GENETIC EXCHANGE IN SALMONELLA NORTON D. ZINDER AND JOSHUA LEDERBERG Department of Genetics, University of Wisconsin, Madison, Wisconsin Reprinted from JOURNAL OF BACTERIOLOGY Vol. 64, No. 5, November, 1952 Printed in U.S.A. Reprinted from JOURNAL OF Bac~emo~oou Vol. 64. No. 5, November, 1952 l'rintpd in U.S.A. GENETTC EXCHANGE IN SALMONELLA' NOR.TON D. ZINDER AND JOSHUA LEDERBERG Departmenf oj Gewfics, University of Wisconsin, Madison, Wisconsin Received for publication April 18, 1952 Genetic investigations with many different bacteria have revealed parallelisms and some contrasts with the biology of higher forms. The successful application of selective enrichment techniques to the study of gene recombination in Es- cherichia coli (Tatum and Lederberg, 1947; Lederberg et al., 1951) suggestred that a similar approach should be applied to other bacteria. This paper presents the results of such experiments with Salmonella typhimurium a.nd other Salmonella serot,ypes. The mechanism of genetic exchange found in these experiments differs from sexual recombination in E. coli in many respect's so as t,o warrant a new descriptive term, transduction. MATERLILS Ah'D METHODS Most of the strains of S. typhimurium were provided by Lilleengen (1948) as representative of his 21 "phage types", LT-1 through LT-22. Most if not all strains of X. typhimukm are lysogenic (Boyd, 1950), and these have provided 12 lines of bacteriophage. Other cultures were obtained from F. Kauffmann, E. K. Borman, and P. R. Edwards. All cultures were maintained on nutrient agar slants. Specific growth factor dependent mutants (auxotrophs) were obtained from ultraviolet irradiated cell suspensions subject'ed to the penicillin method for selective isolat,ion (Davis, 1950a; Lederberg and Zinder, 1948). Similar mutants have been obtained in Salmonella by Plough et al. (1951) and Bacon et al. (1951). Other methods for the isolation and characterization of auxotrophic and fer- mentation mutants have been documented elsewhere (Lederberg, 1950; Leder- berg and Lederberg, 1952). Streptomycin resistant mutants were selected by plating dense, unirradiated cell suspensions into agar containing 500 mg per L of dihydrost)reptomycin. "Complete" indicator medium (EMB) was made up from the same formula as for E. coli (Lederberg, 1950). The defined eosin methylene blue medium ("EML agar") contained in g per L: sodium lactate, 2.5; (NHd)zSO.+, 5; NaCl, 1; MgSOI, 1; K2HP04, 2; methylene blue hydrochloride, 0.05; eosin Y, 0.3; and agar, 15. Difco products, penasksay broth, and nutrient, agar, mere employed as "complete" media. 1 Department of Genetics, paper no. 479. This investigat,ion was supported by research grants (E72) from the Xational Microbiological Institute of the Kational Institutes of Health, Public Health Service, from the Rockefeller Foundation and from the Research Committee of the Graduate School from funds supplied hy the Wisconsin Alumni Research Foundation. This work has been submitted by the senior author to the University of Wis- consin as a dissertation for the degree of Doctor of Philosophy. His present addrws is. Rockefeller Institute for Medical Research, New Tork, Xew York. 679 680 NORTON D. ZINDER .4ND JOSUUA LEDERBERG [VOL. 64 Unless otherwise stated, all cultures were incubated at 37 C, and plates were scored after 24 and 48 hours. EXPERIMENTAL RESULTS Direct crosses: platings of mixed cultures. In E. coli, recombinants were detected selectively by plating various auxotrophs together on minimal a.gar. Both parents are suppressed on this medium and, barring various experimental errors, colony formation is confined to prototrophic recombinant cells. Such errors can be debected by appropriate controls but are best mitigated by t,he use of double NO. LT-2 SW-272 SW-414 (LA-2) LT-22 SW -240 SW-279 SW-307 ;;;;; LA-22 SW-479 1 SW--r43J Type 22 parent Phenylalanine and tyrosineless SW-240 tryptophanless SW-279 galactose-negat,ive SW-307 xylose-negative SW-351 streptomycin-resistant SW-435 mannitol-negative SW-435 maltose-negative LT-7 Type 7 parent SW-lb4 Prolineless SW-188 Methionineless SW-191 Leucinelcss SW-481 SW-184 galactose-negative SW-492 SW-188 galactose-negative SW-503 SW-191 galactose-negative SW -514 LT-7 streptomycin-resistant SW-515 SW-503 streptomycin-resistant TABLE 1 Mutant strains and symbols used -. I6UTATIONS PF,*TINE* SYMBOL __-__~- __~ __.- ~~ __.. -~ ~-. .-__ --__ Type 2 prototrophic Prot Methionineless, auxotrophic Aux SW-272 histidineless Gal- Xyl- Sr Mtl- Mal- nutritional mutants (diauxotrophs). These are obtained by the iterated isola- tion of mutants in previously established auxotroph lines. One of Lilleengen's strains was refractory to our techniques of mutant isola- tion. Two-step mutants with mutually complementary nutritional requirements were prepared from each of the remaining twenty types, Of t'he two hundred possible pairwise combinations, including "selfed" crosses, one hundred were tested. Each combination was studied by mixing and plat.ing log washed cells of t.he two parents on a minimal agar plate. Fifteen mixture plates a,nd five control plates for each parent by itself were inoculated in each test. Fifteen combinations yielded prototrophs in contrast to barren controls. Strain LA-22 was the most "fertile", especially with LA-2 (see table 1). This cross yielded 19521 GENETIC EXCHANGE IN SALMONELLA 681 about one prototroph per hundred thousand parental cells plated. Crosses in which LA-22 was not involved gave prototrophs so infrequently and sporadically as to be of doubtful significance. It has since become evident that LA-22 is genetically a single, st'able mutant although it was derived in two steps and has a complex nutrition. LT-22 is lysogenic for a virus (hereafter referred toas PLT-22) active on LT-2. This virus is capable of inducing lysogenicity in LT-2. Among the lysogenic derivatives of LA-2 three different interaction groups were found: the majority no longer interacted with LA-22 to give prototrophs; a few interacted with impaired efficiency; still fewer were not affected in this respect. These experi- ments indicated that genetic exchanges did occur and that latent bacteriophage played some role in the interaction. Indirect crosses: pihtings of cells andjifiltrates. To test the possible role of filtrable factors in this interaction, a u-tube with an "ultra-fine" sintered Pyrex filter partition was prepared according to Davis's (1950b) design. By alternating suction between the arms of the tube, two intact populations of growing bac- teria could be made to share the same medium. The integrity of the filter was verified in control experiments by leaving one compartment uninoculated. Then lo8 cells of each parent were inoculated into twenty ml of broth and placed in either arm of the tube. Ten ml were flushed from side to side every twenty minutes for four hours while the culture grew to saturation. The two populations were washed and plated upon minimal medium. Prototrophs appeared in the platings of LA-22 but not of LA-2. Sterile filtrates of LA-2 broth cultures did not elicit prototrophs from LA-22. However, filtrates from mixed cultures of LA-2 and LA-22 elicited about one prototroph per million LA-22 cells. Thus LA-2 produced a filtrable agent (PA), under stimulation from LA-22, that could elicit prototrophs from LA-22. Filtrates of LA-22 cultures, containing sub- stantial amounts of phage (PLT-22) active on LA-2, also stimulated FA produc- t,ion from LA-2. The role of this phage will be