A dnaB analog ban, specified by bacteriophage P1: genetic and physiological evidence for functional analogy and interactions between the two products.

Summary Bacteriophage P1 has been shown previously to determine a product ban than can substitute in DNA replication for the protein specified by cistron dnaB of Escherichia coli. However, ban product furnished by P1 bac prophage (ban constitutive) substitutes only poorly for DNA replication in the absence of dnaB product in a strain bearing an unsuppressed amber mutation, dnaB266, as shown by the cryosensitivity of the dnaB266 (P1 bac) lysogen and its unability to support growth. An additional mutation (termed crr) in the P1 bac prophage has been obtained which confers cryoresistance to the sup ⁺ dnaB266 (P1 bac crr) lysogen and restores its ability to support growth. ban product produced in P1 bac crr lysogen fulfills all dnaB roles in vivo, especially in the various instances in which ban product expressed in P1 bac lysogens does not. The ban product is expressed constitutively in P1 crr prophage. The crr-1 mutation is tightly linked to the bac-1 and ban-1 mutations and is dominant over crr ⁺. The nature of the crr mutation is discussed: two hypotheses are considered, that of a mutation in the ban gene rendering the ban product more active or that of a site mutation in the ban operon increasing the level of ban expression. Expression of ban product (wild type or altered) leads to interactions with the variously altered dnaB product. Both positive and negative interactions are described. Genetic results presented here suggest that ban and dnaB subunits interact to form hybrid dnaB-like molecules; the average composition of which depends on the relative quantities of ban and dnaB subunits in the cell.     

Regulation of the ban gene containing operon of prophage P1

Summary A physical map of the ban gene of P1 and sites relevant to its regulation has been deduced from cloning of the appropriate regions of P1 wild-type and of P1 ban regulatory mutants. The cloning required the presence of P1 repressor in the cell confirming the existence of a repressible ban operon (Austin et al. 1978). Evidence for additional member(s) of that operon is presented. Of particular interest for understanding the regulation of ban are the relative positions of a binding site for the P1 repressor and of the regulatory mutations bac and crr that render ban expression constitutive. The results reveal a repressible operon-like structure of about 4 kb within the P1 EcoRI-3 fragment that comprises a c1 repressor binding site/bac additional gene(s) — crr/ban in the clockwise direction of the circular map of P1.     

A dnaB analog specified by bacteriophage P1

Bacteriophage P1 is shown to determine a product that can substitute in DNA replication for the protein specified by cistron dnaB of Escherichia coli. The viral dnaB analog (ban) is repressed in the wild-type P1 prophage and expressed constitutively in plaque-forming mutants, P1bac, described here. A particular P1bac prophage allows lysogens of dnaBts bacteria to survive as colony-formers at temperatures that arrest DNA synthesis in the non-lysogens. The P1bac prophage furthermore permits construction of an otherwise inviable strain bearing the unsuppressed amber mutation dnaB266.P1bac prophages also suppress the groP character which is associated with certain dnaB mutations. The subclass of dnaB mutations called groP are those which prevent the growth of bacteriophage λ⁺ at temperatures permissive for bacterial DNA synthesis, but allow the growth of certain λ mutants (λπ); π mutations have been mapped in gene P. Thus, λ⁺ is enabled to grow in groP hosts by the presence of P1bac-1 prophage. When dnaB protein is absent, however, as in the case of the unsuppressed amber mutant, the ban protein furnished by the P1bac prophage does not support λ growth. Therefore, in the groP(P1bac-1) lysogens both the dnaB and ban products are needed for λ growth, suggesting interactions between these E. coli and P1 proteins or their subunits.Mutations (termed ban) that prevent the expression of the dnaB analog determined by P1 have been obtained. P1bac-1ban-1, unlike P1bac-1, fails to replicate in dnaBts hosts at temperatures non-permissive for bacterial DNA synthesis. Thus, the dnaB protein and its P1-determined analog can interchangeably fulfill an essential role in the replication of both the E. coli and P1 replicons. At permissive temperatures the lysogenization of certain dnaBts strains by P1bac-1ban-1 is very inefficient, probably as a result of negative complementation.Mutations bac-1 and ban-1 are closely linked on the P1 chromosome and their order relative to several amber mutations has been determined. Dominance studies of the alleles in transient diploids show that the ban-1 mutation is recessive to ban⁺. The bac-1 mutation, on the other hand, behaves in dominance tests as a DNA site mutation that permits constitutive expression in cis of the operon to which the ban gene belongs.     

