A novel and conserved cell wall enzyme that can substitute for the Lipid II synthase MurG

ABSTRACT The cell wall is a stress-bearing structure and a unifying trait in bacteria. Without exception, synthesis of the cell wall involves formation of the precursor molecule lipid II by the activity of the essential biosynthetic enzyme MurG, which is encoded in the division and cell wall synthesis (dcw) gene cluster. Here, we present the discovery of a cell wall enzyme that can substitute for MurG. A mutant of Kitasatospora viridifaciens lacking a significant part of the dcw cluster, including murG, surprisingly produced lipid II and wild-type peptidoglycan. Genomic analysis identified a distant murG homologue, which encodes a putative enzyme that shares only around 31% amino acid sequence identity with MurG. We show that this enzyme can replace the canonical MurG, and we therefore designated it MglA. Orthologues of mglA are present in 38% of all genomes of Kitasatospora and members of the sister genus Streptomyces. CRISPR interference experiments showed that K. viridifaciens mglA can also functionally replace murG in Streptomyces coelicolor, thus validating its bioactivity and demonstrating that it is active in multiple genera. All together, these results identify MglA as a bona fide lipid II synthase, thus demonstrating plasticity in cell wall synthesis. IMPORTANCE Almost all bacteria are surrounded by a cell wall, which protects cells from environmental harm. Formation of the cell wall requires the precursor molecule lipid II, which in bacteria is universally synthesized by the conserved and essential lipid II synthase MurG. We here exploit the unique ability of an actinobacterial strain capable of growing with or without its cell wall to discover an alternative lipid II synthase, MglA. Although this enzyme bears only weak sequence similarity to MurG, it can functionally replace MurG and can even do so in organisms that naturally have only a canonical MurG. The observation that MglA proteins are found in many actinobacteria highlights the plasticity in cell wall synthesis in these bacteria and demonstrates that important new cell wall biosynthetic enzymes remain to be discovered.

Download Full-text

Substrate Inhibition of VanA by d-Alanine Reduces Vancomycin Resistance in a VanX-Dependent Manner

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00276-16 ◽

2016 ◽

Vol 60 (8) ◽

pp. 4930-4939 ◽

Cited By ~ 6

Author(s):

Lizah T. van der Aart ◽

Nicole Lemmens ◽

Willem J. van Wamel ◽

Gilles P. van Wezel

Keyword(s):

Cell Wall ◽

Streptomyces Coelicolor ◽

Substrate Inhibition ◽

Model Organism ◽

Vancomycin Resistance ◽

Dependent Manner ◽

Wild Type ◽

Content Type ◽

Lipid Ii ◽

Vancomycin Resistant

ABSTRACTThe increasing resistance of clinical pathogens against the glycopeptide antibiotic vancomycin, a last-resort drug against infections with Gram-positive pathogens, is a major problem in the nosocomial environment. Vancomycin inhibits peptidoglycan synthesis by binding to thed-Ala–d-Ala terminal dipeptide moiety of the cell wall precursor lipid II. Plasmid-transferable resistance is conferred by modification of the terminal dipeptide into the vancomycin-insensitive variantd-Ala–d-Lac, which is produced by VanA. Here we show that exogenousd-Ala competes withd-Lac as a substrate for VanA, increasing the ratio of wild-type to mutant dipeptide, an effect that was augmented by several orders of magnitude in the absence of thed-Ala–d-Ala peptidase VanX. Liquid chromatography-mass spectrometry (LC-MS) analysis showed that high concentrations ofd-Ala led to the production of a significant amount of wild-type cell wall precursors, whilevanX-null mutants produced primarily wild-type precursors. This enhanced the efficacy of vancomycin in the vancomycin-resistant model organismStreptomyces coelicolor, and the susceptibility of vancomycin-resistant clinical isolates ofEnterococcus faecium(VRE) increased by up to 100-fold. The enhanced vancomycin sensitivity ofS. coelicolorcells correlated directly to increased binding of the antibiotic to the cell wall. Our work offers new perspectives for the treatment of diseases associated with vancomycin-resistant pathogens and for the development of drugs that target vancomycin resistance.

