papers in
MICROBIAL GENETICS

bacteria and bacterial viruses

selected by JOSHUA LEDERBERG

 

UNIVERSITY OF WISCONSIN PRESS « MADISON — 1951
ta

 

 

————PREFACE——

THE FirsT purpose of this book is to supplement a lecture course on microbial
genetics with original readings in a form most readily available to the student.

’ At most universities, the papers included in this collection will be represented

by single copies of the original journals. Thus, it is usually difficult for any
number of students to read the papers at all, and impossible for anyone (includ-
ing the instructor) to read and perhaps annotate them at Jeisure. This volume
is not intended to include all the reading on this subject which should be
required for a course. I hope, however, that it will make available most of
the papers requiring the most general or the most intensive study.

The scope of the collection must obviously have some limits in size and
cost. These have led to the restriction to bacteria and bacterial viruses, although
consideration of fungi, protozoa, algae, and viruses is essential to a well-rounded
course, even to one organized, as at Wisconsin, primarily for students majoring
in bacteriology. Within this field, there are a great many important papers
which have necessarily been omitted. No apologies need be offered for a selection
which must be largely arbitrary. The papers selected were those which seemed
to illustrate best certain concepts and methodologies for the benefit of a course
in microbial genetics. In some choices, weight had to be given to other
circumstances such as the length and availability of alternative papers. It must
be emphasized that these selections do not reflect historical priority or scientific
worth as such. A bibliographic list is included to help remedy the unavoidable
deficiencies of the collection. -

This book could not have been produced without the magnanimous coopera-
tion of the publishers and copyright holders of the original papers, to whom

specific acknowledgment is made elsewhere. Special thanks are due to abe

authors who have been very helpful, especially in providing scarce reprints for
use as copy. I must also record an obligation to my colleagues who did their
best to advise me on the proper scope and contents of this collection.

Madison, April 1, 1951

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—— INTRODUCTION

Mutation and adaptation—The problem of organic adaptation in bacteria
has been resolved into two points of view which are closely analogous to “‘neo-
Darwinian” and “Lamarckian” concepts of organic evolution, respectively.
The first holds that adaptive changes occur spontaneously, as sporadic mutations,
not in any specific relationship to environmental conditions, and that natural
selection functions to fix the best adapted genotypes. The Lamarckian viewpoir.
suggests that the adaptive mutation is itself directed by the environment, and
that natural selection plays a subsidiary role. Many of the papers reprinted
show experiments which support the spontaneous mutation hypothesis (1, 3, 5)-
One (6) deals with the end result of bacterial adaptation to chloromycetin
and also argues for spontaneous mutations with accumulative effect by
separating the components by recombination. There is no representative here
of the alternative point of view which is defended strongly by Hinshelwood and
his associates (28, 67, 70). In considering this problem it is essential to keep
in mind physiological adaptations which are nonheritable, but whose interplay
with heritable changes may trap the unwary (97, 120, 45-Lwoff). General
discussions of this problem are included in most of the review articles (32, 54,
81, 82, 87, 121, 129).

Mutations for resistance to bacteriophage had been considered some years
before Luria and Delbriick’s paper (1), notably by Burnet (34). This author
drew the same conclusions concerning the spontaneity of the resistance muta-
tions, mainly through his ability to pick out resistant mutants by their colonial
morphology without the use of phage as a selective agent. Much of the current
work on E. coli phages (43. 44, 21) involves a set of 7 virus strains described
as T1 through T7. Mutation rates for resistance to these phages have bgeae-__
recorded, together with useful information on cross-resistance patterns (48).

The physiological study of mutant characters is known as phenogenetics,
in contrast to formal or cryptogenetics which deals directly with the mechanism
of hereditary transmission. One would reasonably expect that a gene mutation
would require a period of time to work its effects on the phenotype or outward
behavior of the organism. This lag in bacterial mutation effects (phenotypic
or phenomic lag) was first noticed directly with phage-resistance mutations

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JOSHUA LEDERBERG

™,
induced by radiations (45-Demerec). Later, Newcombe postulated a similar
lag for spontaneous mutations to account for discrepancies in the estimation of
mutation rates by different methods, according to whether they counted
mutant individuals or mutant clones (4). In both of these instances, however,
the interpretation is complicated by the cytological structure ef the bacterial
cell (15) which speaks for the concurrence of several nuclei (and therefore of
several replicate gene sets) in each cell. Recessive mutations such as phage

—seset drug resistance would require for their phenotypic expression that mutant

and nonmutant nuciei be segregated, so that there would also be a segregation
lag in the genesis of the mutant phenotype (103, 14, 47). The description of
phenomic lag in the development of nutritionally sufficient “back-mutations”
induced by ultra-violet light (UV) (7) avoids this difficulty, for these mutations
are presumably dominant, and would not require segregation for their expres-
sion, A final caution which must be entered is that we cannot be entirely certain
that the mutation-producing event within the cell is coincidental with the
experimental irradiation (mutation delay hypothesis) (26, 49).

