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  • 1
    Online Resource
    Online Resource
    Fluorescence Detection-Based Functional Assay for High-Throughput Screening for MraY
    American Society for Microbiology ; 2004
    In:  Antimicrobial Agents and Chemotherapy Vol. 48, No. 3 ( 2004-03), p. 897-902
    Details
    In: Antimicrobial Agents and Chemotherapy, American Society for Microbiology, Vol. 48, No. 3 ( 2004-03), p. 897-902
    Abstract: We have developed a novel assay specific to MraY, which catalyzes the first membrane step in the biosynthesis of bacterial cell wall peptidoglycan. This was accomplished by using UDP-MurNAc- N ε -dansylpentapeptide, a fluorescent derivative of the MraY nucleotide substrate, and a partially purified preparation of MraY solubilized from membranes of an Escherichia coli overproducing strain. Two versions of the assay were developed, one consisting of the high-pressure liquid chromatography separation of the substrate and product (dansylated lipid I) and the other, without separation and adapted to the high-throughput format, taking advantage of the different fluorescence properties of the nucleotide and lipid I in the reaction medium. The latter assay was validated with a set of natural and synthetic MraY inhibitors.
    Type of Medium: Online Resource
    ISSN: 0066-4804 , 1098-6596
    URL: Article
    DOI: 10.1128/AAC.48.3.897-902.2004
    RVK:
    XA 24552
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2004
    detail.hit.zdb_id: 1496156-8
    SSG: 12
    SSG: 15,3
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  • 2
    Online Resource
    Online Resource
    Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains
    American Society for Microbiology ; 2015
    In:  mBio Vol. 6, No. 5 ( 2015-10-30)
    Details
    In: mBio, American Society for Microbiology, Vol. 6, No. 5 ( 2015-10-30)
    Abstract: Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.
    Type of Medium: Online Resource
    ISSN: 2161-2129 , 2150-7511
    URL: Article
    DOI: 10.1128/mBio.02327-14
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2015
    detail.hit.zdb_id: 2557172-2
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  • 3
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    Online Resource
    Substrate-Induced Inactivation of the Escherichia coli AmiD N -Acetylmuramoyl- l -Alanine Amidase Highlights a New Strategy To Inhibit This Class of Enzyme
    American Society for Microbiology ; 2009
    In:  Antimicrobial Agents and Chemotherapy Vol. 53, No. 7 ( 2009-07), p. 2991-2997
    Details
    In: Antimicrobial Agents and Chemotherapy, American Society for Microbiology, Vol. 53, No. 7 ( 2009-07), p. 2991-2997
    Abstract: In the eubacterial cell, the peptidoglycan is perpetually hydrolyzed throughout the cell cycle by different enzymes such as lytic transglycosylases, endopeptidases, and amidases. In Escherichia coli , four N -acetylmuramoyl- l -alanine amidases, AmiA, -B, -C, and -D, are present in the periplasm. AmiA, -B, and -C are soluble enzymes, whereas AmiD is a lipoprotein anchored in the outer membrane. To determine more precisely the specificity and the kinetic parameters of AmiD, we overproduced and purified the native His-tagged AmiD in the presence of detergent and a soluble truncated form of this enzyme by removing its signal peptide and the cysteine residue responsible for its lipidic anchorage. AmiD is a zinc metalloenzyme and is inactivated by a metal chelator such as EDTA. Native His-tagged and truncated AmiD hydrolyzes peptidoglycan fragments that have at least three amino acids in their peptide chains, and the presence of an anhydro function on the N -acetylmuramic acid is not essential for its activity. The soluble truncated AmiD exhibits a biphasic kinetic time course that can be explained by the inactivation of the enzyme by the substrate. This behavior highlights a new strategy to inhibit this class of enzymes.
