Molec. gen. Genet. 156, 297-302 (1977) © by Springer-Verlag 1977

Restoration of Immunity in Lysogens Carrying Prophage P2, Derepressed at High Temperature

Eva Nilsson and L. Elizabeth Bertani Microbial Genetics Laboratory, Karolinska Institutet, S-104 01 Stockholm, Sweden

Summary. The possibility that a strain lysogenic for phage P2 could be brought into the so-called "ant i - immune" state in Which the synthesis of phage re- pressor is permanently turned off, was tested in the following way. Two lysogenic strains that could be derepressed at 42°C were prepared. In one, the prophage had, in addition to a temperature-sensitive repressor mutat ion (c5) , a m b e r mutations in the two early genes A and B. In the other, the prophage had an unknown defect that blocked expression of the A and B genes. Both strains could multiply at 42 ° C as well as or almost as well as a non-lysogen. After the strains had grown for several generations at 42 ° C, they were returned to 30 ° and the resynthesis of re- pressor was followed by measuring the restoration of immunity to super-infection. In both cases, the immunity returned slowly over a period of 2 to 3 h. In a strain made doubly lysogenic for two a m A a r a B

c5 prophages, immunity was restored at a more rapid rate, suggesting that the rate of restoration depended mainly on the number of copies of repressor gene present. Attempts to demonstrate "channe l ing" to- wards the lytic pathway in the derepressed lysogens was also negative. The temperature treatment tended instead to increase the frequency of lysogenization of superinfecting P2. Thus, the presence of a system, similar to the cro system described for phage lambda, to regulate repressor synthesis in phage P2 could not be demonstrated.

of the phage. The activity of such a repressor may be antagonized by other phage-coded proteins called antirepressors. Two anti-repressor systems have been described, one in phage lambda (Echols et al., 1973; Reichardt, 1975) and one in phage P22 (Levine et al., 1975; Susskind and Botstein, 1975). These two sys- tems differ from each other in several respects and may serve different functions. In the case of lambda, the anti-repressor is the product of a gene called cro

and it is thought to play a role in promoting the expression of late lytic functions. Mutations in the cro gene result in phage that lysogenize with 100% efficiency. In P22, on the other hand, the entire anti- repressor system can be eliminated with little, if any, effect on either lysogenization or phage multiplica- tion.

The activity of the cro gene in lambda was first demonstrated (Eisen et al., 1970) by cultivating a tem- perature derepressible lysogen at 42 ° C, then return- ing the derepressed bacteria to 30 ° C and observing that they remained in a non-immune (so-called "ant i - immune") state. Restoration of immunity was ob- tained if the prophage had a mutat ion that blocked expression of the cro gene. In order to see if the synthesis of P 2 repressor was regulated in a similar way, we have repeated this experiment using tempera- ture derepressible P 2 lysogens. In all cases examined, the immunity of the lysogen always returned, albeit slowly.

Introduction

All temperate phages described so far code for a reg- ulatory protein, a repressor, that promotes and main- tains the lysogenic state by blocking the lytic functions

For offprints contact: L.E. Bertani

Materials and Methods

Bacteriophages. The temperate phage P2 and many of its derivatives are described in Bertani and Bertani (1971); amber (am) and tem- perature-sensitive (ts) mutations in genes A, B and K in Lindahl (1974) ; deletion mutants P2 vir22 and P2 vir79 in G. Bertani (1975).

