M. & I. May 13, 1950 SUL2iaRY SHEET RG 1445(C2) D UNIVERSITY OF WISCONSIN Joshuc Lodorborg cee Assistant Professor of Genetics ee oe "Gonetics of Salmonolla." Year ’ Requosted Recommonded Provisus Commitmont Application Pericd 3d (C2) $4320 045320 — $4,320 3/1/50-1/31/51 ~. anne RECOMZSNDATION: Approval for 1 year. COMMENTS ¢ The Study Soction considers seme progross has bocn made in this study and approval is recommended in view of tho existing ccmmitmont for furthor support. It is undorstocd thet thu investigetor has boon unabls to demonstrate genotic recombination in the gonus Selmonglle. Priority Scere: 191 Council action: 5 «Gefiction: Pravious and Current Support, this Project Grant No. Roquosted Grented Application Periscd RG 1445 $3700 $3,780 1/1/%8-6/30/49 1445(C) 3,780 3,780 1/1/49-7/32/50 Microbiology & Innunology May 5-6, 1951 SUMMARY SHEET UNIVERSITY OF WISCONSIN Joshua Lederberg Associate Professor of Genetics "Genetics of Bacteria." _ Previous Year - Requested Recommended Commitment Application Period ath (C3) | $9,936 / $4,320 - 9-1-51/8-31-52 5th (C4) 10, 800° | 4,320 9-1~52/8-31-53 6th (C5) 10, 800 \ ~ | th (C6) 11, 880 NU 8th (C7) 11, 880 RECOMMENDATION? Approval ERIORITY SCORE: 192 COGEMTS: Discussion revealed that the investigator's early work was done with E. coli. He then went to Yisconsin and applied sinilar technics to Salmonella species and did not have successe He has now gone off on a tangent but the new approach is extrenely interestings The need for competent investigation in the fiold of bacterial genetics wes expressed, However, it is felt that he should continue at his forner financial level until the new approach is tried and found productive. ” an PINAL ACTION: June 15-16, 1951 PHS Support, this Project Requested Granted Aovlication Period RG 1445 thru (C) $7, 560 $7, 560 7-1 ~48/ 7-31-50 1445(C2) 4, 320 4, 320 8-1~50/8~31—51L L&I Lo april 26-27, 1952 SUILIRY SHINT 3 72(c4S) . THE UIIVERSITY OF “ISCONSIN Madison 6, Wisconsin Joshua Lederberg Associate Trofessor of Genct "Gcnotics of Bactorta," ‘ ‘ Previous Year Requested —— Recormendo Commitment Application Fortod : ee) vtyy600 F< “, 320 4,320 9-1-52/8-31-, 3 045 860 =) 860 - te Gee ar ROCCE DaTION! Approval, pa ¢ PRICRITY SCR 1g pr" COt 72S This professor is a rccomized authority in the ficld of g-nevics, who has dcmonstrated his orisinelity and productivity. The supplemental fund roqu.sted is amply justificd in accordance with the terns of thc project and th. compctonce of the investigator, Indeed, the funds requvsted repr scnt almost the sum by Thich his provious request was rcduecd “until the now cpproach is tricd and found pro- ductive! FINaL sCTIOns Jun., 1952 PES Support Grant lo, . Requested Grantod Grant Trortod = 72 thru (c2) ‘11, 880 jL1, 880 7-1-48/8-3L-51 (03) 9,936 4,520 9-1-51/8-31-52 ") eee ro, eo wie J $s s ; 3 2 eames i rn THE GENETICS OF SALICNELLA RC+1UL15 (62) National Institute of Health, U.S. Fuolic Health Service, Eethesda, Md. Third Annual Progress Report, submitted April 20, 1951. Joshua Lederberg Associate Professor of Genetics University of Wisconsin, Madison, SU, 2iARY Current work suggests thet, under certain conditions, Salmonella cul= tures form reduced ceils which a) reedili; pess filters retaining the usual bacteria; b) are more resistant to disinfection (heat, alcohol, chloroform); end c) remain dormant in the absence of a stimulus from living cells. If verified, these findings might require a reexamineticn of concepts of becteriological sterility. | This result wes unexpectedly encountered in connection with work on the mechanism of genetic recombinetion. Such a procesgq, analogous to that established in Escherichia coli K-12, seems to occur in some strains of S. typhimurium. Whether there is a specific connection between the filtreble agent and genetic recombination, as apnears possible, is the subject of current exneriments. -i-« THE GENETICS OF SALMONELLA RG=1445 (¢2) Third Annual Progress Rerort, submitted April 20, 1951. Recanitulation and Introduction This research project on genetic aspects of the biology of Salmonella is directly