185.
Cavalli-Sforza, L. L., and Lederberg, J. (1953). VI Int. Congr.

Microbiol, p. 108.
Cavalli-Sforza, L. L., and Lederberg, J. (1956). Genetics, 41, 367.
Eagle.. H. (1955). Perspective and Horizons in Microbiology, p. 168.

Rutgers University Press.
Lederberg, J., and Lederberg, E. M. (1952). J. Bad., 63, 399.

DISCUSSION

Hinshelwood: This is a beautiful method with streptomycin, and I
admire the experiment very much. But what level of chloramphenicol
resistance can one get by the indirect method?

Cavalli-Sforza: The degree of resistance that was obtained in the work
with Lederberg was perhaps two or three times the original level. You

Discussion 41

cannot get in one step a high increase in resistance to chloramphenicol,
and you must therefore work on what occurs in nature. In the case of
chloramphenicol, in fact, some trouble was encountered and indirect
selection took a little longer. The level of resistance of the particular
mutant selected indirectly was such that it did not give 100 per cent
survival with the concentration of the drug that would kill all of the
sensitives, it gave only 12-20 per cent survival.

Hinshelwood: As regards chloramphenicol resistance, by means of
growing mass cultures in different biochemical media (in different sugars,
broth and synthetic medium and so on) we can change what may be
called the natural level of resistance in the range from 10 to 20 or 30 parts
per million; and in the same way resistance to proflavine and certain
other drugs changes. On the other hand, the resistance can be raised to
800 or 1000 by the direct action of the drug, so it is a very low-grade
resistance indeed which is selected in your experiment. There is a great
contrast, in any case, between chloramphenicol and streptomycin where
the degree of resistance rises abruptly after adaptation, by whatever
means, to quite a low concentration of streptomycin.

We think that there is a distinction between types of resistance. We
think, moreover, that streptomycin resistance may sometimes be due to
the lack of the power of cells to take up the drug.

Cavalli-Sforza : I think that with chloramphenicol you have to work in
those conditions, because you cannot get higher resistance in just one
step. It is another genetic system. You have not got any single gene
capable of giving high resistance at once. You could get higher resis-
tance only by repeating the entire process of selection on first-step
mutants, and so on.

Hinshelwood: Still, when mutants are selected up to a certain point in
your environment, the further steps should be coming in quite freely.

Cavalli-Sforza: The later cycles of an indirect selection experiment
may be easier than the first ones ; but if not, you can change the method
if you want to, for instance you could go over to replica plating.

Hinshelwood: You would not be inclined to entertain the idea that
there are really two types of adaptation ?

Cavalli-Sforza: What was done here was to test one hypothesis. The
present method can only disprove the genetical hypothesis; if it does
disprove it, it leaves the field open for the alternative one, but it has not
done that.

Hinshelwood: Have these resistant cells obtained by the indirect
method been tested for streptomycin adsorption ? That would be a very
interesting datum to have.

Cavalli-Sforza: I don't think they have been tested.

Dean: Would you consider, if you got no selection, that the genetical
hypothesis is disproved for a particular drug and a particular organism ?
For instance, if one did an experiment with a certain drug and a certain
organism and found no selection at all, would you consider that as
disproving the genetical hypothesis ?

Cavalli-Sforza : Of course ; but you have to be very careful, in using
indirect selection, about those shortcomings that I mentioned, for

42 Discussion

instance that mutants have to multiply in competition with the wild type.
This is not a gratuitous assumption, it is something which you can test
directly; if the mutant multiplies much more slowly than the normal,
then the method is difficult to apply.

Lederberg: A control is needed in such a case, i.e. an artificial recon-
struction of a mutant which you know was produced ; you then put it in
with the wild type to show that 3^ou can select it under those conditions.
If you then fail to obtain similar mutants without making artificial
mixtures you can conclude that no mutants of that type are present in
untreated cultures. I emphasize of that type, because this consideration
of differential growth rate might still come in, but you would certainly
have to stretch the genetical hyjiothesis quite far to get selection.

Yudkin: Prof. Cavalli-Sforza said that mutants would be expected
necessarily to be at a disadvantage, compared with the natural types.
When we are dealing with drugs like some of the antibiotics which may
occur in nature, it is reasonable to suggest that the resistant mutants
grow more slowly than the wild type, for otherwise the sensitive strains
would have disappeared at some time. But when we are dealing with
drugs like proflavine, which the bacteria are most unlikely to have
encountered in nature, then there is no reason to suppose that the
resistant mutants have a gro^vth disadvantage.

Cavalli-Sforza: I don't think that the fact that the particular strain
has had experience before of one particular drug is very important.

Any mutation that arises in an otherwise homogeneous population of
cells has a fitness value relative to the normal type, which of course

^ W ^ \ r ^ \ rr

Fig. 1. (Cavalli-Sforza). An oversimplified picture of the
process of genetic adaptation taking place automatically in a
culture kept under fairly constant conditions, in terms of the
distribution of fitness values of mutations arising in tlie popu-
lation at various stages. It shows why most mutations are
likely to be "unfavourable." The abscissa gives tlie fitness
value of a mutation; the ordinate, the frequency of mutations
having given fitness values. Arrows indicate the lapse of
generations. The stippled area represents the proportion of
mutations which are "favourable", i.e. have positive fitness;
the white area the proportion of "unfavourable" ones.

depends on the specific environment considered. Different mutations
will presumably show different fitness values; a few may have a fitness
value of zero or nearly so (if we thus describe the absence of advantage
or disadvantage in respect to the normal type, e.g. the mutant grows and
dies at the same rate as the normal) ; others may have a positive fitness
value (i.e. they are "favourable" mutations), and the rest a negative
fitness value ("unfavourable" mutations) (Fig. 1)*. If a bacterial strain
* For greater clarity Fig. 1 was added in proof.

Discussion 43

has had a long experience of growth in given conditions, such as normal
laboratory media and transfer routine, which are fairlj^ constant, it must
have adapted genetically to it. Genetic adaptation by natural selection
takes place automatically, in fact, and most of the favourable mutations
nuist have been fixed by it, thus decreasing the proportion of favourable
mutations available to the organism and increasing that of unfavourable
mutations. Of course, the strain must have had time to adapt to the
"usual" conditions, or in other words these must really be "usual";
therefore, the previous history of the strain may have some importance.
On the other hand, the tendency of any new mutation to have a negative
fitness value is not only a theoretical expectation ; it is a fact, as for in-
stance the data in the present paper have show^n.

Davis: It seems to me that we are using the term "drug resistance"
for two different concepts. When we say that one strain is more resistant
than another we mean operationally the following : two families of cells
are both grown under identical conditions and then identically tested to
determine the concentration of drug that brings about a certain degree
either of interference w ith growi;h or of active bactericidal action ; and
one family is found to require for this effect a higher concentration of
drug than the other. But ^vhen we say one cell is more resistant than
another we mean quite a different thing. If a number of cells are plated
on a medium containing a borderline concentration of drug, some cells
will die and others will give rise to colonies. We have a right to conclude
that the ones that died were less resistant, bj^ definition (i.e. if they
w^ould not have died in the absence of the drug). But we do not know
that the more resistant survivors are more resistant in an inheritable
way. They may be. They may also be simply those cells, in the inevi-
table range of physiological variation in a genetically homogeneous popu-
lation, that happened to be able to withstand the borderline concentration
of drug sufficiently to initiate colony formation. And, once initiated, the
microcolony could so modify its environment as to ensure its continued
growth.

No geneticist would deny that such physiological variations can affect
the chance a cell has of resisting a borderline concentration of drug.
Indeed, it w ould be safe to predict that one could shift the average level
of such phenotypic resistance by varying the richness of the medium,
the aeration, the stage in the history of the culture at which the
organisms were harvested, etc. Furthermore, it seems inevitable
that the surviving cell, in beginning to grow in the presence of the drug,
would undergo further physiological changes in adaptive response to the
presence of the drug; such adaptive changes not only might alter the
susceptibility of the cell to the drug ; they also should be passed on to
the progeny as long as these progeny are grown in the presence of the
drug. But such adaptive changes in resistance, in contrast to inheritable
ones, would disappear after a suitable number of generations of growth
in the absence of the drug.

While genetically orientated microbiologists have recognized the pos-
sibility of such adaptive influences on the resistance of a cell, they have
not been much inclined to investigate the problem. I think Sir Cyril

44 Discussion

Hinshelwood has performed a service in focussing on this interesting and
neglected area of biology. However, the real rub comes in his claim that
such reversible adaptive resistance, if carried through enough generations
will gradually develop (by some process other than random mutation
plus selection) into a stable, inheritable resistance. Most biologists
would be sceptical about the existence of such gradual, non-mutational
stabilization of an adaptation; and I feel that the experiments cited in
support of this concept are all compatible with mutation and selection.

One further comment: as Sir Charles Harington pointed out in his
introduction, the general interest in drug resistance and the raison d'etre
of this symposium have arisen from the practical importance of the
problem. I would like to emphasize that the problem to which he refers
is the emergence of strains with an inheritable increase in resistance.
This is what is ordinarily meant by drug resistance. The problem of the
adaptive and other non-inheritable physiological factors affecting the
observed level of resistance of a cell is also interesting — but it is not the
problem of drug resistance.

Pontecorvo: This last point which Prof. Davis has made is precisely
the one I meant when I mentioned what has been done in higher
organisms, particularly by Waddington. The experiment there is to
expose embryos of flies to a certain concentration of drug at certain
critical periods in development : a proportion of them develop into ab-
normal adults. Breeding is selective, i.e. only from the abnormal adults.
After a few generations abnormal adults develop even without treatment
or with a reduced one. In this case it is quite evident from the procedure
of the experiment that what has happened is that there was initially,
let us call it "physiological", variability: some individuals responded,
some did not. A genetic mechanism which can be pin-pointed to particu-
lar regions of the chromosome set has taken over later on. That is
precisely the transition from one mechanism to the other. It would be
important to see whether this transition can or cannot be favoured by
means other than selection by the stimulus ; so far, I am not convinced
that there has been any proof one way or the other.

Gyorffy: To raise a point concerning definition: we need to make a
distinction between the terms "heritable" and "genetic"; they are not
synonymous, and we must take care in using them, because "heritable"
or "hereditary" means transmissible or transmitted from one generation
to another ; and "genetic " implies control by the genotype, by genes ; and
it is well known that all modifications are non-genetic changes. The
"Dauermodificationen", which very often occur in micro-organisms, are
"inherited" through a number of generations although they are not
genetic changes. Another complication is that each bacterial cell in
itself represents one generation, and if it is modified as by environmental
influence it may be "inherited" through a number of generations. That
again is not a real genetic change. I wonder whether we really are able,
in the usual experiments, to differentiate by the criterion of the term
"heritable" between a genetic change and a Dauermodification. The
term "heritable" seems to me somewhat ambiguous, and it will be better
when we no longer use this term in microbial genetics but use instead

Discussion 45

the word "genetic". Then we can make the distinction that the genetic
change is controlled by the genotype.

Pontecorvo: We have, of course, an operational test in some cases. In
Esch. coH we can use segregation and recombination, either by a sexual
process or by transduction. In other organisms, for instance ascomycetes,
we have the ordinary test of sexual reproduction and segregation as well
as "parasexual" segregation, etc. So we can unequivocally distinguish
in the majority of cases, even in micro-organisms, a genetic change from
an inheritable change which is not genetically determined.

Davis: It seems to me that the science of genetics must be concerned
with all mechanisms of inheritance, and not simply those involving
chromosomal genes. Indeed, the term "gene" was surely derived from
"genetics" and not vice versa. I therefore wonder whether it might not
be useful to use the term "genetic" to include all mechanisms of in-
definitely transmitted inheritance, both chromosomal and non-chromo-
somal, and to use the term "genie" for chromosomal mechanisms.

Kunicki-Goldfinger : One should be very careful when differentiating
between genie mutation and physiological, more or less stable, change,
especially if recombination analysis is not possible.

In this connexion some phenomena may be pointed out which are
apparently due to mutation, but which are, in fact, caused by physio-
logical changes in bacterial cells. In an Esch. coli population only a very
small fraction of cells can grow in the presence of lithium chloride. Not
more than 1 per 100,000 cells is capable of forming a colony on media
containing lithium chloride. In the majority of strains these resistant
forms are not stable and their progeny are as susceptible as the parental
strain. Without analysis of population during growth the change may be
interpreted as being due to the selection of pre-existing mutants. In
reality it is caused by a physiological adaptation in a small fraction of
the heterogeneous population.

The characteristic growth curve, which Prof. Cavalli-Sforza dis-
cussed, may also be due to selection of spontaneous mutants, or to
overgrowing of the culture by a new physiological variant induced by
the environmental conditions. This is the case in Brucella grown in syn-
thetic medium. A gro\\i:h curve with many peaks is then obtained. At
least some variants, whose growth resulted in the formation of additional
peaks, were shown to be of non-mutational origin. Some R-variants
could be obtained from homogeneous S-populations in conditions exclu-
ding cell multiplication. In this case the majority of cells were trans-
formed into a new type. If this change is not due to semi-stable physio-
logical adaptation, it may be caused by total mutation of almost the
whole population, induced by environmental factors, which seems to me
less probable.

Hotchkiss: Prof. Davis has pointed out quite clearly what the con-
ceptual disagreement is. I suggest we turn more to the expermiental
inconsistencies. Sir Cyril has mentioned that cultures selected in low
concentrations of streptomycin would be resistant to high levels of
streptomycin. I know that in many organisms one may find streptomycin
resistance also: so I would like to know whether a low resistance is

46 Discussion

found in the same situation; if not, I would be concerned about
possible special selective features.

The other point is rather similar, related to the case of sugar fer-
mentation — the inconsistency between the results of Hinshelwood and
Dean and those of Lederberg and Pollock. I think the cultures that
Lederberg and Pollock have been examining should have a complete
round trip and return to Hinshelwood' s laboratory and be observed
under his conditions again. I would be very pleased, for instance, to see
that cultures which they had submitted to some very brief processing
would also show large colonies in your media. Sir Cyril, and if they did
not, then one could infer that differences in your media have prevented
you from seeing this selection in favour of the more rapidly growing or
rapidly utilizing mutant.

GENETIC ASPECTS OF DRUG RESISTANCE

M. Demerec

Carnegie Institution of Washington, Department of Genetics,
Cold Spring Harbor, New York

If the problem of origin of bacterial resistance to drugs had
arisen recently, instead of about ten years ago, it would have
been a relatively simple matter to plan appropriate experi-
ments to elucidate the role played by genetic mechanisms in
the development of such resistance. A decade ago, however,
genetical research with bacteria was in a very early stage, and
it was still an open question whether or not the hereditary
mechanism operating in bacteria would prove to be the same
as the mechanism that had been demonstrated in higher
organisms, including fungi. Since then, ample evidence has
been accumulated to justify the conclusion that the genetic
mechanism in bacteria is similar to that in higher organisms.
This conclusion is further strengthened by the results of
studies made with bacterial viruses, which can be crossed
more easily than bacteria. In such crosses, using easily dis-
tinguishable markers, one may identify classes of offspring
representing the two recombinant types as well as the two
parental types (Hershey, 1946; Hershey and Chase, 1951).
Further anatysis has shown that the proportions in which the
four classes are represented agree with expectation based on
Mendelian segregation (Visconti and Delbriick, 1953). More-
over, the results of experiments using several markers (Hershey
and Rotman, 1948) reveal the existence of linkage relation-
ships. Taken together, these findings indicate that the basic
genetic process in bacterial viruses — the lowest living organ-
isms known — is similar to that with which we were already
familiar in all higher organisms. Thus it is reasonable to
suppose that the basic genetic mechanism is the same in all
living organisms, not excluding bacteria. This basic genetic

47

48 M. Demerec

mechanism is vested in genes, which are component parts of
threadhke structures known as chromosomes.

Since it is now a well established fact that in every organism
genes carry the primary responsibility for transmission of
all biological properties whose mode of inheritance has been
investigated, and since bacterial resistance to drugs has all
the characteristics of such properties, it is to be expected
a priori that a genetic mechanism is responsible for the origin
and transmission of resistance. It would be remarkable
indeed if bacterial resistance did not conform with what
appears to be a general biological rule. And the experimental
evidence obtained by direct studies of resistance fully confirms
the a priori expectation. Geneticists have directed a greater
than ordinary eff^ort towards this analysis, not only because
in the beginning very few of the methods now available were
at their disposal, but also because the problem was of con-
siderable practical importance for purposes of regulating the
clinical use of antibiotics, and therefore evidence adequate to
convince non-geneticists as well as geneticists was called for.
The results of their work fully support the conclusions reached,
namely, that as a rule genes determine bacterial resistance to
drugs, and that mutations in certain genes are responsible for
its origin. Comprehensive reviews of the literature on micro-
bial drug resistance have been published by Cavalli-Sforza
and Lederberg (1953) and by Bryson and Szybalski (1955).

The most conclusive evidence of genie determination of
resistance to drugs is the fact that in all genetic tests bacterial
resistance has shown the same patterns of inheritance as
other gene-determined characters studied. The tests have
involved three diff*erent approaches: studies of the origin of
resistant variants ; studies of frequencies of spontaneous and
induced mutations; and studies of transmission of resistance
by crossing, by transformation and by transduction.

Origin of resistant variants

Much of the work has been directed toward distinguishing
between two possible modes of origin of bacterial resistance :

Genetic Aspects of Drug Resistance 49

(1) induction, either in non-genie or in genie components of
the bacterial cell, by the drug used in treatment; (2) gene
mutation, independent of the treatment, and subsequent
selection of the mutants by the drug. The primary object
of these studies has been to determine whether or not resistant
variants appear spontaneously in a bacterial population and
give rise to clones of resistant individuals, as would be ex-
pected if they originate by mutation but not if their origin
were dependent upon non-genetic change of phenotype.
Several methods have been used.

The first evidence of genie origin of resistance was obtained
with Staphylococcus aureus and penicillin (Demerec, 1945), by
means of the so-called fluctuation test developed by Luria
and Delbriick (1943) in their studies of the origin of resistance
to bacteriophages. The fluctuation test was later performed
with several other bacteria and several other drugs (Oakberg
and Luria, 1947; Demerec, 1948; Newcombe and Hawirko,
1949), with results that confirmed the genetic origin of resist-
ance. Exceptions that have been reported (Eriksen, 1949;
Welsch, 1952; Banic, 1954) are readily explainable (Cavalli-
Sforza and Lederberg, 1953) in terms of the nature of the
materials used or the techniques employed, and do not
constitute evidence against the genetic origin of resistant
variants. The fluctuation test allows quantitative predictions
of the magnitude of any variance dependent upon mutation.
By means of statistical models (Lea and Coulson, 1949;
Armitage, 1952) it has been possible to predict the exact
distribution and size of clones on the basis of the mutational
theory.

A second method of testing the spontaneous origin of
resistant variants is based on a technique devised by New-
combe (1949) for studies of phage resistance. Newcombe's
technique compared the number of resistant colonies observed
on plates to which phage had been added after the growth of
microcolonies with the number observed on control plates on
which the microcolonies had been dispersed by spreading just
before the application of the phage. By the introduction of

50 M. Demerec

membrane filters, upon which bacterial colonies could be
transferred from one medium to another without disturbing
their position, this technique was adapted for studies of drug
resistance (Bornschein, Dittrich and Hohne, 1951; Dittrich,
1951).

A third method of testing the origin of resistant variants
is the replica-plating method originated by Lederberg and
Lederberg (1952), in which samples of colonies grown on an
agar surface can be transferred to a drug-containing medium,
without serious disturbance of their spatial arrangement, by
stamping consecutive plates with a piece of sterile velveteen.
With this method it has been possible to show the drug-
independent origin of mutants resistant to streptomycin
(Lederberg and Lederberg, 1952) and isoniazid (Bryson and
Szybalski, 1952).

A fourth method, which like the replica-plating method
permits "indirect" selection of resistant variants in the
absence of the drug, was described recently by Cavalli-Sforza
and Lederberg (1956), and its further application has been
discussed by Cavalli-Sforza (this symposium, p. 30). It is
carried out by making subcultures in liquid medium and
choosing the "best" subcultures by testing samples on agar
medium containing the drug. Using this method, Cavalli-
Sforza and Lederberg have supplied additional evidence of
the spontaneous origin of variants in Escherichia coli that are
resistant to streptomycin or to chloramphenicol.

Mutability

Two features usually associated with bacterial resistance to
streptomycin, namely, the appearance of highly resistant
variants in a single step and the appearance of streptomycin-
dependent variants, make it especially suitable for quantita-
tive studies of the occurrence of changes from sensitivity to
resistance or dependence, of reverse changes from dependence
to sensitivity, and of changes from dependence to resistance.
Such quantitative studies have been carried out extensively
with Esch. coli. It has been found that whenever any such

Genetic Aspects of Drug Resistance 51

change occurs it is transmitted to the offspring of the variant
bacteria and behaves as a heritable characteristic. Observa-
tions of changes from sensitivity to resistance (Newcombe and
Hawirko, 1949; Demerec, 1951) have indicated that they
occur at a predictable rate (about 1 X 10"^ to 1 X lO-^^ pej.
bacterium per division) as is expected of changes due to gene
mutations. Reverse changes from streptomycin dependence
to non-dependence also occur spontaneously with a certain
frequency, characteristic in each strain; and the frequency
of occurrence can be increased by treatment with any agent
that has been proved capable of inducing gene mutations in
bacteria (Demerec, 1951; Demerec, Bertani and Flint, 1951).
The frequency of occurrence of changes from dependence to
non-dependence varies considerably among different depen-
dent strains of separate origin. Similar variation has been
detected w ith regard to the frequency of occurrence of changes
from sensitivity to resistance and dependence among sensitive
strains derived from dependent strains by isolation of sensi-
tive variants (Demerec, 1950). These patterns of behaviour
are analogous to those observed in genetic analyses of spon-
taneous and induced mutability in several genes concerned
with biochemical reaction in Esch. coli (Demerec, 1953),
Salmonella typhimurium (Hartman, 1956) and Neurospora
crassa (Giles, 1951), which studies have shown that each gene
as well as each allele of a gene has its own characteristic
mutation rate.

Another way in which the determinant of streptomycin
resistance acts like a gene is evidenced by studies of a mutator
factor in Esch. coli (Treffers, Spinelli and Belser, 1954 ;
Treffers et al., 1956). This factor, whose location on the
chromosome has been determined (Skaar, 1956), affects the
stability of the genome by increasing the rates of mutation
of individual genes. Similar factors have been identified in
Drosophila (Demerec, 1937; Plough and Holthausen, 1937),
and in maize (McClintock, 1951). It has been found that this
mutator factor in Esch. coli has the same general effect on the
determinant of streptomycin resistance as on the other

52 M. Demerec

genetic markers with which it has been tested ; in other words,
in this respect also streptomycin resistance behaves as a gene-
determined character.

Thus the most reasonable conclusion to be reached from
quantitative studies of resistance to streptomycin is that a
genie mechanism is responsible for its origin and nature. It
would be difficult indeed to visualize an adaptive mechanism
that would account for the complex and at the same time
precise patterns of action revealed by the genetical studies.
The predictability with which changes from dependence to
non-dependence occur, and the ease with which they can be
observed experimentally, have made it possible to utilize this
property in developing one of the most efficient methods now
available for the study of induced mutability, and particularly
for the detection of mutagenicity among various chemicals
(Demerec, Bertani and Flint, 1951).

Mendelian analysis

Although there is very little doubt at present that the basic
genetic mechanism in bacteria is similar to that operating in
higher organisms, bacteria as a group are still notoriously
unsuited to simple Mendelian analysis — which continues to
be regarded by many as the only source of indisputable
evidence of genie inheritance of characters. It seems to me
that at the present stage of our knowledge about genes and
their action we can afford, in cases where segregation tests
cannot be carried out because of a lack of suitable techniques,
to accept the results of analysis by other, more recently
developed methods.

Evidence about drug resistance obtained by the recom-
bination method, which closely approaches standard Men-
delian tests, supports the conclusion that in the bacteria
studied by this method a genetic mechanism is responsible for
resistance. The recombination method has been used to
analyse high resistance to and dependence on streptomycin
in strain K 12 of Esch. coli. The results indicate that a single
gene locus is involved in the transmission of both resistance

Genetic Aspects of Drug Resistance 53

and dependence (Demerec, 1950; Neweombe and Nyholm,
1950), and that a linkage relationship exists between strep-
tomycin resistance and a methionine marker (Lederberg et al.,
1952). Other studies made with the K 12 strain have shown
that any one of several gene loci — four or five at least — may
be responsible for the inheritance of a low degree of resistance
to chloramphenicol (Cavalli and Maccacaro, 1950, 1952).
It has also been established that one gene locus is responsible
for a low degree of resistance to azide, and that it is closely
linked with the locus controlling resistance to bacteriophage
Tl (Lederberg, 1947). In this case, further experiments
using the two linked markers have demonstrated segregation
conforming to Mendelian expectation (Cavalli, 1952). Work
done by several investigators has shown that the genes
governing resistance to certain drugs (streptomycin and azide),
as well as those involved in resistance to some phages, are
distributed more or less indiscriminately among other gene
loci; and there is no reason, at least from their positions on
the chromosome, to assume that they are in any way excep-
tional as compared with other bacterial genes.

Transformation and transduction analyses

Two unique mechanisms have been discovered whereby
genetic properties — presumably conveyed in genes or chro-
mosomal fragments — may be transferred from one bacterial
strain to another. In transformation, the transfer is accom-
plished by deoxyribonucleic acid (DNA) extracted from
donor cells and brought into contact with the recipient bac-
teria, whereas in transduction phage particles act as vectors.
Genetic markers that have been used successfully in transfor-
mation experiments include resistance to penicillin, strep-
tomycin, and sulpha drugs (Hotchkiss, 1951; Alexander and
Leidy, 1953); and transduction has been used to study strep-
tomycin resistance (Zinder and Lederberg, 1952; Baron,
Formal and Spilman, 1953; Iseki and Sakai, 1954). Thus, in
this respect also, drug resistance corresponds to other pro-
perties that are controlled by genes.

54 M. Demerec

Discussion

Experimental evidence shows that resistance to drugs
originates in discrete steps, and that a high degree of resist-
ance may either be built up through successive changes or be
attained in a one-step change. The pattern followed is charac-
teristic of the drug. Two patterns have been recognized : the
"penicillin" pattern, in which high resistance is reached only
through multi-step changes; and the "streptomycin" pattern,
in which high resistance may arise either by multiple steps or
in a single step (Demerec, 1948). Recombination, transform-
ation, and transduction analyses all show that the trans-
mission of each successive degree of multi-step resistance is
distinct and independent — as required by genetical theory — •
and leave no room for theories involving the blending inherit-
ance that would result from adaptive changes.

One argument that has been advanced in favour of adaptive
change is based on the observation that bacteria exhibiting
higher resistance than the original strain can readily be
obtained from cultures grown in medium containing a drug
in low concentration, where all the bacteria survive. It is
reasoned that in such cases the drug has no opportunity
to select resistant mutants that might already have been
present in the culture, and therefore that the observed
increase in resistance results from adaptive changes. How-
ever, detailed studies in which Esch. coli was grown in low
concentrations of streptomycin have shown that even under
these conditions the presence of the drug gives an advantage
to resistant variants, which most likely originate as mutants
during the previous growth of the bacteria used in the experi-
ments (Demerec et al., 1950). In the most critical portion of
this study, aliquots containing about one hundred bacteria
were plated onto a series of broth agar plates, half containing
one (jLg. of streptomycin per ml., and the other half — used as
controls — containing no streptomycin. After 24 hours of
incubation the numbers of colonies appearing on the experi-
mental and on the control plates were about equal, indicating
that the presence of streptomycin had not affected survival

Fig. 1. Magnified photograph of an Esch. coli colony grown on broth-agar
medium containing 1 [jig. of streptomycin per ml. and incubated at 37°
for 48 hours. Note the light background region of bacterial growth with a
number of faster-growing secondary colonies on top of it. Bacteria from
these secondary colonies are more resistant to streptomycin than bacteria
taken from the background region.

[ facing page 55

Genetic Aspects of Drug Resistance 55

of the plated bacteria. The colonies on the streptomycin
plates, however, were smaller than those on the plates lacking
streptomycin. After an additional 24 hours of incubation
their size increased, and their shape became irregular because
of the growth of secondary colonies. Microscopic examination
revealed that each was composed of a thin base of background
growth with several fast-growing secondary colonies on top
of it (Fig. 1). Bacteria taken from the secondary colonies
exhibited higher resistance to streptomycin than bacteria
taken from the light background region. Thus it is evident
that, even at low concentrations of the drug, resistance does
not develop adaptively but originates through an abrupt and
discrete change, and that the drug acts as a selective agent by
favouring the growth of resistant variants.

As a rule, a bacterium that becomes resistant to one drug
still remains sensitive to others, which is consistent with the
assumption that mutations in different genes are responsible
for resistance to different drugs, and with the prevailing view
of geneticists that a gene mutation affects primarily one bio-
chemical reaction. One would expect the spontaneous
occurrence of multiply resistant mutants to be very rare,
since in general the probability of double mutation is equal
to the product of the individual mutation rates of the two
genes involved. Experimental tests of that expectation were
made by Szybalski and Bryson (1953), working with resist-
ance to isoniazid and sodium ^-aminosalicylate (PAS) in
Bacillus megaterium. The high mutation rates in this organism
and the single-step development of resistance to those drugs,
without the production of intermediately resistant variants,
made the experimental determination of rate of mutation to
double resistance technically feasible. The rate of mutation
to isoniazid resistance was found to be Q{±1 -5) X 10-^, that to
PAS resistance 1( ±0-3) X 10-6, ^nd that to double resistance
8(dzl-8)xlO-io. Thus the observed rate of mutation to
double resistance, although statistically higher than predicted,
falls well within one order of magnitude of expectation. It is
very likely that a better agreement between the observed and

56 M. Demerec

the calculated values might be obtained if the formula for calcu-
lating probabilities could take into account the variability due
to several biological factors of which geneticists are well aware.

As a logical consequence of the genetic concept of the origin
of bacterial resistance, multiple chemotherapy was suggested
by geneticists as the most effective means of preventing the
development of resistant pathogens. Its successful clinical
use provides good confirmation of the validity of the assump-
tion that gene mutations play the most important role in the
origin of resistant variants.

Very little is known yet about the chemical structure of
genes, but it has been convincingly demonstrated that they
are not protein molecules and thus cannot be enzymes.
Evidence is rapidly accumulating, however, to indicate that
each gene controls one enzymic reaction — in other words,
that there is an intimate relationship between genes and
enzymes. This constitutes a common ground for agreement
between those geneticists who judge that change in a certain
gene is responsible for a particular resistance and those bio-
chemists who consider that resistance is the result of modifica-
tion in a certain enzymic reaction. The striking difference
between the two groups lies in their interpretation of the
nature of this change — the geneticists concluding that it is
brought about by gene mutation, independent of the presence
or absence of a drug, whereas the biochemists believe that it
develops gradually under the influence of the drug. Even here,
however, there is a certain degree of concurrence between
the two groups, since neither denies the possibility that both
kinds of mechanism may operate. Nevertheless, geneticists
have ample experimental evidence to justify the conclusion
that a mechanism involving gradual adaptation must play only
a minor role, if any, in the development of resistant strains.

Summary

During the past decade much time and effort have been
devoted to examination of the role played by genetic mechan-
isms in the origin of bacterial resistance to various drugs.

Genetic Aspects of Drug Resistance 57

When the problem of bacterial resistance first arose, the back-
ground of information about genetic mechanisms in micro-
organisms, and particularly in bacteria, was still very meagre;
consequently the genetical analysis of such resistance pro-
ceeded slowly and encountered a number of difficulties that
would not be met with at the present stage of our knowledge.

The accumulated evidence is ample to show that drug
resistance follows a pattern to be expected of characters de-
termined by genes. This evidence is derived from statistical
analyses of the mode of origin of bacterial resistance; from
genetical studies of recombination between different grades of
resistance, and also between resistance and other genetic
characters ; from studies of the transfer of genetic characters,
including resistance, from one bacterium to another by trans-
formation and transduction techniques; and from studies of
spontaneous mutability and of mutability induced by various
mutagens and by a mutator factor.

None of this evidence excludes the possibility that extra-
genic mechanisms, in addition to genie mechanisms, may
operate in the development of drug resistance. However, if
we can assume that the findings made regarding numerous
other bacterial properties hold true for resistance as well, it
can reasonably be inferred that the role of extragenic factors
is a minor one.

REFERENCES

Alexander, H. E., and Leidy, G. (1953). J. exp. Med., 97, 17.

Armitage, p. (1952). J. R. statist. Soc, B, 14, 1.

Banic, S. (1954). Schweiz. Z. allg. Path., 17, 183.

Baron, L. S., Formal, S. B., and Spilman, W. (1953). Proc. Soc. exp,

Biol, lY.Y., 83, 292.
BoRNSCHEiN, H., DiTTRiCH, W., and HoHNE, G. (1951). Naturd)is-

senschaften, 38, 383.
Bryson, v., and Szybalski, W. (1952). Science, 116, 45.
Bryson, v., and SzYBALSKi, W. (1955). Advanc. Genet., 7^1.
Cavalli, L. L. (1952). Bull. World Hlth. Org., 6, 185.
Cavalli-Sforza, L. L., and Lederberg, J. (1953). VI Int. Congr.

Microbiol., p. 108.
Cavalli-Sforza, L. L., and Lederberg, J. (1956). Genetics, 41, 367.
Cavalli, L. L., and Maccacaro, G. A. (1950). Nature, Lond., 166, 991.
Cavalli, L. L., and Maccacaro, G. A. (1952). Heredity, 6, 311.

58 M. Demerec

Demerec, M. (1937). Genetics, 22, 469.

Demerec, M. (1945). Proc. nat. Acad. Sci., Wash., 31, 16.

Demerec, M. (1948). J. Bad., 56, 63.

Demerec, M. (1950). Amcr. Nat., 84, 5.

Demerec, M. (1951). Genetics, 36, 585.

Demerec, M. (1953). Symp. Soc. exp. Biol., 7, 43.

Demerec, M., Bertani, G., and Flint, J. (1951). Amer. Nat., 75, 119.

Demerec, M., Witkin, E. M., Catlin, B. W., Flint, J., Belser,

W. L., DissoswAY, C, Kennedy, F. L., Meyer, N. C, and

Schwartz, A. (1950). Yearb. Carneg. Instn., 49, 149.
DiTTRiCH, W. (1951). Experientia, 7, 431.
Eriksen, K. R. (1949). Acta path, microhiol. scand., 26, 269.
Giles, N. H. (1951). Cold Spr. Harb. Symp. quant. Biol, 16, 283.
Hartman, Z. (1956). Carnegie Inst. Wash. Publ., 612, 107.
Hershey, a. D. (1946). Cold Spr. Harb. Symp. quant. Biol., 11, 67.
Hershey, a. D., and Chase, M. (1951). Cold Spr. Harb. Symp. quant.

Biol., 16, 471.
Hershey, A. D., and Rotman, R. (1948). Proc. nat. Acad. Sci., Wash.,

34, 89.
Hotchkiss, R. D. (1951). Cold Spr. Harb. Symp. quant. Biol, 16, 457.
IsEKi, S., and Sakai, T. (1954). Proc. Japan Acad., 30, 143.
Lea, D. E., and Coulson, C. A. (1949). J. Genet., 49, 264.
Lederberg, J. (1947). Genetics, 32, 505.

Lederberg, J., and Lederberg, E. M. (1952). J. Bad, 63, 899.
Lederberg, J., Lederberg, E. M., Zinder, N. D., and Lively, E. R.

(1952). Cold Spr. Harb. Symp. quant. Biol, 16, 413.
LuRiA, S. E., and Delbruck, M. (1943). Genetics, 28, 491.
McClintock, B. (1951). Cold Spr. Harb. Symp. quant. Biol, 16, 13.
Newcombe, H. B. (1949). Nature, Lond., 164, 150.
Newcombe, H. B., and Hawirko, R. (1949). J. Bad, 57, 565.
Newcombe, H. B., and Nyholm, M. H. (1950). Genetics, 35, 603.
Oakberg, E. F., and Luria, S. E. (1947). Genetics, 32, 249.
Plough, H. H., and Holthausen, C. F. (1937). Amer. Nat., 71, 185.
Skaar, p. D. (1956). Proc. nat. Acad. Sci., Wash., 42, 245.
Szybalski, W., and Bryson, V. (1953). J. Bad, 66, 468.
Treffers, H. p., Alexander, D. C, Thanassi, F., and Bonner, D. M.

(1956). Microbial Genetics Bull, 13, 35.
Treffers, H. P., Spinelli, V., and Belser, N. O. (1954). Proc. nat.

Acad. Sci., Wash., 40, 1064.
Visconti, N., and Delbruck, M. (1953). Genetics, 38, 5.
Welsch, M. (1952). Bull World Hlth. Org., 6, 173.
Zinder, N. D., and Lederberg, J. (1952). J. Bad, 64, 679.

DISCUSSION

Walker: Dr. Demerec said that the development of drug resistance
takes place quite abruptly. The point of view I would like to suggest
for discussion is this : if we have a template upon which genie material
is being replicated, and the genie material is assembled and peels off as

Discussion 59

it is formed, it comes to a point where the block takes place by the drug ;
if that is at a very late stage in the formation of the genie material this
can split off and convey information for subsequent replication, but is
incomplete in so far as the whole information is not available for subse-
quent replication. In subsequent replication the drug does not enter into
the picture at all. That explains why one can get resistance forming in
one generation and also quite abruptly. There may be many flaws in
that picture, and I am putting it forward only as a basis for discussion :
that it may be the means whereby the information can be transmitted for
subsequent formation of genie material which is not affected by the drug.

Lederberg: This model is one of a number of formal schemes which can
be elaborated to explain the fact that the initial genetic changes (if they
arose) were being produced by the drug. I think that if we had many
examples where we could unambiguously show that these initial "catas-
trophes" w^ere produced by the drug, we would then avidly seek details
of the models in which they would exist. But I think the whole point
we have been discussing is not the ways in which this might happen, if it
happened, but whether it happens at all. Prof. Cavalli-Sforza, for
example, has shown that we can get these alterations in the copies of the
template which occur without the drug ever having been present in
those cells. I know there will not be unanimous agreement on this ; there
will be those here who will continue to insist that there are local effects
which are in fact induced by the drug, but I don't think even that is
Prof. Hinshelwood's model of what is going on, and he has a rather more
subtle understanding of the inductive effects that might come into play.
We may come to a time when we know just what reagent to use, when
we can create specific blocks in replication of particular genes in the
genetic material, but so far we have not found such reagents.

Hotchkiss: It should require a high degree of specificity indeed, to
cause a block at one particular unit, when all of it seems to be made of
DNA in which the chemist can as yet discern no specificity except that
implied in the genie action.

Alexander: I w^onder whether Dr. Demerec's point that he finds this
sharp distinction is as decisive as it seems (between bacteria which have
mutated and those which have not). The time interval in bacterial
development is so short that it might be possible that this is a directed
mutation, directed by the physiological state of the cell. There may be
some similarity in the carcinogenic process, because once a tumour cell
has been produced this is a clear case of mutation, yet there is a long
interval in which we have to produce an unnatural physiological condi-
tion in the animal before this occurs; in some cases this is done with
chemicals which are mutagens. But the most decisive proof that a
mutagen is not always necessary comes from the experiments by the
Oppenheimers, who found that the introduction of an unnatural en-
vironment, namely a thin film of any sort, will eventually after a year or
so create the condition where tumour cells grow. So there one can make
a good case that the mutation which has given rise to the tumour cell
has come as a response to an unnatural physiological environment. The
same situation might also arise in bacteria, but since cell division and

60 Discussion

bacterial growi:h is so quick, one may not detect this intermediate stage
by the methods used. One may only detect the end-effect when the final
mutational response to the changed environment has already taken place.
If the time interval could be narrowed within division times, one might
then find a gradual adaptation finishing eventually in a mutation.

Demerec: The evidence accumulated by geneticists working with vari-
ous organisms indicates that, as a rule, mutations produce discrete rather
than gradual effects ; and the findings of research with antibiotics con-
form with this general picture.

The experiment discussed in my presentation was not designed to
show whether or not the drug, in this case streptomycin, induces muta-
tion. It was designed to show that resistance to a low concentration of
the drug develops in discrete steps. The critical experiments, as men-
tioned by Lederberg, in studying whether mutation to resistance origi-
nates independently of the drug, are those where the drug has not been
used, such as the experiment which was discussed by Cavalli-Sforza this
morning, and also a replica plating experiment which was done by
Lederberg.

Yudkin: Dr. Demerec, I think we all agree that what you have shown
once more is that genetic changes do occur. I don't think that either of
the two examples mentioned prove that induction or adaptation did
not occur, and I suggest that e.g. the outgrowth of resistant papillae or
parts of a colony may indeed have occurred through induction between
the drug and some of the bacteria which, when they become resistant,
obviously grow much faster.

Another argument used in favour of drug resistance originating only
through genetic change is the chemotherapeutic one, i.e. the use of a
combination of drugs is much more effective than the use of the drugs
singly. And surely Sir Cyril would say that for a bacterium to become
adapted to a combination of drugs must be very much more difficult
than for it to become adapted to a single drug.

Davis: I would question Dr. Alexander's statement that cancer is
clearly caused by mutation. I would agree that a cancer results from
inheritable change in a somatic cell; but the inheritable properties of
somatic cells, after all, are determined in part by the process of differ-
entiation, and in this process something in the environment directs
gradual changes that are inheritable. It would therefore seem reasonable
to consider the gradual process of carcinogenesis as an aberration of
differentiation.

As Dr. Pontecorvo pointed out, geneticists would not deny the possi-
bility that the environment can direct an inheritable change, for they
would then be denying the process of differentiation. Unfortunately,
our understanding of differentiation is very primitive compared with
our understanding of gene mutations. It has long been hoped that
unicellular organisms would be helpful, since it is in principle quite
conceivable that one could find chemical or physical agents that would
cause such directed inheritable changes in these organisms. However,
few cases that fit this category have been observed, and none in bacteria.
The burden of proof still rests on those who would claim that drug

Discussion 61

resistance, or any other inheritable change in bacteria, can be brought
about through a directive environmental influence.

Pontecorvo: Those changes, of any type, that we are actually able to
demonstrate as genetic are of course a very selective sample. Any
change which is either not big enough or not sharp enough or perhaps
too much subject to environmental variation cannot be tested; at least
it is very difficult to subject such changes to the test of segregation,
which is the only one that we can use for an unquestionable distinction.

Hinshelwood: Dr. Demerec, what is your opinion about the fact that
the sensitive cells had grown before the ones that you termed resistant ?

Demerec: The reason is that we used a very low concentration of
streptomycin, a concentration that did not kill any of the bacteria
plated on it, as I have shown by comparing the numbers of colonies
obtained on plates containing streptomycin and on control plates without
it. Therefore, the sensitive bacteria were able to divide, but they divided
slowly and formed a very thin layer of gro^vlh. We assume that during
those divisions mutants appeared which were better adapted to gro^vth
on a medium containing this low concentration of streptomycin, and that
thus we had the secondary colonies originating.

Hinshelwood: We have done experiments on the crossing of yeasts and
examined the galactose fermentation, and we got clearcut Mendelian
phenomena, superimposed on which there were adaptive developments
of the segregated characters themselves, so that both adaptive and clear-
cut Mendelian phenomena were present in the same system. We have
also done experiments on the crossing of bacterial strains of certain
drug-resistant cells and got an almost continuous spectrum of resistance
between the levels of the parents. This may, of course, be ascribed to
the fact that the mutation steps are very small, but even so it is hard to
regard the result as evidence of a sharp segregation. However, in refer-
ence to Dr. Demerec's opening remarks, who has ever said that in sexual
recombination or in the segregation phenomena of Mendel there was
other than transmission of basic material and information? And who
would ever have thought of attributing sexual recombination and segre-
gation to adaptation ? I have never heard of anybody who would do it.

