Bacillus subtilis strains lacking penicillin-binding protein 1 (PBP1), encoded by ponA, required greater amounts of Mg2+ or Ca2+ for vegetative growth or spore outgrowth than the wild-type strain and strains lacking other high-molecular-weight (HMW) PBPs. Growth of ponA cells in a medium low in Mg2+ also resulted in greatly increased cell bending compared to wild-type cells or cells lacking other HMW PBPs. The addition of high levels of Mg2+ to growth media eliminated these phenotypes of a ponA mutant. In contrast to the effects of divalent cations, NaCl did not restoreponA cell growth in a divalent-cation-deficient medium. Surprisingly, wild-type cells swelled and then lysed during both vegetative growth and spore outgrowth when 500 mM NaCl was included in a divalent-cation-deficient medium. Again, Mg2+ addition was sufficient to allow normal vegetative growth and spore outgrowth of both wild-type and ponA cells in a medium with 500 mM NaCl. These studies demonstrate that (i) while HMW PBPs possess largely redundant functions in rich medium, when divalent cations are limiting, PBP1 is required for cell growth and spore outgrowth; and (ii) high levels of NaCl induce cell lysis in media deficient in divalent cations during both vegetative growth and spore outgrowth.

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...

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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...

The loss of Bacillus subtilis penicillin-binding protein (PBP) 2a, encoded by pbpA, was previously shown to slow spore outgrowth and result in an increased diameter of the outgrowing spore. Further analyses to define the defect inpbpA spore outgrowth have shown that (i) outgrowingpbpA spores exhibited only a slight defect in the rate of peptidoglycan (PG) synthesis compared to wild-type spores, but PG turnover was significantly slowed during outgrowth of pbpAspores; (ii) there was no difference in the location of PG synthesis in outgrowing wild-type and pbpA spores once cell elongation had been initiated; (iii) outgrowth and elongation of pbpAspores were dramatically affected by the levels of monovalent or divalent cations in the medium; (iv) there was a partial redundancy of function between PBP2a and PBP1 or -4 during spore outgrowth; and (v) there was no difference in the structure of PG from outgrowing wild-type spores or spores lacking PBP2a or PBP2a and -4; but also (vi) PG from outgrowing spores lacking PBP1 and -2a had transiently decreased cross-linking compared to PG from outgrowing wild-type spores, possibly due to the loss of transpeptidase activity.