Mutations That Improve the ANT Promoter of Salmonella Phage P22

Mutations that increase the activity of the promoter for the antirepressor gene of phage P22 were isolated by pseudoreversion of four severe promoter-down mutations. The sequence changes in these pseudorevertants include single base pair substitutions, single base pair deletions, tandem double base pair deletions and multisite mutations. The single base pair substitutions change nonconsensus base pairs to consensus base pairs at positions -14 and -8. The other mutations provide support for the idea that the length of the spacer region between the conserved -35 and -10 hexamers is an important determinant of promoter strength. Deletions of one or two base pairs in the spacer region apparently activate an alternate -10 hexamer by shifting it from a spacing of 19 base pairs to a spacing of 18 or 17 base pairs, respectively.     

Genetic analysis of Escherichia coli integration host factor interactions with its bacteriophage lambda H'' recognition site.

The bacteriophage P22-based challenge phage system was used to study the binding of integration host factor (IHF) to its H' recognition site in the attP region of bacteriophage lambda. We constructed challenge phages that carried H' inserts in both orientations within the P22 Pant promoter, which is required for antirepressor synthesis. We found that IHF repressed expression of Pant from either challenge phage when expressed from an inducible Ptac promoter on a plasmid vector. Mutants containing changes in the H' inserts that decrease or eliminate IHF binding were isolated by selecting challenge phages that could synthesize antirepressor in the presence of IHF. Sequence analysis of 31 mutants showed that most changes were base pair substitutions within the H' insert. Approximately one-half of the mutants contained substitutions that changed base pairs that are part of the IHF consensus binding site; mutants were isolated that contained substitutions at six of the nine base pairs of the consensus site. Other mutants contained changes at base pairs between the two subdeterminants of the H' site, at positions that are not specified in the consensus sequence, and in the dA + dT-rich region that flanks the consensus region of the site. Taken together, these results show that single-base-pair changes at positions outside of the proposed consensus bases can weaken or drastically disrupt IHF binding to the mutated site.     

Second-site suppressors of the bacteriophage P1 virs mutant reveal the interdependence of the c4, icd, and ant genes in the P1 immI operon.

The immI operon of phage P1 contains the genes c4, icd, and ant, which are transcribed in that order from the same constitutive promoter, P51b. The gene c4 encodes an antisense RNA which inhibits the synthesis of an antirepressor by acting on a target ant mRNA. Interaction depends on the complementarity of two pairs of short sequences encompassing virs+ and the ribosome-binding site involved in ant expression. Accordingly, in a P1 virs mutant phage, antirepressor is synthesized constitutively. We have isolated lysogen-proficient, second-site suppressors of P1 virs in order to evaluate the interdependence of the immI-specific genes. From a total of 17 suppressors analyzed, 15 were found to be located in the icd gene. They were identified as frameshift mutations, containing base insertions or deletions in tandem repeats of a single base pair. One suppressor was identified as a P51b promoter-down mutation; the second site of another suppressor was found to be located in the c4 gene. Furthermore, it was shown that virs cannot be suppressed by ant (icd+) suppressors. The results confirm the model that the immI operon is transcribed as a unit, that the icd and ant genes are translationally coupled, and that the constitutive synthesis of Icd protein alone is lethal to the bacterial cell. The existence of a c4 suppressor of virs, whose effect is not yet known, points to a still more complex regulation of antirepressor synthesis than was anticipated from the model.     

Hierarchies of base pair preferences in the P22 ant promoter.

Oligonucleotide-directed mutagenesis was used to complete a collection of mutations in the -35 and -10 hexamers of the ant promoter of Salmonella phage P22. The effects of all 36 single-base-pair substitutions on promoter strength in vivo were measured in strains carrying the mutant promoters fused to an ant-lacZ gene on a single-copy prophage. The results of these assays show that certain consensus base pairs are more important than others; in general, the least-critical positions are among the most poorly conserved. Some mutations within the hexamers have smaller effects on promoter strength than certain mutations outside the hexamers in this and other promoters. Several different patterns of base pair preferences are observed. These hierarchies of base pair preferences correlate well (but not perfectly) with the hierarchies defined by the frequency distribution of base pairs at each position among wild-type promoters. The hierarchies observed in the ant promoter also agree well with most of the available information on base pair preferences in other promoters.     

The c4 repressors of bacteriophages P1 and P7 are antisense RNAs.

The c4 repressors of P1 and P7 inhibit antirepressor synthesis and are solely responsible for heteroimmunity of the phages. We show that c4 is a new type of antisense RNA acting on a target, ant mRNA, that is transcribed from the same promoter. Interaction depends on complementarity of two pairs of short sequences encompassing the ribosome binding site involved in ant expression. We demonstrate that heteroimmunity of P1 and P7 is due to just two substitutions in each of the complementary sequences of c4 and ant mRNA. Based on P1-P7 sequence comparison and a mutant analysis, we propose a secondary structure model for c4 RNA, with the complementary regions in loops as important sites for antisense control.     

Role of antirepressor in the bipartite control of repression and immunity by bacteriophage P22

The bipartite immunity and repression system of the temperate Salmonella bacteriophage P22 has been analyzed by genetic means. Both parts of the immunity system, immI and immC, are necessary to confer upon lysogens immunity to superinfection with P22. The product of the c2 gene (which lies in immC) is a repressor which apparently regulates directly the expression of phage genes in a manner analogous (if not identical) with that found for coliphage λ.The immI region contains three genetic elements. One of these (mnt; Gough, 1968) appears to specify another repressor whose specific activity is continuously required for the maintenance of lysogeny. We have identified two new regulatory elements in immI through the isolation of mutants. Virulent mutations (virA) in the Vy element confer the ability to grow in immune P22 lysogens by destroying or inactivating the repression functions of the lysogen (possibly the c2-repressor itself). The third element in immI is a structural gene (ant) for a protein (antirepressor) which is regulated by mnt (repressor) and Vy (promoter/operator).We have shown that the ability of P22 to grow on immI-deletion lysogens, the dominant virulence of virA virulents, and the requirement for mnt for the maintenance of lysogeny, all depend on an intact ant⁺ gene. It is proposed that P22 antirepressor represents a new type of regulatory protein which acts by controlling other regulatory proteins.     