Mutant characters—Something is known of the phenogenetics of phage
resistance. The extraction of sensitive and resistant cultures results in prepara-
tions differing in the ability to neutralize the phage “in vitro.” This argues
strongly for the concept that phage sensitivity is correlated with the presence
of a specific receptor substance (44, 61). Another metabolic correlate of phage
resistance is less explicable. In certain specific combinations of bacterium and
phage, the resistance mutation simultaneously leads to new nutritional require-
ments, (often for tryptophane or proline). Unfortunately, this correlation has
not been found in strains which can be studied by recombination, so that it
is not certain whether a simple genetic change causes the two effects, (23, 139,
45-Luria).

Phage resistant mutations play such a large part in genetic study because
of the ease with which they can be selected out of large populations, simply by
adding suspensions of the virus under appropriate conditions. This facility is
shared by antibiotics and other antibacterials as sclective agents. The advantage
that the “drug” is not another biological system whose variability must be
watched is counterbalanced by the fact that resistance to most chemicals is a
quantitative rather than an all-or-none phenomenon, and that a great many
genes may be involved. The probable reason for this is that antibiotics interfere
with key metabolic steps that are linked to a wide diversity of other reactions,
so that any one of a number of biochemical changes may influence the growth
response to an inhibitor. The initial reaction between phages and cells is, on the
other hand, directly associated with the presence of a receptor substance with a
high order of specificity.

Current interest in drug-resistance mutations is largely motivated by their
importance in limiting the effectiveness of chemotherapy (95), but they have
provided interesting material for more theoretical genetic studies as well. In

~

4 .

x

r

NERS A wb Sr
INTRODUCTION

most cases, resistance may be developed in a series of individually small steps,
speaking for the cumulative action of a number of gene mutations as postulated
above. The evidence for this comes primarily from kinetic studies of increasing
resistance under selection (5, 107, 135). It has been confirmed by direct crossing
experiments involving chloromycetin resistant mutants of E. colt (6). Progeny
of crosses between a sensitive and a fully-adapted (many-step) resistant parent
showed a segregation of many intermediate grades of resistance, representing
the reassortment of the sensitive and resistant genes in a variety of combinations.

Streptomycin-resistance shows some unique features in contrast to the
agents just summarized. In many species, a mutation conferring full resistance
occurs at a rate, about 107! per fission, which overshadows the smaller step
mutations characteristic of resistance to other agents (5, 28, 100, 135). This
low rate is perhaps the smallest mutation rate to be accurately measured in
any organism. A more striking oddity is the mutation which over-adapts the cell
to streptomycin so that the resistant mutant is dependent upon streptomycin
for growth (95, 100). The function of streptomycin is possibly to regulate an
over-expanded enzyme system, analogous to the precedent of a sulfonamide-
dependent mutant of Neurospora (141).

Amino acids and vitamins are not commonly thought of as antibiotics but
it has long been known that they sometimes interfere with, rather than promote,
bacterial growth (78, 111, 117-Snell, 140). The very ubiquity of these com-
pounds imparts a special interest to them as possible natural regulators. Of equal
interest is the correlation between sensitivity to amino acids and virulence in
Salmonella and Brucella (12, 109) which may reflect a hitherto unsuspected
general principle.