discussed later. To help the further exposition of our experiments, we shall use the term transduction for genetically unilateral transfer in contrast to the union of equivalent elements in fertilization. The working hypothesis that Salmonella FA is an agent of genetic transduction provides a useful frame of reference for our discussion. Assay of FA. Stock FA was prepared by growing LA-22 and LA-2 in mixed culture in broth. After 48 hours, the cells were sedimented and the supernatant passed through a sintered Pyrex filter. The sterility of a filtrate was verified by inoculating samples into broth at the time of preparation and by platings in agar as controls for particular experiments. This precaution was taken although complete sterility is not critical to most experiments since more than a million cells of LA-2 per plate are needed to interact with LA-22 to give prototrophs in the "direct crosses". These preparations have been stored in the refrigerator for several months without loss of activity. A standard procedure for assay of FA was developed for further work. LA-22 was grown on nutrient agar plates and harvested in dense saline suspension. 682 NORTON D. ZINDER AND JOSHUA LEDERBERG [VOL. 64 The viable count was obtained by plating suitable dilutions on nutrient agar. Various dilutions of cells were plated with a constant volume of an FA prepara- tion on minimal agar. Prot,otrophs appeared at 24 hours and were counted after 48 hours. Figure 1 shows that a constant response was found with about log to lOlo cells per plate. The decline at high cell densities was probably due to overcrowding and inhibition of colony formation, and at lower densities to physical separation of cells and agent or to the saturation of susceptible cells. log cells of LA-22 were plated with serial dilutions of F,4. Over a considerable range a linear relationship was found between the yield of protot,rophs and F&ML 0.012 0.025 0.05 0.1 I I I I 1 140 - 120 - I I 100 I09 IO'O IO" CELL COUNT Figure 1. Assay of PA. FA (LT-2) and cells (LA-22) were mixed at various dilutions and plated on minimal :tgar. Prototrophs were counted after 48 hours. amount of FA (figure 1). The effect of higher concentrations of FA will be dis- cussed in a later section. A unit of FA may be defined as the cont,ent of a filtrate that will elicit a single prototroph from an optimum concentration of LA-22 cells. Filtrates from mixed cell prepa,rations usually contain about 2,500 such units per ml. Chemical reactivity of FA. With the development of a standardized assay it was possible to compare the effects of various treatments on FA and bacterial cells. The latter are sterilized by shaking with such agents as chloroform, toluene, alcohol, and formalin. Of these only formalin inactivates FA. The bacteria are sterilized by heating at 56 C for 30 minutes. Temperatures of 70 C are necessary for detectable effects on FA. It is rapidly inactivated only when 100 C is ap- proached. 19521 GENETIC EXCHAh-GE IN SALMOh-ELLA 683 FA is quantitatively precipitated from broth by one to two volumes of cold alcohol or half saturated ammonium sulfate. A heavy floe appears in both cases which, for the most part, remains water insoluble; FL4, however, redisperses. Xone of several enzymes tested affected FA. They were added directly to the wtive filtrates and incubated for two hours. The tests included pancreatin (100 m&ml), trypsin (100 pg/ml), Taka-diastase (100 ma/ml), ribonuclease (10 pg/ml), and desoxyribonuclease (20 pg/ml). The failure of desosyribonuclease to inactivate FA was of particular interest. Enzymat)ic activity was verified by test'ing samples of the reaction mixture for reduction of t)he viscosit,y of thymus nucleic acid (kindly supplied by Dr. R. D. Hotchkiss). Similar cont,rols were not done for the other enzymes. Evocation oj F&4. The propert,ies of phages latent in Salmon,ella have been summarized by Burnet and McKie (1929) and Boyd (1951). Lysogenic phages, i.e., those obtained from lysogenic bacteria, poorly lyse sensitive cultures and readily provoke secondary resistant lysogenic forms. Visible lysis of sensitive cells is observed only at low mult,iplicitics of infection. With higher multiplicities there is little evidence of lysis. When phage is added to brot'h cult'ures, the tube does not clear, and the bacteria grow at a reduced but, significant rate. I'LT-22 is typical of these phages. To determine mhet,her PLT-22 was unique in it,s FA evoking act,irity, a variet,y of treatments was applied to resting and growing cells of TAT-2 strains. F=2 was not detected in the filt'rates of young cultures or after autolysis with benzene, extractJion of dried cells, treatment' with high concentrations of a,nti- biotirs (penicillin, bacitracin, and aureomycin), or complete phage lysis. Dilute antibiotirs, lithium chloride, and crystal violet yielded variable F/l. High act,ivity is most, readily detected in the filtrates of cultures treat)ed mit#h lysogenic phages. These results indicat#e tfhat FA is not released by mechanical, chemical, or biological disruption of cells. However, various deleterious agents elicit its appearance in a may that may parallel the action of latent phage. The most, effective concentrations of these reagent,s were those which only slightly in- hibited the cells. F-4 has also been detect'ed in aged cultures when autolysis sets in. This may be due to the action of mutant lysogenic phage. The production of FA in response to chemical st'imuli has not yet, been suffi- cient,ly controlled to give consistent' yields needed for experimental use. How- ever, when a filtrat,e containing little or no FA is prepared after treatment of LA-2 with such agents as cry&al violet or penicillin and reinoculated into broth with LA-2, FA is released in large amounts. This procedure has been carried through for five cycles. The apparent regeneration of FL4 was probably due to a lysogenic phage which had been released in t'he first treatment,. The lack of a reliable indicator for this phage has hindered t'he analysis of this reaction. However, it has been a useful tool for the evocat,ion of FA from a single st#rain without the introduction of extraneous bact,eria or viruses. Morphological and physical s&dies. FL4 has been qaantjitatively sedimented and recovered in t)he Spinco ult*racentrifuge at 100,000 G for 30 min. Partial sedimentjation occurred in the International centrifuge with mult)ispeed head at 20,000 G. In these preparat,ions FL4 is, therefore, of more than macromolecular 684 NORTOPi D. ZINDER AND JOSHUA LEDERBERG [VOL. 61 dimensions. Filtrat,ion through a series of gradocol membranes (obtained through the courtesy of Dr. S. E. Luria) was also used to estimate the particle size. Ten to twent,y per cent of FA were retained by a membrane of A.P.D. 420 rnp, sevent,y per cent at 230 and 170 rnp, and ninet,y-nine per cent at 120 mp. These results indicate a particle size slight,ly less than 0.1 p (Bawden, 1950). FA preparat,ions exhibit numbers of small, barely resolvable, granules under the phase contrast microscope. Electron micrographs show granules whose size is in rough agreement with the estimates of E'il from filtration experiments (figure 2). Some of the granules agglutinated with anti-0 serum. Visible floccules which can be removed by centrifugat'ion appeared in the react#ion tube. Holvever, the activity remained intact in the supernatant. Upon incubation wit,h anti- serum, some of t,he granules enlarge and by four hours have attained sizes of Figure 