Synthesis of P1 ban protein in minicells infected by P1 mutants

Summary Phage P1 encodes a dnaB analog (ban) protein. Synthesis of ban protein has been studied in minicells infected by P1 mutants and has been identified as a polypeptide of 56,000 molecular weight by immunoprecipitation using antibody directed against E. coli dnaB protein. The amount of ban protein synthesized by P1 mutants increases in the order: P1 wild type, P1bac, P1crr, and P1bac crr. The relative amount of ban protein identified in P1bac- and P1bac crr-infected minicells is approximately the same as that previously found in dnaBsdban heteromultimers isolated from the corresponding P1 lysogens.     

The isolation and characterization of escherichia coli dnaB::Tn10 insertion mutations

Summary Exploitation of the ability of the ban protein encoded by phage P1 to compensate for dnaB-defective host mutations, allowed the isolation of dnaB::Tn10 insertion mutations. The presence of P1bac prophage was required for survival of dnaB::Tn10 mutants, and such lysogens were cryosensitive. The insertions were shown to map in dnaB by transduction and this was confirmed by complementation analysis. The dnaB::Tn10 (P1bac) strains were non-permissive for growth but did support the growth of -dnaB ⁺specialized transducing phage. No antigenically active dnaB product could be detected by immunologic assays using either of two methods. In addition, it was shown that the observed cryosensitivity of P1bac suppression was a direct result of reversible inactivation of the ban protein at low temperature.     

A new gene of bacteriophage P22 which regulates synthesis of antirepressor

Two new mutants of bacteriophage P22 are described which define a new regulatory gene, arc (for antirepressor control). The properties of the arc mutants and of 31 phenotypic revertants indicate that the arc gene codes for a trans-acting protein whose primary role is to depress synthesis of P22 antirepressor protein during the lytic cycle of infection. Failure to regulate antirepressor production apparently leads secondarily to a lethal defect (i.e. failure to produce progeny phage).Although under certain conditions the arc function can be expressed by P22 prophages and can act as a weak barrier to superinfecting homologous phage, the arc product is neither necessary nor sufficient for maintenance of the prophage state or superinfection immunity in lysogens. Instead, as shown previously by others (Levine et al., 1975; Botstein et al., 1975), the prophage mnt gene product is responsible for repressing antirepressor synthesis, both by the prophage and by superinfecting phage.     

Role of the cro gene in bacteriophage lambda development

Previous experiments have shown that the product of the cro gene of baeteriophage λ can exert an anti-repression activity, defined by the capacity of certain “cro-constitutive” defective lysogens to channel a superinfecting λ phage toward lytic development. We have used a combination of biological and biochemical assays to draw two main conclusions concerning this anti-repression activity: (1) after infection of a cro-constitutive cell, the superinfecting phage is unable to establish repression because it is unable to commence synthesis of cI protein (λ repressor) at a substantial rate; (2) the cause of this diminished synthesis of cI protein is the capacity of cro product to repress synthesis of the cII and cIII proteins, which normally activate the cI gene to establish repression in an infected cell. From our experiments and those of others, we suggest that cro product possesses a repression activity which is similar to that of the cI protein itself, but normally exerts a very different physiological role: the turnoff of synthesis of replication, recombination and regulation proteins as the virus enters the late stage of lytic development.     

The activity of ant product of the Salmonella phage P22 against the closely related but heteroimmune phage L.

Summary P22 mutants defective in the early gene 24 are complemented by phage L in mixed infection. P22 12 ^- and P22 23 ^- mutants are not complemented by phage L. Gene function 24 of an L prophage is turned on by a superinfecting P22 24 ^- mutant and complements the missing function of the defective P22 phage. Since this transactivation of prophage gene 24 depends on a functional gene ant in the superinfecting P22 mutant, it indicates derepression for leftward directed gene expression in prophage L. On the contrary neither the rightward directed expression of gene 12 nor of gene 23 in prophage L can be turned on by superinfecting P22 24 ^- 12 ^- or P22 24 ^- 23 ^- mutants (and also not by P22 12 ^- and P22 23 ^-) to a degree sufficient for complementation of simultaneously superinfecting L virB 12 ^- or L virB 23 ^- mutants. The failure to detect release of repression for rightward directed gene expression of prophage L corresponds to the earlier observation (Prell, 1975) that P22 superinfecting L lysogens cannot release replication inhibition for simultaneously infecting phage L. The results are discussed with respect to the mechanism underlying the different action of P22 antirepressor in L and in P22 lysogens.     

Restoration of immunity in lysogens carrying prophage P2, derepressed at high temperature

Summary The possibility that a strain lysogenic for phage P2 could be brought into the so-called antiimmune state in which the synthesis of phage repressor 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 mutation (c5), amber 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 repressor 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 amA amB 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 channeling towards 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.     

