Structure and reconstitution of a hydrolase complex that releases peptidoglycan from the membrane after polymerization

Bacteria are surrounded by a peptidoglycan cell wall that is essential for their survival1. During cell wall assembly, a lipid-linked disaccharide-peptide precursor called Lipid II is polymerized and crosslinked to produce mature peptidoglycan. As Lipid II is polymerized, nascent polymers remain membrane-anchored at one end and the other end becomes crosslinked to the matrix2–4. A longstanding question is how bacteria release newly synthesized peptidoglycan strands from the membrane to complete the synthesis of mature peptidoglycan. Here we show that a Staphylococcus aureus cell wall hydrolase and a membrane protein containing eight transmembrane helices form a complex that acts as a peptidoglycan release factor. The complex cleaves nascent peptidoglycan internally to produce free oligomers as well as lipid-linked oligomers that can undergo further elongation. The polytopic membrane protein, which is similar to a eukaryotic CAAX protease, controls the length of these products. A 2.6 Å resolution structure of the complex shows that the membrane protein scaffolds the hydrolase to orient its active site for cleavage of the glycan strand. We propose that this complex serves to detach newly-synthesized peptidoglycan polymer from the cell membrane to complete integration into the cell wall matrix.

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FtsW is a peptidoglycan polymerase that is activated by its cognate penicillin-binding protein

10.1101/358663 ◽

2018 ◽

Cited By ~ 13

Author(s):

Atsushi Taguchi ◽

Michael A. Welsh ◽

Lindsey S. Marmont ◽

Wonsik Lee ◽

Daniel Kahne ◽

...

Keyword(s):

Cell Wall ◽

Side Wall ◽

Polymerase Activity ◽

Cell Wall Assembly ◽

Peptidoglycan Synthesis ◽

Lipid Ii ◽

Peptidoglycan Cell Wall ◽

Class B ◽

A Cell

AbstractThe peptidoglycan cell wall is essential for the survival and shape maintenance ofbacteria.1 For decades it was thought that only penicillin-binding proteins (PBPs) effected peptidoglycan synthesis. Recently, it was shown that RodA, a member of the Rod complex involved in side wall peptidoglycan synthesis, acts as a peptidoglycan polymerase.2–4 RodA is absent or dispensable in many bacteria that contain a cell wall; however, all of these bacteria have a RodA homologue, FtsW, which is a core member of the divisome complex that is essential for septal cell wall assembly.5,6 FtsW was previously proposed flip the peptidoglycan precursor Lipid II to the peripasm,7,8 but we report here that FtsW polymerizes Lipid II. We show that FtsW polymerase activity depends on the presence of the class B PBP (bPBP) that it recruits to the septum. We also demonstrate that the polymerase activity of FtsW is required for its function in vivo. Our findings establish FtsW as a peptidoglycan polymerase that works with its cognate bPBP to produce septal peptidoglycan during cell division.

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Biochemical reconstitution defines new functions for membrane-bound glycosidases in assembly of the bacterial cell wall

10.1101/2021.03.06.434200 ◽

2021 ◽

Author(s):

Atsushi Taguchi ◽

Suzanne Walker

Keyword(s):

Cell Wall ◽

Amino Acid ◽

Single Amino Acid ◽

Cell Wall Assembly ◽

Lipid Ii ◽

Lytic Transglycosylase ◽

Peptidoglycan Cell Wall ◽

Muramidase Activity ◽

Membrane Bound

ABSTRACTThe peptidoglycan cell wall is a macromolecular structure that encases bacteria and is essential for their survival. Proper assembly of the cell wall requires peptidoglycan synthases as well as membrane-bound cleavage enzymes that control where new peptidoglycan is made and inserted. We are only beginning to understand the roles of peptidoglycan cleavage enzymes in cell wall assembly. Previous studies have shown that two membrane-bound proteins in Streptococcus pneumoniae, here named MpgA and MpgB, are important in maintaining cell wall integrity. MpgA was predicted to be a lytic transglycosylase based on its homology to Escherichia coli MltG while the enzymatic activity of MpgB was unclear. Using nascent peptidoglycan substrates synthesized in vitro from the peptidoglycan precursor Lipid II, we report that both MpgA and MpgB are muramidases. We show that replacing a single amino acid in E. coli MltG with the corresponding amino acid from MpgA results in muramidase activity, allowing us to predict from the presence of this amino acid that other putative lytic transglycosylases actually function as muramidases. Strikingly, we report that MpgA and MpgB cut nascent peptidoglycan at different positions along the sugar backbone relative to the reducing end. MpgA produces much longer peptidoglycan oligomers and we show that its cleavage site selectivity is controlled by the LysM-like subdomain, which is also present in MltG. We propose that MltG’s ability to complement loss of MpgA in S. pneumoniae despite performing different cleavage chemistry is because it can cleave nascent peptidoglycan at the same distance from the lipid anchor.

