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Affinity of P2 rdl for prophage sites on the chromosome of Escherichia coli strain C
VIROLOGY
1%
Affinity
m-387
(1963)
of P2 rd I for
Prophage
Escherichia
Sites on the Chromosome
coli Strain
of
C.’
ERICH SIX Department
of Microbiology, Accepted
State
University
November
of Iowa,
Iowa
City,
Iowa
21, 1962
The choice of sites for the establishment of a P2 prophage at the chromosome of Escherichia coli C was studied using P2 rd 1 phages liberated by two donors, one carrying P2 rd 1 at site I, the other at site II. Values for the following parameters were determined: (i) probability of acceptance of superinfecting W vi? by cells lysogenized with W rd 2, (ii) immunity level of cells lysogenized with PR rd 1, and (iii) probability of coexistence of P2 rd 1 prophage with a P2 Hy dis prophage. The results, together with those of bacterial crosses, indicate that phage from a donor carrying P2 rd 1 at site I is strongly restricted to reoccupation of site I, whereas phage from a donor carrying P2 rd 1 at site II is not restricted to reoccupation of either site II or site I. The results also show a correlation between the immunity level of a cell lysogenic for P2 rd I and the site(s) occupied by its P2 rd 2 prophage(s). INTRODUCTION
The temperate phage P2 can establish itself as prophage at different sites on the chromosome of its host, Escherichia coli (Bertani and Six, 1958). In E. coZi strain C these sites or locations are distinguished by means of crosses between F- and F+ derivatives. The three different sites studied so far (locations I, II, III) show characteristic transfer frequencies under the standard selective conditions employed in the crosses and are nonallelic to each other. Recent work by Kelly (1963) indicated linkage between sites I, II, and III and genesinvolved in the synthesis of histidine, methionine, and tryptophan, respectively. The plaque type mutants and immunity variants of Pd that have been studied can occupy the same sites ‘Parts of the work described in this paper were done at the Max-Planck-Institut fiir Biologie (Tiibingen, Germany) under a fellowship of the MaxPlanck Society, and at the Department of Biology, University of Rochester, supported by the United States Public Health Service grant E-2862. At the State University of Iowa the investigation is supported by the United States Public Health Service grant E-4043.
as P2 wild type. Among the different sites, site I is strongly preferred when nonlysogenie cells are infected with P2 and also when cells already lysogenic for Pg are superinfected with either a homoimmune or a heteroimmune P2 variant (Bertani and Six, 1958; Six, 1960, 1961a). Other sites (II, III) were first recognized in doubly lysogenic cells, which carried one prophage at site I and one at II or III. From these doubly lysogenic strains, singly lysogenic strains carrying a prophage only at II or at III can be obtained either by bacterial crosses (Bertani and Six, 1958) or by partial curing of the doubly lysogenic cells upon superinfection with a heteroimmune phage (Six, 1960). Three explanations have been offered (Six, 1961a) for the observed preference for site I: (1) differences in the affinity of the various sites for P2; (2) existence in the P2 lysates of phages with different site specificities; and (3) a combination of (1) and (2).
The purpose of this paper (see also Six, 1961b) is to distinguish among these explanations by comparison of the “lysogenization patterns” (that is, of the relative affini375
376
ERICH
ties for different sites) of P2 phagc derived from two different donors: strain C-208 = C (P2 rd I) I and strain C-86 = C (P2 rd 011. It will be shown that phage from donor C-86 does not exhibit the preference for site I that phage from C-208 shows. This excludes the possibility that there is-with respect to the site affinity-only one kind of P2 phage and that the lysogenization pattern is controlled solely by the affinity of these sites for Pd. Because an analysis of lysogenization patterns based only on bacterial crosses would be impractical, lysogenization patterns have been investigated using one or more of the following criteria, based on properties of lysogenic cells that depend on the prophage site occupied : 1. The probability of adding as a prophage a superinfecting phage known to prefer site I without loss of a preexisting prophage, is lower if site I is already occupied than if it is free (Six, 1961a). 2. The degree of immunity of a lysogenic cell, measured by challenge with an intermediate virulent mutant of P2, is low for cells carrying a P2 prophage at site I only, high for cells carrying P2 prophages at both sites I and II, and intermediate for some other cases (for example, P2 at site II only). 3. It can be assumed that a site is never occupied by more than one P2 prophage at a time since in all known doubly lysogenic derivatives of E. coli strain C the two prophages can be separated in bacterial crosses (see also Table 7). Therefore, if superinfection of a lysogenic cell carrying a prophage at site N (N = I, II, etc.) leads to establishment of a second prophage without loss of the first one, the second prophage presumably occupies a site *N. Bacterial crosses have been used only to verify the interpretation of results obtained by the above methods. MATERIALS
AND
METHODS
Phages. The weak virulent mutant P2 virl, the intermediate virulent mutant P2 G-1 21irl3, and the strong virulent mutant P2 vir3 have been described earlier, together with the general techniques and media employed in the study of phages of the P2 group (Bertani, 1954, 1957; Six, 1961a). For
SIX
phage P2 rd 1 spontaneous lysa,tes were prepared by growing cultures of the donor strains C-208 (here called strain A) or C-86 (strain B) in L broth or nutrient, broth to reach ca. 5 x lo7 cells per milliliter. The cells were then killed by adding streptomycin (followed in most experiments by chloroform) and were removed by centrifugation. The supernatant fluids were stored in the refrigerator. Bacteria. The nonlysogenic streptomycinresistant indicator strain Sh/s of Shigella dysenteriae and its two lysogenic derivatives Sh-76 (carrying P2 Hyl dis) and Sh-77 (carrying P2 wild type) have been described (Bertani, 1951; Six, 1961a). All derivatives of E. coli strain C used in the experiments are listed in Table 1. Most of them have been described before. Testing for prophages was done by assaying the free phage liberated by growing cultures of lysogenic cells. The cells were killed by adding streptomycin or chloroform or by heating to 58”, then a drop of culture was placed on L agar plates seeded w&h suitable indicator strains. Bacterial crosses were carried out as described by Bertani and Six (1958). Selected markers were: arginine and tryptophan independence (from the F+ parent) and streptomycin resistance (from the F- parent). Bacteria from recombinant colonies were streaked before testing them for prophages. The number of recombinants tested in a cross varied from 58 to 240; in 69 of the 74 crosses it ranged between 80 and 120. Occupation of site I by a P2 rd 1 prophage was determined by testing for allelism with a prophage known to occupy site I in the cross partner. In some cases occupation of site II was determined in the same way. The precision of such test is lower for site II than for site I because the transfer frequency from the F+ parent is lower for site II: according to Bertani and Six (1958) the average transfer frequency is 41% for site I and only 15% for site II. Also, the separability from site I of an unknown site X + I occupied by P2 rd 1 was determined as the proportion of recombinants that have not received site S from the F+ parent among those recombinants that have received site I from the F+ parent. If the frequency was less than 50%.
PROPHAGE
SITE
TABLE
377
OF Pd RD L
AFFINITIES 1
STRAINS OF E. coli USED” Strain C-208
C-86 C-2 C-8 c-is c-29 C-62 C-70 c-79 C-83 C-86
Prophage
P2 rd 1 P2 Id 1
Site
P2 Hyl dis Pbc P2 Hy’ dis P2 c P2 Hyl dis
Use
Origin
I
F+ F+ F+ FSr; FS”; F+ Ss; F+
I
S”; F+
Donor A Donor B For crosses Recipient For crosses For crosses Recipient, for crosses F or crosses
I
Sr; F-
F or crosses
From expt. no. 5, Six (1960)
S”; F’ Sr; F+
Recipient Recipient
Six (1959) Spontaneous
I II
None None
P2 P2 c PI Hy’ dis
Pertinent characteristics
I I
S”; S”; S”; Sr;
Six (1961a) Bertani (1962) Bertani and Six Bertani and Six Bertani and Six Bertani and Six Six (1959)
(1958) (1958) (1958) (1958)
Six (1959)
II
II II
None
mutation
of
of C-8 of C-8 mutation
of
c-2 C-218 c-220 G-211
PB Hy’ dis P2 Hy* dis
C-227
P2 rd 1
C-228
P2 rd 1 P2 rd 1
I I
None
6’1; FSr; FSr; F-
I
Sr; F-
I
13; F-
II
Recipient Recipient Host for tion of sates Immunity standard Immunity standard
producplate lylevel level
Lysogenization Lysogenization Spontaneous C-lb Lysogenization expt. no. 2 Lysogenization expt. no. 2
of
C-8 in
of
C-8 in
Q All prophage sites were confirmed by bacterial crosses testing for allelism with previously lished sites I and II. SB; Sr: Streptomycin sensitive, streptomycin resistant F-; F+: Mating types rd; 1; c: Plaque morphology markers; round; large, and clear, respectively. b C-l is original strain C (see Bertani and Six, 1958).
the site X was classified as belonging to group C (= close to I) ; if the frequency was higher than SO%, as belonging to group D (= distant from I). According to this classification site II belongs to group D. In all these crosses the F- parent was a clone lysogenized with P.2 rd 1. If the F- parent did not also carry PW Hy dis, site I was marked by a P2 Hyl dis prophage in the F+ parent (C-62). If the F- parent was lysogenic for Pf? Hyl dis it was assumed that this prophage still occupied site I and the cross was performed with a nonlysogenic F+ parent (C-2). The distinction between clones C (Pd rd l), (PZrd I)x and C (-)I (PZrd Zjx in a cross with a partner carrying a prophage at site I depends on the absence or presence of nonlysogenic recombinants. If site X is very
estab-
close to I (X = C) such recombinants might be missed, and this would lead to a misclassification of a C (-)I (PZ rd Z)c clone as C (PdrdZ)I (P2rdZ)c. The “immunity” level of cells lysogenic for PZ rd 1 was studied by spotting a loopful of the Iysogenic cultures on a L agar plate seeded with lo7 to lo* Pb vi+ viP3 phages. The degree of lysis exhibited on the spot after incubation allowed a rough classification int,o 3 to 5 immunity leveis ranging from “low” to “high.” Strains C-227 and C-228 served as standards for Yaw” (or: “low 1”) and “high” immunity levels, respectively. [See Bertani and Six (1958) for a comparable test on lysogenic Shigella strains.] Media. Bacteria were grown in L broth?