Download Full-text

Cardiolipin Alters Rhodobacter sphaeroides Cell Shape by Affecting Peptidoglycan Precursor Biosynthesis

mBio ◽

10.1128/mbio.02401-18 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 4

Author(s):

Ti-Yu Lin ◽

William S. Gross ◽

George K. Auer ◽

Douglas B. Weibel

Keyword(s):

Cell Wall ◽

Rhodobacter Sphaeroides ◽

Cell Morphology ◽

Cell Shape ◽

Molecular Mechanisms ◽

Wild Type ◽

Anionic Phospholipid ◽

Content Type ◽

Lipid Ii ◽

Topological Features

ABSTRACT Cardiolipin (CL) is an anionic phospholipid that plays an important role in regulating protein biochemistry in bacteria and mitochondria. Deleting the CL synthase gene (Δcls) in Rhodobacter sphaeroides depletes CL and decreases cell length by 20%. Using a chemical biology approach, we found that a CL deficiency does not impair the function of the cell wall elongasome in R. sphaeroides; instead, biosynthesis of the peptidoglycan (PG) precursor lipid II is decreased. Treating R. sphaeroides cells with fosfomycin and d-cycloserine inhibits lipid II biosynthesis and creates phenotypes in cell shape, PG composition, and spatial PG assembly that are strikingly similar to those seen with R. sphaeroides Δcls cells, suggesting that CL deficiency alters the elongation of R. sphaeroides cells by reducing lipid II biosynthesis. We found that MurG—a glycosyltransferase that performs the last step of lipid II biosynthesis—interacts with anionic phospholipids in native (i.e., R. sphaeroides) and artificial membranes. Lipid II production decreases 25% in R. sphaeroides Δcls cells compared to wild-type cells, and overexpression of MurG in R. sphaeroides Δcls cells restores their rod shape, indicating that CL deficiency decreases MurG activity and alters cell shape. The R. sphaeroides Δcls mutant is more sensitive than the wild-type strain to antibiotics targeting PG synthesis, including fosfomycin, d-cycloserine, S-(3,4-dichlorobenzyl)isothiourea (A22), mecillinam, and ampicillin, suggesting that CL biosynthesis may be a potential target for combination chemotherapies that block the bacterial cell wall. IMPORTANCE The phospholipid composition of the cell membrane influences the spatial and temporal biochemistry of cells. We studied molecular mechanisms connecting membrane composition to cell morphology in the model bacterium Rhodobacter sphaeroides. The peptidoglycan (PG) layer of the cell wall is a dominant component of cell mechanical properties; consequently, it has been an important antibiotic target. We found that the anionic phospholipid cardiolipin (CL) plays a role in determination of the shape of R. sphaeroides cells by affecting PG precursor biosynthesis. Removing CL in R. sphaeroides alters cell morphology and increases its sensitivity to antibiotics targeting proteins synthesizing PG. These studies provide a connection to spatial biochemical control in mitochondria, which contain an inner membrane with topological features in common with R. sphaeroides.

Download Full-text

PepN is a non-essential, cell wall-localized protein that contributes to neutrophil elastase-mediated killing ofStreptococcus pneumoniae

10.1101/313569 ◽

2018 ◽

Author(s):

Charmaine N. Nganje ◽

Scott A. Haynes ◽

Christine M. Qabar ◽

Rachel C. Lent ◽

Elsa N. Bou Ghanem ◽

...

Keyword(s):

