scholarly journals Structure and mutagenic analysis of the lipid II flippase MurJ from Escherichia coli

2018 ◽  
Author(s):  
Sanduo Zheng ◽  
Lok-To Sham ◽  
Frederick A. Rubino ◽  
Kelly Brock ◽  
William P. Robins ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Plasma Membrane ◽  
Cell Biology ◽  
Model Organism ◽  
Structural Data ◽  
Outer Face ◽  
Antibiotic Development ◽  
Cell Wall Assembly ◽  
Lipid Ii

AbstractThe 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 prior to 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 are only available for MurJ 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. Finally, through the use of sequence co-evolution analysis we identify functionally important interactions in the outward-open state of the protein, supporting a rocker-switch model for lipid II transport.

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

2018 ◽  
Vol 115 (26) ◽  
pp. 6709-6714 ◽  
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.

scholarly journals Formation of flavone-based wooly fibres by glandular trichomes of Dionysia tapetodes

2020 ◽  
Author(s):  
Matthieu Bourdon ◽  
Josephine Gaynord ◽  
Karin Müller ◽  
Gareth Evans ◽  
Simon Wallis ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Cell Biology ◽  
Glandular Trichomes ◽  
Thread Formation ◽  
Flavone Derivatives ◽  
Chemical Techniques ◽  
Surface Covering ◽  
Membrane Cell ◽  
Putative Mechanism

AbstractDionysia tapetodes, a small cushion-forming mountainous evergreen in the Primulaceae, possesses a vast surface-covering of long silky fibres forming the characteristic “wooly” farina. This contrasts with some related Primula which instead possess a powdery farina. Using a combination of cell biology and analytical chemical techniques, we provide a detailed insight of wooly farina formation by glandular trichomes that produce a mixture of flavone and substituted flavone derivatives, including hydroxyflavones. Conversely, our analysis show that the powdery form consist almost entirely of flavone. The wooly farina in D. tapetodes is extruded through specific sites at the surface of the glandular head cell, characterised by a small complete gap in the plasma membrane, cell wall and cuticle. The data is consistent with formation and thread elongation occurring from within the cell. The putative mechanism of wool thread formation and its stability is discussed.

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

2016 ◽  
Vol 60 (8) ◽  
pp. 4930-4939 ◽  
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.

scholarly journals The active repertoire of Escherichia coli peptidoglycan amidases varies with physiochemical environment

2020 ◽  
Author(s):  
Elizabeth A. Mueller ◽  
Abbygail G. Iken ◽  
Mehmet Ali Öztürk ◽  
Mirko Schmitz ◽  
Barbara Di Ventura ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Cell Separation ◽  
Antimicrobial Agents ◽  
Model Organism ◽  
Growth Conditions ◽  
Low Ph ◽  
E Coli ◽  
Ph Dependent ◽  
Stimulation Of

ABSTRACTNearly all bacteria are encased in a peptidoglycan cell wall, an essential crosslinked matrix of polysaccharide strands and short peptide stems. In the Gram-negative model organism Escherichia coli, more than forty cell wall synthases and autolysins coordinate the growth and division of the peptidoglycan sacculus in the periplasm. The precise contribution of many of these enzymes to cell wall metabolism remains unclear due to significant apparent redundancy, particularly among the cell wall autolysins. E. coli produces three major LytC-type-N-acetylmuramoyl-L-alanine amidases, which share a role in separating the newly formed daughter cells during cytokinesis. Here, we reveal two of the three amidases exhibit growth medium-dependent changes in activity. Specifically, we report acidic growth conditions stimulate AmiB—and to a lesser extent, AmiC—activity. Combining computational and genetic analysis, we demonstrate that low pH-dependent stimulation of AmiB requires three periplasmic amidase activators: EnvC, NlpD, and YgeR. Altogether, our findings support overlapping, but not redundant, roles for the E. coli amidases in cell separation and illuminate the physiochemical environment as an important mediator of cell wall enzyme activity.IMPORTANCEPenicillin and related β-lactam antibiotics targeting the bacterial cell wall synthesis are among the most commonly prescribed antimicrobials worldwide. However, rising rates of antibiotic resistance and tolerance jeopardize their continued clinical use. Development of new cell wall active therapeutics, including those targeting cell wall autolysins, has been stymied in part due to high levels of apparent enzymatic redundancy. In this study, we report a subset of E. coli amidases involved in cell separation during cell division are not redundant and instead are preferentially active during growth in distinct pH environments. Specifically, we discover E. coli amidases AmiB and AmiC are activated by acidic pH. Three semi-redundant periplasmic regulators—NlpD, EnvC, and YgeR—collectively mediate low pH-dependent stimulation of amidase activity. This discovery contributes to our understanding of how the cell wall remains robust across diverse environmental conditions and reveals opportunities for the development of condition-specific antimicrobial agents.

scholarly journals Antibiotic hypersensitivity signatures identify targets for attack in the Acinetobacter baumannii cell envelope

2020 ◽  
Author(s):  
Edward Geisinger ◽  
Nadav J. Mortman ◽  
Yunfei Dai ◽  
Murat Cokol ◽  
Sapna Syal ◽  
...  
Keyword(s):  
Cell Wall ◽  
Acinetobacter Baumannii ◽  
Cell Envelope ◽  
Opportunistic Pathogen ◽  
Drug Action ◽  
Antibiotic Development ◽  
Cell Wall Assembly ◽  
Combination Strategies ◽  
High Doses ◽  
Wall Assembly

