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 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 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 Role of Peptidoglycan Amidases in the Development and Morphology of the Division Septum in Escherichia coli

2007 ◽  
Vol 189 (14) ◽  
pp. 5334-5347 ◽  
Author(s):  
Richa Priyadarshini ◽  
Miguel A. de Pedro ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Cell Envelope ◽  
Penicillin Binding Protein ◽  
Mature Cell ◽  
Daughter Cells ◽  
Peptidoglycan Synthesis ◽  
A Cell ◽  
Entire Cell

ABSTRACT Escherichia coli contains multiple peptidoglycan-specific hydrolases, but their physiological purposes are poorly understood. Several mutants lacking combinations of hydrolases grow as chains of unseparated cells, indicating that these enzymes help cleave the septum to separate daughter cells after cell division. Here, we confirm previous observations that in the absence of two or more amidases, thickened and dark bands, which we term septal peptidoglycan (SP) rings, appear at division sites in isolated sacculi. The formation of SP rings depends on active cell division, and they apparently represent a cell division structure that accumulates because septal synthesis and hydrolysis are uncoupled. Even though septal constriction was incomplete, SP rings exhibited two properties of mature cell poles: they behaved as though composed of inert peptidoglycan, and they attracted the IcsA protein. Despite not being separated by a completed peptidoglycan wall, adjacent cells in these chains were often compartmentalized by the inner membrane, indicating that cytokinesis could occur in the absence of invagination of the entire cell envelope. Finally, deletion of penicillin-binding protein 5 from amidase mutants exacerbated the formation of twisted chains, producing numerous cells having septa with abnormal placements and geometries. The results suggest that the amidases are necessary for continued peptidoglycan synthesis during cell division, that their activities help create a septum having the appropriate geometry, and that they may contribute to the development of inert peptidoglycan.

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.

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 Mechanism of Action of NB2001 and NB2030, Novel Antibacterial Agents Activated by β-Lactamases

2004 ◽  
Vol 48 (2) ◽  
pp. 477-483 ◽  
Author(s):  
Geoffrey W. Stone ◽  
Qin Zhang ◽  
Rosario Castillo ◽  
V. Ramana Doppalapudi ◽  
Analia R. Bueno ◽  
...  
Keyword(s):  
Antibacterial Agents ◽  
Mechanisms Of Action ◽  
Enoyl Reductase ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Class A ◽  
Biochemical Assays ◽  
Lactam Ring ◽  
A Cell ◽  
Hydrolysis Of

ABSTRACT Two potent antibacterial agents designed to undergo enzyme-catalyzed therapeutic activation were evaluated for their mechanisms of action. The compounds, NB2001 and NB2030, contain a cephalosporin with a thienyl (NB2001) or a tetrazole (NB2030) ring at the C-7 position and are linked to the antibacterial triclosan at the C-3 position. The compounds exploit β-lactamases to release triclosan through hydrolysis of the β-lactam ring. Like cephalothin, NB2001 and NB2030 were hydrolyzed by class A β-lactamases (Escherichia coli TEM-1 and, to a lesser degree, Staphylococcus aureus PC1) and class C β-lactamases (Enterobacter cloacae P99 and E. coli AmpC) with comparable catalytic efficiencies (k cat/Km ). They also bound to the penicillin-binding proteins of S. aureus and E. coli, but with reduced affinities relative to that of cephalothin. Accordingly, they produced a cell morphology in E. coli consistent with the toxophore rather than the β-lactam being responsible for antibacterial activity. In biochemical assays, they inhibited the triclosan target enoyl reductase (FabI), with 50% inhibitory concentrations being markedly reduced relative to that of free triclosan. The transport of NB2001, NB2030, and triclosan was rapid, with significant accumulation of triclosan in both S. aureus and E. coli. Taken together, the results suggest that NB2001 and NB2030 act primarily as triclosan prodrugs in S. aureus and E. coli.