Cell Wall ◽

Streptococcus Pneumoniae ◽

Neutrophil Elastase ◽

Serine Proteases ◽

Signal Sequence ◽

Cell Wall Protein ◽

Aminopeptidase N ◽

Human Neutrophils ◽

Wild Type ◽

Bona Fide

ABSTRACTStreptococcus pneumoniae(Spn) is an asymptomatic colonizer of the human nasopharynx but can also cause invasive diseases in the inner ear, meninges, lung and blood. Although various mechanisms contribute to the effective clearance ofSpn, opsonophagocytosis by neutrophils is perhaps most critical. Upon phagocytosis,Spnis exposed to various degradative molecules, including a family of neutrophil serine proteases (NSPs) that are stored within intracellular granules. Despite the critical importance of NSPs in killingSpn, the bacterial proteins that are degraded by NSPs leading toSpndeath are still unknown. In this report, we identify a 90kDa protein in a purified cell wall (CW) preparation, aminopeptidase N (PepN) that is degraded by the NSP, neutrophil elastase (NE). Since PepN lacked a canonical signal sequence or LPxTG motif, we created a mutant expressing a FLAG tagged version of the protein and confirmed its localization to the CW compartment. We determined that not only is PepN abona fideCW protein, but also is a substrate of NE in the context of intactSpncells. Furthermore, in comparison to wild-type TIGR4Spn, a mutant strain lacking PepN demonstrated a significant hyper-resistance phenotypein vitroin the presence of purified NE as well as in opsonophagocytic assays with purified human neutrophilsex vivo. Taken together, this is the first study to demonstrate that PepN is a CW-localized protein and a substrate of NE that contributes to the effective killing ofSpnby NSPs and human neutrophils.IMPORTANCENeutrophils are innate immune cells needed to effectively clearStreptococcus pneumoniae(Spn). Neutrophil serine proteases (NSPs) are important for killing phagocytosedSpn, however, the identity of theSpnproteins that are degraded by NSPs are unknown. This study identifies aSpncell wall protein, aminopeptidase N (PepN) that is degraded by the NSP, neutrophil elastase (NE). We demonstrate that PepN is abona fidecell wall protein and mutants lacking PepN are significantly more resistant than wild-type to killing by purified NE and human neutrophils. This study demonstrates that PepN is a NE substrate and its degradation contributes to effectiveSpnkilling. By better understanding how neutrophils killSpn, we aim to inform the development of improved therapeutic interventions.

Download Full-text

BceAB-type antibiotic resistance transporters appear to act by target protection of cell wall synthesis

10.1101/835702 ◽

2019 ◽

Cited By ~ 1

Author(s):

Carolin M Kobras ◽

Hannah Piepenbreier ◽

Jennifer Emenegger ◽

Andre Sim ◽

Georg Fritz ◽

...

Keyword(s):

Cell Wall ◽

Antimicrobial Peptides ◽

Gc Content ◽

Critical Factor ◽

Peptide Antibiotics ◽

Cell Wall Synthesis ◽

Gram Positive ◽

Gram Positive Bacteria ◽

Lipid Ii ◽

High Level

ABSTRACTResistance against cell wall-active antimicrobial peptides in bacteria is often mediated by transporters. In low GC-content Gram-positive bacteria, a wide-spread type of such transporters are the BceAB-like systems, which frequently provide a high level of resistance against peptide antibiotics that target intermediates of the lipid II cycle of cell wall synthesis. How a transporter can offer protection from drugs that are active on the cell surface, however, has presented researchers with a conundrum. Multiple theories have been discussed, ranging from removal of the peptides from the membrane, internalisation of the drug for degradation, to removal of the cellular target rather than the drug itself. To resolve this much-debated question, we here investigated the mode of action of the transporter BceAB of Bacillus subtilis. We show that it does not inactivate or import its substrate antibiotic bacitracin. Moreover, we present evidence that the critical factor driving transport activity is not the drug itself, but instead the concentration of drug-target complexes in the cell. Our results, together with previously reported findings, lead us to propose that BceAB-type transporters act by transiently freeing lipid II cycle intermediates from the inhibitory grip of antimicrobial peptides, and thus provide resistance through target protection of cell wall synthesis. Target protection has so far only been reported for resistance against antibiotics with intracellular targets, such as the ribosome. However, this mechanism offers a plausible explanation for the use of transporters as resistance determinants against cell wall-active antibiotics in Gram-positive bacteria where cell wall synthesis lacks the additional protection of an outer membrane.

Download Full-text

Staphylococcus aureusPenicillin-Binding Protein 2 Can Use Depsi-Lipid II Derived from Vancomycin-Resistant Strains for Cell Wall Synthesis

Chemistry - A European Journal ◽

10.1002/chem.201301074 ◽

2013 ◽

Vol 19 (36) ◽

pp. 12104-12112 ◽

Cited By ~ 5

Author(s):

Jun Nakamura ◽

Hidenori Yamashiro ◽

Hiroto Miya ◽

Kenzo Nishiguchi ◽

Hideki Maki ◽

...

