Molecular analysis of BcrR, a membrane-bound bacitracin sensor and DNA-binding protein from Enterococcus faecalis

Autoren

Jonathan C Gauntlett
Susanne Gebhard
Stefanie Keis
Janet M Manson
Klaas M Pos
Gregory M Cook

DOI

10.1074/jbc.m709503200

ISSN

0021-9258

Ausgabe der Veröffentlichung

13

Zeitschrift

Journal of Biological Chemistry

Sprache

en

Paginierung

8591 - 8600

Datum der Veröffentlichung

2008

Status

Published

Herausgeber

Elsevier BV

Herausgeber URL

http://dx.doi.org/10.1074/jbc.m709503200

Datum der Datenerfassung

2021

Titel

Molecular Analysis of BcrR, a Membrane-bound Bacitracin Sensor and DNA-binding Protein from Enterococcus faecalis

Ausgabe der Zeitschrift

283

Data source: Crossref

Abstract

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.

Addresses

Department of Microbiology and Immunology, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand.

Autoren

Jonathan C Gauntlett
Susanne Gebhard
Stefanie Keis
Janet M Manson
Klaas M Pos
Gregory M Cook

DOI

10.1074/jbc.m709503200

eISSN

1083-351X

Externe Identifier

PubMed Identifier: 18227063

Open access

false

ISSN

0021-9258

Ausgabe der Veröffentlichung

13

Zeitschrift

The Journal of biological chemistry

Schlüsselwörter

Cell Membrane
Enterococcus faecalis
Bacitracin
ATP-Binding Cassette Transporters
DNA-Binding Proteins
Gene Expression Regulation, Bacterial
Amino Acid Sequence
Protein Binding
Mutation
Molecular Sequence Data
Promoter Regions, Genetic

Sprache

eng

Medium

Print-Electronic

Online publication date

2008

Paginierung

8591 - 8600

Datum der Veröffentlichung

2008

Status

Published

Publisher licence

CC BY

Datum der Datenerfassung

2008

Titel

Molecular analysis of BcrR, a membrane-bound bacitracin sensor and DNA-binding protein from Enterococcus faecalis.

Sub types

Research Support, Non-U.S. Gov't
Journal Article

Ausgabe der Zeitschrift

283

Data source: Europe PubMed Central

Abstract

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.

Autoren

Jonathan C Gauntlett
Susanne Gebhard
Stefanie Keis
Janet M Manson
Klaas M Pos
Gregory M Cook

Autoren-URL

https://www.ncbi.nlm.nih.gov/pubmed/18227063

DOI

10.1074/jbc.M709503200

ISSN

0021-9258

Ausgabe der Veröffentlichung

13

Zeitschrift

J Biol Chem

Schlüsselwörter

ATP-Binding Cassette Transporters
Amino Acid Sequence
Bacitracin
Cell Membrane
DNA-Binding Proteins
Enterococcus faecalis
Gene Expression Regulation, Bacterial
Molecular Sequence Data
Mutation
Promoter Regions, Genetic
Protein Binding

Sprache

eng

Country

United States

Paginierung

8591 - 8600

PII

S0021-9258(20)52816-0

Datum der Veröffentlichung

2008

Status

Published

Datum, an dem der Datensatz öffentlich gemacht wurde

2008

Titel

Molecular analysis of BcrR, a membrane-bound bacitracin sensor and DNA-binding protein from Enterococcus faecalis.

Sub types

Journal Article
Research Support, Non-U.S. Gov't

Ausgabe der Zeitschrift

283

Data source: PubMed

Molecular analysis of BcrR, a membrane-bound bacitracin sensor and DNA-binding protein from Enterococcus faecalis

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