    Type of Medium: Online Resource
    ISSN: 0066-4804 , 1098-6596
    URL: Article
    DOI: 10.1128/AAC.01520-07
    RVK:
    XA 24552
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2009
    detail.hit.zdb_id: 1496156-8
    SSG: 12
    SSG: 15,3
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  • 4
    Online Resource
    Online Resource
    MppA, a Periplasmic Binding Protein Essential for Import of the Bacterial Cell Wall Peptide l -Alanyl-γ- d -Glutamyl- meso -Diaminopimelate
    American Society for Microbiology ; 1998
    In:  Journal of Bacteriology Vol. 180, No. 5 ( 1998-03), p. 1215-1223
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 180, No. 5 ( 1998-03), p. 1215-1223
    Abstract: Mutants of a diaminopimelic acid (Dap)-requiring strain of Escherichia coli were isolated which failed to grow on media in which Dap was replaced by the cell wall murein tripeptide, l -alanyl-γ- d -glutamyl- meso -diaminopimelate. In one such mutant, which is oligopeptide permease (Opp) positive, we have identified a new gene product, designated MppA (murein peptide permease A), that is about 46% identical to OppA, the periplasmic binding protein for Opp. A plasmid carrying the wild-type mppA gene allows the mutant to grow on tripeptide. Two other mutants that failed to grow on tripeptide were resistant to triornithine toxicity, indicating a defect in the opp operon. An E. coli strain whose entire opp operon was deleted but which carried the mppA locus was unable to grow on murein tripeptide unless it was provided with oppBCDF genes in trans . Our data suggest a model whereby the periplasmic MppA binds the murein tripeptide, which is then transported into the cytoplasm via membrane-bound and cytoplasmic OppBCDF. In assessing the affinity of MppA for non-cell wall peptides, we have found that proline auxotrophy can be satisfied with the peptide Pro-Phe-Lys, which utilizes either MppA or OppA in conjunction with OppBCDF for its uptake. Thus, MppA, OppA, and perhaps the third OppA paralog revealed by the E. coli genome sequence may each bind a particular family of peptides but interact with common membrane-associated components for transport of their bound ligands into the cell. As to the physiological function of MppA, the possibility that it may be involved in signal transduction pathway(s) is discussed.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.180.5.1215-1223.1998
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 1998
    detail.hit.zdb_id: 1481988-0
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  • 5
    Online Resource
    Online Resource
    Identification of the UDP-MurNAc-Pentapeptide: l -Alanine Ligase for Synthesis of Branched Peptidoglycan Precursors in Enterococcus faecalis
    American Society for Microbiology ; 2001
    In:  Journal of Bacteriology Vol. 183, No. 17 ( 2001-09), p. 5122-5127
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 183, No. 17 ( 2001-09), p. 5122-5127
    Abstract: Many species of gram-positive bacteria produce branched peptidoglycan precursors resulting from the transfer of various l -amino acids or glycine from amino acyl-tRNA to the ɛ-amino group of l -lysine. The UDP-MurNAc-pentapeptide: l -alanine ligase and alanyl-tRNA synthetase genes from Enterococcus faecalis were identified, cloned, and overexpressed in Escherichia coli . The purified enzymes were necessary and sufficient for tRNA-dependent addition of l -alanine to UDP-MurNAc-pentapeptide in vitro. The ligase belonged to the Fem family of proteins, which were initially identified genetically as factors essential for methicillin resistance in Staphylococcus aureus .
