Resistance to the antibiotic bacitracin in Enterococcus faecalis strain AR01/DGVS is conferred by the genes bcrABD that are under the control of the regulatory protein BcrR. The N-terminal domain of BcrR has similarity to the helix-turn-helix motif of DNA-binding proteins and topological modelling predicts that the C-terminal domain contains four transmembrane α-helices. These data have led to the hypothesis that BcrR is a novel transmembrane transcriptional regulator which acts to both sense bacitracin and initiate transcription of bcrABD. The transcription of bcrABD in the presence of bacitracin results in the expression of a putative ABC transporter, BcrAB, that may function by the efflux of bacitracin from the cell membrane.
The aim of this study was to further characterise the mechanism of bacitracin resistance in E. faecalis and to conduct structural and functional studies with BcrR. A series of bcrA-lacZ transcriptional fusions were created to investigate the regulation of bcrABD transcription by BcrR and map the bcrABD promoter. We determined that BcrR activates bcrA-lacZ expression in response to bacitracin directly and not indirectly via the build-up of cell wall stress or cell wall precursors. A 69-bp region of the bcrABD promoter was required for activation of bcrA-lacZ expression. The introduction of mutations into this sequence demonstrated that two inverted repeat regions (viz. 5�-CTGACA(N)₆GTGTC-3�) were required for bcrA-lacZ expression. Furthermore, the creation of a bcrR::kan insertion mutant and a bcrR-lacZ transcriptional fusion revealed that bcrR is required for high-level bacitracin resistance in AR01/DGVS and that BcrR does not act to auto-regulate bcrR-lacZ expression.
To study BcrR function, we overproduced BcrR with a C-terminal hexa-histidine tag in Escherichia coli membranes, extracted the protein with n-dodecyl-β-D-maltoside, and subsequently purified it via Ni�⁺-NTA affinity and gel filtration chromatography to apparent homogeneity. Purified BcrR was reconstituted into liposomes and BcrR binding to bcrABD promoter DNA was analysed using gel-shift assays. We demonstrated that BcrR binds the bcrABD promoter in both the absence and presence of Zn�⁺-bacitracin and that the presence of the inverted repeat regions is necessary for binding. Purification of BcrR has also enabled us to commence structural studies toward obtaining the three dimensional structure of BcrR. We obtained crystals of BcrR that diffracted poorly to a resolution of 19 Å.
To characterise the function of the putative ABC type transporter (exporter) of bacitracin, BcrAB, we conducted transport assays with purified bacitracin A, fluorescently labelled FITC-bacitracin A, and radioactive ⁶�Ni�⁺-bacitracin. Transport assays conducted with radioactive ⁶�Ni�⁺-bacitracin and inverted membrane vesicles of E. coli, in which BcrABD had been recombinantly overproduced, displayed uptake of ⁶�Ni�⁺-bacitracin, suggesting that BcrAB functions as a transporter of bacitracin.
We conclude that BcrR is a transmembrane DNA-binding protein that functions as both a bacitracin sensor and regulator of bcrABD expression. We propose that BcrR binds to inverted repeat regions in bcrABD promoter to regulate bcrABD expression. Expression of bcrABD results in the production of BcrAB, which appears to be capable of bacitracin efflux (i.e. uptake in inverted membrane vesicles), conferring high-level bacitracin resistance to E. faecalis.
| Identifer | oai:union.ndltd.org:ADTP/266208 |
| Date | January 2008 |
| Creators | Gauntlett, Jonathan C, n/a |
| Publisher | University of Otago. Department of Microbiology & Immunology |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Jonathan C Gauntlett |
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