A comparison of the DNA binding and bending capacities and the oligomeric states of the immunity repressors of heteroimmune coliphages P2 and WPhi.

Bacteriophages P2 and WPhi are heteroimmune members of the P2-like family of temperate Escherichia coli phages. Temperate phages can grow lytically or form lysogeny after infection. A transcriptional switch that contains two con-vergent promoters, Pe and Pc, and two repressors regulate what life mode to enter. The immunity repressor C is the first gene of the lysogenic operon, and it blocks the early Pe promoter. In this work, some characteristics of the C proteins of P2 and WPhi are compared. An in vivo genetic analysis shows that WPhi C, like P2 C, has a strong dimerization activity in the absence of its DNA target. Both C proteins recognize two directly repeated sequences, termed half-sites and a strong bending is induced in the respective DNA target upon binding. P2 C is unable to bind to one half-site as opposed to WPhi, but both half-sites are required for repression of WPhi Pe. A reduction from three to two helical turns between the centers of the half-sites in WPhi has no significant effect on the capacity to repress Pe. However, the protein-DNA complexes formed differ, as determined by electrophoretic mobility shift experiments. A difference in spontaneous phage production is observed in isogenic lysogens.

P2 growth restriction on an rpoC mutant is suppressed by alleles of the Rz1 homolog lysC.

Escherichia coli strain 397c carries a temperature-sensitive mutation, rpoC397, that removes the last 50 amino acids of the RNA polymerase beta' subunit and is nonpermissive for plating of bacteriophage P2. P2 gor mutants productively infect 397c and define a new gene, lysC, encoded by a reading frame that extensively overlaps the P2 lysis accessory gene, lysB. The unusual location of lysC with respect to lysB is reminiscent of the Rz/Rz1 lysis gene pair of phage lambda. Indeed, coexpression of lysB and lysC complemented the growth defect of lambda Rz/Rz1 null mutants, indicating that the LysB/C pair is similar to Rz/Rz1 in both gene arrangement and function. Cells carrying the rpoC397 mutation exhibited an early onset of P2-induced lysis, which was suppressed by the gor mutation in lysC. We propose that changes in host gene expression resulting from the rpoC397 mutation result in changes in the composition of the bacterial cell wall, making the cell more susceptible to P2-mediated lysis and preventing accumulation of progeny phage sufficient for plaque formation.

Capsid size determination in the P2-P4 bacteriophage system: suppression of sir mutations in P2's capsid gene N by supersid mutations in P4's external scaffold gene sid.

The sid gene of the P2-dependent phage P4 provides an external scaffold so P2 N gene encoded protomers assemble as T = 4 capsids rather than as P2's T = 7 capsids. Mutations (sir) in the middle of N interfere with Sid's function. We describe a new P4 mutant class, nms ("supersid") mutations, which direct also P2 sir to provide small capsids. Three different nms mutations were located near the sid end, commingled with sid(-) mutations. Suppression of sir by nms is not allele-specific. Our results favor this interpretation of capsid size control: (i) sir mutations reduce pN protomer flexibility and thereby interfere with the generation of T = 4 compatible hexons; (ii) the C-termini of Sid molecules link up when forming the scaffold; nms mutations strengthen these Sid-Sid contacts and thus allow the scaffold to force even sir-type protomers to form T = 4 compatible hexons. Some related findings concern suppression of N ts mutations by P4.

Escherichia coli rpoC397 encodes a temperature-sensitive C-terminal frameshift in the beta' subunit of RNA polymerase that blocks growth of bacteriophage P2.

Escherichia coli 397c is temperature sensitive for growth at 43.5 degrees C and unable to plate bacteriophage P2 at 33 degrees C. The mutation conferring these phenotypes was mapped to the rpoC gene. RNA synthesis is temperature sensitive in the mutant strain, and the beta' subunit of RNA polymerase isolated from this strain exhibits increased electrophoretic mobility. DNA sequence analysis revealed that the mutation is a deletion of 16 bp, resulting in a frameshift that leads to truncation of the beta' subunit at the carboxy terminus.

Functional characterization of the P2 A initiator protein and its DNA cleavage site.

The A protein of bacteriophage P2 initiates DNA replication by a single-stranded cut at the origin, and the DNA replication proceeds unidirectionally by a modified rolling circle type of replication. The P2 A protein belongs to a family of proteins involved in the initiation of rolling circle DNA replication, and the prototype for this family is the well-characterized A protein of phage phi X174. One of the common motifs of this family contains two conserved tyrosine residues, which have been shown to be able to alternate in catalyzing the cleavage as well as joining reactions in the phi X174 A protein. We investigated the role of the conserved tyrosine residues in P2 A protein by in vitro mutagenesis. Only one of the two conserved tyrosine residues was found to be involved in the cleavage reaction. The tyrosine residue dispensable for cleavage and ligation is, however, required at some other stage of the P2 growth cycle, since viable recombinants containing this mutation could not be obtained. The sequence requirements for cleavage of the target site were analyzed with a set of oligonucleotides having single base alterations in the nick region, and the results indicate that only five core nucleotides need to be conserved for efficient cleavage.

Bacteriophage P2 and P4 morphogenesis: assembly precedes proteolytic processing of the capsid proteins.

