The pbpB gene, which encodes penicillin-binding protein (PBP) 2B of Bacillus subtilis, has been cloned, sequenced, mapped, and mutagenized. The sequence of PBP 2B places it among the class B high-molecular-weight PBPs. It appears to contain three functional domains: an N-terminal domain homologous to the corresponding domain of other class B PBPs, a penicillin-binding domain, and a lengthy carboxy extension. The PBP has a noncleaved signal sequence at its N terminus that presumably serves as its anchor in the cell membrane. Previous studies led to the hypothesis that PBP 2B is required for both vegetative cell division and sporulation septation. Its sequence, map site, and mutant phenotype support this hypothesis. PBP 2B is homologous to PBP 3, the cell division protein encoded by pbpB of Escherichia coli. Moreover, both pbpB genes are located in the same relative position within a cluster of cell division and cell wall genes on their respective chromosomes. However, immediately adjacent to the B. subtilis pbpB gene is spoVD, which appears to be a sporulation-specific homolog of pbpB. Inactivation of SpoVD blocked synthesis of the cortical peptidoglycan in the spore, whereas carboxy truncation of PBP 2B caused cells to grow as filaments. Thus, it appears that a gene duplication has occurred in B. subtilis and that one PBP has evolved to serve a common role in septation during both vegetative growth and sporulation, whereas the other PBP serves a specialized role in sporulation.
Penicillin-(cloxacillin-) resistant mutants of Bacillus subtilis were isolated in a stepwise fashion and the five penicillin-binding components (PBCs) in each were examined to determine which of the proteins, if any, corresponds to the penicillin killing site. PBCs III and V were previously eliminated as the likely penicillin target. In the present work, PBC IV showed no change in sensitivity to cloxacillin in any of the resistant mutants isolated. PBC I did not change until the fifth-step mutant, in which it could not be detected by penicillin binding. Since PBC I did not bind penicillins that are lethal for this mutant, it also cannot be the lethal target. PBC II showed increased resistance to cloxaciffin in three discrete steps, i.e., in mutants 1, 4, and 5, accompanied by changes in its electrophoretic mobility. However, the sensitivity of PBC II to penicillin C changed very little. Correspondingly, the cloxacillinresistant mutants were unaltered in their sensitivity to penicillin G in vivo. (12).Preparation of Membranes and Isolation of PBCs. Membranes were prepared by grinding cells with glass beads as described previously (9). The penicillin-binding components were isolated by affinity chromatography as described (10) except the Sepharose 4B200 (Sigma) was coupled with 6-aminohexanoic acid (Aldrich) prior to 6-aminopenicillanic acid (6-APA) (Sigma) substitution.Penicillin Binding Assays.[14C]Penicillin G (AmershamSearle, 53 mCi/mmol) was bound to B. subtilis membranes as described (9). Sensitivity of the purified PBCs to a variety of penicillins was measured by first prebinding with an unlabeled penicillin at different concentrations and then adding a saturating concentration of [14C]penicillin G. The protocol was similar to that described elsewhere (8). The binding reaction was stopped by adding a 250-fold excess of unlabeled penicillin G, followed by precipitation of the protein with 80% acetone. The protein precipitate was dried with a stream of air, boiled in sample buffer (see below), and applied to a gel.Sodium Dodecyl Sulfate (NaDodSO4) Polyacrylamide Gel Electrophoresis. Tube gels were run as described by Weber and Osborn (13). The gels were 8 cm long and subjected to electrophoresis for 6 hr at 8 mA per gel. Discontinuous NaDodSO4 polyacrylamide slab gels (2 mm) were run as described (14,15). The upper stacking gel was 3% acrylamide and the running gel was 7.5% acrylamide. The sample buffer contained 10% (vol/vol) glycerol, 5% mercaptoethanol, 3% NaDodSO4, 0.0625 M Tris-HCl, pH 6.8, and 0.002% bromphenol blue.Samples, suspended in 30 ;J of sample buffer and boiled for 5 min, were subjected to electrophoresis at 110 V for 3.5 hr. The gel was stained with Coomassie brilliant blue (16) for 2 hr at 370 followed by overnight destaining with 5% methanol and 7.5% acetic acid. "C-Labeled proteins in the dried slab gel were detected by fluorography (17). To quantitate the results, the x-ray film (after 1-4 days' exposure) was scanned with a double-beam recording microdensitometer manufactured by ...
