October 25, 1952 The Genetics of Bacteria (E72-C4) Anmal Progress Report submitted to the Microbiological Institute, National Institutes of Health Public Health Service, Bethesda 14, Maryland by Joshua Lederberg, Associate Professor of Genetics Department of Genetics, University of Wisconsin This report covers the period from February 1, 1952 to October 20, 1952. A. Summary In Salmonella, the agent of genetic transfer (transduction) has been tentea- tively identified as bacteriophage. Under certain conditions, it appears that phage particles may incorporate fragnents of the genetic material of the host bacterium. When these phages infect a second host they may transduce these genetic factors to it. In previous studies, the genetic markers involved mutritional, fermentative and drug-resistance factors. Special attention has now been given to the transduction of flagellar antigens, which are the basis of serotypic clas- sification in this group. Several new serotypes have been built up, as well as presently recognized species, by the recombination of antigenic factors: for ex- ample, S. paratyphi B (b: 1, 2) from S. typhimurium (4: 1, 2) X- S. abony (b: enx). The genetic basis of nonflagellated 0-forms and of monophasic Salmonella types has also been explored, ani should lead te useful applications in the simplified prepar- ation of diagnostic sera and in the typing of otherwise untypable isolates. The genetic mechanism of antigenic phase variation has also been studied. In E, coli, the genetic and physiological control of "sexual" fertility have been further explored. Stocks showing extremely high frequencies of recombination have been found, and some preliminary studies have been made on the cytological basis of recombination. These do not yet point to any definite conclusion. lyso- genic bacteriophage has been found to behave as if it were a genetic factor, and an isolated instance of genetic transduction by phage has been found. These observations proviée further support for the previously unsuspected genetic functions of symbiotic phages. Immunogenetic studies with different fertile lines of E. coli have shown that serotypic factors are under direct genetic control much as in higher organisms. ~ Qe THE GENETICS OF BACTERIA E72-C(3)-C(4) B. Full statement of progress 1. Salmonella. Previous studies summerized in the last report (February, 1952) have brought to light a new mechanism of genetic exchange in Salmonella typhimurium: genetic treneduction. Varicus hypotheses have been entertained on the nature of the trans- ducing agent, including the possibility of its relationship to "L-forns," but were abandoned as new evidence was developed. The agent (PA--filtrable agent) was most consistently found in lysates provoked by phage and appeared to be associated with particles about 0.1 micron in diameter. These particles are evidently bacterio- phage itself. The identity of FA with phage has deen supported by the similarity of FA and phage titrations after filtration through gradocol membranes, high speed centrifugation, and most pertinent, adsorption on specific Salmonella hosts. I[t has not yet beon proven that the same particles which transduce can also lyse; possibly, the transducing particles represent immature or imperfect phage. However, the more generel question has not been settled whet determines whether a bacterium will lyse or will become symbiotically infected after it adsorbs a particle of the "temperate" phages used in these investigations. Whether lytic phages also potential- ly transduce cannet be tested as the new host cells will be destroyed. Whether ten- perate phages in other bacterial species may have similar genetic functions is a point that we hope to examine when the occasion permits. Aside from the theoretical implications of the mediation of genetic transfer by phage, the characterization of FA has been very important in guiding the prepar- ation of FA from other Salmonella serotypes for studies on antigenic recombination. fhe host range of the phage that has been used in most of the studies to date, PLT-22, ie confined to serotypes carrying the XII2 somatic antigen, i.e. most members of groups B and D. Temperate phages with different or overlapping host ranges would be useful to extend the range of these experiments, especially if one could apply a phage with cosmopolitan range, specific for the "R" antigens. Dr, C. C, Spicer (supported by a W.H.O. fellowship) is currently working with the principal investigator, but the phages tested have not yet been brought to a sufficiently high titre. FA has been prepared from a considerable