\ FURTHER DISCUSSIONS ON THE EFFECTS OF PHASE DIFFERENCE ON THE H- ANTIGEN TRANSDUCTION IN SALMONELLA DIPH&SIC STRAINS. Report by Tetsuo Iine (Jane 28, 1955) In the previous report, the effect of phase differences on the H-antigen transduction in Salmonella was tested. The data are summarized in Table 1 and Table 2. From these results, the hypothesis has been propoged on the mechanism aan variation; that is, each phase is controled by the different locus, phase 1 by H, and phase 2 by Hp, and Hy suppresses the action of His ani phase variation occurres by the mutation of H, to inactive allele h,. In other words, phase 1 has genotype Hho and phase 2 has genotype H,Ho. In the present paper, ( Tall 3 aed [nb 4) the data offered by Dr. Lederberg ,will be discussed, ,by comparing them with those of the previous report and with the results deduced from above hypothesis. Before beginning the discussion of the data, the expected results from the above hypothesis will be considered theoretically, which will be convenient to discuss the data quantitatively. In general, phdpe/1/pAL1/bAn/ tFApAAnE S/H /Pt/Po. ApA/ DRABB/?/PAA/HL/SE/Hb/ AA FABATA/ED/ENE/ALTAPE SEA phage can transduce Eycor hy framephace:l cell, and Hy or Ho from phase 2 cell in regard to the H-factors, so the frequencies of the phages which carry H,, Ho or ho in total H-carrying phages are as follows: FA(Hy )----- 1, (a; +d) = 1y Fa(H,) ---=- 1,d5 FA(ho)----- 19d, 4.) - | lhe Ay) 1, = Probability of H,-incorporation by phage in total H-incorporation.” ~~ v Ws 1» = Probability of H,-incorporation by phage in total H-incorporation, A 1j*1, =i] d, = Frequency of phase 1 cells in donor culture, d, Frequency of phase 2 cells in donor cultures djtd2 = 1 These phages are transduced into recipient cells and produce following four combinations: Byho, HH, BH, and Hyfio, and the frequency of each type is as follows, Bho wnre- 17047] €1) HB, ---- lyeado(rytrp) = 1y¢,8, (2) EH, ---- 1,%79 (3) Hy hi. ---- 1,¢54, (ry +rg) = 12¢p44 (4) ry = Frequency of phase 1 cells in recipient culture. Po = Frequency of phase 2 cells in recipient culture. c, = Efficiency of H,-incorporation by recipient cells. Cp = Efficiency of H,-incorporation by recipient cells. For the simplification, le is replaceé by t ( transductien coefficient of locus ), then, Bho o-——-— tyr) (1') * BE, --— tf, 2") * HLH, ——= $12 (3') mi, ——~ tod, (uty When antiserum for the H-antigens of the recipient cells are used as selective agents, type (3) and type (4) are selected away, so the ratio of phase 1 trans- duced type soc phase 2 transduced type 4s H: Hy & tyr, + tod, (5). Thus, when antiserums are used as selective agents, the frequency of the transduct tion of phase 1 increasé with the increase of the frequency of the phase 1 cells in recipient culture regardless the frequency in donor cultures, whenas the frequency of the transduction of phase 2 increases with the ingrease of the phase 2 cells in donor cultures regardless the frequency in recipient culture. The transduced types,when donor, recipient or both contain only one ofvabternative phases,are shown in Table 5. Now, we shall turn back to the experimental results. The data shown in table 3 coingide well with results deduced from the hypothesis. Only one contra- diction is the appearance of thec2 Ho-transduced types in phase 1 -x phase I combination, Ag indicated by formula (5) and Table 5, Ho-transduced type appears only when donor contains phase 2 cell, so the possible explanation of this discrepancy may be the contamination of phase 2 during the course of preparation of phase l-lysate by the phase variation (ho to Ho). The same consequences have been observed also in the phase 1 -x phase 2 and phase 2 -x phase 2 combinations in Table 1, though the direction of the variation is werent These few contami- nations are very liable to occur as the mutation rat? between each phase are very high and the preparation of the lysate