Rod Shape Determination by the Bacillus subtilis Class B Penicillin-Binding Proteins Encoded by pbpA and pbpH

ABSTRACT The peptidoglycan cell wall determines the shape and structural integrity of a bacterial cell. Class B penicillin-binding proteins (PBPs) carry a transpeptidase activity that cross-links peptidoglycan strands via their peptide side chains, and some of these proteins are directly involved in cell shape determination. No Bacillus subtilis PBP with a clear role in rod shape maintenance has been identified. However, previous studies showed that during outgrowth of pbpA mutant spores, the cells grew in an ovoid shape for several hours before they recovered and took on a normal rod shape. It was postulated that another PBP, expressed later during outgrowth, was able to compensate for the lack of the pbpA product, PBP2a, and to guide the formation of a rod shape. The B. subtilis pbpH (ykuA) gene product is predicted to be a class B PBP with greatest sequence similarity to PBP2a. We found that a pbpH-lacZ fusion was expressed at very low levels in early log phase and increased in late log phase. A pbpH null mutant was indistinguishable from the wild-type, but a pbpA pbpH double mutant was nonviable. When pbpH was placed under the control of an inducible promoter in a pbpA mutant, viability was dependent on pbpH expression. Growth of this strain in the absence of inducer resulted in conversion of the cells from rods to ovoid/round shapes and lysis. We conclude that PBP2a and PbpH play redundant roles in formation of a rod-shaped peptidoglycan cell wall.

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A Mother Cell-Specific Class B Penicillin-Binding Protein, PBP4b, in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.186.1.258-261.2004 ◽

2004 ◽

Vol 186 (1) ◽

pp. 258-261 ◽

Cited By ~ 10

Author(s):

Yuping Wei ◽

Derrell C. McPherson ◽

David L. Popham

Keyword(s):

Bacillus Subtilis ◽

Rna Polymerase ◽

Mother Cell ◽

Binding Proteins ◽

Binding Protein ◽

Specific Class ◽

Penicillin Binding Protein ◽

Penicillin Binding Proteins ◽

Peptidoglycan Structure ◽

Class B

ABSTRACT The Bacillus subtilis genome encodes 16 penicillin-binding proteins (PBPs), some of which are involved in synthesis of the spore peptidoglycan. The pbpI (yrrR) gene encodes a class B PBP, PBP4b, and is transcribed in the mother cell by RNA polymerase containing σE. Loss of PBP4b, alone and in combination with other sporulation-specific PBPs, had no effect on spore peptidoglycan structure.

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Two Class A High-Molecular-Weight Penicillin-Binding Proteins of Bacillus subtilis Play Redundant Roles in Sporulation

Journal of Bacteriology ◽

10.1128/jb.183.20.6046-6053.2001 ◽

2001 ◽

Vol 183 (20) ◽

pp. 6046-6053 ◽

Cited By ~ 33

Author(s):

Derrell C. McPherson ◽

Adam Driks ◽

David L. Popham

Keyword(s):

Cell Wall ◽

Bacillus Subtilis ◽

Germ Cell ◽

Double Mutant ◽

Binding Proteins ◽

Penicillin Binding Proteins ◽

Class A ◽

Mutant Strains ◽

Spore Walls ◽

Multiple Class

ABSTRACT The four class A penicillin-binding proteins (PBPs) ofBacillus subtilis appear to play functionally redundant roles in polymerizing the peptidoglycan (PG) strands of the vegetative-cell and spore walls. The ywhE product was shown to bind penicillin, so the gene and gene product were renamedpbpG and PBP2d, respectively. Construction of mutant strains lacking multiple class A PBPs revealed that, while PBP2d plays no obvious role in vegetative-wall synthesis, it does play a role in spore PG synthesis. A pbpG null mutant produced spore PG structurally similar to that of the wild type; however, electron microscopy revealed that in a significant number of these spores the PG did not completely surround the spore core. In a pbpF pbpG double mutant this spore PG defect was apparent in every spore produced, indicating that these two gene products play partially redundant roles. A normal amount of spore PG was produced in the double mutant, but it was frequently produced in large masses on either side of the forespore. The double-mutant spore PG had structural alterations indicative of improper cortex PG synthesis, including twofold decreases in production of muramic δ-lactam and l-alanine side chains and a slight increase in cross-linking. Sporulation gene expression in the pbpF pbpG double mutant was normal, but the double-mutant spores failed to reach dormancy and subsequently degraded their spore PG. We suggest that these two forespore-synthesized PBPs are required for synthesis of the spore germ cell wall, the first layer of spore PG synthesized on the surface of the inner forespore membrane, and that in the absence of the germ cell wall the cells lack a template needed for proper synthesis of the spore cortex, the outer layers of spore PG, by proteins on the outer forespore membrane.