Demerec: I agree with you. Fortunately, there is no serious disagree-
ment at present about the mechanism of Mendelian inheritance. Un-
fortunately, however, until very recently we did not know about sexual
recombination in bacteria. And when our first reports on genetic
research with bacteria were published, the question was raised by
bacteriologists and geneticists: Have bacteria a genetic mechanism
similar to that found in higher organisms? Proof of the existence of
such a mechanism came only recently.

Lederberg: In any experiment where bacteria grow in the presence of
a drug it is possible to attribute special significance to this and to say
that perhaps the drug played some role in inducing and fixing mutation.
That type of discussion was quite profitable ten years ago. At the present
time we have a number of sharply delineated cases where, on the one
hand, we can demonstrate the occurrence of structural changes leading
to resistance in the absence of the drug, and these are mutations; on the

62 Discussion

other hand, we have a much smaller sample, but nevertheless a few quite
unambiguous cases, of kinetic changes in enzyme balance which are
sufficiently self-regenerative so that they fit into the type of picture that
Prof. Hinshelwood elaborated. Therefore, there is no need to argue any
further as to the fact that these mechanisms do exist. If there is any
point in the controversy, it is : What happens in this specific situation,
what happens in the next specific situation? I suggest that if we are
going into details of experiments, we decide beforehand that there will
be two or three chosen as exemplifying the type of analysis that must be
done, and then we can concentrate on trying to see to what extent
mutational occurrence has been ruled out in any one case; to what
extent have directed adapted changes (which may have more or less
permanence) been ruled out or ruled in. But to say that something might
have happened here and something else there is no longer the point
since we have mutual agreement of examples of both extreme types of
behaviour.

Yudkin: We are really having this discussion on two levels. Dr. Ponte-
corvo said that we are agreed — and I wish we were — that we accept that
these two phenomena which are most under discussion can occur. What
I am a little concerned about is that from time to time we read that "It
has now been proved " that mechanism A has occurred and that therefore
we may assume that mechanism B does not.

Slonimski: It cannot be said that mechanism A has been proved; all
that one can say is that mechanism B has been disproved. On logical
grounds a hypothesis can only be disproved, but not proved.

Yudkin: I take exactly the opposite view: I think nothing has been
disproved, but certain things have been proved.

Pollock: I agree with Prof. Yudkin's last remark. I do not know of
anj^one who has denied the existence of so-called spontaneous mutations
appearing in certain cases, but I am not so sure about examples of
induced heritable adaptive changes.

Lederberg : Novick has been carrying the two states of lactase formation
in Esch. coli for what could be considered to be an indefinite number^
many hundreds — of generations.

Pollock: I feel fairly certain that they do exist, but if we are going to
adopt your suggestion of taking a few isolated examples, then it is
important to find one which we can get to grips with on the adaptation
side.

Lederberg: The one that has satisfied me without any doubt that there
can be such a mechanism is the one that Cohn and Novick showed.
That one can be carried on indefinitely.

Davis: Dr. Demerec has pointed out that in the presence of a concen-
tration of streptomycin that did not kill the cells, but allowed them to
grow extremely slowly, after a while resistant mutants appeared. In
trying to decide whether the amount of background growth would be
sufficient to account for the number of resistant clones, it might be
important to take into account some work reported by Novick and
Szilard a few years ago. It is generally assumed that the rate of appear-
ance of a given mutation in a given strain has a fixed value per generation.

Discussion 63

However, when these investigators varied the growth rate by controlling
the supply of a required amino acid, they found that the mutation rate
remained constant per unit time rather than per generation. Thus, when
an organism was growing at one-twelfth its normal rate it produced
twelve times as many phage-resistant mutants per generation as when
growing at the full rate. I don't know that anyone has carried out
parallel experiments on the effect of slowing growth inhibitors, but this
work raises an interesting possibility ; when we are dealing with border-
line drug concentrations that slow growth but do not kill the organisms,
mutation rates per generation may be very much higher than what we
are accustomed to.

INHERITANCE IN SINGLE BACTERIAL CELLS

W. Howard Hughes

Wright-Fleming Institute of Microbiology, St. Mary's Hospital, London

When Demerec (1948) presented his views on stepwise
mutations he stimulated a number of independent workers to
challenge them (Hughes, 1952: Eagle, Fleischman and Levy,
1952; Mayr-Harting, 1955). While agreeing with the general

005

0-01 0-02 0-03 004
Penicillin concentration (u./ml.)

Fig. 1. Titration of sensitivity of single-cell strains of staphylococci using

small implants. Key to curves : , parent strain ; , single-cell strain

I ; •-•-•, single-cell strain II. Parent strain and strain I give a value of

0-0325 u./ml. for 0% survivors. Strain II gives a value of 0-035 u./ml. under

identical conditions. (Hughes, W. H. (1952), J. gen. Microbiol, 6, 175.)

picture of the emergence of resistance as he painted it, the
present author objected to his conclusions that there were
clearcut steps in the process, believing the steps might be
artifacts imposed by the limitations of titration methods
generally. It seemed that only by working throughout with
single cell isolates or by maintaining a continuous photo-
graphic observation would it be possible to settle this question

Inheritance in Single Bacterial Cells

65

to the satisfaction of other workers who had a bias in favour
either of adaptation or of major mutation as the basis for
bacterial variation.

If single cells are cultured and from the clone further single
cells are selected these will differ from one another in anti-
biotic sensitivity (Fig. 1). The differences here are so small

0-05 0-5 5-0

log penicillin concentration (u./ml.)

Fig. 2. Results of tube titration of sensitivity of single-cell strains of staphylo-
cocci. Key to curves: , strain I; , strain Via; — O — » strain

XXX« ; , strain XXXIa. The four subcultures show a similar distri-
bution of sensitivity, although the average sensitivity of each is at a different
level. Each curve on this is built up from 11 separate points, the only 3 points
not falling on their line are marked «. (Hughes, W. H. (1952), J. gen. Microbiol.,

6, 175.)

that had the titration not been repeated on several occasions
with different batches of penicillin over intervals of many
months the author would not have been convinced that they
were not due purely to experimental error.

If now from the tail of the population the more resistant
cells are picked, new populations with increased resistance are
obtained. Fig. 2 shows the stages of the investigation. It will
be seen that the increase is fairly steady. This impression
can be misleading since only the most resistant population is

DRUG RES. — 3

66 W. Howard Hughes

drawn. If five or six single cells were picked the individual
populations derived from them would vary, occasionally
being more sensitive than the parent but usually being inter-
mediate between the parent and this most extreme example.
The apparent increase in resistance by even steps is here due
to selection having been made and is not an expression of any
law of variation.

Cavalli-Sforza saw these results before they were completed
and pointed out that the pattern was that of a normally distri-
buted population curve rather than that of mutation as ordi-
narily understood (Cavalli-Sforza, personal communication). It
should be noticed that the sensitive moiety of the population is
killed off by the methods used and that there is no evidence from
this type of experiment of cells more sensitive than the parent.

Yudkin considered this type of experiment on antibiotics
and his own work with flavin, and advanced his theory of
resistance (Yudkin, 1953). This is still the most likely
explanation of the work of the present author, and of that
of Eagle and his associates. No subsequent experiments have
thrown any serious doubt on it.

Yudkin's theory postulates that when a cell divides, the
daughter cells will be unlike each other and unlike the parent
cell. Support for this hypothesis was provided by studies of the
behaviour of a strain of Escherichia coli B when grown under
conditions of diminished oxygen supply (Figs. 3-5). The
long organism was produced at the first division of the parent
cell, the sister cell gave a normal colony. This inability
to divide is frequently seen in fresh isolates of most rod-
shaped organisms. If these long cells are selected and sub-
cultured in broth and then a fresh test is made it will be found
that they form an increasingly large proportion of the entire
culture and strains can be bred composed of cells all of which are
sensitive to the environment. With this phenomenon in mind
the reactions of bacteria to antibiotics can be reconsidered.*

* A film that illustrates and contrasts the action of penicillin and of a wide
spectrum antibiotic, in this case chloramphenicol, was shown at this point,
and can be obtained from the Photographic Department, St. Mary's Hospital,
London.

4r

/^

9

\'*

Figs. 3-5. Development of the mixed colony from
a sintrle organism. Fig. 3. Stage of third division
Fiff 4. Stage of fifth division. Fig. 5. Stage ot
seventh division. The solitary L-form is now
vacuolated. (Hughes, W. H. (1953), J. gen. Micro-
biol., S,'W7.)

[ facing page 66

^

Fig. 6. Differences in related single cells. (Hughes, W. H. (1955/>), J. gen.
Microbiol., 12, 269.)

Inheritance in Single Bacterial Cells

67

Now taking the action of penicillin first, Fig. 6 shows a
group of organisms transferred to penicillin-agar. Just as the
morphological effect of the drug varies with concentration, so
if the concentration is kept constant individual cells in a
culture will be influenced differently. The cell on the right of

m

I

rp^^irp

'iW^ ^A^^

Key .

(K Long form alive A L°"g ^^^'^ ^^^^ • ^° growth, autolysed

Fig. 7. The effect of 10 u. penicillin /ml. on the individual cells of nine micro-
cultures, grown from nine members of a clone of sixteen cells. (Hughes W. H.
(19556), J. gen. Microbiol., 12, 269.)

68 W. Howard Hughes

Fig. 6 is already dead and lysis is occurring ; that at the top is
inhibited for division but not for growth and on transfer to
penicillinase-agar might recover; the group on the left is
dividing. By using the morphological changes and tests for
viability to assess the effect on the individual cell it is possible
to build up a kind of family tree of resistance and sensitivity.
A single cell is taken and allowed to divide to give a colony of
from 4 to 16 individuals. These are transferred separately to
penicillin-agar and their fate followed for a fixed period of time,
anything from 3-5 hours is suitable. At the end of this time all
cells which have not autolysed are transferred to penicillinase

\

CCCC CCC* CC«C 9 9 9 9
5 6 7

ccce#ccc ccc*cco ccc cccccc

• Organism died C Organism f&rmed colony

Fig. 8. Diagram showing fate of eight microcolonies derived from the same

single cell when plated out on 7-5 u. penicillin/ml. agar. (Hughes, W. H.

(19556), J. gen. Microbiol, 12, 269.)

broth and their viability tested. Fig. 7 shows typical results.
Cell 3 gives among its descendants almost all variations. Cell 1,
on the other hand, gives a uniformly sensitive group. This
strain in the presence of penicillin gives daughter cells which
differ from one another in resistance (Hughes, 19556). Fig. 8
shows a complete population exposed and tested for viability.
The selection of sensitive populations as well as of resistant
ones from the small cell was first done on spontaneous charac-
ters in Esch. coli B (Hughes, 1955a). It was noticed that in
a strain repeatedly subcultured from single cells the growth
rate of microcolonies still varied. The difference between
slow and quick growing colonies was shown before there was
any depletion of nutrients in the medium. Fig. 9 shows the

Diameter of colonies
Parent strain no. 1

4: 0-1 mm. }

Fig. 9. Histocrram showing rates of growth of colonies from
single cells of Escherichia coli grown under anaerobic condi-
tions. Measurements of colony diameter made after 3 hr.
incubation at 37°. Re-selection of the subcultures indicated
by arrows. (Hughes, W.H. {1955 a), J. gen. Microbiol., 12,205.)

70

W. Howard Hughes

selection of populations starting with a single cell. The
differences between the two lower populations is even greater
than appears since many of the "colonies" given an arbitrary
diameter of 1 actually failed to divide at all.

This method can be applied to the wide spectrum anti-
biotics. Streptomycin has been used, since Demerec had

Rl

Rll

ON 8 UNITS

ON lO UNITS

ON 12 UNITS

DIAMETER OF COLONIES
Fig. 10. Stages in the selection of streptomycin
resistance.

chosen this to contrast with penicillin in his original paper
(Demerec, 1948). Klebsiella pneumoniae was used in the
present series of experiments, as it forms a neat colony which
is easy to measure. The steps in re-selection for resistance are
given in Fig. 10. At each level of antibiotic, up to 12 sub-
cultures of the largest colonies were necessary before the
diameter of the strain on normal medium was reached. At
this point the new strain was able to survive and divide, at

Inheritance in Single Bacterial Cells 71

any rate for 4 hours, in a strength 20 per cent above that
which had originally been used. At the present rate of pro-
gress it will require about 40 subcultures to double the
resistance of the strain, but the number of cells being ex-
amined at each level is from 100 to 300 only.

It appears that the transition from sensitivity to resistance
can be made gradually by very large numbers of small steps
made possible by the organisms differing from one another
just as do the units in any other population.

REFERENCES

Demerec, M. (1948). J. Bad., 56, 63.

Eagle, H., Fleisciiman, R., and Levy, M. (1952). J. BacL, 63, 623.

Hughes, W. H. (1952). J. gen. Microbiol., 6, 175.

Hughes, W. H. (1955«)- J- gen. Microbiol., 12, 265.

Hughes, W. H. (19556). J. gen. Microbiol., 12, 269.

Mayr-Harting, a. (1955). J. gen. Microbiol., 13, 9.

YuDKiN. J. (1953). Nature, Lond., Ill, 541.

DISCUSSION

Siocker: Dr. Hughes' approach is, I beUeve, a very valuable one. In
micromanipulation experiments, one is confined to examining small
populations. This means that one can only observe frequent mutations.
One is apt to think of spontaneous mutations as being very infrequent
events; but in bacteria, in regard to certain quantal or all-or-none
changes, there are a number of examples of spontaneous changes of
heritable character at rates of the order of IO-2 or 10-^ per bacterium
per generation. In most cases no analysis has been made to see whether
or not these changes result from changes of chromosomal genes. In
Salmonella, spontaneous changes of flagellar antigenic phase occur in
each direction at about this rate ; and this change has been shown to be
in fact something occurring at a chromosomal gene (Lederberg, J., and
lino, T. (1956). Genetics, 41, 743). Hughes' demonstration of heritable
differences in antibiotic sensitivity between two cells picked at random
from the same population is formally analogous to the differences in
antigenic phase which one may find between two Salmonella colonies
obtained by plating out a single colony; and might, like it, result from a
'change of state ' at a chromosomal locus.

Hughes has examined cells of a single clone grown in a common
environment, and has shown that even a pair of sister cells may differ in
their ability to grow in a particular antibiotic concentration. Until a
direct test has been made one should not assume that such differences
are due to differences in the genetic constitution of the cells concerned,

72 Discussion

for one can show that in some systems there is heterogeneity within a
clone which is due neither to genetic differences nor to detectable
differences in the environments of the differing individuals. For in-
stance, in some Salmonella strains only a characteristic proportion
(e.g. 10-^) of the cells of a clone can synthesize locomotor apparatus,
even when the clone has been grown in a shaken flask of broth, so that
all the cells grew in an identical environment (so far as it is possible to
provide one). A direct comparison of the progeny of synthesizing and of
non-synthesizing individuals (isolated by micromanipulation) shows no
difference : therefore the two kinds of cell do not differ genetically. The
difference between them can therefore be attributed neither to differ-
ences in their genotypes, nor to (detectable) differences in their environ-
ments. Thus, the genotype and the environment determine only the
relative probabilities of the two alternative phenotypes in any particular
individual. This is presumably true also in the case of drug resistance ; a
common example is that under some conditions of selection (for instance,
an antibiotic concentration which permits colony formation by say one
per cent of the cells plated) it is not infrequent to find that the anti-
biotic resistance of the population of these colonies is indistinguishable
from that of the parent strain ; all one can say is that the original pro-
genitors of these colonies differed in their phenotype from the mode, but
that particular variation of phenotype is not heritable.

Dr. Hughes, would you please explain in more detail the actual
procedure by which you observed these small steps with apparent
increasing resistance to the drug. Did these cells, for instance, go through
an intermediate period of cultivation in the absence of the drug to
show that something other than a phenotypic adaptation had occurred?

Hughes: Yes ; you take a single cell or microcolony, grow up a manage-
able amount of culture, about 500-1000 cells, then transfer to broth.
From this you plant out again in the test medium, which in this case is
agar with a given level of streptomycin. You weed the cells out so that
there is a clear area to each. You then cultivate for a standard length
of time at a standard temperature and measure the colony diameter.
Having measured up the whole of the diameters, you pick the smallest
and the largest colonies, make new emulsions and repeat the procedure.

In the technique for testing two daughter cells separately, you take a
single cell and put it into a drop of broth. You wait until it has divided,
then transfer each individual into a drop of broth. You then transfer the
drop to a marked position on a small block of agar containing the in-
hibitor (I always put one on each corner of the block). Then you can
follow them at any interval ; you recover them after your standard time
of exposure and put them into normal nutrient medium, or if you wish,
into a penicillinase medium if you are working with penicillin, to see
whether they recover and give living organisms or not. You can repeat
this as often as your patience will allow.

Davis: In the experiments where drug resistance was building up,
were they always cultivated in that concentration of the drug ?

Hughes: No, they had one subculture in between each test in normal
broth.

Discussion 73

Yudkin: Have these strains which gradually progress toward resist-
ance always been derived in the presence of the drug ?

Hughes: In the particular case of streptomycin they were so derived.
They were then grown out away from the drug, and tested again. On
the other hand, I believe it would be possible to pick from the sensitive
side, for instance in chloramphenicol ; but in streptomycin one can only
pick the large colonies, not the small, because in streptomycin a micro-
colony of about 16 organisms will be dead within 4 hours.

Yudkin : That would still be in the presence of the drug ; but I wonder
if it would be possible to select toward sensitivity in the absence of drug.

Lederberg: What were the population sizes?

Hughes: You have an overnight subculture; 10 ml. building up to the
full population which you would expect of an overnight culture. Then
you start again from that.

Eagle: How do you interpret these results? In the specific case of
antibiotic resistance, are you suggesting that in these cultures there is a
tremendously broad spectrum of mutated organisms, differing in degree
of resistance, or are you suggesting that these are indeed adaptive
changes ?

Hughes: I believe that every time a bacterial cell divides, just as every
time you sow a seed of any other plant, you get individuals. I believe
that all bacteria in a culture differ from all the other bacteria in a culture ;
and that is the only explanation that I can see for the very wide differ-
ences ; and that the so-called mutant is the product of killing off 99 • 999
per cent of them and taking the one that survives. Then, it is quite
impossible to tell whether you have selected from a very broad spectrum
or whether you have got a genuine mutant. I don't deny the existence
of mutants, I am sure they exist. But I do say that there is a great deal
of variation which, if you are going to use the term "mutant" for it,
means that every time a bacterial cell divides, the two daughter cells
are mutants. I don't think we should use the term in that way. I think
that something very much more clearcut should happen — an entirely
new characteristic, not a gradation — to merit the term "mutant". I
think they are all individuals.

Lederberg: We are all prepared to accept the conclusions which have
just been stated. But I wonder if the situation might not be slightly more
complex. Firstly, that there might be individual responses on the part
of different cells to a drug like penicillin is almost to be expected and you
certainly have given us a very beautiful demonstration of it. As your
work shows, there is reason to believe that the action of penicillin is
concerned with the manufacture of cell walls, and it would be no surprise
if the rate at which new walls form were related to the amount already
there. One would not be surprised to see that there are individual
responses on the part of individual cells. I just raise the question, not
because the conclusion you give is in any sense unlikely, but this does
mean that every cell division gives rise to a transmissible difference
between the two cells. It seems quite likely that when you do finally
make measures of the norm of reaction of two clones, that may reflect
to some extent mutations which are transmissible. But is it certain

74 Discussion

that those transmissible differences, that you find as distinguishing the
two clones that you finally isolate, occur at the time that you separate
these two cell? Could there not have been occasional large differences
in the norm of reaction arising by larger changes during the growth of
the large clones; which would mean that you might have physiological
individuality occurring in every cell division, and genetical changes
occurring perhaps less frequently but with high probability at some time
in the growth of the clone (although considering the very small magni-
tude of the physiological change which is involved here, I would be
willing to accept their occurrence during cell division)?

Hughes: In certain stated cases with some particular cells, the daughter
cells differ from each other sufficiently for one to be a lethal and the other
to survive. That happens with sufficient frequency, and could be bred
for. My view is that if you can show that a single individual cell is
different from its twin sister, then mutation must be a very frequent
thing since it is picked up in my populations of from 100-300 cells.

Lederberg: A difference between sister cells is not necessarily a trans-
missible difference. Although such transmissible differences may arise,
they may be superimposed upon the much more frequent physiological
fluctuation — one cell gets more cell wall than the other. We know that in
cell division the two cells are not necessarily exactly the same size. You
don't know that every step of selection involves a genotypic difference;
only an occasional one need have done so.

Hughes : In the case of sensitivity to the environment whereby the cell
becomes long in form, there is only one division — that of the parent cell.
In that case you subculture the actual cell which varied, and not a clone.

Demerec: I agree with Dr. Hughes that practically no two bacterial
cells are the same as far as their genetic constitution is concerned. In
our laboratory, in keeping stocks of different mutants we try to avoid
passing the stock through a single-colony transfer, because we are afraid
that by so doing we might change the other properties of the bacteria.
Each bacterial cell has a large number of genes, any one of which may be
able to mutate; some cells mutate with higher and some with lower
frequency. If we had a test sensitive enough to pick up all the gene
mutations that occur, we should get a mutation rate very much higher
than that obtained when we observe mutations of a single gene.

Cavalli-Sforza: I am rather worried that Dr. Hughes found such a
great deal of variation in using the micromanipulator to select single
cells. When you use the much simpler method of isolation by plating
you don't find such a tremendous variation. I wonder. Dr. Hughes, if
you can give an explanation for that. Do you think that one would
perhaps get the same amount of variation from cell to cell if isolation
were made in the standard way ? Also, w hat is the accuracy of measure-
ment that can be obtained with your method ? You grow your colonies
directly in an oil-chamber, so there may be considerable variation in
local conditions that may affect the estimation of growth. Also, can you
say exactly how many cells die in your selection experiments with
penicillin? Do the majority die?

Hughes: It depends on what level you choose; you can choose a 20 per

Discussion 75

cent mortality or higher or lower than that. In the case of the "family
trees", about 80 per cent died.

Ledcrberg: In regard to this matter of considering a genotype as a
norm of reaction, all geneticists have been worried about what is called
residual variation in supposedly pure lines. We never know if we can
ascribe this type of statistical variability to uncontrollable variation in
the environment, with maternal carry-over effects that have no genetic
significance, or very minor minimal changes in the genetic material
itself. Most genetic work is purposely concerned with changes so large
that they can easil}^ be scored, and in the case of bacteria, usually with
changes large enough and persistent enough to be scored in clones. We
have here quite a different dimension of analj^sis from that to which we
were accustomed, although it does recall the work of Dr. Jennings with
isolated rhizopods (Jennings, H. S. (1929). Bibliogr. genet., 5, 105). We
must seriously consider the possibility that there is another dimension
in genetic change besides the one to which we are accustomed. Here I
agree with Dr. Hughes that there may be, together with the fixed
continuity of polynucleotides which give the essential structure, other
chemical variations in the chromosome which could control the level of
activity of genetic material. Dr. Stocker has already alluded to phase
variation; here it was possible to show that the difference in antigenic
expression is due to oscillations in state between the expressivity and the
non-expressivity of a given specific antigenic determinant (Ledcrberg J.,
and lino, T., (1956), Genetics, 41, 743) ; and this was a major change. We
would certainly not want to exclude the possibility that this is going on
all the time, and onty if you have the most refined methods of analysis
are you going to be able to pick up the changes in local activity of the
genetic material. We must, therefore, try to translate this type of ob-
servation into the possibility of conventional genetic analysis by recom-
binational methods, and see whether we can localize that sort of change
that you have described. Secondly, we should see whether we can control
these changes environmentally from the outside. There is no indication
as to the inherent nature of these changes, whether they are purely
metabolic accidents, or whether the type of medium they are in may have
something to do with them.

Pontecorvo: If instead of studying bacterial cells, which seem to be
locked into a tough membrane, you looked at mammalian cells in tissue
culture, you would find there is an enormous amount of variation in
shape, size, etc. Each cell has an individualitj?^, so to speak.

Eagle: There are large differences with respect to resistance to drugs.

Lederberg: You might also find a very wide variation in chromosome
number from one cell to another. But is that residual variation wholly
genetic in the sense that Dr. Gj^orffy was using the term, or are there
other methods of variation?

Hayes: Cells oiEsch. coH in young culture as a rule contain four nuclear
analogues. This means that two generations are required for segregation
of a cell which is under the sole control of one of the four nuclei of the
initial cell. There is good evidence to suggest that one normal nucleus
within a cell is able to support normal growth. If Dr. Hughes" non- viable

76 Discussion

cells, which express their defect on the first division, are due to something
analogous to a lethal nuclear mutation, then this change must either
have occurred simultaneously in two adjacent nuclei of the initial cell,
or else in one nucleus one generation back. In a scheme of this sort,
under conditions such as Dr. Hughes used where the behaviour of cells
in a population which had been carried through several generations was
studied, one would expect to find a number of non- viable cells which
failed to divide ab initio, as well as cells which segregated a non-viable cell
only at the second generation. What is your opinion on this, Dr. Hughes?

Hughes: I don't know what happens inside the cell. But the type
which gives one lethal and one normal cell exists, and so does the one
that is immediately a lethal. On the first isolate we have 3 per cent of
this lethal, and 12 per cent of a cell that divides to give one lethal and
one normal; on re-selection from these we get up to 17 per cent direct
lethals and 57 per cent of mixed ones. At no time did I see this second
generation type you anticipate. It was first division or nothing. I don't
know the explanation.

Hayes: Stress has been laid on the fact that these changes are probably
either mutational, or else have something to do with enzymic equilibria
and so on in the cytoplasm.

There is another kind of genie change which could have profound
effects on the cell, which is not mutational in the sense of altering the
direct function of a particular gene, and which is not connected with the
cytoplasm, and this is the question of the rearrangement of genes within
the chromosome, the position effect. It has been shown recently by
Jacob, at the Pasteur Institute, that when selection is made for Hfr
derivatives of Esch. coli K 12 donor strains, which have a very high
fertility, the genes of different Hfr isolates from the same witd-type
strain may appear in quite different orders on the chromosome. It is
not known whether the change to the Hfr state is directly associated
with, and possibly due to, rather rare chromosomal rearrangements, or
whether the rather rare Hfr mutations selected for are simply super-
imposed upon chromosomal reorganizations which may occur quite
frequently. There is some vague morphological evidence of nuclear
fusion, suggestive of autogamy, in bacterial cells ; such a process could
possibly result in rearrangement of the order of genes on the chromo-
somes when they separate out again. Position effects of that sort might
greatly influence the viability or the biological efficiency of individual
bacterial cells.

Hughes: I should mention that Avhile you can select out for a high
proportion of lethals, you cannot select out a lethal-free strain. You can
get rid of the cells which die immediately, you can get the percentage
down to a fraction of one per cent, but you cannot lose entirely the cells
which divide into one lethal and one normal. They persisted however
long I have gone on with this type of experiment.

Lederberg: Do I understand correctly that these lethal-type cells only
appear during the zero or first generation of transfer from broth or agar
and not subsequently in the growth of the clone ?

Hughes: That is correct as far as I know.

Discussion 77

Lederberg: That would suggest one of two possibilities: either (1) that
during the course of growth in broth you accumulate a higher frequency
of alteration, depending on the nuclear configuration of the cell that you
begin with and that shows up either in both or in only one of the first
daughter progeny; or (2) that the noxious stimulus is not agar but the
switch from broth to agar, which may cause some imbalance in the
relative rate of formation of cell wall or cell substance. Once the cells
start to grow on agar they might become well adjusted to those particu-
lar circumstances. Have you got the observational evidence that would
bear on this question?

Hughes: I don't think so. I have always thought that there was a
mutation involved in this, and that in broth it was no disadvantage to
have this particular character, and that the population built up in broth
would be detected when transferred to solid medium. ^Vhether it is the
shock of transfer to solid medium, or something else, I don't know.

Lederberg : Is there any indication that the time of cultivation in broth
has an influence on the proportion of lethals which appear when the
clones are transferred back on agar ? This would be one line of evidence
relative to the question of accumulation of these mutations in broth.

Hughes: My impression was that a short subculture, i.e. building up
a microcolony of perhaps 200 or 300 in broth, gave much the same per-
centages as an overnight broth culture. I would like to repeat that.

Lederberg: That would favour the conception that it is the sharp
change of medium that is the noxious stimulus.

Ultraviolet light induces a possibly analogous abnormality. After a
week of exposure of Esch. coli K12 to ultraviolet 60-80 per cent of the
cells may divide to give one normal, rapidly dividing offspring, and
another abnormal, swollen and probably in viable one. This is almost
certainly a reflection of the multinucleate structure of the cells (Leder-
berg, J., et al. (1951), Cold Spr. Harb. Symp. quant. Biol., 16, 413).

lerusalimsky : Dr. Hughes mentioned in his interesting report a very
important point on the inequality of bacteriological cells arising from
division. This question has not been investigated sufficiently. It is not
known whether inequality of cells is random and non-regular or whether
one of them always resembles the mother and the other the daughter.
Malek in Czechoslovakia and Streshinsky in the USSR support the
second point of view, and our experiments also tend to support it. Our
experiments are directed towards explaining the causes of the varying
resistance of individual cells in a population, and discovering the relation
between the degree of resistance and the physiological age of cells.
Unless these individual differences are taken into consideration, it is very
difficult to distinguish adaptive phj^siological changes from mutations.
It seems to me that Dr. Hughes' film has proved that resistance to peni-
cillin arises mostly in young daughter cells, and therefore this pheno-
menon is probably a physiological adaptation.

PENICILLIN-INDUCED RESISTANCE TO
PENICILLIN IN CULTURES OF BACILLUS CEREUS

M. R. Pollock

National Institute for Medical Research, Mill Hill, London

Although the mechanisms of drug resistance in bacteria
are, in general, not well understood, there are certain strains
whose ability to multiply after addition of penicillin can be
confidently ascribed to their production of an enzyme, peni-
cillinase, which hydrolyses the drug to the antibiotically
inactive penicilloic acid. This is true for the naturally occurring
penicillin-resistant staphylococci (Barber, 1953) and several
different species in the Bacillus genus. This report is concerned
solely with the mechanism of the development of this peni-
cillinase-type of penicillin resistance in cultures of Bacillus
cereus, and it will be shown that the change can take place
in two quite distinct ways, both of which require the addition
of penicillin for their full expression. In both mechanisms,
the change involves a purely quantitative increase in the
ability to form a single, enzymically active protein. Such a
relatively simple system naturally lends itself to a more de-
tailed and more accurate analysis at the biochemical level
than most types of drug resistance.

In the first mechanism, the resistance is acquired by the
process of enzyme induction. A greatly increased rate of
penicillinase formation can be very rapidly acquired, within
the space of a few minutes, in all or most of the individual
cells in the population by brief contact with low concentra-
tions of penicillin (1 unit/ml. or less). This acquired ability
to produce penicillinase at increased rate is biochemically
stable but is not inherited by daughter cells as a genetically
stable character, being gradually lost as the cells grow in the
absence of the antibiotic. After 7 to 12 generations the popu-

78

Penicillin-induced Penicillin Resistance 79

lation has returned to the original state of (relative) penicillin
sensitivity. The phenomenon of induced penicillinase form-
ation occurs in most strains of B. cereus, and in Bacillus
suhtilis and Bacillus megaterium, and has been studied in
great detail in B, cereus strain 569 (Pollock, 1953). It is not
proposed to discuss it in much further detail here except to
point out that even the uninduced cells (untreated with peni-
cillin) produce small quantities of penicillinase ("basal"
enzyme) and that maximal induction results in its formation
at about 300 times the basal rate. It has not hitherto been
found possible to test the penicillin-sensitivity of cells in the
absence of penicillinase induction. This is probably because
penicillin, except in very high concentrations, does not itself
appear to inhibit induced penicillinase synthesis, which takes
place very rapidly after addition of penicillin in sensitivity
tests. Thus although the increase in penicillin resistance of
individual cells on penicillinase induction is probably con-
siderable, only a relatively slight increase can be actually de-
monstrated. It could, however, be shown (by the experiment
summarized in Table I) that preinduction of cells with peni-
cillin at subinhibiting doses does considerably increase their
chances of survival when treated later with much higher con-
centrations of the drug. Spores of strain 569 incubated for
5 hours in plain nutrient agar (i.e. having grown to produce
microcolonies of 16 to 32 cells) were unable to survive addition
of penicillin to a concentration above 60 units/ml., while the
same inoculum grown in penicillin-agar suffered only slight
reduction in viability after addition of 100 units/ml., and
a considerable proportion of microcolonies (11 out of 42) were
still able to grow into normal colonies after addition of 160
units penicillin/ml. (No such protective effect of induction
occurs with the penicillinase-constitutive mutant strain 569/H
which forms penicillinase at maximal rate without need for
previous treatment with penicillin.)

The second process by which B. cereus populations may
acquire the penicillinase type of penicillin resistance is the
(perhaps) more familiar one of mutation followed by selection.

80

M. R. Pollock

Very rarely there may appear mutant cells, which give rise
to clones able to form penicillinase spontaneously at high
rates ("penicillinase-constitutive" mutants). These muta-
tions occur by a process which is independent of the presence
of penicillin. Their cause in unknown. Penicillin, however,
is necessary for the full expression of resistance within the

Table I

Bacillus cereus : Effect of previous treatment with penicillin on the

PENICILLIN resistance OF (o) THE PENICILLINASE-INDUCIBLE STRAIN
569 (b) THE CONSTITUTIVE MUTANT STRAIN 569/H.

No. of colonies/ plate after 16 hr. incubation at 35°

^inal concentration

•'

11' J

,^

■penicillin {unitsj

ml.) added 5 hr.

after inoculation

of plates

Strain 569 inoculated into :

Nutrient ^^^^^^'^^ «^«^
i\umeni j^^unitpeni-

«^«^ cUlinlml.

Strain 5Q9IH

Nutrient
agar

inoculated into :

Nutrient agar

+ 1 unit peni-

cillinlml.

35

42

37

43

20

30

51

21

26

40

17

37

27

28

60

4

16

14

15

100

29

13

18

120

9

9

5

140

8

1

9

160

11

2

3

Spores of 569 and 569/H were inoculated into a series of nutrient agar plates
with and without added penicillin at 1 unit/ml. After 5 hr. incubation at 35°
layers of agar containing different concentrations of penicillin were poured on
the surface such that final concentrations of the penicillin, after diffusion
through the plate, ranged from to 160 units/ml. Incubation at 35° was then
continued and the colonies counted after a further 16 hr.

culture, in order to permit the selective growth of the few
resistant cells and so allow the evolution of the population
towards one in which all or most of the individuals form the
enzyme spontaneously at high rate. Resistance developed in
this way is genetically stable and may persist undiminished
during repeated subcultures in the absence of penicillin. Such
"spontaneous" penicillinase-constitutive mutants (569/H)
can be detected at an incidence of about 1 in 10^ in cultures
of strain 569 (derived from a single spore) — i.e. amongst the

Penicillin-induced Penicillin Resistance 81

same population as that which undergoes penicilHnase-
induction under the direct influence of penicilhn (Kogut,
Pollock and Tridgell, 1956). However, a culture of strain 569
cannot be induced to evolve into a culture of 569/H by growth
in the liquid penicillin-containing medium. There are three
possible reasons for this. (1) The margin of diff'erence between
the penicillin resistance of 569 and of 569/H is not large
because (as explained above) increased penicillinase forma-
tion is induced so rapidly in 569 that cells take only a few
minutes before they are able to produce the enzyme almost
as fast as 569/H. (2) 569/H grows more slowly than 569. Its
specific growth rate in broth or casein hydrolysate is about
80 per cent that of 569 in the same media. (3) Penicillinase is
a communal bacterial weapon against penicillin, especially
because 85-90 per cent of the total enzyme is liberated into
the medium by both strains and therefore its production by
one section of a mixed population of cells might be expected
also to protect, to some extent, other individuals in the
culture.

The mutation-plus-selection method of developing peni-
cillin resistance can, however, be studied satisfactorily in
another group of B, cereus strains, none of which are suscept-
ible to penicillinase induction. These will be referred to as
the "5" group. B. cereus strain 5 was found (Sneath, 1955)
to be unique amongst Q5 strains oiB. cereus tested, in having
a very high penicillin sensitivity (the growth of single spores
being inhibited by only 0-01 units penicillin/ml.). No peni-
cillinase production [as tested by the standard manometric
method of Henry and Housewright (1947)] could be detected
in strain 5, either before or after treatment with penicillin.
After overnight growth of a fairly large inoculum (approx.
10^ cells per ml.) into broth containing 100 units penicillin/ml.,
strain 5 was found to evolve into a practically homogeneous
population of penicillin-resistant cells (5/B) capable of form-
ing penicillinase spontaneously at very high rate. By applica-
tion of the velvet pad technique of Lederberg and Lederberg
(1952), Sneath was able to demonstrate very beautifully that

82 M. R. Pollock

strain 5 mutated to 5/B "spontaneously" (i.e. in the absence
of penicillin). The demonstration in this instance was par-
ticularly satisfying because it was possible to show the pro-
duction of penicillinase (acid-formation — due to production
of penicilloic acid — in nutrient agar containing a pH indicator,
after addition of a high concentration of penicillin) by 5/B
cells which had never been in contact with penicillin, even
for the test itself. This is possible simply because the enzyme
diffuses out from the colony on or in nutrient agar and can
be demonstrated in the medium after the colony itself has
been removed. This completely disposes of any objection that
the mutation, in the first place, might be a relatively non-
specific event resulting in susceptibility to a subsequent
specifically induced change.

Subsequently, it was discovered that spore suspensions of
strain 5 contained, as well as 5/B mutants, another type (5/P)
of cell, with intermediate penicillin resistance, which formed
penicillinase at approximately one-eighth of the rate of 5/B.
Its "spontaneous" mutative origin was never formally proved,
but it seems likely that it arose in the same way as 5/B. The
specific penicillinase activities (units enzyme/mg. dry bac-
terial weight) were measured on whole cultures growing
logarithmically in 1 per cent gelatin-broth and found not to
vary much within a single strain. All 5/P cells were able to
form colonies in agar containing penicillin up to a concentra-
tion of 0-1 units/ml. but not higher; whereas 5/B cells could
develop into colonies in penicillin up to 1 • units/ml. It was
thus possible to make accurate viable counts of mixtures of
5, 5/P and 5/B cells by plating out in suitable dilutions into
agar containing different concentrations of penicillin. The
proportions of these three types found in the spore suspension
studied were 1,000,000 : 6 : 4. Cultures of 5 from single spore
inocula were regularly found to contain this 1 : 250,000 pro-
portion of 5/B mutants, but the 5/P strain has so far only
been found in one spore suspension and must presumably
therefore result from a very rare mutation. On plates con-
taining 0-1 units penicillin/ml. there was no sure way of

Penicillin-induced Penicillin Resistance 83

distinguishing 5/P colonies from those of 5/B, but cultures from
separate colony isolates were found to form penicillinase at
rates characteristic either of the 5/P or 5/B prototypes with
slight variation and no overlap (see Fig. 1). Since these
isolates were obtained from the same plate, it could hardly
be argued that 5/B colonies were simply examples of the
extreme limits of a wide variation normally shown by 5/P
clones, or vice versa. It could be confidently concluded that
they represented two distinct strains. If, therefore, there is
any clonal variation within populations of 5/P and 5/B of

o '

in

1

Jj

/•O 2-0 J-0 4-0 SO

log specific penicillinase activity
pl./hr./mg.

Fig. 1. Distribution of specific penicillinase activities amongst

single-colony isolates of penicillin-resistant mutants from cultures

of B. cereus 5.

the continuous type postulated by Yudkin (1953) affecting
the rates of penicillinase formation by individual cells, it must
have very low genetic stability and/or a very narrow range
and would therefore be of little significance in the evolution
of a more resistant population.

Addition of penicillin to cultures of members of the "5"
group causes no significant increase in rate of penicillinase
formation. This lack of inducibility is probably the explana-
tion of why it is easy (in comparison with the 569 group) to
exert differential selection pressure by growth in appropriate
concentrations of penicillin.

It was at first thought that the 5— >5/B mutation involved
the acquisition of a completely new enzyme. However,

84 M. R. Pollock

concentrates of the supernatant fluid obtained after spinning
off cells from cultures of strain 5 were found to contain traces
of penicillinase activity. A technique for micro-assay of peni-
cillinase in untreated culture supernatant fluid was eventually
evolved which permitted accurate measurement of activity
with a sensitivity up to 100 times that of the manometric
method. Since this method has not been published before,
it is described here in detail.

Micro -assay of Penicillinase Activity

Two-ml. samples of culture supernatant fluid were diluted 1 : 5 in 1 %
gelatin broth containing 20 units penicillin/ml. and incubated at 30°.
One-ml. quantities were withdrawn every 15 min. and added to 9 ml.
ice-cold 0-01 M potassium phosphate buffer (pH 7-0). The residual
penicillin in the diluted sample was assayed in octuplicate by cup-plate
assay using the ICI strain of B. subtilis as indicator organism, by com-
parison with penicillin standards of from 0-8 to 2-0 units/ml. (Hum-
phrey and Lightbown, 1952). After addition of samples, the plates were
stored for 2 to 4 hr. at 0° to allow diffusion of the penicillin (and so
increase the sensitivity of the test) before incubation overnight at 35°.
The amount of enzyme preparation added was adjusted such that not
more than 50% of the penicillin had been destroyed before at least
3 samples had been taken.

Zero-point estimation of penicillin made in this way proved that it
was possible to make accurate assays in the presence of enzyme. This
was feasible because of the decrease in enzyme activity on sampling,
due to a combination of 2 factors: (1) the lowering of temperature to 0°
until the antibiotic had time to diffuse away from the enzyme in the
cups and (2) the 1 : 10 dilution of substrate from a concentration which
was, initially, well below enzyme saturation level; this must have
caused a nearly proportional decrease in enzyme activity.

For the purposes of this particular assay, the two following assump-
tions had to be made: (1) The activity in the culture supernatant fluid
corresponded approximately with total activity — as with all the other
strains of B. cereiis so far tested. (2) The Michaelis affinity constant of
the strain 5 enzyme was identical with that of the 5/B penicillinase. In
view of the other similarities between 5 and 5/B penicillinases (to be
discussed later) this seems an entirely reasonable assumption.

The enzyme activity was finally calculated by plotting the residual
penicillin concentration against time, measuring the initial rate of peni-
cillin destruction and adjusting the value to what it would be were the
enzyme saturated with substrate. An accurate figure for the Km of
penicillinase is thus required — and can in fact be calculated, using the
same technique, by varying substrate concentration and having samples
with rather higher enzyme activities than those available from cultures
of strain 5.

Penicillin-induced Penicillin Resistance 85

In this way it was established that cultures of strain 5
produce penicillinase consistently at approximately one five-
thousandth the rate shown by strain 5/B. Although this
activity was from 25 to 250 times (in different cultures) that
expected from the rather variable content of 5/P and 5/B
cells, it seemed essential to eliminate the possibility that these
mutants might, when growing in the presence of large numbers
of 5, produce much more enzyme than that expected from
activities measured in pure culture, and thus themselves be
responsible for the activity observed.

The effect on penicillinase production of supplementing
the "natural" 5/P and 5/B content of 5 cultures by a known
number of added cells and subsequently growing the mixed
culture for 5 hours at 35°, with careful differential counts of
all 3 types, is illustrated in Table II. It can be seen that only
when the 5/P and 5/B content of the culture reached propor-
tions approximately 200 times the normal level, was there any
significant increase in the amount of penicillinase produced;
and the extent of this increase corresponds to the value ex-
pected from the additional number of 5/P and 5/B cells present.