number of serotypes in groups B and D, and investigated in respect to the basis of 0-forms, monophasic types, the mechanism of phase variation, and the synthesis of new serotypes. Te studies on 0-forms wore carried out in association with Dr, B. A. D. Stocker (a Commonwealth Fund fellow). Salmonella types iacking flagella and H antigens were received from several sources. In some cases, they were Known to originate from a definite type; the history and type of others were unknown. In addition, a phage was received from N. Boulgakow (Paris) that had been de- scribed in 1936 as specific for flagellated species of Salmonella. This old report had been forgotten or discredited, but we were able to confirm his prin- cipal findings. The flagellotropic phage has been a convenient reagent for selecting nonflagellated 0 forma from the occasional susceptible H strains. There is no obvious relationship between the flagellar serotype and susceptibility. FA prepared from motile Salmonellas was applied to various 0 forms in semi- solid agar, so as to select for any transductions of motility. Swarms of motile bacteria were obtained in every case. Wherever the original type was known, the tranginduced motile forms conformed to it, rather than to the source of the FA. “ = =< in another instance, the sercdiagnosis of the transinduced H form agreed with previous suspicions from the biochemical behavicr of the original 0 (S. dublin-- P, R, Edwards, priv. comm.). One exception to this rule wes found in a strain provided by F. Kauffmann, an 0 ferm described as originating from a monophasic S. paratyphi b. The motile swarms obtained from this O form were of two types! the first b, conforming with the original etrain, and the second conforming te the source of the FA e.g., 4, from S&S typhimurium. Farther genetic studies on these i transinductions support the conclusion that they are exceptional trans- fers of two closely linked fectors, one for motility, the other for the specificity ef the E antigen whereby b is substituted by i. Several genetic loci ere concerned in the immotility of different 0 forms. FA wae prepared from each of them, whenever feasible, and applied to each of the others. Each FA conferred motility on each of tne other 0 forms, showing that each 0 form could complement the genotype of any other. Mo 0 form could trans- duce itself, but FA from spontaneous motile reversions (which occur infrequently 4n the strains examined) conferred motility on the parent 0. At least five or six loci for flegella have thus been identified, but only one of then is linked to a determinant of H antigens. It seems reasonadle that hitherto untypabdle strains shovld be typed by transducing motility to them from a motile Salmonella. A rere flagellar type shovld be used as the source of FA to facilitate the detection of exceptional cases where the transinduced H may conform to the FA rahter than tis own original type. Efferts to transduce notility to the natural 0 serotype, 5. pullorum~gallinarum, ware unsuesessful. This may be due partly to the strong lytie action of the phage 68 these hosts. In addition, the immotility may be due to the coincidence of saveral ganetic changes which cannot be restored all at once by transduction. Another category of imactile cultures was received from B, Leifsen--thees are “paralysed,” i.e., they carry a distinctive H antigen {(S, typhimurium) but the flagella are nonfunctiozel. FA from 0 forms conferred normal motility on the peralysed strains, and vice ‘rerga. In addition, two paralysed strains wers found thet were complementary to each other. It 48 concluded that two additional Loci, distinct from these characterised in the 0 forms, are involved in flagellar funetion, Thus, genetic control over Salmonella flagella is exercised at three Levels: (1) the determination of whether flagella will be formed at all (several loci), (2) whether they will function in motility (two loci) and (3) their anti- genic constitution (one locus so far). The genetic loci involved appear to ba entirely separate except for an unusual "linkage" of one of the factors of the first group with the third. Subsequent studies have been concerned primarily with immumogenetic problems. It may be pointed out, if further evidence needs to be adduced, that these genetic studies are entirely inconsistent with Pijper's theory of motility, according to which the flagella ere accidental concomitants of motion, rather than the locomotor organs themselves. Monophasic strains of Salmonella types have been recognized as wreful re- agente for the preparation eof diagnostic antisera which will ba uncontaminated bir nke uniibelies for abbernative phases. Several mengphsaele cultures have beon collseted & tm ar frgm the field by Eéwards and Keuffmann, and are used for thie murpace. 