Society of American Bacteriologists

San Francisco

August 13, 1953
This is, I believe, the first time that the Society has assigned

a geneticist to the pleasant burden of this AERIS

THis way—be-read—

   

 

First,

the extension of elective enrichment as a fundamental tool for the iso~
lation and enumeration of specific microbial types within initially pure
cultures, as well as from the mixed flora of natural habitats; Second,

wn
the demographic , treatment of bacterial cultures and colonies as aggregates
of individual cells, whose homogeneity must be explicitly assessed, usually

by just such selective methods; and, Third, the realization that the very

individuality of a bacterial type poses a problem in heredity, which mst

be met and answered in terms of some genetic theory, —Phe—-eorrespondence—of—

a—eompetent theory fr bacteria with tie structure that has been developed ~

 
to related-inquirtes-with-vartous—bacteria,-

These three methodological principles have been fruitfully applied
—~

in bacterial genetics as in the analysis of different modes of adaptation
and of agents which will induce genetic mutations, but I mst confine my
remarks today to their elucidation of the exchange of hereditary determi-
nants among bacterial teyhen—

Even today, there is no compelling morphological evidence of sexual
fusion in bacterta--a remark that applies a forteriori to the pictures I /

other
will show myself, later, Many published Claims have been supported by

#

highly suggestive but not irrefutable photographs, My own account starts
about seven years ABO, ost thoughtful students had coneluded that a
purely morphological approach was unlikely to be decisive » and the
desultory attempts to detect crossing by genetic techniques had given
results either negative or incredible, At the least, the burdens of
proof devolved upon any affirmative claims of bacterial sexuality,

Some favorable circumstantial evidence did, in fact, encourage the

first experiments. In one of the most thoroughly categorized groups of

bacteria, the Salmonellas, the patterns of the various somatic and flagellar
-3-

antigens represented in the Kauffmann-White scheme are scarcely intelligible
except in terms of recurrent recombination, As we shall see, this conjecture
has been confirmed, though not quite as expected,

Escherichia cold wae a preferred species for the first investigations,
In 19h), E. L, Tatum (and, independently, R. R. Roepke and his associates)
had isolated mtritionally exacting, what we now call "auxotrophic," mutants
from strain K-12 of E. coli, In 196, I went to Professor Tatum's Laboratory
at Yale University to join in experiments we had previously discussed to
test the possibility of genetic exchange with the help of such mutants,
We ‘heped to exploit the selective property of a synthetic » minimal medium
to suppress auxotrophic mutants, and the to select for prototrophic bacterla--
those with wild type nutrition, amd lacking differential growth-requirements,
t-nochanismof-(fenetic exchange could be efficiently detected by culturing
different auxotroph mutants together in various media, and then plating
them into minimal agar, The parental auxotrophs would be suppressed, but
any crossing should also engender, among others, prototroph recombinants
which would be readily detected and recovered, The selective efficiency
vould enna crossing experiments previously reported (for example by

Gowen and Lincoln), that these negative results would not necessarily
be discouraging.
The first experiments gave an inescapable result: mixtures of
various auxotrophs would vongensise prototrophs in the ratio of about a
million-to-one, Clearly, the key to this affirmation is the selective
method; otherwise, it would have taken far more time than you would care
to listen about to comiuct the experiment to a single defivite results,
or more likely it would have been given up as a hopeless task, like its
predecessors,
avd possible
It was, poootbiey in various ways, to confirm that the prototrophs
were pure cultures, and that they could not be explained as aupartefact of
spontaneous variation of either parent by itself, But the generation of
pretotrophs was only the first stage of the analysis, showing thet some
ww
form of genetic interaction between different bacterla was possible, The
behavior of other specific traits or genstic markers and the physical and
cultural conditions of its occurrence must next be recounted,
E. coli is one of the most ubiquitous of bacteria, and the chan

acteristics of any typical strain will be familiar to each of you. Except

that during the thirty years since it was firet isolated it may have lost
characteristic 0 ani K antigens, strain K-12 conforms to type. In particular,
it exhibits no special growth requirements, it ferments a variety of sugars
(glucose, lactose, maltose, xylose, mannitol, and so forth, but not sucrose
or cellobiose), it is susceptible to many "coli-phages" and colicins and other
antibiotics, including streptomycin, These characters are enumerated only to

indicate the traits which in suit of genetic variation, have furnished the

genetic markers for further studies, Buring—the peat severs)_yeare special

P Uitte “ieee DEE Sere Leper “tC eT lTtatz AO PEO OR_O SLOGIeH ei TaRLanee
a Le > riz 5 Pt TU erty
wee Rs BS aL) GB Suro o Lat 0 ets ‘