378
ERICH
SIX
which contains 10 g tryptone (Difco), 5 g tion about the lysogenization pattern since yeast-extract (Difco), 10 g NaCl, and 1 g P2 vir’ presumably prefers site I. [This asglucose per liter of water. When noted, nu- sumption was not tested by bacterial crosses,but it was shown that the probabiltrient broth (Difco) was used. L agar and LO agar contain L broth and ity of adding P2 vi+ as prophage to a cell 1% agar; L agar contains also 2.5 X 10W3M already carrying a P2 prophage is much lower if site I is occupied than if it is free CaClz . (Six, 1961a).] H ence, cells carrying both RESULTS P2 rd 1and P2 vi+ should be less frequent if P2 rd 2 itself prefers site I. 1. Direct Infection with Spontaneous LyIn one control experiment, cells of strains sates A and B were infected with P2 vi9 under Since P2 is a noninducible phage, one can- conditions similar to those of the experinot obtain directly from the lysogenic do- ments described below. For both strains nors high titer lysates than can infect a ma- about 83% of the cells survived and there jority of recipient cells. Hence, the low titer was no indication that P2 virl would select spontaneous lysates obtained from donors for or against cells carrying P2 rd 1 at site A (= C-208) and B (= C-86) were used to I. Under the same conditions less than infect nonlysogenic recipient cells at low 10P3% of nonlysogenic cells of strain C-85 multiplicities, and the not lysogenized cells survived. In experiment no. 1 the recipient cells were then eliminated by infection with the from a growing culture of nonlysogenic weak virulent P2 vir’ at high multiplicity. This second infection can furnish informa- strain C-85 were suspended in spontaneous lysates of donor strains A and B to give a cell titer of about 5 x lo7 per milliliter. The TABLE 2 phage to cell ratio was ca. 5 x lo-” for lyACCEPTANCE OF P2 vi+ BY CELLS LYSOGENIZED sate A and 2 x 1O-3 for lysate B, respecWITH P.9 rd 1 FROM Two DIFFERENT DONORS tively. CaC12was added (final concentration 1 j 2 Experiment no.. . . . .j of 5 x 1O-3 M) and the mixtures were incubated for 30 minutes at 37” to allow phage C-85 C-8 Recipient.. . . . . . . . . . adsorption. The infected cultures were then diluted 1: 100 in 20 ml of nutrient broth conDonor’.. . .... . A B A B taining streptomycin, and incubated at 37” ~--with aeration for 2 hours to reach cell titers Numbs; of survivors of ca. 4 X lo6 cells per milliliter. CaClz was 38 99 94 . . . . . . . . . . . . . . . 40 then added (5 x 1O-3 M), and O.l-ml portions of the cultures were mixed with 0.1 ml Number of lysogenic survivors.. .~T~,/,~T of a P2 vi+ suspension (titer: 5 X lo8 per milliliter), incubated at 37” for 10 minutes Prophages carried in to allow adsorption of the phage, and plated subclone pairs from Frequency among lysogenic on L agar (agar layer method). Colonies the survivors survivors (in 70) were isolated, streaked on nutrient agar PZ vi+ PZrdl plates, and 2 subclones from each streak were tested for prophages. The results, m o/2 94 376 91 45 shown in Table 2, indicate that cells lysogew o/2 0 0 5.5 3 nized by lysate B give a much higher fre5s l/2 6 19 5.5 9 quency of addition of P2 vi+ than cells 2/2 w 0 44 0 43 lysogenized by lysate A. Experiment no. 2, also included in Table a A: C-808 = C(P2 rd Z)i ; B: C-86 = C(P2 2, was conducted in a similar way. Strain rd 1111 . C-8, nonlysogenic, was used as recipient. b Including one isolate with one subclone carrying P2 rd 1; the other P2 f-t. Again, cells that received their P2 rd 1
PROPHSGE
SITE AFFINITIES
OF P2 RD L
379
TABLE 3 from lysate B showed a higher frequency of P2 wit-l prophages than cells whose P2 rd 1 IMMIUNITY LEVELS OF C-8 CELLS LYSOGENIZED WITH Pd rd 1 AND SUPERINFECTEDWITH Pd came from lysate A. The nonlysogenic survirl (EXPERIMENT NO. 2) vivors were all found to be completely P2 resistant (= resistant to P2 vir3), Frequency of The results of experiments nos. 1 and 2 immunity levels indicate that phage P2 rd 2 from donor B is (in %I I ‘rophages Lysate” nb -less restricted to site I than is phage from carried donor A. I JOW Internediate High In experiment no. 2 one subclone from ___-each of the pairs of lysogenic isolates that 0 1 P2 rd 1 A 81 99 had given identical results (see Table 2) P2 rd 1 and 0 0 0 was also tested for immunity level by chalPb vi+ lenge with P2 vi+ vir13. Among 81 such clones lysogenized by lysate A, 80 showed a 3.5 2 B PR rd 1 46 57 low immunity level; instead 7 out of 57 3.5 3.5 Pt rd 1 and 42 clones lysogenized by lysate B (= 12%) P2 vi+ - showed intermediate or high immunity levels. These results, shown in Table 3, suggest a A: donor C-208 = C(P2 rd l)~ ; B: donor C-86 = C(P2 rd I)II . a difference in the lysogenization pattern b n: Number of clones with two identical subbetween phages from donors A and B, presumably a higher tendency of phage from clones. donor B to form double lysogens as compared with phage from donor A.2 ceived their P2 rd 1 from donor B. The presFor five clones from experiment no. 2 car- ence of P2 vi+ complicates the interpretarying only P2 rd 1, the P2 rd 1 sites were tion of the observed transfer frequencies for studied by crossing with strain C derivatives the P2 rd 1 sites. In particular it can be asC-29, C-62, and C-70, carrying prophages sumed that recombinants carrying only P2 at site I or at I and II. Two low immunity vi+ or P2 vi+ together only with P2 Hyl dis level clones both were found to be C (P2 rd cannot be recovered. Therefore, classifica1)I. One of them had been lysogenized with tion of sites different from I as C or D can donor A phage, the other (C-227) with do- only be tentative. One low immunity level nor B phage. One intermediate immunity clone appeared to carry P2 rd 1 presumably level clone lysogenized by donor B phage at a site C, another presumably at a site D. was doubly lysogenic, carrying P2 rd 1 pro- A high immunity level clone was found to phages at sites I and C. Two high immunity carry one P2 rd 1 at site I and a second P2 level clones were also found to be doubly rd 1 at a site D different from II. lysogenic carrying P2 rd 1 prophages at sites These results agree with, and lend further I and II. One of them had been lysogenized support to, the assumption that in most of with donor A phage, the other (C-228) with the clones that carry P2 vi+ the P2 rd 1 is donor B phage. Three more clones from ex- established at a site different from I, and periment no. 2 carrying P2 rd 1 and P2 virl that in most clones without P2 virl, site I is were analyzed in the same way. All had re- occupied by a P2 rd 1. In summary, the evidence obtained in the * One may ask whether, in the four isolates that) direct infection experiments leads to the folshow the presence of P2 wilj and have intermediate lowing conclusions: P2 rd 1 derived from or high immunity levels, the P2 vi? contributes to site I (donor A) has a strong tendency to the higher immunity level. This is unlikely, beseek again establishment at that site; this cause the data of Table 3 do not reveal any corredoes not exclude a rare occupancy of a diflation between presence of P2 vi? and level of immunity, and because P2 vi? is a mutant that has ferent site. P2 rd 1 derived from site II (dolost the ability to produce immunity substance nor B) does not have such a strong preference for site I; whether it has any preference (Six, 1961a).
380
ERICH
for site II cannot be decided from these experiments.
SIX
among clones lysogenic for P2 rd 1. This parameter will be called the probability of coexistence with a heteroimmune prophage established at site I (symbol P,). 2. Plaque Analysis Experiments Usually strain C-218 was chosen as reGeneral procedure. Further information cipient since it yields larger plaques than on lysogenization patterns was sought using the other strains. The results, summarized a different experimental approach. Phage in the upper part of Table 4, show that P2 rd 1 liberated by donor A (= C-208) or P2 rd 1 stemming from donor B (average B (= C-86) was used to produce plaques on Pr = 64%) has a higher probability of coon a lawn of recipient cells. From these existence than P2 rd 1 from donor A (averplaques, bacterial clones lysogenic for P2 rd age PI = 7%). 2 were isolated. Presumably, these “plaque In 11 other experiments (to be reported clones” had been lysogenized with P2 rd 1 elsewhere) Pr was determined for donor A only after the phage liberated by the donor phages and similar values were found as in had undergone several rounds of lytic re- the experiments detailed in Table 4. Lysogenization of strains C-220 (experiproduction in the recipient cells. The plaque clones were then tested for immunity level, ment no. 3b) and C-62 (experiment nos. 8a, 9a) with P2 rd 1 led to somewhat different or for the presence or absence of a preexistvalues for Pi ; yet, a higher value for Pi was ing heteroimmune P2 Hy dis prophage (if again obtained for phages from donor B the recipient had been lysogenic for this than for those from donor A (Table 4, lower phage) , or for both properties. In the experiments reported below, phages part). For an interpretation of the PI values, it produced by donors A and B were plated on L agar plates together with recipient cells, must be kept in mind that persistence of mixing 2 drops of a fully grown culture of P2 Hy dis at site I indicates establishment the recipient strain with one or two drops of of P2 rd 1 at a different site, but that loss of spontaneous lysates suitably diluted (usu- P2 Hy dis from site I is not proof that P2 ally 1: 100). Streptomycin was added if the rd I is carried at this site. Hence PI gives only a lower limit for the probability of P2 recipient cells were streptomycin resistant. From the plaques formed on these plates, a rd 1 occupying a site *I. Further tests, to be discussed below, will show, however, that mixture of phage and cells was transferred with a sterile wire to tubes containing 1 ml most of the clones that had lost P2 Hy dis of L broth. After 4-5 hours of incubation at upon lysogenization with P2 rd 1 from donor A were indeed C (P2 rd Z)I. 37”, streaks were prepared on LO agar. From Probability of coexistence of P2 Hy dis at colonies isolated from these streaks, a second set of streaks was prepared, and one site II with P2 rd 1. Since phages from docolony isolated from each of the second-set nor A, but not those from donor B, have a streaks was transferred to L broth and strong preference for site I, does the phage tested for prophages carried. The subclones from donor B show a strong preference for that carried no P2 rd 1 prophage and those site II? This question was investigated by recognizable as unsegregated with respect using recipient strain C-88, which carries P2 Hyl dis at site II. The results of two exto the prophages carried were discarded. Probability of co-existence of P2 Hy dis periments (Table 5) give a negative answer. The probability of coexistence with P2 Hy’ at site I with P2 rd 1. For these experiments a recipient strain carrying a P2 Hy dis pro- dis at site II (symbol Pn) is high not only phage at site I was employed. From each of for P2 rd 1 from donor A, as expected, but the plaques used, one clone lysogenic for also for P2 rd 1 from donor B, and the difference in the Pn values for phages from the P2 rd 1 was tested for presence of the P2 Hy two donors is not significant. Again it should dis prophage. The frequency of establishbe emphasized that loss of P2 Hy dis does ment of P2 rd 1, at a site presumably different from I, could then be estimated at the not prove that P2 rd 1 was established at the proportion of clones still carrying P2 Hy dis site previously occupied by P2 Hy dis.