~ It is likely that a biochemical basis will be found for the effects of ail tvpes
of mutations (29), but microorganisms have been especially prolific in the
production of mutants with overt effects on metabolism. For this reason, micro-
bial and biochemical genetics are intimately associated and often confused.
Gene mutations affecting anabolic processes are usually detected as nutritional
or auxotrophic mutants, whose growth depends upon an external supply of
the missing metabolite. Auxotrophs have been used for the exploration of many
biosynthetic pathways, which are remarkably similar in bacteria, molds, and
mammals. Davis’ paper (7) outlines this methodology, which is based on the
pioneer investigations of Tatum, Beadle, and other workers on the production
and characterization of auxotrophic mutants in fungi (30, 69, 117-Tatum)
and in bacteria (63, 126, 127, 112). Mutations leading to catabolic defects
have been especially useful in bacterial work, both as genetic markers and
in the analysis of fermentation pathways (53) -

Mutations to auxotrophy do not lend themselves to quantitative estimation
of rates, despite more or less efficient selective methods for their isolation (88).
On the other hand, mutations from auxotrophy to the nutritionally wild type
or prototrophic state are suitable for selective counting methods, but due care
JOSHUA LEDERBERG

must be taken to minimize residual growth of auxotrophic inocula at the
expense of growth factors carried over with the cells or resident as impurities in
the test medium (114, 116).

Serological variation is of key importance in medical bacteriology, but its
genetic study is barely under way. The work of Kauffmann and of Edwards
and Bruner on Salmonella illustrates the provocative information now at hand

= zaudBy 9, 10, 71). A compendium of the serological variation of bacteria generally

xi

would be inappropriate here, but the subject has been treated exhaustively in
excellent reviews and books (66, 36, 54, 32)-

Given two alternative forms of a gene—say a and A, each of which mutates
into the other at a definite rate—it is easy to show that an equilibrium will
eventually be established such that the ratio of a : A will be equal to the ratio
of the mutation rates to the respective conditions (42). Most mutation rates
are so low (of the order of one per million or billion cell divisions) that muta-
tional equilibria would take too long for human observation, even if the neces-
sary constancy of the environment were possible. Mutations involving the
antigenic structure of the flagella of Salmonella have, however, been found
to have unusually high rates (accounting for the readiness with which they
have been found), and Stocker (124) has described mutational equilibrium
as approached from inocula of either form.

Induced mutations——-Since Muller's announcement in 1928 that X-rays
would induce mutations in fruitflies, an extensive segment of genetic research
has concerned the discovery of mutagenic agents and the conditions of their’
effect. Higher organisms like Drosophila and maize are indispensable in the
finer analysis of the cytological basis of induced genetic alterations, but micro-
organisms are very useful tools in the screening of new agents for mutagenic
activity, and in the study of the gross quantitative aspects of such activity.
The same types of mutants already mentioned as best enumerated on a selective
basis are particularly useful here. Dose-response data have been published
for mutations induced by X-rays (113, 45) and by UV (49, 104) but their
interpretation, especially for UV, is far from simple.

It has been thought that radiations induced mutations by direct photo-
chemical processes, i.e., that the gene itself might be activated by the absorption
of a quantum of UV, or by collision with a secondary electron following an
X-ray quantum absorption, (60, 80). Some revision of this concept is now
necessary on the basis of recent research. The effectiveness of X-rays is
potentiated by the presence of oxygen, and there may be a tenfold difference
between the doses required for a given effect in oxygen as against an inert atmos-
phere (47). This argues for a radiochemical intermediate, possibly some free
radical (peroxide?) which depends upon oxygen for its production under the
influence of X-radiation.

Owing to the powerful penetrability of X-radiation, it has been used in
Drosophila studies more extensively than UV, which penetrates through living
material so poorly that there are serious experimental difficulties in its ap-
INTRODUCTION

plication in the genetics of plants and animals. The small size of bacterial cells
ideally suits them for experiments with this agent, and UV is probably the
most convenient and widely used mutagenic treatment in microbial experiments.
The analysis of UV-effects has been stimulated by the discovery of photore-
activation (72, 55). A number of workers have found that a treatment of
various types of cells (actinomycete spores, bacteria, bacteriophage, mold
spores, Arbacia eggs) with visible light partially cancels both the lethal and
the mutation-inducing effects of a previous dose of UV (56, 73, 104, 105, 134).
This might suggest that a light-sensitive substance is produced by UV, but the
possible nature of this substance and its locus within the cell are obscure. Bac-
teriophage inactivated with UV is photoreactivated by visible light only
following its absorption into sensitive cells. The effect of UV in inhibiting
enzyme synthesis is also subject to photoreactivation (125) but no systems
simpler than intact cells have been shown to give such an effect.