2. h pnrtinlly purified act,ive filtrate, 40,000 X. (Electron micrograph by Dr. I'd line&erg.) 5 to 8 p (see figure 11, Lederberg et al., 1951). These "large bodies" formed mixed floccules with added bacterial cells. Treatment of SaZ,rnoneZZa with FA-eliciting phage or penicillin results in the formation of chains after one and one-half hours of incubation, and by three hours only "snakes" with swollen bulbular central portions are present (Fleming rt al., 1950). Debris and small granules are also seen. FL4 has also been produced by t,his time. The supernatants of these cultures were difficult to sterilize by conventional means. Filtratjion t.hrough eight or fourteen pound test Mandler candles resulted in filtrat,es with a viable count of about, 100 per ml. Comparable filtrates of untreated cultures have regularly proven to be sterile. Sintered pyrex "UF" filters were found to be suitable for sterile filtration of active fil- t'rates. These observations are reminiscent of the L-forms of bacteria particularly as interpreted by Klieneberger-Nobel (1961), Dienes and Weinberger (1951), and 19521 GENETIC EXCHANGE IN SALMONELLA 685 Tulasne (195 1). There is, however, no evidence of a functional relationship bet,ween L-forms and transduction. We have not yet succeeded in obtaining L-colony growth from our cultures t,hat would permit more direct tests, nor have other workers made genet,ic analyses of L-t,ype growth to fortify specula- tions on their role in a life cycle. Sources and range of activity of FA. FA has been defined t,hus far as a specific product of strain LA-2 with the single capacity of transducing a particular mutant of LT-22. However, ot,her direct crosses involving LA-22 had given prototrophs. To determine if FL4 could be obtained from ot,her strains, a simplified test was applied, involving the selection of streptomycin resist'ant' prototrophs, "SRP" (Lederberg, 195la). SW-435 (LA-22 Sr) was grown in mixed culture with each of fifty different wild type (streptomycin sensitive prototrophs) S. typhimurium strains, and the mixture plat,ed on minimal agar containing 500 mg per L of strept'omycin. Twenty-eight of the crosses yielded evident recom- binants, showing that FA could probably be produced by many strains. FA has been isolated from each of twent,y-five tested strains of S. typhimurium when the proper stimulus was found. PLT-22 served for the many strains SUS- ceptible to it, which probably explains the success of the SRP crosses, while other lysogenic phages (from the Lilleengen series) stimulated other strains resistant t,o PLT-22. In general, inoculation of log cells of S. typhimwium and 10" to log particles of a lysogenic phage to which it is suscept,ible into 10 ml of fresh broth will yield FA aft#er four hours of incubation. Penicillin in low con- cent'rations (one to five units per ml) was successful for some culbures. A demonstration of recombination in Salmonella was initially sought' and found in terms of t,he recovery of prototrophs from mixed platings of auxotrophs. A more complete proof of typical sexuality would depend upon the occurrence of new combinations of `Lunselected markers" (Lederberg, 1947). SW-478 (LA-22 Gal - , Xyl- , Mt#l- , S*) was crossed with SW-414 (LA-2 Gal+, Xylf , R/ITl+, Ss) on EML agar containing one of the various sugars so t'hat one unselected fermentative character could be scored directly on the cross plate. Of some 20,000 prototrophs screened, none differed from SW-478 except in their nutrihion. In addition t,o mutational differences, L,4-22 and LA-2 differed intrinsically in abilit,y to utilize malat,e, alanine, or succinate as the sole carbon source required for growth. All of the prototrophs resembled LA-22. Wit,h a t#otal of eight un- selected markers there was no evidence of co-segregation. These experiments were repeated with active filtrates from LT-2 and gave the same result. Genetic t,ransfers for each of three markers (one nutrit,ional and t,wo fer- mentat,ive) were observed when experiments were set, up in such a way as to select for t,hem. SW-435 (Aux, Gal-, Xyl--, Sr) was plated with FL4 (from LT-2 Prot), Gal+, Xyl+, Sg) on minimal, EMB galactose and EMB xylose agar. Upon the EMB media there first appeared a thin film of growth (pink and henre nonfermenting) and t,hen small outcroppings or papillae which fermented the galactose or xylose. These papillae grow quit,e large (figures 3 aud 4) because of their utjilizatjion of the sugar when other nutrients are depleted. The sylose-negat,ive mutant gave some papillae due to spontaneous reversion 686 KORTOA' D. ZINDER AND JOSHUzk LEDERBERG [VOL. 64 but not enough to interfere with the scoring of the test. The galactose negative mutant is more stable and has only rarely reverted. The number of papillae on EMB or prototrophs on minimal agar (table 2) \vas approximately the sa,me so t,hat the efficiency of transduction for different selected charwters may be uni- form. However, the miselected markers remained unaltered; that, is, all prot,otroph selections were nonfermenters and t'he papillae selections acted only upon the XEDIUI FA Minimal 120 prototrophs 1 EMB galactose 111 papillae EMB xylose 138 papillae ~ Figures nre colonies or papillae per plate. 0 colonies 0 papillae; film of bacteria 15 papillae; film of bacteria - Figwes 3 and 4. SW-135 plated on EMB galnctose agnr with heat innct,ivnted (3) and active (4) FA. one sugar and were auxotrophic. All of the t,ransduced ~11s were still strept,o- mycin resistant. The foregoing experiment was repeated on double sugar agar. Individual papillae fermented either galactose or xylose and were all auxot,rophic. Because of a slight difference in texture it was possible to differentiate the two kinds of papillae directly on t,he indicator plate. Entire papillae were picked and trans- ferred t,o the alternative sugar and to minimal agar. Among t,he many tested, no mixed papillae were found. Any such could be detected by this rigid selec- tion. 19521 GENETIC EXCHANGE IN SALMONELLA 687 From these experiments, we conclude that an FA filtrate has many activities, producing many different transductions (but no more than one per cell) that result in singly transduced clones. We have observed no linked segregations such as had been found in E. coli recombination. The singular activity of FA might still be reconcilable with a gametic interpretation if the failure to show linkages were due to structural differences in the chromosomes of the parents. Alternatively, FA might have been considered in terms of a nonspecific mutagen with independent action on different factors. Further experiments have disqualified both of these views beyond reasonable doubt. LT-7 served as an efficient, donor and recept,or of FA and was chosen for the study of the intrastrain transfers and to test these considerations (see table 1 for its markers). To be certain of the source of the FA employed, it was prepared (as described previously) without external bacterial or viral influences. FA was prepared from SW-184 (prolineless), SW-188 (methionineless), and SW-191 (leucineless). Each preparation was assayed for transduction from auxotrophy t,o TABLE 3 The effect of PA from LT-7 and ifs derivatives u.pon LT-7 derivatives -. __- .-~ _I__ / ! SW-188 I CELLS/PA Ix-7 s-iv-184 ) /------ i SW-191 FOILED FA -- --__, -- -~ SW-184 203 247 j 2t?