PROPHAGE
TABLE PROBABILITY
-
OF P2 RD L
SITE AFFINITIES
381
4
OF COEXISTENCE WITH A P.2 Hy dis PROPHAGE AT SITE I (PI) FOR P9 rd 1 PHAGE PRODUCED BY Two DIFFERENT LYSOGENIC DONORS
Lysate A
Lysate B Donor
Recipient
Expt. no.
C-208 = C(P2 rd 1)I N” C-218 C-218 C-218 C-218 C-21 8
3a 4 5 6 7
C-86 = C(P2 rd I)11 N
PI
77
0.08
71
0.08 0.06 0.07 0.02 0.07 0.0-0.08
34 68 47
Overall : 297
PI
_86 66 47b
0.67 0.58 0.62”
118b 31
0.65”
348
Total of 11 other experiments
C-21 8
3b 8a 9a
C-220 C-62
59
0.17
91
50
C-62
51
0.02 0.04
44 27
513
Average 0.04 0.65 0.43 0.26
-
a N = number of clones carrying P2 rd 1 tested. b Combining data of two subclones of C-86 (see text). There was no statistically ence between the PI values obtained for the two subclones. TABLE PROBABILITY
0.68 0.64
significant
differ-
5
OF COEXISTENCE WITH A P2 Hyl dis PROPHAGE AT SITE II (Pri) FOR P2 rd 1 PHAGE PRODUCED BY TWO DIFFERENT LYSOGENIC DONORS
Lysate A
Lysate B Donor
Expt. no.
Recipient C-208 = C(P2 rd I)I
8b 9b
C-83 C-83
C-86 = C(P2 rd 1)~
N”
RI
N
PI1
55 44
0.62 0.50
41 29
0.54 0.55
o N: number of clones carrying P2 rd 1 tested.
Immunity level of plaque clones. In some of the experiments described above (nos. 3a, 3b, 4, 8a, 8b), the immunity level of the clones lysogenic for P2 rd 1 was also determined by challenge with P2 vi+ vir13. This was done also in experiment no. 10, using the nonlysogenic strain C-8 as recipient and employing the same donor lysates as in experiments nos. 8a and b. As shown in Table 6, the difference between donors A and B is now reflected in a difference of the distribu-
tion of immunity levels: in each experiment lysogenization with P2 rd 1 from donor B resulted in a higher percentage of clones with intermediate or high immunity level than lysogenization with P2 rd 1 from donor A. This finding is in qualitative agreement with the results of the direct-infection experiment no. 2 (Table 3). Correlation between immunity level and occupied prophage sites. Experiment no. 2 shows that the immunity level is correlated
382
ERICH
SIX
TABLE DISTRIBUTION
OF IMMUNITY
LEVELS
LYSOGENIZED
-
WITH
6 AMONG
DESCENDANTS
OF CELLS
PR rd 1 IN PLAQUES
Lysate A
Lysate B Donor
Expt. no.
C-208 = C(PB rd Z)I
Recipient
Frequency of immunity InterInternediate 1 mediate 2 10” 3a 4 8a 3b 8b
C-8 C-818 C-818 C-69 c-110 C-83
0.02 0.08 0.03 0.00 0.15 0.09
C-86 = C(PZ rd l)rr
levels
Frequency of immunity
High
0.04 0.07 0.03 0.02 0.08 0.05
0.03 0.03 0.10 0.04 0.03 0.24
High
0.09 0.17 0.16 0.06 0.27 0.38
0.26 0.17 0.06 0.09 0.18 0.10
-
3 According to Bertani (1962) the presence of a PZ Hy dis prophage tends to lower the immunity level of cells carrying P2. This could explain why in the presence of P2 Hy’ dis at site I the clones carrying P2 rd 1 at a site C show a low immunity level.
0.08 0.05 0.20 0.16 0.11 0.20
0.69 0.47 0.39 0.68 0.49 0.68
-
a Number of clones carrying Pi? rd 1 tested: 98 for lysate A, 97 for lysate b Sum of three preceding frequencies (intermediate 1, 2, and high).
with the number and possibly also the site of the P2 rd 1 prophages carried by the clones. The nature of this correlation was studied by analyzing in bacterial crosses the P2 occupied sites of 48 clones obtained in experiments nos. 3a, 4, 8a, and 10. The results of these crosses, together with those for 4 clones obtained in experiment no. 2 and not carrying P2 zG1, are summarized in Table 7. If the immunity level was low and no Pt Hyl dis prophage was present, P2 rd 1 was always present at site I. In five clones with a low immunity level and lysogenic for P2 rd 1 and P2 Hyl dis, the P2 rd 1 prophage was present at a site *I, that is, was separable from the P2 Hyl dis prophage in a cross with C-2. Intermediate or high immunity levels were always indicative of the presence of a P2 at a site *I, either alone or if the immunity level was high, together with another PW prophage which as a rule occupied site I.3 These results indicate that the immunity level serves as an indicator for occupation
Not 10~5
~~
I
0.35 0.24 0.14 0.43 0.21 0.39
levels
B.
of a site +I by P2 rd 1, at least in the absence of a P2 Hy dis prophage. Accepting this conclusion the difference found in experiment no. 10 (Table 6) between phages from donor A and B is again recognized as a difference in their ability to establish a prophage at a site +I, as in the other experiments. This agreement supports the proposed interpretation of the immunity level. Evaluation of the prophage coexistence test. The prophage coexistence tests were based on the assumption that addition of P2 rd 1 as a prophage without loss of a preexisting P2 Hy dis prophage indicates that P2 rd 1 occupies a different site than does P2 Hy dis. This assumption was confirmed by the bacterial crosses reported above. It seemed reasonable further to assume that loss of P2 Hy dis was caused by P2 rd 1 replacing it at the same site, leading to C (PC rd 1)I if the recipient was formerly a C (P2 Hy disJI The study of the immunity levels, together with the cross analyses, shows that this interpretation is valid only for clones with a low immunity level. 39 clones carrying P2 rd 1 but not P2 Hy dis were analyzed (see Table 7). All 17 low immunity level clones were C (P2 rd 1)I . Of these clones, 11 were obtained by replacement of a P2 Hyl dis previously occupying site I in recipients
PROPHAGE
SITE AFFINITIES TABLE
OF P8 RD L
383
7
RESULTS OF THE BACTERIAL CROSSEST
-
Number of cloneswith the indicated lysogenic constitution Img;‘ty
Low Intermediate 1 Intermediate 2 High
Donor
Site
Prophages
PZ rd 1 PZ rd I PZ Hy’ dis PZ Hyl dis I. . . . PZ rd 1 PZ Hyl dis P2rd 1 P2rdl PZrdl P2rdl C...... P2rdl P2rdl P2rdl P2rdl D...... ____~ 8 A B 9 5 __----__ ---~ __---_______ -1 1 A 1 2 1 B --_________ __-__________--___ 2 2 2 A 2 3 6 B ___-____ __-___2 A 1 1 3 B
II . C: sites close to I, but C * I (see under Material ~A:C-~O~=C(P??T~Z)~.B:C-~~=C(PB~~Z) and Methods). D : sites distant from I (including site II) (see under Material and Methods). The table summarizes the results for 52 clones isolated in experiments nos. 2 (only clones not carrying P2 sir’), 3a, 4, 8a, and 10, analyzed in F- X F+ crosses with respect to the P2 rd 1 sites occupied. In experiments nos. 2 and 10 a nonlysogenic recipient, C-8, was used: in the other experiments, recipients C-68 or C-818, both carrying P2 Hyl dis at site I. The lysogenic constitutions indicated are the simplest ones consistent with the data. The possibility cannot be excluded that instead of one P2 rd 2 prophage at a site C there are two (or more) at different sites C or that instead of a P2 rd I only at a site D there are also others at sites D or C. Sites C. The three clones C(P2 rd Z)o (all from experiment no. 10) were crossed with C-62. Nonoccupation of site I was indicated by finding 2, 7, and 8 nonlysogenics respectively among 100 recombinants tested in each cross. In crosses involving 15 clones carrying a P2 rd I at a site C, a fraction s15y0 of the recombinants showing transfer of site I did not show also transfer of site C. For one other clone this fraction was 29%. For two other clones [one clone C(P2 Hyl dis)I (P8 rd Z)o with immunity level “intermediate 1” and one clone C(P2 Hyl dis)r (P2 rd Z)c (PR rd Z)n] sites could only tentatively be classified as C. In these cases the classification was based solely on the fact that the transfer frequency of these sites was similar to that expected for site I. Sites D. None of the clones classified as C(P2 rd l)D belonged to experiments 2 or 10. For six clones from other experiments [2 clones C(P2 Hyl dis)I (P2 rd Z)Dand one clone C(P2 rd Z)n with immunity level “intermediate 2 ” 2 clones C(P2 rd Z)r (P2 rd Z)Dand one clone C(P2 Hyl dis)r (P2 rd Z)C (P2 rd Z)n with high immunity level] allelism of site D with site II was tested, and in all cases the results are compatible with D = II. Two of these clones had received their P2 rd 1 from donor A. In crosses involving the 12 other clones carrying a P2 rd Z at a site D, 67% or more of recombinants showing transfer of site I did not show also transfer of site D. Site of P2 Hyl dis. For six clones [two clones C(P2 Hyl dish (PI rd Z)C, three clones C(P2 Hyl dis)I (P2 rd Z)D , and one clone C(P2 Hyl dis)I (P2 rd Z)c (P2 rd Z)n] allelism of the P2 Hyl dis site with site I was tested and confirmed, as expected, since these clones were obtained by superinfection of a C(P2 Hyl dis)I with P2 rd 1. One of these clones had received P2 rd Z from donor A. C-66 or C-218, the remaining 6 by occupation of site I in the nonlysogenic recipient C-8. Among 22 clones with a higher immunity level, 17 were doubly lysogenic carry-
ing one Pb rd 1 at site I and another at a different site. Six of these clones were obtained from recipient C-218, 11 from C-8. In the remaining 5 clones site I remained unoccu-
384
ERICH
SIX
TABLE
8
IMMUNITY LEVEL AND RETAINMENT LYOSOGENIZED
Donor .
..
WITH
OF P9 rd
Ps?Hyl
clis OF 1 IN PLAQUES’”
C-d18 CELLS
I
...
Intermediate
Immunity level.
1 ! 1
Expt. no.