Chemical mutagens.—A byproduct of research on chemical warfare agents
during World War II (as the realization of the possibility of mutagenic activity
of chemicals. The nitrogen and sulfur mustards (Q-chloro-alkylamines and
sulfides) have been studied especially extensively, and found to be potent
mutagens for all organisms studied. In general, their effects are similar to those
of X-rays and UV, but there are differences in details (26). The similarity
of effects is made the more pronounced by the fact that mutants that are
relatively resistant to “mustard” can be selected in E. coli, strain B, and these
mutants also show augmented resistance to UV and X-ray. (33, 138).

" Active programs are under way in several laboratories to screen compounds
for mutagenic activity, with bacteria prominent among the test organisms.
The methodology of such a program, and some of the precautions needed
to justify a positive conclusion, are illustrated in Witkin’s paper (11). There
is a wealth of further literature on this subject, and it is likely to remain an
active field. There is so far no rational basis which can be used to predict
the activity of new compounds, and substances as diverse as formaldehyde,
acriflavine, urethane, caffeine, hydrogen peroxide, and manganous ion are
credibly reported as active in one or another system (26, 49, 58, 65, 47). In
general, reagents with a high reactivity for labile organic H groups, e.g., for-
maldehyde, organic peroxides, acyl halides and sulfates, ethylene oxide, acetic
anhydride, and diazomethane are probably mutagenic (86) but this does
not account for the mutagenicity of such chemically inactive compounds as
caffeine or urethane.

Much more careful work is needed in this field, but so far there is no
convincing evidence for any appreciable specificity in the mutagenic capacities
of any of these chemicals or physical treatments. Each of them induces a wide
diversity of mutations (as far as this has been investigated), and in general,
the results of, e.g., X-ray treatment, would not be readily distinguishable from
that of formaldehyde. On the other hand, it is possible that different gene
forms may differ in the frequency with which they will respond to different
JOSHUA LEDERBERG

mutagenic treatments; but if there is any specificity, it has so far been of a
second order. In this respect, induced resemble spontaneous mutations, whence
some genes may mutate more frequently than others, but not in such a way
that the environment can be said to direct the mutation of a specific gene pre-
ferentially to the exclusion of others.

Spontaneous mutation.—We may return at this point to the mechanism of
“spontaneous mutation,” keeping in mind that the study of experimentally
controlled variables on mutation erases the distinction between spontaneous
and induced. So long as the concept connoted by these terms is kept clearly
in mind as one distinct from that of directed mutation, there need be no
confusion.

Evidence bearing on the relationship between growth and mutation js
especially paradoxical, for mutations to phage resistance, for instance, ap-
parently do not accumulate in a resting culture (1). On the other hand, cultures
whose growth is regulated in a special steady-state, controlled-flow culture
vessel (“chemostat”) mutate at nearly constant rates per unit time, whether
the cells are proliferating slowly or rapidly (106). One interpretation is that
spontaneous mutations are due not to intramolecular accidents or reproductive
errors, but rather to the action of intracellular chemical mutagens formed by
metabolic processes. In this connection jt is worth noting that formaldehyde
and hydrogen peroxide are both fairly common metabolic intermediates, and
that two other mutagens, caffeine and allyl isothiocyanate (mustard oil), are
also natural products. Information on the effect of temperature changes on
mutation rate would be especially valuable if it could be dissected from effects
on growth or metabolism. Under conditions of steady growth a temperature
increment of 10° accelerated mutations in E. coli by a factor of about 2, and a
similar increment is reported in other systems (85).

Bacterial populations—The necessity for thinking of bacterial cultures
always in terms of populations, which may have genotypically diverse com-
ponents, can scarcely be over-emphasized. The process reviewed to this point,
mutation, is the fundamental source of genetic variation, but in view of the
smallness of spontaneous mutation rates, it is obvious that the occasional
change of a cell from one genetic condition to another can make little impression
upon the composition of bacterial populations. The forces that determine which
genetic types will predominate in bacterial cultures are the subject of population
dynamics. In diploid sexual organisms, population genetics is greatly compli-
cated by recombination and by the concealment of genetic variation in the
heterozygous condition, so that the most drastic culling may have to be carried
out for a great many generations to have a marked effect on the relative
frequency of different gene forms. Selection in bacteria is, as a rule, more
straightforward, as shown, for example, by the quantitative isolation of phage-
resistant mutants by a single application of the virus. The physiological inter-
actions of bacteria in dense cultures lead to less trivial problems in population
dynamics. Such interaction may involve an obvious competition for nutrients,
INTRODUCTION

(12). The intricate interrelationships of the production of and sensitivity
to species-specific antibiotics (“colicins’) have been thoroughly analyzed for
E. coli by Fredericq (59). One of the most exhaustive analyses of selection
dynamics in bacteria will serve best to illustrate the complexity of populational
interactions in which several distinct effects may be superimposed (115). A
number of populational interactions have been described with a less complete
analysis (138, 136, 85).

