Phenotypes of Bacillus subtilis mutants lacking multiple class A high-molecular-weight penicillin-binding proteins

Abstract

Examination of Bacillus subtilis strains containing multiple mutations affecting the class A high-molecularweight penicillin-binding proteins (PBPs) 1, 2c, and 4 revealed a significant degree of redundancy in the functions of these three proteins. In rich media, loss of PBPs 2c and 4 resulted in no obvious phenotype. The slight growth and cell morphology defects associated with loss of PBP 1 were exacerbated by the additional loss of PBP 4 but not PBP 2c. Loss of all three of these PBPs slowed growth even further. In minimal medium, loss of PBPs 2c and 4 resulted in a slight growth defect. The decrease in growth rate caused by loss of PBP 1 was accentuated slightly by loss of PBP 2c and greatly by loss of PBP 4. Again, a lack of all three of these PBPs resulted in the slowest growth. Loss of PBP 1 resulted in a 22% reduction in the cell radius. Cultures of a strain lacking PBP 1 also contained some cells that were significantly longer than those produced by the wild type, and some of the rod-shaped cells appeared slightly bent. The additional loss of PBP 4 increased the number of longer cells in the culture. Slow growth caused by a mutation in prfA, a gene found in an operon with the gene encoding PBP 1, was unaffected by the additional loss of PBPs 2c and 4, whereas loss of both prfA and PBP 1 resulted in extremely slow growth and the production of highly bent cells.The peptidoglycan component of eubacterial cell walls is required to maintain cell shape and resist the turgor pressure produced by high concentrations of cell solutes. Polymerization of the peptidoglycan is carried out on the outer surface of the cytoplasmic membrane by the family of penicillin-binding proteins (PBPs) (7). The PBPs have been divided into three classes based on sequence similarities (7). The low-molecularweight class of PBPs generally have D,D-carboxypeptidase activity and have not been found to be essential proteins (7). Some of the class B high-molecular-weight PBPs have been clearly shown to possess a transpeptidase activity involved in cross-linking peptidoglycan chains (9, 10, 12) and are required for cell septation and maintenance of cell shape (29,30,37). The class A high-molecular-weight PBPs appear to be bifunctional proteins with a transglycosylase N-terminal domain and a transpeptidase C-terminal domain (7,11,18,31). The two class A high-molecular-weight PBPs of Escherichia coli have been shown to have partially redundant functions (32, 38); loss of either PBP produces little or no phenotypic effect, whereas loss of both is lethal.Bacillus subtilis is studied as a model for cell differentiation and as a model gram-positive bacterium because of the availability of well-developed genetic tools. We have been studying the PBPs of B. subtilis in order to elucidate their specific roles in growth, division, and sporulation. Among the PBPs found in vegetative B. subtilis cells are the class A high-molecularweight PBPs 1, 2c, and 4, the products of the ponA, pbpF, and pbpD genes, respectively. We have previously described the lack...

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

“…Previously it was shown that cells of a prfA deletion mutant (⌬prfA::spc; strain PS2061) produce normal levels of PBP1 but grow more slowly and vary significantly in cell length compared to wild-type cells (33,34). An identical phenotype was observed for strain PS2123, which contains an in-frame deletion of prfA (33).…”

“…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 ϳ50% 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).…”

“…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.…”

“…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).…”

“…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).…”