related to previous and concurrent studies on genetic recombi~ nation in the releted group of coliform bacteria, It will help to out- line progress and aims of the Salmonella project if the results from Escher= ichia coli are briefly recapitulated,. About five years ago, BE. L. Tetum and the undersigned (at Yale University), discovered that genetic recombination occurs among cells of &. coli strain Km12 (1,2). This was demonstrated by mixing cells from different nutrition= ally execting or auxotromhnic mutant cultures on a synthetic agar mediun, }By sunvressing the auxotropnic verents, this medium selects for a small pro- portion of cells which have become nrototrovhic, i.e., able to form colonies in the absence of supnplementery growth fectors. With appropriately devel= oped mutant stocks it was possible to rule out the possibility thet these prototrovhs erise from intrinsic instability of the mutant parents; they could arise only from the interaction of the distinct mutents: e.g., Ab with aB give rise to AB. This type of recombdinetion process immediztely suggested the possibility of a sexual process in this bacterium, This pos= sibility has been reinforced by a number of experiments wich showed the following: 1) Recombination was not confined to nutritional fectors. Hany other genetic differences introduced with the narents (e.g., fermentetion cheracters, virus resistance, drug resistance) reessort in all possible combinetions emongs the prototronphs. 2) Recombination occurs pairwise. In mixtures of three kinds of mtants, only those recombinants occur which could erise from pairwise exchenges, whereas uniquely tri-partite exchanges are not found. 3) In certain crosses, cultures ("heterozygous diploids") have been isolsted which cerry the genetic fectors from both perents, which may later segregete during the further proliferetion of the culture (3). Single cell studies showed conclusively that tris secregetion involves the separation of intra~celluler units, not entire single cells (4). 4) YWumberous attempts to effect senetic exchanges by means of culture filtrates, cell extracts, or other prereretions not contcining normal viable cells of both nerents have feiled comletely. This requircnent for intact cells from both parents sunvorts the concent thet the fusion of ordinery vegetative bacterial cells is the bt-sis of genic conjunction (as in many other microbes) end thet no special semetic forms need be invoked. However, the actual fusion of cells has not been observed in this meterial, so that our conclusions on this detail of the "sexual" process ere entirely infer= ential, -2= Until recently, evidence for ;ene recombinetion in bacteria was confined to strain K-12 of . coli. Cavalli, woriting et Cambridge, Ingland, has discovered a strain there which can be crossed with K-12 (5) and more re—- cently a considerable number of strains neve been isolated (about 3) of a series of 500 tested) which elso skov this pnenomenon (6). Hany of these new "crossable! strains are quite different from X-12, some being classi- fiable as peracolon or as coliform intermedictes, This result makes it all the more necessary end hopeful to scrutinize other bacteria for similer genetic processes. Two objectives ere preeminent: 2) to provide the besis for genetic analysis’ of problems unique to other bacterial groups, Gcfen— antigenic and pethogenic variation in Salmonella, and b) to find better material for the determineticn of the mechanisms, scope, end ecological role of recombinetion. s te Experimental Resilts with Salmonella Considereble time was svent during the first two years in collecting suitable cultures, developing techniques for producing ciochemical mta~ tions in Salmonella (7), end in perZecting the training of the research assistant assigned to this problem (ir. iorton D. Zinder). After an inter= val during waich mutants were induced in a diversity of types, including S. tynhimurium, poona, madelia, "coli", end others, it wes decided to con= centrate on a coherent set of cultures of S. typhimurium. Such a set was provided by Dr. S, Lillceungen (Stockholm) who hed