PZ Hy’ dis
3a
No Yes
4
No Yes
High
2
Frequency of clones
present
-__
Intermediate
__--__
0.80 0.000.0140.0140.09
Pt
AP
__o.20
0.0140.030.0140.0140.014
a A: C-208 = C(PB rd 1)r.B: C-86 = C(PB rd Z)rr. The “low immunity” class is here split into two subclasses: “low 1” and “low 2,” the immunity level of “low 2” being a little higher, as judged from the degree of bacterial growth in the presence of PB vir1virr3, than that of “low 1.” Clones with a low immunity level carrying Pa rd I and PR Hyl dis usually belong to subclass “low 2.” Pt = frequency of clones not showing a low immunity level and/or carrying PR Hyl dis (presumably at site I) besides a P2 rd 1. AP = Pt - Pr (Pr = probability of coexistence of P2 rd 1 with a PB Hyl dis at site I). AP gives the fraction of clones not recognized as carrying a PZ rd 1at a site *I if only the coexistence test is applied. The data presented in this table correspond to those shown in Tables 4 and 6.
pied and P2 rd 1 was carried at a different site. Two of these clones were obtained from recipients C-62 or C-218, 3 from C-8. The best available estimate of the fraction of clones carrying P2 rd 1 at a site +I may be obtained by combining prophage coexistence and immunity level tests to give the joint probability Pt for either coexistence with P2 Hy dis (at site I) or for establishment of a strain of intermediate or high immunity level. Table 8 shows that most clones that have lost P2 Hyl dis upon lysogenization with P2 rd 1 from donor A, possess a low immunity level and are presumably all C (P2 rd Z)i . Hence, it appears justified to interpret the low Pi values obtained for phage from donor A as reflecting the strong preference of such phage for prophage site I. Homogeneity tests. The values for PI (the probability of coexistence of P2 rd 1 with a P2 Hy dis located at site I) in the preceding section are average values for the spontaneous lysates. The question arises whether these lysates are homogeneous with respect to preference for prophage site I. If the ly-
sates were not homogenous, PI might reflect only the relative frequencies of phages with strong preference for site I and phages with less or no preference for site I. The homogeneity of some of the donor lysates used in the experiments already described was studied by obtaining ten clones from the same plaque for a number of plaques and testing these “sister clones” for prophages carried. The results of this sister-clone analysis (presented in Fig. 1) gave no indication of a heterogeneity of donor A lysates, but for donor B lysates at least two components could be distinguished: one component apparently had the same preference for site I as donor A phage, the other had much less preference, if any, for this site. A heterogeneity of lysates B might reflect a heterogeneity of the cells of donor strain B (= C-86). In testing this hypothesis two subclones were isolated from C-86: one formed colonies of the usual size, the other produced smaller colonies on L agar. The Pi values were determined in the fashion already described in two experiments, nos. 5 and 6. The P, values obtained for the two subclones of
PROPHAGE
SITE AFFINITIES
OF PZ RD L
385
tion, the plate lysates were used to infect growing cells of strain C-85 at a multiplicity of about 5. The infected cell suspension were diluted 1: 100 into nutrient broth and S. Investigation of Plate Lysates incubated at 37” for 4 hours. About lo6 cells The results of the analysis of plaque of the infected cultures were then superinclones imply a certain stability of the de- fected with P2 vi+ and plated onto L agar gree of affinity to site I during lytic growth plates in the same manner as described for of the phage. One would then expect that experiment no. 1. A total of 80 colonies were plate Iysates obtained from the spontaneous examined for prophages carried in two sublysates of donors A and B would still reflect clones. Of 40 clones lysogenized by lysate the difference observed between the spon- A, none had accepted P2 vi+; of 39 clones lysogenized by lysate B, 8 carried P2 virl taneous lysates. The spontaneous lysates used for experi- in both subclones tested, 2 more in only one ment no. 1 were also used to produce plate subclone. In experiment no. 11 strain C-211 was lysates by plating 0.1 ml portions of the unused as host for the preparation of the plate diluted spontaneous lysates together with lysates. The Pr values of these lysates were Sh/s cells on L agar plates. After sterilizadetermined by plating with cells of C-218. The values obtained, 0.04 for lysate from A 25 and 0.63 for lysate from B, lie within the r range of PI values found for the corresponding spontaneous lysates. This indicated that the additional rounds of lytic multiplication of the phages in host C-21 1 had not significantly altered the degree of the affinity of the phages for prophage site I.
donor B did not differ significantly from each other nor from the Pi values determined for donor B itself.
DISCUSSION
Different lines of evidence presented in this paper all lead to the same conclusion: there is a difference in the lysogenization pattern of P2 rd 1 phages depending on the IOL( I I I I I I I lysogenic donor strain used as source of the 3 4 5 6 n= 0 I 2 phage. This difference may be described as FIG. 1. Histogram showing the frequency N of follows: P2 rd 1 from donor C (P2 rd ZJ1has groups of n clones carrying both P2 rd 1 and PS a strong preference for establishing as proHy’ dis among six sister clones isolated from the phage at site I of recipient cells; P2 rd I same plaque and carrying P% rd 1. From individual P2 rd 1 plaques (on a C-218 from donor C (P2 rd Z)n shows neither this preference for site I nor a comparable preflawn) that had already yielded a clone carrying erence for site II. P2 rd 1 when the PI values for the spontaneous The difference between phage from the lysates of donors A (= C-208) and B (= C-86) were determined, ten more sister clones were iso- two donors appears to be one of degree: lated and tested. The histogram shows the numphage from C (P2 rd Z)i may establish a ber N of plaques which, among the first six sister prophage at sites *I (including II), but occlones found to carry P2 rd 1, yielded n clones car- cupation of these second-choice sites both rying also P2 Hy’ dis (0 I n 4 6). close to I (= C) and distant from I (= D) The clone used for the previous Pr determinais observed more often for phage from C (P2 tion was not included, but the results for plaques that had originally yielded a clone carrying P2 rd 1 rd 2)n. The latter phage can also choose and P2 Hy’ dis are shown in dark shading (13 sit.e I for prophage establishment. Judging from the distribution of immunity levels plaques for A, 14 for B), those having originally yielded a clone carrying only P2 rd 1 in light shad- among clones lysogenized with this phage ing (13 plaques for A, 12 for B). and the proposed correlation between im-
386
ERICH SIX
munity level and P2 rd 1 sites it may tentatively be concluded that phage from C (PW rd Z)rr has retained some preference for a certain region on the recipient chromosome, namely the region including sites I and C, but lacks the high specificity for site I. The experimental evidence is also compatible with the following hypothesis, The region that appears to be preferred by phages from both donors is the only one available for establishment of a single prophage in previously nonlysogenic cells; sites D are occupied by P2 rd 1 only in doubly lysogenic cells carrying also a P2 rd 1 prophage at site I, or in cells which, prior to the establishment of a P2 rd 1 prophage, already carried at site I at PW Hy dis prophage, which was either retained or lost upon infection with P2 rd 1. This hypothesis agrees with observations by Bertani (1962) concerning occupation of site II. The question remains why Bertani, using a somewhat different experimental method, did not also find any clones carrying P2 rd 1 at a site C only. The nature of the difference observed between phages produced by the two donors remains unknown. At present it can be stated only that the phages themselves must carry a determinant for site affinity associated with their genetic material. The difference in site affinity persists during phage multiplication in plaques and in plate lysates, that is, during replication in hosts that do not possess whatever modifying factor is contained in the donor strains. Any such factor must become genetically associated with the phage prior to the spontaneous lysis of the donor cells. The only other pertinent information obtained is that phages from C (P2 rd Z)r appear to be uniformly restricted to site I. The observed inhomogeneity of phages from C (P2 rd Z)n might be an artifact caused by the presence of a P2 Hy dis prophage in the recipient. Experiments in progress employing different bacterial strains lysogenized with different P2 variants should help to clarify the role of the donor and to answer the question whether occupation of site II, as opposed to occupation of site I, is a necessary or suffi-
cient condition to produce the difference between phages from C (PW rd 1)r and C (P2 rd
UII
.
It may be noted that P2 rd I, and possibly all P2 variants, are unlike other temperate phages studied so far, such as lambda and Pl, in that they may exist in both a siterestricted and an unrestricted or less restricted form. In this respect P2 rd I resembles another episome, the F factor, shown by Jacob and Adelberg (1959) to exist either in a site-restricted or a site-unrestricted form. Previously (Six, 1961a) a study of the establishment of P2 c as prophage in cells C(-),(P2 rd Z)II and C (P2 Hy dis)I (P2 rd Z)n had indicated a strong steric hindrance by the P2 Hy dis prophage against the establishment of P2 c, presumably at site I. The experiments described in this paper do not reveal any appreciable steric hindrance by P2 Hy dis in recipients C (P2 Hy dis), for establishment of P2 rd I at site I. Phage from C-208 shows a strong preference for site I even if this site is occupied by a P2 Hy dis prophage, and phage from C-86 shows a decreased preference for this site regardless of its occupation. Presumably, the lack of immunity to P2 in recipients C (P2 rather than the difference in the HY dis11, markers of the infecting phage, is responsible for the lack of steric hindrance in t,he present case. The apparent lack of steric hindrance in nonimmune recipients may have several causes : (1) the availability of a large number of preprophages, due to multiplication of the genome of the infecting PW rd 1 in nonimmune (but not in immune) cells (and due also to multiple infection in plaques) ; (2) a removal of the P2 Hy dis prophage from site I by a “curing” effect resulting from infection with P2 rd 1; establishment of P2 rd 1 as prophage could then occur secondarily in the freed site I. Curing of lysogenic cells by superinfection with heteroimmune phage has been observed (Cohen, 1959; Six, 1960). REFERENCES G. (1951). Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escheri&a coli. J. Bacterial. 62,293-300.
BERTANI,
PROPHAGE
SITE AFFINITIES
BERTANI,G. (1954). Studies on lysogenesis. III. Superinfection of lysogenic ShigeZZa dysenteriae with temperate mutants of the carried phage. J. Bacterial. BERTANI,
67,696-707.
G. (1957). A dismune mutant of temperate
phage P2. Bacterial.
Proc. (Sot. Am. Bacteriolo-
gists) 1957, 38.
G. (1962). Multiple lysogeny from single infection. Virology 18, 131-139. BERTANI, G., and SIX, E. (1958). Inheritance of prophage P2 in bacterial crosses. Virology 6, 357381. COHEN, D. (1959). A variant of phage PZ originating in Escherichia coli, strain B. Virology 7, 112126. JACOB, F., and ADELBERG, E. A. (1959). Transfert des BERTANI,
OF PZ RD L
387
caracteres gCnCtiques par incorporation au facteur sexuel d%scherichia coli. Compt. rend. acad. xi. 249, 189-191. KELLY, B. (1963). Localization of PZ prophage in two strains of Escherichia coli. Virology 19, 3239. SIX, E. (1959). The rate of spontaneous lysis of lysogenic bacteria. Virology 7,328-346. SIX, E. (1966). Prophage substitution and curing in lysogenic cells superinfected with hetero-immune phage. J. Bacterial. 80,728-729. SIX, E. (1961a). Inheritance of prophage P2 in superinfection experiments. Virology 14, 226-233. SIX, E. (1961b). Attachment site specificity of temperate coli phage P2. Bacterial. Proc. (SOL Am. Bacteriologists)
1961,162.
