scholarly journals A regulatory pathway that selectively up-regulates elongasome function in the absence of class A PBPs

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Yesha Patel ◽  
Heng Zhao ◽  
John D Helmann
Keyword(s):  
Cell Shape ◽  
Alternative Sigma Factor ◽  
Regulatory Pathway ◽  
Compensatory Mechanisms ◽  
Penicillin Binding Proteins ◽  
Support Cell ◽  
Class A ◽  
Peptidoglycan Synthesis ◽  
Acting In Concert ◽  
Suppressor Analysis

Bacteria surround themselves with peptidoglycan, an adaptable enclosure that contributes to cell shape and stability. Peptidoglycan assembly relies on penicillin-binding proteins (PBPs) acting in concert with SEDS-family transglycosylases RodA and FtsW, which support cell elongation and division respectively. In Bacillus subtilis, cells lacking all four PBPs with transglycosylase activity (aPBPs) are viable. Here, we show that the alternative sigma factor σI is essential in the absence of aPBPs. Defects in aPBP-dependent wall synthesis are compensated by σI-dependent upregulation of an MreB homolog, MreBH, which localizes the LytE autolysin to the RodA-containing elongasome complex. Suppressor analysis reveals that cells unable to activate this σI stress response acquire gain-of-function mutations in the essential histidine kinase WalK, which also elevates expression of sigI, mreBH and lytE. These results reveal compensatory mechanisms that balance the directional peptidoglycan synthesis arising from the elongasome complex with the more diffusive action of aPBPs.

scholarly journals Insights into the role of lipoteichoic acids in Bacillus subtilis- a new function for MprF

2021 ◽  
Author(s):  
Aurelie Guyet ◽  
Amirah Alofi ◽  
Richard A Daniel
Keyword(s):  
Bacillus Subtilis ◽  
Cell Envelope ◽  
Cellular Level ◽  
Penicillin Binding Proteins ◽  
Class A ◽  
Peptidoglycan Synthesis ◽  
A Cell ◽  
Antimicrobial Peptide Resistance ◽  
Lipoteichoic Acids

In Bacillus subtilis, the cell is protected from the environment by a cell envelope, which comprises of layers of peptidoglycan that maintain the cell shape and anionic teichoic acids polymers whose biological function remains unclear. In B. subtilis, loss of all Class A Penicillin-Binding Proteins (aPBPs) which function in peptidoglycan synthesis is conditionally lethal. Here we show that this lethality is associated with an alteration of the lipoteichoic acids (LTA) and the accumulation of the major autolysin LytE in the cell wall. We provide the first evidence that the length and abundance of LTA acts to regulate the cellular level of LytE. Importantly, we identify a novel function for the aminoacyl-phosphatidylglycerol synthase MprF which acts to modulate LTA biosynthesis in B. subtilis and in the pathogen Staphylococcus aureus. This finding has implications for our understanding of antimicrobial peptide resistance (particularly daptomycin) in clinically relevant bacteria and MprF-associated virulence in pathogens, such as methicillin resistant S. aureus.

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

eLife ◽  
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.

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

2020 ◽  
Vol 117 (11) ◽  
pp. 6129-6138 ◽  
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.

scholarly journals Author response: Class-A penicillin binding proteins do not contribute to cell shape but repair cell-wall defects

2020 ◽  
Author(s):  
Antoine Vigouroux ◽  
Baptiste Cordier ◽  
Andrey Aristov ◽  
Laura Alvarez ◽  
Gizem Özbaykal ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Shape ◽  
Binding Proteins ◽  
Author Response ◽  
Response Class ◽  
Penicillin Binding Proteins ◽  
Class A

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

2020 ◽  
Author(s):  
Víctor 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

ABSTRACTPeptidoglycan 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 demonstrate 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 allows 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.

scholarly journals Class-A penicillin binding proteins do not contribute to cell shape but repair cell-wall defects

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Antoine Vigouroux ◽  
Baptiste Cordier ◽  
Andrey Aristov ◽  
Laura Alvarez ◽  
Gizem Özbaykal ◽  
...  
Keyword(s):  
Cell Wall ◽  
Single Molecule ◽  
Cell Shape ◽  
Binding Proteins ◽  
Mechanical Stability ◽  
Cell Envelope ◽  
Cylindrical Part ◽  
Penicillin Binding Proteins ◽  
Class A ◽  
Wall Stiffness

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.

scholarly journals Homologues of the Bacillus subtilis SpoVB Protein Are Involved in Cell Wall Metabolism

