title: Bacillus subtilis as a platform for molecular characterisation of regulatory mechanisms of Enterococcus faecalis resistance against cell wall antibiotics.
abstract: To combat antibiotic resistance of Enterococcus faecalis, a better understanding of the molecular mechanisms, particularly of antibiotic detection, signal transduction and gene regulation is needed. Because molecular studies in this bacterium can be challenging, we aimed at exploiting the genetically highly tractable Gram-positive model organism Bacillus subtilis as a heterologous host. Two fundamentally different regulators of E. faecalis resistance against cell wall antibiotics, the bacitracin sensor BcrR and the vancomycin-sensing two-component system VanSB-VanRB, were produced in B. subtilis and their functions were monitored using target promoters fused to reporter genes (lacZ and luxABCDE). The bacitracin resistance system BcrR-BcrAB of E. faecalis was fully functional in B. subtilis, both regarding regulation of bcrAB expression and resistance mediated by the transporter BcrAB. Removal of intrinsic bacitracin resistance of B. subtilis increased the sensitivity of the system. The lacZ and luxABCDE reporters were found to both offer sensitive detection of promoter induction on solid media, which is useful for screening of large mutant libraries. The VanSB-VanRB system displayed a gradual dose-response behaviour to vancomycin, but only when produced at low levels in the cell. Taken together, our data show that B. subtilis is a well-suited host for the molecular characterization of regulatory systems controlling resistance against cell wall active compounds in E. faecalis. Importantly, B. subtilis facilitates the careful adjustment of expression levels and genetic background required for full functionality of the introduced regulators.
authors.affiliation: Department Biology I, Microbiology, Ludwig-Maximilians-Universität München, Martinsried, Germany.
authors.forename: Chong
authors.initials: C
authors.lastname: Fang
authors.affiliation: Department Biology I, Microbiology, Ludwig-Maximilians-Universität München, Martinsried, Germany.
authors.forename: Emanuel
authors.initials: E
authors.lastname: Stiegeler
authors.affiliation: Department of Microbiology and Immunology, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand.
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Cook
authors.affiliation: Department Biology I, Microbiology, Ludwig-Maximilians-Universität München, Martinsried, Germany.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Mascher
authors.affiliation: Department Biology I, Microbiology, Ludwig-Maximilians-Universität München, Martinsried, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Gebhard
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D013293:Enterococcus faecalis / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D017930:Genes, Reporter; D008826:Microbial Sensitivity Tests; D011401:Promoter Regions, Genetic; D014161:Transduction, Genetic
chemical_list: D000900:Anti-Bacterial Agents