Population complexities may also arise when more than one mutation
occurs, so that the population consists of several Categories of genotypes. One
example has been cited already in the stepwise development of resistance by
the cumulative effect of serial mutations. A second has been described so
recently that its implications are still under discussion. In independent experi-
mental work, three groups of investigators noticed a perplexing sequence of
cycles in bacterial populations under conditions of continuous or reiterated
culture (25, 106, 124). “Marker” mutant cells increased in Proportion as
mutations accumulated, but instead of increasing indefinitely, their ratio was
subject to sporadic downward shifts. The Same interpretation was independently
formulated for each Case: an adaptive mutation increasing the fit of the
bacteria to their rather artificial in vitro environment. Owing to the overwhelm-
ing preponderance of the cells not Carrying the marker, the adaptive mutation
will usually occur in an unmarked cell, the descendants of which will then
displace the rest of the Population, markers and all. After the changeover,
marker mutations accumulate again until a Possible second changeover takes
place to complete another cycle, It is not predicted that this process could
alter the ultimate equilibrium of cell types under mutation “pressure,” but

Interclonal variation: Sexual recombination—The Preceding discussion
of bacterial Populations has supposed that each bacterial cel] is genctically
isolated from its Partners, i.e., that reproduction is exclusively vegetative or
clonal. A large body of evidence is now at hand, however, which shows that
this picture is incomplete, and that some account must be taken of interclonal
processes. One such process was discovered in 1946 by Tatum and Lederberg
(45, 84, 128) and described as genetic recombination, for the experiments are
based on the selective isolation of genetic factor combinations from mixtures
of different kinds of mutant cells. The conditions under which these genctic
exchanges take Place, and the patterns in which they result, led these authors

xv
JOSHUA LEDERBERG

to conclude that recombination in E. coli, strain K-12, results from a sexual
process (13). This conclusion is based upon evidence which is, to date, entirely
negative, indirect, or genetic. It is substantiated, however, by single-cell studies
on heterozygous diploid cultures (14, 142), and by a direct demonstration of
the nonfiltrability of the agents of recombination (40). A variety of selective
techniques can be used for the isolation of recombinants from strain K-12 of
E. coli. In addition to the selection of pretotrophs from mixtures of auxotrophs

-~~>-scaeehich is most commonly used, one may select for dually resistant recombinants

XVI

from mixtures of cells each resistant to one inhibitor (83). A combination of
these methods has been profitably employed to detect new strains crossable to
I\-12; a number were found, some with many properties differentiating them
from each other. Developments in the author’s laboratory to the date of this
writing have been summarized (47—-Lederberg).

Recombination in strain K-12 has been applied by a number of other
workers for various problems (102). The genetics of resistance to chloromycetin
(6) and to streptomycin have been contrasted (46, 101). The very occurrence
of factorial segregation shows, of course, that bacterial enzymes are not to be
identified with the genes required for their formation (98). In fact, it is
controversial whether the patterns of gene-enzyme interrelationships in bacteria
support the “one gene-one enzyme” proposed as a generalization from the
properties of auxotrophs in Neurospora (6a, 47-Lederberg, Horowitz, Bonner),

Transformations.—The history of “type transformation” is often regarded as
dating from Griffith’s classical experiment on pneumococcal types in 1928 (64),
but this work is antedated by a confusing array of studies which date at least
as far back as the controversy over the etiology of typhus fever. When the
rickettsial etiology of this disease was finally established, many workers sug-
gested that the serological reaction of Proteus OX-1g9 with rickettsial antibodies
(Weil-Felix reaction) was due to a transformation of non-reactive Proteus
by rickettsial products, and gave the name “paragglutination” to induced
serological variations such as were supposed to be involved here. In support
of this hypothesis, (to which no credit is now given) many workers reported
that the serological reaction of E. coli and other enteric bacteria could be
modihed by cultivation in filtrates, extracts, or lysates of serologically distinct
cultures (reviewed in 81). Griffith’s demonstration was, however, the first which
proved to be readily reproducible by other workers, and remains a proper
starting point for modern discussions of transformation. His paper is unfortun-
ately too lengthy to be appropriate for reprinting here, but his observations, and
those of other workers leading to the isolation and characterization of the
active principle responsible for the Pneumococcus transformation are sum-
marized in the paper reprinted here (16),