1%
Affinity
m-387
(1963)
of P2 rd I for
Prophage
Escherichia
Sites on the Chromosome
coli Strain
of
C.’
ERICH SIX Department
of Microbiology, Accepted
State
University
November
of Iowa,
Iowa
City,
Iowa
21, 1962
The choice of sites for the establishment of a P2 prophage at the chromosome of Escherichia coli C was studied using P2 rd 1 phages liberated by two donors, one carrying P2 rd 1 at site I, the other at site II. Values for the following parameters were determined: (i) probability of acceptance of superinfecting W vi? by cells lysogenized with W rd 2, (ii) immunity level of cells lysogenized with PR rd 1, and (iii) probability of coexistence of P2 rd 1 prophage with a P2 Hy dis prophage. The results, together with those of bacterial crosses, indicate that phage from a donor carrying P2 rd 1 at site I is strongly restricted to reoccupation of site I, whereas phage from a donor carrying P2 rd 1 at site II is not restricted to reoccupation of either site II or site I. The results also show a correlation between the immunity level of a cell lysogenic for P2 rd I and the site(s) occupied by its P2 rd 2 prophage(s). INTRODUCTION
The temperate phage P2 can establish itself as prophage at different sites on the chromosome of its host, Escherichia coli (Bertani and Six, 1958). In E. coZi strain C these sites or locations are distinguished by means of crosses between F- and F+ derivatives. The three different sites studied so far (locations I, II, III) show characteristic transfer frequencies under the standard selective conditions employed in the crosses and are nonallelic to each other. Recent work by Kelly (1963) indicated linkage between sites I, II, and III and genesinvolved in the synthesis of histidine, methionine, and tryptophan, respectively. The plaque type mutants and immunity variants of Pd that have been studied can occupy the same sites ‘Parts of the work described in this paper were done at the Max-Planck-Institut fiir Biologie (Tiibingen, Germany) under a fellowship of the MaxPlanck Society, and at the Department of Biology, University of Rochester, supported by the United States Public Health Service grant E-2862. At the State University of Iowa the investigation is supported by the United States Public Health Service grant E-4043.
as P2 wild type. Among the different sites, site I is strongly preferred when nonlysogenie cells are infected with P2 and also when cells already lysogenic for Pg are superinfected with either a homoimmune or a heteroimmune P2 variant (Bertani and Six, 1958; Six, 1960, 1961a). Other sites (II, III) were first recognized in doubly lysogenic cells, which carried one prophage at site I and one at II or III. From these doubly lysogenic strains, singly lysogenic strains carrying a prophage only at II or at III can be obtained either by bacterial crosses (Bertani and Six, 1958) or by partial curing of the doubly lysogenic cells upon superinfection with a heteroimmune phage (Six, 1960). Three explanations have been offered (Six, 1961a) for the observed preference for site I: (1) differences in the affinity of the various sites for P2; (2) existence in the P2 lysates of phages with different site specificities; and (3) a combination of (1) and (2).
The purpose of this paper (see also Six, 1961b) is to distinguish among these explanations by comparison of the “lysogenization patterns” (that is, of the relative affini375
376
ERICH
ties for different sites) of P2 phagc derived from two different donors: strain C-208 = C (P2 rd I) I and strain C-86 = C (P2 rd 011. It will be shown that phage from donor C-86 does not exhibit the preference for site I that phage from C-208 shows. This excludes the possibility that there is-with respect to the site affinity-only one kind of P2 phage and that the lysogenization pattern is controlled solely by the affinity of these sites for Pd. Because an analysis of lysogenization patterns based only on bacterial crosses would be impractical, lysogenization patterns have been investigated using one or more of the following criteria, based on properties of lysogenic cells that depend on the prophage site occupied : 1. The probability of adding as a prophage a superinfecting phage known to prefer site I without loss of a preexisting prophage, is lower if site I is already occupied than if it is free (Six, 1961a). 2. The degree of immunity of a lysogenic cell, measured by challenge with an intermediate virulent mutant of P2, is low for cells carrying a P2 prophage at site I only, high for cells carrying P2 prophages at both sites I and II, and intermediate for some other cases (for example, P2 at site II only). 3. It can be assumed that a site is never occupied by more than one P2 prophage at a time since in all known doubly lysogenic derivatives of E. coli strain C the two prophages can be separated in bacterial crosses (see also Table 7). Therefore, if superinfection of a lysogenic cell carrying a prophage at site N (N = I, II, etc.) leads to establishment of a second prophage without loss of the first one, the second prophage presumably occupies a site *N. Bacterial crosses have been used only to verify the interpretation of results obtained by the above methods. MATERIALS
AND
METHODS
Phages. The weak virulent mutant P2 virl, the intermediate virulent mutant P2 G-1 21irl3, and the strong virulent mutant P2 vir3 have been described earlier, together with the general techniques and media employed in the study of phages of the P2 group (Bertani, 1954, 1957; Six, 1961a). For
SIX
phage P2 rd 1 spontaneous lysa,tes were prepared by growing cultures of the donor strains C-208 (here called strain A) or C-86 (strain B) in L broth or nutrient, broth to reach ca. 5 x lo7 cells per milliliter. The cells were then killed by adding streptomycin (followed in most experiments by chloroform) and were removed by centrifugation. The supernatant fluids were stored in the refrigerator. Bacteria. The nonlysogenic streptomycinresistant indicator strain Sh/s of Shigella dysenteriae and its two lysogenic derivatives Sh-76 (carrying P2 Hyl dis) and Sh-77 (carrying P2 wild type) have been described (Bertani, 1951; Six, 1961a). All derivatives of E. coli strain C used in the experiments are listed in Table 1. Most of them have been described before. Testing for prophages was done by assaying the free phage liberated by growing cultures of lysogenic cells. The cells were killed by adding streptomycin or chloroform or by heating to 58”, then a drop of culture was placed on L agar plates seeded w&h suitable indicator strains. Bacterial crosses were carried out as described by Bertani and Six (1958). Selected markers were: arginine and tryptophan independence (from the F+ parent) and streptomycin resistance (from the F- parent). Bacteria from recombinant colonies were streaked before testing them for prophages. The number of recombinants tested in a cross varied from 58 to 240; in 69 of the 74 crosses it ranged between 80 and 120. Occupation of site I by a P2 rd 1 prophage was determined by testing for allelism with a prophage known to occupy site I in the cross partner. In some cases occupation of site II was determined in the same way. The precision of such test is lower for site II than for site I because the transfer frequency from the F+ parent is lower for site II: according to Bertani and Six (1958) the average transfer frequency is 41% for site I and only 15% for site II. Also, the separability from site I of an unknown site X + I occupied by P2 rd 1 was determined as the proportion of recombinants that have not received site S from the F+ parent among those recombinants that have received site I from the F+ parent. If the frequency was less than 50%.
PROPHAGE
SITE
TABLE
377
OF Pd RD L
AFFINITIES 1
STRAINS OF E. coli USED” Strain C-208
C-86 C-2 C-8 c-is c-29 C-62 C-70 c-79 C-83 C-86
Prophage
P2 rd 1 P2 Id 1
Site
P2 Hyl dis Pbc P2 Hy’ dis P2 c P2 Hyl dis
Use
Origin
I
F+ F+ F+ FSr; FS”; F+ Ss; F+
I
S”; F+
Donor A Donor B For crosses Recipient For crosses For crosses Recipient, for crosses F or crosses
I
Sr; F-
F or crosses
From expt. no. 5, Six (1960)
S”; F’ Sr; F+
Recipient Recipient
Six (1959) Spontaneous
I II
None None
P2 P2 c PI Hy’ dis
Pertinent characteristics
I I
S”; S”; S”; Sr;
Six (1961a) Bertani (1962) Bertani and Six Bertani and Six Bertani and Six Bertani and Six Six (1959)
(1958) (1958) (1958) (1958)
Six (1959)
II
II II
None
mutation
of
of C-8 of C-8 mutation
of
c-2 C-218 c-220 G-211
PB Hy’ dis P2 Hy* dis
C-227
P2 rd 1
C-228
P2 rd 1 P2 rd 1
I I
None
6’1; FSr; FSr; F-
I
Sr; F-
I
13; F-
II
Recipient Recipient Host for tion of sates Immunity standard Immunity standard
producplate lylevel level
Lysogenization Lysogenization Spontaneous C-lb Lysogenization expt. no. 2 Lysogenization expt. no. 2
of
C-8 in
of
C-8 in
Q All prophage sites were confirmed by bacterial crosses testing for allelism with previously lished sites I and II. SB; Sr: Streptomycin sensitive, streptomycin resistant F-; F+: Mating types rd; 1; c: Plaque morphology markers; round; large, and clear, respectively. b C-l is original strain C (see Bertani and Six, 1958).
the site X was classified as belonging to group C (= close to I) ; if the frequency was higher than SO%, as belonging to group D (= distant from I). According to this classification site II belongs to group D. In all these crosses the F- parent was a clone lysogenized with P.2 rd 1. If the F- parent did not also carry PW Hy dis, site I was marked by a P2 Hyl dis prophage in the F+ parent (C-62). If the F- parent was lysogenic for Pf? Hyl dis it was assumed that this prophage still occupied site I and the cross was performed with a nonlysogenic F+ parent (C-2). The distinction between clones C (Pd rd l), (PZrd I)x and C (-)I (PZrd Zjx in a cross with a partner carrying a prophage at site I depends on the absence or presence of nonlysogenic recombinants. If site X is very
estab-
close to I (X = C) such recombinants might be missed, and this would lead to a misclassification of a C (-)I (PZ rd Z)c clone as C (PdrdZ)I (P2rdZ)c. The “immunity” level of cells lysogenic for PZ rd 1 was studied by spotting a loopful of the Iysogenic cultures on a L agar plate seeded with lo7 to lo* Pb vi+ viP3 phages. The degree of lysis exhibited on the spot after incubation allowed a rough classification int,o 3 to 5 immunity leveis ranging from “low” to “high.” Strains C-227 and C-228 served as standards for Yaw” (or: “low 1”) and “high” immunity levels, respectively. [See Bertani and Six (1958) for a comparable test on lysogenic Shigella strains.] Media. Bacteria were grown in L broth?
