1887

Abstract

630 possesses a cryptic but functional gene cluster homologous to the operon of . Expression of in the presence of subinhibitory concentrations of vancomycin is accompanied by peptidoglycan amidation on the -DAP residue. In this paper, we report the presence of two potential asparagine synthetase genes named and in the genome whose products were potentially involved in this peptidoglycan structure modification. We found that expression was only induced when was grown in the presence of vancomycin, yet independently from the resistance and regulation operons. In addition, peptidoglycan precursors were not amidated when was inactivated. No change in vancomycin MIC was observed in the mutant strain. In contrast, overexpression of resulted in the amidation of most of the peptidoglycan precursors and in a weak increase of vancomycin susceptibility. AsnB activity was confirmed in . In contrast, the expression of the second asparagine synthetase, AsnB2, was not induced in the presence of vancomycin. In summary, our results demonstrate that AsnB is responsible for peptidoglycan amidation of in the presence of vancomycin.

Funding
This study was supported by the:
  • Héloise Coullon , Ministère de lʼenseignement supérieur, de la recherche et de lʼInnovation
  • Fariza Ammam , Ministère de l'enseignment supérieur, de la recherche et de l'innovation
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2020-04-30
2020-06-02
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References

  1. Vollmer W, Blanot D, de Pedro MA. Peptidoglycan structure and architecture. FEMS Microbiol Rev 2008; 32: 149 167 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  2. Bernard E, Rolain T, Courtin P, Hols P, Chapot-Chartier M-P. Identification of the amidotransferase AsnB1 as being responsible for meso-diaminopimelic acid amidation in Lactobacillus plantarum peptidoglycan. J Bacteriol 2011; 193: 6323 6330 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  3. Levefaudes M, Patin D, de Sousa-d'Auria C, Chami M, Blanot D et al. Diaminopimelic acid amidation in Corynebacteriales: new insights into the role of LtsA in peptidoglycan modification. J Biol Chem 2015; 290: 13079 13094 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  4. Ngadjeua F, Braud E, Saidjalolov S, Iannazzo L, Schnappinger D et al. Critical Impact of Peptidoglycan Precursor Amidation on the Activity of l,d-Transpeptidases from Enterococcus faecium and Mycobacterium tuberculosis . Chemistry 2018; 24: 5743 5747 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  5. Dajkovic A, Tesson B, Chauhan S, Courtin P, Keary R et al. Hydrolysis of peptidoglycan is modulated by amidation of meso-diaminopimelic acid and Mg2+ in Bacillus subtilis . Mol Microbiol 2017; 104: 972 988 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  6. Veiga P, Erkelenz M, Bernard E, Courtin P, Kulakauskas S et al. Identification of the asparagine synthase responsible for D-Asp amidation in the Lactococcus lactis peptidoglycan interpeptide crossbridge. J Bacteriol 2009; 191: 3752 3757 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  7. Figueiredo TA, Sobral RG, Ludovice AM, Almeida JMFde, Bui NK et al. Identification of genetic determinants and enzymes involved with the amidation of glutamic acid residues in the peptidoglycan of Staphylococcus aureus . PLoS Pathog 2012; 8: e1002508 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  8. Münch D, Roemer T, Lee SH, Engeser M, Sahl HG et al. Identification and in vitro analysis of the GatD/MurT enzyme-complex catalyzing lipid II amidation in Staphylococcus aureus . PLoS Pathog 2012; 8: e1002509 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  9. Efron PA, Mazuski JE, colitis Cdifficile. Clostridium difficile colitis.. Surg Clin North Am 2009; 89: 483 500 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  10. Keller PM, Weber MH. Rational Therapy of Clostridium difficile Infections. Viszeralmedizin 2014; 30: 5 309 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  11. Ammam F, Marvaud J-C, Lambert T. Distribution of the vanG-like gene cluster in Clostridium difficile clinical isolates. Can J Microbiol 2012; 58: 547 551 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  12. Peltier J, Courtin P, El Meouche I, Catel-Ferreira M, Chapot-Chartier M-P et al. Genomic and expression analysis of the vanG-like gene cluster of Clostridium difficile . Microbiology 2013; 159: 1510 1520 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  13. Ammam F, Meziane-Cherif D, Mengin-Lecreulx D, Blanot D, Patin D et al. The functional vanGCd cluster of Clostridium difficile does not confer vancomycin resistance. Mol Microbiol 2013; 89: 612 625 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  14. Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning: A Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1982
    [Google Scholar]
  15. Heap JT, Cartman ST, Kuehne SA, Cooksley C, Minton NP. ClosTron-targeted mutagenesis. Methods Mol Biol 2010; 646: 165 182 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  16. Fagan RP, Fairweather NF. Clostridium difficile has two parallel and essential Sec secretion systems. J Biol Chem 2011; 286: 27483 27493 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  17. Heap JT, Pennington OJ, Cartman ST, Minton NP. A modular system for Clostridium shuttle plasmids. J Microbiol Methods 2009; 78: 79 85 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  18. Barreteau H, Bouhss A, Fourgeaud M, Mainardi J-L, Touzé T et al. Human- and plant-pathogenic Pseudomonas species produce bacteriocins exhibiting colicin M-like hydrolase activity towards peptidoglycan precursors. J Bacteriol 2009; 191: 3657 3664 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  19. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001; 29: 45e 45 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  20. Mengin-Lecreulx D, Flouret B, van Heijenoort J. Pool levels of UDP N-acetylglucosamine and UDP N-acetylglucosamine-enolpyruvate in Escherichia coli and correlation with peptidoglycan synthesis. J Bacteriol 1983; 154: 1284 1290 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  21. Mengin-Lecreulx D, van Heijenoort J. Effect of growth conditions on peptidoglycan content and cytoplasmic steps of its biosynthesis in Escherichia coli . J Bacteriol 1985; 163: 208 212 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  22. Glauner B. Separation and quantification of muropeptides with high-performance liquid chromatography. Anal Biochem 1988; 172: 451 464 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  23. Mengin-Lecreulx D, Flouret B, van Heijenoort J. Cytoplasmic steps of peptidoglycan synthesis in Escherichia coli . J Bacteriol 1982; 151: 1109 1117 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  24. Dupuy B, Sonenshein AL. Regulated transcription of Clostridium difficile toxin genes. Mol Microbiol 1998; 27: 107 120 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  25. Monot M, Boursaux-Eude C, Thibonnier M, Vallenet D, Moszer I et al. Reannotation of the genome sequence of Clostridium difficile strain 630. J Med Microbiol 2011; 60: 1193 1199 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  26. Kurka H, Ehrenreich A, Ludwig W, Monot M, Rupnik M et al. Sequence similarity of Clostridium difficile strains by analysis of conserved genes and genome content is reflected by their ribotype affiliation. PLoS One 2014; 9: e86535 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  27. Shen WJ, Deshpande A, Hevener KE, Endres BT, Garey KW et al. Constitutive expression of the cryptic vanG Cd operon promotes vancomycin resistance in Clostridioides difficile clinical isolates. J Antimicrob Chemother 2019
    [Google Scholar]
  28. Soutourina OA, Monot M, Boudry P, Saujet L, Pichon C et al. Genome-wide identification of regulatory RNAs in the human pathogen Clostridium difficile . PLoS Genet 2013; 9: e1003493 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  29. Janoir C, Denève C, Bouttier S, Barbut F, Hoys S et al. Adaptive strategies and pathogenesis of Clostridium difficile from in vivo transcriptomics. Infect Immun 2013; 81: 3757 3769 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
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