scholarly journals The Phospholipid N-Methyltransferase and Phosphatidylcholine Synthase Pathways and the ChoXWV Choline Uptake System Involved in Phosphatidylcholine Synthesis Are Widely Conserved in Most, but Not All Brucella Species

2021 ◽  
Vol 12 ◽  
Author(s):  
Beatriz Aragón-Aranda ◽  
Leyre Palacios-Chaves ◽  
Miriam Salvador-Bescós ◽  
María Jesús de Miguel ◽  
Pilar M. Muñoz ◽  
...  
Keyword(s):  
Cell Envelope ◽  
Innate Immune ◽  
Intracellular Bacteria ◽  
Choline Uptake ◽  
Uptake System ◽  
Innate Immune Responses ◽  
A Cell ◽  
Direct Linkage ◽  
Phosphatidylcholine Synthesis

The brucellae are facultative intracellular bacteria with a cell envelope rich in phosphatidylcholine (PC). PC is abundant in eukaryotes but rare in prokaryotes, and it has been proposed that Brucella uses PC to mimic eukaryotic-like features and avoid innate immune responses in the host. Two PC synthesis pathways are known in prokaryotes: the PmtA-catalyzed trimethylation of phosphatidylethanolamine and the direct linkage of choline to CDP-diacylglycerol catalyzed by the PC synthase Pcs. Previous studies have reported that B. abortus and B. melitensis possess non-functional PmtAs and that PC is synthesized exclusively via Pcs in these strains. A putative choline transporter ChoXWV has also been linked to PC synthesis in B. abortus. Here, we report that Pcs and Pmt pathways are active in B. suis biovar 2 and that a bioinformatics analysis of Brucella genomes suggests that PmtA is only inactivated in B. abortus and B. melitensis strains. We also show that ChoXWV is active in B. suis biovar 2 and conserved in all brucellae except B. canis and B. inopinata. Unexpectedly, the experimentally verified ChoXWV dysfunction in B. canis did not abrogate PC synthesis in a PmtA-deficient mutant, which suggests the presence of an unknown mechanism for obtaining choline for the Pcs pathway in Brucella. We also found that ChoXWV dysfunction did not cause attenuation in B. suis biovar 2. The results of these studies are discussed with respect to the proposed role of PC in Brucella virulence and how differential use of the Pmt and Pcs pathways may influence the interactions of these bacteria with their mammalian hosts.

scholarly journals Genetic Characterization of a Cell Envelope-Associated Proteinase from Lactobacillus helveticus CNRZ32

1999 ◽  
Vol 181 (15) ◽  
pp. 4592-4597 ◽  
Author(s):  
Jeffrey A. Pederson ◽  
Gerald J. Mileski ◽  
Bart C. Weimer ◽  
James L. Steele
Keyword(s):  
Cell Surface ◽  
Deletion Mutant ◽  
Cell Envelope ◽  
Physiological Role ◽  
Lactobacillus Helveticus ◽  
Whole Cells ◽  
A Cell ◽  
Hydrolysis Of

ABSTRACT A cell envelope-associated proteinase gene (prtH) was identified in Lactobacillus helveticus CNRZ32. TheprtH gene encodes a protein of 1,849 amino acids and with a predicted molecular mass of 204 kDa. The deduced amino acid sequence of the prtH product has significant identity (45%) to that of the lactococcal PrtP proteinases. Southern blot analysis indicates thatprtH is not broadly distributed within L. helveticus. A prtH deletion mutant of CNRZ32 was constructed to evaluate the physiological role of PrtH. PrtH is not required for rapid growth or fast acid production in milk by CNRZ32. Cell surface proteinase activity and specificity were determined by hydrolysis of αs1-casein fragment 1-23 by whole cells. A comparison of CNRZ32 and its prtH deletion mutant indicates that CNRZ32 has at least two cell surface proteinases that differ in substrate specificity.

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