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An Antibiotic-Inducible Cell Wall-Associated Protein That Protects Bacillus subtilis from Autolysis

Journal of Bacteriology ◽

10.1128/jb.00403-07 ◽

2007 ◽

Vol 189 (13) ◽

pp. 4671-4680 ◽

Cited By ~ 37

Author(s):

Letal I. Salzberg ◽

John D. Helmann

Keyword(s):

Cell Wall ◽

Bacillus Subtilis ◽

Cell Envelope ◽

Sequence Similarity ◽

Envelope Stress ◽

Class B ◽

Dependent Cell ◽

A Cell ◽

Close Relatives ◽

Minimal Region

ABSTRACT In Bacillus subtilis, antibiotics that impair cell wall synthesis induce a characteristic stress response including the σW and σM regulons and the previously uncharacterized yoeB gene. Here we demonstrate that YoeB is a cell wall-associated protein with weak sequence similarity to a noncatalytic domain of class B penicillin-binding proteins. A yoeB-null mutant exhibits an increased rate of autolysis in response to cell wall-targeting antibiotics or nutrient depletion. This phenotype does not appear to be correlated with gross alterations in peptidoglycan structure or levels of autolysins. Promoter dissection experiments define a minimal region necessary for antibiotic-mediated induction of yoeB, and this region is highly conserved preceding yoeB homologs in close relatives of B. subtilis. These results support a model in which induction of YoeB in response to cell envelope stress decreases the activity of autolysins and thereby reduces the rate of antibiotic-dependent cell death.

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Penicillin-Binding Proteins (PBPs) and Bacterial Cell Wall Elongation Complexes

Subcellular Biochemistry - Macromolecular Protein Complexes II: Structure and Function ◽

10.1007/978-3-030-28151-9_8 ◽

2019 ◽

pp. 273-289

Author(s):

Mayara M. Miyachiro ◽

Carlos Contreras-Martel ◽

Andréa Dessen

Keyword(s):

Cell Wall ◽

Bacterial Cell ◽

Binding Proteins ◽

Bacterial Cell Wall ◽

Penicillin Binding Proteins

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Escherichia coli Mutants Lacking All Possible Combinations of Eight Penicillin Binding Proteins: Viability, Characteristics, and Implications for Peptidoglycan Synthesis

Journal of Bacteriology ◽

10.1128/jb.181.13.3981-3993.1999 ◽

1999 ◽

Vol 181 (13) ◽

pp. 3981-3993 ◽

Cited By ~ 195

Author(s):

Sylvia A. Denome ◽

Pamela K. Elf ◽

Thomas A. Henderson ◽

David E. Nelson ◽

Kevin D. Young

Keyword(s):

Escherichia Coli ◽

Cell Wall ◽

Binding Proteins ◽

Kanamycin Resistance ◽

Open Reading Frames ◽

Penicillin Binding Proteins ◽

E Coli ◽

Peptidoglycan Biosynthesis ◽

Kanamycin Resistance Cassette ◽

Genes Encoding

ABSTRACT The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biological roles. To better understand the contributions these proteins make to the physiology ofEscherichia coli, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two ressites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via λ phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in trans, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bpres site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, thedacD gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the β-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism.

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Dynamic localization of penicillin-binding proteins during spore development in Bacillus subtilis

Microbiology ◽

10.1099/mic.0.27692-0 ◽

2005 ◽

Vol 151 (3) ◽

pp. 999-1012 ◽

Cited By ~ 16

Author(s):

Dirk-Jan Scheffers

Keyword(s):

Bacillus Subtilis ◽

Green Fluorescent Protein ◽

Binding Proteins ◽

Cytoplasmic Membrane ◽

Fluorescent Protein ◽

Spore Formation ◽

The Other ◽

Spore Development ◽

Penicillin Binding Proteins ◽

Green Fluorescent

During Bacillus subtilis spore formation, many membrane proteins that function in spore development localize to the prespore septum and, subsequently, to the outer prespore membrane. Recently, it was shown that the cell-division-specific penicillin-binding proteins (PBPs) 1 and 2b localize to the asymmetric prespore septum. Here, the author studied the localization of other PBPs, fused to green fluorescent protein (GFP), during spore formation. Fusions to PBPs 4, 2c, 2d, 2a, 3, H, 4b, 5, 4a, 4* and X were expressed during vegetative growth, and their localization was monitored during sporulation. Of these PBPs, 2c, 2d, 4b and 4* have been implicated as having a function in sporulation. It was found that PBP2c, 2d and X changed their localization, while the other PBPs tested were not affected. The putative endopeptidase PbpX appears to spiral out in a pattern that resembles FtsZ redistribution during sporulation, but a pbpX knockout strain had no distinguishable phenotype. PBP2c and 2d localize to the prespore septum and follow the membrane during engulfment, and so are redistributed to the prespore membrane. A similar pattern was observed when GFP–PBP2c was expressed in the mother cell from a sporulation-specific promoter. This work shows that various PBPs known to function during sporulation are redistributed from the cytoplasmic membrane to the prespore.