If they are generated by a sexual process, /prototrophs should also
exhibit reconbinations for any additional unselected markers which might
differentiate the two parents, For example » if one auxotroph parent were
lactose-positive and streptomycin-sensitive y While the other auxotroph were
lactose~negative, streptonycin-resistant, the protetrophs should fall into
four classes in respect to these two unselected markers: the parental com-
bina tions--positive-sensitive and negative-resistant-<-and two new combinations s

positive-resistant and negativeesensitive, With three markers, there would be
-~6«-

eight potential classes, and so forth, This prediction has been borne ont
in great detail, some crosses having been carried out with as many as six
and seven differential markers, Moreover, the role of a marker as selected
or unselected is not absolute, but depends on the technical details. Ina
medium supplemented with the appropriate growth factors, selection on the
nutritional markers may be relaxed, while bacteriophages and antibiotics
may be substituted in an obvious way as the specific selective agents, Thus
it has been possible to recover some of the recombinants that would otherwise
by prottligh aslictin,
be missed, such as dual auxotrophs, The regularity with which an unlimited
array of recombinants can be generated, regardless of the particular mode of
selection, refutes their interpretation as any artefact of spontaneous varie
ation, Theory and experience concur again that recombination does not
generate any new variation beyond the reshuffling of markers already embodied
in the parents, In all genetic work it is, of course, essential to scrutinize
any marker for its inherent stability and the regularity with which it can
be classified, Special attention must be given this point in recombination

studies, but if this is satisfied, the unrcstrained reassortment of unselected

markers is the surest testimony of a recombination process,
-7<

If, among the protetrophs from a given cross, the various classes of
combinations of markers are emmerated, it is found that they appear in
characteristic proportions: although, as a rule, every possible class will
be represented, some combinations will be mech more frequent than others,

As a general rule, parental combinations will be more frequent than recom
binations for any small group of markers, Contimed study reveals Wi dit
many very marked correlations of pairs of factors, For example, with lactose-~

fermentation and Té-resistance, the two recombination or "crossover" classes

together made up respectively only 6% and 2 of the total, while the two
parental combinations were 75% and 17%, S4milar » factore for maltose~—

 

 

By an extension of this type

of analysis, it has been shown that at least six or seven markers (four already
mentioned, and one for resistance to phage Tl) can be ordered on a linear
linkage map, On such a map, the probability of recombination between two
markers is propertional to the indicated distance between them; it is linear

insofar as these probabilities are additive. The triumph of genetics has been
the rigorous correlation of the linear linkage map with the linear chromosome,
A proof of equal figor has yet to be accomplished in bacterial cytogeneties,
The mapping of these factors, beyond the pairwise relationships already
mentioned, may be complicated by various anomalies of "chromosome" behavior
which may or may not be of primary importance, Temporarily suppressing any
such anomalies, we may summarize the customary life cycle of strain K-12
(as witnessed by genetic evidence only) as follows: the vegetative cell is
haploid, although other genetic and cytological work supports a two or four
meleated condition aa usual, Among a mildion cells, under ordinary cultural
conditions, a single pair may mate by a process still unobserved, though a full
cell fusion is perhaps less likely than SEG, consucarion, The diploid
stage is evanescent, and persists only long enough to allow reassortment and the
segregation of haploid recombinante to complete the cycle. This will be
recognized as following the same sequence as many other fungi ,--Neurospora
or Zygosaccharomyces,--and dissimilar to the yeast Saccharomyces which hes
a prolonged diploid phase,