P2 areA127 c5, P2 amBl l6 c5, P2 tsB40 virl and P2 tsK60 virl were obtained from G. Lindahl. P2 intl e5 was isolated by crossing P2 tsB40 intl (Lindahl, 1969) with P2 tsD4 c5 (Bertani, 1968). P2

298

Table 1. Bacterial strains used

E. Nilsson and L.E. Bertani: Restoration of Immuni ty in P2 Lysogens

Collection Prophage carried Prophage Origin and Reference number location

C- 1 a None C-I055 None

C- 1278 None C-1497 P2 intl c5 I C-2068 P2 amBl l6 c5 I C-2073 P2 arnA127 c5 I C-2075 P2 arnA 127 arnB116 c5 I C-2096 P2 arnA127 arnBl l6 c5 I C-2097 2 × P2 amA127 arnBll6 c5 I and x C-2098 P2 arnA 127 arnBll6 c5 x C-2099 P2 intl c5 I C-2100 P2 in tl c5 de f4 I C-2801 P2 arnA127 c5 I C-2802 P2 amBl l6 c5 I

Escherichia coli C adapted to grow in synthetic medium (Bertani, 1968) A streptomycin-resistant derivative obtained by mutat ion and recombination

(Wiman et al., 1970) A his-, rnetG- derivative of C- la (Calendar and Lindahl, 1969) By mixed infection of C- la with P2 and P2 intl c5 By infection of C- la By infection of C- la By infection of C- la By infection of C-1278 By superinfection of C-2096 By transduction of C-2097 By mixed infection of C-1278 with P2 and P2 intl c5 From C-2099 (see Materials and Methods for details) By infection of C-1278 By infection of C-1278

Table 2. Complementat ion of temperature-sensitive superinfecting phages in derepressed lysogens ~

Strain Prophage carried Burst size of

P2 tsB40 virl P2 tsK60 virl

C- la None 0.68 2.1 C-1497 P2 intl c5 82 61 C-2073 P2 amA127 c5 116 14 C-2068 P2 amB116 c5 1.1 47 C-2075 P2 arnA127 arnBll6 c5 0.78 8.4

C-1497 P2 intl c5 36 30 C-2073 P2 arnA127 c5 78 8.7 C-2068 P2 arnB116 c5 1.2 34

C-2099 P2 intl c5 52 29 C-2801 P2 arnA127 c5 116 8.3 C-2802 P2 arnBll6 c5 7.7 25 C-2100 P2 intl c5 def4 1.3 11

" Cultures of the indicated strains, growing exponentially at 30 ° C, were concentrated to about l0 s per ml in LB broth contain- ing 5 m M CaC12. Aliquots were placed at 42 ° C and infected with either P2 tsB40 virl or tsK60 virl at multiplicities of 2-6 phage per bacterium. After 10 min, the infected bacteria were diluted 10,000-fold into LB broth at 42 ° C. Phages able to form plaques on the non-permissive indicator C-1055 at 30 ° C were assayed at 60min and 80min after infection. The figures given are based on averages of these two assays

amA127 amBl l6 c5 was prepared by first crossing P2 tsB40 c5 (Bertani, 1968) with P2 arnA127 or P2 arnB116 c5 with P2 tsA54 to obtain P2 amA127 tsB40 c5 and P2 tsA54 arnBll6 c5, respec- tively. The latter two strains were then crossed with each other, selecting for ts + recombinants. The genotype of the double amber recombinants was verified in marker rescue experiments (the wild- type alleles of arnA127 and arnBll6 cannot be rescued), in back- crosses with P2 arnA127 c5 and P2 arnB116 c5 (no am ÷ recombi- nants obtained) and by complementat ion (Table 2).

Bacteria. The bacterial strains used are listed in Table 1. The defec- tive strain C-2100 was obtained by plating C-2099, which carries P2 intl c5, at 42 ° C. It was one of a few survivors that was immune to P2 virl at 30 ° C, but not at 42 ° C. No phage is produced by the strain at any temperature, not even when it has been super- infected with int + phage to supply the missing int ÷ gene product. The original strain can be cured of the prophage by heteroimmune superinfection and the resultant non-lysogen has the properties of C-1278. Thus, the defect is on the prophage.

