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.     

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.     

Identification of the repressor and repressor bypass (antirepressor) polypeptides of bacteriophage P1 synthesized in infected minicells

Summary P1 infected minicells synthesize approximately 50 phage-encoded polypeptides. Phage expression is temporally controlled, demonstrating phage polypeptides synthesized both early and late after infection. The P1 repressor, gpc1 ¹ (Mr=33,000), repressor bypass polypeptide, gprebA (Mr=27,500) and cistron 10 product, (gp10) (Mr=64,000), have been identified by infection of minicells with P1 amber mutants. The beta-lactamase gene product (gpbla) carried by the closely related phage P7 and the chloramphenicol acetyl-transferase gene product (gpcat) carried by P1 Cm (in Tn9) have been demonstrated. Infection of minicells by P1vir ^s or P1c4 mutants results in increased synthesis of gprebA and a second polypeptide designated gprebB (Mr=40,000). The P1vir11 mutation leads to increased synthesis of a small polypeptide (Mr=3,500) but does not affect the amount of gpc1 synthesized.     

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.     

Effects of mutations in the immunity system of bacteriophage P1.

A mutant of bacteriophage P1 that made an altered c1 repressor is described. The mutant c1 product had two configurations: in lysogens, at high temperatures, it permitted constitutive expression of the normally repressed DNA replication function ban and was insensitive to the action of ant, a product expressed by the virulent mutant P1virs and by the heteroimmune phage P7 (formerly phiamp+) and normally able to overcome c1 repression; in mutant lysogens at low temperatures, the mutant repressor was apparently normal (able to repress ban and sensitive to ant action). Genetic studies of this mutant led to the isolation of a derivative that formed unstable lysogens. These studies suggested that the ban product was normally under c1 control; they further showed that ant overcame c1 repression by inactivating c1 rather than by creating a bypass of repressor activity.     

Dual control of lysogeny by bacteriophage P22: An antirepressor locus and its controlling elements

Two distantly linked clusters of genes on the Salmonella typhimurium phage P22 chromosome are involved in the control of lysogeny and superinfection immunity. One cluster consists of genes c1, c2, and c3, which are primarily responsible for the establishment and maintenance of lysogeny. It has been proposed that the second cluster consists of three loci which are responsible for the synthesis and control of an antirepressor substance which overcomes the repression mediated by the c2 gene product. This paper reports the isolation of mutants in a locus designated “ant” having characteristics expected of antirepressor mutants. Evidence is presented that the other loci in this second immunity region, mnt and virA, control the expression of the ant gene as represser and promoter/operator, respectively. The interactions of these three loci with each other and with the other immunity region are discussed.     

Organization of the early region of bacteriophage phi 80. Genes and proteins

An EcoRI segment containing the early region of bacteriophage phi 80 DNA that controls immunity and lytic growth was identified as a segment whose presence on a plasmid prevented growth of infecting phi 80cI phage. The nucleotide sequence of the segment (EcoRI-F) and adjacent regions was determined. Based on the positions of amber mutations and the sizes of some gene products, the reading frames for five genes were identified. From the relative locations of these genes in the genome, the properties of some isolated gene products, and the analysis of the structures of predicted proteins, the following phi 80 to lambda analogies are deduced: genes cI and cII to their lambda namesakes; gene 30 to cro; gene 15 to O; and gene 14 to P. An amber mutation by which gene 16 was defined is a nonsense mutation in the frame for gene 15 protein, excluding the presence of gene 16. An amber mutation in gene 14 or 15 inhibits phage DNA synthesis, as is the case with their lambda analogues, gene O or P. Some characteristics of proteins from the early region predicted from their primary structures and their possible functions are discussed.     

Escherichia coli deoxyribonucleic acid synthesis mutants: their effect upon bacteriophage P2 and satellite bacteriophage P4 deoxyribonucleic acid synthesis.

Escherichia coli C strains containing different deoxyribonucleic acid (DNA) synthesis mutations have been tested for their support of the DNA synthesis of bacteriophage P2 and its satellite phage P4. Bacteriophage P2 requires functional dnaB, dnaE, and dnaG E. coli gene products for DNA synthesis, whereas it does not require the products of the dnaA, dnaC, or dnaH genes. In contrast, the satellite virus P4 requires functional dnaE and dnaH gene products for DNA synthesis and does not need the products of the dnaA, dnaB, dnaC, and dnaG genes. Thus the P2 and P4 genomes are replicated differently, even though they are packaged in heads made of the same protein.     

The c1 repressor of bacteriophage P1: I. Isolation of the c1 protein and determination of the P1 DNA region to which it Binds

A bacteriophage P1-specific DNA binding protein has been partially purified from P1-infected Escherichia coli and identified as the P1c1 repressor. This protein is absent from non-suppressing cells infected with a P1c1 amber mutant. The binding activity of the protein isolated from cells infected with a c1ts mutant is thermolabile in vitro, so the repressor protein is the product of the c1 gene. Studies on P1 DNA fragments generated by restriction endonuclease digestion indicate that the c1 repressor binds preferentially in vitro at a site or sites located close to the c1 gene itself.