The experiments so far tell nothing of the chemistry or morphology

of the mating process, ‘Two alternatives merit the closest consideration:
a bona-fide union of two cells, or something akin to the pneumococcus

transformation, This second alternative would mean that one of the parental,

gametes would be replaced by a sub-cellular fragment. However, extensive

studies, in several laboratories, have uniformly failed to substantiate the

second alternatives the only conditions which permit recombination are those
a

in which direct access is permitted between the parental cella. Hayes has

found that cells that have been, so to speak, "killed" with streptomycin

may function (with reduced efficiency) in recombination, A separation of

the capacity for colony formation from other signs of vital function has,

however, many precedents in disinfection studies. ant Morphological changes

in the streptomycin-treated material hat oma substantiate a gametic role

for any element other than the entire cell have not been presented, Other

antibacterial agents do not markedly discriminate betweer sexual and vegetative

functions, It ispchowavem, very difficult to evaluate many of the experiments

that have been published on these effects, As predicted from the mating

theory, recombination has been shown, by T. C, Nelson, to fit the kinetics

of a bimolecular reaction, and rates of recombination can be compared

quantitatively only when the rate w constants can be inferred, But no

amount of negative evidence can add up to an affirmative picture of the
details of the mating mechanism, Until a morphological demonstration is
completed the hypothesis of a conjugal union te explain genetic recombination
has this weight only: that it is consistent with every datum so far adduced,
Until recently, morphological study had no encouragement whatever,
Nelson's kinetic constants could be read as counting about 5000 random

collisions for every mating, a rate simply toolow for any but speculative

Subse uently, , &-6.
cytology, More—recentiy; owes, Cavalli discovered a much more fertile

with whietr

SQ

atrain, *

   

with~thie-sitraim the ratio of matings to ramiom collisons approaches one,
80 that constructive cytology is now possible, though still difficult, and
the microscopic approach has been resumed, In some very early attempts,
we have had some encouragement from seeing pictures like this,
LANTERN SLIDE 1

What such figures may have to do with the mating process is purely
conjectural, It is tolerably certain from phase-contrast microscope obser-
vations that cells may be attached in pairs while living, but their further
history has not been followed up, We have seen nothing else, so far, with
any suggestive quality, The outcome of this study will, we feel, be of public

~

interest as exposing either an artefact of which we must beware in all such
studies, or as some aspect of the sexual mechanism, I want to emphasize
that, by itself, any picture such as this stands as very meager evidence
indeed,

I have already indicated that the postulated diploid, sygote phase
is short-lived, and is not propagated as such at all in the usual sequence
of the life eycle, This diploid phase, is in fact, a figment of neasens ind active
dtude inference from the facts of recombination. Fortunately, exceptional
deviations from the standard life cyele have substantiated this reasoning,
for un-separated (or non-disjunctional) diploids have been found among the
progeny of a certain mutant stock, "Het." When a Het Lact is crossed with
a lactose-negative parent, many of the progeny are typical prototropha,
stable both for their mtritional and their fermenting qualities, though,
of course some will be "Lac+" and others "Lac-," A few percent, however,
prave to be persistently segregating for all of these qualities (and indeed
for almost any marker that may distinguish the parents), This is show
tn-part>x their appearance on an indicator mediums

_ Fig 2 1 P- 47-0
LANTERN SLIDE 2 = SH. 5)

which is typical. of the colonies stemming from a single cell, To abbreviate

a long story, the heterozygous diploid cell is segregating for several. markers,
The stable, haploid segregants usually display the combination of markers
of one or other parent; less often, new combinations are seen, Hewownm,
Fhe markers always segregate at the same time, The facts of recombination
agree wheeag with the hypothesis that the recombinants stem from intermediate
diploids like these. What is exceptional here is the tendency of the diploid
cell to propagate as such, in about 19 out of 20 fissions, in M, BR, Zelle's
single cell pedigrees, It is thus possible to compare the meclear cytology
of haploid and diploid cells:
LANTERN SLIDES 3 - / = ae ea
About all that can be surely claimed in the present state of

bacterial cytology ia that the diploid cells have distinctly more complex

moclei, I would not venture, as yet, to quote chromosome counts on this

material, Sorattinete-poswthie-misunlerstanting, [t should be emphasized

that the diploid hybrids are not merely unstable for a single marker, but
show a bloc-wise separation of (the momerous markers differentiating the
two parents, Two markers have to be excepted from this rule: maltose-

fermentation, and streptomycin-resistance, These have been invariably

hemi-zyrous, 1.6., only once represented, in the diploid progeny of Het
-13-

crosses, although sometimes one, sometimes the other parent's markers are
preserved, in any given diploid, This raises the question whether the gamete
was already defective, or whether there has been a later elimination of a
chromosome segnent carrying these linked markers, The second interpretation
faveuk

that the gametes are intact and the aberration secondary 1s supperted by the-
~cecunrenceofsome diploids in which the Mal marker comes from one parent, 7Ae-
streptomycin marker from the other,

