Penicillin-Binding Protein-Related Factor A Is Required for Proper Chromosome Segregation in Bacillus subtilis

Abstract

Previous work has shown that the ponA gene, encoding penicillin-binding protein 1 (PBP1), is in a two-gene operon with prfA (PBP-related factor A) (also called recU), which encodes a putative 206-residue basic protein (pI ‫؍‬ 10.1) with no significant sequence homology to proteins with known functions. Inactivation of prfA results in cells that grow slower and vary significantly in length relative to wild-type cells. We now show that prfA mutant cells have a defect in chromosome segregation resulting in the production of ϳ0.9 to 3% anucleate cells in prfA cultures grown at 30 or 37°C in rich medium and that the lack of PrfA exacerbates the chromosome segregation defect in smc and spoOJ mutant cells. In addition, overexpression of prfA was found to be toxic for and cause nucleoid condensation in Escherichia coli.Penicillin-binding proteins (PBPs), which catalyze the polymerization and cross-linking of bacterial peptidoglycan, can be divided into three classes based on their amino acid sequence: the low-molecular-weight PBPs and the high-molecular-weight (HMW) class A and class B PBPs (8). The HMW class B PBPs are monofunctional transpeptidases, some of which have essential functions in septation and maintenance of cell shape (8), while the HMW class A PBPs have both transglycosylase and transpeptidase activities (14, 42) and appear to be somewhat functionally redundant (17, 34). The Bacillus subtilis ponA gene, coding for the HMW class A PBP1, is transcribed predominantly during log-phase growth (34). Previous work with B. subtilis mutants lacking one or several of the three known HMW class A PBPs (PBP1, PBP2c, and PBP4) showed that (i) lack of PBP1 results in slower growth, increased cell length, and decreased cell diameter and (ii) PBP1 is functionally more important than PBP2c and PBP4 (34). It was also recently demonstrated that PBP1 localizes to cell division sites and plays an important role in the formation of the peptidoglycan division septum in vegetative cells of B. subtilis (30).The ponA gene is part of a two-gene operon that also includes prfA (PBP-related factor A [note that in the B. subtilis genome database, prfA is called recU]), which is located immediately upstream of and cotranscribed with ponA (33). prfA codes for a putative 206 residue, basic protein (pI of 10.1), which has no significant sequence homology to proteins with known functions. However, DNA sequencing has indicated that genes encoding similar proteins are present in a large number of gram-positive bacteria: a BLAST search (2) of prfA against completed and unfinished microbial genomes (preliminary sequence data were obtained from The Institute of Genomic Research Website at http://www.tigr.org) produced sequences with significant homology from 10 different grampositive organisms, while no prfA homologs were detected in gram-negative bacteria by this analysis. The former organisms include Enterococcus faecalis (54% identity in a 192-aminoacid overlap), Staphylococcus aureus (58% identity in a 167-amino-acid overlap), Streptococcus p...

The RuvAB Branch Migration Translocase and RecU Holliday Junction Resolvase Are Required for Double-Stranded DNA Break Repair in Bacillus subtilis

“…Unlike Streptococcus pneumoniae in which the recU gene (TIGR SP0370) is apparently essential (Thanassi et al 2002), a B. subtilis DrecU mutant is viable, although it grows poorly (Table 2) and accumulates suppressor mutations at a high frequency (Pedersen and Setlow 2000;Carrasco et al 2002Carrasco et al , 2004. Therefore, the DruvAB null mutation was transferred into representatives from the different epistatic groups (a, recF15 and DrecO; b, addA5 addB72; g, recH342; e, recD41; and z, DrecS, DrecQ, and DrecJ strains), the double and triple mutants were exposed to MMS, 4NQO, or MMC, and their phenotypes were recorded.…”

“…Previously we reported the construction of ruvA2 recU40 double-mutant strains (Alonso et al 1992) and here report the construction of the recD41 DruvAB strain. However, the recU40 strain is proficient in plasmid transformation and shows a doubling time similar to the wild-type strain, whereas a DrecU is significantly impaired in plasmid transformation and shows a marked growth defect (Fernandez et Pedersen and Setlow 2000;Carrasco et al 2002; Table 2). It is likely that the recU40 allele may encode only a partially defective HJ resolvase.…”

Genetic Recombination inBacillus subtilis168: Contribution of Holliday Junction Processing Functions in Chromosome Segregation

“…Using an indirect measurement (measurement of repair centers as a measurement of blocked replication forks), we assumed that replication fork reloading might occur with a similar frequency in B. subtilis cells (25). The rate of formation of RecN-RecOF repair centers in the absence of any exogenous DNA-damaging agent was found to be about 35 and 5% in exponentially growing ⌬recA and ⌬recU cells, respectively (25).A defect in the HJ resolvase RecU (3) (7,20,35,42,45,56). The ⌬recU ⌬smc double mutant does not seem to be viable.…”

“…A defect in the HJ resolvase RecU (3) (also termed penicillin-binding protein [PBP]-related factor A [designated PrfA]) or in the DNA organizer SMC complex (formed by the Smc, ScpA, and ScpB proteins) in an otherwise wild-type (wt) background, leads to the accumulation of anucleate cells (ϳ3 and 10%, respectively) (7,20,35,42,45,56). The ⌬recU ⌬smc double mutant does not seem to be viable.…”

“…A defect in the HJ resolvase RecU (3) (7,20,35,42,45,56). The ⌬recU ⌬smc double mutant does not seem to be viable.…”