195°-1953

Application for reasearch support to
the RFS"ARCH COMMITTYF OF THY ONIVYRSITY OF WISCONSIN
submitted by Joshua Lederberg, Aasocizte Professor of Genatics
January 12, 1952
TITLE AND REASCHS FOR STUDY

Bacterial Genetics. One of the most importent, but least known
aspects of the biology of bacteria ie their genetics. In two species s0
far well studied in thie laboratory, Escherichia coli has given accumlated
evidence for a sexual cycle resewbling that of higher forms; more recently,
the related Sulmonella typhimurium has exhibited a mechaniem of transfer of
single or isolated genetic factore that is quite unique, though voorly
understood, We can achieve adecuate control over microorganiems, whether
from a technologies!, agricultural, or medical point of view only when
their genetics is understood, along with their biochemistry, physiology
and morphology.

RESULTS OBTAINED 10 DATE AND PROCEDURE for prejeeted atudy.

This preject is a continuing one, and {t will not be possible to
separate future procedures from past reeulte. The accompanying progress
report therefore also outlines the directions of work planned for the
future.

A. Formal genetics of Escherichia coli. ‘the analysis of formal
genetics of Escherichia coli 1a being continued. Special emphasis is be-
ing given to discrepancies between experimental data and the theory of a
linear chromosome aa the basia of heredity in this bacterium (as it is in
higher forms). Single cell studies on segregating diploids suggeet that
some discrepancies may not be inherent in the actual genetic ratios, but
in differential growth of different types. Thies work is necessarily slow,

and definite conclusions will not be available until it is completed.
A study of the genetic effects of radiations on diploid E. goli
has been initiated. The most significant finding to date is that single
cells treated with UV give progenies with a variety of effects: either the
cell (probebly the nucleus as well) ia not e unit target, or the effects
of radiations persist as a blolegical disturbance despite the apparent
gerowth-recovery of the cell. These effects then later give rise to the
variety of changes in different offspring.

fhe isolation of new strains of F. golf that can be crossed with ex-
isting fertile strains was a major subject thie yeer. Farlier estimates
were optimistic: of about 1500 strains tested, only 30, or 2% could be
crossed. These 30 strains sre being studied closely for (a) their linkage
patterns, (b) the possibility of compatibility groups and (c) characters
of interest for genetic study. So far as can de determined, the new strains
resemble the original 2. coli K-12 in their fundamental biology and life
cycle, However, many of them are culturally and serologically distinct.
We are now planning studies on the genetic basia of the natural (se opposed
to laboratory-created) differences between the strains. The serological
differences, in particular, permit of an immunogenetic analysis . For a
variety of reasons, bacteria provide excellent experimental aaterial to
study the biological bases of gene-controlled aatigenic differences such
as are the foundation of blood-typing in man, cattle, and other wammale.
In additicn, although FE. coli is "not pathogenic," the genetic basis of
eerotypes ie of intrinsic interest to the student of infections diseases.
This program was initicted on RF51:430 until definitive support could be
secured from the Rockefeller Foundation (effective Janusry 1, 1952).
Dr. P. D. Skser has undertaken its immediate direction, and has completed
preliminary experiments and preparation of sntiseral reagents.

B. A pew mechanism of genetic transmission in becteria. Salmonella
typhimurium (mouse-typhoid; enteric fever grovp) hes been the subject of
-3-

genetic study in this laboratory since 1947. During the past year, a
reasonably clear picture of its behavior has developed for the first
time. The Salmonella bacteria were chosen for this study because they
provide good experimental material for studies on virulence, because they
are readily cultured in the laborxtory and are related to the F. coli
familiar te us, and because their serological characters (presumably
under genic contrel) are important in pudlic health bacteriology.