Prophage repression as a model for the study of gene regulation. I. Titration of the lambda repressor

Wiesmeyer, Herbert (Vanderbilt University, Nashville, Tenn.). Prophage repression as a model for the study of gene regulation. I. Titration of the lambda repressor. J. Bacteriol. 91:89-94. 1966.-The concentration of lambda repressor molecules within a lambda lysogenic cell was estimated from the multiplicity of superinfecting homologous phage necessary to permit replication and release of plaque-forming units. A multiplicity of 20 superinfecting phage was found sufficient to permit replication to occur in the normal lambda lysogen. The phage released after lysis of the superinfected lysogen was composed of both prophage and superinfecting phage types. Superinfection of the lysogen at lower multiplicities resulted in the lysis of only a small percentage of infected cells and is thought to represent a possible heterogeneity of repressor concentration in the lysogenic population. Viability of the superinfecting particle was found to be unnecessary for titration of the repressor. The repressor concentration in three lysogens of the nonultraviolet-inducible mutant of lambda, lambda(ind-), was found to be greater than 20 regardless of the host bacterium. However, the number of cells yielding phage after superinfection was found to vary with the particular host. The specificity of the lambda repressor was shown to be limited to homologous phage, as determined following heterologous superinfection experiments with phages T6r, 82c, 434c, 434hy, and 424. In all instances except that of superinfection with phage 434hy, only heterologous phage replication occurred. Superinfection by phage 434hy resulted in the release of both prophage and superinfecting phage types. The latter type represented approximately 80% of the total phage released.     

Superinfection Exclusion by P22 Prophage and the Replication Complex

Wild-type sie(+) P22 prophage converted Salmonella typhimurium lysogens to exclude deoxyribonucleic acid (DNA) injected by superinfecting phage. DNA from a P22 superinfecting virulent phage associated with the replication complex in a sie(-) lysogen but not in a sie(+) lysogen.     

Prophage substitution and prophage loss from superinfected Escherichia coli recA(P1) lysogens.

It is shown that the plasmid prophage P1 can be displaced by a superinfecting P1 phage in Escherichia coli recA(P1) lysogens. Six widely separated phage markers were used to distinguish between residual recombination and total substitution. It is further shown that superinfection of recA lysogens can lead to loss of both phage (curing). These two phenomena, previously reported in Rec+ strains, are thus independent of host recombination and may result from perturbations of some function involved in plasmid maintenance.     

Gene responsible for superinfection exclusion of heteroimmune corynebacteriophage.

Wild-type beta and gamma corynebacteriophages are heteroimmune and infect lysogens of each other productively. Unlike their wild-type counterparts, the bin mutants of each phage are excluded in lysogens carrying the heteroimmune phage. The wild-type phages overcome exclusion by means of the bin gene product which appears to act as an antirepressor. When repression is lifted, exclusion of bin mutants is abolished (N. Groman and M. Rabin, J. Virol. 28:28-33, 1978; J. Virol. 36:526-532, 1980). It has not been clear whether the excluding compound is the immune repressor itself or one whose synthesis is positively regulated by repressor. We have isolated beta exclusion mutants (xcl) that as prophage exhibited normal immune repression but no longer excluded gamma-bin mutants. Furthermore, we have shown that an xcl phage with an active immune repressor acted in trans to continue the positive regulation of exclusion by a second xcl+ prophage whose immune repressor was inactivated. From these results it was concluded that there is a gene distinct from the imm gene which is directly or indirectly responsible for exclusion. The xcl gene, mapped in prophage crosses, was located between imm and bin, that is, in the regulatory region of the phage genome. The simplest hypothesis compatible with the established observations is that beta immune repressor regulates the expression of the xcl and bin genes, the former positively and the latter negatively. It is likely that an analogous regulatory model applies to gamma phage since it has already been shown that both beta and gamma have bin alleles.     

ban operon of bacteriophage P1. Mutational analysis of the c1 repressor-controlled operator

The repressor of bacteriophage P1, encoded by the c1 gene, represses the phage lytic functions and is responsible for maintaining the P1 prophage in the lysogenic state. The c1 repressor interacts with at least 11 binding sites or operators widely scattered over the P1 genome. From these operators, a 17 base-pair asymmetric consensus sequence, ATTGCTCTAATAAATTT, was derived. Here, we show that the operator, Op72 of the P1ban operon consists of two overlapping 17 base-pair sequences a and b forming an incomplete palindrome. Op72a matches the consensus sequence, whereas Op72b contains two mismatches. The evidence is based on the sequence analysis of 27 operator mutants constitutive for ban expression. They were identified as single-base substitutions at positions 2 to 10 of Op72a (26 mutants) and at position 8 of Op72b (one mutant). We conclude from gel retardation and footprinting studies that two repressor molecules bind to the operator and that positions 4, 5 and 7 to 10 of the operator play an essential role in repressor recognition.     