worked out 2. procedure for becteriophege t)ning of tnis snecies, He kindly placed et our disrosal representative cultures of each of his 22 tynes of world- vide origin. In this wey we could te feirl; sure of covering a compre— hensive sample of S. typhimurium cultures without unnecessarily reduplicating our worl, Auxotrovhic mutants heve been induced in 20 of these tines. The present moterial has alloved crossing tests (lixe those in Z. coli K-12) to be made in ebout half of the 200 possible c-nbinations (inter- end intra-strein crosses); the mutants required to comlete all of the possible combiné tions ere being produced from day to da’. it snould be pointed ont thet, desrite technical advances, the production end cherecterizeticn of at least ti0 double auxotrophic mitants in eech of 20 strains represents @ considerable tiplication of effort and hes occuried the lerger pert of the time svent on this project to date. Of the 99 combinetions so fer tested, 9 heve more or less consistently inimal agar mediun, while the perents seneretely do not form colonies under these conditions, This is preliminary presumptive evicence for recombination in Selmonella tymhinurium. Some of the combi- nations heve given very low yields of pvrototrophs, @ result which hinders the further study of the mecianisms of the enperent recombinétion. Our effort hes been focussed on one p2rticuler combineticn wiich proved to be excentionally fertile. Culture "A" is a mutant derived in tvo stens from Lilleeungen's type 2, "B" from tyne 22. "a" requires histidine end methionine iB" phenylalanine “plus® tyrosine, and tryptophane. Cultures of A or of B by themselves have never been founc to vroduce prototrorhs, even when very dense suspensions were pleted on nininal ager. However, mixtures of A with given prototronhs on nm -3- B heve given yields of prototrophs of at least 1075 of the perental inoculum, (considerably higher than has been found in z. coli). Since this combina= tion gives the highest yield of presumably recombinant prototrophs, it was selected for further study, Two directions ere being followed a) the genetic rules of recombination, and b) its biological mechenism, With respect to a) certain peculiarities have been noted already. At- tempts to induce fermentation mutants in a have been mostly unsuccessrul; 2 number of mutants heve been induced in 3, and such stocks as "B" Gal- Xyl~= (salactose-, xylose+negative) developed; in distinction to the Gal + XyI + cheracteristic of the original A and 3. "Gal" and "Xyl" are here used as unselected markers, is€e» the distribution of + end — qualities among nrototrophs of A Gal+ Xyl+ x B Gal- Xyl- is followed. So far, the proto= trophs neve been almost all Gel- Xyl- like the "BM" parent, and a like re- sult has been obtained with other merkers. However, a very small nronortion of Galt+ Xyl- and Gal- Xyl+ have been formed. The disproportion of types might be due to genetic linkage, but more work will be needed to cleer this Up. It has also been noticed thet many fermentation mutants lose the "fertility" characteristic of the original culture. This probebly rerre- sents an inherent instability in tue cepacity to react wito other strains wiich mast be given close ettention in our surveys The most unexpected results deal with the mechanism of genetic inter- ection, and heve been purposely iezt to the lsst. In view of their rather heterodox character, they will have to be subjected to more than usual scrutiny, and tested in other laboratories pefore they can be entirely ac- ceptable to any lerge »vody of yvorkxers, including ourselves. Tiese experiments began with one modelled ofter 2 revort by Be De Davis (8). A U-tube was constructed with an ultra-fine sintered Pyrex filter in the horizontal arm. The tube was sterilized and filled with broth. wan ves inoculeted in one 46rm, 13" in the other. 