* i 253 31* SW-188 1 62 76 0 0 SW-191 I 198 210 / 236 I 6S ; E3+ 10* LA-22 (control) I 230 ( 242 202 I 275 ' 0 --- * Presumably spontaneous reverse mutations. Figures are transductions from auxotrophy to prototrophy per plate. prototrophy of each of the three LT-7 auxotrophs and LA-22 (control for the presence of any activity). The preparations had fairly uniform activity on LA-22. However, FA from each of the three LT-7 auxotrophs could transduce the other two but not its source culture (table 3). FA thus conforms to the genotype of the cells from which it comes. Several galactose-negative mutants were obtained in each of the three auxotrophs. None of several thousand transduced proto- trophs was galactose positive. FA, from SW-184 (prolineless), when plated with SW-188 (methionineless) on minimal agar supplemented with proline, resulted only in proline independent colonies (prototrophs). Comparable results have been obtained with each of the three auxotrophs in similar experiments. In the course of transduction, there was no linked segregation or association of these three nutritional markers with each other or with fermentative markers. Streptomycin resistance provided still another marker that remained unaltered in cells transduced for other characters. Several galactose-negative mutants were transducible to galactose-positive by FA from their parental wild type. FA from these mut'ants gave diverse results. The mutants were never transduced by their own FA, but they could be trans- 688 NORTON D. ZINDER AND JOSHUA LEDPRBERG [VOL. 64 duced by FA from some of the other mutants. These interactions provided a basis for grouping the mutants with respect to allelism or genie identity (table -4). All of the transductions discussed thus far have been in the direction of mutant to wild t,ype. It is difficult, as a rule, to screen for changes in the other direction owing to the lack of adequate selective methods. This can be done with streptomycin resistance since the wild type condition is sensitive (S") and the mustant is resistant (S*). Freshly harvested cells were exposed to FA from streptomycin resistant and sensitive "parents" and then plated upon EMB galactose. After two hours of incubation (to allow for phenomic lag, Davis 1950a) the plates were sprayed with a concent~rated solution of streptomycin (0.1 g per ml). Table 5 shows that the transduction did occur but only when a TABLE 4 The effect of FA from several galactose-negative mutants upon these same mutants CELLS/PA LT-7 I GAL-1 -_____ -__ __- Gal-l +* -1 DAL-2 GAL-3 I GAL4 f--- Gal-2 + / ;t 1 1 ; i Gal-3 + / i - j ; Gal-4 I + j +' j : j _ - ( - ~--- * Galactose positive papillae produced. t No more papillae than on control. TABLE 5 Comparison of the e#ect of FA from streptomycin resistant and sensitive cells on sensitive cells CELLSIFA j m-7 (Gal +, Se) I SW-514 (Gal f, ST) j SW-191 (Gal -, S") ) SW-515 (Gal -, S') LT-7 / (Gal+, S*) SW-191 I (Gal-, Sa) I ! 203 Gal+ j 0 174 Gal+ j 1 228 Gal- 0 158 Gal- I I Figures are the number of streptomycin-resistant colonies per plate. streptomycin resistant mutant was the source of the FA employed. No associated changes were found. The stability of the transduced cells was verified by tests of many daughter colonies by replica plating (Lederberg and Lederberg, 1952) to normal and streptomycin containing media. It is now evident that the particular FA for which an assay has been defined is just one of several coexisting functions of a given filtrate. We are entitled to refer to FA for any of the genetic factors so far studied, and the range of action of a given filtrate can be designated in the same way as the genotype of the culture from which it is obt'ained: e.g., Prot, Gal+, Xyl+, Sr for SW-514 (figure 5), as well as for the FA derived from it. Unless otherwise qualified, however, FA will continue to refer to the transduction assayed on LA-22. .Idsorption of FA. The first, step in transduction must be the adsorption of FA 19521 GENETIC EXCHANGE IN SALMONELLA 689 on compet,ent cells. LA-22 was harvested from nutrient agar plates. Aliquots were suspended in one ml of an active filtrate for various intervals. The cells were sedimented and plated on minimal agar to determine the number of ex- changes. After a heat shock at 56 C bo destroy any unsedimented cells, the supernatants were assayed with LA-22 for unadsorbed FA. Moderate amounts of FA were completely adsorbed within the time necessary for centrifugation (15 minutes) and were recovered quantitatively in the precipitated cells. All tested smooth strains of S. typhimurium adsorbed FA. Cells of the donor strain adsorbed as efficiently as the others, consistently wit,h the success of intrastrain transfers. Disinfection by boiling or ultraviolet irradiation (to leave an extremely small viable fraction) did not affect adsorption. Rough cultures, selected by aging in broth (Page et al., 1951) did not adsorb. These results indicated that the site of adsorption is heat stable, is not affected by the death of the cell, and may hc related to t,he somatic antigen. fA ew-514, PRO,, Q*L+, XYL+, 5') OR SW-351 IAUX. QAL;XYL-. 5') PROT *UY AUY IUX QAL- QALt S&L- QAL- XIL- *IL- XYL+ XYL- _' S' S' S' Figzcre 5. Multiple potentialities of an active filtrate With the amounts previously used, FA sssays were directly proportional to FA concentration. Cells of L-4-22 were harvested from nutrient agar. Aliquots cont'aining 1O'O cells were sedimentfed in each of ten centrifuge tubes and the supernates discarded. Multiple aliquots of FA (one to ten ml) were added and 15 minutes at 37 C allowed for adsorption. Supernates and cells were collected and assayed on EML galactose. Xo concordant changes (i.e., galactose positive) were observed among the prototrophs. Figure 6 shows that a maximum number of transductions occurred with about eight ml of FA. The saturated sediments adsorb no more FA from larger aliquots. Except for a small systematic loss, probably mechanical, all unit,s of FA are accounted for either in the supernatant or the sediment. The interference in adsorption implied by saturation was demonstrated more explicitly in a blocking experiment. SW-188 (methionineless, M-) was exposed to an excess of FA from M- cells for fifteen minut,es. FA from LT-7 (&I+) was then added to the sediment,ed cells for an additional fifteen minutes before the cells were again scdiment,ed. The M+ FA was not bound, nor was the SW-188 transduced. This verifies the blocking concept and indicates t'hat, the adsorption is irreversible after the fifteen minut#es allowed for sa,turation. 690 SORTON D. ZINDER .4ND JOSHUA LEDERBERG [VOL. 64 Since adsorption of FA is so rapid it appears safe to assume that the large proportion of the individual bacteria are capable of adsorbing it. We can make an approximate minimum estimate of the number of adsorbable particles per ml of this filtrate by dividing the total number of bacteria by the number of ml required to saturate them (one particle per bacterium); lOlo/ or 1.3 X log. A maximum number of particles per ml is set by the fact that the active filtrate showed no turbidity as might be expected with more t.han 10" particles per ml. I 2 3 4 5 6 7 8 9 IO FA, ML LEGEND O--O SEDIMENT X-X SUPERNATANT .