2% 205 tkoly thet monephasicity is Guntrolled by the genetic backsrouni of $he s67aln, sather than by the particular antigenic facter involved. Snéc is Derne oat oF tho traumluetions sumbered 1-10, AL antigenic transductions to the mencphasic a) ce ey pe a ly S. typhi, or to @ monophasic variont S. paratyphi 2 have resulted in types which are thonselves monophasic. If the technique can be extended to species carrying very infrequent somatic antigens, the transinductions should be especially useful for the preparation of disgnostic gera which will be effectively pure without absorption. Work has been initiated en the genetic mechanism of phase variation. A con- sideration of transductions # 11 and 12, tegether with other data, has led to the following paradox? when a culture is in the specific phase, the antigenic factors for the second phase cannot be detected cither in the FA from this phase, or vy its serological reactions. The second phase is, however, latent in the bacteria be- cause, for example in # 11, after b has been substituted for i, the second phase continues to be l, 2. The sane argument holds for the latency of the specific Phase, conversely. The following hypothesis is suggested: phase 2 and phase 2 are determined by different factors at each of two loci, respectively. The functioning of the two factors is mutually exclusive, so that one locus is some- how suppressed. Ina comparable situation in Paramecium, Sonnebdorn has shown that the expression of latent scrotypes is a matter of the cytoplasmic state, but it appears more likely here to concern a mechanism more closely connected with the locus itself. There are few analopies for this behavior in‘ the genetics of higher forms, except that many workers have made similar speculations for the differ- ential functioning of different genes at different times and places during embryonic development. Further studiés may be expected to furnish a solid basis for the con- firmation ov refutation co? this hypothesis. As a by-product of these studies, many new serotypes have been reconstructed, as shown in the accompanying table. In addition, familiar serotypes such as S. paratyphi B have been recovered, as in #11, The principal investigator is planning to initiate a more systematic program of development of Salmonella types in col- laboration with Dr. P. R. Hiwards at the Public Health Service Communicable Diseases Center at Chamblee, Ga., in the course of a forthcoming visit to his laboratory. New Salmonella serotypes are being isolated from the field nearly every month. That transduction plays a likely role in the evolution of these types seems almost certain: the gut enviroment is known potentially 'to contain all of the essential elements-~-Salmonella bacteria, phages and copro-antibodies--of the laboratory model. It may also be argued a forteriori that care should be taken against the indiscriminate use of antibiotics in chemotherapy or in nutrition which may encourage a similar diffusion of drug-resistance from resistant variants that may be innocent in themselves to nore serious pathogens inhabiting the same environment. 2. Escherichia coli. The discovery of the genetic control of compatibility was outlined in the previous report. By an extension of this work, special combinations of parents have been shown to give a recombination frequency of as high as ten percent in four hours of mixed culture. This is still a relatively low rate for direct cytological observations, especially as optimal rates of recombination are associated with high rates of growth, which make it difficult to follow individual cells under the microscope.. Some observations have been made from which bigh frequency strains (Hfr) seem te be associated with a poorly staining material that forms blebs or droplets near the cell surface. In a few instances, cells have been seen under phase microscopy that appeared to be stuck together, as if by this material, but it has not yet been possible to work out their subsequent behavior either in living = 5 or in muclear stained proparations. No suggestions of holocellular fusion or Copulation have been scen. As a working hypothesis, it is suggested that the parent cells become temporarily attached to each other, and that a nucleus is transmitted from one to the other, followed by the separation cf the parent cells. This would be comparable to the conjugatien observed in ciliates, and