37 elternating suction on the tvo sides, tne medium was flushed from one compartment to the other until the cells had become so dense es to clos tne filter. Several experi= ments in which one side only wes {nocaulsted with A or B confirmed the in- tecrity of the filter, and the stebility of the perent culturese The cells in each comertment were hervested and washed seneratelye either A nor B cells from control exmerinments geve any nrototrovhs on minimal egere However, the 2 cells from the U-tubes in waich the opposite «rm conteined A, repeatedly geve numberous prototrophs; the A cells did note &s in the -exrerinents in wiich the cells vere nixed directly, most of the prototrowhs cerriea the unselected mericers of D, vut otuer ty. es herve elso peen noticed. Evidently, tne interacsion of A with 3.involves en "arent" sroduced by A which cen pass @ filter taet reteins the typical cells or a end Be The egent has been stucied farther in culture filtretes end other preperetions. It is not produced (excert to # very linited extent in eged cultures) by A or B cultures seperstely. fined cultures of A and 3 grown for severel hours vere sedimented snd tre superne tents passed through two pendler filters, one medium, one extre fine, (the first of these usually — ~~ suffices for sterile filtration). 0.1 ml. of such a filtrate plated with iu? cells of B usually yielded ca. 100-200 »rototrophs. The filtrates themselves were sterile by the usual criteria (no colonies on synthetic or comlete agar medium; no turbidity in yeast extract broth). Tyo filtrable factors are denonstreted by these experiments: 1) from B which stimulates A to form the agent 2) which reects with 3 to form pro= totropus. The first factor is probvebly a latent lysogenic bacteriopnege secreted by B, as it can be propageted on A concomitantly with phege lysis. & can be replaced by sublethal concentretions of crystal violet; other deleterious treatments ere being studied, The egent is readily assayed by pl-ting test samples with washed B cells on minimal egar. The number of prototronis formed is proportional to the volume of a given filtrate tested, i.e., the assay 4s linerr. The esent is more resistont than ordinery cells to inectivetion by heet, chlioro= form, benzene, or alcohol, end is relatively uneffected by exposures which effectively sterilize the ceils of B. It is apperently nondialyzable. It is precipitated by €0-70% ethanol or 60% saturated ammonium sulfate; the sediment redisperses reedily in water. The azent hes also been sedi- mented directly from filtrates by vltre-centrifugetion in the Spinco cen= trifuge. These findings simplify the concentration end preperation of the agente We have not succeeded in extracting the egent fron cells of 3B killed by heat, or subjected to autolysis under conditions proven harmless to the agent itself, This and leter findings sug:est thet the egent is 2 biological product rather than an intrecelluler component of 3. The most obvious interpretations of the egents ere: \ 1. as a "trensforming arent" siniler to those of pneumococci or Hemo— — philus influenzae. 2, as a minute cell product, a "gamete", or 2 form similar to the I~ forms reported vy severel other autiors (see 9). Tre following results are especially tentative but incline to the letter hypothesis. Microsconic examinetion of active sedizents shows berely visible grenules end rods resembling ordinery bacteria exceyt for their greatly diminished size. Probebly more immortent, pletings of anparently sterile filtretes with verious kinds of ceils, inclucing Escherichia ccli, heve re- sulted in colonies with the seme cultural cacrecteristics and matant merkers as the originel Be Tis suggests thet the agent consists of reduced cells ‘which ere a) "filtreble!! b) ere more resistent to antisentic treatments, ang c) will remain dormant except in the vresence of livirs vecteria, The nossible implications of this tentctive resvlt for brozd problems of enti~ sepsis end "sterility" ere obvious. In eCuivion, it shovld be telten into account in reviewing tne mecuenisms of ttrensformations" renorted for verious | bacteria. Whether they have 2 unique sexucl or gemetic function is prble= — metical., In preliminery experiments, novever, tne filtrates may heve evoked prototropns more reedily end frome vider range of mutent cultures than did _dntect cells of Be l. 9. References Tatum, E. Le, and Lederberg, Joshua 1947 Gene Reconbination in the Bacterium Escherichia coli, J. Bact. 53: 673-537 OE Lederberg, Joshua 1947 Gene Recombination and Linked Segregations in Escherichia coli. Genetics 32:505-525. Lederberg, Joshua 1949 Aberrant Heterozygotes in Escherichia coli. Proc. Nat. Acad. Sci. U.S. 35:178-184, —* Zelle, Me R. and Lederbers, Joshua 1950 Single-cell Isolations of Diploid Heterozygous Escherichia coli. J. Bact. 61:351-355. Cavalli, L. L. and Heslot, =. 