-' TOTAL RECOVERY - INPUT Figure 6. Adsorption of FA. 10'0 cells of LA-22 were exposed to FA (LT-2) for 15 to 30 minutes. Supernatants and sediments were collected after centrifugation and assayed, respectively, for residual FA and for transductions already initiated. So many unverified postulates are required that a detailed discussion of possible models for the kinetics of adsorption would be unprofitable here. It may be pointed out, however, that the low or zero frequency of double exchanges does not imply that one species of FA particle excludes another. If most of the bacteria are competent to be transduced, the frequency of a particular transduc- tion will be the probability that any of the particles adsorbed will have a par- ticular effect. Double transductions will occur in t,he same ratio to single ex- changes as the absolut'e frequency of the lat,ter, and this is too low (ca 10-5) for double exchanges to be detected in our experiments. However, if transduc- tion is limit'ed to a small proportion of competent cells, dual transductions would 19521 GENETIC EXCHASGE IN SALMONELLA 691 not have independent probabilities, and further assumptions such as mutual exclusion would be required to account for the low frequency of observed dual events. The following picture appears to be most consistent with the observations to date. An active filtrat,e contains a population of numerous species of granules, each corresponding to a genetic effect although some may be intrinsically inert. Each bacterium may absorb a limited number of particles, in the possible range from one to perhaps one hundred. Each adsorbed particle has a fixed, inde- pendent probability of exerting its particular transductive effect. The low fre- quency of single, and particularly of double transductions, is limited by t,he total number of particles that may be adsorbed as well, perhaps, as by the low probability that an adsorbed particle will complete its effect. Serial transduction. Dual transduction has never been observed in a single experiment. That this is due to the considerations described previously rather than some intrinsic limitation is shown by serial transfers. Once a cell has been transduced it can be grown out, reexposed to FA, and selected for other changes. SW-351 (Aux, Gal-, Xyl-) has been serially transduced from auxotrophy to prototrophy, from galactose negative to positive, and from xylose negat(ive to positive. The order in which these transfers were accomplished made no differ- ence. There was no loss of efficiency with the iterated transduct,ions as compared to t.he single transduction of SW-351 for any of the characters. SpeciJicity of adsorption of FA. The adsorption experiments had indicated a correlat,ion of adsorptive ability and immunological specificity. Preliminary experiments with some dozen Salmonella serotypes confirmed and narrowed this correlation to the presence of somatic antigen XII. Broth cultures of the serotypes to be tested were sedimented and one ml of FA was added. Adsorption proceeded for fifteen minutes, and then the reaction t,ubes were heat shocked at 56 C for one hour to sterilize the cells. Preliminary experiments with known adsorbing cells had shown that FA once adsorbed was not eluted by this procedure. The mixtures were assayed on LA-22 for free FA. Some fifty different serotypes have been tested in this manner. Although some types with XII are inert, none of the types without XII adsorbed. This correlation is maintained with the "Salmonella coli" types. The XII carrying strains that adsorbed were: S. paratyphi B, S. typhi-murium (25 strains), S. Stanley, S. heidelberg, S. Chester, S. san-diego, S. abortus-ovis, S. typhi W, S. typhi V, S. enteritidis, S. moscow, S. blegdam, S. eastbourne, S. sendai, S. abony, E. coli 3, E. coli 4, S. kaapstad, S. salinatis, S. pullorum, and S. gallinarum. The following XII types did not adsorb: S. paratyphi A and S. abortus-bovis, presumably owing to the absence of the XII2 component. The nonadsorbing, non-XII types tested were: S. typhi-murium (rough variant), S. cholerae-suis, S. newport, S. london, S. senftenberg, S. aberdeen, S. poona, S. Worthington, S. hvittingfoss, S. kentucky, S. Wichita, S. urbana, S. habana, S. altendorf, S. vejle, S. montevideo, E. coli 1, E. coli 2, E. coli 5, E. coli K-12, S. bonariensis, S. jlorida, and S. madelia. Inter-type transductions. It is not known whether t,he adsorption of FA is sufficient to indicate susceptibility to genehic transfer, hut. preliminary data 692 NORTON D. ZINDER 4ND JOSHUA LEDERBERG (VOL. 64 identify a possible receptor group, among which inter-type transductions may be possible. S. typhi and S. typhimurium differ in a number of cultural and serological characters. The latter ferments both arabinose and rhamnose while the former does not ferment and is inhibitred by either of these sugars. S. typhi Watson V was exposed t,o FA from S. typhimurium and inoculated into Durham fermenta- tion tubes containing one per cent of either sugar in nutrient broth. After 24 hours a more luxuriant growth appeared in the FA treated cultures, and acid was produced by 48 hours. From these tubes cultures were isolated that differ from S. typhi only in their ability to ferment these sugars. The control cultures, without FJ, show little evidence of growth and no evidence of fermentation. Although S. typhimurium produces gas from rhamnose and arabinose, these new forms remain typically anaerogenic. The experiment has also been conducted on agar. Treated cells were plated on EMB arabinose and EMU rhamnose. S. typhi occasionally mutates to a noninhibited form (Kristensen, 1948) which was represented by white papillae which were observed on both the experimental and control plates. However, the purple (fermenting) papillae were observed only on Zhe experimental plates. Culturally they resembled the fermenting strains isolated after transduction in broth. These results have been repeated with two other strains of S. kyphi. Usin g a streptomycin resistant mutant of S. typhimurium as the source of FA, it has been possible to transfer t.his character to S. typhi. Attempts to produce aerogenic fermentation of glucose by S. typhi by treatment with F-4 have all met with failure, possibly owing to insufficiently selective conditions to detect cells transduced for this character. S. typhi is antigenically characterized IX, XII: d, - (monophasic) while S. typhimurium is I, IV, V, XII: i - 1, 2, 3. S. typhi was exposed to F.4 from S. typhimurium, and transduction of the flagellar antigen was selected for. A tube based upon the mycological growth tube (Ryan et al., 1943) was half filled with soft agar containing diluted anti-d serum (l/ZOO of serum t,itrating to l/5,000). The cells were heavily inoculated at one side of the tube and watched for migration. In one experiment, two out of four experimental tubes showed migration while the three control tubes showed complete fixation of the inoculum. There was a sharp delineation between the migrating cells and t'he fixed inoculum. The former were fished from the uninoculated end of the tube and tested cul- turally and serologically. Both of the isolat,es culturally resembled S. typhi. One of them reacted with anti-i serum while t,he other did not react with either S. typhi or S. typhimurium flagellar antiserum and was diagnosed as a j phase (Kauffmann, 1936). The analysis of these two st,rains was confirmed by Dr. P. R. Edwards. Transduction of the i antigen was obtained from twelve of thirty-one tested inocula of lo* FA saturated S. typhi cells. "j" phases have appeared occasionally in both experimental and control tubes. No i phases were detected in 50 control tests withouta FA. The complete antigenic analysis of the "hybrids" is IX, XII: i, -. Unlike S. typhimurium, from which the i flagellar phase was derived, phasic variation has not been found in these `Lhybrids". Experiments are now in progress seeking transduction of other flagellar and somatic antigens. 