The penicillinase activity of strain 5 cultures cannot there-
fore be ascribed to the presence of 5/P and 5/B mutants. It
is not, however, certain whether this low penicillinase pro-
duction is a property distributed evenly among the rest of the
population. A search was made, without success, for any
evidence of further heterogeneity. But it is not known
whether colonies consisting of cells producing the enzyme, say
at one-fifth the rate of 5/P, would necessarily be detected by
the penicillin-sensitivity or penicillinase production tests used,
though this seems likely. Such a hypothetical strain would
have to form 1 per cent of the total population of strain 5 in
order to account for the observed activity. Direct testing of
cultures by manometric assay of penicillinase from indi-
vidual colonies has yielded only 5/P and 5/B types and nega-
tives. We have therefore provisionally concluded that the
wild type forms penicillinase, constitutively, at a rate corre-
sponding to the production of about 15 molecules per cell —

86

M. R. Pollock

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Penicillin-induced Penicillin Resistance

87

or about 1 molecule per cell every 3 minutes — in logarithmic-
ally growing cultures. Its penicillin sensitivity would suggest
that such a very low level of formation is not of much benefit
to the organism under conditions of the test.

A chart summarizing most of the properties of these dif-
ferent strains with respect to penicillinase production has been

Table III

Penicillinase activity and penicillin resistance of strains of B. cereus.

NON- INDUCIBLE STRAINS

5 ("microconstitutive")

( parvo-
constitutives' )

569/h/i

[oTI V ("000

[oT] ^^^ [m]

s/b ("magncKonstitutive")

I 3.0 I (75,000)

INDUCIBLE STRAINS

569 ("inducible") Induction *- 569

' \ penicillin / (25,000)

'Deadaptation'by

growth without

penicillin

("semi -constitutive")

569/a Induction >569/A

(K^OOo) penicillin (35^00o)

S69/H ("pleno-constitutive";
[lol (75^000)

Strains are denoted by numbers, e.g. 569, and presumed mutants therefrom
by added letters, e.g. 569/H. Approximate rate of penicillinase production is
given in circles as number of molecules per cell (assuming mean \vt. of one
cell = l -3 X 10~i2 g )^ Figures in squares indicate the maximum concentration
of penicillin (in units per ml.) allowing colony formation from at least 50 per
cent of viable spores inoculated into nutrient agar. M= proved mutation.
[M]= presumed mutation.

compiled as illustrated by Table III, and a system of nomen-
clature suggested. It is more or less self-explanatory; but
certain comments are necessary.

(1) Within the 5 group, the relation between rates of
penicillinase production and penicillin resistance is obvious.
There can hardly be any doubt that the one is responsible for
the other. The reason for the relatively slight difference
between the penicillinase resistance of 569 and 569/H has been
ascribed to the very rapid induction of penicillinase formation
by penicillin in 569. Were it possible to inhibit penicillinase
induction without affecting growth, this point could be tested

88 M. R. Pollock

directly. However, there is every reason to believe that were
it not for this rapid induction, 569 would be highly sensitive
to penicillin; and this conclusion is supported by the rela-
tively low penicillin resistance of the non-inducible double
mutant strain 569/H/l derived from 569/H. In the absence
of penicillin, 569/H/l produces 15 times as much enzyme as
569; 569/H/l is however strictly non-inducible so that, after
addition of penicillin, 569 forms penicillinase approximately
25 times as fast as 569/H/l. It is therefore not difficult to
understand why 569 can develop in concentrations of peni-
cillin up to 30 times the highest levels permitting growth of
569/H/l. The significance of enzyme induction as a means of
developing specific drug resistance is that it is (usually) adap-
tive and therefore economical. At the expense of a slightly
lower penicillin resistance than 569/H, 569 avoids using up to
1 per cent of its protein-synthesizing ability on a normally
useless enzyme — except under just those conditions where that
enzyme is essential for survival. It should be noted that
enzyme induction may also contribute to the development of
drug resistance (of a genetically more stable type) by per-
mitting survival and multiplication of cells after addition of
the drug and so increasing the possibility of mutations towards
higher resistance (e.g. constitutive mutants, or mutants with
more efficient induction systems).

The difPerence in penicillin resistance of 5/B and 569/H,
whose rate of total penicillinase production is the same, may
possibly be due to the very much larger amount of intra-
cellular enzyme [" y-penicillinase " : see Pollock (1956^)]
present in the latter.

These two strains have been referred to respectively as
"magno-" and "pleno-" penicillinase-constitutive types.
There is really nothing apart from their origin to justify such
a distinction. It was made with the intention of emphasizing
that the pleno-constitutive strain 569/H was derived from an
inducible parent strain which was able to undergo a series of
mutations involving different increases in the quantity of
basal enzyme. The semi-constitutive strain 569/A which still

Penicillin-induced Penicillin Resistance 89

retains some inducibility might be regarded as an example of
this type of change; and 569/H could be considered as its
apogee. However, isolation of the non-inducible 569/H/l
from a culture of 569/H which had grown some time in casein
hydrolysate rather suggests that the mutation: 569->569/H
might be considered as "development of constitutivity " and
the difference between 569/H and 569/H/l might be similar
to that between the magno- and parvo-constitutives of the
5 series. No change from non-inducible to inducible peni-
cillinase production has yet been found.

(2) Strains of the " 5 group " are completely insusceptible to
penicillinase induction. They form a family which are indis-
tinguishable from one another by any known criterion apart
from rates of penicillinase production. But they are physio-
logically distinct from the 569 family, differing slightly in
colonial morphology, in rates of fermentation of salicin and
starch (Sneath, personal communication) and in the type of
penicillinase produced.

(3) The 5/B and 569/H constitutive penicillinases and the
569 (induced) penicillinase have been purified and their physico-
chemical and biochemical characteristics compared (Pollock,
Torriani and Tridgell, 1956; Kogut, Pollock and Tridgell,
1956). The 5/B penicillinase differs slightly but significantly
from the 569/H enzyme by all criteria examined (molecular
weight, sedimentation constant, molecular activity, electro-
phoretic mobility and salt solubility) while the induced 569
and 569/H enzyme are indistinguishable. An immunological
comparison [based on slopes of enzyme neutralization curves
in the presence of increasing quantities of a specific antibody
preparation partially absorbed with the induced 569 enzyme —
see Pollock (1956a)] has shown the 5/B and 569 enzymes to be
related though distinguishable, while the basal 569 (unin-
duced) penicillinase gives the same slope as the 569 (induced)
569/H and 569/H/l enzymes.

There is clearly not enough of the " microconstitutive "
5 penicillinase to allow its isolation and physicochemical
characterization. But, as with the basal 569 enzyme, an

90

M. R. Pollock

immunological comparison with 5/B penicillinase is possible.
Fig. 2 shows how a partially absorbed anti-569 antibody pre-
paration neutralizes the 5/B enzyme and a preparation of
5 penicillinase (obtained from a culture supernatant fluid
concentrate) to give identical neutralization slopes. The 5/P
enzyme is likewise indistinguishable from 5 and 5/B ; whereas
the 569 penicillinase gives a quite different slope. Results
obtained (in a diff*erent experiment) with culture supernatant

B.megateri um 496
749

B.cereus

'04 '06

ml. antiserum
Fig. 2. Neutralization of different penicillinases by partially-absorbed
anti-penicillinase serum.

fluids from penicillinase-producing strains of B. suhtilis and
B. megaterium show no neutralization whatever and indicate
that their enzymes are far less closely related to 569 penicil-
linase than those of the 5 group.

This immunological test can therefore be regarded as a very
sensitive indication of probable identity. It appears reasonable
to conclude that within each family of strains the change in
rates of increase of penicillinase activity — whether occurring
by enzyme induction or by mutation— reflects a purely quanti-
tative alteration in the rates of formation of the same protein.

Penicillin-induced Penicillin Resistance 91

Speculative Discussion

This symposium will undoubtedly contain many discus-
sions on the extent to which drug resistance may depend in
the first place upon a spontaneous change within an organism
(followed by selection) or upon a change specifically induced
in the organism by some factor in the environment. A study
of the development of the penicillinase type of penicillin
resistance in B. cereus, where changes in resistance can be
followed accurately at a biochemical level, has shown that
both types can occur, even within the same population of
individuals. It is vital, however, to recognize that the speci-
fically induced change (enzyme induction) is due to an
increase in the activity of an enzyme-forming system which
itself does not appear to play any part — however indirect —
in stimulating its own production. It is biochemically stable,
but genetically transitory. Although the antibiotic antidote
(penicillinase) developed in both types is identical, its mode of
acquisition and its genetic stability are quite different. Once
this distinction is accepted (but not before), it is permissible
to ask whether after all there may be some connexion between
the two types of phenomenon.

In a speculative discussion (Pollock, 1953) ways have been
suggested by which enzyme induction by specific environ-
mental factors might theoretically lead to a stable heritable
change in an organism. It was argued that a necessary pre-
requisite for such an event would be that the external inducer
should, by some means (however indirectly), stimulate the
cell to form more of the inducer itself (or its biological equi-
valent). This attempt was influenced by what was felt to be a
need to adapt the theoretical speculations of Hinshelwood
(1946) towards the established facts of induced enzyme
synthesis. Now although the synthesis or activation of bio-
logically important molecules (e.g. formation of certain
polysaccharides or activation of many proteinase precursors)
in some subcellular systems often runs an autocatalytic course
because the reaction is stimulated by its own product, it

92 M. R. Pollock

must be admitted that good evidence for "self-reproducing"
enzyme-forming systems is so far non-existent.

It is true that there have been a number of interesting
claims to have demonstrated that heritable changes can be
specifically induced in micro-organisms by environmental
factors [acquisition of ability to ferment sucrose in yeast
(Kossikov, 1950); development of streptomycin resistance in
Pseudornonas (Linz, 1950); loss of penicillin resistance in
staphylococci after treatment with chloramphenicol (Voureka,
1952); lactose-induced development of ability to ferment
lactose in Escherichia coli ("mutabile") (Dean and Hinshel-
wood, 1954); proflavine-induced proflavine resistance in
Aerobacter aerogenes (Dean, 1955) etc.]. But either these
have been open to theoretical objections or the phenomena
themselves have not yet been fully confirmed by other workers.
It would appear that specifically induced heritable change in
individual cells has so far been firmly substantiated only for
cases of transformation of bacterial strains by deoxyribo-
nucleic acid-containing extracts from other types of bacteria.
Nevertheless, other instances of "specifically directed muta-
tions" may well exist — and indeed should be looked for with
increasing persistence.

It has already been emphasized that the changes in peni-
cillin resistance of B, cereus reported have involved simple
quantitative changes in amounts of penicillinase produced.
Similar, though less marked, quantitative changes (due to
single gene mutations) have been reported by Markert and
Owen (1954) and Owen and Markert (1955) for Glomerella
tyrosinases and by Yanofsky (1952) for Neurospora trypto-
phan synthase. Different strains of staphylococci (from the
same cultures and therefore probably derived one from
another by mutations) were found by Rogers (1953) to fall
into distinct groups with widely differing rates of hyaluroni-
dase production. Even the penicillin-sensitive parent strain 5
of the penicillin-resistant 5/B mutant forms minute amounts
of the same enzyme which, when produced in large quantities,
is responsible for the resistance of 5/B. There has been no

Penicillin-induced Penicillin Resistance 93

change — either by mutation or by enzyme induction — in the
kind of protein produced. It is therefore perhaps permissible
to wonder how frequently truly qualitative changes at a
molecular level may in fact occur. Qualitative changes —
both from mutations and interactions of genes — have of course
often been described [e.g. hybrid antigens in red cells (a) of
pigeons (Irwin, 1947), (b) of rabbits (Cohen, 1956), atebrin
resistance in pneumococci, said to be due to alteration in a
flavo-protein (Sevag and Gots, 1948); two types of j9-nitro-
benzoic acid resistance in Esch. coli said to be due to altera-
tion in affinity of an enzyme for the drug (Davis, 1951)] but
only rarely have attempts been made to show that such
changes are due to intramolecular alteration of a protein
which can be properly isolated and characterized.

The best instances of possibly qualitative, heritable
changes in proteins, due to single gene mutations or interac-
tions, are those involving alterations in the thermostability
of an enzyme [e.g. the pantothenate-synthesizing enzyme of
Esch. coli (Maas and Davis, 1952); and the tyrosinase of
Neurospora (Horowitz and Fling, 1953, 1956)]. Unfortunately,
thermostability is one of the properties of enzymes which is
known to vary considerably with variations in composition
of the medium and on association with other molecules
(Lawrence and Halvorson, 1954; Stewart and Halvorson,
1954).

Other interesting instances are: (1) two types of glutamic
acid dehydrogenases (apparently differing in their extent of
reversible inactivation) isolated by Fincham (1957) from
strains of Neurosjjora differing by a single gene, and (2) the
aureomycin-resistant nitratase found by Saz and co-workers
(1956) and Saz and Martinez (1956) in an aureomycin-resistant
strain of Esch. coli (origin not studied genetically), and
reported to have a conjugated flavin moiety more firmly
bound than had the corresponding sensitive enzyme obtained
from the sensitive strain. In neither case have the enzymes
been isolated and compared after full purification.

In cases where types of a well-characterized protein species

94 M. R. Pollock

can be clearly shown to differ qualitatively in the same and
closely related organisms (e.g. the human haemoglobins,
cattle p-lactoglobulins, etc.) it is often too readily assumed
that the respective synthesizing systems have arisen from one
another by a single mutation. So far there is no direct evidence
to show that this is so.

Again, in cases where some enzymic activity appears to
be lost (or gained) on mutation, it is becoming increasingly
apparent that the loss or gain is not absolute and that the
"loss" strain (whether mutant or wild type) is suffering from
a block that (to use Bonner's terminology) is "leaky". Even
in cases where none of the relevant biochemical activity can
be detected [e.g. the apparent lack of glutamic acid dehydro-
genase in an a-amino acid-requiring mutant of Neurospora
(Fincham, 1954)] some hesitation is justified before conclud-
ing that the loss is absolute. Quite apart from the possibility
that the assay technique is insufficiently sensitive, even a
demonstration that there is less than one molecule per cell
of a certain enzyme, has no special significance (in relation to
this particular point) in organisms which reproduce by binary
fission — as long as the synthesizing system is genetically
stable.

Now, there can hardly be any doubt that during the course
of evolution qualitative changes in enzymes do occur. Unless
there is some quite inconceivable degree of convergent
evolution, the systems synthesizing human haemoglobins A,
S, C, D and E, for instance, must have evolved either from
each other or some common ancestor. The question is there-
fore not whether qualitative heritable changes in protein
occur; but how frequently and by what means do they occur?

So far, the proved results of single mutations as expressed
in terms of proteins seem to be purely quantitative. Is it
therefore not possible that qualitative changes (at the protein
molecular level) never in fact result from single mutations as
studied in the laboratory? To use Beadle's terminology: can
single mutations ever be (at a molecular level) neo-morphic? —
or are they always hypo- or hypermorphic? Or do single

Penicillin-induced Penicillin Resistance 95

mutations result in minute qualitative alterations (minor
changes in folding of the molecule or displacement of a single
amino acid within the chain) too slight to be detected by most
available techniques? If this is so, perhaps a series of successive
mutations and alterations to the protein molecule may be
necessary before the change is detectable. Or, perhaps, a
single mutation may never result in more than a relatively
slight shift in the distribution of properties amongst a micro-
heterogeneous population of very closely related but different
individual molecules within a molecular species, all of which
have a similar function [see Colvin, Smith and Cook (1954),
for evidence of " microheterogeneity " amongst populations of
a single species of protein molecules]. The slow evolution,
from cultures of penicillin-sensitive staphylococci, grown for
very long periods in low penicillin concentrations, of strains
apparently capable of producing minute traces of penicil-
linase (Barber, this symposium, p. 262) is of special interest
in this connexion. Naturally occurring qualitative evolution
at a molecular level may proceed very much more slowly
than might be at first suspected from consideration of the
mutation rates and selection pressures observed within the
laboratory.

REFERENCES

Barber, M. (1953). Symp. Soc. gen. Microbiol., 3, 235.

Cohen, C. (1956). Science, 123, 935.

Colvin, J. R., Smith, D. B., and Cook, W. H. (1954). Chem. Rev., 54,

687.
Davis, B. D. (1951). ColdSpr. Harb. Symp. quant. Biol., 16, 73.
Dean, A. C. R. (1955). In Origins of Resistance to Toxic Agents, p. 42.

New York : Academic Press.
Dean, A. C. R., and Hinshelwood, C. N. (1954). Proc. roy. Soc. B,

142, 225.
FiNCHAM, J. R. S. (1954). J. gen. Microbiol, 11, 236.
Henry, R. J., and Housewright, R. D. (1947). J. biol. Chem., 167, 559.
Hinshelwood, C. N. (1946). The Chemical Kinetics of the Bacterial

Cell. Oxford: Clarendon Press.
Horowitz, N. H., and Fling, M. (1953). Genetics, 38, 360.
Horowitz, N. H., and Fling, M. (1956). Proc. nat. Acad. Sci., Wash.,

42, 498.

96 M. R. Pollock

Humphrey. J. H., and Lightbown, J. W. (1952). J. gen. Microbiol, 7,

129.
Irwin, M. R. (1947). Advanc. Genetics, 1, 133.
KoGUT, M., Pollock, M. R., and Tridgell, E. J. (1956). Biochem. J.,

62, 391.
KossiKOV, K. V. (1950). Dokl. Akad. Nauk., USSR, 73, 381.
Lawrence, N. L., and Halvorson, H. O. (1954). J. Bad., 68, 334.
Lederberg, J., and Lederberg, E. M. (1952). J. Bad., 63, 399.
LiNZ, R. (1950). Ann. Inst. Pasteur, 78, 105.
Maas, W. K., and Davis, B. D. (1952). Proc. nat. Acad. Sci., Wash.,

38, 785.
Markert, C. L., and Owen, R. D. (1954). Genetics, 39, 818.
Owen, R. D., and Markert, C. L. (1955). J. Immunol., 74, 257.
Pollock, M. R. (1953). Symp. Soc. gen. Microbiol., 3, 150.
Pollock, M. R. (1956«). J. gen. Microbiol., 14, 90.
Pollock, M. R. (19566). J. gen. Microbiol., 15, 154.
Pollock, M. R., and Torriani, A. M. (1953). C.R. Acad. Sci., Paris,

237, 276.
Pollock, M. R., Torriani, A. M., and Tridgell, E. J. (1956). Biochem.

J., 62, 387.
Rogers, H. J. (1953). J. Path. Bad., 66, 545.
Saz, a. K., Weiss Brownell, L., and Slie, R. B. (1956). J. Bad., 71,

421.
Saz, a. K., and Martinez, L. M. (1956). J. biol. Chem., 223, 285.
Sevag, M. G., and Gots, J. S. (1948). J. Bad., 56, 737.
Sneath, p. H. a. (1955). J. gen. Microbiol., 13, 561.
Stewart, B. T., and Halvorson, H. O. (1954). Arch. Biochem. Biophys.,

49, 168.
VouREKA, A. (1952). J. gen. Microbiol, 6, 352.
Yanofsky, C. (1952). Proc. nat Acad. Set., Wash., 38, 215.
YuDKiN, J. (1953). Nature, Lond., 171, 541.

DISCUSSION

Pontecorvo: The very important question that Dr. Pollock has raised,
of whether in gene mutations one can get qualitative changes, has been
in the minds of all of us for many years. Some years ago I would have
agreed with him that they were mainly quantitative, but the fact that
out of not more than ten cases which have been properly studied, one
(i.e. tyrosinase) is of that kind is more than I would have expected.

Pollock: I don't think a thermostability change is at all a good
criterion.

Pontecorvo: It is a difference in the enzyme itself.

Pollock: No, I don't think this is necessarily so. I think it could be
due to association with some other molecule, which changes its thermo-
stability. You can get changes like that, as Halvorson has shown with
enzymes derived from spores. There is a profound change in thermo-
stability, which seems to go hand in hand with solubilization of the

Discussion 97

enzyme. I don't say that cases previously quoted do not involve qualita-
tive chan<yes but simply that they are not at all good examples, for that
reason. One wants a purified enzyme in which one can characterize
tilings like the molecular weight and the specific enzyme activity. Such
criteria are much better than thermostability.

Davis: I think that at a sufiiciently refined level of analysis one can
never answer the question of whether or not a temperature-sensitive
mutant, which produces an apparently temperature-sensitive enzyme,
is really producing an altered enzyme. One can always ask the question :
What is a true enzyme and what is an enzyme with something attached
to it ? It is true that changes in the environment of a protein, such as
solubilization or even changes in the concentration of various solutes in
the medium, can change its thermostability. Thus, Dr. Maas and I
observed that the thermostability of pantothenate synthase, whether
from the wild-type or from the temperature-sensitive mutant, is several
degrees higher in the intact cell than in the extract. However, I think
that the results of the mixture experiment excluded the possibility that
the difference in thermostability of the two extracted enzymes depended
on differences in their respective environments; for in this experiment
the fraction that had been contributed by the mutant underwent
irreversible denaturation, just as when the mutant extract was tested by
itself, while the other fraction of the total enzyme activity remained
unchanged. Therefore, in such a mixture, whatever the factor may be
that makes one of the enzymes more stable, it is not present in an excess
that can affect the other. It is a stoichiometric change in the enzyme.

Pollock: Even if you did get an association between some relatively
quite small molecular weight compound which conferred thermostability
on the enzyme at an earlier stage, it might well be irreversible and not
be affected by the subsequent extract.

Davis: This would be a different enzyme.

Pollock: I quite accept your point here. I would only say that it is
very easy to change the thermostability of enzymes by treatment. It is
not the best criterion.

Slonimski: Emil Smith, in his work on papain, showed that you can
chop off something like 18 amino acids without- changing the enzyme
activity. Therefore, you can have a partial protein molecule without
any detectable change, as judged by the usual criteria of enzymic activity.
It will be interesting to see whether, in such a case, there is a change in
thermostability.

Lederberg: There is really a much more fundamental difficulty in
trying to distinguish between qualitative and quantitative changes. The
cell produces what it chooses, not necessarily all that it can produce.
Even in the case of the human haemoglobin the wild genotj^DC is perfectly
capable of making foetal haemoglobin and does so during embryonic life.
But in the course of normal development there is a transition to the
formation of typical adult haemoglobin. There are certain mutations
which prevent this changeover and the result is that in the adult indi-
vidual of mutant genotype you then get large amounts of foetal haemo-
globin instead. If you did not know all the developmental details you

DRUG EES. — 4

98 Discussion

would say that this gene was responsible for a shift in the production of
one haemoglobin as against another, whereas in fact it is responsible for
suppressing one potentiality and the persistence of another one. This is
a difficulty which can be applied to any possible case of mutation
affecting the specific properties of protein which have been produced.
You have no way of telling whether the original genotype could have
produced this new protein if it had "wanted" to, perhaps changing the
conditions under which it may "want" to. If we disregard that, we can
allude to a considerable series of mutational changes of antigenic speci-
ficity in the protein of the flagella of Salmonella, which are rather readily
selected for by means of antiserum. Here we have unusually favourable
conditions for the detection of mutational changes affecting the specific
configuration of the protein, but a detailed analysis of the chemical
basis of the observed changes in immunological specificity remains to be
done.

Pollock: That may be what the trouble is. Is it not possible that there
you have simply got a change in the position of the antigens ?

Lederberg: Absolutely, but this consideration and the one I stated
before will apply to any experiment of this kind. Until we know how
two types of protein are being made, so that we can say that one is being
made as the direct causal alternative to the other, we shall always be
able to say that we are shifting the quantitative or even topographical
relations of the products of a cell.

Pollock: You mean that even if an enzyme of type A is formed in a
particular strain, and on mutation a functionally similar enzyme, but
of type B, is formed; and that if then one were quite unable to detect
any trace of type A enzyme in the mutant, one could always say that
there were traces there but they were too feeble to be detected.

Lederberg: This would apply to haemoglobin in adult man.

Pollock: I agree, but I was visualizing the possibility of there being
a change in the structure of the system that was responsible for forming
the protein ; that one might substitute itself for the other and that they
could not really exist together.

Lederberg: I am not proposing that any bacterium is potentially
capable of producing any enzyme that humans do. I am proposing that
this is rather a sterile question as far as experimental procedures at the
present time are concerned, until we understand more directly the way in
which specific proteins are formed and how they are determined by the
genetic material.

Barber: Was it possible, by a simple process of enzyme adaptation,
to increase penicillinase production in strain 5 ?

Pollock: No, the 5 and 5P and 5B strains are not inducible under any
conditions; nor do they give rise to inducible mutants.

Alexander: Since in successive generations the amount of penicillinase
per bacterium goes down, have you proved that this is because the
amount produced by each individual organism goes down, or could it be
that they are just getting diluted out, i.e. that some retain the full
activity and some don't produce any?

Pollock: It could be the latter, yes. It would be very interesting to

Discussion 99

know, but we have not succeeded in getting a test critical enough to
decide this.

Alexander: The type of dilution technique which Prof. CavalU-Sforza
described this morning could presumably be used.

Pollock: No, because it is not a genetically stable phenomenon. It is
only possible to test indirectly the penicillinase-forming ability of indi-
vidual cells. Theoretically that could be done by penicillin sensitivity,
but I am not sure that it is sufficiently sensitive to allow one to decide
whether the enzyme-forming property is homogeneously distributed or
whether it is confined to the original cells induced.

Barber: The staphylococcus penicillinase-producing culture will quite
frequently throw off variants which produce practically no penicillinase.
Is that the case with your 569 strain?

Pollock: No, we never have a complete negative. I should point out
that we could show, by a reconstruction test, that the low activity of 5
was not due to the presence of 5P and 5B mutants in the 5 culture. We
don't know how homogeneously the property forming small amounts of
penicillinase is distributed amongst individual clones of strain 5 ; but
5 cultures are never penicillinase-negative.

Pontecorvo : You would not be able to identify negative strains if you
got them. Suppose a strain, instead of having 50 molecules per cell, had
none : the only way would be to test millions of single cell isolates.

Pollock : You cannot completely eliminate the possibility of there being
some true negatives. But the sensitivity of the micro-assay method is
much more than 50 molecules per cell. You can get it down to 50 times
that sensitivity quite easily. There may not be a stable genetic distri-
bution of the property amongst individual cells, but you get a very
constant level of activity from different 5 colonies. If you go on isolating
from individual colonies you always find about the same amount of
enzyme, so I suspect it is fairly evenly distributed.

Alexander: Would you get the same rate of penicillinase production
if you irradiated an enzyme so as to stop it from dividing ?

Pollock: Yes, you can give doses of ultraviolet which will almost
completely stop cell division and cause 99 • 9 per cent reduction in viable
count, with very little effect on the enzyme-forming ability, at least for
an hour or so.

Alexander: That would indicate that it is probably the ability to
produce penicillinase that is diluted out and that you have the same
number of penicillinase-producing bacteria at the beginning of the hour
as at the end of the hour, when there are very many more organisms.

Pollock: I don't see how you can draw any conclusions about the
distribution of the enzyme-forming ability from that.

Stocker: Is the slower gro^\i;h of the constitutive mutant true only of
569?

Pollock: No, 569/H devotes between 1 and 2 per cent of its synthetic
powers to forming an apparently useless enzyme. One would like to
think that it makes it up by not growing so rapidly, but it does not fit in.
For one thing that would be a very small change in relative gro^\i:h rate,
whereas in fact there is a difference of about 20 per cent. Furthermore, it

100 Discussion

certainly does not apply to the 5-^5 /B mutation; 5/B grows at exactly
the same rate as 5, although there is roughly the same relative difference
between these two strains as between 569 and 569 /H, in rate of peni-
cillinase formation.

Lederberg: Is there a difference in growth rate between 569 in the
presence and in the absence of penicillin ? This would be another basis
for comparison of cells that are and are not making enzymes. It could be
used as a method of inducing penicillinase formation to see if that slows
down the growth rate, in an inducible strain.

Pollock: No, there is not.

Knox: How do you measure sensitivity to penicillin?

Pollock : We plate either vegetative cells or spores in plates containing
different concentrations of penicillin and titre at the concentration which
will be necessary to prevent, on an average, 50 per cent of the spores
developing into colonies.

Knox: What is the inoculum size?

Pollock: Such that you can count individual colonies on a plate quite
easily. If j'ou had a lot you would destroy the penicillin.

Stocker: Is it the case that strain 569 can be grown, for instance, under
conditions in which it is unable to respond to penicillin? One might then
find an enhanced difference in sensitivity to penicillin, for instance, by
plating preadapted and non-preadapted spores on penicillin agar at 44°,
or anaerobically.

Knox: We followed up Pollock's observation about temperature, and
we found, as you would expect, that penicillin sensitivity did enormously
depend on the temperature at which the test was carried out (Knox, R.,
and Collard, P. (1952), J. gen. Microbiol, 6, 369). At 42° the organisms
were highly sensitive, at 37° they appeared resistant.

Stocker: Did it also depend on the previous history of the inoculum, as
to exposure to penicillin when tested at the high temperature ?

Knox: It depended both on the size of the inoculum and on the pre-
vious history. At 42° adapted cells were able to grow in 10-100 times
greater concentrations of penicillin than unadapted cells.

Pollock: It is very difficult to get dissociation between induction of
penicillinase formation and growth.

Slonimski : Do you have a specific inhibitor for the induction?

Pollock : All one gets is competition between two inducers with different
inducing powers. It is rather doubtful whether one can look on that as
specific inhibition of induction, but there does appear to be competition
for a specific site on the cells.

Slonimski : The difficulty in testing the penicillin sensitivity of strain
569 lies in the fact that it forms an enzyme so quickly that you cannot
measure its sensitivity. What if you were to add to the culture of 569
the inhibitor of penicillinase formation?

Pollock: But there is only a competitive interaction between two in-
ducers both of which are very active in inducing the enzyme, one more
than the other, and if you mix them together you get a competitive
interaction.

Slonimski: It might be worth a trial. Would it not be of interest for

Discussion 101

the problem of seeing whether resistance is exactly proportional to the
rate of synthesis of penicillinase?

Pollock: Yes, one mi«fht possibly do that.

Hayes: What about low pH's, to which I understand penicillinase-
producing staph ^^lococci are sensitive, and the enzyme is not produced?

Pollock: I admit I have not exhausted all the possibilities.

Knox: With regard to the relation between penicillin sensitivity and
the type of penicillinase, have you tested all these strains at say 42° or
43°?

Pollock: No, I have not, but it might be interesting.

Knox: You might get a better correlation between penicillin sensitivity
and the amount of enzyme initially present.

Hotchkiss: Does the amount of penicillinase in your unit correspond
approximately to the amount which could destroy an appreciable part
of the penicillin in the medium? In other words, I assume that when
you say that a strain having penicillinase has resistance to 10 units you
must be measuring under conditions in which penicillin is maintained at
approximately 10 units. This must mean that you do not destroy very
much by this sj^stem.

Pollock: It is rather surprising that you can produce such a lot of
enzyme and still not have a very resistant organism ; also that you can
find a strain producing, for instance, 15 molecules of penicillinase per
cell and have an organism almost as sensitive as the most sensitive of
staphylococci.

Hotchkiss: You can see that the question has a general bearing on the
survival of the cell; whether for other systems, too, the amount of
inducible protein can be such that it can change response to drugs.

DIRECTED HEREDITARY CHANGES OF

FERMENTATIVE PROPERTIES OF YEAST BY

A SPECIFIC SUBSTRATE

K. V. KossiKOV

Institute of Genetics, Academy of Sciences of the U.S.S.R., Moscow

Investigations into the directed changes of micro-organ-
isms are in full swing in the Soviet Union. At the end of 1951,
a conference devoted to this problem was organized under the
auspices of the Institute of Microbiology of the Academy of
Sciences of the U.S.S.R., the transactions of which were pub-
lished in 1952. Monographs on the variability of micro-
organisms were published by Kalina (1952) and Muromzev
(1953). Investigations are under way on the adaptation of
micro-organisms to various toxic substances (Imshenetsky,
1953; Planelies and Moroz, 1956; and others).

The extensive experimental data obtained in recent years
have shown more clearly the inconsistency of the mutation
selection theory in explaining the phenomena of adaptation
of micro-organisms to various bactericidal substances and to
new sources of nutrition. The nature and speed of the
adaptational changes occurring in these organisms, the
quantitative and qualitative dependence of these changes on
the dose and length of action of the substance to which the
organism became adapted, and also a number of other factors
observed in the study of these phenomena cannot be accounted
for by mere selection of spontaneous mutations.

Dean and Hinshelwood (1953), discussing the main issues
of the theory of spontaneous mutations, and of the selection
that follows them, came to the conclusion that in some cases,
at any rate, changes may occur in populations — according to
this theory — which do not take place in reality.

The need to explain these phenomena, starting from other

102

Induced Mutational Changes in Yeast 103

theoretical assumptions, is becoming more and more urgent.
It is reasonable to base these assumptions on the direct
influence of environmental factors on the variability of micro-
organisms; and by so doing we can easily explain the above-
mentioned regularities in adaptive changes, and the reversion
in certain cases of the adapted forms to the original type,
when the adapting factors cease to exert an influence.

However, the question arises as to whether the adaptive
changes can, in nature and stability, equal mutational changes,
which are usually considered to be spontaneous. In other
words, the question (which applies both to vegetative and
sexual reproduction) is whether there appear in the process
of adaptation changes, other than these already mentioned,
which will be inherited even if the adapting substrate is
removed from the medium.

This paper is concerned with the elucidation of these
questions, and in particular with the directed changes of the
fermentative properties of yeasts of some species of the genus
Saccharomyces, and with the inheritance of new properties
developed in them.

Adaptation of Saccharomyces globosus to
Fermentation of Sucrose

The present author first observed adaptation of S. globosus
to fermentation of sucrose in 1948, when the phenomenon
occurred in a control test-tube on the sixty-seventh day after
seeding. At the time, this aroused the interest of the present
author because it could not be readily accounted for by the
appearance of the adaptive enzyme (cf. fermentation of
galactose by certain yeasts), and it led to the systematic
investigation of the variability of fermentative properties of
S. globosus and other yeasts (Kossikov, 1948, 1951, 1952,
1954).

We investigated cultures obtained from single spores of S.
globosus 349. During the germination of spores, diploidization
occurred resulting in the development of homozygous diploid

104 K. V. KossiKOv

cultures. Each culture was first tested for its ability to fer-
ment sugars. It was established that none of the cultures
selected for the experiment fermented sucrose or maltose
during 30 days of cultivation on the nutrient medium, in the
presence of 2 per cent of these sugars (these sugars being the
only carbohydrate source).

Special experiments were carried out with the aim of investi-
gating the influence of cells which failed to ferment sucrose,
on those capable of fermenting this sugar (in a mixed culture).
It was found that the presence of non-fermenting cells con-
siderably retarded the fermentation of sugar. It is reasonable
to expect that, during the process of adaptation, there first
appears in the mass of yeast cells a single cell (or a very
limited number of cells) which can ferment a specific sugar.
If non-fermenting cells retard the reproduction of these
particular cells, and thus hinder the demonstration of ferment-
ative abilities of the progeny, how long will it take such a cell
to propagate sufficiently in these conditions and cause the
fermentation of sucrose determinable in the experiment?

To answer these questions, additional experiments were
carried out which enabled us to find the maximal period
necessary for detecting — in the mass of sucrose-nonfermenting
cells — this one sucrose-fermenting cell.

These experiments were carried out in test-tubes with
gas-traps, on a medium containing 2 per cent sucrose and • 3
per cent autolysed yeast extract. S. glohosus 349 was used as
the sucrose-nonfermenting culture. The sucrose-fermenting
culture was obtained from the adapted cell from the same
strain, S. glohosus 349, by cultivating this strain on the
medium with sucrose. Preliminary tests showed that the
ability to ferment sucrose, developed by this cell, was very
strongly heritable.

In the first series of experiments there were three variants
of yeast dilutions, the yeasts being of the sucrose-nonferment-
ing type. In the first variant the test-tubes were filled with
350-400 million, in the second variant with 3-4 million and in
the third variant with 35-40 thousand sucrose-nonfermenting

Induced Mutational Changes in Yeast 105

cells. The fourth variant was the control, where no sucrose-
nonfermenting cells were present. After the sucrose-non-
fermenting cells had been introduced into the test-tubes, one
sucrose-fermenting cell was added to each test-tube of each
variant (20 test-tubes per variant). Observations of fermenta-
tion of experimental and control cultures were continued for a
period of 45 days. A second series of experiments was carried
out along the same lines. The results of both series of experi-
ments are shown in Table I.

Table I shows that the increase in concentration of sucrose-
nonfermenting cells corresponds with an increase in the time
which elapsed between the addition of one sucrose-fermenting
cell and the appearance of the first signs of fermentation. If
in the absence of sucrose-nonfermenting cells fermentation
begins on the third or fourth day, then in the presence of
sucrose-nonfermenting cells we observe a retardation of
fermentation by approximately 15-20 days. In one case,
fermentation commenced only on the twenty-fourth day.
However, no very long delay was noted although observations
were continued over a period of up to 45 days.

Experiment 1, The above data demonstrate that in order to
see spontaneous mutation, a 10-day period of observation of
the process of fermentation is not long enough. In the condi-
tions of our experiments the maximal period was 24 days.
The above results were taken into account in our first experi-
ment, and we increased the content of sucrose in the medium
to 6 per cent, adding • 75 per cent glucose to the sucrose. It
was suggested that, firstly, the yeast cells were propagating
and fermenting glucose to form a fermentative type, and that
thus the transition to sucrose-fermenting cells was made
easier — provided these cells can produce invertase. Secondly,
the increased sucrose concentration in the medium could also,
to some extent, lead to an increased effect on variability —
provided that the presence of this sugar in the medium stimu-
lates the formation of invertase by the cell. A medium con-
taining 6 per cent lactose and • 75 per cent glucose was also
prepared. The number of cultures used in the experiment was

106

K. V. KossiKov

h

Pi

w

o

>

H
<!

H

pj
«

H

°^

W W

;5 D

Q

I

1

1

i

1 1 I 1 1 1 1 1

- - I I - 1 1 1

CO

7

I— 1

1 1 ^ 1 01 (M r^ 1

2

1 1 r-l 1 1 rH 1

00

1— 1
1

05

1 1 1 1 1 1 - 1

^

I

MM M ^ 1

CO

CO

111^ 1 1 rH 1>

01

Number of
cultures that

started
fermenting

rHrHlMTjl COCO(Ml>

CO
CO

Number of
test-tubes

in
experiment

S^gg gggg

r-(

Number of sucrose-
nonfermenting cells
per single sucrose-
fermenting cell

350-400 million

3 • 5-4 million
35-40 thousand

450-500 million
4-5-5 million
45-50 thousand

&

1^

r^ (M

Induced Mutational Changes in Yeast 107

221. The test-tubes in the experiment were kept under
observation for 225 days.

During the first three days shght fermentation was ob-
served in all the test-tubes, as evidenced by a slight evolution
of carbon dioxide resulting from glucose fermentation. On the
fourth to fifth day fermentation ceased. A certain amount of
carbon dioxide accumulated in the gas-traps of almost every
test-tube (occupying not more than one-twentieth the volume
of the gas-traps).

Further observation of fermentation was made, the control
starting-point being a notch on the wall of the test-tube. If
amount of carbon dioxide in the gas-trap tended to increase,
it was assumed that fermentation had occurred due to
adaptation of a culture to fermentation of sucrose or lactose,
respectively. On the twenty-eighth day following seeding, an
increase was noted in the amount of carbon dioxide in the gas-
traps of two of the test-tubes which contained sucrose.
Another culture began to ferment sucrose on the eighty-fourth
day. In order to ascertain whether what had occurred in this
experiment was really the adaptation of cells of a given
culture to the fermentation of sucrose, the fermented culture
was reseeded onto the medium with sucrose, without adding
glucose. It was believed that in the three-stage consecutive
fermentation of sucrose by the adapted culture, certain cells
developed which transmit this quality of fermentation to the
progeny. In the course of 208 days, another 21 cultures of
cells were seen to have started fermenting. None of the cul-
tures trained on lactose became adapted to fermentation of
this sugar during the same period of time.

Fresh medium (2-0 ml.), consisting of 6 per cent sucrose
and 0-4 per cent autolysed yeast extract, was added to the
test-tubes containing lactose, 208 days after commencement of
the experiment. Fresh medium (2-0 ml.), consisting of 6 per
cent lactose and 0-4 per cent autolysed yeast extract was
added to the test-tubes containing sucrose. This was the
commencement of tests for spontaneous changes. It was
assumed that if even one single cell in a culture maintained on

108 K. V. KossiKOV

lactose acquired the capacity to ferment sucrose, the addition
of sucrose to the medium would cause reproduction to occur.
The same might occur in the corresponding case of a culture
maintained on sucrose.

On the seventeenth day after addition of fresh medium to
the test-tubes (during which time four more cultures had
become adapted to fermentation of sucrose), cells from these
test-tubes were transferred to those provided with gas-traps
and fresh medium. The cultures which were kept initially on
sucrose medium, and to which we added 2-0 ml. of a medium
with lactose, were now transferred to the medium containing
6 • per cent lactose and • 4 per cent autolysed yeast extract.
Cultures initially kept on lactose medium, and to which we
had added 2 • ml. of a medium with sucrose, were now trans-
ferred to the medium containing 6 per cent sucrose and 0-4
per cent autolysed yeast extract. Observation of fermentation
was continued for a further 28 days. Therefore, each culture
initially seeded into the sucrose medium could develop on the
medium in the presence of lactose for a 45-day period following
the 208-day cultivation on sucrose. If lactose-fermenting
cells had developed in such cultures, they would have started
fermenting this sugar; however, no cultures were detected
which had adapted to fermentation of lactose.

Somewhat different results were obtained in the work with
cultures initially kept on the lactose medium and later
transferred to the sucrose medium. It was to be expected
that, in this case, during the 45 -day cultivation period of
experimental cultures on the medium with sucrose, cells
would develop which would be adapted to fermentation of
sucrose. The essential question, however, was: on which day
after the contact with sucrose did fermentation begin, and
how many fermenting cultures appeared? In fact, of the 221
cultures, only 2 were found to be adapted to fermentation of
sucrose; and one of these started fermenting sucrose on the
twenty-eighth day, and the other on the thirtieth day after
contact with sucrose. This experiment shows that, during
cultivation on lactose, no cells developed which were capable

Induced Mutational Changes in Yeast 109

of fermenting sucrose. If, in the mass of cells, there had been
even one cell capable of fermenting sucrose, fermentation
would have begun and would have been evident not later
than 24 days after contact with sucrose (see Table I).

The two cultures, which began fermenting sucrose after
having been maintained on lactose, should be considered to
have become adapted to fermentation of sucrose during the
period of their cultivation on sucrose, i.e. due to the influence
of sucrose. The results of this experiment are summarized
in Table II. This table shows that the ability of yeast cells to
ferment sucrose is associated with the presence of this sugar
in the medium.

Experiment 2. The previous experiment showed that the
addition of glucose to the medium leads to some acceleration
of adaptation of experimental cultures to fermentation of
sucrose. It was of some interest to do the experiment without
adding glucose to the medium. In contradistinction to
Expt. 1, one diploid culture of S. globosus (obtained from a
single spore) was used. The culture was seeded into Petri
plates containing wort-agar. The 2 -day colonies were re-
seeded into test-tubes, with gas-traps, containing a medium
which consisted of 4 per cent sucrose and • 5 per cent auto-
lysed yeast extract. Altogether, 454 test-tubes were seeded
with the same number of colonies. The aim of this experiment
was to demonstrate spontaneous mutation in response to
sucrose-fermenting capacity during the reproduction of cells
on wort-agar on Petri plates. The experiment was carried out
over a period of 160 days. During this period, out of a total
of 454 cultures only 5 started to ferment sucrose ; the first one
on the seventy-seventh day, and the other four on the eighty-
sixth, 120th, 122nd and 122nd day, respectively. Taking into
account the data shown in Table I, this experiment can be
said to confirm completely the previous ones, and it demon-
trates that, out of a great number of cultures, here again not
a single case of sucrose fermentation can be explained by the
theory of spontaneous mutation.

It should be pointed out that cells which do not ferment

110

K. V. KossiKOV

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Induced Mutational Changes in Yeast 111

sucrose when seeded into the medium containing sucrose and
autolysed yeast extract, propagate more or less intensively,
especially within the first few days after seeding. In an
experiment where 32-35 million cells were introduced into the
test-tubes, within 6 days the number of cells had increased to
150-200 million. Later on, the reproduction of cells slowed
down considerably, and only when sucrose-fermenting cells
appeared in the test-tubes did the numbers of cells increase
rapidly.