For some time, K~12 was the only E, coli strain in which recombination
could be demonstrated, others having been tested with negative results,
but in a later survey about one wild-type strain for every 25 tested was
found to be fertile with strain K-12, The fertile strains encompass a wide
varlety of serological and cultural subtypes, but all of them are included
in E. coli as presently understood, The taxonomic delineation of this
species is therefore supported by concrete genetic evidence, Whether the
other 2 out of 25 strains form additional intra-compatible groups is not
known, but entirely possible,

One purpose of this survey was to see whether compatibility preferences

could be found by ranging over a large group of strains, But meanwhile, it

was found that the seclal structure of K-12 was not so simple either, AlL
- i -

of the crossing stocks originally had been developed from the same clone of
Kel2, aml since they were all crossable with each other it was concluded that
the strain was not (to speak loosely) sexually differentiated, 4.e,, it was
homothallic, Later, Cavalli (in Milan) ani Mrs, Lederberg (in Madison) dis-
covered certain K-12 strains to be both self- and mtually incompatible,
We called the wild type, compatible strains, F+y the incompatible » Fe,
and. Yo recapitulate, P~ x F- is sterile, while F+ x F+ and F+ x F- are
both fertile. Therefore, it was not until two F- testers showed up that
the incompatibility system could be uncovered, About the same time, Hayes
had fount his differential effect of streptomycin, and a comparison of all
our results indicated ales that F~ cells were completely inactivated by
streptomycin, while F+ cells retain some sexual function, The speculation
that the F~ cell donates the larger part of the cytoplasm to the zygote,
i.e., that it may be a sort of oo~gamete, is still tenable, Other studies
have also shown that the polarity of a cross with respect to F status also
determines the trend of eliminatimgtion of the deficient maltose- and
streptemycin-markers from the diploid sygote.

If compatibility were inherited like other murkers, it would have been

detected long since. But, remarkably, it is contagious, for when properly
-15-

marked F~ and F+ cells are simply grown together, after a few hours most
of the F~ are converted to F+, This conversion is rather mysterious,
Although 1t occurs about as frequently as the calculated collisons of
the two kinds of cells, no infective agent has been separated from either
the F+ or F~ sources, There is a rough (but not a detailed) agreement of
the circumstances under which this conversion eccurs, and the circumstances
of genetic recombination, and indeed both may require a contact of cell
surfaces. The supposition, advanced elsewhere, that the so-called F+ agent
1S rmupregnadse

rather than the cell itself is the vehicle of genetic transfer, sannet~oo-
added until the two have been seperated physically. Other differences

» , w
can be accomodated by postulating a variable’ competence of the F+ agent
or p@ ite infectivity and maintenance,

Among the new crossable strains about half are Fe and can be converted
to F+ by growth with K-12; others are F+ ami will convert K-12? Fe stocks to
an F+ state of variable permanence, But we are far from knowing the whole
picture; some strains show no signs of compatibility differentiation, or of

re
the F+ agent, although thes can be "infected" with it, The only sign of

this eryptic infection is that the infected strain can reconvert a K-12

tester, But the evidence / for an F+ virus is purely epi-bacteriological,
~16-

and a term such as infection may be an unwarranted extrapolation, however
convenient it 1s as a laboratory shorthand,

Despite the gaps in our kmowledge, the foundations of recombination
are already secure enough to allow applications to many problems of general
interest. For example, numerous genetic factors are concerned with the
economy of single bacterial enzymes, ani vice verga, which show in turn
that the one-gens t one-enzyme hypothesis.is a useful, but fictitious
approximation, In a preliminary study of antigenic factors in Escherichia