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Staphylococcus aureus Mutants with Increased Lysostaphin Resistance

Journal of Bacteriology ◽

10.1128/jb.00457-06 ◽

2006 ◽

Vol 188 (17) ◽

pp. 6286-6297 ◽

Cited By ~ 70

Author(s):

Angelika Gründling ◽

Dominique M. Missiakas ◽

Olaf Schneewind

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Cell Wall Assembly ◽

Bacteriolytic Enzyme ◽

Transposon Insertion ◽

Transposon Mutants ◽

Altered Cell ◽

Cross Bridges ◽

Polytopic Membrane Protein ◽

Wall Assembly

ABSTRACT Staphylococcus simulans secretes lysostaphin, a bacteriolytic enzyme that specifically binds to the cell wall envelope of Staphylococcus aureus and cleaves the pentaglycine cross bridges of peptidoglycan, thereby killing staphylococci. The study of S. aureus mutants with resistance to lysostaphin-mediated killing has revealed biosynthetic pathways for cell wall assembly. To identify additional genes involved in cell wall envelope biosynthesis, we have screened a collection of S. aureus strain Newman transposon mutants for lysostaphin resistance. Bursa aurealis insertion in SAV2335, encoding a polytopic membrane protein with predicted protease domain, caused a high degree of lysostaphin resistance, similar to the case for a previously described femAB promoter mutant. In contrast to the case for this femAB mutant, transposon insertion in SAV2335, herein named lyrA (lysostaphin resistance A), did not cause gross alterations of cell wall cross bridges such as truncations of pentaglycine to tri- or monoglycine. Also, inactivation of LyrA in a methicillin-resistant S. aureus strain did not precipitate a decrease in β-lactam resistance as observed for fem (factor essential for methicillin resistance) mutants. Lysostaphin bound to the cell wall envelopes of lyrA mutants in a manner similar to that for wild-type staphylococci. Lysostaphin resistance of lyrA mutants is attributable to altered cell wall envelope properties and may in part be due to increased abundance of altered cross bridges. Other lyr mutants with intermediate lysostaphin resistance carried bursa aurealis insertions in genes specifying GTP pyrophosphokinase or enzymes of the purine biosynthetic pathway.

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Reconstitution of Membrane Protein Complexes Involved in Pneumococcal Septal Cell Wall Assembly

PLoS ONE ◽

10.1371/journal.pone.0075522 ◽

2013 ◽

Vol 8 (9) ◽

pp. e75522 ◽

Cited By ~ 11

Author(s):

Marjolaine Noirclerc-Savoye ◽

Violaine Lantez ◽

Luca Signor ◽

Jules Philippe ◽

Thierry Vernet ◽

...

Keyword(s):

Cell Wall ◽

Membrane Protein ◽

Protein Complexes ◽

Cell Wall Assembly ◽

Membrane Protein Complexes ◽

Wall Assembly

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Structure and mutagenic analysis of the lipid II flippase MurJ fromEscherichia coli

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1802192115 ◽

2018 ◽

Vol 115 (26) ◽

pp. 6709-6714 ◽

Cited By ~ 26

Author(s):

Sanduo Zheng ◽

Lok-To Sham ◽

Frederick A. Rubino ◽

Kelly P. Brock ◽

William P. Robins ◽

...

