P2 Growth Restriction on an rpoC Mutant Is Suppressed by Alleles of the Rz1 Homolog lysC

Abstract

Escherichia coli strain 397c carries a temperature-sensitive mutation, rpoC397, that removes the last 50 amino acids of the RNA polymerase ␤ 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 . Indeed, coexpression of lysB and lysC complemented the growth defect of 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.During bacteriophage infection, the machinery for macromolecular synthesis in the cell is recruited to serve the needs of the virus, and systematic changes in viral gene expression take place in a defined sequence. Bacterial DNA-dependent RNA polymerase (RNAP; subunit composition, a 2 ␤␤Ј), the enzyme responsible for most host transcription, is a major target of this regulation (14). Thus, mutations in RNAP rpo genes often specifically prevent bacteriophage development. To date, mutations that interfere with bacteriophage development have been identified in genes coding for all RNAP subunits except the smallest subunit, (reviewed in reference 34). Some of these mutations define RNAP sites that interact directly with viral regulators (25,26,40), while others affect phage gene expression indirectly by altering the properties of RNAP, such as the efficiency of transcription termination (39). The power of phage genetics, allowing isolation of suppressor mutations that overcome the blocks conferred by the changes in RNAP, has been invaluable in the study of these phenomena, and the results have greatly enriched our understanding of the basic processes of transcription (27).From this perspective, the block against plaque formation for phages P2 (10) and N4 (25) conferred by the rpoC397 mutation has long been provocative. This mutation removes 16 bp close to the end of rpoC, resulting in the replacement of the last 50 amino acids of RNAP subunit ␤Ј with 23 incorrect residues (10). In addition to being nonpermissive for the two phages, Escherichia coli strain 397c, carrying rpoC397, has a gross temperature-sensitive (Ts) growth phenotype. Based on biochemical experiments, the N4 block appears to be due to the loss of a contact between N4 SSB (single-strand DNA binding protein) and ␤Ј that is required for activation of viral late transcription (25). However, the mechanism by which the rpoC397 mutation prevents P2 growth has not been determined. Here, we report genetic and physio...

The Spanin Complex Is Essential for Lambda Lysis

“…The cation-dependent spherical cell phenotype has also been demonstrated for mutants in the two-component spanin genes of diverse phages, including the coliphage P2 (19) and the cystovirus PRD1 (15). Although videomicroscopy of infections in the absence of stabilizing cations needs to be done for a few of these systems, the nearly universal presence of either two-component or single-component spanin genes in the genomes of phages that grow on Gram-negative hosts (25) makes it likely that the lessons derived from lysis will be generally applicable.…”

“…In fact, the diversity and interchangeability of holins, endolysins, and spanins indicate that direct interaction is unlikely. For example, the holin and endolysin functions equally well with the phage P2 LysBC twocomponent spanins, which are encoded by an overlapped spanin gene architecture and have no sequence similarity with the lambda proteins (19). Likewise, the P22 endolysin, a glycosidase of the T4 lysozyme family, can substitute for the lambda endolysin, a transglycosylase, although the lambda and P22 holins are nearly identical (7).…”

Colicin Biology

“…It is localized in the outer membrane and plays a role in the timing of lysis in lysogenic cells following induction (328,700). Similar lipopeptides have been shown to be encoded by the lysis genes of other bacteriophages such as P2 and N4, which form heteromeric complexes and are required for host lysis (436).…”

Rz/Rz1 Lysis Gene Equivalents in Phages of Gram-negative Hosts

“…Nested open reading frames (ORFs) encoding a type II membrane protein and an outer membrane lipoprotein are found in the distal position of the lysis clusters of the two well-studied but unrelated phages T7 4,5 and P2 6,7 and several of their relatives (Figure 1(a)). The T7 Rz1 equivalent, 18.7, is located entirely within the Rz homolog, 18.5, in the + 1 reading frame.…”