1949 Recombinetion in Bacteria: Oute Crossing E. coli Z-i2, Nature 164: 1057-53. Lederberg, Joshua 195la Prevelence of Escherichia coli Strains Ex- hibiting Genetic Recombination. In Fress (Science). Lederberg, J. Isolation and Cheracterizetion of Biochemical HNutants of Bacteria, Meth. in hed. Res. 3:5—22. Davis, Be D. 1950 Nonfiltrability of the Agents of Genetic Recombina~ tion in Escherichia coli. J. Eact., 60:507~503. Stempen, Eenry and Hutchinson, . G. 1952 The Formation end Develop— ment of Large Eodies in Froteus Vulgeris 0i-19. I. Bright Phese Contrast Observations of Living Becteria. J. Bact. 61:321~-335. February 11, 1952 THLE COPY, Tne Genetics ef Pacteria (B72-63) annual Progress Zenort submitted to the Microbiological Institute, Tstionel Institutes of Health Public Fealth Service, Bethesda 14, Maryland by Joshue Lecerterz, isscciate Professor of Genetics Department of Genetics, University of Wisconsin Tais renort covers the period frem Avril 20, 1951 to February 1, 1952 A. Summary r Salmonella typhimurium, = nev mechanism of genetic exchange hes been discovered: transductisn. Many oc ltures engender a filtreble egert (FA) charecterized ty its geretic activity. FA transmits individual cherzcteristics to individual celis, in contrast to the linkege comslexes exchanzed in sexual recombination. All of the traits studied (nutritionel requirements; fermertations of suger : resistence to strentomycin; and flagellsr serotype) were transduci vl Tor the most part, leboretory mutetions of S. tynhimurium were studied. In addition, the natural differences of $ tyoni and S, tychimurium were exchanged resulting, for exomple, in the serological "nytria" of the antigenic formula: IX, XII, it--, (somatic antigen of typri; flagella of tynhimurium). Detailed studies of the genetic and kinetic cronerties of the TA have been initietved. It may be related to varticles etout .1l micron in diameter visualized with the electron microscope in nartially yurified active preparations. y In Escherichia coli, the sexusl »rocess underlying genetic recombination has been studied for some time. Merny new interfertile strains have been found, For the first time, evidence of svecific compatibility relatior— ships, ieee, of sexual or heterothallic differentiation, has been uncovered. Direct evidence of the sronteneity of drug-resistance mutation has been secured by a new method: replica vlat =~ 2 THE GLONCTICS OF BACTERIA -E72-C(3) Be Full Stetement of Progress 1. Genetics of Salmonella. The last renort presented first evidence for a process of genetic exchange in Salmonella tynphimuriun, This renort also gave a brief review of previous wor!: ~ con to these studies. At this time, it can be seen thet certein of «..e conclusions of the 1951 renort were incorrect, par- ticularly those which attempted to rationalize genetic excharge in Salmonella in terms of "reduced cells" or gametes. The mechanism of recombination in Selmonella appeers to be furdamertally different from the sexual processes - coli, To emphasize this distinction, the term "transduction" will oO Fy fla be anplied to the Salmonella system, Most, if not all, strains of S. tychimuriuz are lysogenic, carrying one or more latent phages, acting on other typhimurium or other serotypes, ncer avpropriate conditions irnvelving either attack by a latent rhage from another strain, or the {rether obscure) activation of its own latent nage, almost all strains have teen found to literate a filtrable agent preg "A" with remarkable genetic proverties. Sterile, cell-free preparations of FA ore capable of transferrinz individual traits from the donor