P, D, H, O, euhK antigens
coli, Dr, /Skaar in our laboratory has shown that hemi may be recombined

in the same way as other genetic markers differentiating different strains,

And in the field of drug resistance, Newcombe and Cavalli have shown,

weepectivety, that the response to streptomycin is achieved, im the_mein,_

by a single genetic mutation which confers full resistance, while resistance
to chloramphenicol is governed by the interaction of great many separate
mutations with cumlative effects, To findinge were powerful confime-
tion of previous anticipations, The results of a recent investigation

by Mrs, Lederberg were less predictable, We had thought thet symblotically

carried bacteriophage would behave like a cytoplasmic genetic factor, that is,
like one of the plasmagenes which ere the topic of a good deal of geneticists!
discussions these days, However, the results of crosses uf lysogenic and
sensitive sub-strains of K-12 showed that the trait of lysogenicity was
inherited like any other marker, and that it was in fact linked to markers
for galactose fermentation, The clinghing evidence here was the isolation
of diploids heterozygous for these markers, so that a hybrid which was
lysogenic and galactose positive would engender segregants some of which
are of the other parental type, sensitive to the potentially symbiotic
virus and gelactose-negative, together with a small proportion of the
other two combinations. F, H. Burnet had long since foreseen thet the
lysogenic complex embodied the integration of the latent phage in the
hereditary make-up of the bacterium, but it is difficult to see how this
concept could have been substantiated more firmly than by recombination
analysis, But I would std11 conclude that the largest contribution of
this approach 4s the impetus it gives to the unification of bactertology
within a more coherent comperative blLology.

This is not to say that there is nothing unique in bacterial genetics:

stood t
since 1928 the pneumococons transformation has refute any such complacency,
-~ 18 .

As soon as the outlines of the K~12 story became visible, it was important
to learn whether sexual recombination occurred more generally among bacteria,
The Salmonella group was the next choice of material, My early experiments
with S. typhimurium gave the tantalising result that various auxotroph
mixtures appeared to give protetroph recombinants, but that attempts to
secure evidence of recombination of unselected markers all failed,

Therefore, I drew what later turned out to be the skeptical but incorrect
conclusion, that these apparent prototrophs were artefacts, andi sit more
different strains had tobe studied, When N, Zinder Joined the program,

this was his experience also for over two years, and crosses of over a
hundred pairs of parents. It was only when we incorrectly interpreted

a two-step mutant as a two-factor mutant that we drew the correct con-
clusion, that a recombination mechanism was in fact sporating. te

system proved to be very different from the sexual recombination that

we had been looking for along the lines of K-12 work, Instead, eeu
transfer here is mediated by a filtrable agent, namely certain potentially
lysogenic bacteriophages, When this phage is grown on one Salmonella strain,

some of its markers can be transferred by the phage to a second bacterium,
amd there replece the previous markore of the recipient strain, In
general, only a single marker is carried by a given phage particle, and
the over-all efficiency is rather low: about one marker per million

1S
phages bat this eeeiiy made up for by the efficienc¥pe of selectiest-
techniquey, However, any marker is capable of being transferred, inde-

pendently of the other as may be illustrated by this lantern slide,

LANTERN SLIDE S = fg. Sect
where the donor strain is galactose positive, xylose positive, and
so forth, and the recipient 1s negative for these markers, If 100
million phage particles are adsorbed on an equal mmber of recipient
cells, about 100 positive papillae can be selected on galactose agar,
and the same for xylose and any other marker, But the galactose-
positive cells are still xyLose-negative, and vice versa, Thus, two

sharp

features of the Salmonella system emerge in contrast with E, colt
KeL2s in Salmonella, the agent of recombination is a filtrable phage
particle, not the whole cell, and the unit of recombination is a small
fragment, not the whole genotype, The analogy with the "transformations"

of the pneumococcus and other bacteria is obvious, ani suggests that these

phenomena be classified together as what I have called "transductions,"
The common feature of genetic transduction ia thet a small fragment of
the total genotype is transferred, In the pnoumococcus, Avery, MacLeod
and MeCarty could disrupt the donor bacterla by various chemical procedures,
and isolate a principle plausibly, if not rigoronsly, shown to consist of
desoxyribomeleic acid, In Salmonella, a phage particle verforms this
delicate operation as a by=product of its own nefarious syntheses, hut
while it saves us these labors, and assumes the burden of transporting
the fragment snd iajecting it into the new host, it has so far also succeeded
dn denying us the access to the fragmeat jbeded for biochomical analysis,

The most plausible view of transduction seems to mec to be that the
fragments are indeed pieces of chromosomes, usually so short as to en-
compass only one marker of the several followed in any one experiment, A
few exceptional cases have been found, however, which are best interpreted
as the correlated transfer of two markers; these would then he factors,
closely linked on the sane chromosome,