378
ERICH
SIX
which contains 10 g tryptone (Difco), 5 g tion about the lysogenization pattern since yeast-extract (Difco), 10 g NaCl, and 1 g P2 vir’ presumably prefers site I. [This asglucose per liter of water. When noted, nu- sumption was not tested by bacterial crosses,but it was shown that the probabiltrient broth (Difco) was used. L agar and LO agar contain L broth and ity of adding P2 vi+ as prophage to a cell 1% agar; L agar contains also 2.5 X 10W3M already carrying a P2 prophage is much lower if site I is occupied than if it is free CaClz . (Six, 1961a).] H ence, cells carrying both RESULTS P2 rd 1and P2 vi+ should be less frequent if P2 rd 2 itself prefers site I. 1. Direct Infection with Spontaneous LyIn one control experiment, cells of strains sates A and B were infected with P2 vi9 under Since P2 is a noninducible phage, one can- conditions similar to those of the experinot obtain directly from the lysogenic do- ments described below. For both strains nors high titer lysates than can infect a ma- about 83% of the cells survived and there jority of recipient cells. Hence, the low titer was no indication that P2 virl would select spontaneous lysates obtained from donors for or against cells carrying P2 rd 1 at site A (= C-208) and B (= C-86) were used to I. Under the same conditions less than infect nonlysogenic recipient cells at low 10P3% of nonlysogenic cells of strain C-85 multiplicities, and the not lysogenized cells survived. In experiment no. 1 the recipient cells were then eliminated by infection with the from a growing culture of nonlysogenic weak virulent P2 vir’ at high multiplicity. This second infection can furnish informa- strain C-85 were suspended in spontaneous lysates of donor strains A and B to give a cell titer of about 5 x lo7 per milliliter. The TABLE 2 phage to cell ratio was ca. 5 x lo-” for lyACCEPTANCE OF P2 vi+ BY CELLS LYSOGENIZED sate A and 2 x 1O-3 for lysate B, respecWITH P.9 rd 1 FROM Two DIFFERENT DONORS tively. CaC12was added (final concentration 1 j 2 Experiment no.. . . . .j of 5 x 1O-3 M) and the mixtures were incubated for 30 minutes at 37” to allow phage C-85 C-8 Recipient.. . . . . . . . . . adsorption. The infected cultures were then diluted 1: 100 in 20 ml of nutrient broth conDonor’.. . .... . A B A B taining streptomycin, and incubated at 37” ~--with aeration for 2 hours to reach cell titers Numbs; of survivors of ca. 4 X lo6 cells per milliliter. CaClz was 38 99 94 . . . . . . . . . . . . . . . 40 then added (5 x 1O-3 M), and O.l-ml portions of the cultures were mixed with 0.1 ml Number of lysogenic survivors.. .~T~,/,~T of a P2 vi+ suspension (titer: 5 X lo8 per milliliter), incubated at 37” for 10 minutes Prophages carried in to allow adsorption of the phage, and plated subclone pairs from Frequency among lysogenic on L agar (agar layer method). Colonies the survivors survivors (in 70) were isolated, streaked on nutrient agar PZ vi+ PZrdl plates, and 2 subclones from each streak were tested for prophages. The results, m o/2 94 376 91 45 shown in Table 2, indicate that cells lysogew o/2 0 0 5.5 3 nized by lysate B give a much higher fre5s l/2 6 19 5.5 9 quency of addition of P2 vi+ than cells 2/2 w 0 44 0 43 lysogenized by lysate A. Experiment no. 2, also included in Table a A: C-808 = C(P2 rd Z)i ; B: C-86 = C(P2 2, was conducted in a similar way. Strain rd 1111 . C-8, nonlysogenic, was used as recipient. b Including one isolate with one subclone carrying P2 rd 1; the other P2 f-t. Again, cells that received their P2 rd 1
PROPHSGE
SITE AFFINITIES
OF P2 RD L
379
TABLE 3 from lysate B showed a higher frequency of P2 wit-l prophages than cells whose P2 rd 1 IMMIUNITY LEVELS OF C-8 CELLS LYSOGENIZED WITH Pd rd 1 AND SUPERINFECTEDWITH Pd came from lysate A. The nonlysogenic survirl (EXPERIMENT NO. 2) vivors were all found to be completely P2 resistant (= resistant to P2 vir3), Frequency of The results of experiments nos. 1 and 2 immunity levels indicate that phage P2 rd 2 from donor B is (in %I I ‘rophages Lysate” nb -less restricted to site I than is phage from carried donor A. I JOW Internediate High In experiment no. 2 one subclone from ___-each of the pairs of lysogenic isolates that 0 1 P2 rd 1 A 81 99 had given identical results (see Table 2) P2 rd 1 and 0 0 0 was also tested for immunity level by chalPb vi+ lenge with P2 vi+ vir13. Among 81 such clones lysogenized by lysate A, 80 showed a 3.5 2 B PR rd 1 46 57 low immunity level; instead 7 out of 57 3.5 3.5 Pt rd 1 and 42 clones lysogenized by lysate B (= 12%) P2 vi+ - showed intermediate or high immunity levels. These results, shown in Table 3, suggest a A: donor C-208 = C(P2 rd l)~ ; B: donor C-86 = C(P2 rd I)II . a difference in the lysogenization pattern b n: Number of clones with two identical subbetween phages from donors A and B, presumably a higher tendency of phage from clones. donor B to form double lysogens as compared with phage from donor A.2 ceived their P2 rd 1 from donor B. The presFor five clones from experiment no. 2 car- ence of P2 vi+ complicates the interpretarying only P2 rd 1, the P2 rd 1 sites were tion of the observed transfer frequencies for studied by crossing with strain C derivatives the P2 rd 1 sites. In particular it can be asC-29, C-62, and C-70, carrying prophages sumed that recombinants carrying only P2 at site I or at I and II. Two low immunity vi+ or P2 vi+ together only with P2 Hyl dis level clones both were found to be C (P2 rd cannot be recovered. Therefore, classifica1)I. One of them had been lysogenized with tion of sites different from I as C or D can donor A phage, the other (C-227) with do- only be tentative. One low immunity level nor B phage. One intermediate immunity clone appeared to carry P2 rd 1 presumably level clone lysogenized by donor B phage at a site C, another presumably at a site D. was doubly lysogenic, carrying P2 rd 1 pro- A high immunity level clone was found to phages at sites I and C. Two high immunity carry one P2 rd 1 at site I and a second P2 level clones were also found to be doubly rd 1 at a site D different from II. lysogenic carrying P2 rd 1 prophages at sites These results agree with, and lend further I and II. One of them had been lysogenized support to, the assumption that in most of with donor A phage, the other (C-228) with the clones that carry P2 vi+ the P2 rd 1 is donor B phage. Three more clones from ex- established at a site different from I, and periment no. 2 carrying P2 rd 1 and P2 virl that in most clones without P2 virl, site I is were analyzed in the same way. All had re- occupied by a P2 rd 1. In summary, the evidence obtained in the * One may ask whether, in the four isolates that) direct infection experiments leads to the folshow the presence of P2 wilj and have intermediate lowing conclusions: P2 rd 1 derived from or high immunity levels, the P2 vi? contributes to site I (donor A) has a strong tendency to the higher immunity level. This is unlikely, beseek again establishment at that site; this cause the data of Table 3 do not reveal any corredoes not exclude a rare occupancy of a diflation between presence of P2 vi? and level of immunity, and because P2 vi? is a mutant that has ferent site. P2 rd 1 derived from site II (dolost the ability to produce immunity substance nor B) does not have such a strong preference for site I; whether it has any preference (Six, 1961a).
380
ERICH
for site II cannot be decided from these experiments.
SIX
among clones lysogenic for P2 rd 1. This parameter will be called the probability of coexistence with a heteroimmune prophage established at site I (symbol P,). 2. Plaque Analysis Experiments Usually strain C-218 was chosen as reGeneral procedure. Further information cipient since it yields larger plaques than on lysogenization patterns was sought using the other strains. The results, summarized a different experimental approach. Phage in the upper part of Table 4, show that P2 rd 1 liberated by donor A (= C-208) or P2 rd 1 stemming from donor B (average B (= C-86) was used to produce plaques on Pr = 64%) has a higher probability of coon a lawn of recipient cells. From these existence than P2 rd 1 from donor A (averplaques, bacterial clones lysogenic for P2 rd age PI = 7%). 2 were isolated. Presumably, these “plaque In 11 other experiments (to be reported clones” had been lysogenized with P2 rd 1 elsewhere) Pr was determined for donor A only after the phage liberated by the donor phages and similar values were found as in had undergone several rounds of lytic re- the experiments detailed in Table 4. Lysogenization of strains C-220 (experiproduction in the recipient cells. The plaque clones were then tested for immunity level, ment no. 3b) and C-62 (experiment nos. 8a, 9a) with P2 rd 1 led to somewhat different or for the presence or absence of a preexistvalues for Pi ; yet, a higher value for Pi was ing heteroimmune P2 Hy dis prophage (if again obtained for phages from donor B the recipient had been lysogenic for this than for those from donor A (Table 4, lower phage) , or for both properties. In the experiments reported below, phages part). For an interpretation of the PI values, it produced by donors A and B were plated on L agar plates together with recipient cells, must be kept in mind that persistence of mixing 2 drops of a fully grown culture of P2 Hy dis at site I indicates establishment the recipient strain with one or two drops of of P2 rd 1 at a different site, but that loss of spontaneous lysates suitably diluted (usu- P2 Hy dis from site I is not proof that P2 ally 1: 100). Streptomycin was added if the rd I is carried at this site. Hence PI gives only a lower limit for the probability of P2 recipient cells were streptomycin resistant. From the plaques formed on these plates, a rd 1 occupying a site *I. Further tests, to be discussed below, will show, however, that mixture of phage and cells was transferred with a sterile wire to tubes containing 1 ml most of the clones that had lost P2 Hy dis of L broth. After 4-5 hours of incubation at upon lysogenization with P2 rd 1 from donor A were indeed C (P2 rd Z)I. 37”, streaks were prepared on LO agar. From Probability of coexistence of P2 Hy dis at colonies isolated from these streaks, a second set of streaks was prepared, and one site II with P2 rd 1. Since phages from docolony isolated from each of the second-set nor A, but not those from donor B, have a streaks was transferred to L broth and strong preference for site I, does the phage tested for prophages carried. The subclones from donor B show a strong preference for that carried no P2 rd 1 prophage and those site II? This question was investigated by recognizable as unsegregated with respect using recipient strain C-88, which carries P2 Hyl dis at site II. The results of two exto the prophages carried were discarded. Probability of co-existence of P2 Hy dis periments (Table 5) give a negative answer. The probability of coexistence with P2 Hy’ at site I with P2 rd 1. For these experiments a recipient strain carrying a P2 Hy dis pro- dis at site II (symbol Pn) is high not only phage at site I was employed. From each of for P2 rd 1 from donor A, as expected, but the plaques used, one clone lysogenic for also for P2 rd 1 from donor B, and the difference in the Pn values for phages from the P2 rd 1 was tested for presence of the P2 Hy two donors is not significant. Again it should dis prophage. The frequency of establishbe emphasized that loss of P2 Hy dis does ment of P2 rd 1, at a site presumably different from I, could then be estimated at the not prove that P2 rd 1 was established at the proportion of clones still carrying P2 Hy dis site previously occupied by P2 Hy dis.
