The sporulation-specific penicillin-binding protein 5a from <i>Bacillus subtilis</i> is a <scp>dd</scp>-carboxypeptidase <i>in vitro</i>

Todd, John A.; Bone, Eileen J.

doi:10.1042/bj2300825

Cited by 26 publications

(14 citation statements)

References 13 publications

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“…Among the structural features that PBP 5* has in common with most other low-molecular-weight PBPs are a cleavable NH2-terminal signal sequence, a carboxy-terminal sequence that can form a potential membrane anchor as an amphilphilic a-helix, three conserved activesite domains, and overall sequence identity of at least 30%. These new data are consistent with other features that were already known, namely, that PBP 5* is membrane bound in B. subtilis, reacts enzymatically with ,B-lactam antibiotics, and has D,D-carboxypeptidase activity in vitro (10, 43,49,50).…”

Section: Discussionsupporting

confidence: 79%

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Isolation and sequence analysis of dacB, which encodes a sporulation-specific penicillin-binding protein in Bacillus subtilis

Buchanan

Ling

1992

J Bacteriol

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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...

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Section: Discussionsupporting

confidence: 79%

“…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).…”

mentioning

confidence: 99%

Isolation and sequence analysis of dacB, which encodes a sporulation-specific penicillin-binding protein in Bacillus subtilis

Buchanan

Ling

1992

J Bacteriol

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“…Several spore-specific penicillin-binding proteins which have been proposed to have a role in cortex biosynthesis have been identified either biochemically or by sequence homology (12,47,53). PBP 5* from B. subtilis was purified by affinity chromatography and found to have DD-carboxypeptidase activity in vitro (51). This enzyme is located primarily in the outer forespore membrane, and its transcription is under the control of the mother-cell-specific sigma factor, E (7,8).…”

Section: Discussionmentioning

confidence: 99%

Structural analysis of Bacillus subtilis 168 endospore peptidoglycan and its role during differentiation

et al. 1996

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The structure of the endospore cell wall peptidoglycan of Bacillus subtilis has been examined. Spore peptidoglycan was produced by the development of a method based on chemical permeabilization of the spore coats and enzymatic hydrolysis of the peptidoglycan. The resulting muropeptides which were >97% pure were analyzed by reverse-phase high-performance liquid chromatography, amino acid analysis, and mass spectrometry. This revealed that 49% of the muramic acid residues in the glycan backbone were present in the ␦-lactam form which occurred predominantly every second muramic acid. The glycosidic bonds adjacent to the muramic acid ␦-lactam residues were resistant to the action of muramidases. Of the muramic acid residues, 25.7 and 23.3% were substituted with a tetrapeptide and a single L-alanine, respectively. Only 2% of the muramic acids had tripeptide side chains and may constitute the primordial cell wall, the remainder of the peptidoglycan being spore cortex. The spore peptidoglycan is very loosely cross-linked at only 2.9% of the muramic acid residues, a figure approximately 11-fold less than that of the vegetative cell wall. The peptidoglycan from strain AA110 (dacB) had fivefold-greater cross-linking (14.4%) than the wild type and an altered ratio of muramic acid substituents having 37.0, 46.3, and 12.3% ␦-lactam, tetrapeptide, and single L-alanine, respectively. This suggests a role for the DacB protein (penicillin-binding protein 5*) in cortex biosynthesis. The sporulationspecific putative peptidoglycan hydrolase CwlD plays a pivotal role in the establishment of the mature spore cortex structure since strain AA107 (cwlD) has spore peptidoglycan which is completely devoid of muramic acid ␦-lactam residues. Despite this drastic change in peptidoglycan structure, the spores are still stable but are unable to germinate. The role of ␦-lactam and other spore peptidoglycan structural features in the maintenance of dormancy, heat resistance, and germination is discussed. Dormant bacterial endospores formed by the genera Bacillus and Clostridium are the most resistant living structures known and are able to survive thousands if not millions of years (9). During the quiescent state, spores exhibit high-level resistance to many treatments, including heat, UV light, desiccation, and the action of deleterious chemicals. As a result of their resistance properties, spores are able to survive many food preservation and pasteurization procedures and thus cause huge problems to the food industry (11).Endospores are characterized by a relatively dehydrated protoplast encased in integument layers (20). The most prominent of the integuments are the spore coat layers which determine the physical properties of the spore surface and are responsible for resistance to enzymatic assault (56). However, the spore coats are not involved in the maintenance of dormancy and heat resistance (56). Between the spore coats and the protoplast membrane is a thick layer of bacterial peptidoglycan, consisting of two sublayers. Innermost is the ...

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“…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).…”

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confidence: 99%

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

1997

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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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The sporulation-specific penicillin-binding protein 5a from Bacillus subtilis is a dd-carboxypeptidase in vitro

Abstract: The sporulation-specific penicillin-binding protein 5a was purified from Bacillus subtilis and shown to possess dd-carboxypeptidase activity in vitro.

Cited by 26 publications

References 13 publications

Isolation and sequence analysis of dacB, which encodes a sporulation-specific penicillin-binding protein in Bacillus subtilis

Isolation and sequence analysis of dacB, which encodes a sporulation-specific penicillin-binding protein in Bacillus subtilis

Structural analysis of Bacillus subtilis 168 endospore peptidoglycan and its role during differentiation

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

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