A number of experiments, in which the defective strain was superinfected with various derivatives of P2 at 42°C indicated that the mutat ions intl and c5 can be rescued from the prophage by recombination, as well as the wildtype alleles of the mutat ions cox4, amA127, arnB116, vir3 and vir24 (in the latter case, assuming that the virulent mutat ions are epistatic to c5; that is, that a recom- binant with c5 phenotype must be vir+). The c5 mutat ion can be rescued from the prophage by all point mutants tested, but not by the deletion mutants P2 vir22 and P2 vir79. Since vir79 is the smaller of the deletions, this suggests that the defect lies within the vir79 deletion, which covers about 1% of the chromo- some in the region of the C gene and includes the c5 mutation. The defect has been designated def4.

The old and fun genes of the C-2100 prophage are active, as judged by the ability of the lysogen to exclude phage lambda (Lindahl et al., 1970) and its sensitivity to 5-fluorodeoxy-uridine (Bertani, 1964). The strain is a poor host for the satellite phage P4 (Barrett et ai., 1974), however, suggesting that the late genes of the prophage are not expressed. The activity of the early pro- phage genes A and B was tested by superinfecting the derepressed strain with phages having mutat ions in either the early gene B or the late gene K and measuring the extent of complementat ion (Table 2). Both P2 tsB40 virl and P2 tsK60 virl are complemented in lysogens that carry P2 intl c5 as prophage. P2 tsB40 virl is also complemented well in lysogens carrying an arnA127 prophage, whereas P2 tsK60 virl is not. This is expected since the transcription of late functions in P2 depends on genes A and B (Lindqvist and Bovre, 1972; Geisselsoder etal . , 1973) and the product of gene A is cis-acting (Lindahl, 1970). Thus, the prophage cannot utilize the gene A product of the superinfecting phage to turn on its own K gene. Results similar to these have been obtained using P2 3"34 virl instead of the K virl mutant . The defective pro- phage is unable to complement either of the superinfecting phages

E. Nilsson and L.E. Bertani: Restoratiou of Immuni ty in P2 Lysogens 299

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Fig. 1. Stability of lysogens at 42 ° C. Cultures of the non-lysogenic strain C-1278 (curve A, crosses), the same strain made singly lysoge- nic (C-2096) (curve B, circles), or doubly lysogenic (C-2097) (curve C, triangles) for P2 areA127 a m B i 1 6 c5, and the defective strain C-2100 (curve D, squares), growing exponentially in LB-broth at 30 ° C, were diluted 1000-fold into the same medium at 42 ° C ( = time 0). The ability of the bacteria to form colonies at 30°C was assayed at the indicated time intervals. DupIicate data are given for C-1278 and C-2100

very well. Since the K gene is far away from the A and B genes, which are adjacent to each other, the defect in the prophage of C-2100 may block transcription of genes A and B and consequently of all other late genes.

Media. Cultures were grown in LB-broth and assays were made on LB-agar (G. Bertani, 1951) using the soft agar-layer technique. Fur ther details are given in the legends.

Results

Irreversible Inactivation of P2 Repressor. When bac- teria lysogenic for P2 c5 are incubated at 42 ° C, they lose their immunity to superinfection and their colony-forming ability, although they produce no phage (Bertani, 1968). Bacterial killing and loss of immunity also occur in the presence of 50 lag per ml chloramphenicol, added 10 rain before the transfer to 42 ° C, and even if the treated bacteria are further incubated for 1 h at 30 ° C in chloramphenicol. Thus, unlike the case for some mutants of lambda (Lieb,

1966), P2 c5 repressor activity cannot be restored in the absence of protein synthesis. We assume then, that restoration of immunity in a derepressed P2 c5 lysogen requires synthesis of new repressor.