PROPHAGE
TABLE PROBABILITY
-
OF P2 RD L
SITE AFFINITIES
381
4
OF COEXISTENCE WITH A P.2 Hy dis PROPHAGE AT SITE I (PI) FOR P9 rd 1 PHAGE PRODUCED BY Two DIFFERENT LYSOGENIC DONORS
Lysate A
Lysate B Donor
Recipient
Expt. no.
C-208 = C(P2 rd 1)I N” C-218 C-218 C-218 C-218 C-21 8
3a 4 5 6 7
C-86 = C(P2 rd I)11 N
PI
77
0.08
71
0.08 0.06 0.07 0.02 0.07 0.0-0.08
34 68 47
Overall : 297
PI
_86 66 47b
0.67 0.58 0.62”
118b 31
0.65”
348
Total of 11 other experiments
C-21 8
3b 8a 9a
C-220 C-62
59
0.17
91
50
C-62
51
0.02 0.04
44 27
513
Average 0.04 0.65 0.43 0.26
-
a N = number of clones carrying P2 rd 1 tested. b Combining data of two subclones of C-86 (see text). There was no statistically ence between the PI values obtained for the two subclones. TABLE PROBABILITY
0.68 0.64
significant
differ-
5
OF COEXISTENCE WITH A P2 Hyl dis PROPHAGE AT SITE II (Pri) FOR P2 rd 1 PHAGE PRODUCED BY TWO DIFFERENT LYSOGENIC DONORS
Lysate A
Lysate B Donor
Expt. no.
Recipient C-208 = C(P2 rd I)I
8b 9b
C-83 C-83
C-86 = C(P2 rd 1)~
N”
RI
N
PI1
55 44
0.62 0.50
41 29
0.54 0.55
o N: number of clones carrying P2 rd 1 tested.
Immunity level of plaque clones. In some of the experiments described above (nos. 3a, 3b, 4, 8a, 8b), the immunity level of the clones lysogenic for P2 rd 1 was also determined by challenge with P2 vi+ vir13. This was done also in experiment no. 10, using the nonlysogenic strain C-8 as recipient and employing the same donor lysates as in experiments nos. 8a and b. As shown in Table 6, the difference between donors A and B is now reflected in a difference of the distribu-
tion of immunity levels: in each experiment lysogenization with P2 rd 1 from donor B resulted in a higher percentage of clones with intermediate or high immunity level than lysogenization with P2 rd 1 from donor A. This finding is in qualitative agreement with the results of the direct-infection experiment no. 2 (Table 3). Correlation between immunity level and occupied prophage sites. Experiment no. 2 shows that the immunity level is correlated
382
ERICH
SIX
TABLE DISTRIBUTION
OF IMMUNITY
LEVELS
LYSOGENIZED
-
WITH
6 AMONG
DESCENDANTS
OF CELLS
PR rd 1 IN PLAQUES
Lysate A
Lysate B Donor
Expt. no.
C-208 = C(PB rd Z)I
Recipient
Frequency of immunity InterInternediate 1 mediate 2 10” 3a 4 8a 3b 8b
C-8 C-818 C-818 C-69 c-110 C-83
0.02 0.08 0.03 0.00 0.15 0.09
C-86 = C(PZ rd l)rr
levels
Frequency of immunity
High
0.04 0.07 0.03 0.02 0.08 0.05
0.03 0.03 0.10 0.04 0.03 0.24
High
0.09 0.17 0.16 0.06 0.27 0.38
0.26 0.17 0.06 0.09 0.18 0.10
-
3 According to Bertani (1962) the presence of a PZ Hy dis prophage tends to lower the immunity level of cells carrying P2. This could explain why in the presence of P2 Hy’ dis at site I the clones carrying P2 rd 1 at a site C show a low immunity level.
0.08 0.05 0.20 0.16 0.11 0.20
0.69 0.47 0.39 0.68 0.49 0.68
-
a Number of clones carrying Pi? rd 1 tested: 98 for lysate A, 97 for lysate b Sum of three preceding frequencies (intermediate 1, 2, and high).
with the number and possibly also the site of the P2 rd 1 prophages carried by the clones. The nature of this correlation was studied by analyzing in bacterial crosses the P2 occupied sites of 48 clones obtained in experiments nos. 3a, 4, 8a, and 10. The results of these crosses, together with those for 4 clones obtained in experiment no. 2 and not carrying P2 zG1, are summarized in Table 7. If the immunity level was low and no Pt Hyl dis prophage was present, P2 rd 1 was always present at site I. In five clones with a low immunity level and lysogenic for P2 rd 1 and P2 Hyl dis, the P2 rd 1 prophage was present at a site *I, that is, was separable from the P2 Hyl dis prophage in a cross with C-2. Intermediate or high immunity levels were always indicative of the presence of a P2 at a site *I, either alone or if the immunity level was high, together with another PW prophage which as a rule occupied site I.3 These results indicate that the immunity level serves as an indicator for occupation
Not 10~5
~~
I
0.35 0.24 0.14 0.43 0.21 0.39
levels
B.
of a site +I by P2 rd 1, at least in the absence of a P2 Hy dis prophage. Accepting this conclusion the difference found in experiment no. 10 (Table 6) between phages from donor A and B is again recognized as a difference in their ability to establish a prophage at a site +I, as in the other experiments. This agreement supports the proposed interpretation of the immunity level. Evaluation of the prophage coexistence test. The prophage coexistence tests were based on the assumption that addition of P2 rd 1 as a prophage without loss of a preexisting P2 Hy dis prophage indicates that P2 rd 1 occupies a different site than does P2 Hy dis. This assumption was confirmed by the bacterial crosses reported above. It seemed reasonable further to assume that loss of P2 Hy dis was caused by P2 rd 1 replacing it at the same site, leading to C (PC rd 1)I if the recipient was formerly a C (P2 Hy disJI The study of the immunity levels, together with the cross analyses, shows that this interpretation is valid only for clones with a low immunity level. 39 clones carrying P2 rd 1 but not P2 Hy dis were analyzed (see Table 7). All 17 low immunity level clones were C (P2 rd 1)I . Of these clones, 11 were obtained by replacement of a P2 Hyl dis previously occupying site I in recipients
PROPHAGE
SITE AFFINITIES TABLE
OF P8 RD L
383
7
RESULTS OF THE BACTERIAL CROSSEST
-
Number of cloneswith the indicated lysogenic constitution Img;‘ty
Low Intermediate 1 Intermediate 2 High
Donor
Site
Prophages
PZ rd 1 PZ rd I PZ Hy’ dis PZ Hyl dis I. . . . PZ rd 1 PZ Hyl dis P2rd 1 P2rdl PZrdl P2rdl C...... P2rdl P2rdl P2rdl P2rdl D...... ____~ 8 A B 9 5 __----__ ---~ __---_______ -1 1 A 1 2 1 B --_________ __-__________--___ 2 2 2 A 2 3 6 B ___-____ __-___2 A 1 1 3 B
II . C: sites close to I, but C * I (see under Material ~A:C-~O~=C(P??T~Z)~.B:C-~~=C(PB~~Z) and Methods). D : sites distant from I (including site II) (see under Material and Methods). The table summarizes the results for 52 clones isolated in experiments nos. 2 (only clones not carrying P2 sir’), 3a, 4, 8a, and 10, analyzed in F- X F+ crosses with respect to the P2 rd 1 sites occupied. In experiments nos. 2 and 10 a nonlysogenic recipient, C-8, was used: in the other experiments, recipients C-68 or C-818, both carrying P2 Hyl dis at site I. The lysogenic constitutions indicated are the simplest ones consistent with the data. The possibility cannot be excluded that instead of one P2 rd 2 prophage at a site C there are two (or more) at different sites C or that instead of a P2 rd I only at a site D there are also others at sites D or C. Sites C. The three clones C(P2 rd Z)o (all from experiment no. 10) were crossed with C-62. Nonoccupation of site I was indicated by finding 2, 7, and 8 nonlysogenics respectively among 100 recombinants tested in each cross. In crosses involving 15 clones carrying a P2 rd I at a site C, a fraction s15y0 of the recombinants showing transfer of site I did not show also transfer of site C. For one other clone this fraction was 29%. For two other clones [one clone C(P2 Hyl dis)I (P8 rd Z)o with immunity level “intermediate 1” and one clone C(P2 Hyl dis)r (P2 rd Z)c (PR rd Z)n] sites could only tentatively be classified as C. In these cases the classification was based solely on the fact that the transfer frequency of these sites was similar to that expected for site I. Sites D. None of the clones classified as C(P2 rd l)D belonged to experiments 2 or 10. For six clones from other experiments [2 clones C(P2 Hyl dis)I (P2 rd Z)Dand one clone C(P2 rd Z)n with immunity level “intermediate 2 ” 2 clones C(P2 rd Z)r (P2 rd Z)Dand one clone C(P2 Hyl dis)r (P2 rd Z)C (P2 rd Z)n with high immunity level] allelism of site D with site II was tested, and in all cases the results are compatible with D = II. Two of these clones had received their P2 rd 1 from donor A. In crosses involving the 12 other clones carrying a P2 rd Z at a site D, 67% or more of recombinants showing transfer of site I did not show also transfer of site D. Site of P2 Hyl dis. For six clones [two clones C(P2 Hyl dish (PI rd Z)C, three clones C(P2 Hyl dis)I (P2 rd Z)D , and one clone C(P2 Hyl dis)I (P2 rd Z)c (P2 rd Z)n] allelism of the P2 Hyl dis site with site I was tested and confirmed, as expected, since these clones were obtained by superinfection of a C(P2 Hyl dis)I with P2 rd 1. One of these clones had received P2 rd Z from donor A. C-66 or C-218, the remaining 6 by occupation of site I in the nonlysogenic recipient C-8. Among 22 clones with a higher immunity level, 17 were doubly lysogenic carry-
ing one Pb rd 1 at site I and another at a different site. Six of these clones were obtained from recipient C-218, 11 from C-8. In the remaining 5 clones site I remained unoccu-
384
ERICH
SIX
TABLE
8
IMMUNITY LEVEL AND RETAINMENT LYOSOGENIZED
Donor .
..
WITH
OF P9 rd
Ps?Hyl
clis OF 1 IN PLAQUES’”
C-d18 CELLS
I
...
Intermediate
Immunity level.
1 ! 1
Expt. no.