AbstractAcinetobacter baumannii is an opportunistic pathogen that is a critical, high-priority target for new antibiotic development. Clearing of A. baumannii requires relatively high doses of antibiotics across the spectrum, primarily due to its protective cell envelope. Many of the proteins that support envelope integrity and modulate drug action are uncharacterized, largely because there is an absence of orthologs for several proteins that perform essential envelope-associated processes, impeding progress on this front. To identify targets that can synergize with current antibiotics, we performed an exhaustive analysis of A. baumannii mutants causing hypersensitivity to a multitude of antibiotic treatments. By examining mutants with antibiotic hypersensitivity profiles that parallel mutations in proteins of known function, we show that the function of poorly annotated proteins can be predicted and used to identify candidate missing link proteins in essential A. baumannii processes. Using this strategy, we uncovered multiple uncharacterized proteins with critical roles in cell division or cell elongation, and revealed that a predicted cell wall D,D-endopeptidase has an unappreciated function in lipooligosaccharide synthesis. Moreover, we provide a genetic strategy that uses hypersensitivity signatures to predict drug synergies, allowing the identification of β-lactams that work cooperatively based on the cell wall assembly machineries that they preferentially target. These data reveal multiple pathways critical for envelope growth in A. baumannii that can be targeted in combination strategies for attacking the pathogen.

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

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.

Microcompartments within the yeast plasma membrane

2013 ◽  
Vol 394 (2) ◽  
pp. 189-202 ◽  
Author(s):  
Hans Merzendorfer ◽  
Jürgen J. Heinisch
Keyword(s):  
Plasma Membrane ◽  
Cell Biology ◽  
Model Organism ◽  
Dynamic Interaction ◽  
Eukaryotic Cells ◽  
Classical Concept ◽  
Yeast Plasma Membrane ◽  
Bud Neck ◽  
Microscopic Studies ◽  
Membrane Compartments

Abstract Recent research in cell biology makes it increasingly clear that the classical concept of compartmentation of eukaryotic cells into different organelles performing distinct functions has to be extended by microcompartmentation, i.e., the dynamic interaction of proteins, sugars, and lipids at a suborganellar level, which contributes significantly to a proper physiology. As different membrane compartments (MCs) have been described in the yeast plasma membrane, such as those defined by Can1 and Pma1 (MCCs and MCPs), Saccharomyces cerevisiae can serve as a model organism, which is amenable to genetic, biochemical, and microscopic studies. In this review, we compare the specialized microcompartment of the yeast bud neck with other plasma membrane substructures, focusing on eisosomes, cell wall integrity-sensing units, and chitin-synthesizing complexes. Together, they ensure a proper cell division at the end of mitosis, an intricately regulated process, which is essential for the survival and proliferation not only of fungal, but of all eukaryotic cells.

scholarly journals The mycobacterial cell wall partitions the plasma membrane to organize its own synthesis

2019 ◽  
Author(s):  
Alam García-Heredia ◽  
Takehiro Kado ◽  
Caralyn E. Sein ◽  
Julia Puffal ◽  
Sarah H. Osman ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Membrane Domains ◽  
Peptidoglycan Biosynthesis ◽  
Cell Wall Assembly ◽  
Peptidoglycan Synthesis ◽  
Cell Wall Biogenesis ◽  
Mycobacterial Cell Wall ◽  
Wall Assembly ◽  
Made In

AbstractMany antibiotics target the assembly of cell wall peptidoglycan, an essential, heteropolymeric mesh that encases most bacteria. Different species have characteristic subcellular sites of peptidoglycan synthesis that they must carefully maintain for surface integrity and, ultimately, viability. In rod-shaped bacteria, cell wall elongation is spatially precise yet relies on a limited pool of lipid-linked precursors that generate and are attracted to membrane disorder. By tracking enzymes, substrates and products of peptidoglycan biosynthesis in Mycobacterium smegmatis, we show that precursors are made in plasma membrane domains that are laterally and biochemically distinct from sites of cell wall assembly. Membrane partitioning is required for robust, orderly peptidoglycan synthesis, indicating that these domains help template peptidoglycan synthesis. The cell wall-organizing protein DivIVA and the cell wall itself are essential for domain homeostasis. Thus, the peptidoglycan polymer feeds back on its membrane template to maintain an environment conducive to directional synthesis. We further show that our findings are applicable to rod-shaped bacteria that are phylogenetically distant from M. smegmatis, demonstrating that horizontal compartmentalization of precursors is a general feature of bacillary cell wall biogenesis.

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

2020 ◽  
Author(s):  
Kaitlin Schaefer ◽  
Tristan W. Owens ◽  
Julia E. Page ◽  
Marina Santiago ◽  
Daniel Kahne ◽  
...  
Keyword(s):  
Cell Wall ◽  
Membrane Protein ◽  
Transmembrane Helices ◽  
Cell Wall Assembly ◽  
Lipid Ii ◽  
Peptidoglycan Cell Wall ◽  
The Matrix ◽  
Peptide Precursor ◽  
Polytopic Membrane Protein ◽  
Wall Assembly

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.

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

2018 ◽  
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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