Testing of experimental cultures for their ability to ferment
maltose: The sediment of yeast cells from each test-tube was
transferred to new test-tubes, with gas-traps, containing a
maltose medium. One of these cultures fermented maltose
very well; this was culture 72/349, which had previously
adapted to fermentation of sucrose on a sucrose medium.
Subsequent reseedings showed that the ability to ferment
sucrose and maltose, developed by this culture, was very
strongly heritable.

The appearance of yeast cells capable of fermenting maltose
in a sucrose medium was of special interest for the elucidation
of the question of the nature of such changes. In the first
place, it was necessary to find out how often these changes
occur. In a series of experiments, 55 cultures were developed
which were adapted to fermentation of sucrose, when culti-
vated on a sucrose medium. Out of these 55 cultures, 42 were
developed from single spores of S. globosus, and 13 from single
spores of the second generation of hybrids from S. ellipsoideus
X S, globosus. These hybrids, prior to their adaptation to
fermentation of sucrose, did not even ferment sucrose, let
alone maltose. Many of the cultures mentioned in these
experiments became adapted to fermentation of sucrose (in
2-3 seedings or more).

All 55 cultures were tested for their ability to ferment
maltose. It was found that as well as culture 72/349, culture
73/349 also fermented both sucrose and maltose. Therefore,
out of 42 cultures of S, globosus, 2 cultures became adapted
on the sucrose medium to fermentation, not only of sucrose,

112 K. V. KossiKOv

but also of maltose. Attention should be drawn to the fact
that both these cultures developed from the same cell, which
formed a 4-spore ascus. All 4 spores of this ascus germinated
and produced cultures designated as cultures 72/349, 73/349,
74/349 and 75/349, respectively.

During a series of four variants of experiments on adapt-
ation to fermentation of sucrose, culture 72/349 became
adapted to fermentation of this sugar. However, it developed
the ability to ferment both sucrose and maltose in only one
of the four variants. During a series of six variants of our
experiments, culture 73/349 became adapted to fermentation
of sucrose, and in all six cases it developed the ability to
ferment both sucrose and maltose. Cultures 74/349 and
75/349, in one instance each, became adapted to fermentation
of sucrose, and in both instances failed to ferment maltose.
Out of 13 hybrid cultures, only one (153/3), when cultivated
on sucrose, proved to have become adapted to fermentation of
both sucrose and maltose.

But one cannot, in this case, speak of an accidental varia-
tion of fermentative properties of yeast cells, independent of
the substrate. The fact is that the effect of sucrose on the
yeast cell is conditioned by the biochemical structure of the
sugar. Sucrose is composed of 2 monosaccharides, glucose and
fructose. Invertase acts in the same way as p-D-fructosidase;
but sucrose can be split also by a-glucosidase (maltase) : under
certain conditions, maltase can hydrolyse not only maltose
but also sucrose. Therefore, it is reasonable to expect that
sucrose, which consists of glucose and fructose, can stimulate
the formation not only of fructosidase but also of glucosidase.
This is confirmed by experimental data obtain by the present
author. Yeast cultures which fermented both sucrose and
maltose failed to ferment raffinose, indicating that the enzyme
a-glucosidase was present in the cells of these cultures. Cultures
which fermented only sucrose (and not maltose) fermented one-
third of the total amount of raffinose, indicating that the enzyme
P-D -fructosidase was present in the cells of these cultures.
These data led us to conclude that in the yeasts investigated

Induced Mutational Changes in Yeast 113

by us, when cultivated on a sucrose medium, the frequency of
formation of the enzyme p-D-fructosidase was eighteen times
that of the formation of the enzyme a-glucosidase.

Experiment 3. In this experiment, unhke the previous ones,
adaptation to fermentation of sucrose was carried out on a
soKd medium with agar. The medium consisted of 4 per cent
sucrose, 3 per cent glucose and 0*3 per cent autolysed yeast
extract. It was poured into shallow glass plates of a capacity
of 1000 ml., as much as 200 ml. being poured into one plate.
The inoculum used was the 48-hour culture of S. globosus 349.
After 3-4 days the surface of the agar was entirely covered
with a layer of yeast cells multiplying in the presence of
glucose. It was assumed that secondary colonies would be
formed during the development of cells adapted to fermenta-
tion of sucrose. Such secondary colonies did start to appear
after approximately 30-40 days of cultivation in a thermostat
at 25-26° C. After 58 days, cells from 4 secondary colonies
were tested for ability to ferment sucrose, and in 3 of those
colonies adaptation of cells was found to have occurred.
Numerous reseedings of these cells, on fresh medium contain-
ing sucrose, showed that their adaptation to fermentation of
sucrose was strongly retained in all 3 cases, and is inherited by
the progeny.

Adaptation of Saccharomyces globosus to
Fermentation of Maltose

Fewer experiments were carried out to elucidate the
adaptation ofS. globosus to fermentation of maltose than were
carried out in the case of adaptation to fermentation of
sucrose; our data show that it is much more difficult to
develop the former type of adaptation than the latter.

Experiment 1. In this experiment we used 12 cultures ob-
tained from single spores of S. globosus 349, and 20 cultures
obtained from single spores of second-generation hybrids
(S. globosus X S. ellipsoideus). Preliminary tests showed that
neither the hybrid cultures nor the S. globosus cultures

114 K. V. KossiKOV

fermented maltose during a period of 30 days on a medium
containing maltose. The experiment was carried out in flasks
fitted with Maysle gas-seals, each flask containing 50 ml. of
medium. One-half of each type of culture was seeded on a
medium containing 2 per cent maltose, • 75 per cent glucose
and • 3 per cent autolysed yeast extract ; and the other half
was seeded on beer-wort (saccharimeter: 14°). Maltose-
fermenting cultures of S. ellipsoideus 169 were seeded on the
same media, as a control. Sugar-fermentation was registered
by a decrease in the weight of the flasks due to evolution of
carbon dioxide. During the first 3-4 days there was a slight
decrease in the weight of all the experimental flasks, due in
the one case to fermentation of glucose and in the other to
fermentation of the beer- wort monosaccharides. At the end of
this period (3-4 days), fermentation ceased in the experi-
mental flasks due to the inability of cells of S. glohosus to
ferment the maltose in the medium; while in the control
flasks, fermentation proceeded (as a one-peak curve) and all
sugars, including maltose, were largely fermented at the end
of 4-5 days. It was assumed that, in the experimental flasks,
in the case of the development of cells capable of fermenting
maltose, secondary fermentation would arise resulting from
fermentation of maltose.

The flasks were weighed daily for a period of 107 days; and
it was found that in this time fermentation began in two
cases in the experimental flasks — in the one case in the flask
seeded with S. glohosus 72/349, and in the other in the flask
seeded with the hybrid culture 217/3. Reseeding of cells from
both these flasks, into fresh beer- wort medium (containing
maltose), showed that in both cases cells had become adapted
to fermentation of maltose, and this maltose-fermenting
ability was retained even during numerous reseeding into
wort-agar.

Experiment 2. In this case, the solid medium, wort-agar, was
used for the adaptation of yeast to fermentation of maltose.
The medium was poured into 4 shallow glass plates, of a
capacity of 1000 ml., each plate containing 200 ml. of medium

Induced Mutational Changes in Yeast 115

in a 10-12 mm. -thick layer. A 48-hour culture of S. globosus
was seeded onto the surface of the agar ; and in 3-4 days this
surface was covered with a thin layer of yeast cells multi-
plying in the presence of the wort-monosaccharides. It was
expected that if cells became adapted to fermentation of
maltose, they would propagate and form secondary colonies;
such secondary colonies did develop after 200-300 days of
cultivation at 25° C. Cells from these colonies were seeded
into a medium containing maltose, in test-tubes with gas-
traps. In 3 of the 14 secondary colonies tested, cells were
detected which were adapted to fermentation of maltose.
Numerous reseedings of these cells into liquid wort and wort-
agar showed that the maltose-fermenting ability is retained in
all 3 cases, and is inherited by the progeny.

Experiment 3. In this experiment on adaptation to fermenta-
tion of maltose, we used cultures of S. globosus which had pre-
viously become adapted to fermentation of sucrose. We used
20 such cultures, each of which was obtained from a single
spore. These cultures did not differ in fermenting ability from
cultures of S. paradoxus; both these species fermenting
monosaccharides and sucrose to the same degree.

The experiment was carried out in Maysle flasks, with
gas-seals, each flask containing 100 ml. beer-wort (sacchari-
meter: 14°) at 25-26° C. As in Expt. 1, adaptation to fer-
mentation of maltose was determined by secondary fermenta-
tion. The flasks were weighed daily for the first ten days and
then every tenth day. During the first 3-4 days there was a
slight decrease in the weight of all the flasks, due to fermenta-
tion of the wort-monosaccharides. At the end of this period
fermentation ceased, and only 200 days later did secondary
fermentation begin in one of the flasks. Reseeding of cells
from these flasks into fresh medium, containing maltose,
showed that in this case cells had become adapted to fermenta-
tion of maltose. Numerous reseedings of these cells into
liquid wort and wort-agar showed that this fermenting
ability is strongly retained and is inherited by the progeny.
The remaining 19 flasks were weighed over a period of a further

116

K. V. KossiKov

400 days, and not one of the 19 showed secondary fermenta-
tion. The results of experiments on adaptation of yeast to
fermentation of maltose are shown in Table III.

Table III

Adaptation of S. globosus to fermentation of maltose

Medium and

conditions of

cullivalion

Number of
cultures tested

Number of
cidtures adapted
to fermentation

'of maltose

Duration of

experiment

{in days)

Beer-wort

12 (strain 349)

1

107

(saccharimeter : 14°)

50 ml. in Maysle gas-

20 (hybrid)

1

107

seal flasks

Wort-agar in glass

14 (strain 349;

3

400

plates

secondary
colonies)

Beer-wort

20 (strain 349 ;

1

600

(saccharimeter : 14°)

cultures

100 ml. in Maysle

previously

gas-seal flasks

adapted to

ferment

sucrose)

Adaptation of S. paradoxus to fermentation of
maltose and simple dextrins of malt -wort

S. paradoxus readily ferments monosaccharides and sucrose,
but does not ferment maltose and dextrins of malt-wort.

Experiment 1. Cells of S. paradoxus 37 were seeded into 8
Maysle flasks, with gas-seals, each flask containing 200 ml.
beer- wort (saccharimeter: 15°). The control flasks were seeded
with S. ellipsoideus. The flasks were weighed daily, and when
primary fermentation had ceased they were weighed every
tenth day. In one of the experimental flasks, secondary
fermentation began on the 350th day after the experiment
was begun. Seeding of cells from this flask into fresh medium,
containing maltose, showed that cells had become adapted to

Induced Mutational Changes in Yeast 117

fermentation of maltose. The adapted culture was designated
as 37,6 m-1. At the end of 490 days, secondary fermentation
was observed in a second flask. Seeding of cells from this
flask into fresh medium, containing maltose, showed that cells
had become adapted to fermentation of maltose. The adapted
culture was designated as 37,7 m-1. The remaining 6 flasks
were weighed over a period of a further 270 days, and not one
of the 6 showed secondary fermentation.

Experiment 2. This experiment, in parallel with the previous
one, was carried out on adaptation of S. paradoxus 37 to
fermentation of maltose, on solid medium (wort-agar). The
methods used and the experimental conditions were much the
same as those used in adaptation of S. globosus to maltose-
fermentation on wort-agar.

After approximately 350 days of cultivation, secondary
colonies began to appear. Five secondary colonies were
observed, 3 of which were found to be adapted to fermentation
of maltose. Out of the maltose-fermenting and asci-forming
colonies, we selected (by means of a micromanipulator) 29
cultures obtained from single spores, and these were tested
for ability to ferment maltose. These cultures, which were
designated as A37-9/2m-l-N, all fermented maltose ; however,
in 7 of them growth ceased and the cultures died on reseed-
ing into wort-agar. This cell-death of adapted S. paradoxus
on wort-agar was quite unexpected, because all cultures of
S. globosus, which had become adapted to fermentation of
maltose and sucrose, and which had also been obtained from
single spores, propagated very well when reseeded into wort-
agar, and retained their fermenting ability quite strongly.
Besides the 7 cultures of S. paradoxus which died, in 3 other
test-tubes almost all cells died, and in only one place — i.e. on
slanting agar — did there develop one colony the cells of which
propagated normally. This phenomenon observed during
reseeding of maltose-fermenting cultures of S. paradoxus was
most interesting. Cell-death did not occur immediately on
seeding; the seeded cells multiplied slightly, then ceased to
grow and acquired first a light brown and then a dark brown

118 K. V. KossiKOV

colour. Reseeding of such cells into fresh liquid and solid
media of different contents showed that the cultures could not
be revived.

These data testify to the fact that different forms of
S. paradoxus are developed as a result of their adaptation
to fermentation of maltose. In one case the developed form
retained its acquired maltose-fermenting property not only
on reproduction in conditions of fermentation but also during
reseeding into various nutrient media. In another case, the
developed form retained its acquired maltose-fermenting
property on reproduction in conditions of fermentation, but
its vitality was lower. In inadequate conditions, not only does
it lose this property (reversion), but it dies. It is quite possible
that in this case alterations connected with the adaptation to
fermentation of maltose led to a disturbance of some physio-
logical processes in the cell and to disorders in cell-metabolism,
under the conditions of the experiment. However, this dis-
turbance is eliminated if a new qualitative alteration occurs
and the cells become capable of propagating on wort-agar, as
well as on media containing other sugars, the ability to fer-
ment maltose being retained. This statement is confirmed by
the above-cited three cases of the appearance of separate
colonies in the mass of dead cells on wort-agar. When
cultures selected from these 3 colonies were later reseeded,
they did not differ from the other 19 cultures which were
stable and quite viable from the very moment of development.

Experiment 3. In this experiment, adaptation of yeasts to the
fermentation of simple dextrins of malt-wort was observed.
Experimental details have already been published (Kossikov
and Rayevskaya, 1956); the main results are as follows:

S. paradoxus, previously adapted to fermentation of
maltose, was adapted to fermentation of simple dextrins of
malt-wort by means of long training on this medium without
reseeding. The ability to ferment simple dextrins was in-
creased in further reseedings of the various cultures into fresh
malt-wort. Some of the adapted cultures, obtained from
single cells, began to ferment malt-wort at the same depth as

Induced Mutational Changes in Yeast 119

S. cerevisiae XII; the intensity of fermentation of the former
was somewhat less than that of the latter, during the first 4-5
days.

The scheme of directed changes of the fermentative
properties of yeasts under the influence of a specific substrate

J n:>c>

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HOCHO^^

XH,OH

-Jilt (S

H^H,OH

Activation of
substrate by enzyme

Fig. 1. Scheme of directed changes of fermentative properties of
yeast under the influence of the specific substrate.

Acting
specific substrate

Active enzyme appearing
in the cell

(Fig. 1) shows that on the medium containing maltose, forms
of yeast have been obtained which readily ferment maltose
and less readily ferment sucrose. The enzyme developed in
these cells is a-glucosidase, which is known to activate maltose
and sucrose.

Two forms of yeast have been obtained on the medium with
sucrose. One form is analogous to that obtained on maltose;

120 K. V. KossiKOv

cells of this type readily ferment maltose and less readily
ferment sucrose. Therefore, the enzyme developed in these
cells must be a-glucosidase. Cells of the second altered form
readily ferment sucrose, and ferment one-third of raffinose,
but do not ferment maltose; therefore, the enzyme developed
in these cells must be p-D-fructosidase. Thus, sucrose, as the
specific substrate, may cause the appearance in yeast cells of
two enzymes — a-glucosidase and p-D-fructosidase — and these
enzymes can activate not only sucrose and maltose but also
one-third of raffinose, i.e. three different carbohydrates.

It follows from the scheme shown in Fig. 1 that the forma-
tion in yeast cells of the active enzymes, a-glucosidase and
p-D-fructosidase, is due to the presence of certain carbo-
hydrates in the cultural medium, and the biochemical speci-
ficity of these carbohydrates conforms completely to the
specificity of the enzymes which they cause to appear in the
cell. The mechanism of formation of these enzymes is not
known.

Inheritance of Fermentative Properties developed by
Yeasts as a result of Directed Changes

Because of the specificity of the reproduction of yeasts
(sexual reproduction), in making a detailed study of the
inheritance of fermentative properties developed in these
yeasts we carried out experimental investigations of the
following questions:

(a) Inheritance of sugar-fermenting properties in sexual re-
production {spore formation) : Cells which fermented a specific
sugar were isolated from the previously adapted culture and
seeded into wort-agar, where they formed asci. The spores
from these asci were selected by means of a micromanipulator,
and cultures obtained from single spores were tested for their
ability to ferment their corresponding sugars. Observations
were carried out over a period of one month.

Five different cultures adapted to fermentation of sucrose
were studied. All five gave similar results showing that in

Induced Mutational Changes in Yeast 121

spore formation there occurred segregation in respect of the
altered character. If we take into account the 4-spore asci,
all the spores of which germinated and produced separate
cultures, then all 16 asci obtained from these cultures showed
segregation in the ratio of 2 : 2, i.e. 2 spores of each ascus
formed sucrose-fermenting cultures and 2 formed sucrose-
nonfermenting cultures. It should be pointed out that, in all
cases, the difference in sugar-fermenting capacity of the
different cultures was very wxll pronounced: in fermenting
cultures fermentation began on the first or second day and
ended on the third or fourth day; in nonfermenting cultures
no sign of fermentation was observed for 30 days.

The same results have been obtained in experiments with
cultures adapted to fermentation of maltose. In two cultures
obtained from cells adapted to fermentation of maltose, 11
asci were investigated : all of these showed segregation in the
ratio of 2 : 2, and here also the difference between fermenting
and non-fermenting cultures was clearly marked.

Another type of heredity was detected in S. paradoxus 37,
adapted to fermentation of maltose. Out of 60 isolated spores
(nine 4-spore and eight 3-spore asci), 29 germinated and formed
cultures. All of these fermented maltose. However, among
the 4-spore asci not a single one was found in which all the
spores w^ould germinate and form cultures. Only 3 asci
formed 3 viable cultures each, and in 2 cases the third culture
died when reseeded into wort-agar. In one case, one viable
colony was formed in a culture among the mass of dead
cells. As regards the remaining asci, only 1 or 2 spores proved
to be viable.

The fact that all 29 cultures obtained from single spores of
the altered cells formed maltose-fermenting cultures gives us
ground for suggesting that, in these cultures, segregation does
not occur in respect of the altered character. The most likely
assumption, in this case, would be that adaptation to fermenta-
tion of maltose occurs in the haploid phase of cell develop-
ment, i.e. in the time between germination of spores and
formation of the haploid cell, and fusion of this with a similar

122 K. V. KossiKOV

haploid cell. Since the culture of S. paradoxals readily pro-
duced spores, and adaptation occurred on a solid medium
(wort-agar), the presence of a sufficient number of spores has
thus been provided for. However, a different explanation is
also possible: cells adapted to fermentation of maltose were
heterozygous as regards this capacity ; in the process of spore-
formation they formed spores which yielded not only viable
maltose-fermenting cultures but also maltose-fermenting
cultures of low viability. It was these cells that died during
reseeding on wort-agar.

(b) Stability of fermentative properties developed during cultiva-
tion on agar medium containing other sugars : Cultures obtained
from single spores were inoculated into wort-agar after it had
been found that they fermented their corresponding sugar.
When yeasts adapted to fermentation of maltose were re-
seeded into wort-agar, the conditions of existence were
changed (aerobic instead of anaerobic), but the mass of the
nutrient substrate contained the same sugar, i.e. maltose.
In the case of yeasts adapted to fermentation of sucrose not
only were the conditions of existence changed, but also the
substrate, since the wort-agar contained a very slight per-
centage of sucrose.

So as to exclude completely from the medium the sugar to
which the cells had become adapted, the experimental
cultures (after having been trained on wort-agar) were
reseeded into agar with galactose (3 per cent galactose; 0-4
per cent autolysed yeast extract). Here, the altered cells did
not need to use their newly acquired ability to form an active
enzyme, since galactose can be fermented by the original
unaltered yeast.

Sixty-six of the cultures, adapted to fermentation of sucrose,
were tested. They were cultivated on wort-agar for 261-628
days. None of the cultures tested lost their ability to ferment
sucrose; in fact, this ability was retained undiminished.
When the same cultures were cultivated on the medium
containing galactose for 53-54 days, they retained their
ability to ferment sucrose and only in 8 of them was this

Induced Mutational Changes in Yeast 123

ability diminished. Twenty-seven of the cultures adapted to
fermentation of maltose were tested. All of these after having
been cultivated on wort-agar (for 268-509 days) and after
having been cultivated on the medium containing galactose
(for 54-56 days) completely retained their ability to ferment
maltose.

(c) Inheritance of fermentative properties, developed by yeasts,
when crossed with their original forms : When cultures adapted
to fermentation of sucrose were crossed, by coupling of spores,
with the original S. glohosus 349, a hybrid was obtained which
readily fermented sucrose; this experiment was indicative of
the dominance of the sugar-fermenting property in the first
generation. In the second sexual generation we tested twenty
4-spore asci, all 80 spores of which germinated and produced
viable cultures. The tests for the ability to ferment sucrose
were continued over a period of one month. It was found that
nineteen asci showed very pronounced segregation as regards
the ability to ferment sucrose, i.e. in the ratio 2 : 2, and one
ascus in the ratio 3:1.

When cultures adapted to fermentation of maltose were
crossed with the original S. glohosus 349, a hybrid was ob-
tained which readily fermented maltose. In the second sexual
generation we tested thirteen 4-spore asci, all of which showed
segregation as regards the ability to ferment maltose, in the
ratio 2 : 2.

{d) Comparative analysis of fermentative properties of original,
altered and some other forms of yeasts having a close affinity
between them: As a result of experiments carried out by the
author on adaptation of S. globosus to fermentation of
sucrose and maltose, and of S. paradoxus to fermentation of
maltose and simple dextrins of malt-wort, new forms were
obtained which differed from the original strains as regards
their fermentative properties ; those obtained from S. globosus
are divided into 4 groups:

Group 1. In contradistinction to S. globosus, these forms
ferment not only monosaccharides, but also sucrose and

124 K. V. KossiKov

one-third of raffinose. Therefore, they acquired the abihty to
produce the active enzyme p-D-fructosidase.

Group 2. These forms ferment not only monosaccharides, but
also maltose. Therefore, they acquired the ability to produce
the active enzyme a-glucosidase.

Group 3. These forms ferment maltose and sucrose but fail
to ferment raffinose. As has already been mentioned, in this
case sucrose is hydrolysed by the enzyme maltase. These
forms do not differ from the preceding ones in the type of
enzyme produced. However, since the ability to produce the
active enzyme maltase was developed in this case on the
medium containing sucrose, these forms readily ferment
sucrose already within the first 10 days, while the cultures of
the preceding group failed to ferment sucrose within the
same period of time.

Group 4. These forms ferment not only monosaccharides, but
also sucrose, maltose and one-third of raffinose. They were
obtained in two stages: first a culture oiS. glohosus 74/349 was
adapted to fermentation of sucrose ; then this was adapted to
fermentation of maltose on a medium containing maltose.

The forms obtained from S. paradoxus are divided into 2
groups, based on differences in fermentative properties:

Group 1. In contradistinction to S. paradoxus, these forms
ferment maltose. Therefore, they acquired the ability to
produce the active enzyme a-glucosidase.

Group 2. After long cultivation on beer-wort, cultures pre-
viously adapted to fermentation of maltose (group 1) became
adapted to fermentation of simple dextrins of malt-wort.

The above-mentioned altered forms, obtained from S.
glohosus and S, paradoxus, were studied in comparable condi-
tions, with the aim of estimating their sugar-fermenting
capacity and the depth at which beer- wort is fermented. In
parallel experiments, we also studied in comparable conditions
cultures of original forms of S. glohosus, S. paradoxus, S.
chodati, S. ellipsoideus and S. cerevisiae. The last three species
were used for comparison with the altered forms.

Induced Mutational Changes in Yeast 125

The fermenting medium contained 10 per cent of the
required sugar and yeast extract. The substances tried
were gkicose, galactose, sucrose, maltose, raffinose and the

Culii

S.globo5USM9

72 /349 m- I -6 -21

72/349M-4-73

74/349-I10S-I8
74/349-I-I05-I8mI-4

S. paradoxus 37

M37-9/2M-/

S.chodati m

S.ellipsoideus m

Fermentation of Sugars (s. /m I.)

Glucose I Galactose \ Sucrose | Raffinose

357 270 000

000 0-59

I I I — 1^ I I I
198 A 30S

0-27 i 411 ^^-^

Maltose \Ma/t-wort

2-33 K 324

0-00 0-70

I- 60 1-92

000 073

1-39 0-89

Fig. 2. Fermentative properties of original, altered and
some other types of yeast. Upper borderline of shaded
portions = curve of fermentation of sugars. Numbers
above the curves of fermentation indicate the amount
of CO2 produced during fermentation (in g./lOO ml.
medium). Only one-third, if any, raffinose was fermented.

usual beer- wort (saccharimeter : 13-5°). The experiments
were carried out at a temperature of 25-26° C, over a period
of 10 days; results are shown in Fig. 2. Characteristic curves

126

K. V. KossiKOV

show the intensity of fermentation for each day. The capacity
to ferment rafhnose is shown in comparison with the raffinose-
fermenting capacity of S. carlsbergensis.

A 37' 9/2 M- 1-29 D

* simple dextrins
of malt-wort

A 37-9/2 M-l-29

+ maltose

5. cerevisiae

o

+ simple dextrins
of malt-wort

S.ellipsoicleus 74/349-H0S-i6M-h4

maltose

+ sucrose

S.q lobosuS

"O

■hgalactose
i- glucose
+ fructose
+ mannose

+ maltose

S. paradoxus 74/349^ios-i8

Fig. 3. Fermentative properties of some species of
Saccharomyces (centre) and of forms obtained experi-
mentally (left and right).

The existing classification of species of yeast of the genus
Saccharomyces is based on behaviour toward various sugars,
i.e. they are classified mainly according to their ability to
ferment a specific sugar. On comparison of the data on
fermentation of various sugars by altered forms of yeasts

Induced Mutational Changes in Yeast

127

with the data on species of the genus Saccharomyces (which
species are rather close to the altered forms of yeasts), we con-
cluded that it has been possible to obtain experimentally,
from S. glohosus, new forms which may be referred to the
following 3 species: S. paradoxus (group 1); S. chodati
(group 2) and S. ellipsoideus (group 4). The forms obtained

Fig. 4, Fermentation of malt-wort by certain species of
Saccharomyces, and by new forms obtained experimentally.
1. S. globosus; 2. S. paradoxus; 3. S. ellipsoideus; 4. S.
cerevisiae XII. Altered forms produced from .S'. globosus:
lA-74/349-1-105-18; lB-74/349-l-105-18m-l-4. Altered
forms produced from S. paradoxus: 2A-A37-9/2m-l-29;
2B-A37-9/2-l-29d.

from S. paradoxus may be referred to 2 species : S. ellipsoi-
deus (group 4) and S. cerevisiae (group 2).

These data testify to the fact that by cultivating yeasts of
the genus Saccharomyces on their specific substrates, it is
possible to induce changes in species characters. The scheme
of variation is shown in Fig. 3. Fig. 4 shows the intensity of
fermentation and the depth at which malt-wort is fermented

128 K. V. KossiKOV

by the altered forms, and also by the original forms and by
S. ellipsoideus and S. cerevisiae.

Discussion

The experimental data quoted above enable us to give a
positive reply to the question on the stability of directed
adaptive changes, which was raised at the beginning of this
paper. These changes take place under the direct influence of
the specific substrate, and are not only quantitative but also
qualitative, i.e. they lead to the development of a new
quality. This quality is expressed in the capacity to produce
active invertase (or maltase), hydrolyse and ferment sucrose
(or maltose), retain this capacity and transmit it to the
progeny. It should be pointed out that in the investigations
of Oparin, Helman and Elpiner (1954), on extracts of S.
glohosus cells subject to the action of supersonic waves, it was
possible to detect very small quantities of invertase. This
experiment confirmed the data obtained by Yurkevitch (1950),
that active invertase is not found in intact cells and after
short autolysis. The appearance of invertase due to the
influence of supersonic waves is explained by Oparin, Helman
and Elpiner as the freeing of invertase from stable compounds
of the protoplasm. The hydrolytic activity of the enzyme is
completely inhibited in the process of growth and reproduction
of cells. Oparin, Helman and Zhukova (1954), in further
investigations on cells previously adapted by the present
author to fermentation of sucrose, but trained on a medium
without this sugar, and subject to the action of supersonic
waves, found an invertase activity 20 times as great as that
which was found in the original cells not adapted to fermenta-
tion of sucrose but subject to the action of supersonic waves.
It should be noted that great changes occurred in the carbo-
hydrate content of the adapted cells.

If we 'Consider the variations occurring in the organisms as
being directed ones, i.e. caused by changes in the conditions
of their development, this does not mean that we should

Induced Mutational Changes in Yeast 129

agree that there exists only one type of mechanism of develop-
ment of these variations, and that we should consider them
either as being unstable (bearing on quantitative shifts in the
enzymic organization of cells) or stable ones of the mutation
type. In each and every case, the character of variation will
be determined by the nature of the organism undergoing
change and by the peculiarities of the factor which exerts its
influence in those organisms. As regards the variation in
fermentative properties of yeasts, one can speak of four types
of adapted variations based on existing experimental data :

The first type of variation involves the fermentative system
of the cell. An example of this type of variation is the capacity
acquired by the yeast cell to split sucrose by means of maltase.
This is not a case of qualitative change in the fermentative
system, but of alterations in the permeability of the cell
(Oparin, Helman and Zhukova, 1955). As our investigations
have shown, a most important factor here is the substrate on
which the cell acquires the ability to produce the active enzyme
maltase. If this ability is acquired on a medium containing
maltose then the splitting of sucrose usually becomes difficult.
Cultures obtained in this way from S. glohosus start to ferment
sucrose usually after a lapse of 10 days, but ferment maltose in
1-2 days. The same cultures oiS. glohosus, if they acquire the
ability to produce the active enzyme maltase on a medium
containing sucrose, will ferment sucrose within the first 2-3
days. In both cases hydrolysis of sucrose is effected by
maltase. The time of cultivation of such cells on the sucrose
medium is also of great significance. S. heterogenicus, S. pro-
stoserdovi and S. chodati were tested for ability to ferment
maltose and sucrose (but not raffinose), and it was demon-
strated (Kossikov, Helman and Rayevskaya, 1956) that all
of these hydrolyse sucrose by means of maltase, since maltase
but not invertase was detected in the cells after fermentation
of sucrose. Fermentation of sucrose in these species began at
varying times (from 2 to 5 days).

The second type of variation was detected in the adaptation
of S. paradoxus to fermentation of maltose. In this case, the

130 K. V. KossiKOv

adapted form did not prove to be very viable, but propagated
more or less normally for some time in very strictly defined
conditions. In inadequate conditions, not only does this form
lose its acquired property (reversion), as one would expect,
but it dies off altogether. This form can become viable only
if some new quaUtative change occurs in it. To the same type
of variation belong 10 cultures out of 29 obtained from single
spores ofS. paradoxus 37, adapted to fermentation of maltose;
3 out of these 10 cultures developed (on wort-agar) stable
colonies which readily ferment maltose.

The third type of variation is characterized by the appearance
in the cells (under the influence of the specific substrate) of the
so-called adaptive enzymes which are produced only when
there is an adequate specific substrate in the medium. If the
specific substrate is removed from the medium the cells no
longer produce the enzyme, and they lose their acquired
property. This type of variation is known to exist among
yeasts of the genus Saccharomyces during their adaptation to
fermentation of galactose and mehbiose. One would think
that during prolonged cultivation on the medium with the
adequate sugar (as the only source of carbohydrate), cells
would develop which could retain the abihty to produce an
adequate enzyme, the specific substrate being absent from the
medium.

The fourth type of variation is that where cells retam the
acquired abihty to produce an active enzyme, ferment an
adequate sugar and transmit this capacity to the progeny
after reseeding into media not containing this sugar. In sexual
reproduction (spore formation) the altered cells become segre-
gated. An example of this is found in the adaptation of
S, globosus to fermentation of sucrose and maltose, and of
S. paradoxus to fermentation of maltose.

Experiments to determine quantitatively the sucrose-
fermenting activity of invertase in original cultures of S. glo-
bosus, in heterozygous cultures adapted to fermentation of
sucrose and in homozygous cultures (these last being obtained
from heterozygous cultures through spore formation), gave

Induced Mutational Changes in Yeast 131

very interesting results. It was found that in the cells of the
heterozygous cultures, the activity of invertase was only one-
half of that in the homozygous cultures. Not only the original
S. glohosus cultures, but also those obtained from heterozygous
cells, but failing to ferment sucrose, have no active invertase
in the intact cell (Kossikov and Rayevskaya, 1957).*

The experimental data on the inheritance of sucrose-
fermenting ability during spore formation and on the activity
of invertase in heterozygous and homozygous cells, give us
grounds to believe that, in this case, in the process of adapta-
tion to fermentation of sucrose the ability to produce the
active enzyme invertase appears in the cell. As yet, it is
difficult to say whether this capacity is related to the nuclear
or other structural elements of the cell. The detection of an
invertase activity in the homozygous diploid culture, twice
as high as that in the heterozygous diploid culture, leads
us to conclude that the production of invertase is related
to biochemical reactions and structures characteristic
of the haploid cell. Fig. 5 shows the scheme of inheritance,
by S. glohosus cells, of the ability to ferment sucrose during
sexual reproduction.

Can one speak in this case of the heritability of acquired
characters? The sum-total of our data bearing on the stability
and heritability of new enzymic properties of yeast, developed
in the process of adaptation to fermentation of sugars (fourth
type of variation), leads us to answer this question in the
affirmative. However, one should bear in mind that not every
single character acquired by the organism is inherited. What
we are referring to is the heritability of those characters which
arise as a result of changes in the modus vivendi, this change
leading to stable variations in cell metabolism and to alter-
ations in cell function.

* The activity of invertase was determined by the Berthran method. Cultures
failing to ferment sucrose had no active invertase if they were obtained from
cells cultivated for a long time in the presence of sucrose or from the spores of
heterozygous cultures. The activity of invertase adapted to fermentation of
sucrose in the heterozygous cultures was 17 and in the homozygous cultures
was 34 (expressed in mg. of glucose per 10 mg. of pressed yeast during 60 min.).

132

K. V. KossiKOv

If we accept that the cell structure is the manifestation of
the specificity of metabolic processes occurring in this cell, and
that alterations in metabolism are the principal or really the
sole cause of the development of new forms, we can then
clearly understand the (usually negative) action of irradiation,

^^a

Fig. 5. Scheme of inheritance, by S. glohosus cells, of newly-
acquired ability to ferment sucrose during sexual repro-
duction (spore formation).

a: non-fermenting sucrose, invertase activity = 0;

6: fermenting sucrose, invertase activity = + ;

c: fermenting sucrose, invertase activity = -|--j-.

leading to disturbances in structure and function, and the
creative role of induced change in metabolism in the process of
adaptation to the changed modus vivendi.

Certainly, the substrate cannot cause changes, in all cases
and under all conditions, which will remain stable in the
progeny. We believe that, as a rule, it is those changes con-
nected with the necessity of adaptation to new sources of

Induced Mutational Changes in Yeast 133

nutrition and to changed conditions of existence that lead to
alterations in the chain of biochemical reactions and in the
corresponding structural elements of the cell. This conclusion
is very simple and at the same time natural, since the con-
stantly observed close interrelationship of the organism with
the conditions of its existence testifies to the fact that these
conditions are not only the most essential source of develop-
ment but are also the cause of changes in the organism. The
experimental data on the increase of the fermentative
properties of yeast, the increase being connected with the
acquisition of new hereditary stable functions (under the
influence of the specific substrate), confirms this assumption.
These are, undoubtedly, positive hereditary changes. The evolu-
tional significance of this type of change is unquestionable.

Directed changes in the ability to ferment galactose under
the influence of the substrate (galactose) were observed by
Lindegren and Pittman (1953). Lindegren (1956) believes that
what happened, in this case, was the restoration of the
recessive locus which evidently had formerly been active but
had been damaged and inactivated ; so that the non -functioning
recessive gene again became functional and active (dominant).
Although in this case we speak about restoration of the lost
function and, according to Lindegren, restoration takes place
due to mutations under the influence of galactose but not of
lactose, we failed to detect this type of mutation occurring
spontaneously. These data, if considered along the lines
already mentioned, can be considered as confirmation of the
directed hereditary changes of the fermentative properties of
yeast, under the influence of the specific substrate, which
were observed in our experiments (Kossikov, 1948, 1951,
1952).

Conclusion

(1) Our experiments carried out in the course of the last
eight years, on directed changes of fermentative properties of
S. globosus, S. paradoxus and other yeasts under the influence
of a specific substrate, have shown that in these cases single

134 K. V. KossiKOV

cells arise which are capable of fermenting an adequate
sugar. In control experiments we found no changes of a kind
which could be explained as the appearance of spontaneous
mutations.

(2) The study of inheritance by yeast cells of the properties
which arose as a result of adaptation to fermentation of sucrose
and maltose has shown that these properties are transmitted
to offspring not only in vegetative but also in sexual repro-
duction (spore formation). In the latter case segregation was
observed. One-half of the spores formed by altered cells
usually give cultures which ferment sugar, the remainder form
cultures which do not ferment an adequate sugar. In S.
jyaradoxus forms were also obtained, adapted to fermentation
of maltose, which on sexual reproduction did not show
segregation.

(3) The control of the stability of fermentative properties
acquired by yeast when resown in agar media containing other
sugars has shown that the capacity to ferment both sucrose
and maltose remains stable.

(4) Stability of fermentative properties which arose in the
cells is clearly evident, also, in crossing altered strains with
original (unaltered) strains. We obtained a dominant capacity
to ferment an adequate sugar in the first generation and a
segregation by the same characteristics in the second sexual
generation.

All these experimental data testify to the fact that directed
hereditary changes were obtained under the influence of the
substrate.

(5) As a result of our experiments we obtained new forms.
From S. globosus we obtained: (i) forms additionally ferment-
ing sucrose and one-third of rafiinose; (ii) forms additionally
fermenting maltose, and (iii) forms additionally fermenting
sucrose, maltose and one-third of raffinose. These forms may be
classified, according to their fermentative capacities, as species
of S. paradoxus, S. chodati and S, ellipsoideus respectively.

From S. paradoxus we obtained: (i) forms additionally
fermenting maltose and (ii) forms additionally fermenting

Induced Mutational Changes in Yeast 135

maltose and simple dextrins of malt- wort. These forms may
be classified as species of S. ellipsoideus and S. cerevisiae,
respectively.

(6) Biochemical investigation has shown that the active
enzyme invertase is found in intact cells, adapted to fermenta-
tion of sucrose and grown on a medium containing glucose,
whereas this enzyme is totally absent from original S. glohosus
cells. Homozygous adapted cells have twice as much invertase
as heterozygous ones.

REFERENCES

Dean, A. C. R., and Hinshelwood, C. (1953). In Adaptation in
Micro-organisms, p. 21. Cambridge University Press.

Imshenetsky, a. (1953). VI Int. Congr. Microbiol., 1, 685.

Imshenetsky, a., and Perova, K. (1955). Microbiology, 24, v. 2, 148.

Kalina, G. p. (1952). Vegetative Hybridization and Directed Mutabil-
ity of Bacteria. Kiev.

KossiKOV, K. V. (1948). C. R. Acad. Sci., U.R.S.S., 63, No. 5, 573.

KossiKOV, K. V. (1951). C. R. Acad. Sci., U.R.S.S., 80, No. 1, 105.

KossiKOV, K. V. (1952). Proc. Inst. Genetics, Acad. Sci., U.R.S.S., No.

19, 199.

KossiKOV, K. V. (1954). Genetics of Yeast and Methods of Selection of

Yeast Cultures. Moscow.
KossiKOV, K. V. (1956). Proc. Acad. Sci., U.R.S.S., Biol. Series, No. 5,23.
KossiKOV, K. v., Helman, N. S., and Rayevskaya, O. G. (1956).

C. R. Acad. Sci., U.R.S.S., 111, No. 6, 1369.
KossiKOV, K. v., and Rayevskaya, O. G. (1956). Proc. Inst. Genetics,

Acad. Sci., U.R.S.S., No. 23, 326.
KossiKOV, K. v., and Rayevskaya, O. G. (1957). C. R. Acad. Sci.,

U.R.S.S., 112, No. 1, 141.
LiNDEGREN, C. C. (1956). C R. Lab. Carlsberg. Ser. physioL, 26, No. 1,

253.
LiNBEGREN, C. C, and PiTTMAN, D. (1953). J. gen. Microbiol., 9, 494.
MuROMZEV, S. N. (1953). Mutation of Micro-organisms and Problems of

Immunity. Moscow.
Oparin, a. I., Helman, N. S., and Elpiner, I. E. (1954). C. R. Acad.

Sci., U.R.S.S., 97, No. 2, 293.
Oparin, A. I., Helman, N. S., and Zhukova, I. G. (1954). C. R. Acad.

Sci., U.R.S.S., 99, No. 4, 593.
Oparin, A. I., Helman, N. S. and Zhukova, I. G. (1955). Biochemistry,

20, V. 5, 572.

Planelies, H. H. (1956). J. Acad. med. Sci., U.R.S.S., No. 6, 17.
Planelies, H. H., and Moroz, A. F. (1956). Antibiotics, No. 6, 30.
YuRKEViTCH, V. V. (1950). Thesis Inst. Biochem., Acad. Sci., U.R.S.S.
YuRKEViTCH, V. V. (1954). C. R. Acad. Sci., U.R.S.S., 94, No. 2, 329.

136 Discussion

DISCUSSION

Harington: Prof. lerusalimsky, I believe you have some data which
you would like to report now.

lerusalimsky : The experimenters working on adaptation of micro-
organisms did their best to distinguish physiological non-hereditary
changes from spontaneous mutations. Nature, however, does not know
any hard and fast rules. It is possible to admit the existence of cate-
gories intermediate between non -hereditary modifications and hereditary
changes. It is common knowledge that the precious qualities developed
in cultivated plants are inherited by them during an unlimited length
of time in vegetative reproduction. However, some of these qualities
can very easily disappear in the case of reproduction by means of seeds.

Bacteria undergo adaptive changes which could be transferred to one
or to many generations of vegetative cells. However, it seems to me
that only those changes that are transferred through sexual or asexual
reproductive cells could be considered truly hereditary. To my mind,
a certain kind of analogy of the latter are spores. The life cycle of
bacteria ends in spore formation; and ince versa, germination of spores
starts the next life cycle. During the formation of the spore, essential
changes occur in the cellular structure and the greater part of the cellular
content is not included in the spore. One should assume that it is the
most stable properties, which can be considered hereditary, that will be
transferred through spores. Unstable changes, which are retained in
vegetative reproduction alone and disappear in spore formation, should
be considered non-hereditary. To quote an example: strain CL aceto-
hutylicimi, which we had at our disposal, ferments xylose after a certain
period of adaptation. Proof of this can be obtained by seeding the spores
into glucose and xylose media (Fig. 1). The cells, once they start to
produce enzymes, continue to propagate and they ferment xylose at
almost the same rate as glucose.

However, the above is true only in those cases where the cells seeded
are vegetative ones. As soon as the cells in the xylose medium form
spores, their newly acquired capacity disappears, and again a certain
amount of time will be needed to produce adaptive enzymes (Fig. 1).
Consequently, the ability to produce these enzymes at an increased rate
is not transferred through spores and therefore cannot be considered
hereditary.

Could the changes which were induced by environmental factors be
so strengthened and stabilized as to be transferred through spores, and
thus become hereditary?