PZ Hy’ dis
3a
No Yes
4
No Yes
High
2
Frequency of clones
present
-__
Intermediate
__--__
0.80 0.000.0140.0140.09
Pt
AP
__o.20
0.0140.030.0140.0140.014
a A: C-208 = C(PB rd 1)r.B: C-86 = C(PB rd Z)rr. The “low immunity” class is here split into two subclasses: “low 1” and “low 2,” the immunity level of “low 2” being a little higher, as judged from the degree of bacterial growth in the presence of PB vir1virr3, than that of “low 1.” Clones with a low immunity level carrying Pa rd I and PR Hyl dis usually belong to subclass “low 2.” Pt = frequency of clones not showing a low immunity level and/or carrying PR Hyl dis (presumably at site I) besides a P2 rd 1. AP = Pt - Pr (Pr = probability of coexistence of P2 rd 1 with a PB Hyl dis at site I). AP gives the fraction of clones not recognized as carrying a PZ rd 1at a site *I if only the coexistence test is applied. The data presented in this table correspond to those shown in Tables 4 and 6.
pied and P2 rd 1 was carried at a different site. Two of these clones were obtained from recipients C-62 or C-218, 3 from C-8. The best available estimate of the fraction of clones carrying P2 rd 1 at a site +I may be obtained by combining prophage coexistence and immunity level tests to give the joint probability Pt for either coexistence with P2 Hy dis (at site I) or for establishment of a strain of intermediate or high immunity level. Table 8 shows that most clones that have lost P2 Hyl dis upon lysogenization with P2 rd 1 from donor A, possess a low immunity level and are presumably all C (P2 rd Z)i . Hence, it appears justified to interpret the low Pi values obtained for phage from donor A as reflecting the strong preference of such phage for prophage site I. Homogeneity tests. The values for PI (the probability of coexistence of P2 rd 1 with a P2 Hy dis located at site I) in the preceding section are average values for the spontaneous lysates. The question arises whether these lysates are homogeneous with respect to preference for prophage site I. If the ly-
sates were not homogenous, PI might reflect only the relative frequencies of phages with strong preference for site I and phages with less or no preference for site I. The homogeneity of some of the donor lysates used in the experiments already described was studied by obtaining ten clones from the same plaque for a number of plaques and testing these “sister clones” for prophages carried. The results of this sister-clone analysis (presented in Fig. 1) gave no indication of a heterogeneity of donor A lysates, but for donor B lysates at least two components could be distinguished: one component apparently had the same preference for site I as donor A phage, the other had much less preference, if any, for this site. A heterogeneity of lysates B might reflect a heterogeneity of the cells of donor strain B (= C-86). In testing this hypothesis two subclones were isolated from C-86: one formed colonies of the usual size, the other produced smaller colonies on L agar. The Pi values were determined in the fashion already described in two experiments, nos. 5 and 6. The P, values obtained for the two subclones of
PROPHAGE
SITE AFFINITIES
OF PZ RD L
385
tion, the plate lysates were used to infect growing cells of strain C-85 at a multiplicity of about 5. The infected cell suspension were diluted 1: 100 into nutrient broth and S. Investigation of Plate Lysates incubated at 37” for 4 hours. About lo6 cells The results of the analysis of plaque of the infected cultures were then superinclones imply a certain stability of the de- fected with P2 vi+ and plated onto L agar gree of affinity to site I during lytic growth plates in the same manner as described for of the phage. One would then expect that experiment no. 1. A total of 80 colonies were plate Iysates obtained from the spontaneous examined for prophages carried in two sublysates of donors A and B would still reflect clones. Of 40 clones lysogenized by lysate the difference observed between the spon- A, none had accepted P2 vi+; of 39 clones lysogenized by lysate B, 8 carried P2 virl taneous lysates. The spontaneous lysates used for experi- in both subclones tested, 2 more in only one ment no. 1 were also used to produce plate subclone. In experiment no. 11 strain C-211 was lysates by plating 0.1 ml portions of the unused as host for the preparation of the plate diluted spontaneous lysates together with lysates. The Pr values of these lysates were Sh/s cells on L agar plates. After sterilizadetermined by plating with cells of C-218. The values obtained, 0.04 for lysate from A 25 and 0.63 for lysate from B, lie within the r range of PI values found for the corresponding spontaneous lysates. This indicated that the additional rounds of lytic multiplication of the phages in host C-21 1 had not significantly altered the degree of the affinity of the phages for prophage site I.
donor B did not differ significantly from each other nor from the Pi values determined for donor B itself.
DISCUSSION
Different lines of evidence presented in this paper all lead to the same conclusion: there is a difference in the lysogenization pattern of P2 rd 1 phages depending on the IOL( I I I I I I I lysogenic donor strain used as source of the 3 4 5 6 n= 0 I 2 phage. This difference may be described as FIG. 1. Histogram showing the frequency N of follows: P2 rd 1 from donor C (P2 rd ZJ1has groups of n clones carrying both P2 rd 1 and PS a strong preference for establishing as proHy’ dis among six sister clones isolated from the phage at site I of recipient cells; P2 rd I same plaque and carrying P% rd 1. From individual P2 rd 1 plaques (on a C-218 from donor C (P2 rd Z)n shows neither this preference for site I nor a comparable preflawn) that had already yielded a clone carrying erence for site II. P2 rd 1 when the PI values for the spontaneous The difference between phage from the lysates of donors A (= C-208) and B (= C-86) were determined, ten more sister clones were iso- two donors appears to be one of degree: lated and tested. The histogram shows the numphage from C (P2 rd Z)i may establish a ber N of plaques which, among the first six sister prophage at sites *I (including II), but occlones found to carry P2 rd 1, yielded n clones car- cupation of these second-choice sites both rying also P2 Hy’ dis (0 I n 4 6). close to I (= C) and distant from I (= D) The clone used for the previous Pr determinais observed more often for phage from C (P2 tion was not included, but the results for plaques that had originally yielded a clone carrying P2 rd 1 rd 2)n. The latter phage can also choose and P2 Hy’ dis are shown in dark shading (13 sit.e I for prophage establishment. Judging from the distribution of immunity levels plaques for A, 14 for B), those having originally yielded a clone carrying only P2 rd 1 in light shad- among clones lysogenized with this phage ing (13 plaques for A, 12 for B). and the proposed correlation between im-
386
ERICH SIX
munity level and P2 rd 1 sites it may tentatively be concluded that phage from C (PW rd Z)rr has retained some preference for a certain region on the recipient chromosome, namely the region including sites I and C, but lacks the high specificity for site I. The experimental evidence is also compatible with the following hypothesis, The region that appears to be preferred by phages from both donors is the only one available for establishment of a single prophage in previously nonlysogenic cells; sites D are occupied by P2 rd 1 only in doubly lysogenic cells carrying also a P2 rd 1 prophage at site I, or in cells which, prior to the establishment of a P2 rd 1 prophage, already carried at site I at PW Hy dis prophage, which was either retained or lost upon infection with P2 rd 1. This hypothesis agrees with observations by Bertani (1962) concerning occupation of site II. The question remains why Bertani, using a somewhat different experimental method, did not also find any clones carrying P2 rd 1 at a site C only. The nature of the difference observed between phages produced by the two donors remains unknown. At present it can be stated only that the phages themselves must carry a determinant for site affinity associated with their genetic material. The difference in site affinity persists during phage multiplication in plaques and in plate lysates, that is, during replication in hosts that do not possess whatever modifying factor is contained in the donor strains. Any such factor must become genetically associated with the phage prior to the spontaneous lysis of the donor cells. The only other pertinent information obtained is that phages from C (P2 rd Z)r appear to be uniformly restricted to site I. The observed inhomogeneity of phages from C (P2 rd Z)n might be an artifact caused by the presence of a P2 Hy dis prophage in the recipient. Experiments in progress employing different bacterial strains lysogenized with different P2 variants should help to clarify the role of the donor and to answer the question whether occupation of site II, as opposed to occupation of site I, is a necessary or suffi-
cient condition to produce the difference between phages from C (PW rd 1)r and C (P2 rd
UII
.
It may be noted that P2 rd I, and possibly all P2 variants, are unlike other temperate phages studied so far, such as lambda and Pl, in that they may exist in both a siterestricted and an unrestricted or less restricted form. In this respect P2 rd I resembles another episome, the F factor, shown by Jacob and Adelberg (1959) to exist either in a site-restricted or a site-unrestricted form. Previously (Six, 1961a) a study of the establishment of P2 c as prophage in cells C(-),(P2 rd Z)II and C (P2 Hy dis)I (P2 rd Z)n had indicated a strong steric hindrance by the P2 Hy dis prophage against the establishment of P2 c, presumably at site I. The experiments described in this paper do not reveal any appreciable steric hindrance by P2 Hy dis in recipients C (P2 Hy dis), for establishment of P2 rd I at site I. Phage from C-208 shows a strong preference for site I even if this site is occupied by a P2 Hy dis prophage, and phage from C-86 shows a decreased preference for this site regardless of its occupation. Presumably, the lack of immunity to P2 in recipients C (P2 rather than the difference in the HY dis11, markers of the infecting phage, is responsible for the lack of steric hindrance in t,he present case. The apparent lack of steric hindrance in nonimmune recipients may have several causes : (1) the availability of a large number of preprophages, due to multiplication of the genome of the infecting PW rd 1 in nonimmune (but not in immune) cells (and due also to multiple infection in plaques) ; (2) a removal of the P2 Hy dis prophage from site I by a “curing” effect resulting from infection with P2 rd 1; establishment of P2 rd 1 as prophage could then occur secondarily in the freed site I. Curing of lysogenic cells by superinfection with heteroimmune phage has been observed (Cohen, 1959; Six, 1960). REFERENCES G. (1951). Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escheri&a coli. J. Bacterial. 62,293-300.
BERTANI,
PROPHAGE
SITE AFFINITIES
BERTANI,G. (1954). Studies on lysogenesis. III. Superinfection of lysogenic ShigeZZa dysenteriae with temperate mutants of the carried phage. J. Bacterial. BERTANI,
67,696-707.
G. (1957). A dismune mutant of temperate
phage P2. Bacterial.
Proc. (Sot. Am. Bacteriolo-
gists) 1957, 38.
G. (1962). Multiple lysogeny from single infection. Virology 18, 131-139. BERTANI, G., and SIX, E. (1958). Inheritance of prophage P2 in bacterial crosses. Virology 6, 357381. COHEN, D. (1959). A variant of phage PZ originating in Escherichia coli, strain B. Virology 7, 112126. JACOB, F., and ADELBERG, E. A. (1959). Transfert des BERTANI,
OF PZ RD L
387
caracteres gCnCtiques par incorporation au facteur sexuel d%scherichia coli. Compt. rend. acad. xi. 249, 189-191. KELLY, B. (1963). Localization of PZ prophage in two strains of Escherichia coli. Virology 19, 3239. SIX, E. (1959). The rate of spontaneous lysis of lysogenic bacteria. Virology 7,328-346. SIX, E. (1966). Prophage substitution and curing in lysogenic cells superinfected with hetero-immune phage. J. Bacterial. 80,728-729. SIX, E. (1961a). Inheritance of prophage P2 in superinfection experiments. Virology 14, 226-233. SIX, E. (1961b). Attachment site specificity of temperate coli phage P2. Bacterial. Proc. (SOL Am. Bacteriologists)
1961,162.