Stability o f Lysogens at 42 ° C. In order to look for restoration of immunity in derepressed P2 c5 lyso- gens, it was desirable to have strains that could un- dergo unlimited multiplication at 42 ° C. It was known that the killing of P2 c5 lysogens could be at least partly overcome if the prophage had additional muta- tions in either of genes A or B (Lindahl, 1974). We found, however, that single mutations in either of these genes were not sufficient to maintain unin- terrupted multiplication at this temperature. Bacterial survival was improved by using a lysogen, C-2096, carrying the double amber recombinant P2 areA127 a m B l l 6 c5 as prophage, but the only strain that was found to multiply just as well as the non-lysogen C-1278 was the defective strain C-2100 (Fig. 1). This strain was isolated from a lysogen carrying P2 intl c5, as a derivative able to form colonies at 42 ° C. As described under Materials and Methods, the pro- phage carried by the strain cannot form plaques. The defect appears to be localized to a portion of the C gene and blocks the expression of the early genes A and B of the prophage, although the wildtype alleles of the mutations amA127 and a m B l l 6 are still pre- sent.

The difference in growth rates between strains C- 2100 and C-2096 is also reflected in the ability of the superinfecting phage, P2 virl, to multiply in these strains after they have been derepressed at 42 ° C. There is no restriction on the multiplication of P2 virl in the defective strain. In the amA araB c5 lyso- gen, the proportion of infective centers reaches 100% immediately after derepression, but decreases again somewhat as the bacteria continue to be incubated at 42 ° C. According to one-step growth curves, the latent period of P2 virl in areA araB c5 lysogens that have been incubated at 42°C for 90 rain and then returned to 30 ° C is prolonged by 15 rain as compared to a non-lysogenic host, C-1278. The prolongation of the latent period is, for unknown reasons, even more pronounced for the immunityinsensitive mutant P2 vir3.

Thus, in the defective strain, the superinfecting phage that is used to measure restoration of immunity can multiply unrestrictedly, but the genotype of the prophage carried by the strain is not exactly known, whereas in the other strain, the prophage genotype is known very precisely, but there is some interference with the multiplication of superinfecting phages. The reasons for the difference between the strains will be discussed further in a later section.

300 E. Nilsson and L.E. Bertani: Restoration of Immunity in P2 Lysogens

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Fig. 2. Restoration of immunity in derepressed lysogens. Cultures of C-2096, singly lysogenic in loc I (curve A, circles), C-2097, doubly lysogenic (curve B, triangles) and C-2098, singly lysogenic in an unknown location (curve C, crosses) for P2 areA127 amB116 c5 and the defective strain C-2100 (curve D, squares), all of which had been growing in LB-broth at 42 ° C for 90 rain, were shifted to 30°C at time 0. The bacteria were maintained at 2-4 × 107 per ml by diluting them approximately 2-fold every hour with LB-broth prewarmed to 30 ° C. Samples were taken at the times indicated, concentrated to 1-2 x 108 per ml by centrifuga- tion and resuspended in LB-broth containing 5 mM CaC12. The samples were infected with P2 virl at a multiplicity of 0.2 and plated using C-1055 as indicator. The number of virl plaques ob- tained at 30 ° C, as a proportion of the input virl particles is plotted on the ordinate. Duplicate data are given for some of the strains

Restoration of Immunity in Derepressed Lysogens. Cul- tures of the two lysogenic strains just described, incu- bated at 42°C for 90 rain, were returned to 30°C and samples were taken at various times to measure the proport ion of bacteria sensitive to the challeuge phage (Fig. 2). The bacteria were diluted every hour to keep them growing exponentially. Essentially the same result was obtained with each of the strains. ~During a period of 5 to 6 h, the proport ion of sensitive bacteria decreased from 100% to between 2 and 15% in various experiments. About 50% of the bacteria had regained their immunity after 2 to 4 h at 30 ° C. Samples of bacteria, taken at the end of the experi-

ments were fully sensitive to P2 virl at 42°C and do not represent a genetically changed population. Similar results have been obtained using strain C-la carrying the amA araB c5 prophage.

In the first experiments, the inocula for the 42 ° C cultures were taken from cultures that had grown overnight at 42 ° C. Since these gave the same results as those described in Figure 2 and since there is a selective disadvantage for the amA amB c5 lysogens at 42 ° C, we used inocula grown at 30 ° C in the re- maining experiments.