Keyword(s):

Cell Wall ◽

Plasma Membrane ◽

Cell Biology ◽

Model Organism ◽

Structural Data ◽

Outer Face ◽

Antibiotic Development ◽

Cell Wall Assembly ◽

Lipid Ii ◽

Peptidoglycan Cell Wall

The peptidoglycan cell wall provides an essential protective barrier in almost all bacteria, defining cellular morphology and conferring resistance to osmotic stress and other environmental hazards. The precursor to peptidoglycan, lipid II, is assembled on the inner leaflet of the plasma membrane. However, peptidoglycan polymerization occurs on the outer face of the plasma membrane, and lipid II must be flipped across the membrane by the MurJ protein before its use in peptidoglycan synthesis. Due to its central role in cell wall assembly, MurJ is of fundamental importance in microbial cell biology and is a prime target for novel antibiotic development. However, relatively little is known regarding the mechanisms of MurJ function, and structural data for MurJ are available only from the extremophileThermosipho africanus. Here, we report the crystal structure of substrate-free MurJ from the gram-negative model organismEscherichia coli, revealing an inward-open conformation. Taking advantage of the genetic tractability ofE. coli, we performed high-throughput mutagenesis and next-generation sequencing to assess mutational tolerance at every amino acid in the protein, providing a detailed functional and structural map for the enzyme and identifying sites for inhibitor development. Lastly, through the use of sequence coevolution analysis, we identify functionally important interactions in the outward-open state of the protein, supporting a rocker-switch model for lipid II transport.

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The discovery and biosynthesis of nucleoside antibiotics Inhibiting cell wall assembly

Planta Medica ◽

10.1055/s-0032-1320238 ◽

2012 ◽

Vol 78 (11) ◽

Author(s):

SG Van Lanen

Keyword(s):

Cell Wall ◽

Nucleoside Antibiotics ◽

Cell Wall Assembly ◽

Wall Assembly

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Faculty Opinions recommendation of A widespread family of bacterial cell wall assembly proteins.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.13363962.14734062 ◽

2011 ◽

Author(s):

Terry Roemer

Keyword(s):

Cell Wall ◽

Bacterial Cell ◽

Bacterial Cell Wall ◽

Cell Wall Assembly ◽

Wall Assembly ◽

Assembly Proteins

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The role of the exocytic pathway in cell wall assembly in yeast

Yeast ◽

10.1002/yea.3659 ◽

2021 ◽

Author(s):

Qingguo Guo ◽

Na Meng ◽

Guanzhi Fan ◽

Dong Sun ◽

Yuan Meng ◽

...

Keyword(s):

Cell Wall ◽

Cell Wall Assembly ◽

Wall Assembly ◽

Exocytic Pathway

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Properties of Arabinogalactan Proteins (AGPs) in Apple (Malus × Domestica) Fruit at Different Stages of Ripening

Biology ◽

10.3390/biology9080225 ◽

2020 ◽

Vol 9 (8) ◽

pp. 225

Author(s):

Agata Leszczuk ◽

Justyna Cybulska ◽

Tomasz Skrzypek ◽

Artur Zdunek

Keyword(s):

Cell Wall ◽

Malus Domestica ◽

Arabinogalactan Proteins ◽

Ex Situ ◽

Morphological Properties ◽

Mature Stage ◽

Fruit Storage ◽

Cell Wall Assembly ◽

Wall Assembly

Arabinogalactan proteins (AGPs) are constituents of the cell wall–plasma membrane continuum in fruit tissue. The aim of the study was to characterise AGPs contained in fruit by determination of their chemical structure and morphological properties. The results were obtained from in and ex situ investigations and a comparative analysis of AGPs present in Malus × domestica fruit at different stages of ripening from green fruit through the mature stage to over-ripening during fruit storage. The HPLC and colorimetric methods were used for analyses of the composition of monosaccharides and proteins in AGPs extracted from fruit. We have found that AGPs from fruit mainly consists of carbohydrate chains composed predominantly of arabinose, galactose, glucose, galacturonic acid, and xylose. The protein moiety accounts for 3.15–4.58%, which depends on the various phases of ripening. Taken together, our results show that the structural and morphological properties of AGPs and calcium concentration in AGPs are related to the progress of ripening, which is correlated with proper fruit cell wall assembly. In line with the existing knowledge, our data confirmed the typical carbohydrate composition of AGPs and may be the basis for studies regarding their presumed properties of binding calcium ions.

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