Streptococcus pyogenes YtgP (Spy_0390) complements Escherichia coli strains depleted of the putative peptidoglycan flippase MurJ.
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ABSTRACT:
SUBMITTER: Ruiz N
PROVIDER: S-EPMC2715597 | biostudies-literature | 2009 Aug
REPOSITORIES: biostudies-literature
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Streptococcus pyogenes YtgP (Spy_0390) complements Escherichia coli strains depleted of the putative peptidoglycan flippase MurJ.
Antimicrobial agents and chemotherapy 20090615 8
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Project description:Peptidoglycan is a cell-wall glycopeptide polymer that protects bacteria from osmotic lysis. Whereas in gram-positive bacteria it also serves as scaffold for many virulence factors, in gram-negative bacteria, peptidoglycan is an anchor for the outer membrane. For years, we have known the enzymes required for the biosynthesis of peptidoglycan; what was missing was the flippase that translocates the lipid-anchored precursors across the cytoplasmic membrane before their polymerization into mature peptidoglycan. Using a reductionist bioinformatics approach, I have identified the essential inner-membrane protein MviN (renamed MurJ) as a likely candidate for the peptidoglycan flippase in Escherichia coli. Here, I present genetic and biochemical data that confirm the requirement of MurJ for peptidoglycan biosynthesis and that are in agreement with a role of MurJ as a flippase. Because of its essential nature, MurJ could serve as a target in the continuing search for antimicrobial compounds.
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Project description:The peptidoglycan (PG) cell wall is an essential component of the cell envelope of most bacteria. Biogenesis of PG involves a lipid-linked disaccharide-pentapeptide intermediate called lipid II, which must be translocated across the cytoplasmic membrane after it is synthesized in the inner leaflet of this bilayer. Accordingly, it has been demonstrated that MurJ, the proposed lipid II flippase in Escherichia coli, is required for PG biogenesis, and thereby viability. In contrast, MurJ is not essential in Bacillus subtilis because this bacterium produces AmJ, an unrelated protein that is functionally redundant with MurJ. In this study, we investigated why MurJ is not essential in the prominent gastric pathogen, Helicobacter pylori. We found that in this bacterium, Wzk, the ABC (ATP-binding cassette) transporter that flips the lipid-linked O- or Lewis- antigen precursors across the inner membrane, is redundant with MurJ for cell viability. Heterologous expression of wzk in E. coli also suppresses the lethality caused by the loss of murJ. Furthermore, we show that this cross-species complementation is abolished when Wzk is inactivated by mutations that target a domain predicted to be required for ATPase activity. Our results suggest that Wzk can flip lipid II, implying that Wzk is the flippase with the most relaxed specificity for lipid-linked saccharides ever identified.
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Project description:Although peptidoglycan synthesis is one of the best-studied metabolic pathways in bacteria, the mechanism underlying the membrane translocation of lipid II, the undecaprenyl-disaccharide pentapeptide peptidoglycan precursor, remains mysterious. Recently, it was proposed that the essential Escherichia coli mviN gene encodes the lipid II flippase. Bacillus subtilis contains four proteins that are putatively homologous to MviN, including SpoVB, previously reported to be necessary for spore cortex peptidoglycan synthesis during sporulation. MviN complemented the sporulation defect of a DeltaspoVB mutation, and SpoVB and another of the B. subtilis homologs, YtgP, complemented the growth defect of an E. coli strain depleted for MviN. Thus, these B. subtilis proteins are likely to be MviN homologs. However, B. subtilis strains lacking these four proteins have no defects in growth, indicating that they likely do not serve as lipid II flippases in this organism.
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Project description: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.