19521 GEiNETIC EXCHANGE IN SALMONELLA 693 The transduced cell. Prototrophs produced by transduction [FA (LA-2) on LA-221 have been tested for their stability both in vegetative reproduction and further transduction. After isolation from the experimental plate they were purified by streaking. Five single colonies were grown in complete broth and plated. Two hundred colonies from each were picked and retested on minimal agar: all were prototrophic. The transduced culture was reexposed to FA, and another change was selected (galactose negative to positive). Of some 1,500 colonies tested by replica plating, all retained the inibial transduction to proto- trophy. The transduced culture does not release FA during its growth nor is FA obt,ainable from it by any other means than those employed for the parent culture. Some difficulty has been encountered in this respect with the products of intrastrain transduction. They were all resistant carriers of the phage as- sociat'ed with active filtrates and some new phage was needed to evoke FA from them. Phage resistance also reduces the efficiency of iterated transduction, presumably because of impaired adsorption of FA. Spontaneous reverse-mutations regain the ability to transduce their mutant parents as do transduced reversions. That is, when a cell goes from A- to A+ by either means, it can again produce A+ agent. Mut,ation in free FA has not yet been studied. The relationship between bacteriophage and FA. Several recent convergent lines of evidence point to the identity of FA particles and bacteriophage. FA and phage have a common filtration end point; ninety-nine per cent of both are retained by a membrane of A.P.D. 120 rnp. They have a common specificity of adsorption on Salmonella serotypes, correlated with somatic antigen XII. In adsorption on S. typhimukm both reach saturation at the same point, and the phage to FA ratio remains constant. During the course of purification, FA and phage remain together. In short term experiments, FA and phage are released simultaneously from phage infected bacteria. Electron micrographs show a morphological similarity of particles of proper size. That the phage particle can be only a passive carrier of the transductive genetic material is shown by the following experiments. From single phage particles grown on bacterial cells there are obtained high titered phage and a population of FA encompassing the entire genotype of the parental cells but capable of only one transduction per bacterial cell. Single phage particles, from this filtrate, can be grown on bacterial cells from the same original parent but of different genotype. The FA produced is comparable to the genotype of the secondary donor. In the section on the evocation of FA, mention was made of the apparent regeneration of FA by transfer. This was explained as being due to the associa- tion of FA with phage which served to continuously stimulate its production. To test this, A-, Bf, C+ cells were treated with penicillin. The filtrate was transferred with the same cells to yield FA (A-, B+, C+) and a phage which could be assayed on these same cells. When added to A+, B-, C+ cells (from the same original parent,), the FA obt.ained was A+, B - , C+ . All of the Bf agent was adsorbed and lost, and agents paralleling the genotype of the B - 694 NORTON D. ZINDER AND JOSHUA LEDERBERG [VOL. 64 cells obtained. FA had not propagated as such but rather was associated with the necessary stimulus for further production, the phage. DISCUSSION Genetic exchange in S. typhimurium is mediated by a bacterial product which we have called FA (filtrable agent). An individual active fihrate can transfer (transduce) many hereditary traits from one strain to another. Although the total activity of this filtrate encompasses the genotype of its parental culture, each transduction transmim only a single trait per bacterium. This contrasts with genetic exchange in E. coli, strain K-12, where there is unrestrict,ed re- combination of the several markers that differentiate two parental lines. FA may be considered as genetic material which enters the fixed heredity of the transduced cell. We may ask whether this transfer is a simple super-addition or a substitutive exchange and replacement of the resident genetic factors. If streptomycin resistance is a recessive mutation, as inferred from studies of heterozygous diploids in E. coli (Lederberg, 1951b), the transduction of re- sistance disqualifies the simple addition mechanism. Two aspects of FA must be carefully distinguished: the biological nature of the particles themselves and their genetic function. There is good reason to identify the particle with bacteriophage. Nevertheless, the phage particle would function as a passive ca,rrier of the genetic material transduced from one bac- terium to another. This material corresponds only to a fragment of the bacterial genotype. For example, when F-4 from a marked prototroph is plated with an auxotroph on minimal agar, the genotype of the presumed "donor nucleus" is not observed among the transduced prototrophs. The hypothesis of FL4 as a genetic complex rather than a unit might be maintained if the singular effects produced depended on a small chance of release of a,ny particular activity from a complex particle or on some localized nonheritable happenstance in the cell that ordinarily left only one function sensitive to t'ransduction. Still the originally singly transduced cell develops as an isolated clone. Since the clone is composed of some 10' bacteria, one might expect that a complex residuum of an FA particle, if viable, would transduce some one of the daughter cells for another character during the growth of t.he clone. However, each Fll particle produces only a single transduced clone. This speaks for the simplicity of its constitution as well as of its genetic, effect. When LA-22 is transduced from auxotrophy (phenylalanineless and tyrosine- less; tryptophanless) to prototrophy, we have an apparent dual change. If this mutant is plated on minima,1 agar supplemented with phenylalanine and tyrosine, it occasionally reverts to the first step auxotrophic condition. However, when LA-22 is transduced on this medium, no more first step auxotrophs are found than can be explained by spontaneous reversion. The majority of the selected colonies are prototrophs. We have not been able to affect more than one trait in any other inter- or intrastrain transductions. It seems likely that the nutrition of LA-22 was determined by two successive mutations at the same genetic site. Davis' (1951) scheme for aromatic biosynthesis corroborates this notion. .41- 19521 GENETIC EXCHANGE IS SALMONELLA 695 though the mutant LA-22 can revert spontaneously to an intermediate allele, transduction brings about a substitution of the wild type gene for full synthesis. The most plausible hypothesis for the FA granules is that they are a hetero- geneous population of species each with its own competence-in other words, each carries a "single gene" or small chromosome fragment. Regardless of the nature of the FA particles, some mechanism must be postu- lated for the introduction of the transduced genetic material to the fixed heredity of the recipient cell. Muller's (1947) analysis of type transformation in the pneumococcus is apropos here: ". . . there were, in effect, still viable bacterial chromosomes, or parts of chromosomes, floating free in the medium used. These might, in my opinion, have