Identification and characterization of pbpC, the gene encoding Bacillus subtilis penicillin-binding protein 3

Abstract

Penicillin-binding proteins (PBPs) are enzymes involved in the synthesis of peptidoglycan structures inBacillus subtilis such as the vegetative cell wall and the spore cortex. The B. subtilis sequencing project has identified a gene (orf16, EMBL accession number D38161) which exhibits significant sequence similarity to genes encoding class B high-molecular-weight PBPs. We have found that orf16 encodes PBP3 and have renamed this locus pbpC. Transcriptional fusions to lacZ were used to demonstrate that pbpC is transcribed primarily during log-phase growth, with lower amounts expressed during sporulation. During spore germination and outgrowth, pbpC expression resumes coincident with an increase in the optical density of the culture. The major promoter for pbpC is located just upstream of the gene; a low level of expression during sporulation appears to originate from much further upstream. Loss of PBP3 does not produce any detectable change in phenotype with respect to cell morphology, growth, sporulation, spore heat resistance, or spore germination and outgrowth. This was also true when the pbpC mutation was combined with mutations affecting other PBP-encoding genes to produce double mutants. These findings are consistent with previous evidence that many PBPs of B. subtilis have redundant functions within the cell.Peptidoglycan synthesis is required for the elongation of the vegetative cell wall, septation, and formation of the spore cortex in gram-positive bacteria such as Bacillus subtilis. A class of proteins known as penicillin-binding proteins (PBPs) have been shown to exhibit the enzymatic activities involved in the polymerization and cross-linking of peptidoglycan (11). The penicillin-binding domain of PBPs has three highly conserved protein motifs, including SxxK, which contains the active-site serine that covalently binds penicillin and other ␤-lactam antibiotics (11). As this covalent interaction inhibits the activity of these enzymes, it is not surprising that modification of these proteins has been responsible for one type of ␤-lactam antibiotic resistance. One example is the synthesis of the novel lowaffinity PBP2Ј by methicillin-resistant Staphylococcus aureus (12).We have undertaken the characterization of the genes encoding PBPs involved in the synthesis and modification of peptidoglycan in B. subtilis. To date, 11 different PBPs have been identified biochemically, and the genes encoding 9 of these PBPs are known (5, 6, 8, 9, 14, 26-29, 35, 37, 38, 40). However, the genes encoding two relatively abundant PBPs, PBP2a and PBP3, have yet to be identified. In addition, three genes, dacF (39) and two genes identified by the B. subtilis genome sequencing project (EMBL accession numbers Z34883 and D38161), appear to encode PBPs, but the protein products of these genes remain to be elucidated. The product of one of the genes identified by the B. subtilis genome sequencing project (orf16 [2], EMBL accession number D38161, entered as the ycsM gene in SubtiList [18]) has significant amino acid sequence simil...

FemABX Peptidyl Transferases: a Link between Branched-Chain Cell Wall Peptide Formation and β-Lactam Resistance in Gram-Positive Cocci

“…The low-affinity PBPs of enterococci and staphylococci are closely related and form PBP subclass B1, which stands apart from the unmodified class B PBPs and also from S. pneumoniae PBP2x (39). As an exception, Bacillus subtilis PBP3 groups in the B1 cluster but has not been linked to any low-affinity property (70). Class B PBPs must cooperate with either a monofunctional transglycosylase or the transglycosylase domain of a class A PBP to synthesize cross-linked peptidoglycan.…”

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).…”

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

“…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).…”