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.183.17.5122-5127.2001
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2001
    detail.hit.zdb_id: 1481988-0
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  • 6
    Online Resource
    Online Resource
    Contribution of the P mra Promoter to Expression of Genes in the Escherichia coli mra Cluster of Cell Envelope Biosynthesis and Cell Division Genes
    American Society for Microbiology ; 1998
    In:  Journal of Bacteriology Vol. 180, No. 17 ( 1998-09), p. 4406-4412
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 180, No. 17 ( 1998-09), p. 4406-4412
    Abstract: Recently, a promoter for the essential gene ftsI , which encodes penicillin-binding protein 3 of Escherichia coli , was precisely localized 1.9 kb upstream from this gene, at the beginning of the mra cluster of cell division and cell envelope biosynthesis genes (H. Hara, S. Yasuda, K. Horiuchi, and J. T. Park, J. Bacteriol. 179:5802–5811, 1997). Disruption of this promoter (P mra ) on the chromosome and its replacement by the lac promoter (P mra ::P lac ) led to isopropyl-β- d -thiogalactopyranoside (IPTG)-dependent cells that lysed in the absence of inducer, a defect which was complemented only when the whole region from P mra to ftsW , the fifth gene downstream from ftsI , was provided in trans on a plasmid. In the present work, the levels of various proteins involved in peptidoglycan synthesis and cell division were precisely determined in cells in which P mra ::P lac promoter expression was repressed or fully induced. It was confirmed that the P mra promoter is required for expression of the first nine genes of the mra cluster: mraZ ( orfC ), mraW ( orfB ), ftsL ( mraR ), ftsI , murE , murF , mraY , murD , and ftsW . Interestingly, three- to sixfold-decreased levels of MurG and MurC enzymes were observed in uninduced P mra ::P lac cells. This was correlated with an accumulation of the nucleotide precursors UDP– N -acetylglucosamine and UDP– N -acetylmuramic acid, substrates of these enzymes, and with a depletion of the pool of UDP– N -acetylmuramyl pentapeptide, resulting in decreased cell wall peptidoglycan synthesis. Moreover, the expression of ftsZ , the penultimate gene from this cluster, was significantly reduced when P mra expression was repressed. It was concluded that the transcription of the genes located downstream from ftsW in the mra cluster, from murG to ftsZ , is also mainly (but not exclusively) dependent on the P mra promoter.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.180.17.4406-4412.1998
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 1998
    detail.hit.zdb_id: 1481988-0
    SSG: 12
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  • 7
    Online Resource
    Online Resource
    X-Ray Structure and Site-Directed Mutagenesis Analysis of the Escherichia coli Colicin M Immunity Protein
    American Society for Microbiology ; 2011
    In:  Journal of Bacteriology Vol. 193, No. 1 ( 2011-01), p. 205-214
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 193, No. 1 ( 2011-01), p. 205-214
    Abstract: Colicin M (ColM), which is produced by some Escherichia coli strains to kill competitor strains from the same or related species, was recently shown to inhibit cell wall peptidoglycan biosynthesis through enzymatic degradation of its lipid II precursor. ColM-producing strains are protected from the toxin that they produce by coexpression of a specific immunity protein, named Cmi, whose mode of action still remains to be identified. We report here the resolution of the crystal structure of Cmi, which is composed of four β strands and four α helices. This rather compact structure revealed a disulfide bond between residues Cys31 and Cys107. Interestingly, these two cysteines and several other residues appeared to be conserved in the sequences of several proteins of unknown function belonging to the YebF family which exhibit 25 to 35% overall sequence similarity with Cmi. Site-directed mutagenesis was performed to assess the role of these residues in the ColM immunity-conferring activity of Cmi, which showed that the disulfide bond and residues from the C-terminal extremity of the protein were functionally essential. The involvement of DsbA oxidase in the formation of the Cmi disulfide bond is also demonstrated.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.01119-10
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2011
    detail.hit.zdb_id: 1481988-0
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  • 8
    Online Resource
    Online Resource
    Human- and Plant-Pathogenic Pseudomonas Species Produce Bacteriocins Exhibiting Colicin M-Like Hydrolase Activity towards Peptidoglycan Precursors
    American Society for Microbiology ; 2009
    In:  Journal of Bacteriology Vol. 191, No. 11 ( 2009-06), p. 3657-3664
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 191, No. 11 ( 2009-06), p. 3657-3664
