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Bacterial cell wall. MurJ is the flippase of lipid-linked precursors for peptidoglycan biogenesis.

ABSTRACT: Peptidoglycan (PG) is a polysaccharide matrix that protects bacteria from osmotic lysis. Inhibition of its biogenesis is a proven strategy for killing bacteria with antibiotics. The assembly of PG requires disaccharide-pentapeptide building blocks attached to a polyisoprene lipid carrier called lipid II. Although the stages of lipid II synthesis are known, the identity of the essential flippase that translocates it across the cytoplasmic membrane for PG polymerization is unclear. We developed an assay for lipid II flippase activity and used a chemical genetic strategy to rapidly and specifically block flippase function. We combined these approaches to demonstrate that MurJ is the lipid II flippase in Escherichia coli.

SUBMITTER: Sham LT

PROVIDER: S-EPMC4163187 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Bacterial cell wall. MurJ is the flippase of lipid-linked precursors for peptidoglycan biogenesis.

Sham Lok-To LT Butler Emily K EK Lebar Matthew D MD Kahne Daniel D Bernhardt Thomas G TG Ruiz Natividad N

Science (New York, N.Y.) 20140701 6193

Peptidoglycan (PG) is a polysaccharide matrix that protects bacteria from osmotic lysis. Inhibition of its biogenesis is a proven strategy for killing bacteria with antibiotics. The assembly of PG requires disaccharide-pentapeptide building blocks attached to a polyisoprene lipid carrier called lipid II. Although the stages of lipid II synthesis are known, the identity of the essential flippase that translocates it across the cytoplasmic membrane for PG polymerization is unclear. We developed an ...[more]

PMID: 25013077

Similar Datasets

MurJ and a novel lipid II flippase are required for cell wall biogenesis in Bacillus subtilis.

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Bioinformatics identification of MurJ (MviN) as the peptidoglycan lipid II flippase in Escherichia coli.

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The bacterial lipid II flippase MurJ functions by an alternating-access mechanism.

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Structure and Mechanism of the Lipid Flippase MurJ.

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A Burkholderia cenocepacia MurJ (MviN) homolog is essential for cell wall peptidoglycan synthesis and bacterial viability.

Project description:The cell wall peptidoglycan (PG) of Burkholderia cenocepacia, an opportunistic pathogen, has not yet been characterized. However, the B. cenocepacia genome contains homologs of genes encoding PG biosynthetic functions in other bacteria. PG biosynthesis involves the formation of the undecaprenyl-pyrophosphate-linked N-acetyl glucosamine-N-acetyl muramic acid-pentapeptide, known as lipid II, which is built on the cytosolic face of the cell membrane. Lipid II is then translocated across the membrane and its glycopeptide moiety becomes incorporated into the growing cell wall mesh; this translocation step is critical to PG synthesis. We have investigated candidate flippase homologs of the MurJ family in B. cenocepacia. Our results show that BCAL2764, herein referred to as murJBc, is indispensable for viability. Viable B. cenocepacia could only be obtained through a conditional mutagenesis strategy by placing murJBc under the control of a rhamnose-inducible promoter. Under rhamnose depletion, the conditional strain stopped growing and individual cells displayed morphological abnormalities consistent with a defect in PG synthesis. Bacterial cells unable to express MurJBc underwent cell lysis, while partial MurJBc depletion sensitized the mutant to the action of ?-lactam antibiotics. Depletion of MurJBc caused accumulation of PG precursors consistent with the notion that this protein plays a role in lipid II flipping to the periplasmic compartment. Reciprocal complementation experiments of conditional murJ mutants in B. cenocepacia and Escherichia coli with plasmids expressing MurJ from each strain indicated that MurJBc and MurJEc are functional homologs. Together, our results are consistent with the notion that MurJBc is a PG lipid II flippase in B. cenocepacia.

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Visualizing conformation transitions of the Lipid II flippase MurJ.

Project description:The biosynthesis of many polysaccharides, including bacterial peptidoglycan and eukaryotic N-linked glycans, requires transport of lipid-linked oligosaccharide (LLO) precursors across the membrane by specialized flippases. MurJ is the flippase for the lipid-linked peptidoglycan precursor Lipid II, a key player in bacterial cell wall synthesis, and a target of recently discovered antibacterials. However, the flipping mechanism of LLOs including Lipid II remains poorly understood due to a dearth of structural information. Here we report crystal structures of MurJ captured in inward-closed, inward-open, inward-occluded and outward-facing conformations. Together with mutagenesis studies, we elucidate the conformational transitions in MurJ that mediate lipid flipping, identify the key ion for function, and provide a framework for the development of inhibitors.

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A feedback control mechanism governs the synthesis of lipid-linked precursors of the bacterial cell wall.

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Project description: Not available

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Detection of Transport Intermediates in the Peptidoglycan Flippase MurJ Identifies Residues Essential for Conformational Cycling.

Project description:Bacterial cell wall synthesis is an essential process in bacteria and one of the best targets for antibiotics. A critical step on this pathway is the export of the lipid-linked cell wall monomer, Lipid II, by its transporter MurJ. The mechanism by which MurJ mediates the transbilayer movement of Lipid II is not understood because intermediate states of this process have not been observed. Here we demonstrate a method to capture and detect interactions between MurJ and its substrate Lipid II by photo-cross-linking and subsequent biotin-tagging. We show that this method can be used to covalently capture intermediate transport states of Lipid II on MurJ in living cells. Using this strategy we probed several lethal arginine mutants and found that they retain appreciable substrate-binding ability despite being defective in Lipid II transport. We propose that Lipid II binding to these residues during transport induces a conformational change in MurJ required to proceed through the Lipid II transport cycle. The methods described to detect intermediate transport states of MurJ will be useful for characterizing mechanisms of inhibitors.

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