penetrated the capsuleless bacteria and in part taken root there, perhaps, after having undergone a kind of crossing-over with the chromosomes of the host." In a preliminary report on the Salmonella recombination system (Lederberg et al., 1951) it was suggested that FA might be related to bacterial L-forms (Klieneberger-Nobel, 1951). The occurrence of swollen "snakes", filtrable gran- ules, and large bodies in response to certain agents is characteristic both of FA and L-forms. Except for the suggestion of viable filter passing granules we have not repeated the reported cycles. The visible agglutinable granules and the antiserum-induced swollen form are not necessary for FA activity. However, this failure to fit all of the elements to a simple scheme may be due to a system more complex than we are now aware. The bacteriological literature has numerous reports of results which might be interpreted as transduction (see reviews by Luria, 1947, and Lederberg, 1948). These experiments have been criticized or neglected because of difficulties in their reproduction and quantitization but might now be reinvestigated in light of the findings presented. A citation of Borne of the more pertinent ones should suffice at this t'ime. Wolhnan and Wollman (1925) reported the acquisition of Salmonella immunological specificity by E'. coli via filter passing material. Similar material (which can be obtained by phage lysis) has been implicated in the change of penicillin resistant staphylococci and sbreptococci to relative penicillin sensitivity (Voureka, 1948; George and Pandalai, 1949). Shigella paradysenteriae (Weil and Binder, 1947) acquired new immunological specificity when treated with extracts of heterologous types. Boivin (1947) reported a similar change in E'. co&. Unfortunately his strains have been lost and con- firmation is impossible. Bruner and Edwards (1948) in a report of variation of somatic antigens of Salmonella grown in the presence of specific serum commented on the possibility that ba&erial products dissolved in the serum were responsible for the changes. These syst,ems, provocative as they are, are insufficiently documented for detailed comparison with Salmonella transduction. The transformations in the pneumococcus (Avery et al., 1944; McCarty, 1946) and Hemophilus inJEuenzae (Alexander and Leidy, 1951) have been studied more completely. The genetic "transformation" of the capsular character of t,he pneumococcus depends on a specific bacterial product (pneumococcus transforming principle, 696 NORTON D. ZINDER AND JOSHUA LEDERBERG [VOL. 61 PTP). Originally interpreted as a directed mutation it, is now regarded as a variety of genetic exchange (Ephrussi-Taylor, 1950). Thus far transformations have been achieved for the full capsular character (Griffith, 1928), a series of intermediate capsular characters (Ephrussi-Taylor, 1951), M protein character (Austrian and MacLeod, 1949), and penicillin resistance (Hotchkiss, 1951). As in SaZmoneZZa each character is transformed independently. However, there are several differences between the t,wo systems. FA must be evoked while the PTP is extract.ible from healthy cells. The resistance of FA to various chemi- cal treatments has given only negative evidence of its chemical nature. The role of desoxyribonucleic acid in the PTP was verified by its inactivation by des- oxyribonucleic acidase. Retention of activity by gradocol membranes has given comparable estimates for the size (about 0.1 FL) of the FA particles affect ing two different characters. On the other hand, while the particle size of the PTP has been variously estimated from an average centrifugal mass of 500,000 (Avery et aE., 1944) to an ionizing irradiation sensitive volume equivalent to a molecular weight( of 18,000,OOO with high asymmetry (Fluke et a.!., 1951), it is considerably smaller than the FA particle. Pneumococci must be sensitized by a complex serum system for adsorption of PTP. The low but poorly determined frequency of transformations has been thought to be due to the low competence of the bacteria. In the absence of adsorption experiments a system similar to Salmonella has not been ruled out. Important informat,ion is still lacking in both systems and time may resolve these apparent differences. The relationship of transduction in Salmonella to sexual recombination in E. coli is obscure. Transduction has not been found in crossable E. coli nor sexual recombination in Salmonella. These genera are extremely closely related t.axonomically but seem to have entirely different modes of genetic exchange. Sexual recombination was first demonstrated in E'. coli, strain K-12. With the development of an efficient screening procedure, two to three per cent of E. coli isolates were proved to cross with strain K-12 (Lederberg, 195la). The agent, of recombination in E. coli is almost certainly the bacterial cell. The cells ap- parently mat'e, forming zygotes from which parental and recombinant cells may emerge following meiosis, in which linkage is a prominent feature (Lederberg, 1947). The combination of genomes within a single cell has been confirmed by the exceptional occurrence of nondisjunctions which continue to segregate both haploid and diploid complements (Zelle and Lederberg, 1951). Alt,hough lyso- genicity plays a critical role in t,ransduction in Salmonella, all combinations of lysogenic and nonlysogenic cultures of E. coli cross with equal facility (Lederberg, E. M., 1951). Owing to the lack of recombination of unselected markers, transduction is a less useful tool than sexual recombination for certain types of genetic analysis. However, as FA may correspond to extracellular genet,ic material, such problems as gene reproduction, metabolism, and mutation may be more accessible to attack. Sexual systems usually provide for the reassort,ment of genetic material and given an important source of variation for the operation of natural selection in organic evolution. Both sexual recombination and t,ransduction, because of 19521 GENETIC EXCHANGE IN SALMONELLA 697 their low frequency, allow only limited gene interchange in bacteria. Trans- ductive exchange is limited both in frequency and extent. It is too early to assess the role t.hat transduction may have played in the development of the immunologically complex Xalmonellu species. White (1926) speculated that the many serotypes evolved by loss variation from a single strain possessing all of the many possible antigens. Bruner and Edwards (1948) obtained specific examples of loss variation with contemporary species. Trans- duction provides a mechanism for transfer of some of the variation developed spontaneously and independently between the "descending" lines. The genus Salmonella includes a group of serotypes which share a receptor for S. typhi- murium FA. Other receptor groups have yet to be sought. Within such groups it should be possible to evolve in the laboratory other new serotypes comparable to the antigenie hybrid of S. typhi and S. typhimurium. Several different bacterial genera have been intensively studied with regard to modes of genetic exchange. Each of the several known systems differs in details that enlarge our notions of bacterial reproduction and heredity. ACKNOWLEDGMENTS The authors are indebted to a number of workers cited in the text for pro- viding cultures and other materials. They are expecially obligated to Dr. P. R. Edwards, Public Health Service Communicable Diseases Center, Chamblee, Georgia, for patiently providing