requires the growth pt the bacterial cells until the numbers which allow the occurrence of the mutation H, 2 h,- The experiments shown in Table 2 and Table 4 were performed with same donor (TM2, 1:1,2) ani recipient (Sal. abony, b:enx) strain but with different lysates and cultures. The results of phase 1 -x ni phase coincide with each other and also with the theoretical expectation (Table 5). One phase 2 tyansduced type in the combination of phase 1 -x mixeé phase in Table 4 may be explained as the regsulté@ of the mtation from phase 1 to phase 2 during the preparation of lysate. In the results of phase 2 -x mixed phase, marked confliction is found out. According to the theoretical expectation, phase l1-transduced type and phase 2-transduced type must appear at the rate of t,r)/t>. The result indicated in Table 2 is explained as t rj, = to, and ry) = 0.46 as reported in the previous paper, so 0.46t1 = to. That is, the transduction efficiency of phase 2 is about half of phase 1 locus. ( t,/t2 can be calculated also from phase 2 -x phase 1 experiment in Table 4, where the ratio of phase 1 transduced type to phase 2 transduced type coincide with the ratio of t1 to to. So, t,/ty = 42/1, 0.25, = ty. Thus the efficiency of transduction of phase 2 locus is about one fourth of the phase 1. ) While Table 4 showes no phase 1 transduced type in the experiment phase 2 -x mixed phase , which is expressed by the following formula, t17r,] = 0 or +37) « to. as suggested by Dr. Lederberg, r= 0.5, So the remained possibility is t, « te This is completely reverse condition with previous pases in regard to the efficiency of transduction, and it is required to assume the great variability of t)/t,-ratio to explain these results without contradiction. Thus, it may be most important to test in what extent the t;/to-ratio is vaeiable by the experimental condition ( e.g. the concentration of donor or recipient culture, wor the difference of the strain ), in order to proceed the discussion about the mechanism of phase variation on the basis of the proposed hypothesss. fable 1. Transductions between single phase cultures of diphasic strains. Sal. avony (Fla, b:enx) -x Sal. heidelberg (Fla~, ri1,2). Phase Phase Antigen types of Fla*-transformed cells Ratio of of of Unlinked type Linked type Total linked donor recipient r: (2,2) (r):1,2 Total b:(1,2) (b):1,2 Total transduction 1 (b) 1 (r) 21 0 21 22 0 22 43 0.51 1 (b) 2 (1,2) 0 7 7 1 42 43-50 0.86 2 (enx) 1 (r) ‘ll 0 11 = 30 0 30 Al 0.73 2 (enx) 2 (1,2) 1 10 11 1 38 39 50 0.78 Total 33 17 50 5h 80 134 (184 0.73 X2 (ratio of linked transduction) = 13.69, 2s 3 P = 0.01 Table 2. Transduction of H, and H, from single phase culture to mixed phase culture in the diphasic strains ----(1), Transformed cells were selected by anti-i and -1,2 serum. Sal. typhimurium -x Sal. abony. tio, of the transformed cells Donor Recipient i:(enx) _(b):1,2 TM-2 phase 1 = SW-803 19 0 (4) (b: enx) T™-2 phase 2 " 4 14 (1,2) Table 3. Transduction of the H-antigen factors between single phase cultures of diphasic strain (Sal. abony, bremx -x Sal. typhimurium TM-2, i: 1,2). Transformed cells were selected by anti-i and -1,2 serun. Phase of Phase of No. of the transformed cells Donor Recipient bz (1,2) (i): enx 1 (b) 1 (4) 10 2 2 (enx) 1 (4) 4 2 11 1 (0) 2 (1,2) 0 0 2 (enx) 2 (1,2) 0 17 Table 4, Transductions of H, and H, from single phase culture to mixed phase culture in the diphasic strains ~---(2). Transformed cells were selected by anti-i and -1.2 serum. Sal. typhimurium -x Sal. abony. No. of the transformed cells Donor Recipient i: (enx) (b):1.2 (™-2 phase 1 SwW-803 33 1 (1) (b: enx) T-2 phase 2 " 0 . 12 (2,2) The results of H-transduction between diphasic strains, expected from the proposed hbpothesis when selected by the antiserum for the antigen of the Table 5. recipients. Phase of Phase of a r Ratio of H,- transduced type donor recipient 2 1 to Ho transduced type 1 2 1&2 1&2 3 0