Temperate Coliphage HK022: Clear Plaque Mutants and Preliminary Vegetative Map

Wild type phage HK022 was mutagenized by N-methyl-N′-nitro-N-nitrosoguanidine to induce clear plaque mutants. A total of 225 clear plaque mutants were isolated and 198 of these were assignable to one or the other of the two complementation groups of the corresponding cistrons which have been designated as cI and cII, respectively. Approximately 25% of the c mutants were found to be temperature-sensitive (cts); producing turbid plaques at 32 C and clear plaques at 38 C and above. From complementation tests involving cI and cII mutants, bacteria lysogenic for cII prophage were frequently obtained. Double lysogens harboring a cI and a cII prophage were infrequently found and single lysogens harboring only a cI prophage have not been recovered. Bacterial lysogens harboring a prophage carrying a cts mutation in the cI cistron were readily obtainable. However, such lysogens show a lethal phenotype at 40 C and above, although they appear to be fully viable at 32 C. It is shown that by incubation of lysogens harboring a cts mutant of the cI cistron at 42 C, it is possible to isolate cryptic lysogens which are non-immune but harbor at least one of the phage sus⁺ alleles. Genetic data involving cI, cII, and two complementing sus mutants of essential genes are presented. From these data the following vegetative map is deduced: sus4–cII-cI-sus3.     

Ant-mediated inactivation of Salmonella phage L-specified repression at OR of prophage L.

Summary Ant product of phage P22 inactivates repression of prophage L at the right-hand operator o_R and allows for transactivation of prophage gene 12. The transactivation efficiency observed with a series of phage and prophage recombinants, using single superinfection of a lysogenic bacterium, is about the same as that recently observed at o_L of prophage L. This finding is in contrast to the failure to demonstrate derepression at o_R of prophage L in an experimental system employing double superinfection (Prell, 1978a). The reasons for the differing results are discussed and it is shown that derepression by the ant product in trans at o_R of the prophage is not modified to any significant degree by the immunity specificity (L or P22) of the prophage or of the superinfecting phage.     

The antirepressor needed for induction of linear plasmid-prophage N15 belongs to the SOS regulon

The physiological conditions and molecular interactions that control phage production have been studied in only a few families of temperate phages. We investigated the mechanisms that regulate activation of lytic development in lysogens of coliphage N15, a prophage that is not integrated into the host chromosome but exists as a linear plasmid with covalently closed ends. We identified the N15 antirepressor gene, antC, and showed that its product binds to and acts against the main phage repressor, CB. LexA binds to and represses the promoter of antC. Mitomycin C-stimulated N15 induction required RecA-dependent autocleavage of LexA and expression of AntC protein. Thus, a cellular repressor whose activity is regulated by DNA damage controls N15 prophage induction.     

Phage T4 infection restricts rRNA synthesis byE. coli RNA polymerase

Summary Complementation for the maintenance of lysogeny was studied by superinfecting cIts lysogens at 34° C, and then heating to 43° C. With certain exceptions,ts mutants with defects in the left half of the repressor complementedts mutants with defects in the right half to produce a less heat-labile repressor (Fig. 3). AllcIamber mutants failed to complementcIts mutants. ThecI mutantc50 complements allts mutants. Mutations in Pre (cy) or genescII andcIII do not significantly affect the expression ofcI by a superinfecting genome in an immune lysogen. Mutants with very heat-labile repressors failed to complement cy42 for the establishment of lysogeny at elevated temperatures, while those with less heatsensitive repressors apparently did complementcy.According to a suggested model, the left side of thecI product is concerned primarily with subunit aggregation, while operator binding is the function of the right side of the oligomer.     

Integration defective mutants of bacteriophage P2

Summary Mutants of phage P2 unable by themselves to be integrated as prophages have been isolated. These mutants (int) are complemented by the wild type allele and may then yield stable lysogenic strains carrying an int prophage at location I in Escherichia coli C. These lysogens produce either no phage or little phage, depending on the int mutant used. All int mutants isolated appear to belong to a single complementation group.Exceptional lysogens carrying two or more int prophages may be obtained: they may produce spontaneously even more phage than normal lysogens, and they segregate out defective, singly lysogenic clones at low frequency. These exceptional lysogens carry both prophages in location I, presumably in tandem.Strains carrying two or more int prophages but defective in phage production were also isolated. One of these carries its prophages at two different, not closely linked, chromosomal locations.