Keyword(s):

Cell Wall ◽

Binding Protein ◽

Cell Wall Synthesis ◽

Lipid Ii ◽

Resistant Strains ◽

Vancomycin Resistant

Download Full-text

An Association Between Peptidoglycan Synthesis and Organization of the Streptococcus pyogenes ExPortal

mBio ◽

10.1128/mbio.00485-13 ◽

2013 ◽

Vol 4 (5) ◽

Cited By ~ 12

Author(s):

Luis Alberto Vega ◽

Gary C. Port ◽

Michael G. Caparon

Keyword(s):

Cell Wall ◽

Protein Secretion ◽

Streptococcus Pyogenes ◽

Secretory Pathway ◽

Cell Wall Synthesis ◽

Gram Positive ◽

Content Type ◽

Peptidoglycan Synthesis ◽

Lipid Ii ◽

Protein Components

ABSTRACTThe ExPortal ofStreptococcus pyogenesis 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 pathogenStreptococcus pyogenesis 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.

Download Full-text

Lipid II: A central component in bacterial cell wall synthesis and a target for antibiotics

Prostaglandins Leukotrienes and Essential Fatty Acids ◽

10.1016/j.plefa.2008.09.020 ◽

2008 ◽

Vol 79 (3-5) ◽

pp. 117-121 ◽

Cited By ~ 68

Author(s):

Ben de Kruijff ◽

Vincent van Dam ◽

Eefjan Breukink

Keyword(s):

Cell Wall ◽

Bacterial Cell ◽

Bacterial Cell Wall ◽

Central Component ◽

Cell Wall Synthesis ◽

Lipid Ii

Download Full-text

Tropomyosin is essential in yeast, yet the TPM1 and TPM2 products perform distinct functions.

The Journal of Cell Biology ◽

10.1083/jcb.128.3.383 ◽

1995 ◽

Vol 128 (3) ◽

pp. 383-392 ◽

Cited By ~ 92

Author(s):

B Drees ◽

C Brown ◽

B G Barrell ◽

A Bretscher

Keyword(s):

Sequence Analysis ◽

Wild Type ◽

Purification And Characterization ◽

Reading Frame ◽

Over Expression ◽

Sequence Identity ◽

Bona Fide ◽

Essential Function

Sequence analysis of chromosome IX of Saccharomyces cerevisiae revealed an open reading frame of 166 residues, designated TPM2, having 64.5% sequence identity to TPM1, that encodes the major form of tropomyosin in yeast. Purification and characterization of Tpm2p revealed a protein with the characteristics of a bona fide tropomyosin; it is present in vivo at about one sixth the abundance of Tpm1p. Biochemical and sequence analysis indicates that Tpm2p spans four actin monomers along a filament, whereas Tpmlp spans five. Despite its shorter length, Tpm2p can compete with Tpm1p for binding to F-actin. Over-expression of Tpm2p in vivo alters the axial budding of haploids to a bipolar pattern, and this can be partially suppressed by co-over-expression of Tpm1p. This suggests distinct functions for the two tropomyosins, and indicates that the ratio between them is important for correct morphogenesis. Loss of Tpm2p has no detectable phenotype in otherwise wild type cells, but is lethal in combination with tpm1 delta. Over-expression of Tpm2p does not suppress the growth or cell surface targeting defects associated with tpm1 delta, so the two tropomyosins must perform an essential function, yet are not functionally interchangeable. S. cerevisiae therefore provides a simple system for the study of two tropomyosins having distinct yet overlapping functions.

Download Full-text

Visualizing Conformation Transitions of the Lipid Flippase MurJ

10.1101/552745 ◽

2019 ◽

Author(s):

Alvin C. Y. Kuk ◽

Aili Hao ◽

Ziqiang Guan ◽

Seok-Yong Lee

Keyword(s):

Mass Spectrometry ◽

Cell Wall ◽

Bacterial Cell ◽

Structural Information ◽

Conformational Transitions ◽

Cell Wall Synthesis ◽

Lipid Ii ◽

Bacterial Peptidoglycan ◽

Lipid Flippase ◽

Conformation Transitions

AbstractThe 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 cysteine accessibility, mass spectrometry, and complementation studies, we elucidate the conformational transitions in MurJ that mediate lipid flipping, identified the key ion for function, and provide a framework for the development of inhibitors.

Download Full-text