I shall mention only one example confirming the possibility of the last
statement. We adapted CI. aceto-butylicum to increased concentrations
of butanol. The bacteria were cultivated in an apparatus for continuous
cultivation, the concentrations of butanol increasing gradually (by • 2-
0-5 per cent at a time). After each increase of concentration the rate
of bacterial propagation dropped considerably; and only after a long
period could the rate be increased again to that of propagation of the
control culture, after which time we increased the concentration of

Discussion

137

butanol again. Simultaneously the culture was inoculated into the media
with different doses of butanol, to show the degree of resistance developed
by it.

The stock culture could not survive a butanol concentration of more
than 0-8 per cent, the adapted one survived a 2-5 per cent butanol
concentration and propagated normally at a 2 per cent butanol concen-
tration. The experiment took 200 days, which is equivalent to 4,300

ID 20 20f I

Sporosfromxy?. / /pin)re5fromgPu.med.
x^e. // xyeosQ

Z- .

20 30 AO 50 60 hr

Fig. 1. Adaptation of CI. aceto-butylicum to
fermentation of xylose.

successive cellular generations. Fig. 2 shows that the resistance to
butanol increased gradually together with the increase in its concen-
tration. No abrupt increase was observed, which might be expected had
spontaneous mutations appeared in the population. At the same time
the conditions of the experiment were extremely favourable for the
selection of resistant mutants. A very simple calculation shows that
had there been only one mutant per million cells, the coefficients of
propagation of which would exceed that of original cells by a mere 0-1,
then after 200 generations, i.e. in approximately 8-9 days, the number

138

Discussion

of mutants would have equalled the number of initial cells. Had the
propagation coefficient of mutants been twice as large, the number of
both groups would have become the same after 40 generations, i.e. in less
than 2 days. However, to attain full adaptation to every consecutive

NJ

\Adgpt.2ldaifs

Controf^

06 10 1-5 20 25

B u t a n 8 . Vo

Fig. 2 : Adaptation of CI. aceto-

hutylicum to increased concentrations

of butanol. Before sporulation.

concentration of butanol, it required from 460 to 1,500 generations,
i.e. from 20 to 70 days (Table I).

At first, the increased resistance was not transferred through spores.
Later on it became so stable that it was retained by cells growing from
the spores on the butanol-free medium and then seeded in the vegetative
state in media containing different concentrations of butanol (Fig. 3).

Owing to a shortage of time I shall not refer to other experiments
which consisted of adapting Bad. megaterium to greater concentrations
of norsulphazol. In this case adaptation took place more easily and more
quickly than in the case of butanol. Only 70 reseedings were needed to
obtain a fivefold increase in resistance. Simultaneously, sulphazol-
dependent forms could be detected in the population. In the case of
butanol no such phenomenon could be seen. An increased resistance
began to be transmitted through spores.

In our opinion, these experiments show the possibility of stabilizing
adaptive, non-hereditary changes and of their gradual transition to
stable hereditary changes.

Discussion

139

Table I

Adaptation of CI. aceto-butylicum to butanol
IN continuous culture.

Butanoe

timg 0^

adaptation

days

averagQ
numBQr
ofgQDQrations

06

21

460

0-8

28

610

10

36

780

1-5

45

Q80

20

70

1530

Totaes

200

4360

06 10 1-5 20 2-5

B u t a n 8, %

Fig. 3, Adaptation of CI. aceto-hutylicum

to increased concentrations of butanol.

After sporulation.

140 Discussion

In what way do non-hereditary adaptive changes gradually turn into
genotypic properties? To answer this question, special and more pro-
found investigations are required. One can well imagine that the ability
to synthesize a particular enzyme, trained for a long time, will finally
touch on some more remote enzymic systems, which pass from the
vegetative cell into the spores formed in it. Resulting from this, the
vegetative cell developed from the spore will have a greater ability to
synthesize a given enzyme prior to coming in contact with the external
inductor. This will result in hereditary fixing of this ability.

I should like to note that the aim of my report is to draw more
attention to the concept — shared by many of my countrymen — of the
possibility of gradual transformation of acquired adaptive changes into
stable hereditary changes. The experimental testing of this hypothesis
might yield important results, which would have been scarcely possible
had this theory been disposed of a priori.

It would be equally wrong to disagree, without sufficient proof, with
the widely acknowledged theory that mutations are the only form of
hereditary changes. Facts must be the highest arbiter in all scientific
disputes.

MULTIPLE MECHANISMS OF ACQUIRED
DRUG RESISTANCE

Margaret J. Thornley, Jehudith Sinai
AND John Yudkin

Queen Elizabeth College, University of London

There are two possible ways of reconciling the different
theories which have been suggested for the origin of drug
resistance. The first is that the different mechanisms occur
in different micro-organism-drug systems. The second is that
the different mechanisms may occur in a single system and
that the different theories derive from different experimental
approaches.

We have attempted to demonstrate the latter possibility in
the system Escherichia co/i-proflavine. We began with the
usual type of experiments on "training" and "reversion".
Each culture so produced was tested against a range of con-
centrations of proflavine, so as to measure its distribution of
resistance. In a culture grown in broth from a single cell in
the absence of drug, there was a slight increase in the propor-
tion of resistant cells during the first few subcultures.

"Training*' and "Reversion"

When grown even once in the presence of drug there w^as a
great increase in the number of resistant cells, able to grow in
much higher concentrations. Cultures derived from different
single cells gave significantly different numbers of resistant
cells after the same number of subcultures in the same drug
concentration.

After subculture in the presence of drug, repeated sub-
cultures were made in the absence of drug. The degree of
reversion depended on the number of subcultures in drug,
on the concentration of the drug, on the strain of organism

141

142 M. J. Thornley, J. Sinai and J. Yudkin

and on the number of subcultures in the absence of drug. But
complete reversion rarely occurred even after one subculture
in fairly low drug concentration followed by many subcul-
tures in the absence of drug. Conversely, when several sub-
cultures were made in the presence of drug, considerable
reversion still occurred and to about the same extent after
12 subcultures in the presence of drug and after 84.

These results are not in conformity with the suggestions
of Hinshelwood. First, we do not find that the degree of
resistance achieved corresponds to the concentration of drug
in which a culture is grown. Second, we find that reversion
is usually incomplete even after short contact with the drug,
and yet it still occurs after very prolonged contact with drug.
We conclude that this approach does not promise to reveal
much as to the nature of the origin of drug resistance.

Phenotypic adaptation

Baskett (1952) has claimed a rapid increase in resistance to
proflavine in a growing culture of Bacterium lactis aerogenes
to which the drug was added at short intervals. We carried
out this type of experiment with Esch. coli but growth ceased
soon after the additions of proflavine were begun.

However, since Baskett's work seemed to be a conclusive
proof of rapid phenotypic adaptation, we repeated his experi-
ments with the same strain of Bad. lactis aerogenes, kindly
supplied by Sir Cyril Hinshelwood. We confirmed Baskett's
observation that additions of proflavine at intervals of ten
minutes to growing cultures of this organism only slightly
decreased the rate of growth, even though a high concentra-
tion of proflavine of up to 100 [xg./ml. was finally achieved.
The same concentration of proflavine added at once inhibited
growth completely. We found, however, that the cells them-
selves showed only a small increase in resistance. On the other
hand, the presence of filtrate from a culture grown in the
absence of proflavine allowed growth of an inoculum when a
high concentration of proflavine was added (Fig. 1). We
therefore looked for a factor produced in a culture during

Multiple Mechanisms of Drug Resistance 143

growth which antagonizes the inhibitory action of proflavine.
The experiments showed that such a factor was the acidity

Time (mins

Fig. 1. Effect of bacterial filtrate from a sensitive culture on the
inhibitory action of proflavine on BacL lactis aerogenes.

1. Filtrate 40 ml. + fresh medium 10 ml., to which proflavine
was added gradually then inoculated with washed ceils m
logarithmic phase

2. Proflavine added gradually from 210 min. after inoculation

3. Proflavine added at once at 210 min. after inoculation

4. No proflavine added

gradual proflavine addition

66 [ig.lml. proflavine present

no proflavine added

Time from addition of cells in (1) and 210 min. after inoculation
in others.

produced during the growth of the culture. Thus, if the pH
of a growing culture was kept neutral, growth ceased when the

144 M. J. Thornley, J. Sinai and J. Yudkin

proflavine additions were made (Fig. 2). On the other hand, if
a growing culture was acidified, growth continued even if all

.o 3

P 2

•o I

60 I20 I80 24.0

Time (mins)

Fig. 2. Effect of maintaining neutral pH in culture during pro-
flavine additions, and of acidifying the culture when large single
addition of proflavine made, on growth of Bad. lactis aerogenes.

1. pH maintained neutral throughout incubation, proflavine
added gradually

2. Culture acidified to pH 4-9, then all proflavine added at once

3. Proflavine added graduaUy

4. No proflavine added

gradual proflavine addition

65 ^g./ml. proflavine present

no proflavine added

Time from beginning of proflavine additions.

the proflavine was added at once. Finally, acidified fresh
medium with the whole amount of proflavine added supported

Multiple Mechanisms of Drug Resistance 145

the growth of the sensitive cells. That the phenomenon did
not occur with Esch. coli may be due to the difTerences in
the culture medium. With Bad. lactis aerogenes, this was
an inorganic medium which contained glucose, whilst with
Esch. coli the medium was peptone broth only.

It is, however, possible to obtain with Esch. coli a small
increase in resistance in growing cultures if small amounts of
proflavine, giving a final concentration of not more than the
threshold inhibitory concentration of 5 [xg./ml., are added at

Table I

Increase in resistance in culture of Escherichia coli 36 by

ADDITION OF PROFLAVINE IN LOGARITHMIC PHASE

Initial

Proflavine

Control

1

1

3

3

After 270 min.
Proflavine Control

Increase in resistance
Gradient plates
Counts 6-6 fig. I jn\.

10 Atg./ml.
20 /tig./ml.

3

3-2x102

and
2x105 (s)
1-5x102
1-5x102

1-3
1X102

3X10
2-8x10

(s) small colonies

longer intervals of 30 minutes. The increase in resistance
occurred in up to 1 per cent of the cells (Table I). It was
somewhat less in degree than that of the first-step mutants
(see below). It occurred only when the additions of drug
were made in the logarithmic phase, and not when they were
made in the lag or stationary phases (Fig. 3). The cells show-
ing the increased resistance to proflavine were not cross-
resistant to chloramphenicol, aureomycin, erythromycin or
terramycin, drugs to which proflavine-resistant mutants show
cross-resistance. They lost their resistance to proflavine
when grown in the absence of the drug. For these two

146

M. J. Thornley, J. Sinai and J. Yudkin

9-

o6

c 0-

j

3

1

O 7-

1

O

1

<V 6-

/

■^ '^-

/ °'

o

> 4-

/ /'

(N 3^

/ '

cn

/ /

3 =

1

1 ,

j/

0;
o

C 3

I

2 3 4 5 6 7

Time (hours

Fig. 3. Effect of proflavine additions at different phases of cultural
growth on resistance of Esch. coli 36.

Flask 1 — Proflavine additions start in lag phase 30 min. after

inoculation
Flask 2 — Proflavine additions start in lag phase 45 min. after

inoculation
Flask 3 — Proflavine additions start in late logarithmic phase 5J hrs.

after inoculation
Flask 4 — Proflavine additions start in late logarithmic phase 4 hrs.

after inoculation
Flask 5 — Proflavine additions start in middle logarithmic phase 2|

hrs. after inoculation
Flask 6— Control for flask 1
Flask 7 — Control for flasks 2, 3, 4, and 5
Flask 8 — Control for flasks 3 and 4

Log2 of optical density and increase in resistance plotted against time

proflavine added gradually

# flask 5

growth in flask 1 and 6 expressed as logg viable count
no proflavine added

Time from inoculation.

Multiple Mechanisms of Drug Resistance 147
reasons, we believe that the increase is due to phenotypic
adaptation.

lOO

I CYCLE

I CYCLE

4.0

60

80

lOO

I CYCLE

15 40 60 60 80 lOO I20

Time(min5) Timelmins)

A cooling starts after 120 min. from inocu ation
B cooling starts after 90 min. from moculation
C cooling starts after 70 min. from inoculation
D cooling starts after 60 min. from inoculation
Time from returning the flasks to 37°C after cooling.

As well as this moderate increase in resistance of a large
proportion of the cells, there was a rise in the number of cells

148 M. J. Thornley, J. Sinai and J. Yudkin

of high resistance. These cells were cross-resistant to the other
drugs. We believe that they arise not by division of pre-
existing mutants but either by clonal variation or by direct
(Lamarckian) induction.

We now investigated whether a variation in this phenotypic
adaptability might explain the continuous distribution of
resistance in a culture. We tested the possibility that the
variation might be a function of the various stages of the
division cycle. Synchronization of division was attempted by
cooling for a few minutes and then returning to 37°. The
degree of synchronization depended on the time from inocula-
tion of the culture, and the temperature and period of cooling.
In appropriate conditions, it was possible to obtain a reason-
able degree of synchronization, in which a burst of divisions
occurred during the first 10 minutes after cooling, followed
by cycles of 30 minutes with half of the divisions occurring
in the first 20 minutes and half in the next 10 minutes
(Fig. 4).

The cultures with synchronized division cycles also showed
cyles of fluctuation in resistance. These cycles lasted for about
20 minutes (Fig. 5). The variation in resistance during the
cycles was demonstrated by taking samples at intervals and
testing on plates with varying concentrations of proflavine.
The variation showed itself in three ways. First, with appro-
priate concentrations of about 6 jxg./ml. of drug, there was a
great difference at different times in the number of cells able
to produce colonies; this difference was as high as 1,000-fold,
compared with the maximum of less than tenfold in samples
from a non-synchronized culture. Second, the differences at
different times became progressively less with lower concen-
trations of drug on the testing plates, so that when the con-
centration was about 4 [xg./ml. proflavine, the same number
of colonies grew throughout the cycles. This concentration
thus appears to be the inherent resistance of the cells. Third,
the period out of the cycles during which the cells were
able to grow on proflavine plates was longer with the lower
concentrations of drug.

Multiple Mechanisms of^Drug ^Resistance 149

An important conclusion is derived from a consideration of
the technique used for testing varying resistance during the

lOO MO I20

Time(mins)

Fig. 5 a. Resistance to various concentrations of proflavine of cells

in cultures of Esch. coli 36. Non-synchronized cultures.
A Tested from 80 min. after inoculation, on proflavine 4 (Jig./nil.
B Tested from 70 min. after inoculation, on proflavine O — 4-5 [xg./ml.

#—5 {ig./ml.

A— 5-7(^g./ml.

X — 6-6 [JLg. I ml.
Time from inoculation.

division cycles. We consider a cell to have a higher resistance
at one stage of the division cycle when it is able to produce
a colony on a plate with a higher concentration of proflavine.

150 M. J. Thornley, J. Sinai and J. Yudkin

But in order to do so, its daughter cells must have gone
through a stage in the division cycle when their resistance
was low. Nevertheless, they continue to grow and divide.

Fig. 5B. Resistance to various concentrations of

proflavine of cells in cultures of Esch. coli 36. Syn-

ehxonized culture. Synchronization by cooling after

90 min. from inoculation at 12° for 15 min.

O total viable count expressed as number

of generations
# number of cells in 0-1 ml. resistant to

proflavine 5 {j.g./mg., expressed as log^^

Time from returning to 37° C at the end of cooling.

This must mean that the apparently higher resistance is
really a higher adaptability, which is retained in the presence
of the drug throughout the whole of the subsequent division
cycles. Thus, the variation we find in the cycles is not a

Multiple Mechanisms of Drug Resistance 151

variation in resistance but a variation in adaptability. If
this is so, then the addition of proflavine to a synchronized

A

B

c

D

E

8-

7-

^---'

2

1

e*

.-' '

2

,' /

Ip?

2

6

/ '

^5

u

\-^''

/' /

,ol

d

c

V

/'

03-

^

o

*

-J

^«

2-
!■
O

. . r^

Idd2
2 . . ■ 1

60 I20 ISO 60 I20 180 60 I20 I80 60 I20 ISO 60 I20 ISO

Time (mins)

Fig. 6. Effect of proflavine 4 (i.g./ml. on increase in resistance
and growth inhibition when added at different times of a division
cycle in a synchronized culture of Esch. coli 36.

1 — proflavine added at once at 24 min. after returning the

culture to 37°C
2 — proflavine added at once at 56 min. after returning the

culture to 37°C
A — total viable count
B — cells resistant to 5 [jLg./ml. proflavine
C — cells resistant to 5-7 [jig./ml. proflavine
D — cells resistant to 6-2 [i.g./ml. proflavine
E — cells resistant to 10 ptg./ml. proflavine

O — normal colonies 4 y.g.fm\. proflavine present

# — small colonies no proflavine present

Time from end of cooling.

culture should result in no increase in resistance at some
stages and a significant increase at other stages. This predic-
tion we have confirmed experimentally (Fig. 6).

152 M. J. Thornley, J. Sinai and J. Yudkin

The variation in adaptability during the division cycles
can explain the rise in resistance in a growing culture to which
proflavine is added. It can also explain the continuous curve
of resistance in a culture, which will have cells at all stages of
the division cycle and so with the complete range of degrees
of adaptability. We cannot, however, exclude the possibility
that part of the explanation for the continuous curve of
resistance is a superimposed clonal variation.

Lamarckian inheritance

The work of Akiba (1954) and of Szybalski (1955) indicates
that Esch. coli may acquire resistance to streptomycin by
direct induction, and that the resistance so acquired is in-
herited. We may therefore speak of this type of induction as
Lamarckian inheritance. Our own experiments on Lamarc-
kian inheritance of proflavine resistance in Esch. coli were
made both with growing cultures and with non-dividing
organisms.

Most of the work was carried out with rough strains derived
from our original smooth strain of Esch. coli. In some of these
strains, growing cultures in the presence of proflavine, cell
extracts and tap water gave a high increase in resistance in
up to 10 per cent of the cells. A much smaller increase occurs
if cell extracts are not present, recafling the experiments on
phenotypic adaptation. Since extracts from sensitive or from
resistant cells are equally effective, the increase in resistance
is not due to transformation. We believe that it is due to
Lamarckian induction, though we were not able to exclude
the possibility of selection of pre-existing mutants.

In non-dividing cells of some rough strains, washed and
suspended in phosphate buffer, the presence of small con-
centrations of proflavine produced, in 10-14 days, levels of
resistance of about that of first-step mutants in up to 80 per
cent of the surviving cells (Table II). A somewhat lower
increase was achieved with the original smooth strain. A mini-
mal concentration of proflavine was needed; with increasing
concentrations, there was an increase in the rate of induction.

Multiple Mechanisms of Drug Resistance 153

Table II

Induction by proflavine 1 /ig./mt.. for 10 days in non-dividing cells of

Escherichia coli R„

Final

Initial

Control

Proflavine

Total percentage of sur-

vivors

—

0-6

01

Percentage of survivors

with resistance, jLtg./ml.

20

—

0-6

—

50

—

—

100

—

—

200

—

Percentage of survivors

with resistance, jug./ml.

20

004

0-005

80

50

0-002

37

100

0001

1-7

200

in the number of cells induced and in the level of resistance
achieved (Fig. 7). No induction occurred at 5° or 20°, or
when the cell density was low, or in the absence of tap water.
It was found that the presence of proflavine for only two days
was enough to give a substantial rise in the number of resistant
cells when they were suspended in the absence of proflavine
during the following 7 days (Fig. 8). Induction resulted not
only in a considerable rise in numbers of cells with pre-
existing levels of resistance, but also in the emergence of cells
of much higher resistance which previously were not present.
During the induction, there was initially a considerable fall
in the number of viable cells, which then increased. We
believe that cells become temporarily non-viable whilst under-
going induction. The induced cells retained their resistance
when subcultures were made in the absence of drug. They
were cross-resistant to chloramphenicol and aureomycin. In
these ways, and in the much higher levels of resistance
achieved, these cells differed from those in which increased
resistance (phenotypic adaptation) occurred during growth
when small amounts of proflavine were added.

154 M. J. Thornley, J. Sinai and J. Yudkin
A

4 6 8 lO 12

Time (days)
Fig. 7a

Multiple Mechanisms of Drug Resistance 155
C

Fig. 7. Induction to proflavine resistance by 1 • 5
{xg./ml. and 5 pig. /ml. proflavine in non-dividing
suspensions of Esch. coli Rg. Changes in numbers
of sensitive cells, and of cells resistant to different
concentrations of proflavine, at different times
during 14 days incubation at 37°C.

A — control, no proflavine in suspension

B — induced by 1 -5 jxg./ml. proflavine

C — induced by 5 {xg./ml. proflavine

1 — total viable count

2 — count of cells resistant to 20 (i,g. /ml. proflavine

3 — count of cells resistant to 50 [jLg./ml. proflavine

4 — count of cells resistant to 100 [xg./ml. proflavine

5 — count of cells resistant to 200 (jt.g./ml. proflavine

Time from beginning of incubation of the induc-
tion suspension.

156

M. J. Thornley, J. Sinai and J. Yudkin

B

2345678 56789 789

Time (days)
Fig. 8. Induction to proflavine resistance by 2 (i,g./ml. proflavine in a non-
dividing suspension of Esch. coli R^. Samples of the cells washed and resuspend-
ed in buffer and tap water without proflavine at different periods during 9 days
of incubation at 37°C. The resuspended cells incubated further at 37°C to
complete the 9 days incubation period.

A — suspension incubated 1 day in presence of proflavine and 8 days in absence
B — suspension incubated 2 days in presence of proflavine and 7 days in absence
C — suspension incubated 5 days in presence of proflavine and 4 days in absence
D — suspension incubated 7 days in presence of proflavine and 2 days in absence
1 — total viable count
2 — cells resistant to 20 [i.g./ml. proflavine
3 — cells resistant to 50 [jtg./ml. proflavine
4 — cells resistant to 100 (Jig./ml. proflavine

Time from beginning of incubation of the induction suspension.

In similar experiments, we were also able to induce to strep-
tomycin resistance but not to chloramphenicol resistance.

Demonstration and isolation of pre-existing mutants

The fluctuation test of Luria and Delbriick (1943) was
performed on the sensitive strain of Esch. coli. We found a
significantly higher variance in the number of colonies on
proflavine plates arising from several small cultures than in
the number arising from one larger culture. This suggests
that there is a spontaneous mutation in the culture to pro-
flavine resistance.

Multiple Mechanisms of Drug Resistance 157

The existence of these was confirmed by the repHca plating
technique of Lederberg and Lederberg (1952), modified
shghtly by us. We were able to isolate two first-step mutants,
and from one of these two further mutants with successively
increased resistance. If we take as a measure of resistance
the concentration of proflavine which allows one cell in a
thousand to grow, then the increase in resistance of the first-
step mutants was X8 and Xl8. The two further steps from
the latter had further increases of resistance of x3-2 and
Xl-8. Thus, the increase in resistance of the third-step
mutant, compared with the sensitive strain, was by a factor
of about 100.

From the same sensitive strain of Esch. coli, we have also
been able to isolate, in the same way, two first-step mutants
to chloramphenicol resistance. In addition, we have isolated
a proflavine-resistant mutant from the rough strain in which
we had carried out most of our induction experiments.

There was a general relationship between resistance to
proflavine and resistance to chloramphenicol in all of these
mutant strains. In only one instance, between the first- and
second-step proflavine mutant, was there an increase in
resistance to one drug — proflavine — with no change in
resistance to the other drug.

Transformation to proflavine resistance

We attempted transformation of sensitive cells to resist-
ance by deoxyribonucleic acid (DNA) from resistant cells, pre-
pared with modifications according to the methods of Boivin
(1947), McCarty and Avery (1946) and Myers and Spizizen
(1954).

The preparations all gave a positive Stumpf reaction,
indicating the presence of DNA. They were tested by dilut-
ing the preparation with broth and inoculating 0-1 ml. of
diluted 3-hour culture of the sensitive organism. After
24-48 hours incubation, the numbers of resistant and sensitive
cells were determined on plates with and without proflavine.

Preparations made by toluene treatment according to the

158 M. J. Thornley, J. Sinai and J. Yudkin

method of Boivin were not able to transform sensitive cells,
although various modifications of the conditions of prepara-
tion were made.

The method of McCarty and Avery consists of lysis with
deoxycholate in the presence of citrate, removal of protein
with chloroform and amyl alcohol and precipitation of the
transforming principle by absolute alcohol. Four rough
strains, derived from the original smooth strain, were tested
for competence to undergo transformation. In one of these,
there was a slight increase in the number of proflavine-
resistant cells. We thought that this might be due to the
existence of small numbers of a substrain consisting of com-
petent cells. We therefore devised a method of "double
replica plating", by which we might isolate them. A master
plate of one of the rough strains was replicated on plates con-
taining the transforming principle. After incubation, this in
turn was replicated on plates containing proflavine. Colonies
on this plate were traced back to corresponding areas of the
master plate which had been kept at 5°. From ten of these,
subcultures were made and transformation attempted. In
one of them, transformation was achieved as indicated by a
200-fold increase in the number of resistant cells in presence
of DNA from resistant cells, but no increase in presence of
DNA from sensitive cells. Two further experiments with the
same rough strain, and one with a different rough strain, gave
similar results.

The third method, that of Myers and Spizizen, was used by
these authors to produce a highly polymerized DNA, although
they did not carry out transformation experiments with it.
The method consists of lysing with sodium dodecyl sulphate
(duponol), removal of protein with sodium acetate and pre-
cipitation of DNA with acidified alcohol. Transformation
was tested on the original smooth strain of Esch. coli. The
DNA preparation alone did not cause transformation, the
protein precipitate caused transformation in about 0-3 per
cent of the cells, and both preparations together caused trans-
formation in ten times as many cells. No transformation

Multiple Mechanisms of Drug Resistance 159

occurred with similar preparations made from sensitive cells.
Transformation by preparations from resistant cells was lost
after treatment with deoxyribonuclease (DNAse). The level
of resistance reached in the transformed cells was about that
of a first-step mutant, whether the DNA was made from a
first-step or a second-step mutant.

Table III

Transformation to proflavine resistance in Escherichia coli 36

Culture

Cell fraction

Protein or
substitute

CellsjlQ-^
20 yig.lml.

resistant to
50 yig.lml.

1

10

2

—

PrR

2-5x10* (s)

5xl02(s)

3

—

PrS

20

4

—

duponol

10

5

—

serum

5

6

—

deoxycholate

6

7

TPR

—

5

8

TPR

PrR

3X105

5X10*

9

TPR

duponol

2-6x105

2X10*

10

TPR

serum

4

11

TPR

deoxycholate

5

12

TPS

6

13

TPS

PrS

23

14

TPS

duponol

15

15

TPS

serum

12

16

TPS

deoxycholate

7

TPR, Pr

R— DNA and

protein fractions

from resistant mutant.

TPS, Pr

S— DNA and

protein fractions

from sensitive Esch. coli 36.

(s) small

colonies.

We studied the role of the protein fraction, which con-
tained some DNA, by substituting other substances for it and
adding to the DNA preparation. We found that its place could
be taken by duponol, but not by deoxycholate or by rabbit
serum (Table III). The protein fraction was found to carry
duponol with it, so that its activation of DNA presumably
depends on its duponol content. Duponol was shown to

160 M. J. Thornley, J. Sinai and J. Yudkin

inhibit DNAse; it seems therefore that it might act by pre-
venting the destruction of transforming DNA by the enzyme
of the recipient cells.

Conclusion

We have demonstrated, in the one system Esch. coli-pro-
flavine, the origin of resistance through: (1) mutation and
selection, (2) phenotypic adaptation to a low level of resist-
ance at some stages in the division cycle, (3) Lamarckian
inheritance and (4) transformation. We have not been able
to exclude the possibility that clonal variation also occurs.
We believe that in natural situations, the environments and
micro-environments to which the cells are exposed are so
complex that it is profitless to attempt an assessment of the
relative importance of these mechanisms in determining the
emergence of a resistant population. Nevertheless, in the
simpler conditions existing in laboratory experiments, we can
readily visualize the ways in which such a population may
emerge by the simultaneous or sequential occurrence of these
different modes of origin. We believe also that it is possible,
through an extension of the unitary theory of enzyme induc-
tion (Cohn and Monod, 1953; Pollock, 1953), to bring together
these various mechanisms into a unitary theory of the origin
of drug resistance.

REFERENCES

Akiba, T. (1954). In Origins of Resistance to Toxic Agents, p. 82.

New York: Academic Press.
Baskett, a. C. (1952). Proc. roy. Soc. B, 139, 251.
BoiviN, A. (1947). Cold Spr. Harb. Symp. quant. Biol, 12, 7.
Cohn, M., and Monod, J. (1953). Symp. Soc. gen. Microbiol., 3, 182.
Lederberg, J., and Lederberg, E. M. (1952). J. Bad., 63, 399.
LuRiA, S. E., and Delbruck, M. (1943). Genetics, 28, 491.
McCarty, M., and Avery, O. T. (1946). J. exp. Med., 83, 89, 97.
Myers, V. L., and Spizizen, J. (1954). J. biol. Chem., 210, 876.
Pollock, M. R. (1953). Symp. Soc. gen. Microbiol, 3, 150.
SzYBALSKi, W. (1955). Antibiotics Annual 1954-1955, p. 576, New

York: Med. Encycl. Inc.

Discussion 161

DISCUSSION

Dean: We have frequently said that permanence of training is never
absolute. Our general thesis is that the longer the strain is trained the
more stable does the adaptation become ; you can eventually reverse it if
you subculture it for a sulFiciently long time in a drug-free medium, but
it may take a very long time (Hinshelwood, C. (1953), J. chem. Soc,
p. 1947; Dean, A. C. R., and Hinshelwood, C. (1954), Proc. roy. Soc.,.B,
142, 45).

With regard to the accelerated adaptation experiments, I have quoted
cases where I got the concentration up to 43 mg./l. without any change
in the pH of the medium ; the pH was 7 at the beginning and 7 at the end.
I have also done a set where the concentration was gradually increased
up to 63 mg./l., and there pH began at 7 and ended at 6-8. But if a
control was put on at pH 6-8 it did not grow. Furthermore, I think
nobody w^ould question that one would get variations in adaptability
during the gro%\i:h cycle, at least with proflavine. In most of the training
experiments of Davies, Hinshelwood and Pryce, training was done by
subculture in the logarithmic phase.

Yudkin : The point I was making about reversion was that there seemed
to us, at any rate, to be no relationship between the number of times a
culture has been trained and the degree of reversion ; and that very large
numbers of subcultures in the absence of the drug may produce degrees
of reversion no different from those produced by a few subcultures. It
would not be useful to discuss the pH experiment; our findings are just
different. I don't see how the pH stayed constant because in fact there
is a continuous fall in pH, as I have shown. You have a glucose medium
and the culture is growing in logarithmic phase, and I find it difficult
to imagine that the pH stayed constant while the experiment went on.
We used exactly the same medium as you. I should emphasize that
what we are discussing has two aspects : one is the cultural phase, and
the other is the cell-division phase.

Walker: If proflavine is added to a culture at the beginning, is it still
proflavine after two days ?

Dean: It is oxidized in the presence of light and air. One has to
protect against that.

Rose: With these antibacterial agents that have amino groups present,
one imagines that the amino groups could easily be replaced by hydroxy!
through the action of deaminases which would give substances that are
only very feebly antibacterial.

Yudkin: In the induction experiments where they were left in con-
tact with proflavine for several days, after taking them out of proflavine
or its degradation product, as the case may be, there was then a continu-
ing large increase in the number of resistant cells.

Rose: They may be resistant now because they have the ability to
remove one or both amino groups.

Yudkin: I think that may be the basis of resistance.

Slonimski: It has been possible to show with euflavine (2:8 diamino-
iV-methylacridine) that the compound which can be extracted from the

DRUG RES, 6

162 Discussion

yeast cell is the same as the one used to induce mutation. To do this one
extracts with HCl-ethanol, then one gets rid of the extracting medium.
There is no change in the spectrum (measured at different pH's) and
there is no appreciable change in the biological activity. One must,
however, have taken special precautions to ensure preservation of eufla-
vine. The work must be done in the dark, at constant pH, and in a well
defined chemical medium.

Walker: Is the same chemical species present over the pH range in
which you carried out your first experiment, Prof. Yudkin ?

Yudkin: No, probably not. We wanted to know whether the change
which allows bacteria to grow in high concentrations of proflavine is a
change in the cells or a change in the medium. We think we have estab-
lished that it is a change in the medium, and that it is simply a matter
ofpH.

Slonimski: Albert and his collaborators have shown that 12 years ago;
but this is quite an interesting phenomenon in itself because firstly, you
have a competitive ratio of 1 hydrogen ion per 500 to 1000 ions of
acridine, which is quite surprising; and secondly, this is not always
a question of dissociation of acridine. Marcovich (1953, Ann. Inst.
Pasteur, 85, 199) has shown with euflavine (which has a pKa of more
than 12, while proflavine has 9) that the antimutagenic effect of H+ ions
had a pKa around 5 • 5, i.e. it cannot be explained by the dissociation of
the drug.

Davis: If one is concerned with determining the mechanism by which
a resistant strain becomes sensitive on growth in the absence of the
drug, I am not sure I understand the rationale underlying the extensive
experiments of Yudkin, and of Dean and Hinshelwood, designed to
reveal how many culture passages are necessary to bring about this
return of sensitivity. It is known that in mixtures of resistant mutants
and the sensitive parental strain one can find differences in relative rate
of growth that lead to selection — in favour respectively of the resistant
strain in the presence of the drug and the sensitive strain in the absence
of the drug. Hence, whether a resistant strain becomes sensitive in the
absence of the drug after 5 passages — or only after 500 — this observa-
tion per se does not help us to decide whether the change was based on
mutation and selection or on a physiological adaptation.

Yudkin: Speaking solely from our own point of view, what we were
trying to do was to see whether we got the same phenomenon as Hin-
shelwood. We wanted to know what would happen in the Hinshelwood
conditions, but measuring resistance in the more orthodox way of sur-
vival curves. We concluded that you don't seem to get anywhere with
that sort of experimentation.

Dean: In their experiments, Davies, Hinshelwood and Pryce (1945,
Trans. Faraday Soc, 41, 163) were investigating the adaptation of
Bact. laciis aerogenes to proflavine. They trained organisms at certain
concentrations, and then did lag-concentration curves with these strains,
and found that the resistance was continuously graded to conform to the
concentration at which training has been carried out. Then the interest-
ing question arose : Is this resistance stable ? The conclusion is that the

Discussion 163

strain reverts eventually, but Hinshelwood has often stated that the
reversion can be very unpredictable.

Yudkin : What we said was that it makes no difference if you had 12 or 80
subcultures, for example. Indeed we have got a culture which we used
in our transformation experiments, which was trained to a high level of
resistance in that way, and it still is resistant 5 years later.

Dean: Of course there may be quite a variation in technique.

Hughes: In your culture, which is not multiplying in the presence of
proflavine, how satisfied are you that there is not a turnover of cells ?

Yudkin: When we did these experiments, we were rather impressed
by the work of Szybalski and Akiba. We took even more precautions
than they did, but until we go back now and look at the same sort of
things that Szybalski looked at, we cannot be absolutely certain.

Fredericq: You get transformation with DNA extracted from a resis-
tant mutant. Did you try to transfer the resistance conferred by pro-
longed induction.

Yudkin: No, that is one of the things we shall have to do.

Fulton: How difficult is it to get DNA out of bacteria? Can you hope
for purity of the specimen without prolonged chemical operations ?

Yudkin: We did not attempt to make entirely pure preparations. We
made preparations which had DNA activity. It involved lysing, extrac-
tion, precipitation of protein, then taking up in saline. We were content
at this stage, to show (a) that, prepared in this way, the extracts are
active if they come from resistant organisms — whether from a mutant
isolated by replica plating, or from a trained culture — and not w^hen
prepared in exactly the same way from a sensitive culture ; and (b) that
the activity is lost on treatment with DNAse.

Pollock: You said that there was a cycle of 20 minutes in changes in
proflavine resistance, 30 minutes in the actual division time; yet later
on you seemed to imply that there was a relationship between the two.
Could you clarify that?

Yudkin: I don't know the answer. This is an odd situation. It was
Dr. Hotchkiss who started this. He got changes in transformability
which were longer than the division cycles. We get changes in resistance
levels which are shorter. There are elaborate ways in which you can
possibly explain this by imagining that the cooling process interrupts at
more than one level and so on. But I feel there is no explanation at the
moment. I still feel that it is likely that these changes in cycles of
resistance are in some way tied up with cell division.

Hotchkiss: We have the same problem. The two factors we tried to
correlate were the cyclical transformability, i.e. the percentage of cells
which respond to DNA; and the number of cells undergoing division,
exemplified by colony formation. The lack of correspondence of the
cycles might be because cell division is not the same as nuclear division,
so that the time at which nuclear processes are going on may well precede
the time at which cells become separate enough to form single colonies.
Furthermore, when we are measuring cell division, we are measuring a
property of all of the cells; what 1000 cells do in 5 minutes, and in the
succeeding 5 minutes, and so on. We found steps of division such as

164 Discussion

Yudkin has shown. But when we are measuring transformabiHty, we
are measuring a property of only a few of the cells, and these few could
be gradually deviating from the general mode of behaviour of the popu-
lation and so be gradually acquiring a longer and longer true cell division
time, which would not show up except as a slight damping effect on the
overall properties of the divisions of the average cells.

Pollock: If this is a regular change in drug resistance I would have
thought that Yudkin' s evidence would suggest that one should look for
another cause and not try to fit it in with the cell cycle. Might it not for
instance be due to the rate of production of an enzyme which is formed
at a very low rate, so that you really might have a quantal effect due to
only one molecule being formed at a given time ? The time it takes for a
cell to form one molecule would not necessarily be related to cell division,
but it would produce a cyclic effect if that molecule was necessary for the
development of drug resistance.

Yudkin: Yes, I find it perfectly possible. The important thing here
is that there are cycles of adaptability to resistance. If one accepts that
there are these very considerable cycles of resistance, and if one accepts
that an ordinary culture will have cells in all stages of these cycles, then
clearly if one tests such a culture one will find them having what you call
resistance, but which I would suggest is adaptability to resistance, over
a whole range. It seems to us that one of the explanations, if not the
only explanation, of the range of resistance in a sensitive culture, ex-
pressed in survival curves, or distribution curves, is the fact that the cells
show the whole range of cycles of adaptability.

PHYSIOLOGICAL (PHENOTYPIG) MEGHANISMS
RESPONSIBLE FOR DRUG RESISTANCE

Bernard D. Davis

Department of Pharmacology , New York University College oj Medicine

For the most part this symposium has been concerned with
the genetic aspect of the development of drug resistance: i.e.
the mechanism or mechanisms by which a hereditary change
in resistance to an inhibitor arises in a cell and becomes trans-
mitted to succeeding generations. But the problem of drug
resistance has another side that also merits attention: the
nature of the physiological changes, regardless of origin, that
are directly responsible for the decreased susceptibility of the
cell to inhibition by the drug. In other words, for present
purposes I am interested in changes in those units in the cell
that do its everyday work, such as its enzymes and membranes,
rather than in changes in the more intellectual units which
govern, in interaction with the environment, the pattern of
formation of these working units. Furthermore, I should
like to emphasize the distinctness of the two problems. For
regardless of the origin of drug resistance, an analysis of its
phenotypic mechanism, at a biochemical level, would in-
evitably lead us to measure such parameters as the level of
various metabolites and enzymes, the ability of the cell to
concentrate or bind various compounds, and the affinity of
enzymes and transport systems for the drug.

As the physiological problem has so far been much less
extensively explored than the genetic one, it may offer a
greater challenge at the present time. The literature on the
subject has recently been comprehensively reviewed (Abraham
1953; Schnitzer and Grunberg, 1957), and so the present paper
will be restricted to selected aspects of the problem.

165

166 Bernard D. Davis

Possible physiological mechanisms of drug resistance

For purposes of orientation it might be well to start by list-
ing the theoretically possible changes in the function of a cell
that could be expected to increase its resistance to an inhibitor.
These would include the following (Davis and Maas, 1952);
and there might well be others that we are missing.

(1) Alternative metabolic pathway bypassing the inhibited

reaction.

(2) Increased concentration of a metabolite that antagon-

izes the inhibitor.

(3) Increased concentration of the enzyme that the drug

inhibits.

(4) Decreased requirement for a product of the inhibited

metabolic system.

(5) Increased destruction of the inhibitor (or decreased

conversion of an administered compound into a more
active inhibitor).

(6) Formation of an altered enzyme, with decreased affinity

for the inhibitor or with increased relative affinity
for the substrate compared with a competitive inhibitor.

(7) Decreased permeability of the cell (or of subcellular

units) to the inhibitor. (As a special case pointed out by
Mr. Anton Kris, a medical student at Harvard, this could
include increased affinity of a permeation system for
other compounds which interfered with transport of the
inhibitor.)

I should like to comment briefly on these mechanisms, first
pointing out that there is no reason to anticipate that any
one mechanism will ultimately be found to tell the whole
story. If mutations can give rise to an increment of resistance
through various mechanisms, the inhibitor would select them
aU; and there is little doubt that nature would avail itself of
all the means at its disposal to fill such an ecological niche.

The first mechanism, involving an alternative metabolic
path, seems to have long been a speculative favourite, especi-
ally since drug resistance has been shown in many cases to

Physiological Mechanisms of Resistance 167

arise by mutation, and since certain other mutations were
shown to have an all-or-none effect on the formation of a given
enzyme. However, it has become abundantly clear that
the biochemical consequence of mutations is by no means
restricted to such an all-or-none effect, and so this mechanism
has lost much of the basis for its appeal. Furthermore, there
are definite reasons to doubt whether this mechanism occurs
at all. For in a biosynthetic sequence proceeding from com-
pound A to C via B, the appearance of a new route bypassing
B would surely have to involve the insertion of more than one
new enzyme in the sequence. Of course, it is possible that one
or more of these enzymes, though new in the sequence,
might already be present in the cell for other purposes. The
theoretical objection to this mechanism is therefore not
absolute ; but it should be added that the mechanism has not
been clearly demonstrated in any case with which the present
author is familiar.

Mechanism 2, increased concentration of a competitive
metabolite, has also had widespread appeal ever since sul-
phonamides were shown to act by competing with ^-amino-
benzoate (PAB). However, in the few cases where increased
formation of PAB has been demonstrated the effect has been
too slight to explain more than a trivial increase in resistance.
Furthermore, despite much effort none of the antibiotics have
been shown to act by competing with a metabolic intermediate.

The next two mechanisms, increased concentration of the
enzyme or decreased requirement for its product, could also
hardly be expected to produce more than a modest increase in
resistance.

Mechanism 5, destruction of the inhibitor, has been shown
to be important in some penicillin-resistant strains, which
form and excrete the enzyme penicillinase. The behaviour of
such strains is dealt with in detail elsewhere in this symposium
by Dr. Pollock (p. 78) and by Dr. Barber (p. 262).

And now we come to the last two mechanisms, which
involve principles that have become established in micro-
biology only in the past few years : the ability of mutations to

168 Bernard D. Davis

lead to the formation of qualitatively altered enzymes, and
the presence in bacteria of mutable stereospecific permeation
systems. Since these developments are so recent it may be
profitable here to consider their experimental basis as well as
their application, as yet limited, to the problem of drug
resistance.

Qualitatively altered enzymes

While evolutionary considerations have long made it
evident that mutations must be able to affect the nature as
well as the amount of the various enzymes (and other proteins)
formed by an organism, this phenomenon was first demon-
strated less than ten years ago by Pauling and co-workers
(1949), and in mammals rather than microbes. They showed
that patients with sickle-cell anaemia, a hereditary disease,
formed haemoglobin with different electrophoretic mobility
from that of normal human haemoglobin. Since then a number
of different kinds of human haemoglobin have been discovered.

