VIROLOGY 60,603%605 (1972)

Short Communications

Location of the Prophage Conversion Gene of P22

Five genes are known to be active in prophage P22. Two, e2 (1) and mnt (2), are required for maintenance of lysogeny; sic A and sic B (8, 4) are involved in exclusion of superinfecting phages, and al (5) is respon- sible for expression of antigen 1 in Sal- monella typhimurium (P22). Gene al was mapped between the plaque morphology genes m3 and c2 (5) in the region where mnt was subsequently mapped (2).

As a basis for construction of models for control of the prophage active genes, we have mapped al against m3, m,nt, and 9, the tail gene.

Phage crosses were carried out as pre- viously described (6). Exponential cultures of S. typhimurium were infected with mix- tures of genotypically different P22. The multiplicity of infection was 10 for each parent. The infected cells were diluted and incubated at room temperature for 2 hr. Chloroform was added and the lysates were plated at 37” on indicator plates to score plaque morphology (1). On these plates P22 m3 mutants display a golden halo against the green agar; P22m+ does not form a halo. The phenotypes of these two phages are called M and &I+, respectively. These plates also permit discrimination between h21 and hf phages. P22hf produces a plaque with a broad green band; h21 makes a narrow yellow ring.

Centers of plaques of the desired pheno- types were picked to tryptone agar plates and incubated at 25” for about 2 days. The resulting colonies were tested for al antigen by slide or microtiter agglutination tests.

Immune serum was prepared in rabbits by injection of formalin-killed S. typhimurium (P22af). Because prophage P22 evokes no antigen response in Salmonella other than al, Dhe antiserum was made monovalent to

antigen al by repeated absorptions with nonlysogenic bacteria. For slide agglutina- tion bacterial colonies were suspended in a drop of M9 buffer (2) on a microscope slide and a drop of antiserum was added. Bacteria which harbored an a+ prophage agglutinated within 1 min. When microtiter agglutina- tion was used, colonies were picked into 0.15 ml MS-glucose medium (2) in a micro- titer well. After overnight incubation at 25”, the cultures were dilut.ed 2- and 4-fold in microtiter wells; antiserum was diluted >$c in M9 buffer and added to each well. The cultures were scored for agglutination after 8-12 hr at 25”. Known al and a+ lysogens were included on each microtiter plate to serve as controls. We did not observe phase variation in al expression (5). This may be because we did not go through a single- colony isolation; in picking the centers of plaques we picked many bacteria which subsequently grew into the colonies we tested for al antigen. Some colonies were tested by bot’h slide and microtiter agglutination. Re- su1t.s were t’he same with t,he two procedures.

The doubly mutant phage P22 mnt-tsl al was constructed by crossing P22 mnt-tsl with P22 al. The progeny from the cross were plated on tryptone agar at 37”. Under these conditions P22 mnt-tsl displays a “bulls-eye” plaque morphology (2). Such plaques were picked and scored for al by slide agglutination. A phage which was not agglutinated by al antiserum and displayed the plaque morphology of mnt-tsl was chosen as the desired recombinant. Mutation ts 9.1 is a conditional lethal allele of gene 9, the “tail” gene of P22. The order m3 rnd 9 has been established (2).

Because of its location between m3 and ~2, there are three possible sites for al in the cross P22 al mnt-tsl X P22 m3 ts9.1 (Fig. 1). Plaques that displayed the M

603

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604 SHORT COMMUNICATIONS

Predictions for m3-9+ reccwbinants:

MO&l1

+ al mnt + al will be predominantly mnt

m3 + + 9 CL' will be mni and mnt'

Model 2 + mnt al + al will be ING! and mat'

m3 + + 9 a+ will be predominantly m&t'

Model 3

+ mnt + al al will be mnf and mnt'

m3 + 9 + a+ (at least a double crossover) will be mnt and tit

FIG. 1. Possible locations for al in the cross P22 al mnt ts 1 X P22 m3 ts9.1. Predictions for segregation patterns of al and mnt are based on the respective models.

TABLE 1 RESULTS OF VEGETATIVE CROSSES TO LOCATE al Croes 1 P22 al mnt-tsl X P22 m3 ts9.1

m3-9+

a+ mnt-tsl a+ mntf al mnt-tsl al mnt+

No. of rccombinants

97

4 10 27 56

Cross 2 P22 al X P22 m3 ts9.1 h21 (Recombinants between m3 and 9+ were ex-

amined)

a+ h21 11 Frequency of 11/48 = 0.23 a+ h21 + al h21 48 a+ h21 re-

combinants a+ h+ 8 Frequency of 8/190 = 0.04

a+ h+ + al h+ 190 a+ h+ re- combinants

phenotype at 37” were selected as recom- binants between m3 and 9+. AI1 were tested for al antigen. Of 97 m3-9f recombinants, 14 were a+. Recombinants were assayed in two ways to determine whether they were Mnt or Mnt+. Colonies were picked onto indicator plates (I) at 39”; mnt-ts 1 lysogens turn green under these conditions; mnt+ re- main white (2). Colonies were also streaked on tryptone agar plates at 39” and over- laid with wild-type S. typhimurium. The

spontaneously liberated phage were then inspected and determined to be Mnt or Mnt+ on the basis of plaque morphology. The two methods for scoring Mnt produced identical results. Of the models in Fig. 1 only number three predicts that the ratio m3 mnt 9f/m3 mnt+ 9f will be equal in both al and a+ recombinants. In this model a recombinational event between 9+ and a+ occurs as a second crossover and has no effect on the segregation of mnt and mntf in m3 9+ recombinants. The other two models predict a predominance of mnt or mnt+ de- pending on gene orders (Fig. 1). These argu- ments ignore the possible role of negative interference which has not been determined.

