1887

Abstract

Bacitracin is a cell wall targeting antimicrobial with clinical and agricultural applications. With the growing mismatch between antimicrobial resistance and development, it is essential we understand the molecular mechanisms of resistance in order to prioritize and generate new effective antimicrobials. BcrR is a unique membrane-bound one-component system that regulates high-level bacitracin resistance in Enterococcus faecalis . In the presence of bacitracin, BcrR activates transcription of the bcrABD operon conferring resistance through a putative ATP-binding cassette (ABC) transporter (BcrAB). BcrR has three putative functional domains, an N-terminal helix–turn–helix DNA-binding domain, an intermediate oligomerization domain and a C-terminal transmembrane domain. However, the molecular mechanisms of signal transduction remain unknown. Random mutagenesis of bcrR was performed to generate loss- and gain-of-function mutants using transcriptional reporters fused to the target promoter P bcrA . Fifteen unique mutants were isolated across all three proposed functional domains, comprising 14 loss-of-function and one gain-of-function mutant. The gain-of-function variant (G64D) mapped to the putative dimerization domain of BcrR, and functional analyses indicated that the G64D mutant constitutively expresses the P bcrA-luxABCDE reporter. DNA-binding and membrane insertion were not affected in the five mutants chosen for further characterization. Homology modelling revealed putative roles for two key residues (R11 and S33) in BcrR activation. Here we present a new model of BcrR activation and signal transduction, providing valuable insight into the functional characterization of membrane-bound one-component systems and how they can coordinate critical bacterial responses, such as antimicrobial resistance.

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2019-02-19
2024-03-29
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References