Shortly thereafter a mutational change in the nature of a
protein was shown for an enzyme, and in micro-organisms, by
Maas and Davis (1952) working with pantothenate synthase
in Escherichia coli, and by Horowitz and Fling (1953) working
with tyrosinase in Neurospora crassa. In each case the mutant
studied was a temperature-sensitive one: i.e. it lacked the
enzyme in question when grown at ordinary temperatures,
but formed it when grown at low temperatures. When the
enzyme was extracted from the cells grown at low temperature,
it was found to differ strikingly from the corresponding enzyme
of the wild-type strain in one respect: it was irreversibly
denatured at moderate temperatures which did not denature
the wild-type enzyme. When tested at low temperature the
enzymes were indistinguishable in other respects : the reaction
catalysed, cofactors required, pH optimum and Michaelis
constant. Evidence for the thermal instability of the mutant
pantothenate synthase is presented in Fig. 1 (from Maas and
Davis, 1952), in which the triangles represent for the wild-
type extract, and the circles for mutant (99-lt) extract, the

Physiological Mechanisms of Resistance 169

residual enzyme activity (tested at 15°) after incubation of the
extract at the temperatures and for the times indicated. A

2.0

60

MINUTES

120

Fig. 1. Effect of temperature on the stability of the pantothenate-
synthesizing enzyme in extracts of mutant 99-1 < and of the wild type.
Acetone-powder extracts of the two strains were incubated at the
temperatures noted. At the times indicated samples were removed,
cooled to 15° and tested for enzymic activity. In these tests, each tube
received extract from 40 mg. of acetone powder of the mutant or
extract from 3 mg. of acetone powder of the wild type. In addition,
the testing tubes contained in mM concentrations : (3-alanine 20, potas-
sium pantoate 20, KgATP 10, KCl 100, MgS04 10> tris-(hydroxy-
methyl)-aminomethane (Tris) buffer pH 8 • 5 100 ; total volume 1 • ml.
After incubation for one hour samples were assayed for pantothenate
as described in the text. 100 per cent residual activity equals 86 m[jL
moles of pantothenate per ml. per hour for 99-lt, 178 mjji. moles of
pantothenate per ml. per hour for the wild type. O-Q- = mutant
99-lt ; A- A- = wild type. (Reproduced by permission of University
of Chicago Press.)

large difference in thermal stability is seen. Furthermore, the
fact that the difference resides in the enzymes themselves,
rather than in their environments in the respective extracts,

170 Bernard D. Davis

was shown by the results of another experiment in which wild-
type extract, mutant extract, and a mixture of the two were
incubated at an intermediate temperature, i.e. one that caused
destruction of essentially all of the enzyme in the mutant
extract and none in the wild-type extract. The mixture lost a
fraction of its activity corresponding to the mutant component
of the total.

It is evident that mutations can result in subtle alterations
in the nature of the enzyme performing a given reaction.
Those resulting in increased thermal sensitivity appear to be
frequent, probably because they are easy to select. Mutants
with temperature-sensitive blocks in a number of biosynthetic
reactions have been detected in our laboratory by starting
with an auxotroph which lacks a given reaction at all tempera-
tures, selecting reversions at 15°, and then finding which of
these fail to grow at 37° (^laas, unpublished).

Another kind of qualitative alteration, which completely
destroys the activity of an enzyme, has been observed by
Suskind and co-workers (Suskind, Yanofsky and Bonner,
1955; Yanofsky, 1956). They found that certain mutants
which had lost the power to form tryptophan synthase con-
tinued to form a protein that reacted serologically with anti-
body to tryptophan synthase. Other mutants blocked in the
same biosynthetic reaction failed to form the serologically
crossreacting protein. It therefore appears that the former
mutants form an "inactive enzyme" corresponding to the
altered gene, while the latter mutants are even more drastic-
ally altered.

It seems reasonable to expect that mutations can lead to all
sorts of qualitative changes in enzymes, temperature sensitiv-
ity and loss of catalytic activity being recognized first because
of the ease of their selection. These findings encourage the
search for other qualitative changes that would lead to drug
resistance.

Indirect evidence for such a phenomenon has been provided
by a study (Davis and INIaas, 1952) of analogues of two struc-
turally related but metabolically distinct bacterial vitamins.

Physiological Mechanisms of Resistanxe 171

p-aminobenzoic acid (PAB) and j9-hydroxybenzoic acid (POB)
(Fig. 2). As is well known, PAB utilization is competitively
inhibited by analogues in which the carboxyl is replaced by a
substituted or unsubstituted sulphonamide group. Similar
competition between POB and its sulphonamide analogues
was observed. Furthermore, p-nitrobenzoic acid (PNB), an

•Fig. 2. Schematic representation of competition by analogues

of PAB and POB. X = 2-thiazolylamino. (Reproduced by

permission of University of Chicago Press.)

analogue involving replacement at the other end of the molecule
(amino or hydroxyl), competes with both vitamins. It was
observed that mutants resistant to PNB competition with
either vitamin were not altered in respect to its competition
with the other vitamin. Furthermore, with either vitamin
there was no crossresistance between PNB competition and
sulphonamide competition. Without presenting in detail the
arguments involved or the subsidiary evidence (cf. Davis and
Maas, 1952), I should like to summarize by stating that this

172 Bernard D. Davis

concatenation of facts appeared to exclude all but one of the
seven mechanisms listed above. The conclusion, reached thus
by exclusion, was that the mutants were resistant by virtue of
producing an enzyme with decreased affinity for the inhibitor
relative to the competitive metabolite.

The ratiocination involved in this study provided the author
with a good deal of entertainment, much like that involved in
trying to solve a detective story before the last chapter. But
as with such a story, one cannot be sure of the relevance of
the conclusion to real life. It would be highly desirable to
demonstrate directly, with extracted enzymes, the inferred
change in affinity. Unfortunately, though the PAB/sulphon-
amide interaction is the prototype of competitive inhibition,
it has been studied only with intact cells; the biosynthetic
reaction of which PAB is substrate is still unknown. The
same is true of POB. And while growth-inhibiting analogues
are known for a variety of other metabolites, including amino
acids and vitamins, the biosynthetic reactions in which these
metabolites participate are also by and large not enzymically
defined.

Alterations in an extracted enzyme, nitro reductase, have
been reported in bacteria resistant to chlortetracycline
(aureomycin) (Saz, Brownell and Slie, 1956). It is not certain,
however, that inhibition of this enzyme is the basis of action
of the drug.

Specific permeation systems

Shortly after EhrHch developed the effective chemotherapy
of trypanosomiasis with arsenicals he encountered the
phenomenon of drug resistance. Furthermore, he showed
that resistant strains took up less of the drug than sensitive
ones, and concluded that resistance might be based on
decreased permeability to the drug or on a decreased number
of receptors that can bind the drug. However, whether one
is measuring arsenic or modern radioactive antibiotics, a
decreased uptake of drug by resistant cells does not alone
distinguish these two mechanisms. Furthermore, our thinking

Physiological Mechanisms of Resistance 173

about cell permeability was long dominated by a physico-
chemical approach to the kinetics and thermodynamics of
a passive membrane, with the cell viewed as a sort of cello-
phane bag filled with enzymes. It is therefore hardly surpris-
ing that studies based on such a naive model failed to lead to
much enlightenment. Indeed, it would be difficult to explain
with such a model how quantitative decreases in permeability
could lead to corresponding decreases in uptake. For with a
non-metabolizable drug, decreased permeability should lead
to a decreased rate of approach to equilibrium, but not to a
change in the distribution at equilibrium.

In the past few years, however, our picture of the perme-
ability properties of bacteria has altered drastically. The
cellophane bag now possesses a variety of permeation systems,
each stereospecific for a structurally related group of sub-
strates; and the number of units of each kind per cell not
only affects the rate of equilibration between intracellular and
extracellular substrate, but also affects the value of the ratio
reached at equilibrium.

This development has arisen not only from direct studies of
the intracellular concentration of various substances, but also
from studies of the phenomenon of "crypticity" — i.e. the
fact that certain enzymic activities can be demonstrated only
after disruption of the cells. It has long been suspected that
a permeability barrier prevented the added substrate from
reaching the enzyme in such cells ; but it could also be argued
that the enzyme might be present in the cell in an inactive or
latent form which became activated by the process of extrac-
tion. The question could be resolved if it could be proved that
the enzyme was active in the intact cell.

This demonstration has now been accomplished in two cases
by the use of auxotrophic mutants to demonstrate that the
enzyme in question was essential for biosynthetic purposes,
and hence must be present in active form. Thus it has been
established by nutritional, isotopic, and enzymic methods that
5-dehydroquinic acid (DHQ), 5-dehydrosliikimic acid (DHS),
and shikimic acid (SA) are successive intermediates in the

174

Bernard D. Davis

biosynthesis of a group of aromatic metabolites (Davis,
1954-55).

HO COOH COOH

COOH

/ 1 X

O^ljOH

O^OH

5-Dehydroquinic 5-Dehyroshikimic
acid (DHQ) acid (DHS)

■A-

hOqIjOH

Shikimic
acid

Tyrosine
-> Phenylalanine
Tryptophan
p-Aminobenzoic

acid
p-Hydroxybenzoic
acid
-> 6th factor

Yet mutants blocked before DHQ, though they contain the
normal amount of the enzyme converting DHQ to DHS, are
able to grow on DHS but not on DHQ. However, a secondary
mutation of these strains, selected for by exposing large
populations to DHQ, permits them to grow on DHQ (Davis
and Weiss, 1953). Since the enzyme is there all the time, and
is essential for biosynthesis, it is difficult to escape the con-
clusion that the secondary mutation in question has created a
mechanism for the permeation of DHQ. The specificity of such
permeation systems is shown by the fact that some mutations
promote the penetration of DHQ and others similarly affect
its close structural relative DHS ; but neither mutation affects
the other compounds (Davis and Weiss, 1953).

A permeability barrier has been similarly demonstrated for
citrate, which has long been known to be inert for many
organisms that contain the enzymes for its utilization. Much
as in the case of DHQ and DHS it has been shown, with
mutants of Esch, coli and Aerobacter aerogenes blocked before
the compound, that citrate is an essential intermediate in
glutamate formation (Gilvarg and Davis, 1956). Hence the
enzymes between citrate and glutamate, which are readily
demonstrated in extracts of these mutants, must be present
in active form, and the inability of the organisms to utilize
citrate as a replacement for glutamate must be due to a
permeability barrier.

An important development in the study of bacterial

Physiological Mechanisms of Resistance 175

permeation systems has been the demonstration that certain
of these systems are adaptive, i.e. they appear only when the
cells are grown in the presence of the substrate or a related
inducer. This adaptability has been demonstrated for the
citrate system in Pseudomonas (Barrett, Larson and Kallio,
1953; Kogut and Podoski, 1953) and Aerobacter (Green,
1956), and for the p-galactoside transport system in Esch. coli
(Davis, 1956; Monod, 1956). The systems resemble adaptive
(inducible) enzymes in two further respects: the adaptation
requires conditions that permit protein synthesis; and it is
blocked by the presence of glucose or other carbohydrates
which are known, in contrast to lactate or succinate, to block
formation of many adaptive enzymes (Green, 1956; Davis,
1956; Monod, 1956; Rickenberg et ah, 1956).

The kinetics of the formation and action of an adaptive
permeation system have been elegantly analysed by Cohen,
Monod, and co-workers (Monod, 1956; Rickenberg et ah, 1956),
employing p-galactosides. This system has the advantage
that a substance is available, p-thiomethyl galactoside
(TMG), which induces and is transported by the permeation
system but is not metabolized; hence it is possible to study the
ability of the system not only to transport but also to concen-
trate substrate. It would be inappropriate to review here all
this work; but two further properties of the permeation
systems should be noted. They resemble enzymes in their
kinetics, which can be analysed in terms of a Michaelis
constant, and they exhibit typical competition between
structural analogues. Finally, the same group have also
studied in detail analogous systems for concentrating various
amino acids (Cohen and Rickenberg, 1956). These systems
resemble the one for p-galactosides except that they appear to
be constitutive rather than adaptive.*

* Monod has referred to stereospecific permeation systems as permeases
(Rickenberg et al., 1956), a term which seems to imply an enzymic nature. It
seems preferable to avoid such a mechanistic term at this time, since future
work will have to determine whether or not the action of these systems involves
enzymic conversion of the substrate to another compound in the course of
transport.

176 ' Bernard D. Davis

And now, what can we say about the relation of this work
to drug resistance?

It is clear that a variety of specific permeation systems
exist in bacteria ; only a few have been sought, and they have
been readily found. It therefore seems unlikely that non-
specific pores in the membrane are important in bacteria
except for perhaps the smallest molecules and possibly
lipophilic substances. Furthermore, permeation systems, like
enzymes, can be gained or lost by mutation, as has been shown
for all the systems noted above. Finally, the number of
permeation units per cell varies under different conditions, as
could be shown by measuring the ratio of internal to external
TMG. Applying these facts to the problem of drug resistance,
it is easy to imagine that mutations, as well as physiological
adaptations, could alter the number of units for transporting a
drug, and hence could establish various characteristic ratios
of internal to external free drug. Thus, though the passive
model of cell permeability was quite unsatisfactory as a basis
for explaining various degrees of drug resistance, "perme-
ability" in the active sense described here makes possible a
theoretically satisfactory solution. However, these concepts
are so new that they have only begun to be applied to the
problem of drug resistance. For example, it has been shown
that some penicillin-resistant bacteria take up less of the
inhibitor than sensitive strains while others do not. Perhaps
Dr. Eagle, who has done much of this work, will discuss it
here.

Some work of my colleagues (Maas and Frosch, unpublished)
provides rather direct evidence for decreased permeability as a
mechanism of resistance to the inhibitory action of D-serine
on Esch. coli. This inhibitor has the advantage that its mode
of action has been established as competitive inhibition of a
biosynthetic enzyme, pantothenate synthase, whose activity
can readily be measured in intact cells and in extracts (Maas
and Frosch, unpublished; Maas and Davis, 1950).

The effect of D-serine is shown in Table I, in which wild-
type Esch. coli is compared with a D-serine-resistant mutant

Physiological Mechanisms of Resistance 177

with respect to the ability of a resting cell suspension to form
pantothenate from its precursors, p-alanine and pantoic acid.
With intact cells the mutant showed less inhibition of this
reaction by D-serine than did the wild type. However, after
treatment with toluene to destroy permeability barriers the
two cell suspensions were equally susceptible to the inhibition.
Furthermore, in growing cultures, studies with radioactive

Table I

Inhibition by d-serine of pantothenate production by resting cell
SUSPENSIONS OF Esch. coU

D-serine
lig.lml.

Wild type

Resistant mutant

Pantothenate

produced

ixg.lml.

Percentage
inhibition

Pantothenate

produced

lig.lml.

Percentage
inhibition

None
250
500

61
1-2
0-9

Intad

80
85

t cells

1-4
1-3

0-8

7
48

None
1000

21
10

Toluene-tr
52

eated cells

3-6
1-7

53

D-serine showed considerably less uptake by the mutant
strain than by the wild type (Table II). Finally, inhibition
of pantothenate synthesis required about 5 (xg./ml. with
growing sensitive cells, 25 [xg./ml. with growing resistant
cells, and 1000 (jig./ml. with toluenized cells. These results
provide strong evidence that a decrease in permeability to
D-serine is responsible for resistance in the mutant studied.

Decreased permeability also appears to be responsible for
chloramphenicol resistance in Pseudomonas, since intact
resistant cells are less susceptible than the sensitive parental

178

Bernard D. Davis

cells to inhibition of oxidation of a variety of substrates,
whereas disrupted cells of the two strains fail to show this
difference (Kushner, 1955).

In closing, I should like to emphasize several consequences
of these recent developments for the study of antimicrobial
action and of drug resistance. First, compounds that reverse
an inhibition are not necessarily metabolites ; they can also be
analogues that interfere with penetration of the inhibitor.
This concept finally furnishes a plausible explanation, for
example, for the previously puzzling observation (Davis and

Table II

Uptake of i^C-d-sertne during growth

Generation

Total counts jmin. in bacteria

Wild type Mutant

1
2
3

915 136
2160 450
5000 775

Final medium

2690 7880

Maas, 1949) that D-serine inhibition, though clearly located at
pantothenate synthesis, could also be reversed by a variety of
metabolically unrelated amino acids. Secondly, alterations in
permeability are not necessarily restricted to the cell mem-
brane, since subcellular particles are also present in bacteria,
and might be the site of a permeability barrier. Hence, when
studies of drug concentration in resistant cells fail to show a
marked decrease in permeation into the cell as a whole, the
possibility of a change in a permeability barrier has still not
been excluded. Finally, in a diploid bacterium, streptomycin
resistance has been shown to be genetically recessive to sensi-
tivity (Lederberg, 1951). This observation would be difficult
to understand if resistance were due to a qualitative or quanti-

Physiological Mechanisms of Resistance 179

tative change in an intracellular enzyme, and if each of the
paired allelic genes exhibited the expected autonomous control
over the corresponding protein. However, recessive resistance
would be easily explained if the sensitive allele were providing
the cell with normal permeation units.

REFERENCES

Abraham, E. P. (1933). In Adaptation in Micro-organisms, Eds.,

Davies, R., and Gale, E. F. Cambridge University Press.
Barrett, J. T., Larson, A. D., and Kallio, R. E. (1953). J. Bad., 65,

187.
Cohen, G. N., and Ricicenberg, H. V. (1956). Ann. Inst. Pasteur, 91,

829.
Davis, B. D. (1954-55). Harvey Lect., 50, 230.
Davis, B. D. (1956). /n Enzymes: Units of Biological Structure and

Function, p. 509. Ed., Gaebler, O. H. New York: Academic Press.
Davis, B. D., and Maas, W. K. (1949). J. Amer. chem. Soc, 71, 1865.
Davis, B. D., and Maas, W. K. (1952). Proc. nat. Acad. Sci., Wash., 38,

775.
Davis, B. D., and Weiss, U. (1953). Arch. exp. Path. Pharmak., 22, 1.
GiLVARG, C, and Davis, B. D. (1956). J. hiol. Chem., 222, 307.
Green, H., cited in Davis (1956).

Horowitz, N. H., and Fling, M. (1953). Genetics, 38, 360.
KoGUT, M., and Podoski, E. P. (1953). Biochem. J., 55, 800.
Kushner, D. (1955). Arch. Biochem. Biophys., 58, 347.
Lederberg, J. (1951). J. Bact., 61, 549.
Maas, W. K., and Davis, B. D. (1950). J. Bact., 60, 733.
Maas, W. K., and Davis, B. D. (1952). Proc. nat. Acad. Sci., Wash., 38,

785.
MoNOD, J. (1956). In Enzymes: Units of Biological Structure and

Function, p. 7. Ed. Gaebler, O. H. New York: Academic Press.
Pauling, L., Itano, H. A., Singer, S. J., and Wells, I. C. (1949).

Science, 110, 543.

RiCKENBERG, H. V., COHEN, G. N., BUTTIN, G., and MONOD, J. (1956).

Ann. Inst. Pasteur, 91, 829.
Saz, a. K., Brownell, L. W., and Slie, R. B. (1956). J. Bact., 71, 421.
ScHNiTZER, R. J., and Grunberg, E. (1957). Drug Resistance. New

York : Academic Press.
SusKiND, S. R., Yanofsky, C, and Bonner, D. M. (1955). Proc. nat.

Acad. Sci., Wash., 41, 577.
Yanofsky, C. (1956). In Enzymes: Units of Biological Structure and

Function, p. 147. Ed. Gaebler, O. H. New York: Academic Press.

180 Discussion

DISCUSSION

Pontecorvo: I suggest we add another group of organisms, namely the
filamentous fungi. You mentioned dominance and recessiveness. In
Aspergillus we can use classical techniques to identify and locate genes.
Roper has an acraflavine-resistant mutant at one locus which is partially
dominant in the heterozygote, i.e. the heterozygote is intermediate in
resistance. Then he has, at a different locus, a completely recessive
mutant resistant to the same drug. If permeability is responsible in
these two cases, as you pointed out there must be quite a number of
different ways of altering it.

Davis: I am interested to learn of this work. I wonder whether the
mutation to resistance at one of these loci might not involve altered
permeability while that at the other locus caused resistance through
quite a different mechanism.

Pontecorvo : But the question of recessiveness that you raised is more
likely to fall into this type of category.

Eagle: There are two systems in which there is an indication that
differences in resistance reflect an alteration in a cell component, rather
than differences in permeability. One is the recent demonstration by
Saz that Esch. coli contains a nitro reductase which is strongly inhibited
by aureomycin, and which contains both a protein and flavin moiety.
In the nitro reductase of the parent sensitive cell, the flavin dissociates
readily; while in the enzyme complex deriving from aureomycin-
resistant cells the flavin and protein have a greatly increased affinity.
The relationship of this difference to aureomycin resistance, specifically,
is not entirely clear; but what seems to be quite clear is that in the resis-
tant cell there is a nitro reductase which is no longer inhibited by aureo-
mycin, and the flavin component of which is strongly bound to the
protein. The development of resistance is associated with a specific and
qualitative change in an enzyme normally vulnerable to aureomycin.

The other system relates to penicillin resistance. Bacterial species as
they occur in nature differ markedly in their binding affinity for peni-
cillin, and in direct proportion to their penicillin-sensitivity. The sensi-
tive organism is one which has a high binding affinity for penicillin, so
that lethal concentrations are attained in the cell with a relatively low
concentration in the environment. That lethal cellular concentration
was of the same order of magnitude for all bacteria examined. Here
again we are not dealing with a permeability difference; for the same
difference in penicillin-binding capacity between sensitive and resistant
organisms is evident in cell-free sonates. Macromolecular components of
those sonates differ, as do the whole cells, in their affinity for the drug.
Now the curious thing is that this relationship between binding affinity
and penicillin sensitivity applies only to strains as they occur in nature.
If one takes a sensitive strain and makes it resistant by appropriate
selection, that development of resistance is not associated with changes

Discussion 181

in binding affinity, which may increase, decrease or remain unchanged.
The mechanism of resistance in these variant cultures is totally obscure.
Pollock: Prof. Davis, would you care to expand on this case of Cohn
and Novick in which, apparently, there is an almost perpetual inheritance
of an acquired character?

Davis: The essential facts are these. As I noted earlier, when p-thio-
methylgalactoside (TMG) is added to a growing culture of Esch. coli the
cell is induced to form two new entities: one is the well known intra-
cellular p-galactosidase, and the other is the inore recently discovered
system which transports various (3-galactosides into the cell, and even
concentrates some of them (e.g. TMG). When the concentration of TMG
is sufficient — say 10"^m — induction is maximal, and within a minute or
two exponentially growing cells start forming the new components at a
constant rate per unit of new cellular material synthesized. AVhen the
concentration of TMG is too low — between 10"^ and 10-^m — there is no
induction. However, Melvin Cohn found that such a low concentration
of TMG will maintain induction in cells that had been previously grown
in a sufficient concentration to bring about induction. Furthermore, it
is known that at intermediate concentrations of TMG the culture is
induced gradually, the rate of enzyme synthesis per cell rising for many
generations. Aaron Novick and Milton Weiner have recently found that
under these circumstances the rate of synthesis is not increasing gradually
in each cell. Instead, the population is heterogeneous, cells being either
fully induced or uninduced. This is shown by transferring single cells
from such a population to tubes of medium containing a maintenance
concentration of inducer. The induced cells formed fully induced clones ;
the others yielded uninduced clones. Evidently at intermediate concen-
trations of inducer a cell has a small chance of being induced to form its
first permeation unit. This w ill concentrate the TMG, which will increase
the effectiveness of induction, and in this autocatalytic way the cell will
soon be fully induced. Then even low external concentrations of TMG
will provide sufficient internal TMG to maintain full induction.

This system has a close formal resemblance to an environmentally
directed mutation. On growth in an intermediate concentration of TMG
a fraction of the cells are altered (induced). The difference between these
and the uninduced cells can then be transmitted indefinitely through
future generations, provided the medium contains a maintenance con-
centration of inducer. A difference between these cells and mutants,
however, is that the pseudomutations can be uniformly reversed by a
few generations of growth in medium with no inducer at all.

Whether this phenomenon will be relevant to problems of drug
resistance remains to be seen.

Knox: Suppose you were to produce resistance with a drug acting as
an inducer of an adaptive enzyme which enabled the organism to grow
in the presence of the drug either by destroying it as with penicillinase
or in some other more indirect way. It is conceivable that some normal
metabolite present in very low concentrations inight, by being concen-
trated in some such way as you suggest, also act as a permanent inducer
of the same enzyme, so that the organism would remain permanently

182 Discussion

resistant to the drug even when repeatedly subcultured in its absence,
and without any genetic change having occurred. In other words, there
might be some normal metabolite which could maintain drug resistance
by some concentrating mechanism of the kind you described.

Davis: Yes, I can imagine that a drug might induce an altered capacity
of the cell, or of a subcellular particle, to concentrate both the drug and
some substance in the environment other than the drug. Then on further
growth in the absence of the drug the other substance, which might be
a normal metabolite, could conceivably maintain the induced state.

GENETIC AND METABOLIC MECHANISMS

UNDERLYING MULTIPLE LEVELS OF

SULPHONAMIDE RESISTANCE IN PNEUMOCOCCI

ROLLIN D. HOTCHKISS AND AuDREY H. EvANS

The Rockefeller Institute, New York

Development by bacteria of resistance toward a drug to
which they are normally sensitive not only challenges the
medical men, sworn enemies of bacteria, but has lately com-
manded the attention of those whose interest in bacteria is
that of admirers, the biochemists, as well as those historians,
the geneticists. Demonstration by Demerec (1945) that drug
resistance in bacteria could be the result of discrete muta-
tional events, led us to seek stable sulphanilamide resistance
in Pneumococcus which might be transferred to sensitive
cells by the technique of transformation. Heritable transfer
of specific capsule synthesis had been discovered by Griffith
(1928) as an in vivo transformation. Langvad-Nielsen (1944)
could not demonstrate transformation of pneumococci to
sulphonamide resistance, but one must recall that in using
the Griffith procedure, he was requiring transfer of both drug
resistance and encapsulation (or resistance and mouse
virulence) at once, an event we now have reason to consider
unlikely. Even after the classic discovery of Avery, MacLeod
and McCarty (1944) that the capsule -transforming activity
was associated with cell deoxyribonucleate (DNA), our first
attempts in 1948 to induce sulphonamide resistance failed for
various reasons. It was penicillin resistance and strepto-
mycin resistance which were the object of the first quanti-
tative transformations (Hotchkiss, 1951), and since that time
this type of marker has been much used. Sulphonamide
resistance transformations were successfully achieved by the
present authors in 1952, but although mentioned a number

183

184 ROLLIN D. HOTCHKISS AND AuDREY H. EvANS

of times (e.g. Hotchkiss and Marmur, 1954) they have not
been described in any detail.

Of the fifteen drug resistance traits that have by now been
transformed with DNA into Pneumococcus (and one or two
into Hemophilus influenzae) all have so far been transferred
in essentially the normal form as first encountered. In the
case of penicillin-resistant pneumococci, the DNA from
multiple-step highly resistant donor strains gave trans-
formation to unit resistance steps (Hotchkiss, 1951), but none
of the transformants detected bore mutant properties other
than those which had been encountered in the history of the
donor strain. Essentially the same result was obtained with
the first sulphonamide resistance transformations and a
number of others.

In 1955, however, a highly resistant pneumococcal mutant,
designated Fn, was isolated after selection in a single ex-
posure to sulphanilamide. The indications were that a rare
single-step mutation had occurred, resulting in a resistance
to more than 600 [ig. sulphanilamide per ml. of standard
medium. Like the somewhat analogous single-step mutant
obtainable from many bacterial species and highly resistant
to streptomycin, this new pneumococcal strain is stable and
can be propagated indefinitely without change of resistance
level.

Disseminative Transformations

By contrast, when the strain Fn was used as donor of DNA
to transform the sensitive parent strain, only a very few
transformants displayed the high resistance of the donor.
A far greater number of transformants were obtained which
were resistant only to lower concentrations of sulphanil-
amide. Furthermore, when examined at a series of drug con-
centrations, several fairly distinct classes of transformants
could be identified, having quantitatively different resistance
toward sulphonamide, and all but one had a lower resistance
than the donor. By this direct observation and isolation,
four classes could at once be recognized in a first trans-

SULPHONAMIDE RESISTANCE IN PnEUMOCOCCI 185

formation, as indicated in Table I. This result has been
repeatedly observed, with different recipient strains and
different DNA preparations.

Further genetic analysis of the system was made by isolat-
ing and testing several typical transformants of each class,
both as donors and recipients in further transformations.
All proved to be stable on propagation in culture, but those

Table I

DiSSEMINATIVE TRANSFORMATION OF A RESISTANCE PROPERTY
NORMALLY INHERITABLE

Standard sulfonamide resistance level (//g/ml.) of pneumococcal clones

Progeny

Donor

a single-step

mutant

TiPSt- stage
transformants

Second-stage
tpansfopmants

10

40

10

■* 40

-*- 300

600

■*- 600

Identified
genotypes

10

a

40

d

300

ad

10

a

40

d

300

ad

600

Qdb

120

120

120

120

which resembled the donor, Fn, gave the same multiple
classes of transformants, and the one having resistance to
800 (jLg./ml. also gave several transformants, when used as
donors. Certain other types seemed to transfer their geno-
types intact by transformation and accordingly were assigned
the unit marker designations Fa and Fd (or simply a and d),
as indicated in Table I.

Starting from this basis, it was possible to seek and dis-
cover a third unit marker, b. By transforming one unit
strain with the DNA from another, it was possible to create
the paired combinations ab, bd, and ad, and from each of
these with the appropriate single marker DNA to prepare

186 ROLLIN D. HOTCHKISS AND AuDREY H. EvANS

transformants like the original highly resistant strain Fn, now
therefore considered to be Fadb or Fbad. Each of the pair
combinations as DNA donors in transformation gave rise to
the expected transformants of their own phenotype and also
of the unit types of which they are "composed".

Determining quantitatively the proportions of the various
transformants when Fn is the donor, it has been inferred that
its units have the probable sequence adb, in which the pair
ad is linked closely and the pair db is loosely linked. The
linkage between a and b seems to be lower and is an indication
that the donor Fn with a similar linkage has the genetic
composition adb. All seven possible type of recombinants
have been made by transformation of the sensitive strain,
and the relative frequencies of their production are in accord-
ance with this arrangement. It is probable that frequencies of
linkage in such minute recombinations are determined not
only by distances but also by preferential breakage points,
and limitations upon the size of the fragment of DNA can be
incorporated.

Here, then, is a strain, Fn, whose high resistance to sul-
phonamides is determined by its genie substance; in parti-
cular, its DNA. This DNA is passed on intact from mother
to daughter cells at division, but on incorporation into new
cells in transformation is usually fragmented. The three
regions {a, d, b) of alteration in the mutant DNA represent
another example of genetic fine structure (see Demerec,
Blomstrand and Demerec, 1955; Benzer, 1955) which seems
to be within a single gene. Of interest in the present case is
the fact that this linkage of subunits is displayed by chemic-
ally purified DNA. Furthermore, it is a welcome feature in
this instance that each of the genotypes a, d, b, ad, etc.
obtained by disseminative or dispersive transformation has
a distinctive phenotype providing its own basis for quantita-
tive recovery. In previously investigated cases, the recom-
bining variants all have the same phenotype, a certain bio-
chemical or physiological lack, and cannot be distinguished
from each other.

SULPHONAMIDE RESISTANCE IN PnEUMOCOCCI 187

Nature of Sulphonamide Resistance

The possibility of defining the physiological mechanism
which implements such a subtle series of genetic controls,
has led us to study the effect of sulphonamides upon these
mutants and transformants. It is possible to infer from the
literature that the drug acts upon a system utilizing p-amino-
benzoate (PAB) and leading to the production, first of com-
pounds related to folic acid, and through them, of cell sub-
stance (Fig. 1). In support of this inference, it can be shown

Competition between
p -amino -ben zoic and sulfonamide drugs

NHg/ \c00H JiH/^ \5O2NHR

^^ ^ ^^

Unknown
enxyme system

I

TOLIC ACID

coenzyme
form I

1- carbon
intermediates

^glycine *- serine

+ homocysteine >- methionine

+ precursors »- thymine

+ imidazol deriv. >- purines

Fig. 1.

— *' proteins

nucleic
acids

that these sensitive and resistant strains of Pneumococcus
all withstand more sulphanilamide (SA) in the presence of
added PAB. The drug and metabolite compete in the classical
way defined by Woods (see e.g. Woods, 1950), and SA/PAB
molar ratios giving partial growth inhibitions are essentially
constant over 50-fold ranges of absolute concentration. The
retention of resistance over wide ranges of absolute concen-
tration serves to eliminate hypotheses based upon possible
changes in the rates of supply or utilization of metabolite or
drug.

188 ROLLIN D. HOTCHKISS AND AuDREY H. EvANS

There would seem to remain four principal classes of hypo-
theses which might rationalize this family of three sulphon-
amide resistance levels and their various combinations: (l)that
there are several independent PAB-using enzyme systems
essential for cell growth and three of these can become re-
sistant to inhibition; (2) that the affinity of a single enzyme for
sulphonamide relative to PAB is being cumulatively reduced
by each unit of mutation; (3) that the permeability of the cells
to sulphonamide is quantitatively altered as the result of muta-
tions; or (4) that an alternative metabolic pathway, which is
PAB-sparing, becomes available in each resistant strain.

Although a priori any one mutant might arise as above,
the behaviour of the series of mutants makes some of the
hypotheses improbable. It is difficult to understand the
independence of the factors a, b, and d if they determine
separate and essential systems (hypothesis 1) — how, for ex-
ample, could an enzyme Ea, the one altered to give mutant a
its resistance to SA, be unchanged in both the comparatively
resistant mutant d and the sensitive wild-type strain? Such
an independence would seem more in keeping with a series of
independent enzymes which provided alternative pathways
to the same end-products (hypothesis 4). On the other hand,
the cumulative effects of the marker pairs suggest that in
their phenotypic effects they are even more co-operative than
any independent determinants would be. For example, a
(giving a resistance to 10 (xg. SA/ml.) and d (40 (jig./ml.), when
recombined by transformation give a strain ad resistant to
300 (jLg. SA/ml. It would seem most likely that the co-operat-
ing factors a and d are acting upon the same enzyme (hypo-
thesis 2) or the same permeability-determining system
(hypothesis 3) since they potentiate each other so notably.

Little is known about factors controlling permeability, but
it seems clear that a time rate cannot be the limiting one,
since near-infinite time is available as an inhibited cell slows
down and stops growing. Furthermore, if a concentration
rate is the limiting one for permeability, the properties of a
permeability-determining substance which mutates to states

SULPHONAMIDE RESISTANCE IN PnEUMOCOCCI 189

giving different internal concentrations of SA within the cell,
are formally very much like those of an enzyme which mutates
so that it responds differently to the same concentration
of drug. In both cases a single entity is inferred which can
exist in several states having different affinities for the
sulphonamide.

Sulphonamide Inhibition of Cell Growth

There is no great difficulty in assessing accurately the level
of SA which just permits or slightly inhibits growth of a
pneumococcal strain. In such an experiment one is testing

/^q = /^q/inl ©[sulfanilamide

Fig. 2.

12345678
Hours
Inhibition of pneumococcal growth by sulphanilamide
in excess.

for indefinitely continued division of virtually every cell
within the culture. Such a threshold concentration of drug,
however, does not at first sight seem to have much effect
upon cell metabolism. Even when so gross a measure of
growth as the total turbidity of the culture is followed, the
effect of SA seems to be slight. Such turbidity curves as those

190 ROLLIN D. HOTCHKISS AND AuDREY H. EvANS

shown in Fig. 2 seem confusing when it is reahzed that tur-
bidity increases almost as much as in the control when 10 to
20 times the inhibitory concentration of SA is present.

The explanation lies in the limited number of divisions
observed in the experiment. As indicated in Fig. 3, different
growth media can supply different samples and quantities of
the eventual products of PAB metabolism. In addition, the
cells themselves can accumulate and later use substantial
amounts of the catalytically effective product(s) of PAB, folic

Environment

Metabolism

Measurement

cSi?i;?s4Tctori-'-Cf^""i'>^p-=--o-

COOH

Sulfonamide
inhibition

streptococcal assay

TOLIC ACID -'

cell substance
(nucleic acid; protein)

Fig. 3. Determination of inhibitory effects of sulphonamides.

medium supplies
non- competitive products
of folic acid system

- pneumococcal
endogenous assay

-->- turbidity

acid(s). Therefore, when placed under conditions fully
inhibitory to folic acid synthesis the cells nevertheless are
able to complete a limited number (usually three to five) ter-
minal divisions. When using such measures of growth as
turbidity, one may purposely start with an initial culture
heavy enough to measure. An increase of 10- to 30-fold may
be possible in sulphonamide so that the measuring instru-
ment may only reveal the late stages of inhibition or none at
all, as in Fig. 2.

It is clear, therefore, that the true ability to survive in
sulphonamide is only assessed when indefinite propagation is
demanded, as when single organisms are required to produce
colonies. A second difficulty is the PAB-sparing effect of end-

SULPHONAMIDE RESISTANCE IN PnEUMOCOCCI 191

products of PAB metabolism such as the purines, pyrimidines
and amino acids, normal constituents of the complex media
used for pneumococci. Not only the number of terminal
divisions but the limiting steady growth of pneumococci in
sulphonamide is modified by such metabolites. Accordingly,
the inhibitory levels of SA or the SA/PAB ratios for limiting
growth can be made to vary in absolute value when such
factors in the media are varied.

A strong indication that only a single site of action is
involved is found when one relates these indices for mutant
strains and wild types to each other, as the medium is altered
deliberately in this fashion. It was found that the half
inhibitory SA concentrations and SA/PAB ratios could be
altered 5- to 10-fold in magnitude through changing the
medium, and yet the relation between the indices for mutant/
wild type remained constant. It appears reasonable therefore
that the actual basis for the sulphonamide resistance in the
mutants, at least for the two or three so far tested, is an
altered affinity of sites on some single enzyme (or possibly
a single permeability-determining concentrating system) for
SA relative to PAB. If this proves to be true, it may be hoped
that we are now in possession of a system in which a series of
interrelated alterations within a DNA particle exerts genetic
control over corresponding properties at a site within a pro-
tein molecule, altering its affinities for a known metabolite
and drug.

The further definition of the phenotypic modifications
existing in these resistant mutants seems to be possible when
folic acid synthesis is measured, somewhat along the lines of
the method of Nimmo-Smith, Lascelles and Woods (1948).
The sulphonamide inhibition of this function is, in contrast
with growth, not greatly modified by constituents of the
medium, and we have little doubt that more fundamental
SA affinities will soon be established for each strain. The
details of these studies and of a considerably simplified auto-
genous pneumococcal assay for folic acid w^ill be published
elsewhere.

192 ROLLIN D. HOTCHKISS AND AuDREY H. EVANS

General Remarks

By way of more general observation, it should be pointed
out that there may be many other drug systems for which
storage of end-product metabolites, or inhibitors, or their
presence in the medium can give temporary independence
from drug-inhibition. In such systems, many cell divisions
could occur in the presence of the drug. If, during these ter-
minal divisions any adaptive process reduces the requirement
for end-metabolite, then of course an adaptive resistance will
have been developed. If these terminal divisions have
allowed the development of a genetic mutation towards
resistance, then that too may have seemed to be favoured by
the presence of drug. In general, it should however have
appeared in the control populations of equivalent size, and the
actual observation would only be an increased proportion of
resistant cells among the survivors when drug is present.
The familiar variance test should be capable of showing that
the presence or absence of the mutant is determined by chance,
and that only its accumulation during the period of terminal
divisions of the non-resistants is influenced by drug.

The danger exists that in such cases not all cells rated as
"resistant" will be really so, since merely replacing with
fresh medium or shifting the medium or mode of test between
the "adaptation" stage and the challenge for resistance can
lead to the result that any cells which may have stored much
metabolite during "adaptation" will grow for a long time
relatively independently of drug when given a new oppor-
tunity in the challenge situation. Clearly in such a case it is
important to use subtransfers and lineage tests to inquire
to what extent a more or less persistent drug resistance has
been achieved.

REFERENCES

Avery, O. T., MacLeod, C. M., and McCarty, M. (1944). J. exp. Med.,

79, 137.
Benzer, S. (1955). Proc. nat. Acad. Sci. Wash., 41, 344.
Demerec, M. (1945). Proc. nat. Acad. Sci. Wash., 31, 16.

SULPHONAMIDE RESISTANCE IN PnEUMOCOCCI 193

Demerec, M., Blomstrand, I., and Demerec, Z. E. (1955). Proc.

nat. Acad. Sci. Wash., 41, 359.
Griffith, F. (1928). J. Hyg., 27, 113.

HoTCiiKiss, R. D. (1951). Cold Spr. Harb. Symp. quant. Biol., 16, 457.
HoTCHKiss, R. D., and Marmur, J. (1954). Proc. nat. Acad. Sci. Wash.,

40, 55.
Langvad-Nielsen a. (1944). Acta path, microbiol. scand., 21, 362.
Nimmo-Smitii, J., Lascelles, J., and Woods, D. D. (1948). Brit. J.

exp. Path., 29, 264.
Woods, D. D. (1950). Ann. N.Y. Acad. Sci., 52, 1199.

DISCUSSION

Stocker: It has been suggested that strains of Neisseria, resistant to
sulphonamides, have an increased abihty to synthesize PAB. Is there
any suggestion of that kind of resistance in Pneumococcus in addition to
the more coinplex one?

Hotchkiss: We don't see it in these strains, and I have never seen it.
The argument against it is that when we swamp the PAB which the cells
are endogenously making, by supplying PAB in the outer medium, we
still retain the same relative resistance level, the relation between
different mutants remains the same, even when we add so much that it
takes 150 times as much sulphonamide to block growth.

Pontecorvo: Do I remember correctly that in a case of Neiirospora
sulphonamide resistance, there is an increase in endogenous production
of PAB?

Davis: I don't know about simple resistance, but the sulphonamide-
dependent Neurospora mutant does not make more PAB. It is inhibited
by an imbalanced biosynthesis resulting from abnormal responsiveness
to its normal amount of PAB.

Lederberg: In the control of resistance to penicillin is there a funda-
mental difference in the interaction of the genes ?

Hotchkiss: The higher level of penicillin never gave rise to any low
level other than those particular low steps known to be reached during
its derivation, in a certain sequence.

Lederberg: So that the interactions are presumably specific? Are you
distinguishing the levels purely by phenotype ? Because it may be that
there are several different mutations which simply give the same level of
resistance when you have only one of them, but which may interact to
give higher levels, which could be looked for by transformations among
the first ones.

Hotchkiss: We tried transformations among them. We took highly
resistant donors, recovered all of the low-level transformants that they
produced as a mixed culture, made DNA from the mixed culture and
generally tested both mixed culture and DNA to see if either contained
anything from a higher level; whatever it may have contained was too
little to show. It may well be that there are hidden members of the
population that have acquired the factor b or c, but that show no penicillin
resistance.

DRUG RES. — 7

194 Discussion

Lederberg: You had a similar evidence for the specificity of interaction
with chloramphenicol, Cavalli-Sforza.

Cavalli-Sforza: Specificity of interaction is of some interest. As a conse-
quence of it, every process of selection, leading to multistep resistance, is
in itself more or less unique, because much depends, for the later stages,
on which is the first resistant gene that came in. The later ones are
bound to interact with that one ; they have to increase the resistance of
that one. Therefore, you may even find situations where the second gene
that gives second-step resistance does not give resistance by itself; if you
isolate it by recombination, you may find that that second gene is only a
modifier of the first. It may have an effect on resistance only in combina-
tion with the first.

Lederberg: It should then be possible to cross two strains obtained
separately, but of equal resistance, and get progeny which are more
sensitive than either parent.

Cavalli-Sforza: Exactly, that is what is happening.

Dernerec: In the work you have completed so far. Dr. Hotchkiss, have
you obtained any evidence that throws light on the mechanism by which
transformation is accomplished? In this particular case dealing with
transfer of high resistance to sulphurs, you assume that you have three
genes which are linked together and which would be carried in one
transforming unit of DNA. You assume the order of these genes to be
adb. Have you any evidence of a combination of ab ? Can you tell if that
may be transferred when you start with a donor which carries all three ?