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FmhA and FmhC of Staphylococcus aureus incorporate serine residues into peptidoglycan cross-bridges

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014371 ◽

2020 ◽

Vol 295 (39) ◽

pp. 13664-13676 ◽

Cited By ~ 1

Author(s):

Stephanie Willing ◽

Emma Dyer ◽

Olaf Schneewind ◽

Dominique Missiakas

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Binding Proteins ◽

Penicillin Binding Proteins ◽

Daughter Cells ◽

Cross Bridge ◽

Resistant Strains ◽

The Cross ◽

Cross Bridges ◽

Adjacent Wall

Staphylococcal peptidoglycan is characterized by pentaglycine cross-bridges that are cross-linked between adjacent wall peptides by penicillin-binding proteins to confer robustness and flexibility. In Staphylococcus aureus, pentaglycine cross-bridges are synthesized by three proteins: FemX adds the first glycine, and the homodimers FemA and FemB sequentially add two Gly-Gly dipeptides. Occasionally, serine residues are also incorporated into the cross-bridges by enzymes that have heretofore not been identified. Here, we show that the FemA/FemB homologues FmhA and FmhC pair with FemA and FemB to incorporate Gly-Ser dipeptides into cross-bridges and to confer resistance to lysostaphin, a secreted bacteriocin that cleaves the pentaglycine cross-bridge. FmhA incorporates serine residues at positions 3 and 5 of the cross-bridge. In contrast, FmhC incorporates a single serine at position 5. Serine incorporation also lowers resistance toward oxacillin, an antibiotic that targets penicillin-binding proteins, in both methicillin-sensitive and methicillin-resistant strains of S. aureus. FmhC is encoded by a gene immediately adjacent to lytN, which specifies a hydrolase that cleaves the bond between the fifth glycine of cross-bridges and the alanine of the adjacent stem peptide. In this manner, LytN facilitates the separation of daughter cells. Cell wall damage induced upon lytN overexpression can be alleviated by overexpression of fmhC. Together, these observations suggest that FmhA and FmhC generate peptidoglycan cross-bridges with unique serine patterns that provide protection from endogenous murein hydrolases governing cell division and from bacteriocins produced by microbial competitors.

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Class A PBPs have a distinct and unique role in the construction of the pneumococcal cell wall

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1917820117 ◽

2020 ◽

Vol 117 (11) ◽

pp. 6129-6138 ◽

Cited By ~ 9

Author(s):

Daniel Straume ◽

Katarzyna Wiaroslawa Piechowiak ◽

Silje Olsen ◽

Gro Anita Stamsås ◽

Kari Helene Berg ◽

...

Keyword(s):

Cell Wall ◽

Peptidoglycan Hydrolase ◽

Unique Role ◽

Penicillin Binding Proteins ◽

The Core ◽

Class A ◽

Peptidoglycan Synthesis ◽

Resistant Form ◽

Class B ◽

Open Question

In oval-shapedStreptococcus pneumoniae, septal and longitudinal peptidoglycan syntheses are performed by independent functional complexes: the divisome and the elongasome. Penicillin-binding proteins (PBPs) were long considered the key peptidoglycan-synthesizing enzymes in these complexes. Among these were the bifunctional class A PBPs, which are both glycosyltransferases and transpeptidases, and monofunctional class B PBPs with only transpeptidase activity. Recently, however, it was established that the monofunctional class B PBPs work together with transmembrane glycosyltransferases (FtsW and RodA) from the shape, elongation, division, and sporulation (SEDS) family to make up the core peptidoglycan-synthesizing machineries within the pneumococcal divisome (FtsW/PBP2x) and elongasome (RodA/PBP2b). The function of class A PBPs is therefore now an open question. Here we utilize the peptidoglycan hydrolase CbpD that targets the septum ofS. pneumoniaecells to show that class A PBPs have an autonomous role during pneumococcal cell wall synthesis. Using assays to specifically inhibit the function of PBP2x and FtsW, we demonstrate that CbpD attacks nascent peptidoglycan synthesized by the divisome. Notably, class A PBPs could process this nascent peptidoglycan from a CbpD-sensitive to a CbpD-resistant form. The class A PBP-mediated processing was independent of divisome and elongasome activities. Class A PBPs thus constitute an autonomous functional entity which processes recently formed peptidoglycan synthesized by FtsW/PBP2×. Our results support a model in which mature pneumococcal peptidoglycan is synthesized by three functional entities, the divisome, the elongasome, and bifunctional PBPs. The latter modify existing peptidoglycan but are probably not involved in primary peptidoglycan synthesis.

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