Mr, Norton Zinter undertook this problem in 1948, an¢@ is using the
experimental date for his Ph.D. dissertation, The plan was to parallel
the work with EZ, coli. Biochemical mutanta were made in a large number
of straine, and attempts were made te detect croasing by plating mixed
cultures cn a selective, minimel agar medium, ‘The results obtained were
very confusing, as illustrated in previous reports, until it was realized
that the recombination mechaniem here ie quite different from thet in F.
goli. In B. Qoli, all the evidence points to » typical sexual proceas,
wherein recombination results from the fusion, and later segregetion, of
two intact auclel. In Salmonella, under certain eenditions, the cells
Telease fragments of genetic material into the medium. These fragments
may be either single "genes" or emall aggregates, but much less then sn
entire nucleus. Other cells may absorb these fragments. The next steps
we can only guess at, but an end result is that s "racombinsnt" cell is
sometimes formed in which the absorbed fregment beeemes a part of the
genetic mechanism of ite new host cell. To diatingeuieh this process from
fertilisation, which 42 the union of two essentially equil genctyres, ve
have designated it as genetic transduction.

franeduction le an infeetious process, if regarded in a certain light,
and one may ask whether it does not merely depend on the tranemission of
a virus "diaesse" from one cell to another, However, a great many characters

have been examined, and avery one of them is treneducible in the same Way.
These include many 4ifferent nutritional Feouirements, fermentation dif-
ferences, reeistznca to streptomycin, and type-diagnoatic antigens, the
same groupe of markers thet sre inherited as if cerried on a chr onc some
in EB. cold. the converse possibility, that Senic traneduction may throy
light on the origin of viruses, should be given close consideretion.

From the point of view of adartive plasticity, trensduction is not
ao efficient as fertilization, In this experiments, only a single fuector
is trensducible te s enll et one time, so thet "eroseea" of celle Ciffering

in many factore result in only a small frection of 223 the poenible gene

combinetions,

The narsantt ties vee subjected to very clone serological stu¢y,
acd 2 very lerge number of antigenic tyres or "species" are recognised
in the disgnoetic scheme. Theee types reoresent different combinetions
of somstic antigens (designated by roman numsrale) and flagellar antigens
(arabic numerals and lover case letters), Por example, S. typhimurium
is designated se IV, V XII; i; 1, 2, 3, while 8. Syphi is given as Ix,
XII; 4--. Bacteriologists have often apesulsted on the evolution of the
Salzonelia groun, and the origin of the different antigenic combinations,
Wat owing to the lack of convincing precedents, recombination was not im-
plicated. By transduction, however, a hybrid of $. typhi x typhimorina
has been obteined, with the antigenic formula IX, XII; 4--. this hybrid
hae not vreviously been described ag a Salmonella type. If £t had been
isclated (and it might well be antictoated to occur in e patient suffering
from a double infection) from @ earrter or patient, 1¢ would certainly
have been recognised aa a new species. On thia precedent, we may predict
that Salmonella types have arisen, and will ariga efein, from tha recom-
bination of factors of previcnely aatablished forna.

Transduction ia so different from the hereditery patterns familiar to

geneticists that 1% im difficult te reconcile it with the oytological
-~ 5-

observations that suggest the presence of similar nuclear structuras in
EZ. gold and in Salmonella. We will have to learn a great deal more about
genetic transduction before we can evaluate its eignificence for our con-
cepts of the nature of the gene and its relationship to the cell.

C. Replica plating and indirect selection. A method has been developed
which should be useful in any of a variety of microbiological screening
programs, in which a large number of cultures must be tected in a veriety
of media, This method, replica plating, has already become indispensable
in this laboratory for the isolation of nutritional mutants, and testing
genetic segregates, In principle, replica-plating is a simple printing
precess. A sheet of velveteen is fastened te a woeden support. An agar
plate carrying the colonies to be sersened is then pressed down, trans-
ferring on imprint ef each colony on the velveteen. Plates of various
agar media can then be pressed on the same velvet, and each of them will
be imprinted with a replica of the original growth, The advantage of the
method is, of course, that upwards of 200 colonies on a plate oan be trans-
ferred in one operation to a series of other plates with accurate registration
of position.