One interpretation of the above results is that P2 prophage has a cro-like gene product that is not as efficient as that of lambda so that repressor synthesis eventually escapes from its effect. Another possibility is that the slow return of repressor activity simply reflects the normal rate of resynthesis from a pro- phage. It should be possible to distinguish between these alternatives by studying the effect of gene dos- age.

The Effect of Gene Dosage. If the rate at which re- pressor activity returns is normal for resynthesis from one prophage, then one might expect to be able to increase the rate by increasing the number of copies of prophage present. On the other hand, if the phage does have a cro-like gene product, increasing the number of copies of phage would increase all genes proportionately.

A double lysogen was prepared from C-2096 by superinfecting it with more of the same phage and isolating colonies that had a higher level of immunity that the original strain (G. Bertani, 1962). The pres- ence of two prophages in the strain, C-2097, was confirmed by transduction. Removal of the location I prophage, resulted in a single lysogen with corre- sponding lower immunity level (C-2098).

When the double lysogen was incubated at 42 ° C for 90 rain and then returned to 30 ° C, 50% of the bacteria regained their immunity after only 45 rain as opposed to 3 h for the single lysogen (Fig. 2). Thus, the presence of an additional prophage did increase the rate of restoration of immunity. These results suggest that we have not produced an anti-immune state in the P2 lysogens and that the rate at which immunity is restored is dependent mainly on the number of copies of repressor gene present.

Channeling. In lambda, the cro gene product not only blocks repressor synthesis of the prophage, but also of the superinfecting phage. This results in the phe- nomenon of "channel ing" of the superinfecting phage toward lysis (Neubauer and Calef, 1970). Thus, if the bacteria in the anti-immune state are super- infected, the frequency of lysogenization of the second

E. Nilsson and L.E. Bertani: Restorat ion of Immuni ty in P2 Lysogens 301

Tabie 3. Lysogenization of a derepressed lysogen by phage P2 ~ D i s e u s s i o l l

Strain Moi Number of Number of P2 % P2 plaque-formers lysogens lysogens

C-1278 0.87 1.2x108 5 .4x 107 32 C-1278 0.50 5 .9x 107 4 .2x 107 42 C-1278 0.83 7 .4x 107 5.1 x 107 39 C-1278 1.l 5.9× 107 2.9× 107 33

C-2098 1.1 3 .6x 10 v 1.0×108 71 0.85 2.8 x 107 6.2 x 107 69

C-1278 0.74 1.2 x 108 2.0 x 107 14

Cultures of the non-lysogenic strain, C-1278, and strain C- 2098, which carries P2 areA127 amBll6 c5 in a secondary attach- ment site, were incubated for 90-120 rain at 41 ° C, except for the last experiment where C-1278 was kept at 30 ° C throughout . The bacteria were then concentrated to 1-2 x l0 s per ml by centrifuga- tion, resuspended in LB-broth containing 5 m M CaCI2, and infected at 30°C with wildtype P2 at the given multiplicities of infection (Moi). After 10 rain to allow for adsorption, the infected bacteria were diluted 10 x into LB-broth containing phage neutral- izing anti-serum, K-1 per rain. After another 10 min, they were diluted and assayed for plaque-formers on C-1055 and for colony- formers by spreading on LB-agar plates. After the plates had devel- oped at 30 ° C, 100-200 colonies were picked and tested for lysogeny either, in the case of C-1278, by immuni ty to P2 virl or, in the case of C-2098, by production of P2

phage is reduced as compared to its frequency in a sensitive host. We looked, therefore, for a similar channeling effect in the P2 system.

In the previous section, we described the prepara- tion of a lysogen carrying two copies of P2 areA127 araB116 c5 and the subsequent transduction of this strain to remove the prophage occupying location I. Such a strain, C-2098, is suitable for studying lyso- genization by P2, since location I is the favored at- tachment site for this phage. The single lysogen carry- ing P2 areA127 a m B l l 6 c5 in a secondary location is like the previously described single lysogens in other respects. When derepressed at 42 ° C and returned to 30 ° C, repressor activity returns at the same rate (rig. 2).