The genetic interpretations of this transformation are currently a subject of
lively discussion. Its description as a specific induced mutation is probably less
fruitful than as a transfer of hereditary material from one cell to another
(47-Taylor). The main questions which still have to be answered include
 

a yt

INTRODUCTION

a) the further physical and chemical characterization of the agent, b) its
genetic complexity (that is, whether it involves single characters, the full
hereditary material of the pneumococcus, or something intermediate), and c)
its cytological relationships to nuclear or extranuclear structures in these cells.
The current approaches to these problems are summarized in a number of
papers and reviews, (27, 91, 93, 130, 131).

In addition to the volume of older literature which must be supposed
to carry some grain among the chaff, there are a number of more recent reports
of transformations in various organisms. These include E. coli (31), Hemophilus
influenzae (22), Shigella paradysenteriae (133), Alkaligenes radiobacter (39),
and staphylococcus (38). Discussion of these and other transformations should
take into account complications which might arise from bacterial life cycles
more complex than usually regarded.

This presentation has emphasized the contrasting features rather than
the similarities of the phenomena described as genetic recombination vs. trans-
formation. Both phenomena tend to the same genetic result: the elaboration
of the cells whose hereditary traits are derived from more than one parent.
When more is known of the morphological basis of “sexual” genetic recombina-
tion on one hand, and of the genetic properties of transformations, on the other,
a more profitable synthesis of these contrasting concepts may issue.

The recrudescence of interest in bacterial cytology has been largely inde-
pendent of, though contemporary with the development of bacterial genetics
documented in this book (77). The geneticist notes at least two fields where
cytological information is indispensable to him: a) the form and behavior of
the bacterial nucleus, and b) the possible existence of complex “life cycles.”
Reliable information on both these subjects is relatively meagre, but the
evidence of at least the existence of nuclei in bacterial cells is relatively convinc-
ing. Robinow’s paper reprinted here (15) may be taken as one point of de-
parture for the more recent work on this subject (41, 74). With the improvement
of techniques, we may look forward to rapid progress in the establishment of
the details of the nuclear cycle at cell division, segregation, etc. (g9, 118, 92,
122, 47).

The expression “life cycle’ has come to carry many connotations in
bacteriology which hinder a careful discussion of the often conflicting observa-
tions of generations of bacteriologists. At least one type of cycle is indisputable,
the formation of highly resistant endospores by many bacteria. Whether
endosporogenesis has any genetic significance is controversial. At the least,
they may be supposed to represent the stage at which the presence of only one
nucleus per cell (in contrast to the two, four, or more, characteristic of most
rods) is the most likely (see 54), and on this basis may be particularly useful
for certain types of genetic experiments. More than one student of the mode of
nuclear segregation into the endospore has proposed a sequence of nuclear
fusion and meiotic reduction (i.e., autogamy). However, the cytological figures
are not easily interpreted, especially in view of the pitfalls of logical recon-

XVii
XViil

JOSHUA LEDERBERG

struction of fixed cells, and in this form autogamy is a blind alley without the
possibility of genetic novelty, and therefore not amenable to genetic test, (74).

The genetic significance of another cycle, the “L-forms,” is equally obscure.
It is expected that this problem will be clarified in the very near future to an
extent that makes a detailed discussion of its present status here premature. The
reader is referred to provocative discussions published elsewhere (50, 51, 52,

75, 76, 132, 123) These studies reopen the possibility of the existence of

diminutive forms of many bacteria which may be capable of passing through
ordinary bacterial filters. These forms may also be highly resistant (like the
endospores) to heat and antiseptics. Since they also may have special nutritional
requirements, they may remain dormant in ordinary bacteriological media
which support the normal bacterial form. If these observations are confirmed,
the filtrable L-forms might well play a role in some of the transformation
experiments cited earlier. However, the transforming agent would consist not
of a single genetic factor transferred from one cell to another, but of a diminutive
form of one cell which germinates under the influence of another. The analogy
between the diminutive forms and the gametes of other plants and animals
is obvious on a physical plane; the possibility that they may play a similar
function in bacterial biology is a provocative one which will require careful
study.

