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

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 gram-positive 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-amino-acid overlap), Staphylococcus aureus (58% identity in a 167-amino-acid overlap), Streptococcus pyogenes (51% identity in a 195-amino-acid overlap), S. pneumoniae (49% identity in a 124-amino-acid overlap), Deinococcus radiodurans (49% identity in a 124-amino-acid overlap), and Mycoplasma genitalium (32% identity in a 187-amino-acid overlap). For at least two of these species, namely, S. pneumoniae and S. aureus, the prfA genes are also located upstream of ponA homologs (25, 32). Although the prfA gene product has not yet been identified in B. subtilis, it is known that inactivation of prfA results in cells that grow ∼50% more slowly than do wild-type cells and vary significantly in cell length. This phenotype is exacerbated greatly by the additional loss of PBP1 but not by the loss of either PBP2c or PBP4 (34). Finally, it has been shown that a mutation in prfA (recU::cat) renders cells more sensitive to DNA-damaging agents and decreases the efficiency of transformation, suggesting a possible role for prfA in DNA repair and homologous recombination (7). Given that PBP1 plays an important role in cell division in B. subtilis (30) and that a prfA mutation has a clear phenotype (34), we have investigated the function of prfA in detail. Our results indicate that prfA is required for proper chromosome segregation in B. subtilis.