P22 Repressor Research Articles

Cleavage of the lambda and P22 repressors by recA protein.

The site of recA cleavage of the phage lambda and P22 repressors has been determined. Each repressor is cut once by the recA enzyme, lambda repressor between residues 111 and 112, and P22 repressor between residues 94 and 95. recA cleavage occurs at identical alanyl-glycyl sequences in both repressors, and in both repressors, the cleavage separates the repressor's NH2-terminal DNA-binding domain from its COOH-terminal oligomerization domain. A papain-generated proteolytic fragment of lambda repressor consisting of repressor residues 93-236 is also efficiently cleaved by recA protein. Moreover, the recA cleavage of radioactive lambda repressor can be inhibited by certain COOH-terminal proteolytic fragments of lambda repressor which do not contain the alanyl-glycyl cleavage sequence. These facts suggest that recA cleavage of lambda repressor requires an intact COOH-terminal domain but not an intact NH2-terminal domain.

Primary structure of the phage P22 repressor and its gene c2.

The amino acid sequence of the Salmonella phage P2 repressor and the DNA sequence of its gene c2 have been determined. Sequential Edman degradations on intact P22 repressor and repressor peptides generated by proteolytic and chemical cleavages have been overlapped to give approximately 97% of the complete protein sequence. Additionally, the nucleotide sequence of the P22 c2 repressor gene has been determined by DNA sequencing techniques. The DNA sequence and partial protein sequence are collinear and together define the complete amino acid sequence of P22 repressor. The repressor is a single-chain 216 amino acid polypeptide. Basic residues in the sequence tend to be clustered, and residues 9-20 are highly basic, containing five arginyl and three lysyl residues. The carboxyl-terminal 133 amino acids of the c2 repressor are homologous to the carboxyl-terminal sequence of the coliphage lambda cI repressor. The amino-terminal sequences of these two repressors show little similarity.

Kinetics of recA protein-directed inactivation of repressors of phage λ and phage P22

Escherichia coli recA protein directs the inactivation of the repressor of Salmonella typhimurium phage P22 in vitro. As is true for repressor of the E. coli phage λ, inactivation of P22 repressor is accompanied by proteolytic cleavage of the repressor into two detectable fragments. We have investigated the kinetics of inactivation of the λ and P22 repressors in vitro. The fraction of λ repressor inactivated per unit time decreases as its concentration in the reaction is increased. However, high concentrations of λ repressor do not inhibit the inactivation of P22 repressor. Thus, it does not appear that the inactivation system is saturated by λ repressor, but rather that λ repressor is a less efficient substrate at higher concentrations.

Establishment mode repressor synthesis blunts phage P22 antirepressor activity

Phage P22 repressor is synthesized in two modes: the establishment mode requires the pre site, the repressor gene, and the products of two auxiliary phage genes; the maintenance mode, used in established lysogens, requires only the prm site and the repressor gene. P22 antirepressor is a protein that inactivates P22 repressor. A model is presented here in which antirepressor activity is restricted to repressor molecules made in the maintenance mode; repressor made by the establishment mode is postulated to blunt antirepressor activity. Phage P22 vir A constitutively produces antirepressor. The model is used to explain the pattern of phage production from P22 vir A infections of sensitive cells and from P22 vir A superinfection of P22 lysogens. DNA binding activity (repressor) was measured in lysogens superinfected with P22 vir A. Following superinfection, elevated levels of repressor were detected in those cells in which establishment mode repressor synthesis was possible. This observation supports the idea that establishment mode repressor synthesis decreases the activity of antirepressor.

Regulation of Bacteriophage P22 DNA synthesis and repressor levels in P22cly infections

A rifampin-resistant mutant of Salmonella typhimurium carries an altered RNA polymerase. Wild-type (c+) phage P22 displays clear plaques and a reduced lysogenization frequency on this mutant host. The cly mutants of P22 were isolated on the basis of their ability to lysogenize such mutant hosts. Two classes of regulatory events, both of which are dependent on P22 gene c1 activity, are necessary for the establishment of lysogeny in P22. The positive events culminate in repressor synthesis; the negative events cause a retardation in phage DNA synthesis. Neither the positive nor the negative events are observed in P22c+ infections of the mutant host. Both effects are found in P22cly infections of the mutant host. Observable results of both the negative and the positive events are exaggerated in P22cly infections of wild-type hosts as compared to P22c+ infections. The cly mutation apparently increases the positive and negative regulatory events so that they are detectable in the mutant host and exaggerated in wild-type hosts. Possible mechanisms that result in the high frequency of lysogenization that characterizes the cly mutation and the nature of the cly mutation are discussed.

UV sensitivity of a nonrepressor regulatory protein of bacteriophage P22.

The product of phage P22 gene c1 has two functions: it promotes synthesis of P22 repressor and it retards expression of some lytic genes. We present evidence that this product is inactivated in UV-irradiated hosts. The conditions for inactivation of c1 product include a functional DNA recombination system involving the host recA gene.

Px, a hybrid between the serologically unrelated and heteroimmune Salmonella bacteriophages P22 and Py. I. Some properties of Py and Px, genetic evidence for the hybrid nature of Px, and phenotypic mixing between P22, Py and Px.

Two new temperateSalmonella bacteriophages, Py and Px are described. Py is serologically unrelated to P22 and heteroimmune to it. Px is shown to be a hybrid phage containing genetic material from the c-region of P22 and the host range gene of Py. Px is heteroimmune to Py. Px phages seem to be formed only during vegetative replication of P22 in Py lysogenic cells. Independent events of hybrid formation may lead to genetically different types, called Px variants. The Px variants are sensitive to the P22 repressor whereas P22 is not repressed in Px lysogenic cells. The latent periods of P22 and the variant Px1 are about 30 min, whereas Py has a latent period of 60 min. Px1 recombines with P22 or Py only within the regions of genetic homology. Phenotypic mixing between P22 and Px or Py yields in different frequencies all possible types of mixed particles except Py-DNA with a P22 protein coat.

Transduction by phage P22 in a recombination-deficient mutant of Salmonella typhimurium

Recombination-deficient (Rec −) mutants of Salmonella typhimurium were used to study special transduction by phage P22 in the absence of the usual background of general transduction. Rec − strains could not be transduced for any marker with a lysate arising by infection. When the same strains were infected with a lysate obtained by induction, low but significant levels of transduction were observed for the pro A and pro B loci (located to one side of the prophage attachment site), but not for pro C (located to the other side of the attachment site), leu, or thy. Integration-deficient ( int) phage lysates were unable to transduce the pro A and pro B loci in Rec − cells unless int + helper phage were present. These results suggest that although Rec − cells cannot undergo general transduction, special transduction (by lysogenization) for the pro A and pro B loci is found. A mixture of int − and int + phage were unable to transduce the pro C locus despite the large number of pro C-transducing particles present in an int lysate. The inability of the int + phage genomes to promote successful lysogenization of pro C-transducing particles in Rec − cells may indicate a requirement for a structural element near the left prophage end for integration of phage P22. Alternatively, the deletion of c 2, the structural gene for P22 repressor, from most pro C-transducing particles may prevent successful lysogenization by these particles. Finally, the pro C locus may lie too far away from the prophage site to be included in special transducing particles containing any phage DNA.