The results shown in Table I, Cross 1 are consistant with model three. In both the a+ and al classes, about one-third the recom- binants are mnt, the remainder are mnt+.

Figure 2 illustrates the two possible loca- tions of al in the cross P22 m3 Ls9.1 h21 X P22 al. Predictions of segregation patterns of a+ and alleles of h are also listed in Fig. 2. The cross was run and progeny phage were plated at 37”. The centers of M plaques were picked, and antigen al was assayed with microtiter plates. If the order of markers deduced from the cross in Fig. 1 and Table 1, Cross 1 is correct, m3 9+ a+ recombinants will arise as double crossovers and most will be h21. The results in Table 1, Cross 2 show that a+ is associated with h21 about 5.5 times more frequently than with h+. These results are in agreement with those shown in Fig. 1 and Table 1, Cross 1, and we con-

SHORT COMMUNICATIONS 605

Most connon a' recombinant

Model 1

+ + al +

T----- -----I -_________ I 83 9+ h21

m3 9 + h21

Model 2

+ al + +

:-------- -- --_____ -I

m3 + 9 h21

m3 9+ h’

FIG. 2. Predictions of the linkage between alleles of al and h21 depending upon the location of al to the left or right of ts9.1 when m3-9+ recombinants are examined.

pro Am c2 m3mt-d mpmC Lu Lu

Prophage P22

c2 -------- DB 5057

c2 D8 5201 ------------

----------___ mJ DB 136

--- ------ -- m3 mnt D8 120

m3 mnt LIB 147 - ------ -_

FIG. 3. Deletion mutants of prophage P22. Strains lysogenic for DB 5057 and 5201 did not produce antigen al. Lysogens of DB 136, 120, and 147 produced antigen al. Deleted regions are indicated by dashed lines.

eluded that the order of genes is m3 mnt 9 al.

The position of al found in vegetative crosses is compatible with its being at either end of the prophage map, depending upon whether the attachment site is located to the right of al or between genes 9 and al. Using deletion mutants of prophage P22 (kindly provided by David Botstein) we located gene al on the prophage. The results of agglutination tests using the strains shown in Fig. 3 establish that the mnt-utt region of the P22 genome contains the al gene. Prophage deletions DB 5057 and 5201 were negative when tested for al. Strains DB 136, 120, and 147 were all positive.

Genes mnt and al are “on” as prophage genes. Because they flank gene 9, which is involved in the production of phage tails, we considered the possibility that gene 9 is also “on” in P22 lysogens. We concentrated liquid cultures of S. typhimurium (P22) by centrifugation, lysed them according to the procedure of Botstein (‘7), and assayed for “tails” (8) and serum blocking power.

Neither assay was positive and we concluded that gene 9 is “off.” A possible control system to explain these results, is that mnt and al are on the prophage DNA strand that is transcribed and gene 9 is on the opposite strand.

ACKNOWLEDGMENTS

We thank Roger Rossen who showed us how to prepare antiserum and Vedpal S. Malik who did some preliminary work on this problem. This re- search was supported by National Science Foun- dation Grant GB 12190 and American Cancer

Society Grant IN-27M P-13. J.V.S. is supported by National Science Foundation Training Grant

NSF-62-1730.

1. 2. 3.

4.

6.

6. 7. 8.

REFERENCES

LEVINE, M., Virology 3, 2241 (1957). GOUGH, M., J. Viral. 2,992-998 (1968). RAO, R. N., J. Mol. Biol. 35, 607-622 (1968). SUSSKIND, M. M., WRIGHT, A., and BOTSTEIN,

D., Virology 45, 638-652 (1971). YOUNG, B. G., FUKAZAWA, Y., and HARTMAN,

P. E., Virology 23, 279-283 (1964). GOUGH, M., J. Viral. 6, 32Ck325 (1970). BOTSTEIN, D., J. iMoZ. BioZ. 34,621-641 (1968). ISRAEL, V., ANDERSON, T. F., and LEVINE, M.,

Proc. Nat. Acad. Sci. USA 57, 284-291 (1967).

MICHAEL GOUGH’ J.ONE V. SCOTT

Department of Microbiology Baylor College of Medicine Houston, Texas 77026

Accepted July 31, 1972

1 Present address : Department of Microbiology, Health Science Center, S.U.N.Y. at Stony Brook, Stony Brook, NY 11790.