When this strain was incubated at 42°C for 90 min and then returned to 30 ° C and infected with wildtype P2, the frequency of attachment at location I was found to be unusually h i g h - 7 0 % of the infected bacteria as compared to 36% obtained for a non-lysogen treated in the same way (Table 3). Thus, if anything, the phage was channeled towards lysogenization rather than towards lysis. The temper- ature shift also had an effect on lysogenization of the sensitive host C-1278 (Table 3), increasing it from 19%, obtained with bacteria kept always at 30°C to 36% following the temperature shift.

The aim of this work was to demonstrate in P2 the existence of a system for the control of repressor synthesis similar to the cro system described for lambda. We have done two types of experiments; in the first, we have tried to reproduce the antiimmune state in P2 lysogens by incubating suitable strains at 42 ° C to see if subsequent restoration of immunity could be blocked. In the second type of experiment, we looked for channeling of a superinfecting wildtype phage towards lysis in derepressed lysogens. The re- sults of these experiments were the opposite of what would be expected if P2 had a functional cro system - the immunity of the lysogens always returned and the channeling was towards lysogenization rather than towards lysis. Thus, we can find no evidence for a cro-like system in P2.

These experiments could be criticized on the basis that neither of the lysogenic strains used was entirely suitable. The defective strain has the right properties, but one could argue that the defect that blocks expres- sion of the A and B genes, also blocks expression of the cro gene and makes the prophage cro- . This criticism cannot be applied to the strain, carrying P2 areA127 a m B l l 6 c5, whose genotype is precisely known. Incubation of this strain at 42 ° C, however, leads to the derepression of some phage gene product that interferes with both the multiplication of the strain itself and that of a superinfecting phage. Again, one could argue that expression of the cro gene is also affected. Nevertheless, these diverse and imper- fect materials give essentially the same result.

It would be interesting to know why multiplication of the strain carrying an area araB c5 prophage is restricted at 42 ° C. Perhaps the amber fragments from genes A and/or B or an occasional suppressed mole- cule from this region are inhibitory, although the inhi- bition would have to be non-specific, affecting both bacterial host and phage. Alternatively, the inhibition might be due to another gene in the AB operon, which is being transcribed. The defect in the strain carrying P2 int l c5 def4 may turn off the transcription of these genes completely.

At first glance, the rate at which repressor activity is restored to a P2 lysogen after derepression might seem to be unusually slow for a cro- phage. Accord- ing to published data (Eisen et al., 1970) for lysogens carrying cro- lambda prophage, immunity is restored to 50% of the bacteria after only 30 min at 30 ° C. In the P2 system, the rate seems to be dependent on the number of copies of prophage present, since the rate is more rapid in a double than in a single lysogen (Fig. 2). The limiting phage product might be the repressor itself or some factor necessary to stimulate repressor synthesis.

302 E. Nilsson and L.E. Bertani: Restoration of hnmunity in P2-Lysogens

The type of temperature-sensitive repressor muta- tion used might also play a role. As already men- tioned, the c5 mutation of P2 is of the "irreversible" type, whereas in the case of lambda, the mutation used was ci857, which has a reversible effect on lambda repressor. That is, any molecules of repressor that are synthesized at 42 ° C, regain activity again when the strain is shifted to 30 ° C, a process that may proceed more rapidly than resynthesis of re- pressor. Unfortunately this difference cannot be checked by us, because all of the 25 tsC mutants independently isolated for P2 are of the irreversible type (unpublished results of L.E.B.).

Acknowledgements. This work was supported by Research Grant 72 from the Swedish Medical Research Council. E.N. was supported by a scholarship from the University of Stockholm.

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Communicated by W. Arber

Received June 27, 1977