A novel penicillin-binding protein (PBP 5*) with D,D-carboxypeptidase activity is synthesized by Bacilus subtilis, beginning at about stage m of sporulation. The complete gene (dacB) for this protein was cloned by immunoscreening of an expression vector library and then sequenced. The identity of dacB was verified not only by the size and cross-reactivity of its product but also by the presence of the nucleotide sequence that coded for the independently determined NH2 terminus of PBP 5*. Analysis of its complete amino acid sequence confirmed the hypothesis that this PBP is related to other active-site serine D,D-peptidases involved in bacterial cell wall metabolism. PBP 5* had the active-site domains common to all PBPs, as well as a cleavable amino-terminal signal peptide and a carboxy-terminal membrane anchor that are typical features of low-molecular-weight PBPs. Mature PBP 5* was 355 amino acids long, and its mass was calculated to be 40,057 daltons. What is unique about this PBP is that it is developmentally regulated. Analysis of the sequence provided support for the hypothesis that the sporulation specificity and mother cell-specific expression of dacB can be attributed to recognition of the gene by a sporulation-specific sigma factor. There was a good match of the putative promoter of dacB with the sequence recognized by sigma factor E (oE), the subunit of RNA polymerase that is responsible for early mother cell-specific gene expression during sporulation. Analysis of PBP 5* production by various spo mutants also suggested that dacB expression is on a oE-dependent pathway.The penicillin-binding proteins (PBPs) are a family of membrane-bound enzymes that are active in the metabolism of prokaryotic cell walls (16). They are evolutionarily related to some of the soluble P-lactamases and D,D-peptidases secreted by bacteria and also have some structural features in common with a penicillin-binding transmembrane protein involved in signal transduction (24,46,62). The active-site serine of all of these penicillin-interactive proteins is part of the conserved domain S-X-X-K, which is located near the amino terminus of the 1-lactamases and the mature form of those PBPs with a molecular mass of less than 50 kDa but more towards the middle of the sequence in the larger PBPs (24). Closer to the carboxy terminus, all of these proteins also have the sequence K-T-G, H-T-G, or K-S-G, which is an essential part of the tertiary structure of the active site (24). The class A ,-lactamases and the PBPs also have a third domain (S-X-N) in common, which is located between the other two (46).Our studies have focused on PBP 5*, which is different from all of the other PBPs described so far because it is developmentally regulated. This protein, which has D,Dcarboxypeptidase activity in vitro, is not synthesized by Bacillus subtilis until about stage III of sporulation (43,49,50). Although it has a lower molecular weight than any of the six vegetative PBPs in this species, it is not a derivative of one of them (10, 50). Its locatio...
The penicillin-binding proteins (PBPs) of Bacillus subtilis were examined in samples collected at various times from sporulating cultures and compared with the PBPs in a presporulation sample. Large increases in vegetative PBPs 2B and 3 and the appearance of at least one new PBP (42,000 daltons) occurred at reproducible times during sporulation. In some strains a second new PBP (60,000 daltons) was also produced. By comparing the PBP activities in sporulating cells and two spoO mutants we have classified these changes as sporulation-related events rather than the consequences of stationary-phase aging. The other vegetative PBPs (PBPs 1, 2A, 4, and 5) decreased during sporulation, but not in sufficient amount or at the appropriate time to account for the appearance of the new proteins. A possible connection between specific PBP changes and the penicillinsensitive stages of sporulation is suggested.
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