strain to other susceptible strains. For the most part, we have relied unon nutritional requirements (obtained as uitroviolet-light induced muterts, and isoleted with the help of the penicillin method) as the genetic "nerkers" for these experiments. We hove also used fermentative differences (both natural and mutetional), resistance and susceptibility to streptomycin and the natural serological differences between serotypes (S. typhi and S. typhimurium). Since the 6 frequeacy of transduction for a perticuler character is only about 107 to 1079 of the treated cells, selective media must be used to detect the ' Ww ! chenges. For nutritional changes tris simniy involves pietings on syntaevic ager medium. Fermernt-tive or resistarce transductions were detected by platings on suger oF strevtomycin-containing agar. Flagellar antigenic changes were selected ty Gord's technique (inoculation of semisolid agar containing specitic ear. rviseruz). For technical reasons, the genetic changes involving ratritionel requirements have been emphasized for guartitative studies. Sever:: a2 the auxotrophic mutants are sufficiently steble that snontaneous variatic:s could not be detected; for some, the spontaneous reversions did cccur frequentiy enough +o require corrections for their retes. Perhans the most jmnortant distinctive feature of trans- duction (aside from the filtrability of the egent) has to do with the un- correlated behavior of different marxers. In severol different detailed experiments in wiich the TA-dcenscr aifvered from tne Fa-recipient strain in several respects (e.2., nutrivion; fermentation of xylose; fermentation of galactose; resistance to strervtenycin), eack. of the individual traits was sutzect to transductior, tut independently of all the others. Theat is, @ege, 2ll of the streptomycin-resistan ct ct ~ersduceé cells remained like the parent cells in respect to ferments jon ard nutrition, and so on. This is in morked contrast to the unrestricted excnonge of several markers in the non-filtrable Z. coli systex.. ‘fe have therefore defined "transduction as a genetically unilateral exczense, in contrast to the union of equivalent elements in fert: lizetion™, In the lest report, we were uncertain whether the Salmonelle FA might rot corsist of sone sort of "reduced cell! or "gamete", but the transfer of single elements is trcorsistent with such en interpretation. ‘We were also concerned abouts the persistence of occasional,vossidly dorment, celis of the donor strain in the FA filtrates. Ye have since found tnaet filtr«tion through UF sintered Fyrex discs con~ | sistentiy removes 511 contaminating cells without impairing transductive -~- activity. The greeter filtrability (through Mandler diatomaceous earth candles) of bacteria in FA prenerations compered to ordinary cultures is of questionable relevance to the present problem, although it may .still be significant in other connections lof. '-fornst). Most of the FA preparations usedin our present worl: show ro evidence of the persisterce ef the doror cells ir. ony organized form. Since individual traits only are transferred, transduced times are usually readily distinguishable from both the donor and the recipiert strain. Therefore, the sterility of the bay fm preparations (althouga well verified) is not critical for the validity £ their effects. Some thirty different markers, in severel different °o b trains, have been tested for their trensmission by FA, and gave comparable results in eacn case. There can be no doubt that the transductive system applies to most or all of the genetic material of Snilmornella. Reproducible, linear assays for FA have teen develoned, based upon the yield of trototropns from suspensions of auxotropric ceils plated with the test samples on minimal agar. This has allowed studies of the stability ard purification of FA, and of its edsorption on to susceptible cells. Concentreted preparations assaying close to 10° units/ml have been obtained by fractional centrifugation end nrecipitation with alcohol and ammonium sulfate. Such prenerations are visibly opalesecent, and show, under