Hotchkiss: As far as we can see we don't get the ab's from the donor
which has adb. We get either the whole piece or fragments. If we create
ab by using the a and the b separately, then the ab transfers approxi-
mately as often as the adb. So the frequencies fit, in this case, and it is
the rareness of this type that is the basic argument for the sequence, the
db's and the ad's are much more frequent than the ab's.

Davis : Is the linking of these close enough for you to have any idea as
to whether these different units are likely to be concerned with a single
genetic unit in terms of physiological function ? Do you know any other
cases in which a single mutation can give you divisible changes?

Hotchkiss: Mutations can be inversions of whole regions and trans-
positions and so on. It would require many markers to find them in
bacteria, but it would not be out of the question. We tend to think of
mutations in the purest case as more or less point mutation.

Demerec: There are large numbers of mutations known which affect a
region involving several gene loci, but this is the first case, as far as I am
aware, where such mutation is divisible. In most other cases, probably
we are dealing with deficiencies involving a part of a gene or adjacent
genes.

Davis: Prof. Cavalli-Sforza published evidence some years ago that
in multiple-step resistance several different loci can be concerned, each
making its contribution. That result may perhaps have lent support to
the impression, which seems widespread, that it is hard to picture muta-
tions as giving rise to the enormous numbers of degrees of resistance you
can get, because you would have to have so many different loci involved.

Discussion 195

However, we know that with auxotrophic mutants you can get not only
mutations giving rise to an all-or-none appearance or disappearance of
an enzyme, but also mutations causing wide quantitative variations in
the amount present, and other mutations causing qualitative variations
in the value of an enzyme. Hence, the number of loci affecting resistance
to a given drug does not have to be nearly as large as the number of
steps of resistance that can be distinguished, since various alleles at a
single locus could be expected to produce different degrees of resistance.

Cavalli-Sforza : There is a widespread impression that the gradual type
of adaptation is likely to be physiological rather than genetic. I tend to
hold another view : I don't see any reason why the gradual adaptation
should be easily physiological. Dr. Demerec, in the early stages of his
work when he started the genetic analysis of bacterial drug resistance,
called the gradual and the abrupt types of adaptation "penicillin and
streptomycin patterns". Whether some drug tested on some organism
is going to show one or the other pattern depends entirely on the relative
frequency of the various types of resistance mutations that can occur.
If mutations that have a small effect are more frequent, then you are
more likely to have the penicillin type, i.e. the gradual type of adapta-
tion; and that is likely to be the most frequent case, because it seems
reasonable to expect that mutations having small effects are more fre-
quent than those having large effects. There has been an accumulation
of data showing that gradual adaptation also is indeed of genetic origin.
There is evidence from indirect selection, such as the data on
chloramphenicol resistance — first step only — and Yudkin's evidence on
multistep adaptation to proflavine, which shows that in the case of the
chloramphenicol and proflavine systems of "gradual" adaptation you
do have genetic adaptation. There is all the evidence from recombina-
tion, and transformation data, showing that "gradual" adaptation is
due to nuclear genes having small effects, which effects can add together
to give a big one ultimately.

Hotchkiss : We have studied what we call lysis transformation, in a
mixed culture having penicillin-sensitive organisms which were strepto-
mycin-resistant, mixed with penicillin-resistant organisms which were
streptomycin-sensitive. When this mixed culture was grown in peni-
cillin, the penicillin-sensitive cells were killed. Thereupon they lysed,
DNA was released, and this DNA interacted with the surviving resistant
culture so that streptomycin resistance was introduced into that culture.
Therefore, we had a compiling of information; when these cells died,
they passed on a high proportion of their information. Now consider
what couM happen if this were one of the cases such as Prof. Cavalli-
Sforza has seen, for let us say a drug which kills. Suppose one compo-
nent is penicillin-resistant to 5 [ig. and another is penicillin-resistant to
10 [ig. If we introduce 10 [ig. of the drug, the former will be killed, and
if these are independent factors, there could now appear transformant
"tens plus fives", that might well be resistant to 200 ij.g. This would
now be a mechanism for fairly efficient compilation, within a culture, of
all the properties that were present. If we bear in mind that some of
these might be of the latent type, they would not be easily recognized

196 Discussion

and a lysis transformation such as we have demonstrated might easily
produce a dramatic increase in resistance in such a mixed culture.

Lederberg: Since Dr. Hotchkiss has brought this up, a word of caution
may be added. There has been some misunderstanding in the literature
about the possible role of transformation in the development of resistance
in bacterial cultures. It has been suggested, for example, that an initial
resistant cell might arise by some unspecified process, perhaps by spon-
taneous mutation, and that this quality could then spread through the
population by a transformational process. In all of these cases you are
very lucky indeed if you get anywhere near one new resistant for each
resistant cell which has died and released its DNA. In most cases that
figure is very low, and in no case does it exceed one in any practical
situation. For this reason, transformation is not a means by which the
proportion of resistant cells in a culture can rapidly increase.

THE PHENOTYPIG EXPRESSION OF GENES
DETERMINING VARIOUS TYPES OF DRUG
RESISTANCE FOLLOWING THEIR INHERIT-
ANCE BY SENSITIVE BACTERIA

W. Hayes

Department of Bacteriology, Postgraduate Medical School, Loudon

When a gene in a bacterial cell is changed to an allelic state
by mutation, or is replaced by an allele during recombination
in parasexual systems, the altered genotype of the cell will
not immediately be expressed in a corresponding change of
phenotype. This delay of expression, known as phenotypic
lag, can theoretically be accounted for by the operation of
one or more of several circumstances. Firstly, genes can only
manifest their effects through the enzymic potentialities and
organization of the cytoplasm. The new gene finds itself
confronted with a cytoplasm adapted to the expression of the
allele which it has replaced. Thus, if the new gene determines
the synthesis of an enzyme of which the cell is devoid, at least
one molecule of enzyme must be created, and its dependent
synthesis initiated, before expression can occur. Alterna-
tively, if the new gene differs from its allele in being unable
to synthesize a particular enzyme, then its expression will be
delayed until the enzyme molecules already present in the
cytoplasm have been diluted out by successive divisions of
the cell. Secondly, bacteria are peculiar in that each cell
possesses, at least during the logarithmic phase of growth,
two or more nuclear analogues. The occurrence of a mutation
involves a gene in only one of these nuclei so that the mutant
cell initially contains one or more wild-type genes as well as
the mutant gene and therefore resembles a heterokaryon. If,
as appears usually to be the case, the mutant character is
recessive to the wild-type character, then the mutant gene

197

198 W. Hayes

will be unable to express itself phenotypically until it has
become separated from the wild-type genes by nuclear and
cellular division. In this way, although bacteria are haploid
organisms, relationships of dominance and recessiveness can
come into play following mutation. Such relationships are
found, in a more orthodox way, in bacterial parasexual sexual
systems in which a fragment of the chromosome from a
donor cell is introduced into a recipient cell. This fertilized
cell becomes a partial heterozygote within which a process
of recombination occurs. It is not known for certain what
this process involves, but the evidence suggests that the
incoming fragment of donor chromosome and the chromosome
of the recipient cell pair together and then begin to replicate.
During this operation, a replica commenced on one of the pair
may suddenly switch to Topy the other, and thence switch
back to continue copying the chromosome on which it started.
In this way a completely new chromosome is produced which,
although basically that of the recipient cell, incorporates part,
or the whole, of the incoming donor fragment. From this
stage onwards it is likely that the recombinant chromosome
segregates into an autonomous cell in the same manner as a
mutant chromosome, so that something may be learned about
the phenotypic expression of mutations from the study of the
kinetics of segregation and expression in the parasexual
systems of bacteria.

A direct approach to the kinetics of phenotypic expression
of mutations is a difficult matter from both the theoretical
and experimental points of view. In order to study the
sequence of events as a function of time, it is necessary to
have a high degree of synchrony among these events in the
population, and to be able to observe their progress from their
inception. By definition, such synchrony is impossible in the
case of spontaneous mutations which are rather rare events
whose occurrence is determined by chance. Moreover, muta-
tions can usually only be demonstrated by selective tech-
niques so that we can detect only those mutations which have
already become expressed at the time when the selective

Kinetics of Phenotypic Expression 199

agent is applied. These difficulties can be overcome to some
extent by subjecting bacterial populations to the action of
physical or chemical mutagens which not only may greatly
increase the total number of mutations but, at the same time,
will synchronize their initiation. However, the use of such
agents introduces other variables which may have a profound
effect on the sequence of events. For example, it has been
shown that even a low degree of irradiation with ultraviolet
light temporarily arrests the synthesis of deoxyribonucleic
acid of which the genetic material of the cell is constructed
(Kelner, 1953), and leads to marked aberrations in cellular
morphology. The extent to which mutagenic agents may
injure cytoplasmic as well as nuclear function is not clear,
but may be appreciable.

In practice, experiments designed to determine the pheno-
typic lag of induced mutations, involving many different
types of character, have given most conflicting results. In
general, the delay has been found greatly to exceed that
required for segregation of the mutant nucleus or for the
cytoplasmic manifestation of its genotype (Demerec, 1946;
Ryan, 1954). In some systems, on the other hand, a delay of
less than one generation has been suggested (Ryan, 1955).
The work of Witkin (1956) indicates the importance of nutri-
tional and other factors in the environment, during the first
third of the first generation time following irradiation, in
deciding whether or not the mutational change will stabilize
and become expressed.

We are thus faced with the difficulty that the nature of
mutation precludes the direct experimental study of its
kinetics unless the mutations are artificially induced, while
the inducing agents themselves influence the course of events
in such a way as to render the results of such experiments to
a considerable extent invalid. It is clear that the action of
mutagenic agents, as well as the nature of the interaction of
mutant genes with the metabolic processes they determine,
can only properly be evaluated against a background of
knowledge of the kinetics of segregation and phenotypic

200 W. Hayes

expression in uncomplicated systems which can be reasonably
well synchronized. The only available systems of this kind
are the parasexual ones in which genes can be transferred
from one bacterial cell to another of different genotype, and
in which the subsequent behaviour of the resulting recom-
binant chromosome can be studied.

There are three such systems, which differ from one another
mainly in the method whereby the genetic transfer is effected.
In each, a part of the chromosome of a donor cell can be
transferred to a recipient cell and then incorporated into the
recipient chromosome to form a recombinant chromosome. In
transformation, already described by Dr. Hotchkiss (this
symposium, p. 183) the agent of transfer is DNA extracted from
the donor population. In transduction (Zinder and Lederberg,
1952; Zinder, 1953), bacteriophage (i.e. virus) particles of low
virulence, derived from the donor population, act as vectors
of small fragments of the donor chromosome to those recipient
cells which they infect. The frequency with which any par-
ticular gene is inherited by the recipient population in trans-
duction is usually low (ca. 10"^).

The third system, conjugation (Lederberg and Tatum,
1946; Lederberg et at., 1951; Hayes, 1953; Wollman, Jacob
and Hayes, 1956), which is found in Escherichia coli, differs
from transformation and transduction in three respects:

(1) Genetic transfer from donor to recipient cell is effected
directly, by cellular fusion.

(2) A large part of the donor chromosome, comprising
many linked genes, is usually transferred to the recipient cells
and may appear in recombinants.

(3) When a special type of donor strain called Hfr (for
high frequency of recombination) (Cavalli, 1950; Hayes, 1953;
Wollman, Jacob and Hayes, 1956) is employed, the frequency
with which certain recombinant types appear may be as high
as 10-20 per cent of the recipient population (Hayes, 1957).

The work described here has involved a donor Hfr strain
isolated by the present author which, under the experimental
conditions employed, transfers a specific part of its chromo-

Kinetics of Phenotypic Expression 201

some to recipient cells with high frequency, to form partial
zygotes. The transferred fraction of donor chromosome
carries on it, in the order of their arrangement, genes deter-
mining the synthesis of the amino acids threonine and
leucine (T, L), resistance to valine (Val), resistance to sodium
azide (Az), resistance to the virulent bacteriophage Tl and
the ability to ferment lactose (Lac); the recipient cell differs
from the donor in all these characters. The zygotes formed
when cultures of the donor and recipient strains are mixed
together in broth can be represented thus :

Donor T L Val Az Tl Lac

Recipient + + r r r +

When such zygotes are plated on synthetic minimal agar
devoid of threonine and leucine, only those recombinants can
grow which have inherited from the donor chromosomal con-
tribution the two linked genes which control synthesis of
these amino acids; i.e. selection is made for the gene T+L-[-
from the donor parent. The other genes on the transferred
segment of donor chromosome are not selected and w^ill be
inherited among T4-L+ recombinants with a frequency
proportional to their distance from the selective markers
T-I-L4-. In other words, the closer an unselected donor gene
is situated to the selected genes T+L-j-, whose inheritance
is obligatory, the less the probability that it will be separated
from these genes by the occurrence of a cross-over between
them and the greater the probability that it will be included
in recombinants.

The donor genes controlling resistance to valine, sodium
azide and phage Tl are all closely linked to the selective genes
T-f-L+ and are inherited by about 100 per cent, 90 per cent
and 75 per cent of T+L-|- recombinants, respectively. For
this reason, and because they are concerned with very dif-
ferent aspects of cellular function, these markers are well

202 W. Hayes

suited to the study of phenotypic expression. Moreover, in
control experiments, exposure of sensitive recipient cells in
which the zygotes are formed to any one of these drugs pre-
vents further cell division. The assumption can therefore be
made that a resistance gene present in such a phenotypically
sensitive cell is unlikely to be able to express itself after the
appropriate drug has been applied.

The technique used is as follows (Hayes, 1957): Young
broth cultures of donor and recipient cells are mixed and kept
at 37° for 30 minutes to allow zygotes to form. The donor
cells have then fulfilled their fertilizing function and are
killed by adding to the mixture a high multiplicity of the
virulent phage, T6, to which the donor parent is sensitive but
the recipient cells, and the zygotes resulting from their
fertilization, are resistant. In this way, further mating is
prevented and we are left with what is termed a "zygote sus-
pension". To assess the kinetics of segregation and expres-
sion on synthetic minimal agar, two identical series of plates,
warmed to 37°, are inoculated with diluted zygote suspension
so as to yield, after incubation, about 20-30 recombinant
colonies per plate, each colony being composed of the progeny
of a T+L-j- recombinant segregant issuing from a single
zygote. At intervals after inoculation and incubation, the
surfaces of one series of plates are vigorously rubbed, in turn,
with distilled water by means of a glass spreader. This has the
effect of separating the progeny of any T+L+ recombinants
that may have already divided at the time of rubbing so that
the subsequent colony count is doubled for each generation.
Prior to division, of course, the colony count remains con-
stant since the effect of rubbing is simply to alter the position
of the zygotes or segregant s on the plates. At the same time
as plates of the first series are rubbed with distilled water,
plates of the second series are similarly rubbed with an appro-
priate concentration of valine or sodium azide, or with a
washed, high titre suspension of phage Tl. Since these agents
prevent any further division of sensitive organisms, only those
cells, whether zygotes or T+L-1- recombinant segregants.

Kinetics of Phenotypic Expression

203

which have inherited the gene controlhng resistance, and in
which the character of resistance has become expressed, can
produce colonies. When the proportion of recombinant
colonies arising in the presence of the drugs becomes equal
to the proportion of control recombinants which have in-
herited and expressed the gene for resistance, expression is
regarded as complete.

B. Segregation and expression

180 240 300 20 40 60 80100120140160180
Time after dilution (min.)
Fig. 1 . The kinetics of segregation and phenotypic expression.

To study the kinetics of segregation and expression in
nutrient broth, the zygote suspension is simply diluted 1 : 50
into fresh broth at the desired temperature. Samples are
removed as a function of time, appropriately diluted and
plated to synthetic minimal agar as well as to the same
medium containing a suitable concentration of the drug.
Results of two typical experiments are shown in Fig. 1.

The continuous lines represent the kinetics of segregation,
and the interrupted lines the kinetics of phenotypic expres-
sion. With regard to segregation, it will be seen that on
synthetic minimal agar recombinants start to divide about

204 W. Hayes

120 minutes after plating and thereafter multiply with a
generation time of about 60 minutes (Fig. 1 A). In nutrient
broth at 37° (Fig. 1 B), the first division of recombinant
segregants is initiated at 100 minutes after diluting the
zygote suspension into fresh broth, and the generation time is
20 minutes.

When the kinetics of phenotypic expression of either
sodium azide or phage Tl resistance are plotted in terms of the
generation time of the recombinants, closely similar results
are obtained on synthetic minimal agar, nutrient agar and in
nutrient broth. The patterns of expression of these two
characters, however, are very different. Expression of the
character Az'^ (Fig. 1 B) begins at the time of dilution, or
of plating, of the zygotes and then rises exponentially to
become complete just before the recombinants which inherit
it start to divide. There is good evidence that during the
greater part of this period, at least, the genes Az^ from the
donor and Az^ from the recipient parent must be present
together in the partially diploid zygote. In fact, it is likely
that the early stages of expression occur before the process
of recombination proper begins (Wollman, Jacob and Hayes,
1956). From this it follows that the gene Az^ is dominant
to Az^

In contrast, resistance to phage Tl does not begin to be
expressed until after segregation, while full expression is
delayed until the fourth recombinant generation (Fig. 1 A).
This is in conformity with the finding of Lederberg (1949) that
phage Tl resistance is recessive to sensitivity in Esch, coli
diploids. The duration of the phenotypic lag also accords well
with that estimated by indirect methods by Newcombe (1948)
for the expression of phage resistance in spontaneous mutants.
The coincidence of the commencement of expression of this
character with initiation of the first segregant division
strongly suggests that at this time the recombinant cells make
their first appearance as independent units.

The exponential nature of the rise in phenotypic expression
of both characters is reminiscent of Dr. Pollock's curves for

Kinetics of Phenotypic Expression 205

induced enzyme formation (this symposium, p. 78) and prob-
ably reflects the fact that both are the end-result of enzyme
synthesis.

The curve of expression of valine resistance closely follows
that of segregation, so that the gene determining this character
is dominant and, like the selected genes T+L-j-, is fully
expressed very shortly after entry into the recipient cells.

REFERENCES

Cavalli, L. L. (1950). Boll. 1st. sieroter. Milano, 29, 1.

Demerec, M. (1946). Proc. nat. Acad. Sci., Wash., 32, 36.

Hayes, W. (1953). ColdSpr. Harb. Symp. quant. Biol., 18, 75.

Hayes, W. (1957). J. gen. Microbiol., 16, 97.

Kelner, a. (1953). J. Bad., 65, 252.

Lederberg, J. (1949). Proc. nat. Acad. Sci., Wash., 35, 178.

Lederberg, J., Lederberg, E. M., Zinder, N. D., and Lively, E. R.

(1951). ColdSpr. Harb. Symp. quant. Biol., 16, 413.
Lederberg, J., and Tatum, E. L. (1946). ColdSpr. Harb. Symp. quant.

Biol., 11, 113.
Newcombe, H. B. (1948). Genetics, 33, 447.
Ryan, F. J. (1954). Proc. nat. Acad. Sci., Wash., 40, 178.
Ryan, F. J. (1955). Amer. Nat., 89, 159.

WiTKiN, E. M. (1956). ColdSpr. Harb. Symp. quant. Biol., 21, 123.
WoLLMAN, E., Jacob, F., and Hayes, W. (1956). Cold Spr. Harb.

Symp. quant. Biol., 21, 141.
Zinder, N. D. (1953). ColdSpr. Harb. Symp. quant. Biol., 18, 261.
Zinder, N. D., and Lederberg, J. (1952). J. Bad., 64, 679.

DISCUSSION

Lederberg: There is one assumption that has to be considered further
in this type of analysis, and that is whether the fact that the inhibitor
prevents a cell from undergoing a further div^ision likewise prevents it
from completing that process of phenotypic development which can lead
to the resistant phenotype. In other words, I very strongly suspect that a
cell which has both an azide-resistant and an azide-sensitive gene may
still be at least partly sensitive to azide in terms of division, but never-
theless be capable of completing those particular synthetic processes
which are required for the development of azide resistance. In our tests
on the dominance of azide resistance in diploid, we ran into some trouble
because the markers could not be scored on synthetic medium and had
to be scored only on a complete medium. My impression was that there
was an intermediate degree of sensitivity.

Hayes: I have been worried about this very point regarding the azide.

206 Discussion

I am, however, quite happy about the phage Tl, which rapidly kills
sensitive cells.

Lederberg: An analogous example may be seen in that if you take
diploids which are heterozygous for streptomycin resistance and sensi-
tivity, these can be prevented from measurable growth by about 5
units of streptomycin. Nevertheless, if those cells are plated in medium
containing that level of streptomycin, there is an appreciable yield of
segregants, which are streptomycin-resistant, and can grow out, which
suggests that they can escape into this new genotype, so to speak.
What I am not certain of here is how precisely that multiplication is
inhibited.

Hayes: The only way to work with sodium azide in this kind of
experiment was to make the assumption that a dose which inhibited
division of the sensitive parent also prevented further development of
the heterozygote. I quite agree that this assumption may be incorrect.
The only check was that on an agar slide, with the drug included, no
multiplication of any sensitive cell was observed.

Lederberg: With phage the action is all-or-none, i.e. you have a bac-
tericidal effect of phage if it is absorbed at all.

Pollock: It would be interesting to do this kind of analysis with a
mutation which involves loss of the power to form an enzyme. You
postulated there being a dilution out of an enzyme after it ceased to be
produced. It is quite possible that, following a loss mutation, there
might arise an inability to form an enzyme-forming system ; which would
mean that it would be the enzyme-forming system rather than the
enzyme molecule which would be diluted out, and it might take a very
long time indeed before cells lost their resistance.

Hayes: The trouble about this kind of experimentation is that you
are restricted to certain characters; for instance, you could not esti-
mate the phenotypic expression of sulphonamide resistance under these
circumstances because, as Hotchkiss has already pointed out, the cells
can still continue to divide after application of the drug. You must have
something at the very least which will stop any further division.

Lederberg: Have you some information on the expression of strepto-
mycin resistance?

Hayes: No, but we are going to study this.

Davis: There are some definite advantages in using streptomycin, in
that it is the one agent that is bactericidal without any growth of the cell.
In addition, the rate of bactericidal action increases practically linearly
in proportion to the concentration of the drug.

Hayes: Yes, it is ideal; and there are also Lederberg's results with
diploids to serve as comparison. We just have not been able to obtain an
Hfr strain yet which will transfer the streptomycin locus to the zygote.

Davis: Are you at all surprised at the speed with which the phenotypic
delay is overcome in these cases? For instance, in phage resistance
presumably a cell is resistant when it no longer has on its surface any
units which the phage can attack. And from electron micrographs a
sensitive cell appears to have a great many such units. Yet you find that
resistance is phenotypically expressed within a generation or two. This

Discussion 207

rapid expression would seem difficult to account for if the units for
sensitivity were lost, during growth after the mutation to resistance,
simply by dilution.

Hayes: I had visualized a state of affairs in which, after segregation,
the genes started functioning, producing the necessary system to manu-
facture new sites on the cell wall; and that you would have a hetero-
geneous population of cells at various stages, some of which had a certain
proportion of sites, and others a different proportion. It is just a matter
of chance; the more sites a particular cell had synthesized at any
particular time, the greater the chance that under the experimental
conditions it would be able to absorb that phage and be killed.

Cavalli-Sforza: An incidental point is that I do not agree entirely with
the genetic map which you showed. Dr. Hayes. I think valine resistance
should be between T and L, according to my experience. You said that
valine resistance is expressed immediately and more rapidly than azide ;
do you find any difficulty in scoring for azide resistance in minimal?

Hayes: No, but this is a technical point. I use sodium aspartate in
my minimal medium, and if I use m/1500 azide there is no difficulty at
all in scoring colonies of resistant segregants. This level suppresses the
growth of sensitive prototrophs, but allows resistant prototrophs to
grow. The colonies are smaller than on minimal agar without azide, but
this makes counting rather easier. The colonies come up after overnight
incubation at 37°.

Fredericq: When you add your T6 phage to destroy the donor cells,
this is at a time when the two cells are sticking together. Do you some-
times observe a transfer of T6 from the donor into the recipient cell ?

Hayes: This would be hard to observe directly, but by inference, no.
After this treatment one gets exactly the same number of recombinants,
within the experimental error, as one gets when the mating pairs are
simply separated, diluted and plated out. There is no reduction.

One could assume that if the phage was transmitted, then it would
probably multiply and lyse the otherwise resistant cells, and this is
not so.

Stacker: With regard to the time needed for expression of strepto-
mycin resistance, one can make an analogous experiment either in DNA
transformation or phage transduction. Dr. Hotchkiss and others have
described the results in the DNA experiment. In transduction experi-
ments, there is a considerable delay after the phage is added before the
cells first become streptomycin-resistant, and there is then a plateau
before the number of streptomycin-resistant clones begins to increase.
There is also a delay in the case of transduction of motility (where one
knows by inference that motility is dominant to absence of motility).
But the delay in the first appearance of streptomycin-resistant cells is
greater than the delay in the appearance of the first motile cells.

Hayes: Do you know anything about the actual number of generations ?

Stacker: In the motility case the delay is of the order of two and a half
generation times. I don't recollect the figure for streptomycin.

Hotchkiss: In the DNA transfer of streptomycin resistance, the situ-
ation is approximately as follows. There is a delay of about one division

208 Discussion

time before any streptomycin-resistant cells can be detected. This could
be some kind of recessiveness of resistance, but we feel that the resistance
is dominant, because if those newly arising cells are treated with strepto-
mycin you are left with cells that survive streptomycin, but that never-
theless in subsequent generations segregate out sensitive daughters.
The rate at which the tiansformants develop resistance has been shown
by Fox in our laboratory to be the summation of a more or less normal
time-distribution. The first ones appear one division time after DNA,
and the last ones are finished in the course of that next cell-division
period.

Barber: Perhaps Dr. Hayes or Prof. Lederberg would comment on the
possible applicability of the Esch. coli type of recombination for other
bacterial species.

Hayes: A system which may have something in common with it has
been described in Pseudomonas by Hollo way in Australia (Hollo way,
B. W. (1955), J. ge7i. Microbiol., 13, 572), but this is in the very early
stages of working out. Recombination occurs, of course, as Prof. Leder-
berg has shown, in many other strains of Esch. coli, but I don't know of
any other example of this kind of system in other bacterial genera, apart
from Pseudomonas.

Lederberg: Luria has been able to cross some Esch. coli strains with
Shigella. I don't think that sexual recombination is necessarily very rare
in bacteria ; it is the investigations of it that are rare.

We have been talking of dominance as if this were an all-or-none
affair, and I would ask Dr. Hayes and Dr. Hotchkiss to say what happens
if higher concentrations or different concentrations of the antibacterial
agent are used ? Does one get the same pattern of expression as with the
one discussed here?

Hayes: I have not done this with valine, and with azide it is difficult.
There is only a rather critical range of concentration of the drug which
will stop the growth of sensitive cells and allow the resistant cells to grow.

Hotchkiss : As for the transformation, the transformant is resistant to
approximately 2,000 [ig. of streptomycin, so we have a wide range to
cover. At any one time during the process, there are more cells resistant
to say 10 [ig. than there are to 100 fxg., and more are resistant to 100 [ig.
than there are to 200 [xg., and so on; but if the time-course for any one
concentration is obtained, these show very parallel curves and the time
displacement of these curves is almost precisely that of the killing rate
of the respective concentrations. The higher concentration kills more
quickly and therefore it stops any further expression ; 200 [ig. may allow
further expression for perhaps three minutes, 50 [ig. may allow further
expression for five or six minutes.

Lederberg: So there is some indication from this type of experiment that
the levels of streptomycin do allow a progression of the phenotypic
development of resistance for a short interval of time.

Hotchkiss: The time is easily reconcilable with the time of the killing
period.

Pontecorvo : A minor matter of terminology should be raised here in
order to avoid misunderstanding. Prof. Lederberg and I have agreed

Discussion 209

on the following terms. One could describe as a "sexual system" any-
one that involves more or less a total fusion of two genomes ; with the
subdivisions "eusexual" for unadulterated sexual reproduction, and
*'parasexual" for cases of less complete orthodoxy. "Transduction",
on the other hand, is a system in which only part of the genome from a
donor cell goes to a recipient. Under the term "transduction" Prof.
Lederberg also includes transformation, but since I have no vested
interest either in transformation or in transduction I would not like to
go further!

Hayes: In a recent paper in conjunction with Drs. Wollman and Jacob
(Wollman, Jacob and Hayes 1956, loc. cit.) we have referred to these
bacterial systems involving partial zygotes as " merozygotic " systems;
but I was very careful to refer to them today as "parasexual" ones, as I
thought this would please Dr. Pontecorvo!

Pontecorvo: That is very gratifying, but we have agreed with Prof.
Lederberg that it is not legitimate to call them "parasexual".

SPECIFIC POLYHYDROXY COMPOUNDS AS

COFACTORS OF ENZYMIC ADAPTATION AND

ITS INHERITANCE

P. P. Slonimski and H. de Robichon-Szulmajster

Laboratoire de Genetique Physiologique du CNRS, Gif-sur- Yvette

It is a part of the definition of the word "truism", and not
the least important one, that it is accepted as something self-
evident. A truism probably as old as genetics is the one
exemplified by the following quotation from a well known
textbook: "The most important property of a gene is that it
reproduces itself, that is, forms a copy of itself from material
present in the cell. Furthermore, genes may form and give ofP
substances which influence specific reactions, first within the
cell and subsequently elsewhere in the organism. These two
properties or functions of genes, that of autocatalysis and of
heterocatalysis, may prove to be two aspects of the same
process : the specific gene products given off* in the cell may be
merely byproducts of the reactions of gene synthesis" (Sinnot,
Dunn and Dobzhansky, 1950). This statement applies not
only to genes but to all types of genetic material whether
nuclear or cytoplasmic. According to the authors' preferences,
the autocatalytic function is referred to as autoreproduction,
self-duplication, copying, covariant replication or transmission
of information, most probably from nucleic acid to nucleic
acid, while the heterocatalytic function is generally described
as control, determinism or information transfer from nucleic
acid to proteins and enzymes in particular. A purely formal
scheme can represent these two functions:

NA ► NA — >► NA >►

I > I

I ■ I

I y V

Enzyme Enzyme Enzyme
210

COFACTORS OF EnZYMIC ADAPTATION 211

The purpose of the present paper is to introduce a third
member into this scheme. This member will be referred to as
"cofactor" and the two arrows added to the scheme symbohze
that it is specifically involved in the autocatalysis and in the
heterocatalysis of a special type of the genetic material.

NAWjsr-*- NA— *-NA — >
r'^^Cof actor
Enzyme

At the present time the evidence concerning autocatalysis
is based on the study of the non-Mendelian genetic material
responsible for cytochrome oxidase synthesis in yeast. The
evidence concerning heterocatalysis is based on the study of the
oxygen-induced synthesis of the cytochrome system and of
the induced synthesis of maltozymase in yeast. Whether
cofactors play any role at the genie level in yeast or any role
in other organisms remains open for investigation.

The discovery of cofactors is due to a systematic study of
conditions that are required for the induced synthesis of
cytochrome oxidase in yeast and to a fortuitous observation.
Although rather of historical interest, it may be of some use to
report it briefly. One of the present authors (Slonimski) has
been working for the past seven years on the mechanism of the
synthesis of cytochrome systems, and was faced quite early
by the fact that cells harvested at a certain phase of growth
cycle were unable to adapt under the usual conditions. This
observation is quite common to students of induced bio-
synthesis of enzymes. A perusal of the literature shows
numerous examples of decreased adaptability according to the
"physiological state" of the cells (Gale, 1951; Pinsky and
Stokes, 1952). There is no general rule and depending on the
organism, enzyme, and medium employed the modifications
occur during lag, exponential or stationary phase of growth.
Undoubtedly there may be numerous causes, but in general
they have not been properly investigated, the authors being
satisfied with a plausible and ad hoc explanation. This author

212 P. P. Slonimski and H. de Robichon-Szulmajster

has committed himself to one of those, in an effort to explain
the poor adaptability to oxygen of exponential anaerobic
yeast by a deficiency in the free amino-acid pool (Slonimski,
1956). Subsequent experiments have shown that this explana-
tion was wrong. Anaerobic incubation in glucose-containing
buffer may be sufficient to restore adaptability. We started
naively to investigate the eff'ect of some carbohydrates and

C HO C HO

I I

HO— C— H H— C— OH

I I

HO— C— H H— C— OH

I I

CHg- OH CH2— OH

L Erythrose d

CHO CHO

I I

H— C— OH HO— C— H

I I

HO— C— H H— C— OH

I I

CH2— OH CH2— OH

L Threose d

Fig. 1. C4 Sugars.

their derivatives and found a batch of deoxyribose that had a
very strong stimulatory effect on adaptation.

We have eliminated the 2-deoxy-D-ribose which is without
effect, and tracing impurity after impurity we have arrived
at C4 sugars : tetroses. Four members of this class are known
(Fig. 1). Three of them are found to be inactive; the fourth,
D-threose, has not yet been investigated. However, it is
possible to synthesize from pure tetroses by a relatively mild
chemical treatment certain compounds that have novel
biological activity. We shall call them cofactors, those
deriving from erythrose will be designated by E and those

COFACTORS OF EnZYMIC ADAPTATION 213

derived from threose by T. The treatment consists in heating
an acidified aqueous solution of the pure sugar, and the
chemical structure of these derivatives is actually being
investigated by Dr. Asselineau and Prof. Lederer. Further-
more, certain batches of commercial preparations of tetroses
are contaminated by substances that have biological pro-
perties analogous to those obtained by synthesis from pure
C4 sugars.

To verify the hypothesis that tetrose derivatives are in-
volved in the two functions of the genetic material, the
autocatalytic and the heterocatalytic one, we have studied
their action in four biological systems listed in Table I. Every
system has its own particular advantages and drawbacks, and
can provide adequate answers only to a certain type of ques-
tion. A general conclusion can be drawn if a reasonably
coherent picture is obtained by comparing results of the en-
semble. The principal information we can gain from the first
system is whether the cofactors may be involved in the trans-
mission, from the mother cell to the daughter cell, of the
genetic material responsible for cytochrome oxidase synthesis ;
to be more precise, whether they interfere with the interrup-
tion, brought about by euflavine, of the normal transmission
process. Analogous information can be obtained from the
study of the fourth system, with the advantage that the
mutation occurs spontaneously. This last system has a con-
siderable drawback, however, because of a possible effect on
the selection of mutants which precludes any rigorous inter-
pretation as to the mutation process. The study of mutation
by these two methods is relevant to autocatalysis but gives us
no information in respect to heterocatalysis. Data on this
point are given by the study of the second and third systems,
where enzyme synthesis takes place against a constant
genetic background. Furthermore, comparison of the first
with the second system enables us to study the two functions
of the same genetic material, while comparison of the second
with the third permits us to follow the heterocatalytic func-
tions of two different genetic materials.

214 P. P. Slonimski and H. de Robichon-Szulmajster

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COFACTORS OF EnZYMIC ADAPTATION 215

Induced "petite" mutation

Ephrussi and his collaborators have shown that clones of
normal yeast during their growth constantly give rise to
respiration-deficient mutants ("vegetative mutants" or
"vegetative petites") stable in vegetative reproduction. The
respiratory deficiency was shown to be due to lack of several
enzymes (including cytochrome oxidase) firmly bound, in
normal yeast, to particles which can be sedimented by
centrifugation and to behave as a non-Mendelian character in
crosses between normal and mutant yeast. Addition of euflav-
ine (2 : 8-diamino-iV-methylacridine) in sufficient concentra-
tion induces mutation in almost every newly formed bud, i.e.
the mutation rate is close to 1. It was suggested that the
mutation consists of a loss, or irreversible functional inactiva-
tion, of a particulate cytoplasmic autoreproducing factor, and
the question of the possible identity of this genetic material
with subcellular units, defined by various biochemical and
cytological criteria, was discussed. Vegetative mutants do
contain mitochondria that are morphologically similar to
those of normal yeast in spite of the fact that they do not
contain cytochrome oxidase. References to the various aspects
of this work will be found in Ephrussi (1953), Slonimski
(1953« and b) and Ephrussi, Slonimski and Yotsuyanagi
(1955).

Our experiments were performed in the following way. To a
culture of diploid or haploid yeast exponentially growing in a
synthetic, highly buffered medium with excess of glucose,
euflavine is added at zero time. After 6-6-5 hours, cells are
plated on euflavine-free medium and the number of mutant
clones scored. It can be seen from Table II that the difference
in growth of the induced and the control culture is so small
that the frequency of mutants directly reflects the mutation
rate and selection is excluded. Furthermore, for approxi-
mately the first half of a cellular generation no mutants appear
in the population. The average mutation rate calculated over
the period of 2 • 5 generations being • 73, the actual mutation
rate is close to 1 for the last two cell generations.

216 P. P. Slonimski and H. de Robichon-Szulmajster

Table II

Comparison of growth and mutation of control and

INDUCED cultures

No
indepen-
dent
cultures

No
colonies
counted

Fre-
quency

of
mutants

(%)

No cell
genera-
tions

Mutation rate*
( X 10-3)

minimum

maximum

Control

38

ca. 16000

1-2

2-72

1

2

+ Euflavine
Ito2xl0-6M

68

ca. 30000

67-8

2-52

720

7i0

Data from 12 independent experiments pooled together.

* The mutation rate is defined here as the probability of a bud talien at random giving rise
to a mutant clone (of. Marcovlch, 1951).

15 X 10

Fig. 2. Frequency of respiration-deficient mutants
versus euflavine concentration.

Other conditions as in Table III.

COFACTORS OF EnZYMIC ADAPTATION

217

Fig. 2 gives the frequency of mutants as a function of
euflavine concentration. It is interesting to find that the
relation is not Hnear. The two simplest explanations of this
result are either that two or more molecules of euflavine are
necessary to produce a mutational event, or that we are
dealing here with a threshold phenomenon. This last inter-
pretation means that a concentration of euflavine up to
3 X lO^'^M is rendered ineffective, or neutralized by the cells.

Table III

Competition between euflavine and purines
IN induction of mutation

Mutants

Addition

Frequency {%)

Suppressed (%)

Control

0-3

_

Euflavine 1

•2X10-6M

66-8

j^

+ A004 + G004

60-3

10

+A006 + G006

52-9

21

+A0-08 + G0-08

12-2

82

+A0-08

53-7

20

+A012

15-0

77

+ GO-OS

60-9

9

2

4X10-6M

71-2

—

+A012 + G012

61-3

14

+A016 + G016

41-7

42

Expt. G 12. Concentration of adenine and guanine in mg./ml.

Addition of a mixture of nucleic acid constituents suppresses
the mutagenic action of the dye. Their antimutagenic action
is competitive and may be complete. By studying the sup-
pressive effect of individual nucleic acid bases, either singly
or in various combinations, it was found that adenine is by far
the most effective; guanine and thymine are about three
times less active than adenine. Uracil, cytosine, ribose and
deoxyribose are ineffective (Table III).

218 P. P. Slonimski and H. de Robichon-Szulmajster

We can turn now to the cof actors. Their addition con-
siderably changes the action of euflavine. Addition of E acts
hke that of adenine, i.e. suppresses the occurrence of mutation
(Table IV). On the contrary, the addition of T, which derives

Table IV
Antimutagenic action of cofactor E

ExpL

Addition

Mutants

Fre-
quency
(%)

Sup-
pressed
(%)

G17

Control

Euflavine • 6 x IQ-^m

+ Cofactor E{c-1)
1-5x10-%

-\- Cofactor E{c-1)

0-2
43-8
23-8
73-9
641

46
13

G 19

Control

Euflavine 1 -0 x 10"%

„ „ -\- Cofactor E (c-l)

„ „ + Adenine

+ Cofactor E (c-l)

11

76-8

14-3

35-5

1-3

82

55

100

G27

Control

Euflavine 1 -0 x 10"%

„ + Cofactor E (c-2 )
(J-2)
(J-4)

0-7
63 1
41 1
25-8
12-6

35
59

80

Conditions: Adenine 0-15 mg./ml.

Source of cofactor: commercial erythrose batch c-l : 1 mg./ml.;

batch c~2 : 1 mg./ml.
synthetic derivative of pure d -erythrose
(preparations j-2 and j-4 : equiv. 1 mg./ml.)

from the threo isomer of tetrose instead of the erythro one,
potentiates the action of euflavine. In other words, it acts
against adenine (Table V). Cofactor T by itself does not pro-
duce mutation.

COFACTORS OF EnZYMIC ADAPTATION

Table V

Promutagenic action of Cofactor T

219

Expt.

Addition

Mutants

Fre-

Stimul-

quency

(%)

ation
(%)

G17

Control

0-2

Euflavine • 6 X IQ-^M

43-8

—

+ Cofactor T
1-5x10-%

50
73-9

14

,, „ + Adenine

„ -^Cofactor T

58-1
69-1

19

G19

Control

11

Euflavine 1 -0 x 10"%

76-8

—

,, + Adenine

„ +Cofactor T

35-5
61

72

Conditions: Adenine: 0-15 mg./ml.

Source of cofactor: commercial threose 1 mg./ml.

Table VI gives a list of substances assayed to verify whether
the effect of E can be duplicated with something else. All
were found inactive. For the sake of comparison two results
obtained with synthetic derivatives of pure D-erythrose are in-
cluded. The actual amount of the derivative is unknown and is
probably much smaller than the quantity of the sugar of origin.

Euflavine is a very reactive dye that forms readily additive
complexes with a great number of substances (nucleic acids,
proteins, deoxyribonucleotides etc. ; cf. Peacocke and Skerrett,
1955).

Is it not possible that the interaction of purines and
cofactors with euflavine is a chemical combination occurring
in vitro outside the cells, and bearing no relation to the cellular
receptors? This question can be answered in a negative way,
for the following reasons :

{a) The complex formation in vitro could easily explain the
action of E but only with difficulty the action of T.

220 P. P. Slonimski and H. de Robichon-Szulmajster

Table VI

Substances found inactive as antimutagens
("Petite" induction by 1 to 1-5 xlQ-e m Euflavine)

Substance

Concentration

Mutants

(mg.lml.)

suppressed {%)

D-Glucose

1 to 10

D-Ribose

1

1

2-Deoxy-D-ribose

0-5 to 1

L-Arabinose

1

D-Xylose

0-5 to 1

3

D-Sedoheptulose

1

8

D-Ribulose

1

7

L-Erythrulose

1

DL-Glyceraldehyde

1

5

D-Erythrose

0-5 to 1

L-Erythritol

2

2

Glycerol

2

1

L-Threose

1

3

Gluconic acid lactone

1

3

DL-Glyceric acid

1

Reductone

1

4

Furfural

01

4

Kinetine

01

For Comparison

3

D-Erythrose derivative

No. J-5

equiv. 1

63

No. H-2

1-5

98

(h) The amount of euflavine fixed by the cells in the presence
or in the absence of adenine is practically the same. It can be
measured spectrophotometrically after extraction by HCl-
ethanol. To explain the suppression of mutation the amount
fixed should have been more than halved.

(c) The euflavine spectrum does not change upon addition of
adenine, while a definite change is observed upon addition of
adenine deoxyribonucleotide or nucleic acid (Peacocke and
Skerrett, 1955).

COFACTORS OF EnZYMIC ADAPTATION 221

(d) The results obtained in the presence of euflavine are very
similar to those obtained in the absenee of the dye.

It is concluded that adenine and E favour the transmission
of the genetic material responsible for cytochrome oxidase
synthesis, while T acts in the opposite way.

Cytochrome oxidase adaptation

Cytochrome oxidase synthesis is the heterocatalytic func-
tion of the genetic material sensitive to euflavine. This
synthesis can be easily studied in yeast that has been first
grown anaerobically, then washed, suspended in glucose-
containing buffer and aerated. In the absence of molecular
oxygen there is no formation of respiratory enzymes but the
genetic material remains unchanged even after hundreds of
cellular generations. In such an anaerobically grown yeast,
oxygen induces the formation of the whole chain of haemo-
proteinic enzymes (including cytochrome oxidase) with conse-
quent re-establishment of respiration. The synthesis of these
enzymes takes place in the absence of an external nitrogen
source and in the absence of cellular multiplication. The
references concerning various aspects of this phenomenon can
be found in Ephrussi and Slonimski (1950), Slonimski (1953a
and b; 1956).