  1. Ulrich LE, Koonin EV, Zhulin IB. One-component systems dominate signal transduction in prokaryotes. Trends Microbiol 2005; 13:52–56 [View Article][PubMed]
    [Google Scholar]
  2. Cuthbertson L, Nodwell JR. The TetR family of regulators. Microbiol Mol Biol Rev 2013; 77:440–475 [View Article][PubMed]
    [Google Scholar]
  3. Manson JM, Keis S, Smith JM, Cook GM. Acquired bacitracin resistance in Enterococcus faecalis is mediated by an ABC transporter and a novel regulatory protein, BcrR. Antimicrob Agents Chemother 2004; 48:3743–3748 [View Article][PubMed]
    [Google Scholar]
  4. Gauntlett JC, Gebhard S, Keis S, Manson JM, Pos KM et al. Molecular analysis of BcrR, a membrane-bound bacitracin sensor and DNA-binding protein from Enterococcus faecalis. J Biol Chem 2008; 283:8591–8600 [View Article][PubMed]
    [Google Scholar]
  5. Gebhard S, Gaballa A, Helmann JD, Cook GM. Direct stimulus perception and transcription activation by a membrane-bound DNA binding protein. Mol Microbiol 2009; 73:482–491 [View Article][PubMed]
    [Google Scholar]
  6. Dintner S, Heermann R, Fang C, Jung K, Gebhard S. A sensory complex consisting of an ATP-binding cassette transporter and a two-component regulatory system controls bacitracin resistance in Bacillus subtilis. J Biol Chem 2014; 289:27899–27910 [View Article][PubMed]
    [Google Scholar]
  7. Gebhard S, Fang C, Shaaly A, Leslie DJ, Weimar MR et al. Identification and characterization of a bacitracin resistance network in Enterococcus faecalis. Antimicrob Agents Chemother 2014; 58:1425–1433 [View Article][PubMed]
    [Google Scholar]
  8. Ohki R, Giyanto, Tateno K, Masuyama W, Moriya S et al. The BceRS two-component regulatory system induces expression of the bacitracin transporter, BceAB, in Bacillus subtilis. Mol Microbiol 2003; 49:1135–1144 [View Article][PubMed]
    [Google Scholar]
  9. Bernard R, Guiseppi A, Chippaux M, Foglino M, Denizot F. Resistance to bacitracin in Bacillus subtilis: unexpected requirement of the BceAB ABC transporter in the control of expression of its own structural genes. J Bacteriol 2007; 189:8636–8642 [View Article][PubMed]
    [Google Scholar]
  10. Rietkötter E, Hoyer D, Mascher T. Bacitracin sensing in Bacillus subtilis. Mol Microbiol 2008; 68:768–785 [View Article][PubMed]
    [Google Scholar]
  11. Harwood S, Cutting C. (editors) Molecular biological methods for Bacillus Chichester, England: John Wiley & Sons, Inc; 1990
    [Google Scholar]
  12. Fang C, Stiegeler E, Cook GM, Mascher T, Gebhard S. Bacillus subtilis as a platform for molecular characterisation of regulatory mechanisms of Enterococcus faecalis resistance against cell wall antibiotics. PLoS One 2014; 9:e9316910 [View Article][PubMed]
    [Google Scholar]
  13. Patterson AG, Chang JT, Taylor C, Fineran PC. Regulation of the Type I-F CRISPR-Cas system by CRP-cAMP and GalM controls spacer acquisition and interference. Nucleic Acids Res 2015; 43:6038–6048 [View Article][PubMed]
    [Google Scholar]
  14. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 1989; 77:51–59[PubMed]
    [Google Scholar]
  15. Nesterenko MV, Tilley M, Upton SJ. A simple modification of Blum's silver stain method allows for 30 minute detection of proteins in polyacrylamide gels. J Biochem Biophys Methods 1994; 28:239–242 [View Article][PubMed]
    [Google Scholar]
  16. Webb B, Sali A. Comparative protein structure modeling using modeller. Curr Protoc Bioinformatics 2014; 47:5.6.1–5.6.5 [View Article]
    [Google Scholar]
  17. Radeck J, Kraft K, Bartels J, Cikovic T, Dürr F et al. The bacillus biobrick box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis. J Biol Eng 2013; 7:29 [View Article][PubMed]
    [Google Scholar]
  18. Mondragón A, Subbiah S, Almo SC, Drottar M, Harrison SC. Structure of the amino-terminal domain of phage 434 repressor at 2.0 A resolution. J Mol Biol 1989; 205:189–200 [View Article][PubMed]
    [Google Scholar]
  19. Sevilla-Sierra P, Otting G, Wüthrich K. Determination of the nuclear magnetic resonance structure of the DNA-binding domain of the P22 c2 repressor (1 to 76) in solution and comparison with the DNA-binding domain of the 434 repressor. J Mol Biol 1994; 235:1003–1020 [View Article][PubMed]
    [Google Scholar]
  20. Pervushin K, Billeter M, Siegal G, Wüthrich K. Structural role of a buried salt bridge in the 434 repressor DNA-binding domain. J Mol Biol 1996; 264:1002–1012 [View Article][PubMed]
    [Google Scholar]
  21. Harrison SC, Aggarwal AK. DNA recognition by proteins with the helix-turn-helix motif. Annu Rev Biochem 1990; 59:933–969 [View Article][PubMed]
    [Google Scholar]
  22. Bader MW, Sanowar S, Daley ME, Schneider AR, Cho U et al. Recognition of antimicrobial peptides by a bacterial sensor kinase. Cell 2005; 122:461–472 [View Article][PubMed]
    [Google Scholar]
  23. Li M, Lai Y, Villaruz AE, Cha DJ, Sturdevant DE et al. Gram-positive three-component antimicrobial peptide-sensing system. Proc Natl Acad Sci USA 2007; 104:9469–9474 [View Article][PubMed]
    [Google Scholar]
  24. Otto M. Bacterial sensing of antimicrobial peptides. Contrib Microbiol 2009; 16:136–149 [View Article][PubMed]
    [Google Scholar]
  25. Economou NJ, Cocklin S, Loll PJ. High-resolution crystal structure reveals molecular details of target recognition by bacitracin. Proc Natl Acad Sci USA 2013; 110:14207–14212 [View Article][PubMed]
    [Google Scholar]
  26. Lyon GJ, Novick RP. Peptide signaling in Staphylococcus aureus and other Gram-positive bacteria. Peptides 2004; 25:1389–1403 [View Article][PubMed]
    [Google Scholar]
  27. Mascher T, Helmann JD, Unden G. Stimulus perception in bacterial signal-transducing histidine kinases. Microbiol Mol Biol Rev 2006; 70:910–938 [View Article][PubMed]
    [Google Scholar]
  28. Charlebois A, Jalbert LA, Harel J, Masson L, Archambault M. Characterization of genes encoding for acquired bacitracin resistance in Clostridium perfringens. PLoS One 2012; 7:e44449 [View Article][PubMed]
    [Google Scholar]
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