dark- field ané electron microscony numerous grenules uniformly about .1 micron in diemeter. The association of trensductive activity with these granules is only a working hypothesis, tut the size estimate is consistent with that secured by centrifugation ard filtration exverimerts with gradocol nembranes. The vrenarations are not yet, in ovr judszment, sufficiently pure to warrant hes proved to be resistant to several = rE aa direct chemical analysis. enzvmes, including trypsin end desoxyribonuciease. The last point is the chief difference between the Salmonella transduction and the "transforming" “ 5. systems of the pneumococcus ard Eermcrhilus, but may te a reflection of greater structural organization. The adsorptive prcperties of FA Mphenocopy" is reversible, disanpearing wher the cells ere recultured without aerstion. “whether tuis and other éiffererces of various F+ stocks are due to differer.ces in the Fr agent itself is under investigation. Ir addition to these stuiies, wor has continued on the nuclear cytolosy of haploid end dirloid EB. coli; on formal ard physiological geretics, especially of the loci controlling lactose-fermentation; and on the effects of radiations or the geretic behavior of E. coli. The present state cf these problems is such that a formal statemert of progress would not te very meaningful. Some details sre included, however, in a summary analysis of tuis laborctory's work: "Recombination analysis of bacterial heredity", Lederberg, J., Lederterg, 2,, Zinder, M.D., and Lively, MeRe, 1951, Cold Spring Hartor Symposia quent. Sicl., Vol. 16, in press (80 manuscript pages) of which reprints will te forwarded as soon as they are available, -13- C. Significant Accomplishments to Date. The most significant accomplisament of this research progran is to lay the groundwork for the genetic study of bacteria ty recombination techniques. This has led to the Giscovery in Escherichia coli of a re- contination mechanism thet is ulmost certuinly based on a sexual process, @ mechanism previousiy excluded from bacterial biology. The larger pert of our work since has concerv.ed the details of this gexuai process, its natural distribution, and its applicetion to the geretic investigation of drug resistance, enzyme formation ard antigens. It has been verified a that resistance to streptomycin is a result of gene mutation. Thi mutation resembles genetic variations in higher forms in several detailed respects: it occurs spontaneously; it can te localized on a "chromosome" ty means of linkage tests; it shows Simple dcminance relationshirs in neterozygous diploid cells (sensitivity is dominant to resistance). Investigations or.’Neurosrora have led other investigators to the . conclusion thet gene-enzyme reletionsnips are simple and direct. We have found nc support for this conclusion in studies on bacterial lactase. This. enzyme is sutject to control by any of a lerge number of different genes, sone of which also affect other enzymes, Studies on mutents have contributed to the elucidation of "direct" fermentative pathways of disaccherides (e.g., the amylomeltase of H. coli). In the course of these studies, various methods applicable to biochemical and genetic studies have been developed, e.g.: determination of lactase by a chromogenic substrate o~nitrophenyl~B-[-salactoside: the penicillin method for the selective isolation of biochemical mutants: reciica-nlating method for large scale characterization of cultu-es. Sinilar studies on a secord group of organisms, Salmonella typhinuriun, have led to a very different result. Recombinetion occurs, but not as a -~ la- result of a sexuel process. Instead, irdividual genetic factors are singly transduced from one cell to enower. This process has been ex- perimentelly verified to produce evolutionary novelties, for exemple, ~~ a serological thybriat of S. tryoni and §. typhimurium. These studies are not at a level wnere concrete accomplishments in the form of a new vaccine or artiviotic can be cited. They are concerned with the understanding of the diclezy of microorganisms, the details of waich are necessary to long-range develernent of technological, medical, or epidemiological control, The following publications have presented the moin accomplishments; in most cases the titles are self-expisnatory. Lederberg, J. 1947 Gene reccxnbination and linked segregations in E. coli. Genetics 32: 505-525. 