Permanent URL to this publication: http://dx.doi.org/10.5167/uzh-6289
Gauntlett, J C; Gebhard, S; Keis, S; Manson, J M; Pos, K M; Cook, G M (2008). Molecular analysis of BcrR, a membrane-bound bacitracin sensor and DNA-binding protein from Enterococcus faecalis. Journal of Biological Chemistry, 283(13):8591-8600.
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BcrR has been identified as a novel regulatory protein of high level bacitracin resistance encoded by the bcrABD operon in Enterococcus faecalis. The N-terminal domain of BcrR has similarity to the helix-turn-helix motif of DNA-binding proteins, and topological modeling predicts that the C-terminal domain contains four transmembrane alpha-helices. These data have led to the hypothesis that BcrR functions as both a membrane-bound sensor and transducer of bacitracin availability to regulate bcrABD expression. To characterize the bcrABD promoter and identify the promoter elements to which BcrR binds, a series of bcrA-lacZ fusions were constructed. A 69-bp region was identified that was essential for bacitracin-dependent bcrA-lacZ expression. Mutations that targeted this region were used to identify two inverted repeat sequences, each with the sequence 5'-GACA(N)(7)TGTC-3', on the bcrABD promoter that were required for bcrA-lacZ expression. To study BcrR binding to this region, we over-produced BcrR with a C-terminal hexa-histidine tag in Escherichia coli membranes, extracted the protein with n-dodecyl-beta-d-maltoside, and subsequently purified it via Ni(2+)-nitrilotriacetic acid and gel filtration chromatography to apparent homogeneity. Purified BcrR was reconstituted into liposomes, and BcrR binding to bcrABD promoter DNA was analyzed using electrophoretic mobility shift assays. Both inverted repeat sequences were required for BcrR binding, both in the presence and absence of bacitracin. These data demonstrate that membrane-bound BcrR binds specifically to the bcrABD promoter, irrespective of bacitracin concentration. We therefore propose that bacitracin-dependent induction of bcrABD expression by BcrR occurs after DNA binding.
|Item Type:||Journal Article, refereed, original work|
|Communities & Collections:||04 Faculty of Medicine > Center for Integrative Human Physiology|
04 Faculty of Medicine > Institute of Physiology
07 Faculty of Science > Institute of Physiology
|DDC:||570 Life sciences; biology|
610 Medicine & health
|Deposited On:||02 Dec 2008 12:05|
|Last Modified:||23 Nov 2012 13:28|
|Publisher:||American Society for Biochemistry and Molecular Biology|
|Additional Information:||This research was originally published in Gauntlett, J C; Gebhard, S; Keis, S; Manson, J M; Pos, K M; Cook, G M (2008). Molecular analysis of BcrR, a membrane-bound bacitracin sensor and DNA-binding protein from Enterococcus faecalis. Journal of Biological Chemistry, 283(13):8591-8600. © the American Society for Biochemistry and Molecular Biology.|
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