One of the reasons for including papers on viruses and bacteria in the
same volume is the necessity for considering both the host and the “parasite”
in any physiological studies of their association. Furthermore, it is becoming
apparent that a great many bacterial cultures carry bacteriophages in a symbio-
tic relationship (“lysogenicity”) so that what are taken to be bacterjological
investigations have an unavoidable virological component. A second reason is
that the phage-bacterium complex may be profitably regarded as a unit for
comparison with other cellular systems which carry extranuclear hereditary
components (57, 119). Burnet’s paper illustrates this very clearly (17).

It might be worth pointing out also the formal analogy between the transfer
of a latent virus from a lysogenic to a sensitive bacterium and the transfer
of the capsular attributes from smooth to rough bacteria in the pneumococcus
transformation. Whether this analogy is more than superficial only time will
tell. It is becoming clear, however, that there is a continuous gradation of pro-
perties between systems of cytoplasmic heredity such as the chloroplasts of green
plants, and the metabolic granules of yeast, and infective associations such as
the “killer” factors in Paramecium, rickettsia in the Arthropoda, and viruses in
plants and mammals. In many instances at both extremes, “disinfection” or cure
is possible by the use of appropriate drugs ( streptomycin for chloroplasts, acri-
flavine for yeast granules) (47, 57).

Burnet’s paper also illustrates one of the earliest and clearest cases of muta-
tion in a bacteriophage. In a later study, Luria described host range mutations
in phages which restore the virus’ ability to attack bacteria which had mutated
to resistance to the previous form of the virus (18). This process of compensatory
INTRODUCTION

mutations on the part of bacterium and virus evidently can sometimes be
repeated over a great many cycles, and is of some economic importance in the
acetone fermentation industry. These mutations in bacteriophage have been
used as the basis for further genetic studies which have revealed the occurrence
of genetic recombination among viruses (as well as in bacteria) (19, 20). Two
lines of attack are exemplified in the papers here. One uses patent markers,
such as host range and plaque morphology. For the other, recombination among
lethal mutations induced by UV is used to explain multiplicity reactivation,
or the cooperation of several particles damaged by UV to initiate phage growth,
in contrast to the ineffectiveness of individual damaged particles.

This work has naturally stimulated attempts to show similar phenomena in
animal viruses. At present these are represented by reports by Burnet and
Lind (37) of probable recombination in the influenza virus, and similar investi-
gations are under way in other laboratories. Plant and animal viruses are likely
to be more difficult to study from this point of view, chiefly because of technical
difficulties in initiating infections with single virus particles (35, 62, 79).

Genetic study of bacteria and viruses is closely interwoven with the most
general problems of their biology; this is not surprising, for the same has hap-
pened in other areas of biology. But it is to be hoped that genetics will be re-
garded not as a unique or isolated part of bacteriological study, but as an ele-
ment of all teaching and research in microbiology.

THE REPRINTED papers are numbered from 1 to 20; citations to the literature
at large are numbered from 21 to 142, alphabetized by authors’ names. Choice
of citations has been dictated by the same general considerations as were applied
to the reprints, and many interesting examples of specific mutant types have
necessarily been omitted. Many reviews are available which provide more
detailed and balanced accounts of specific subjects; these are listed with
an asterisk (*).
The subjects treated in the reprinted papers may be outlined as follows:
I. Bacteria
A. Spontaneous mutation

1. Mechanism of adaptive mutation (1, 3, 5, 6)

2. Estimation of mutation rates (1, 2, 4)

3. Categories of mutants

a. Resistance to antibacterial agents (1, 2, 3, 4, 5, 6, 11, 12, 17)

b. Biochemical mutations (7) ao

c. Serological mutations (8, 9, 10, 12)
4. Phenotypic lag (4, 7)
B. Induced mutations (11)
C. Population dynamics (12)
D. Interclonal variation
1. Genetic recombination in E. coli (6, 13, 14)
2. Transfer of symbiotic virus (17)

xix
JOSHUA LEDERBERG

3. Type transformation in pneumococci (16)
E. Nuclear cytology of bacteria (15)
II. Bacterial viruses
A. Spontaneous mutation (15, 18)
B. Recombination (19, 20)
—— BIBLIOGRAPHY

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