A less routine application of replica plating has been made to the
problem of the origin of drug-resistant mutants. Despite considerable
evidence in favor of spontaneous mutations followed by biological selection,
the question has continued to be mooted whether an antibiotic might not
actually induce variations to resistance to 1% on the part of previously
sensitive bacteria. So long as resistant mutants could not generally be
detected or isolated without applying the antidlotic or rather unusual
environmental conditions, only rather indirect and abstruse evidence could
be brought to bear. It is now possible to isolate resistant mautents by
indirect selection in which the cells are not exposed to the antibiotic,

In principle, spontaneous mutations to resistance to, e.g. streptomycin
are loeated in s film of growth on plain agar by meane of replicas to
streptomycin agar, Since such mutations do appear to be inherited, they
occur in clones, members of which remain on the plain agar plate after
their aibe have been removed via velvet to the selective agar, By using
the presumed sitee of realetant clones for fresh inocula again to plain
eger, 2 relative concentration of about 100-feld can be achieved for the
resistant mutants, Appropriate repititions of the process (four or five tines)
eventually result in the separation of the mutant clones in well-isolated
colonies from whieh pure cultures can be made. The enrichment line itself
has never been exposed to the antiblotia; the sibs are used to locate the
resistent mutants. Ina sense, indirect selection parallels the use of
pedigree data to select roosters or bulls for breeding stock fer egg or
milk production. The successful application of this method should remove
any doubt that mutations to drug-resistance occur in a bacterial culture
quite independently ef the presence of the drug itself.

D. Genetic studies on actinomycetes. ‘here are both cractical and
theoretical reasons for commaring the genetics of actinomycetes with that
of the simpler bacteria. On the one hand, one might expact to find nuclear
proceases in a form more readily analyzable, and more closely resembling the
filamentous fungi; on the other, the actinomycetes are now among the most
important technical microorganiems. A study has been initiated in Streptomyces
grisedus, and related forms, A number of biochemical mutants have deen
prepared--these are quite similar to the mtante previously obtained with
Neurospora end dbacteria--with the help of replica plating. Combinetions of
different mutants on minimel agar show various affects. Many of the mutants
exhibit eyntrophiem, or mtual feeding of needed metabolites, in an especi-

eslly dramatic way, but this has been disadventageous in tending to obscure
genetic interactions. Several combinations of mutants which show little

or no eyntrophiem have given mycelia which continued to grow on minimal
medium. The spores, however, give only one or the other parental culture.
Although formal proof is so far lacking, there ean be little question thst

we are dealing here with heterokaryons, that ie an admixture of genetically
different nuclei in a common cytoplamm, It seema likely that heterokaryosis
accounte for the often cited observation that mactinomycete culture may
remain constant when the myceliue i# prepagated, but throw many variants

when spores are plated. Finally, in a few instances, stable propotrophic
cultures have been noted, and these may represent recombinants resulting

from a sexual process. If sco, sexuality is rather sporadic within this one
atrein of S. grigeus; it must be pointed cut that other explanations have

not yet been ruled out. A group of other Streptomyces oultures (S. venesuelaec,
8. goelicolor, &. lavendulae)is being studied in a compamtive and combinatorial
plan,

4, FIBANCIAL SUPPORT requested:

 

Staff:
Assistant, 12-month, lise Cahn 1,320
fl ; 10-month, To be named 1,160
" an “#8 1,100
Kourly helps 5006
4,020
PERSONNEL:

Moat of the present aselstente plan to graduate (1 Ph.D., 2 4.5.)
this June, and replacement candidates are under consideration. MTlise
Cahn already hae ,n M.A. (Indiana) and came here last year with an
undergraduate GPA 2,20 and two years additional experience, She has

worked satisfactorily this past semester, and should ultimetely qualify

for the Phd,