1947 The rutrition of Salmonella. Arch. Biochem, 13: 287-290. 1949 Aberrant heterozygotes in Sscherichi2 coli. ———— ‘ Proc. Net. Acad. Sci. U.S., 35: 178-184, 1950 Isolatior and characterization of biochemical mutents of bacteria. Metrcds in Medical Research 3: 5-22. 1950 The veta-I-gelactosidase of E. coli strain Ke12, J. Bact. 60: 381-392. 1950 The selection of geretic recombinations with bacterial growth inhibitors. J. Zact. 59: 211-215. 1951 Single cell isolations of diploid heterozygous EH. coli, J. Bact. €1: 351-355. (with M3. Zelle). 1951 Frevalence of =. coli strains exhibiting genetic Os recombination. Science il4: 68-69. ‘ 1951 Streptomycin resistance: a genetically recessive +549. mutetion. ~ 1g - 1951 Genetic Studies with Bacteria. pp. 263-289 in Genetics in the 20th Century, edited by LeC. Dunn. MacMillan, N.Y. 1951 Recombination analysis of tacterial heredity. Cold Spr. Harb. Symp., 16: In Press. (with E. Lederberg, N. Zinder, E.R, Lively). 1952 Replica plsting ard indirect selection of bacterial mutants. J. Bact.: In Press (arch 1952) (with E.M. Lederberg) 195- Genetic exchange in Salmonelle. MS ready for sutmission (with 1.D. Zinder, pre 37). D. Plans for Next Year The gereral outline of the next yearts work is already inherent in the current experiments summerized in Part B. In the Salmonella transuc~ tion system, the following aspects snould be given snecial emphasis: 1) The purification of the transducing agent, following procedures worked out in preliminary fashion (esrecially differential cerntrifugetion); 2) morphological and chemical characterization of tke purified material; 3) further exploration of the conditions of formation of Fa, end its relationship to phage; 4) the distribution of FA-v~reduction and response — in Salmonella; 5) exploration of serolozical "hybrids" of other Salmonella types, and the pathogenic properties of such hybrids in experimental animals, S In Escherichia coli, the progren of immunogenetic study is merked for special emphasis. Antiseral reagents are teing prepared against inter- fertile strains; some of them heve beer. fractionated by reciprocal ab- Sorption, The segregetion of ertigeric differences in strain crosses; the examination of recombinants for romwerentel antizens; and the antigenic behavior of diploid hybrids ere the main features. In addition mutetion, J. Bact." 61: “tho: ~16- to somatic agglutinog eas, flesellar antigens and extractatle precipitino~ gens are to be studied, the letter including the enzyme lactase, When the groundwork has been laid, it is hoped to lool for mutations affecting antigenic specificity, Selective retiods should be feasible: immobilization by Serum-agar for flagellar chanves, end bacteriolysis with complement for Somatic antigen mutations. bv The heredity control of fertility by the Te System is also of present interest. The mode of transmission of the Ft egernt, its separation from the donor cells ana the physiologicel tesis of the FR modification by aeration are under present investization, We are also studying the Gistribution of the 7+ agert among bacteria, and will loox further into its correlation with the potentiality of senetic recomdination. In strain K-12, there is already evidence (the greater fertility of Fe x as Compared to F+ x Fe and the sterile “ x 7+) that the system leads to an incipient heterothallism as is choracteristic of many fungi. The F+ agents fron various sources will te comnered to determine whether par ticularly compatible combinations can be found that will lead to a sim plification of the study of recombinstior by genetic metneds. If the fre- quency of recombination can be upgreded, ond there is row the first evidence of rational control, it may be possible to anproach the problem of bacterial sexuality by more direct cytological study, mitetion. “Je” Bac