2009 ◽  
Vol 191 (19) ◽  
pp. 6012-6019 ◽  
Author(s):  
Pradeep Vasudevan ◽  
Jessica McElligott ◽  
Christa Attkisson ◽  
Michael Betteken ◽  
David L. Popham
Keyword(s):  
Bacillus Subtilis ◽  
Integral Membrane Proteins ◽  
High Similarity ◽  
Cell Wall Metabolism ◽  
Penicillin Binding Proteins ◽  
Class A ◽  
Peptidoglycan Synthesis ◽  
Alternate Pathway ◽  
Lipid Ii ◽  
Mutant Strains

ABSTRACT Members of the COG2244 protein family are integral membrane proteins involved in synthesis of a variety of extracellular polymers. In several cases, these proteins have been suggested to move lipid-linked oligomers across the membrane or, in the case of Escherichia coli MviN, to flip the lipid II peptidoglycan precursor. Bacillus subtilis SpoVB was the first member of this family implicated in peptidoglycan synthesis and is required for spore cortex polymerization. Three other COG2244 members with high similarity to SpoVB are encoded within the B. subtilis genome. Mutant strains lacking any or all of these genes (yabM, ykvU, and ytgP) in addition to spoVB are viable and produce apparently normal peptidoglycan, indicating that their function is not essential in B. subtilis. Phenotypic changes associated with loss of two of these genes suggest that they function in peptidoglycan synthesis. Mutants lacking YtgP produce long cells and chains of cells, suggesting a role in cell division. Mutants lacking YabM exhibit sensitivity to moenomycin, an antibiotic that blocks peptidoglycan polymerization by class A penicillin-binding proteins. This result suggests that YabM may function in a previously observed alternate pathway for peptidoglycan strand synthesis.

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

2019 ◽  
Author(s):  
Daniel Straume ◽  
Katarzyna Wiaroslawa Piechowiak ◽  
Silje Olsen ◽  
Gro Anita Stamsås ◽  
Kari Helene Berg ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Wall Synthesis ◽  
Unique Role ◽  
Penicillin Binding Proteins ◽  
Class A ◽  
Peptidoglycan Synthesis ◽  
Resistant Form ◽  
Class B ◽  
Open Question ◽  
First Time

AbstractIn oval shapedStreptococcus pneumoniae, septal and longitudinal peptidoglycan synthesis is performed by independent functional complexes; the divisome and the elongasome. Penicillin binding proteins (PBPs) were long considered as 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 non-PBP glycosyltransferases (FtsW and RodA) 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 to show that class A PBPs have an autonomous role during cell wall synthesis inS. pneumoniae. Purified CbpD was shown to target the septum ofS. pneumoniaecells. Using assays to specifically inhibit PBP2x, we demonstrate that CbpD specifically target 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 or repairs nascent peptidoglycan synthesized by FtsW/PBP2x. Our results support a model in which pneumococcal peptidoglycan is made by three functional entities, the divisome, the elongasome and a peptidoglycan-repairing or -remodelling complex consisting of bifunctional PBPs. To our knowledge this is the first time a specific function has been identified for class A PBPs in bacterial cell wall synthesis.

scholarly journals Peptidoglycan Synthesis in the Absence of Class A Penicillin-Binding Proteins in Bacillus subtilis

2003 ◽  
Vol 185 (4) ◽  
pp. 1423-1431 ◽  
Author(s):  
Derrell C. McPherson ◽  
David L. Popham
Keyword(s):  
Bacillus Subtilis ◽  
Mutant Strain ◽  
Binding Proteins ◽  
Wild Type ◽  
Penicillin Binding Proteins ◽  
Class A ◽  
Peptidoglycan Synthesis ◽  
Quadruple Mutant

ABSTRACT Penicillin-binding proteins (PBPs) catalyze the final, essential reactions of peptidoglycan synthesis. Three classes of PBPs catalyze either trans-, endo-, or carboxypeptidase activities on the peptidoglycan peptide side chains. Only the class A high-molecular-weight PBPs have clearly demonstrated glycosyltransferase activities that polymerize the glycan strands, and in some species these proteins have been shown to be essential. The Bacillus subtilis genome sequence contains four genes encoding class A PBPs and no other genes with similarity to their glycosyltransferase domain. A strain lacking all four class A PBPs has been constructed and produces a peptidoglycan wall with only small structural differences from that of the wild type. The growth rate of the quadruple mutant is much lower than those of strains lacking only three of the class A PBPs, and increases in cell length and frequencies of wall abnormalities were noticeable. The viability and wall production of the quadruple-mutant strain indicate that a novel enzyme can perform the glycosyltransferase activity required for peptidoglycan synthesis. This activity was demonstrated in vitro and shown to be sensitive to the glycosyltransferase inhibitor moenomycin. In contrast, the quadruple-mutant strain was resistant to moenomycin in vivo. Exposure of the wild-type strain to moenomycin resulted in production of a phenotype similar to that of the quadruple mutant.

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