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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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Project description: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 extremophile Thermosipho africanus Here, we report the crystal structure of substrate-free MurJ from the gram-negative model organism Escherichia coli, revealing an inward-open conformation. Taking advantage of the genetic tractability of E. 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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Project description:Biosynthesis of many important polysaccharides (including peptidoglycan, lipopolysaccharide, and N-linked glycans) necessitates the transport of lipid-linked oligosaccharides (LLO) across membranes from their cytosolic site of synthesis to their sites of utilization. Much of our current understanding of LLO transport comes from genetic, biochemical, and structural studies of the multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) superfamily protein MurJ, which flips the peptidoglycan precursor lipid II. MurJ plays a pivotal role in bacterial cell wall synthesis and is an emerging antibiotic target. Here, we review the mechanism of LLO flipping by MurJ, including the structural basis for lipid II flipping and ion coupling. We then discuss inhibition of MurJ by antibacterials, including humimycins and the phage M lysis protein, as well as how studies on MurJ could provide insight into other flippases, both within and beyond the MOP superfamily.
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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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Project description:UnlabelledThe ExPortal of Streptococcus pyogenes is a focal microdomain of the cytoplasmic membrane that clusters the translocons of the general secretory pathway with accessory factors to facilitate the maturation of secreted polypeptides. While it is known that the ExPortal is enriched in anionic lipids, the mechanisms that organize the ExPortal are poorly understood. In the present study, we examined the role of the cell wall in organizing and maintaining the ExPortal. Removal of the cell wall resulted in a loss of ExPortal focal integrity accompanied by the circumferential redistribution of ExPortal lipid and protein components. A similar loss occurred upon treatment with gallidermin, a nonpermeabilizing lantibiotic that targets the lipid II precursor of peptidoglycan synthesis, and this treatment disrupted the secretion of several ExPortal substrates. Furthermore, several enzymes involved in the membrane-associated steps of lipid II synthesis, including MraY and MurN, were found to localize to a single discrete focus in the membrane that was coincident with the focal location of the secretory translocons and the anionic lipid microdomain. These data suggest that the ExPortal is associated with the site of peptidoglycan precursor synthesis and that peptidoglycan biogenesis influences ExPortal organization. These data add to an emerging literature indicating that cell wall biogenesis, cell division, and protein secretion are spatially coorganized processes.ImportanceSince Gram-positive bacteria lack a periplasmic space, they lack a protected compartment to spatially coordinate interaction between newly secreted proteins and the factors required to process them. This represents a significant problem for pathogens that depend on the secretion of toxins and cell wall-associated adhesins to cause disease. Streptococci solve this dilemma by restricting secretion and processing factors to a defined region of the membrane. However, the mechanisms that promote restriction are not understood. In this study, we show that restriction of these factors in the pathogen Streptococcus pyogenes is intimately linked with the presence of the cell wall and its synthesis. Furthermore, several cell wall synthesis proteins are also restricted to the site of protein secretion. This study contributes to our understanding of how the Gram-positive cell is organized to coordinate protein secretion and biogenesis with cell wall synthesis and to the ongoing development of antibiotics that target these processes.
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Project description:The peptidoglycan (PG) cell wall is an essential extracytoplasmic glycopeptide polymer that safeguards bacteria against osmotic lysis and determines cellular morphology. Bacteria use multiprotein machineries for the synthesis of the PG cell wall during cell division and elongation that can be targeted by antibiotics such as the β-lactams. Lipid II, the lipid-linked precursor for PG biogenesis, is synthesized in the inner leaflet of the cytoplasmic membrane and then translocated across the bilayer, where it is ultimately polymerized into PG. In Escherichia coli, MurJ, a member of the MOP exporter superfamily, has been recently shown to have lipid II flippase activity that depends on membrane potential. Because of its essentiality, MurJ could potentially be targeted by much needed novel antibiotics. Recent structural information suggests that a central cavity in MurJ alternates between inward- and outward-open conformations to flip lipid II, but how these conformational changes occur are unknown. Here, we utilized structure-guided cysteine cross-linking and proteolysis-coupled gel analysis to probe the conformational changes of MurJ in E. coli cells. We found that paired cysteine substitutions in transmembrane domains 2 and 8 and periplasmic loops of MurJ could be cross-linked with homobifunctional cysteine cross-linkers, indicating that MurJ can adopt both inward- and outward-facing conformations in vivo Furthermore, we show that dissipating the membrane potential with an ionophore decreases the prevalence of the inward-facing, but not the outward-facing state. Our study provides in vivo evidence that MurJ uses an alternating-access mechanism during the lipid II transport cycle.
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