Several of the structural proteins of phage P2 and its satellite P4 undergo proteolytic processing during development of mature phage particles. Here, we report that uncleaved shell protein, gpN, is present in immature capsids of both P2 and P4, showing that assembly precedes processing. This excludes the possibility that processing of gpN is involved in capsid size determination. We also find that N*, the fully processed version of gpN, produced from a plasmid, can assemble into both P2- and P4-sized particles, implying that the amino-terminal end of gpN is not required for assembly initiation nor for the formation of a T = 4 shell. As may be expected for a scaffolding protein, we find that gpO coexists with gpN in immature P2, as well as P4, capsids. This result supports the conclusion that gpO is required for both phages and strongly suggests that the O derivative, h7 (found in mature capsids), results from proteolytic cleavage after gpN/gpO coassembly.

DNA replication studies with coliphage 186: the involvement of the Escherichia coli DnaA protein in 186 replication is indirect.

The inability of coliphage 186 to infect productively a dnaA(Ts) mutant at a restrictive temperature was confirmed. However, the requirement by 186 for DnaA is indirect, since 186 can successfully infect suppressed dnaA (null) strains. The block to 186 infection of a dnaA(Ts) strain at a restrictive temperature is at the level of replication but incompletely so, since some 20% of the phage specific replication seen with infection of a dnaA+ host does occur. A mutant screen, to isolate host mutants blocked in 186-specific replication but not in the replication of the close relative coliphage P2, which has no DnaA requirement, yielded a mutant whose locus we mapped to the rep gene. A 186 mutant able to infect this rep mutant was isolated, and the mutation was located in the phage replication initiation endonuclease gene A, suggesting direct interaction between the Rep helicase and phage endonuclease during replication. DNA sequencing indicated a glutamic acid-to-valine change at residue 155 of the 694-residue product of gene A. In the discussion, we speculate that the indirect need of DnaA function is at the level of lagging-strand synthesis in the rolling circle replication of 186.

Molecular cloning and characterization of bacteriophage P2 genes R and S involved in tail completion.

The sequences of two previously known tail genes, R and S, of the temperate bacteriophage P2 and the sequence of an additional open reading frame (orf-30) located between S and V, were determined. Amber mutations mapping within R and S, Ram3, Ram42, Ram23, Sam75, and Sam89 were sequenced and found to be within their corresponding open reading frames. We constructed overproducing plasmids for R and S and identified these proteins by SDS-PAGE of whole-cell lysates and Coomassie blue staining. The predicted molecular masses of proteins R and S were M(r) 17,400 and 17,300, respectively, although both polypeptides migrated more slowly during gel electrophoresis than would be expected from the sequence data. orf-30 occupies the strand opposite from RS and V and is preceded by several weak potential sigma 70-RNA polymerase promoters, some of which overlap with the V promoter. A construct that had the putative orf-30 promoter region upstream of the lacZ gene produced low levels of beta-galactosidase activity in vivo. Expression from the orf-30 promoter was not stimulated by the phage P4 transcriptional activator protein, delta, which acts at all the known P2 and P4 late promoters. Insertion mutagenesis showed that orf-30 was not an essential gene for P2 growth in Escherichia coli. None of the gene or protein sequences exhibited extensive homology to sequences in the nucleic acid and protein databases. However, the R protein contains a small region homologous to one in the phage T4 tail protein gp15, which is required for T4 tails to bind heads. We propose that R and S are tail completion proteins that are essential for stable head joining.

Bacteriophage P2 and P4 assembly: alternative scaffolding proteins regulate capsid size.

The capsid protein of bacteriophage P2, encoded by the N gene, can assemble into icosahedral capsids of two possible sizes, with diameters of 60 and 45 nm, respectively. Only the larger capsid is used by P2 itself, but the smaller one is exploited by the satellite phage P4. We have analyzed the assembly products of gpN expressed in vivo from a plasmid, i.e., in the absence of any other phage proteins, and find that gpN alone forms closed shells of both sizes, although with poor efficiency. Coexpressing gpN with gpO, the putative P2 scaffolding protein, increases the efficiency of large particle formation. In contrast, introducing the sid gene by P4 infection stimulates the assembly of small particles. Our results suggest that gpO and gpSid act competitively with respect to capsid size determination. Furthermore, we demonstrate that gpN alone undergoes the normal proteolytic maturation steps, implying that gpN processing is either autocatalytic or mediated by a host enzyme.

Bacteriophage P2 and P4 morphogenesis: identification and characterization of the portal protein.

The portal structure has been implicated in several aspects of the bacteriophage life cycle, including capsid assembly initiation and DNA packaging. Here we present evidence that P2 gene Q codes for the P2 and P4 portal protein. First, microsequencing shows that capsid protein h6 is derived from gpQ, most probably by proteolytic cleavage. Second, antibodies against gpQ bind to the portal structure in disrupted P2 phage virions, as observed by electron microscopy. Third, gpQ partially purified from an overexpressing plasmid assembles into portal-like structures. We also show by microsequencing that capsid protein h7 is encoded by the P2 scaffold gene, O, and is probably derived from gpO by proteolytic cleavage. Previous work has demonstrated processing of the major capsid protein. Thus, all essential capsid proteins of P2 and P4 are proteolytically cleaved during the morphogenetic process.