Identification and characterization of pbpA encoding Bacillus subtilis penicillin-binding protein 2A

Abstract

Amino acid sequence analysis of tryptic peptides derived from purified penicillin-binding protein PBP2a of Bacillus subtilis identified the coding gene (now termed pbpA) as yqgF, which had been sequenced as part of the B. subtilis genome project; pbpA encodes a 716-residue protein with sequence similarity to class B highmolecular-weight PBPs. Use of a pbpA-lacZ fusion showed that pbpA was expressed predominantly during vegetative growth, and the transcription start site was mapped using primer extension analysis. Insertional mutagenesis of pbpA resulted in no changes in the growth rate or morphology of vegetative cells, in the ability to produce heat-resistant spores, or in the ability to trigger spore germination when compared to the wild type. However, pbpA spores were unable to efficiently elongate into cylindrical cells and were delayed significantly in spore outgrowth. This provides evidence that PBP2a is involved in the synthesis of peptidoglycan associated with cell wall elongation in B. subtilis.Penicillin-binding proteins (PBPs) are enzymes involved in a number of reactions of peptidoglycan biosynthesis and have been divided into three classes based on sequence similarity (9). In Escherichia coli, the class A high-molecular-weight PBPs have been shown to possess a transglycosylase activity involved in polymerization of the peptidoglycan's sugar backbone (13,18,37). The class A and class B high-molecularweight PBPs of E. coli exhibit transpeptidase activity, which results in peptide cross-links between adjacent glycan strands (11-13, 18, 37), while the low-molecular-weight PBPs of E. coli and Bacillus subtilis are most often carboxypeptidases (9,15,38).In B. subtilis PBPs are involved not only in the synthesis of the peptidoglycan sacculus but also in the synthesis of both the germ cell wall and the cortex of the dormant spore. The latter structure is different from the vegetative cell wall peptidoglycan in that the spore cortex contains muramic acid lactam residues (39). Alteration of the spore cortex structure can affect spore resistance properties as well as spore germination and outgrowth (3,10,(23)(24)(25).To understand in detail the contribution of individual PBPs to cell and spore properties as well as to peptidoglycan structure in B. subtilis, we have been identifying and characterizing the genes encoding these proteins (17,(26)(27)(28)(29). With the exception of PBP2b, which is an essential protein (41), the loss of individual PBPs in B. subtilis has not been associated with dramatic phenotypic changes (17,(26)(27)(28)(29)40). Indeed, cells lacking as many as three class A high-molecular-weight PBPs (PBP1, PBP2c, and PBP4) are viable, exhibiting only a slight growth defect, indicating a redundancy of function amongst the different high-molecular-weight PBPs (30). Furthermore, while PBP2b is thought to be responsible for septum formation in B. subtilis (41), no single B. subtilis PBP responsible for cell elongation has been identified. In contrast, mutations in pbpA encoding PBP2 of E. coli resul...

Transcriptional responses during outgrowth of Bacillus subtilis endospores

“…During outgrowth of MJH003 (sigM) in NB containing 700 mM NaCl, the spores formed large round cells that failed to elongate (data not shown). A similar morphological defect during outgrowth was observed for a pbpA mutant of B. subtilis (Murray et al, 1997). However, MJH002 (sigM) did not have altered expression of pbpA, compared to 1604 (wild-type), when measured using a pbpA-lacZ fusion (data not shown).…”

Staying in Shape: the Impact of Cell Shape on Bacterial Survival in Diverse Environments

“…These observations imply that coccoid cells result from rods that have lost the ability to elongate. Moreover, examples of rods changing into spheres have been demonstrated both genetically and biochemically with the use of antibiotics, thus lending support to this model (28)(29)(30)(31)(32). Indeed, a recent paper describing the possible evolutionary steps that might have driven the bacillus-to-coccoid shape transition in Neisseria meningitidis provides evidence that the process happened in two distinct genetic steps.…”

Signal Peptide-Dependent Protein Transport in Bacillus subtilis : a Genome-Based Survey of the Secretome

“…In B. subtilis, proteins retained in the cell wall include DNases, RNases (173), proteases (10,163,274), enzymes involved in the synthesis of peptidoglycan (penicillin-binding proteins), and cell wall hydrolases (20,96,97) that are involved in cell wall turnover during cell growth, cell division, sporulation, and germination (181,182,219,335).…”