Altered (copy-up) forms of initiator protein pi suppress the point mutations inactivating the gamma origin of plasmid R6K.

The R6K gamma origin core contains the P2 promoter, whose -10 and -35 hexamers overlap two of the seven binding sites for the R6K-encoded pi protein. Two mutations, P2-201 and P2-203, which lie within the -35 region of P2, are shown to confer a promoter-down phenotype. We demonstrate here that these mutations prevent replication of a gamma origin core plasmid. To determine whether or not the reduced promoter activity caused by these mutations is responsible for their effect on replication, we generated two new mutations (P2-245-6-7 and P2-246) in the -10 hexamer of the P2 promoter. Although these new mutations inhibit P2 activity as much as the P2-201 and P2-203 mutations, they do not prevent replication of the gamma origin core. Therefore, activity of the P2 promoter does not appear to be required for replication. We also show that the inability of the gamma origin to function in the presence of the P2-201 and P2-203 mutations is reversed by the hyperactive variants of pi protein called copy-up pi. This suppression occurs despite the fact that in vivo dimethyl sulfate methylation protection patterns of the gamma origin iterons are identical in cells producing wild-type pi and those producing copy-up pi variants. We discuss how the P2-201 and P2-203 mutations could inhibit replication of the gamma origin core and what mechanisms might allow the copy-up pi mutants to suppress this deficiency.     

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.     

Oligopeptidase A is required for normal phage P22 development.

The opdA gene of Salmonella typhimurium encodes an endoprotease, oligopeptidase A (OpdA). Strains carrying opdA mutations were deficient as hosts for phage P22. P22 and the closely related phages L and A3 formed tiny plaques on an opdA host. Salmonella phages 9NA, KB1, and ES18.h1 were not affected by opdA mutations. Although opdA strains displayed normal doubling times and were infected by P22 as efficiently as opdA+ strains, the burst size of infectious particles from an opdA host was less than 1/10 of that from an opdA+ host. This decrease resulted from a reduced efficiency of plating of particles from an opdA infection. In the absence of a functional opdA gene, most of the P22 particles are defective. To identify the target of OpdA action, P22 mutants which formed plaques larger than wild-type plaques on an opdA mutant lawn were isolated. Marker rescue experiments using cloned fragments of P22 DNA localized these mutations to a 1-kb fragment. The nucleotide sequence of this fragment and a contiguous region (including all of both P22 gene 7 and gene 14) was determined. The mutations leading to opdA independence affected the region of gene 7 coding for the amino terminus of gp7, a protein required for DNA injection by the phage. Comparison of the nucleotide sequence with the N-terminal amino acid sequence of gp7 suggested that a 20-amino-acid peptide is removed from gp7 during phage development. Further experiments showed that this processing was opdA dependent and rapid (half-life, less than 2 min) and occurred in the absence of other phage proteins. The opdA-independent mutations lead to mutant forms of gp7 which function without processing.     

Evidence that SbcB and RecF pathway functions contribute to RecBCD-dependent transductional recombination.

A role for the RecF, RecJ, and SbcB proteins in the RecBCD-dependent recombination pathway is suggested on the basis of the effect of null recF, recJ, and sbcB mutations in Salmonella typhimurium on a "short-homology" P22 transduction assay. The assay requires recombination within short (approximately 3-kb) sequences that flank the selected marker and lie at the ends of the transduced fragment. Since these ends are subject to exonucleolytic degradation, the assay may demand rapid recombination by requiring that the exchange be completed before the essential recombining sequences are degraded. In this assay, recF, recJ, and sbcB null mutations, tested individually, cause a small decrease in recombinant recovery but all pairwise combinations of these mutations cause a 10- to 30-fold reduction. In a recD mutant recipient, which shows increased recombination, these pairwise mutation combinations cause a 100-fold reduction in recombinant recovery. In a standard transduction assay (about 20 kb of flanking sequence), recF, recJ, and sbcB mutations have a very small effect on recombinant frequency. We suggest that these three proteins promote a rate-limiting step in the RecBC-dependent recombination process. The above results were obtained with a lysogenic recipient strain which represses expression of superinfecting phage genomes and minimizes the contribution of phage recombination functions. When a nonlysogenic recipient strain is used, coinfecting phage genomes express functions that alter the genetic requirements for recombination in the short-homology assay.     

Maintenance of bacteriophage P1 plasmid.

Three mutants of bacteriophage P1 affected in their ability to maintain the lysogenic state stably are described here. These mutants were normal in lytic growth, but lysogenic derivatives segregated nonlysogens at abnormally high rates (1 to 30% per division). Cells harboring these mutant prophages were elongated or filamentous. The mutations responsible for this prophage instability fell into two classes on the bases of their genetic location, their effect on the ability to lysogenize recA bacteria, and their suppressibility by ant mutations eliminating antirepressor activity. The two mutants that were able to form recA lysogens showed the same prophage instability and partial inhibition of cell division in recA as in rec+ lysogens. The fact that plasmid-linked mutations can cause prophage instability suggests that P1 codes for at least some of the functions determining its own autonomy and segregation.