innumerable cultures, sera, antigenic diagnoses, and counsel. SUMMARY When Salmonella typhiwlurium is grown in the presence of a variety of mildly deleterious agents, especially weakly lytic phages, it produces a filtrable agent (FA) capable of transferring hereditary traits from one strain to another. Individual filtrates may transduce many different traits, but no more than one in a single bacterium. The activities of a filtrate parallel the characteristics of the donor cells. Nutritional, fermentative, drug resistance, and antigenic characters have been transduced. The new characters are stable after many generations of subcultures. FA is resistant to such bacterial disinfectants as chloroform, toluene, and alcohol and to such enzymes as pancreatin, t,rypsin, ribonuclease, and des- oxyribonuclease. The size of the FA particle, as determined by filtration through gradocol membranes, is about 0.1 micron. Adsorption of FA is rapid and, among various serotypes tested, is correlated with the presence of somatic antigen XII. The maximum frequency of transduction for any one character has been 2 X 10-O, a limit set by saturation during adsorption. Some inter-type transfers have been observed. For example, the i flagellar antigen from Salmonella typhi- murium has been transduced to S. typhi to give a new serotype: IX, XII; i, -. Genetic transduction in Salmonella is compared and contrasted with `%ype transformation" in Hemophilus and the pneumococcus and with sexual re- combination in Escherichia coli. 698 NORTON D. ZINDER AND JOSHUA LEDERBERG [VOL. 64 REFERENCES ALEXANDER, HATTIE E., AND LEIDY, GRACE 1951 Determination of inherited traits of H. injluenzae by desoxyribonucleic acid fractions isolated from type-specific cells. J. Exptl. Med., 93,345-359. AUSTRIAN, R., AND MACLEOD, C. M. 1949 Acquisition of M protein through transforma- tion reactions. J. Exptl. Med., 89,451-460. AVERY, 0. T., MhcLnon, c. M., hm MCCARTY, M. 1944 Studies on the chemical nature of the substance inducing transformation of pneumococcal types. J. Exptl. Med., 79, 137-158. BACON, G. A., BURROWS, W. W., AND Yarns, Maaahanr 1951 The effects of biochemical mutation on the virulence of Bacterium typhosum: the loss of virulence of certain mutants. Brit. J. Exptl. Path., 32.85-96. BAWDEN, F. C. 1950 Plant viruses and virus diseases. Chronica Botanica Co., Waltham, Mass. BOIVIN, A. 1947 Directed mutation in colon bacilli, by an inducing principle of des- oxyribonucleic nature: its meaning for the general biochemistry of heredity. Cold Spring Harbor Symposia Quant. Biol., 12.7-17. BOYD, J. S. K. 1950 The symbiotic bacteriophages of Salmonella &phi-murium. J. Path. Bact., 62,501-517. BOYD, J. S. K. 1951 Observations on the relationship of symbiotic and lytic bacterio- phage. J. Path. Bact., 63.445457. BRUNER, D. W., AND EDWARDS, P. R. 1948 Changes induced in the 0 antigens of Sal- monella. J. Bact., 66,449. BURNET, F., AND MCKIE, MARGOT 1929 Observations on a permanently lysogenic stra.in of B. enteritidis Gaertner. Australian J. Exptl. Biol. Med. Sci., 6, 276-284. DAVIS, B. D. 1950a Studies on nutritionally deficient bacterial mutants isolated by means of penicillin. Experientia, 6.41~50. DAVIS, B. D. 195Ob Nonfiltrability of the agents of recombination in Escherichia coli. J. Bact., 60, 507-508. DAVIS, B. D. 1951 Aromatic biosynthesis. III. Role of p-aminobenzoic acid in the formation of vitamin Bit. J. Bact., 62, 221-230. DIENES, L., AND WEINBERQER, H. J. 1951 The L forms of bacteria. Bact. Revs., 16, 245288. EPRRUSSI-TAYLOR, HARRIETT 1950 Heredity in pneumococci. Endeavor, 9, 34-40. EPHRUSSI-TAYLOR, HARRIETT 1951 Genetic aspects of transformations of pneumococci. Cold Spring Harbor Symposia Quant. Biol., 16, 445-456. FLEMING, A., VOUREHA, AYALIA, KRAMER, I. R. H., AND HUGHES, W. H. 1950 The morphology and motility of Proteus vulgaris and other organisms cultured in the presence of penicillin. J. Gen. Microbial., 4, 257-269. FLUKE, D. F., DREW, R. M., AND POLLARD, C. 1951 The effect of ionizing radiations on the transforming factor of pneumococci. Science, 114,480. GEORQE, M., AND PANDALAI, K. M. 1949 Sensitization of penicillin resistant pathogens. Lancet, 266, 955957. GRIFFITH, F. 1928 The significance of pneumococcal types. J. Hyg., 27, 113-159. HOT~HKISS, R. D. 1951 Transfer of penicillin resistance in pneumococci by the desoxy- ribonuoleate derived from resistant cultures. Cold Spring Harbor Symposia Quant. Biol., 16, 457-462. KATJFFMANN, F. 1936 Ueber die diphasische Natur der Typhusbacillen. Z. Hyg. In- fectionskrankh., 119, 104-118. KLIENEBERGER-NOBEL, EMMA 1951 Filterable forms of bacteria. Bact. Revs., 16, 77- 103. KRISTENSEN, M. 1948 Mutative bacterial fermentation. Acta Path. Microbial. Stand., 26, 244-248. LEDERBERG, ESTHER M. 1951 Lysogenicity in E. coli K-12. Genetics, 36, 560. 1952] GENETIC EXCHANGE IN s~ONELU 699 LEDEBBERG, J. 1947 Gene recombination and linked segregations in Escherichia coli. Genetics, 32, 595-525. LEDERBERQ, J. 1948 Problems in microbial genetics. Heredity, 2, 145-198. LEDERBERG, J. 1950 Isolation and characterization of biochemical mutants of bacteria. Methods in Medical Research, 3,522. LEDERBERG, J. 1951a Prevalence of Escherichia coli strains exhibiting genetic recombina- tion. Science, 14, 68-69. LEDERBERG, J. 1951b Streptomycin resistance: a genetically recessive mutation. J. Bact., 61, 549-550. LEDEEBERG, J., AND LEDIRBERG, ESTHER M. 1952 Replica plating and indirect selection of bacterial mutants. J. Bact., 63, 399-406. LEDERBERG, J., AND ZINDER, N. 1948 Concentration of biochemical mutants of bacteria with penicillin. J. Am. Chem. Sot., 70, 4267. LEDERBERG, J., LEDERRERG, ESTHER M., ZINDER, N. D., AND LIVELY, ETHELYN R. 1951 Recombination analysis of bacterial heredity. Cold Spring Harbor Symposia Quant. Biol., 16, 413-443. LILLEENGEN, K. 1948 Typing Salmonella typhimurium by means of bacteriophage. Acta Path. Microbial. Stand., Suppl. 77. LURIA, S. E. 1947 Recent advance8 in bacterial genetics. Bact. Revs., 11, l-40. MCCARTY, M. 1946 Chemical nature and biological specificity of the substance inducing transformations of pneumococcal types. Bact. Revs., 10, 63-71. MULLER, H. J. 1947 The gene. Proc. Roy. Sot. London, B, 134, l-37. PAGE, L. A., GOODLOW, R. J., AND BFUVN, W. 1951 The effects of threonine on popula- tion change8 and virulence of Salmonella typhimurium. J. Bact., 62, 639647. PL~UGH, H. H., MILLER, HELEN Y., AND BERRY, MARION E. 1951 Alternative amino acid requirements in auxotrophic mutants of Salmonella typhimurium. Proc. Natl. Acad. Sci., U. S., 37, 640-644. RYAN, F., BEADLE, G., AND TATU~I, E. 1943 The tube method of measurement of growth rate of Neurospora. Am. J. Botany, 30,784-799. TATTJM, E. L., AND LEDERBERG, J. 1947 Gene recombination in the bacterium Escherichia coli. J. Bact., 63, 673-684. TULASNE, R. 1951 Lea formes L de8 bacthries. Revue Immunol., 16, 223-251. VOUREHA, AMALIA 1948 Sensitization of penicillin resistant bacteria. Lancet, 264, 62-65. WEIL, A. J., AND BINDER, M. 1947 Experimental type transformation of Shigella pata- dysenteriae (Flexner). Proc. Sot. Exptl. Biol., N. Y., 66, 349352. WHITE, P. B. 1926 Further studies of the Salmonelln group. Med. Res. Council (Britain), Spec. Rept. Ser., no. 103. WOLLMAN, E., AND WOLLMAN, E. 1925 Sur la transmission parahereditaire de caractcres chez les batteries. Compt. rend. sot. biol., Paris, 93, 16681569. ZELLE, M. R., AND LEDERBERO, J. 1951 Single cell isolations of diploid heterozygous Eschcrichia coli. J. Bact., 61, 351-355.