When yeast is harvested during certain phases of anaerobic
growth and exposed to oxygen in glucose-containing buffer, its
cytochrome oxidase adaptation is very sluggish. Addition of
small amounts of E at the beginning of adaptation stimulates
considerably the rate of enzyme synthesis. Addition of T
produces the opposite effect, inhibiting adaptation (Fig. 3 and
Table IX).

In such sluggishly adaptable cells the addition of a mixture
of all nucleic acid bases produces a certain stimulation of
adaptation. There are two important features of this pheno-
menon: firstly, that E acts synergistically with the nucleic
acid bases; secondly, only a complete mixture of bases is
stimulatory. Certain incomplete mixtures, on the contrary,
are inhibitory (Table VII). The synergistic action of E and

222 P. P. Slonimski and H. de Robichon-Szulmajster

individual purine and pyrimidine bases is even more striking
(Table VIII). Here again we find the same situation as in the
mutation study : adenine is the most effective of all the bases
studied and thymine is slightly more effective than uracil.
Adenine and cofactor E act synergistically while cofactor T is
inhibitory. Substances assayed for stimulation and found

100

?o»

.jO co-fT E

9'

CONTROL

^CO-F T

HRS

Fig. 3. Effect of commercial erythrose (0-5

mg./ml.) or threose (1 mg./ml.) on respiratory

adaptation.

Adaptation in aerated phosphate buffer con-
taining 15 mg. glucose/ml.

inactive are the following: D-glucose, D-ribose, 2-deoxy-D-ri-
bose, D-arabinose, L-arabinose, D-xylose, L-fucose, L-rham-
nose, D-erythrose, L-erythrose, L-erythritol, L-threose formate,
glycerol, ethanol, gluconic acid lactone, i-tartaric acid,
furfural, furfuryl alcohol, meso-inositol, ergosterol, tween 80,
yeast extract (Difco), casein hydrolysate (enzymic), vitamin
Bi25 folic acid, haemin, haematoporphyrin, mixture of trace
elements.

COFACTORS OF EnZYMIC ADAPTATION

223

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^.2
o ^

224 P. P. Slonimski and H. de Robichon-Szulmajster

Table VIII

Synergistic action of cofactors and
individual bases in cytochrome oxidase adaptation

Increase in Enzyme

Addition

Adaptation

by cofactor

by base

None

67-4

Cofactor E

74-4

70

—

A

65-6

—

0(-l-8)

C

63-5

—

(-3-9)

T

62-3

—

0(-5-l)

U

64-5

—

0(-2-9)

Cofactor E+A

91-7

261

17-3

c

77-4

18-9

30

T

761

13-8

1-7

u

74-2

9-7

(-0-2)

Average of two experiments (A 222 a and A 224 a).
Conditions: Phos. Pht. Succ. buffer : -21^1 pH 4-5.

Glucose : 15 mg./ml.

Bases : ca. 8 x 10~^m ; A : adenine ; C : cytosine ; T : thymine ;
U: uracil.

Source of cofactors: commercial erythrose 0-25 mg./ml.

Adaptation during 7 hrs.

Table IX

Action of derivatives of d-erythrose in

PROMOTING cytochrome OXIDASE ADAPTATION

Addition

Adaptation

Stimu-
lation (0/^)

Control

71
75

—

D-Erythrose pure 0-25 mg./ml.

74

3

0-50

76

,, derivative No. H-1 equiv. 13

mg./ml.

90

23

„ 0-25

j»

90

H-2 „ 0-25

101

37

„ 0-50

5J

99

,, commercial 0-25 mg./ml.

101

39

0-50

102

Expt. A 230, conditions: Phos. Pht. Succ. buffer : 0-2m pH 4-5
Glucose : 15 mg./ml.
Adaptation during 6 hrs.

COFACTORS OF EnZYMIC ADAPTATION 225

Maltozymase adaptation

It is well known that glucose-grown yeast does not im-
mediately ferment certain disaccharides, e.g. maltose. It has
been shown by a number of workers (cf. Spiegelman, 1950;
Spiegelman and Halvorson, 1953), that fermentation can be
induced in the absence of a nitrogen source in cells suspended
in a buffer solution of the inducer. This phenomenon is
referred to as "maltozymase" adaptation.

The nature of the enzymes, of the inducer and of the genetic
material involved in this system is very different from that of
the cytochrome oxidase system.

The first questions to be asked are:

(a) Do tetrose derivatives affect maltozymase formation?

{b) If so, are they identical with cofactors involved in cyto-
chrome oxidase synthesis?

(c) What is their relation in respect to inducer, energy and
building-block requirements and to maltozymase function?

Our attempts were directed principally to providing ade-
quate answers to the first two questions, which are the basic
ones. Before presenting experimental evidence, it is however
necessary to consider a special feature of yeast growth that
may, at first, seem irrelevant.

Maltozymase induction is carried out as follows. Yeast is
grown on glucose, harvested, washed, suspended in a buffer
solution of maltose and the rate of aerobic fermentation
measured. Now, growth of yeast on glucose is a biphasic
phenomenon. In the first phase glucose, even under maximum
aeration, is mostly fermented to ethanol (Swanson and Clifton,
1948 ; Lemoigne, Aubert and Millet, 1954). In the second
phase (and if oxygen is present) the accumulated alcohol is
oxidized. The inefficiency of respiration during the glucose
phase is the result of inhibition of synthesis of the cytochrome
system by aerobic fermentation brought about by high glucose
concentration (counter Pasteur-effect; Slonimski, 1956). A
detailed study of the growth cycle has shown profound modi-
fications not only in the enzymic constitution but also in the

DRUG RES. — 8

226 P. P. Slonimski and H. de Robichon-Szulmajster

structure of the chondriome and of the perinuclear zone
(Ephrussi et ah, 1956).

If yeast is taken from the glucose fermentation phase it will
rapidly adapt to maltose. If it is taken from the ethanol phase
no maltozymase is formed.

In such cells an addition of E produces a dramatic effect,
restoring completely the maltozymase adaptation (Fig. 4).
Cofactor E does not act as a cofactor of maltozymase function,
as is shown by the following experiments.

300

200

100

Q,

C02ferm

CONTROL, GLUCOSE

-o — -^; — -n — — , , , a - »e

<< HRS 8

Fig. 4. Effect of commercial erythrose (0 • 5
mg./ml.) or glucose (0-5 mg./ml.) on malto-
zymase adaptation.
Adaptation in aerated plios. pht. succ. buffer
containing 10 mg. maltose /ml.

Firstly, addition of E does not provoke an immediate
fermentation of maltose but only permits adaptation to occur.
Depending on the amount of E added at zero time, adaptation
takes place more or less rapidly, but even with saturating
concentrations of E the half-maximal rate of fermentation of
maltose is attained only after ca. 3 hours. Secondly, addition
of E six hours after the addition of maltose in excess does not
bring about an immediate fermentation. This last experiment
is of critical importance. If cofactor E were involved in
permitting the expression of the maltozymase function, its

COFACTORS OF EnZYMIC ADAPTATION 227

addition should have provoked an immediate fermentation.
As this is not the case, it seems most probable that it is in-
volved in the formation of maltozymase. Moreover, addition
oi E ov T is without effect on the fermentation of maltose by
fully adapted yeast.

The addition of E is quite sufficient to transform non-
adaptable cells into normal ones, as judged by the rate and
extent of adaptation. Furthermore, E does not stimulate
adaptation of cells harvested during the glucose fermentation
growth phase. Therefore, it seems difficult to avoid the con-
clusion that the cells from the two phases of glucose growth
cycle differ by the presence or absence of cofactor E or some
substance derived metabolically from E.

Cofactor T added to adaptable cells prevents maltozymase
formation. We are faced, therefore, with a situation com-
pletely parallel to the cytochrome oxidase one. However,
preliminary experiments indicate that erythrose derivatives
active in the cytochrome oxidase system are different from
those involved in maltozymase adaptation.

A certain number of substances have been tested with
respect to their ability to replace cofactor E and found
ineffective. They are listed in Table X. To minimize variation
from one experiment to the other, a standard amount of co-
factor E contained in 100 \Lg. of a given impure preparation of
erythrose was run with every experiment and the results
recorded in relation to it. Perusal of Table X shows that :

(a) E does not act as an energy source. A great number of
compounds listed are actually fermented or respired by yeast
while remaining ineffective. Furthermore, E is not fermented
or respired by yeast, although it is metabolized.

(b) E does not act as a source of carbon units derived by means
of any known metabolic pathway. Representative members of
the glycolytic pathway, of the pentose oxidative cycle and of
the tricarboxylic cycle were found inactive.

(c) It is possible that E or its natural homologue may be
synthesized by the cell from small carbon fragments like

228 P. P. Slonimski and H. de Robichon-Szulmajster

Table X

SiTBSTANCES FOUND inactive IN PROMOTING
MALTOZYMASE ADAPTATION

Substance

Concentration
(mg./ml.)

Stimulation (%)

D-Glucose

003to0-5

D-Fructose

01

L-Sorbose

0-1

( + ) aa-Trehalose

0-1

1

D-Ribose

0-5

2-Deoxy-D-ribose

0-5

L-Arabinose

0-5

D -Xylose

0-5

D-Sedoheptulose

0-3

1

D-Ribulose

0-2

1

L-Erythrulose

0-2

1

L-Erythrose

01 to 0-2

4

D-Erythrose

01 toO-5

L-Threose

0-6

5

Araboketose

0-1

2

D-Sorbitol

01

7

D-Arabitol

0-1

8

L-Arabitol

01

1

L,-Adonitol

01

Meso-inositol

01

1

Erythritol

0-5

Furfural

0-2

Furfuryl alcohol

0-2

6

1:2:3: 4-Diepoxybutane

0-4

Glycerol

0-4 to 10

to 25

i-Tartaric acid

0-5 to 1-0

13

Acetic acid

002 to 0-2

to 27

DL-Lactic acid

002 to 0-2

L-Malic acid

002 to 0-2

15

a-Glycerophosphoric acid

01

DL-Glyceraldehyde

0-02 to 0-4

to 35

Diacetyl

01

For Comparison

14

Erythrose (Commercial)

01

100

glyceraldehyde or acetic acid. But these two compounds
produce only slight stimulation. Moreover, this stimulation
is variable from one experiment to another — in contrast to the

COFACTORS OF EnzYMIC ADAPTATION 229

stimulation brought about by cofactor E which is very much
greater and relatively constant.

Only few experiments were done on the mechanism of
action of E. It can be metabolized by the cells into an inactive
form. This can be demonstrated by adding first the cofactor
and delaying the addition of maltose. The stimulation is much
smaller than the one produced by simultaneous addition of
both compounds. Furthermore, the same experiment clearly
shows that the cofactor acts in a different way from the
inducer and does not replace it.

Spontaneous "petite" mutation

Ephrussi and Leupold (unpublished) discovered certain
yeast strains that present a much higher spontaneous mutabil-
ity than usual. Preliminary experiments were carried out
with one of these (strain C982/19b). The results were rather
unexpected. Adenine and cofactor E seem to increase the
percentage of mutants, while cofactor T seems to decrease it.
It is interesting to note that, although the roles are reversed,
the coupling of cofactor E with adenine is maintained.
However, a possible effect on the selection rather than on the
mutation frequency has yet not been excluded.

Acknowledgement

The authors gratefully acknowledge the continued interest of Prof.
B. Ephrussi and the advice and help of Prof. E. Lederer and Dr. J.
Asselineau. Our thanks are due to Prof. Frerejacque, Drs. B. L.
Horecker, A. S. Perlin, N. K. Richtmyer and F. Skoog, who kindly put
at our disposal samples of rare sugars or their derivatives.

REFERENCES

Ephrussi B. (1953). Nucleocytoplasmic Relations in Micro-organ-
isms. Oxford University Press.

Ephrussi, B., and Slonimski, P. (1950). Biochim. hiophys. acta, 6, 256.

Ephrussi B., Slonimski, P., and Yotsuyanagi, Y. (1955). Nature,
LoncL, 176, 1207.

Ephrussi, B., Slonimski, P., Yotsuyanagi, Y., and Tavlitzki, J.
(1956). C. R. Lab. Carlsberg, Ser. physioL, 26, 87.

Gale, E. F. (1951). Chemical Activities of Bacteria. Cambridge
University Press.

230 P. P. Slonimski and H. de Robichon-Szulmajster

Lemoigne, M., xVubert, J. P., and Millet, J. (1954). Ann. Inst.

Pasteur, 87, 427.
Marcovicii, H. (1951). Ann. Inst. Pasteur, 81, 452.
Peacocke, a. R., and Skerrett, J. N. H. (1955). /// Int. Congr.

Biochem., Abstracts, p. 21.
PiNSKY, M. J., and Stokes, J. L. (1952). J. Bact., 64, 337.
SiNNOT, E. W., Dunn, L. C, and Dobzhansky, T. (1950). In Principles

of Genetics, p. 457. New York: McGraw Hill.
Slonimski, P. (1953«). Formation des Enzymes Respiratoires chez

la Levure. Paris: Masson.
Slonimski, P. (1953fe). In Adaptation in Micro-organisms, Eds.

Davies, R., and Gale, E. F. Cambridge University Press.
Slonimski, P. (1956). /// Int. Congr. Biochem., p. 242. New York:

Academic Press.
Spiegelman, S. (1950). In The Enzymes, 1, New York: Academic Press.
Spiegelman, S., and Halvorson, H. O. (1953). In Adaptation in

Micro-organisms, Eds. Davies, R., and Gale, E. F., Cambridge

University Press.
Swanson, W. R., and Clifton, C. E. (1948). J. Bact., 56, 115.

DISCUSSION

Davis: I gather that these studies on adaptive enzyme formation were
carried out under conditions without growi;h, relying on nitrogen sources
within the cell. Have you studied the effect of your compounds on
adaptation under conditions of gro\\i;h ?

Slonimski: We have started experiments on Esch.coli which is probably
the best organism for such a study.

Davis: Your system in yeast has not been tested with growing cells?

Slonimski: Adaptive enzyme formation (cytochrome oxidase and
maltozymase) was studied in the absence of growth and in the absence
of cellular multiplication. Mutation studies were carried out, however,
with cells growing exponentially in full medium. Cytochrome oxidase
synthesis in yeast has a preferential character. ^Vhen, during adaptation
taking place in glucose buffer, one starts growth by addition of growth
factors and a nitrogen source one gets first a temporary inhibition.
Instead of getting a higher rate of synthesis one gets a lower one. This
inhibition is resumed in about an hour.

Pollock: Did you get a completely parallel effect with these substances,
cofactor E and cofactor T?

Slonimski: T is inhibitory in maltozymase, in cytochrome oxidase and
in the euflavine-induced mutation (i.e. it inhibits the transmission of
the normal genetic material). With respect to erythrose derivatives, we
are not yet certain about this, but the substance that works on malto-
zymase may be different from the one that works on respiratory adapta-
tion or the induced "petite" mutation.

We have preparations that stimulate cytochrome oxidase formation
and are not active on maltozymase ; and vice versa, we have one substance
which stimulates maltozymase adaptation and is inactive on cytochrome

Discussion 231

oxidase. But we have an absolutely parallel effect between respiratory
adaptation and the induced "petite" mutation.

Westergaard: What happened with your fourth system, that of
spontaneous mutation ?

Slonimski: This gave a very curious result, but I did not want to talk
about it because selection is not excluded and the results are preliminary.
Cofactor E does not decrease the percentage of mutants ; on the contrary,
it increases it. Adenine does the same. So, from this point of view, it is
exactly the same as in the euflavine-induced mutation. However, the
situation is reversed because it is E which decreases the percentage of
mutants in the euflavine system, while it increases it in the spontaneous
mutation ; conversely, T, which increases the percentage of mutants in
the euflavine system decreases it in the spontaneous system. Spon-
taneous and induced mutants are both respiratory deficient, but recent
work by Ephrussi, Roman and Hottinguer showed that in many of the
highly mutable strains the "petites" are genetically different from the
acridine-induced ones.

Westergaard: These are the somatic "petites"?

Slonimski: I think that the "petites" of C982/19b are somatic (vegeta-
tive) but they may be suppressive — dominant instead of being recessive.

Davis : How would you contrast the action of these substances in the
mutagenic system with the action of other ordinary mutagens?

Slonimski: "Petite" mutation is genetically something rather unusual
and from this material one should not extrapolate hastily to any other
phenomenon. It shows, among other things, a unique relation between
the process of mutation and the process of adaptation, both processes
being, of course, quite distinct. This common reaction is specifically
inhibited by acridines and involves tetrose derivatives. How they act
is purely h\^3othetical at the present time. It seems to me possible that
a tetrose nucleotide or its polyiner is a part of the genetic material.
Another possibility is that it acts like a co-enzj^me in the synthesis of the
proper genetic material. ^Vllen we start getting down to the molecular
level it may be difficult to distinguish between the immediate product of
the action of a genetic determinant and parts of its structure. The point is
that tetrose derivatives seem to be specifically involved in both.

Westergaard: What is the present status of the mitochondria in the
somatic "petite"?

Slonimski: It contains mitochondria which are morphologically similar
to those from normal cells. "Petite" mitochondria don't contain cyto-
chrome oxidase, of course, therefore they will not be stained by Janus
green-B, but on fixed preparations they can be revealed by Altmann
staining. The present status of this problem has been reviewed by
Ephrussi, Slonimski and Yotsuyanagi (1955, loc. cit.).

Pollock: Have you found any substance or even any conditions which
will affect the mutation rate to "petite" without producing a comparable
effect on adaptation?

Slonimski: Mutation and adaptation can be dissociated by cellular
multiplication. The former requires proliferation, and mutant clones
derive almost exclusively from cells formed in the presence of the drug.

232 Discussion

Respiratory adaptation does not require proliferation. On the other
hand, all the substances showing a specific mutagenesis do produce a
comparable inhibitory effect on adaptation. The action of differently
substituted acridines is quite parallel, and the concentrations of euflavine
necessary to produce a half-maximal effect are very similar for mutation
and for adaptation (6 to 7 X 10-'m). However, the converse is not true.
Several substances like benzimidazole or dinitrophenol inhibit adaptation
without producing mutants. It should be added that Harris in our
laboratory found that a continuous anaerobic culture for about a hundred
cellular generations neither increases the percentage of "petites" in the
population nor diminishes the capacity to form cytochrome oxidase
adaptively (Harris, M. (1956), J. cell. comp. Physiol., 48, 95).

Fulton: How did you measure the cytochrome oxidase present?

Slonimski: The method we use routinely is as follows: we make an
extract of cells, spin down the so-called granules, then either we measure
the oxygen uptake in the presence of the hydrogen donor, which is
ascorbic acid, and in the presence of a saturating amount of cytochrome
c (more precisely in the presence of 4 different concentrations of cyto-
chrome c and extrapolate to saturation); or we measure spectrophoto-
metrically the rate of oxidation of reduced cytochrome c. This is quite
laborious. Not all the experiments were performed in this way, the
majority of the experiments were performed by measuring the rate of
overall respiration of intact cells under the conditions where we have
shown previously that it is proportional to the amount of cytochrome
oxidase, as measured by the first method.

Fulton: Your first method requires a lot of material, and your second
one probably less ?

Slonimski: Much less.

Fulton: Had you any trouble in reducing cytochrome c?

Slonimski: We had a little difficulty at the beginning when we used
palladium.

Fulton: We have always experienced trouble in recovering pure reduced
cytochrome c, after reduction with palladium, unless the metal is removed
in an inert atmosphere, and the method is time-consuming.

Slonimski: The most satisfactory method for reduction is, in my
opinion, the one introduced by Chantrenne (1955, Biochim. biophys.
acta., 18, 58). It consists in reducing cytochrome c by passing it on
Duolite S-10, treated previously with Na2S204. We used this method
with commercial cytochrome c which contains some impurities.

Fulton: It tends to undergo auto-oxidation.

Slonimski: If you have peroxides, yes. However, the cytochrome c
reduced on the ion exchange resin can be kept reduced for months if
frozen. I personally prefer the first manometric method, although it
requires a lot of material; but with yeast we had no trouble.

Davis: Have you tried the mutagenic action of your compound on
more ordinary mutations?

Slonimski: We have already started some experiments on two things:
one is mitotic crossing over in yeast and the second is what is called
gene conversion (non-reciprocal recombination).

Short Communication

DEVELOPMENT OF RESISTANCE TO STREPTOMYCIN
IN SERRATIA MARCESCENS

B. Gyorffy and I. Kallay
Institute of Genetics, Hungarian Academy of Sciences^ Budapest

Our work in the field of bacterial genetics was begun three years
ago, and the results of our studies concerning the problem of the
development of streptomycin resistance in Serratia marcescens are
summarized here.

The bactericidal action of streptomycin was determined by the
proportion of cells surviving on exposure to streptomycin for a
limited time. Our results agreed with those already obtained for
other bacteria (Demerec, 1951; Linz and Lecocq, 1955).

The bacteriostatic action of streptomycin was measured by
counting colonies formed on streptomycin-agar. The overall picture
of the distribution of the surviving fraction was similar to that
reported by other workers (Demerec, 1948; Welsch, 1952). The facts
that repeated experiments gave the same fraction of survivors and
that not all of the cells survived even at low concentrations sug-
gested the pre-existence of resistant variants in the populations
(Sneath, 1956).

Colonies picked at random from streptomycin plates were retested.
Many of these clonal populations, and in particular those from the
faintly pigmented colonies, when grown at 4, 6 and 8 [ig. strepto-
mycin/ml. consisted almost wholly of cells having the same sensitivity
as that of the original population. Some colonies, however, with
normal pigment production, consisted of cells capable of forming
the same number of colonies on plates containing 8 and 16 \xg.
streptomycin/ml., respectively, as they did on control plates; and
giving about 10 per cent survival on plates containing 24 and 36 ]ig.
streptomycin/ml., respectively. This low degree of resistance of
substrains was maintained after several subcultures in drug-free
medium.

Above the threshold concentration, when strong selection had
already taken place, the possibility of resistant colonies occurring
was increased. Although some of the colonies formed on plates con-
taining 16-36 fxg. streptomycin/ml. consisted of " persistors ", many

233

234 B. Gyorffy and I. Kallay

colonies survived the same concentrations of streptomycin, on
retest, in a high percentage ; the cells of some of these colonies even
extended their survival range beyond that of the parental popula-
tion, in some cases up to 50-100 [ig. streptomycin/ml. Nevertheless,
on subsequent retest the colonies formed at higher concentrations
of streptomycin showed many "normal overlaps"; and it is by no
means unlikely that their occurrence can be explained by the
physiological-biochemical heterogeneity of the cell population
within a colony, as suggested by Sevag (1955).

In general, we observed that only a small fraction of the isolates
of colonies formed on plates containing 50 and 80 [ig. streptomycin/
ml. consisted of cells which not only gave survival at above 16 and
24 yig. streptomycin/ml., respectively, but which also tended to
keep this high resistance unaltered during a number of transfers.
It seemed likely, however, that this small proportion of greater
resistance originated from the selection of second- step variants
which had arisen on the plates on retest (Abraham, 1953).

In summary, variable levels of resistance were obtained in a single
exposure to streptomycin, and differences were observed between
the independent isolates from the same plate. The survival dis-
tributions in re tests were highly variable, and we found no adjust-
ment to the concentration at which colonies w^ere isolated (Barer,
1951; Gibson and Gibson, 1951; Eagle, Fleischman and Levy, 1952).
The occurrence of "normal overlaps" of an unstable, readily rever-
sible nature was very frequent but stable variants with low levels
of resistance also developed.

Serial transfers were carried out with different strains of Serratia
marcescens. The discrete steps resulting in progressively higher
levels of survival were clearly evident. The small size of inocula
(10^ — 10* cells) used in the transfers excluded the presence of pre-
existent resistant variants.

The average range of the first steps was less variable than that
of the succeeding ones. In some instances, resistance to 100 \Lg.
streptomycin/ml. was attained by three successive steps; in others,
the number of transfers separating the steps differed, and flat
plateaus also occurred (Oakberg and Luria, 1947). No quantitation
was made as proposed by Treffers (1956).

This stepwise development of higher resistance was also apparent
in serial transfers on solid medium, when the isolated single colonies
were repeatedly retested. The average range of the single steps
was approximately the same as that obtained in transfers in liquid
medium. The outgrowth of second-step variants appearing in
microcolonies on a background region on plates containing strep-
tomycin in concentrations above 16 (i,g./ml. was very definite in the

Drug Resistance in Serratia marcescens 235

series of replica plates. The majority of the cells of colonies isolated
at increased concentrations were resistant to the same range of
concentrations, and the small fraction surviving on plates with
considerably higher concentrations represented the further-step
variants.

In general, the higher the concentration to which the organism
was exposed, the greater was the resistance of a fraction of the
emergent colony; while colonies surviving concentrations of 80-
100 [xg. streptomycin/ml. in the repeated retests, consisted mainly
of fully resistant cells. In some instances, variants resistant to high
levels of streptomycin (100 or 1000 (i-g./ml.) were selected from large
populations of sensitive cells, by plating, and were supposed to have
originated by a single step (Newcombe and Hawirko, 1949).

We do not believe that even a low degree of stable resistance to
streptomycin was acquired by physiological adaptation alone, as
claimed by Gibson and Gibson (1951). Adaptive processes may play
a role in the phenotypie manifestation of the resistant variant, and
unquestionably they favour colony formation of persistors. Thus,
the simultaneous appearance of "normal overlaps" and of resistants
with a range of phenotypie variability always tends to obscure the
stepwise discontinuity, and simulates a "continuous spectrum"
which could be used to support the theory of physiological adapta-
tion (Eagle, Fleischman and Levy, 1952).

"Repetitive training", i.e. repeated subculturing in subthreshold
concentrations (0-1 and 4 \ig. streptomycin/ml.) is now in progress.
Results obtained so far are that, in the series where a concentration
of • 1 \Lg. streptomycin/ml. was used, the survival fraction remained
unaltered after 20 transfers; whereas in the series where 4 {xg.
streptomycin/ml. was used, a moderate increase in the fractions
surviving concentrations of 16-50 [j,g./ml. w^as obtained already after
20 transfers. This is in agreement with results obtained by Eagle,
Fleischman and Levy (1952), Gibson and Gibson (1951) and Akiba
(1955); (see however EngUsh and McCoy, 1951). But, as yet, we
can only speculate as to whether enforced phenotypie modification
or emergence of step variants occurred during the prolonged
subculture.

The mutational origin of streptomycin-resistant variants of
Serratia marcescens was indirectly demonstrated by the "fluctuation
test" of Luria and Delbriick (1943). We are aware of the possibili-
ties of error in analysis by means of this test; variability between
independent cultures is not in itself conclusive evidence of mutation,
and the results should be treated with reserve (Barer, 1951 ; Hinshel-
wood, 1952). In repeated experiments, a highly significant variation
was observed in the number of resistant variants among independent

236 B. Gyorffy and I. Kallay

cultures. This was compatible with the spontaneous occurrence of
resistant variants (Demerec, 1948; Newcombe and Hawirko, 1949).
The actual distribution of numbers of resistant cells in independent
cultures, in three fluctuation tests, was compared with the theoretical
distribution (Lea and Coulson, 1949; Armitage, 1953). The good
correspondence between the observed and theoretical distribution
of resistants was satisfactory ; a better correspondence could not be
hoped for within the limits of experimental error. This result can
be considered as quantitative evidence in support of the spontaneous
occurrence of streptomycin-resistant cells.

The mutation rates were calculated by means of the following
four methods: estimating the number of mutations (1) from the
proportion of cultures with no mutants (Luria and Delbriick, 1943);
(2) from the mean (Luria and Delbriick, 1943), (3) from the median
(Lea and Coulson, 1949) and (4) from the maximum value of the
number of resistants (Newcombe, 1948). The mutation rates were
found to be approximately of the order of 10~^ at a screening level
of 100 (Jig. streptomycin/ml., and progressively lower at screening
levels of 80, 50 and 30 [xg./ml. (in a single experiment).

Indirect-selection experiments, by the replica plating technique
of Lederberg and Lederberg (1952), have been carried out repeatedly
without success ; indirect-selection experiments by the use of enrich-
ment cycles (Cavalli-Sforza and Lederberg, 1956) are now in
progress.

On the whole, our observations seem to support the theory of the
mutational origin of resistance to streptomycin, in the case of
Serratia marcescens . These results will be published in detail.

REFERENCES

Abraham, E. P. (1953). Symp. Soc. gen. Microbiol., 3, 201.

Akiba, T. (1955). In Origins of Resistance to Toxic Agents, p. 82, Ed.

Sevag, M. G., Reid, R. D., and Reynolds, D. E. New York:

Academic Press.
Armitage, P. (1953). J. Hyg., 51, 162.
Barer, G. R. (1953). J. gen. Microbiol., 5, 1.

Cavalli-Sforza, L. L., and Lederberg, J. (1956). Genetics, 41, 367.
Demerec, M. (1948). J. BacL, 56, 63.
Demerec, M. (1951). Genetics, 36, 585.

Eagle, H., Fleischman, R., and Ltlvy, M. (1952). J. Bad., 63, 623.
English, A. R., and McCoy, E. (1951). J. Bad., 61, 51.
Gibson, M. L, and Gibson, F. (1951). Nature, Lond., 168, 113.
HiNSHELWooD, C. N. (1952). Ball. World Hlth. Org., 6, 1.
Lea, D. E., and Coulson, C. A. (1949). J. Genet., 49, 2G4.
Lederberg, J., and Lederberg, E. (1952). J. Bad., 69, 184.

Drug Resistance in Serratia marcescens 237

LiNZ, R., and Lecocq, E. (1955). C. R. Soc. Biol, Paris, 144, 1698.

LuRiA, S. E., and Delbruck, M. (1943). Genetics, 28, 491.

Newcombe, H. B. (1948). Genetics, 33, 447.

Newcombe, H. B., and Hawirko, R. (1949). J. Bact., 57, 565.

Oakberg, E. F., and Luria, S. E. (1947). Genetics, 32, 249.

Sevag, M. B. (1955). In Origins of Resistance to Toxic Agents, p. 370,

Ed. Sevag, M. G., Reid, R. D., and Reynolds, D. E. New York:

Academic Press.
Sneath, p. H. a. (1956). J. gen. Microbiol, 13, 561.
Treffers, H. p. (1956). AntibioL Chemother., 6, 692.
Welsch, M. (1952). Bull World Hlth. Org., 6, 173.

DISCUSSION

Hayes: \Vliat was the mutation rate at the lower level of selection,
i.e. at 30 [xg./ml.?

Gyorffy: That was 2-5 X 10-«.

Stocker: In the case where you used low concentrations to screen, so
that a high proportion of the colonies formed were "persistors", i.e. they
were not resistant on retest, did you find any evidence of fluctuation, i.e.
was there marked variation between replicate cultures grown from small
inocula, as to number of persistor colonies formed ? One theoretically
possible explanation for persistors is that they are cells which result from
a mutation-like process which occurs very rapidly in each direction, so
that there is correlation, persisting for a small number of generations
only, between the phenotypes of the parent and the descendants; in
which case one might perhaps expect to find some degree of fluctuation
in a test of that sort.

Gyorffy: We did not observe fluctuation; but mostly we used the
higher degree of resistance, because repeated retesting gives rise to a
great deal of difficulty at lower concentrations.

Cavalli-Sforza : Dr. Stocker's remark might be amplified by saying
that not only a high back-mutation rate, but also a low selective value,
i.e. a low growth rate of the mutant might easily be responsible for
unstable resistance. It is important to think in terms of gro\vi:h rates also,
because that is so much more likely to be effective from the kinetical
point of view than the mutation rate.

Knox: We have heard a great deal about the role of DNA in deter-
mining genetic characteristics and so on, but we have not heard anything
about the role of the protein in the gene. Is it the view of the geneticists
that a gene is a naked DNA or that it is a DNA with some protein fitted
to it in some specific way? If so, it is conceivable that a good deal of this
controversy as to what is an unstable mutant and what is an adaptive
change might actually be due to some change occurring in the protein
part of the gene itself. If that were the case, then the DNA in such a
system might be normal, but there might be not just a purely adapta-
tional change in the enzyme of the cell, but some change in the protein

238 Discussion

component of the gene, if indeed the gene has got a protein component.
In that case, you would have an abnormal protein which was in fact
a part of the gene ; you would then have a system which could be, up to a
point, capable of repeating itself, but gradually it would revert to nor-
mal as the stimulus which evoked the production of the abnormal protein
disappeared. I should like to know what is thought about this problem of
the relation between the protein and the DNA in the gene.

Hotchkiss: As far as I know, DNA has never exerted its function in a
cell that did not contain protein! We don't know any of the steps in the
process of gene action, but one of them might well be a forming of a
complementary protein-like structure ; but we have not the evidence to
give an answer.

Eagle: As a fairly recent convert to the thesis that most resistant
variants which we see in cultures arise as a result of mutation and selec-
tion rather than physiological adaptation, I confess that I am still
puzzled with respect to terminology. I am referring specifically to so-
called first-, second- and third-step mutations. If one takes a culture not
previously exposed to antibiotics, then, varying with the organism and
with the antibiotic, the number of survivors in an agar plate may
fall off steeply as the concentration of antibiotic is increased, or may
fall very slowly. In either case, if a surviving colony is subcultured, and
the distribution of resistance redetermined, then as Dr. Gyorffy has just
reported, some have essentially the same spectrum of resistance as the
parent population. Usually, however, the average resistance of sample
clones growing out at a given concentration of antibiotic tends to be
related to the concentration to which it had been exposed. A single clone
may therefore give rise to colonies which differ widely in their resistance
to antibiotic ; and the gradations are almost imperceptibly fine. Are all of
these organisms first-step mutants varying widely in resistance, as these
results and those of Dr. Hughes would imply; or are there first-, second-,
third-, fourth- and even fifth-step mutants within a single clone?

Cavalli-Sforza: It refers to the sequence in which you have selected.

Eagle: I think we have to be quite clear on this point. I had assumed
that a first-step mutant represented the first mutational step toward
increased resistance, and it has been so described. If we now redefine the
first-step mutant as that isolated on the first attempt at selection, such a
mutant could be a second- or third-step mutant in terms of what actually
transpired.

Dr. Hughes has reported some observations which indicate that
extremely fine gradations of resistance may occur within a single clone.
Operationally, as Prof. Cavalli-Sforza would define them, these are all
first-step mutants; in fact, they could be second-, third, or fourth-step
mutants. The terms should be used with caution, and perhaps avoided.

Cavalli-Sforza: In relation to the first-step variations observed by Dr.
Hughes, I don't know that they are mutants.

Eagle: The differences we have observed are certainly stable. Whether
Dr. Hughes' extremely fine steps are similarly stable apparently remains
to be determined ; but at least for streptomycin, penicillin and chloram-
phenicol, these small differences are real and stable. Are they all to be

Discussion 239

called first-step mutants merely because they are distinguished in the
first attempt at selection?

Cavalli-SJorza: If that happens after one exposure, I think yes. A
first-step mutant might on the other hand be the result of more than
one mutation: only genetic analysis could tell. This depends of course
on conditions of selection, and on mutation rates.

Lederberg: This terminology was developed when our only method of
analysis was mutational; the first-step was just an operational statement
as to how many sequences of selection were made. From that you might
wish to infer that there was a one-gene change, which is not necessarily
true. We now have methods of finding out in some organisms how many
genes are involved at various levels of resistance. I think that is a much
more important question if what you are interested in is the genetic
structure of the resistant forms. I would not ordinarily rely too implicitly
on the number of steps with which you could get a highly resistant form
to tell how many genes are involved. Dr. Hotchkiss has some contra-
dictory evidence on this point. In one system with penicillin he was able
to correlate them very well, in another with sulphonamide resistance he
had a one-step isolation which has given him at least three loci. Ap-
parently there can be coincidences or accidents which will lead to some
discrepancy between the number of steps you think you made and the
number of mutations which have really accumulated.

Dean : It becomes rather important when you calculate mutation rates
from the Lea-Coulson formulae, because in their mathematical analysis
the distribution is based by hypothesis on one genetic change. It should
not be applied to polygenic systems.

Lederberg: You can make reasonable corrections for it, if you keep in
mind that what you are measuring is the summation of all genetic
changes that have the phenotype for which you are scoring.

Fulton: It is a most difficult problem to isolate DNA in highly poly-
merized form from bacteria, and I should like to know what accompanies
the DNA in the bacteriologists' cell-extracts. Since even the elegant
experiments of McCarty on the isolation of DNA from pneumococci have
been criticized, do you think that products labelled DNA by the
bacteriologist are really DNA ?

Yudkin: The important point is that the transforming activity is
destroyed by DNAse.

Hotchkiss: Then you have another tube in the laboratory which one
labels DNAse ; how good is that DNAse ?

Davis: Those interested in the genetics of drug resistance have
deliberately studied only increments of resistance large enough to permit
clean screening. It seems to me we pose impossible questions when we
take the techniques that were suitable for such large degrees of resistance
and apply them in the region of tiny-step resistance. As Dr. Gyorffy
pointed out, in this region there is an overlap between physiological
variations and mutations. A cell may survive a borderline concentration
of drug for physiological reasons and then continue to grow in a slow,
struggling manner. But before this cell has given rise to a colony of a
million cells the clone may have developed one or more mutations that

240 Discussion

improved the growi^h rate in the presence of the drug and so were
selected. Hence, when Dr. Eagle subcultures such a colony and finds that
it gives a new curve of resistance, this does not tell us whether the
parental cell of this colony was genetically different from sister cells that
failed to yield colonies. It seems to me that it will take an elaborate new
kind of experimentation, along lines initiated by Dr. Hughes, to analyse
the role of mutation and that of physiological adaptation in the region of
small differences in resistance.

Eagle: You have answered my question, at least in terms of your own
opinion: that the term "step" is a misnomer, because we don't know
either how many mutational steps there have actually been or their
individual magnitude.

DISTRIBUTION OF DRUG-RESISTANT

INDIVIDUALS IN CULTURES OF

MYCOBACTERIUM TUBERCULOSIS

R. Knox

Department of Bacteriology, Guy''s Hospital Medical School, London

Tuberculosis shows perhaps more clearly than any-
other disease the dangers associated with the development
of drug resistance. With streptomycin, ^-aminosalicylic acid
(PAS), isoniazid and other less commonly used drugs, initial
success in chemotherapy has often been followed by subse-
quent failure associated with the development of drug-
resistant strains. In vitro tests of drug sensitivity however
are in many respects unsatisfactory, and the information they
give is often difficult to interpret and to correlate with results
obtained in vivo. One of the reasons for this is the difficulty
in obtaining accurate information about the number of viable
tubercle bacilli and the proportions of drug-resistant indivi-
duals present in a given bacterial population.

Recently, we have been using semi-solid (0-125 per cent)
agar as a convenient medium for rapid culture of Mycobac-
terium tuberculosis and for the performance of viable counts
(Knox, 1955; Knox, Swait and Woodroffe, 1956). We have
also found media of this kind very useful for the performance
of drug sensitivity tests and for determining what proportion
of individuals in a given culture is resistant to different drug
levels (Knox and Woodroffe, 1957). Preliminary work
showed some interesting differences between streptomycin,
PAS and isoniazid in the patterns of resistance they showed
in this medium. It seemed, therefore, that it would be interest-
ing to investigate by the use of semi-solid medium the distri-
bution of drug-resistant individuals in different conditions of
incubation, with different drugs separately and together.

241

242 R. Knox

It was found that the apparent rate of development of
drug-resistant colonies in cultures of Myco. tuberculosis varied
both with the drug and with the medium. Some of these
differences have been described elsewhere (Knox and Wood-
roffe, 1957). With isoniazid in Kirchner semi-solid agar, pre-
sumptively resistant colonies of the drug-sensitive H 37 Rv
strain grew in decreasing numbers as the drug concentration
was increased. But colonies which had developed in tubes
w^hich initially contained quite high drug concentrations, up
to 10 [i.g./ml. or more, often appeared to be fully sensitive to
isoniazid when retested in subculture. Sometimes they
behaved as though they consisted of a "mixed" culture,
mainly drug-sensitive but containing more resistant cells
than a fully sensitive strain. This could be explained partly
by the fact that isoniazid decays rapidly in Kirchner medium
and any individual cells which might survive exposure to
isoniazid would be able to multiply when the drug decayed
below a certain level. Such individuals would clearly not be
true drug-resistant mutants. On the other hand, they must
be different in some way from the great majority of the
individuals in a drug-sensitive population, whether w^e seek
to explain them in terms of temporary adaptation to the
drug (Hinshelwood, 1946; Dean and Hinshelwood, 1953),
impermeability, "persistence" (Bigger, 1944) or clonal
variation (Yudkin, 1953). For brevity, such cells may be
described as pseudomutants.

It was felt that more information would be obtained about
the distribution of resistant individuals in semi-solid medium
if cultures were allowed to grow for a few days before adding
drug to the medium. In this way, when a large inoculum was
used (say, 10^ cells/ml. of Dubos culture) innumerable minute
colonies could be seen with a hand lens. When serial dilutions
of isoniazid were added at this stage (say after 3 days of
incubation), the drug diffused rapidly through the medium
and these microcolonies showed no further increase in size.
However, after 2-4 weeks of incubation a few large isolated
colonies could be seen developing from among the innumerable

Drug Resistance in Myco. tuberculosis 243

microcolonies, even in tubes which originally contained up to
50 [xg./nil. Thus, although it was not possible to follow the
fate of individual cells, it was possible to study the fate of
individual small clones in such a population of microcolonies.
Single colonies appearing after 2-4 weeks in tubes to which the
drug was added after 3 days of incubation were found to be
highly resistant on subculture, whereas the pseudomutants
mentioned above were much less numerous.

Thus, by incubating cultures of drug-resistant cells for
3 days so that microcolonies developed before exposure to
the drug, drug-resistant mutants were much more easily
obtained. This phenomenon was unlikely to be simply the
effect of inoculum size ; if it were it would be reasonable to
expect a corresponding increase in the proportion of pseudo-
mutants. This did not occur. It is more likely to be related
to the different physiological state of the cultures. When a
mature culture is inoculated into drug-containing medium it
is possible that some of the cells might survive exposure to
the drug because they were not metabolically active at the
moment of inoculation, and in the new drug-containing
medium they remain in a dormant state until the drug decays.
On the other hand, when the drug is added to actively divid-
ing cells without their transference to new medium it is likely
that more of the cells will be vulnerable to the drug and the
only survivors will be true drug-resistant mutants. This
explanation however is not entirely satisfactory, since pseudo-
mutants did not appear, even in tubes to which the drug was
added immediately, if the inoculum w^as increased five- to
tenfold, whereas true mutants did appear, in increased
numbers, as in the tubes to which drug was added after 3 days
of incubation. From this, it seems possible that a large
population of normal sensitive cells exerts a suppressive
effect on the development of pseudomutants.

It might perhaps be said that by the time we have taken
into account factors such as inoculum size, the physiological
state of the culture, the composition of the medium and the
decay of the drug in it, we have reached a very complex

244 R. Knox

situation which is not easy to analyse, and that until more
precise methods are available for labelling tubercle bacilli the
problem of drug resistance in this group of organisms is not a
profitable field. But while it is true that precise genetic
analyses in tubercle bacilli may be more rapidly advanced
by discoveries going on in other groups of organisms, it is after
all possible that some of the problems are unique to mycobac-
teria, and therefore it is worth while trying to collect more
information about the different patterns of resistance which
they show with different drugs. Szybalski and Bryson (1952),
Middlebrook (1954), Mitchison (1952, 1953) and others have
already contributed much in this field.

The experiments here described show how careful we must
be before we talk about mutation rates in tubercle bacilli.
For example, Middlebrook (1956) has stated that by a simple
plating technique it is possible to show that the frequency of
mutants resistant to high levels of isoniazid is 1 in 10^, and to
streptomycin 1 in 10^, and that the frequency of double
mutants seems to be something like the product of these two
rates. We have repeatedly tried to demonstrate this but have
come up against this phenomenon of pseudomutants. We are
trying to find a medium in which isoniazid does not decay, or
som