Most of the markers so far studied have however shown no trage of
linkage with each other,

The absence of a sexual system (at least none has yet been found)

dn these species has prevented the confirmation of this view by recombination
analysis; one could argue, for example, that the fragments are not just
pieces of chromosomes, but whole chromosomes, This would simplify the
problem of how the transduced fragment is incorporated into the new
genotype, but would not readily explain how the old homologues are
ejected, for which there is samy good evidence, It would also demand

so large a number of chromosomes as to raise doubts as to the genetic

now

stability of such a hypothetical system, However;-studies arc(imer —

 

I mentioned earlier that the serological structure of the Salmonella
group was one a prior’ indication of bacterial recombination. This pre-
monition has been confirmed in studies with P. R. Edwards on the recombi~

nation of flagellar antigens. In most Salmonella types, these antigens
~ 22 «

have a dual potentiality, only ‘ome of which is expressed at any one times
the oscillation wadweemthemis called "phase variation." Dy applying
phage grown on one serotype to cells of another flagellar type » in the
presence of homologous antiserum it is possible toselect against the
existing type, ami recover the results of transduction of flagellar
antigens, Forexample, phage grown on Salmonella abony, which is
ba enx ami applied te Salmonella typhimurium which is i: 1,2,4n the
presence of typhimurium eoren wi evoke two new and perfectly stable
serotypes, b : 1,2 and 1: enx in which one phase of the recipient has
been irreversibly replaced by its homologuefrom the donor. The first
of these happens to be a familiar serotype, that of Salmonella
paratyphi Bs the second has not yet been named in the existing codification,
The host range of the transducing phage permits fairly free exchange of
serotypic determinants among three somatic groups of Salmonella (A, B, D)
and it has therefore been possible to generate a considerable mumber of
new combinations of the diagnostic antigens,

No final theory of phase variation has yet emerged, However, it is
apparent that diphasicity represents the alternating expression of two

definite unlinked loci, What determines which locus will be active and
- 23-

which suppressed at any given stage is not yet known, but some indication

of a local, vereiy-reversibie, and at least partly heritable differentiation

enehe faclen
of the itself is given by the fact that the transducing competences

of the two phases of a given serotype are at least quantitatively different,
We find here a convergence of bacterial immunogenetics with developmental

physiology,

The hopeful remark has been made that the geneticist will arrive Mee

a wear

too late to introduce his jargon into bacterlology--in-fnets—tite-et-the

a“

-sanemesting at which the experimental. resulte leading to the present

ant . 7
without its penalities either, and TE soateination tn.

het wrk
entropy or structural organic chemistry ere commensurate with those of

selective aitforentials/and linkage maps,

If the mrk of scientific progress is an increasing ratio of un-

 

answered questions, bacterial genetics scores very high indeed, But, dea te 3

Aha
of the work that can be seen ahead ie~itttie—chort-of

vy
torkifings there is an underlying theme of the unity of biological y @ JAY

Processes that is indispensable in experimental design, The analogy °

 
with the development of bacterial metabolism is a sound one, where, as
C. 3. van Niel has pointed out the “unitarian approach® of comparative
biochemistry has become so large a part of our thinking during the past
two decades that the very fact of its having once been started is no
longer taken into account. I hope the same can be said twenty years
from now for comparative genetics, But as in metabolic research, we
also have to beware of the fallacy that all orgenisams meet the common
problems of biological existence by precisely the same mechanisms, But
if we accept the monophyletic evolution of life, we are not surprised
that these mechanisms show the stigma of family resemblance, That so
many diverse organisms transfer electrons by means of phosphopyridine
micleotide is testimony of the same parallelisms as are witnessed by the
pecarhmnton Me

universal role of -sesmuek wechanians and chromosomal organization of genetic

material,
The whedon of singling out any individual for an award in scionce
Le (at best) debatable, but I am pleased to have this opportumity to
acimowledge my indebtedness to my former professors, F. J. Ryan and
Edwerd Le Tatam, to my colleaguea and students already mentioned, and
to someone who belonge to each of these categories, my wife.

wy 2 .F
we af % vF 4 ? \ ‘
oe 4. . i
vv Af °
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