eonsistent with the genetic finding that the zygotes appear to occur singly, even though the parent cells are presumably multinucleats. Dr. 7. C. Nelson and the principal investi-~ gator are studying the physiological conditions for optimal frequencies of mating with the Hfr cultures in order to facilitate further cytological study. Dr, E. 4H. Lederberg (Project Associate, Chemical Corps support) and a graduate student, M. L. Morse, are studying the genetic functions of bacteriophage in E. coli. The latent phage is transmitted in crosses os if it were attached to a definite locus in the bacterium, Conversely, transduction by this phage has been observed, but only for one rarker-~a locus very closely linked to the latent phage in crosses. Unlike Salmonella trangduction, no other F genetic factors appear to be tranamissible by this latent phusge. The crose-fertility of new isolates of E. coli has permitter® the development of an immunogenetic program with this organiem (Dr. P. D. Skaar). 0, K, and H reagent gera have been developed for several strains. The results of crosses eo far show that different genetic factors control each of these major antigens, and new combinations are produced by crossing. Unfortunately, the E. coli lines of greatest medical interest (0111234 and 055) have not yet given positive results in crossing tests. Susceptibility to the well known T phages appears to be associated with the absence oither cf the 0 or of the K component. REFERENCES (a) Other workers Sertic, VY. and Boulgakov, N. A. 1936 Bacteriophages specifiques pour les varietes bacteriennes flagelles. C. R. Soc. Biol. 123: 887-888. Edwards, P. R. and Bruner, D. W. 1946 Motes on monophasic Salmonella cultures and their use in the production of diagnostic serums. J, Bact, 522 493-498. Friewer, PF. and Leifson, FE. 1952 Non-motile flagellated variants of Salmonella typhimurium. J. Path. Bact. 64: 223-224. Kauffmann, F. Enterobacteriaceae Munksgaard: Copenhagen, 1951. Sonneborn, T. M. 1950 The cytoplasm in heredity. Heredity 4: 11-36. C, Significant accomplishments 1. The discovery of genetic recombination in E. coli strain K-12, and in a mumber of new isolates. , 2, The discovery of genetic transduction in Salmonella. 3. The demonstration that drug-resistance results from spontenegus mutation. Puller detail is given in this and precssing progress reports, and in the following articles of which reprints heave been furnished: Lederberg, J. et al. 1951 Cold Spring Herbor Symposium 16s 413-443 "Recombination analysie of bacteriel heredity" Lederberg, J. and Lederberg, E, M. 1952 Jour. Bact. 63: 399-406 "Replica plating and indirect selection of bacterial matenta" D, Plans for next year For the immediate future, the following program is planned: de Physiological study ef conditions of "sexval" recombination in EB. coli, and cytological study of ita material basin. cS 2, The relationship between the establishment ef lysogenicity and genetic transduction following infection by temperate phages in Salmonelja, and, EB, coli. 3, The extension of transduction technique to other serotypes of Salmonella, especially for alterations from rough to smooth, and for substitutions of somatic and Vi antigens. © oY , The development of & systematic progran for breeding Salmonella types, especially those useful as antigens for disgnoetic work. ' 5. The investigation of mechanisms of genetic exchange in other bacteria. 6, Problems of gene action in relation to the formation of specifig enzymes and antigens. i. 2. 8. 9. 10, 11, 12. abony Intertype transductions in Salmonella Traneducer IV, V, XII; i: 1, 2 typhizuriun IV, ¥V, XII; b: 1, 2 paratyphi 3 altendorf IV, V, XITs v3 1, 2 heidelberg Iv, V, 327s eh: engis sandiego av, V, XII; 4: 1, 2 typhimurium IV, XIZ3 o: 1, 7 altendorf | IV, V, X22; x: 1, 2 heidelberg I, TX, AIT; ga: - enteritidis I, IX, XIZ3; (gp): dublin 0 ?orn I, IV, XII; Bb: enx abony I, IV, XIIT3 b: onz ank ~—X mond awk ook nk WX francdaces KX, XII: dz ~ typhi typhi ™, ZEIT: a: - typhi IX, XII: d: - typhi IX, XII; ds typhi IV, V, XIZ; (b): -- paratyphi B, 0 fern monophasic IV, ¥, XII; (b): -- IV, ¥, XII3 (b): -- IV, V, XII; (b): - IV, V, XII; (b): - IV, V, XII; 43 1, 2 typhimorion IV, ¥, XIT; i: 1,2 typhimerian Result MX, XII; is - new type m, XII; 6: ~ new type (monophasic S. onarimon) IX, XIIs c3 - nev type new type (monophasic S. shoreditch) TX, XII; ommes e O_ now typo typhimurium, monophasic I¥, ¥, HII; ec: - altender?, menopkasic IV, V, XII; ri - heldelberg, monophasic i, V, XII; gm: - Osson IV, ¥, XIX: ep: - nov type IV, ¥, XIZ+ bs 1, 2 paratyphi 3B IV, ¥V, XII; 4: enz new typo