    Abstract: Genes encoding proteins that exhibit similarity to the C-terminal domain of Escherichia coli colicin M were identified in the genomes of some Pseudomonas species, namely, P. aeruginosa, P. syringae , and P. fluorescens . These genes were detected only in a restricted number of strains. In P. aeruginosa , for instance, the colicin M homologue gene was located within the ExoU-containing genomic island A, a large horizontally acquired genetic element and virulence determinant. Here we report the cloning of these genes from the three Pseudomonas species and the purification and biochemical characterization of the different colicin M homologues. All of them were shown to exhibit Mg 2+ -dependent diphosphoric diester hydrolase activity toward the two undecaprenyl phosphate-linked peptidoglycan precursors (lipids I and II) in vitro. In all cases, the site of cleavage was localized between the undecaprenyl and pyrophospho-MurNAc moieties of these precursors. These enzymes were not active on the cytoplasmic precursor UDP-MurNAc-pentapeptide or (or only very poorly) on undecaprenyl pyrophosphate. These colicin M homologues have a narrow range of antibacterial activity. The P. aeruginosa protein at low concentrations was shown to inhibit growth of sensitive P. aeruginosa strains. These proteins thus represent a new class of bacteriocins (pyocins), the first ones reported thus far in the genus Pseudomonas that target peptidoglycan metabolism.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.01824-08
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2009
    detail.hit.zdb_id: 1481988-0
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  • 9
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    Online Resource
    Purification and Characterization of the Bacterial UDP-GlcNAc:Undecaprenyl-Phosphate GlcNAc-1-Phosphate Transferase WecA
    American Society for Microbiology ; 2008
    In:  Journal of Bacteriology Vol. 190, No. 21 ( 2008-11), p. 7141-7146
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 190, No. 21 ( 2008-11), p. 7141-7146
    Abstract: To date, the structural and functional characterization of proteins belonging to the polyprenyl-phosphate N -acetylhexosamine-1-phosphate transferase superfamily has been relentlessly held back by problems encountered with their overexpression and purification. In the present work and for the first time, the integral membrane protein WecA that catalyzes the transfer of the GlcNAc-1-phosphate moiety from UDP-GlcNAc onto the carrier lipid undecaprenyl phosphate, yielding undecaprenyl-pyrophosphoryl-GlcNAc, the lipid intermediate involved in the synthesis of various bacterial cell envelope components, was overproduced and purified to near homogeneity in milligram quantities. An enzymatic assay was developed, and the kinetic parameters of WecA as well as the effects of pH, salts, cations, detergents, and temperature on the enzyme activity were determined. A minimal length of 35 carbons was required for the lipid substrate, and tunicamycin was shown to inhibit the enzyme at submicromolar concentrations.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.00676-08
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2008
    detail.hit.zdb_id: 1481988-0
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  • 10
    Online Resource
    Online Resource
    Toxicity of the Colicin M Catalytic Domain Exported to the Periplasm Is FkpA Independent
    American Society for Microbiology ; 2010
    In:  Journal of Bacteriology Vol. 192, No. 19 ( 2010-10), p. 5212-5219
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 192, No. 19 ( 2010-10), p. 5212-5219
    Abstract: Colicin M (ColM) is a bactericidal protein that kills sensitive cells by hydrolyzing lipid II, involved in the biosynthesis of cell wall peptidoglycan. It recognizes FhuA on the outer leaflet, and its translocation through the outer membrane depends on the energized Ton complex in the inner membrane. To be active in the periplasm, ColM must be translocated through the outer membrane and then interact with FkpA, a periplasmic protein that exhibits both cis - and trans -peptidylprolyl isomerase (PPiase) and chaperon activities. In an attempt to directly target ColM to the periplasm of the producing bacteria, we fused the presequence of OmpA to ColM (sp-ColM). We found that expression of this hybrid protein in an Escherichia coli strain devoid of ColM immunity protein (Cmi) was bactericidal. We showed that sp-ColM was correctly expressed, processed, and associated with the inner membrane. sp-ColM toxicity was related to its enzymatic activity and did not rely on the TonB import proteins or the FhuA receptor. The presence of both activity domains of FkpA was still required for sp-ColM activity. Analyses of deletion mutants of sp-ColM show that the domain required for toxicity corresponds to the C-terminal last 153 amino acids of ColM. Like the full-length protein, this domain is not active in the presence of the immunity protein Cmi. On the other hand, it does not require FkpA for toxic activity.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.00431-10
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2010
    detail.hit.zdb_id: 1481988-0
    SSG: 12
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