Decision letter: Class-A penicillin binding proteins do not contribute to cell shape but repair cell-wall defects

Cell shape and cell-envelope integrity of bacteria are determined by the peptidoglycan cell wall. In rod-shaped Escherichia coli, two conserved sets of machinery are essential for cell-wall insertion in the cylindrical part of the cell: the Rod complex and the class-A penicillin-binding proteins (aPBPs). While the Rod complex governs rod-like cell shape, aPBP function is less well understood. aPBPs were previously hypothesized to either work in concert with the Rod complex or to independently repair cell-wall defects. First, we demonstrate through modulation of enzyme levels that aPBPs do not contribute to rod-like cell shape but are required for mechanical stability, supporting their independent activity. By combining measurements of cell-wall stiffness, cell-wall insertion, and PBP1b motion at the single-molecule level, we then present evidence that PBP1b, the major aPBP, contributes to cell-wall integrity by repairing cell wall defects.

Download Full-text

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.

Download Full-text

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

Download Full-text

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.

Download Full-text

Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins

eLife ◽

10.7554/elife.61525 ◽

2021 ◽

Vol 10 ◽

Author(s):

Victor M Hernández-Rocamora ◽

Natalia Baranova ◽

Katharina Peters ◽

Eefjan Breukink ◽

Martin Loose ◽

...

Keyword(s):

Real Time ◽

Lipid Bilayers ◽

High Throughput Screening ◽

Binding Proteins ◽

Cell Envelope ◽

Penicillin Binding Proteins ◽

Peptidoglycan Biosynthesis ◽

Class A ◽

Peptidoglycan Synthesis ◽

Osmotic Lysis

Peptidoglycan is an essential component of the bacterial cell envelope that surrounds the cytoplasmic membrane to protect the cell from osmotic lysis. Important antibiotics such as β-lactams and glycopeptides target peptidoglycan biosynthesis. Class A penicillin binding proteins are bifunctional membrane-bound peptidoglycan synthases that polymerize glycan chains and connect adjacent stem peptides by transpeptidation. How these enzymes work in their physiological membrane environment is poorly understood. Here we developed a novel FRET-based assay to follow in real time both reactions of class A PBPs reconstituted in liposomes or supported lipid bilayers and we applied this assay with PBP1B homologues from Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii in the presence or absence of their cognate lipoprotein activator. Our assay will allow unravelling the mechanisms of peptidoglycan synthesis in a lipid-bilayer environment and can be further developed to be used for high throughput screening for new antimicrobials.

Download Full-text

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.

Download Full-text

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.

Download Full-text