This Article Full Text (PDF) Other Versions of this Article: JB.00141-08v1 190/10/3444    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Google Scholar Articles by LEBLANC, J. J. Articles by HOFFMAN, P. S. PubMed PubMed Citation Articles by LEBLANC, J. J. Articles by HOFFMAN, P. S.

 Previous Article  |  Next Article 

J. Bacteriol. doi:10.1128/JB.00141-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

An Ortholog of OxyR in Legionella pneumophila (LpOxyR) is Expressed Post-Exponentially and Negatively Regulates the Alkyl Hydroperoxide Reductase (ahpC2D) Operon

Department of Pathology and Laboratory Medicine, Queen Elizabeth II Health Sciences Center, Halifax, Nova Scotia, Canada; Department of Microbiology and Immunology, and Medicine, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7; Department of Medicine, Division of Infectious Diseases and International Health, and Department of Microbiology, University of Virginia School of Medicine, Charlottesville, Virginia, U.S.A. 22908-1340

^* To whom correspondence should be addressed. Email: psh2n{at}virginia.edu.

	Abstract

Legionella pneumophila expresses two peroxide-scavenging alkyl hydroperoxide reductase systems (AhpC1 and AhpC2D) that are expressed differentially during the bacterial growth cycle. Functional loss of the post-exponentially expressed AhpC1 system is compensated for by increased expression of the exponentially expressed AhpC2D system. In this study, we used an acrylamide capture of DNA-bound complexes (ACDC) technique and mass spectrometry to identify proteins that bind to the promoter region of the ahpC2D operon. The major protein captured was an ortholog of OxyR (LpOxyR). Genetic studies indicated that LpoxyR was an essential gene expressed post exponentially, and only partially complemented an Escherichia coli oxyR mutant (GS077). Gel shift assays confirmed specific binding of LpOxyR to ahpC2D promoter sequences, but not to promoters of ahpC1 or LpoxyR: however, LpOxyR weakly bound to E. coli OxyR-regulated promoters (katG, oxyR, and ahpCF). DNase I protection studies showed that the LpOxyR binding motif spanned the promoter and transcriptional start sequences of ahpC2 and that the protected region was unchanged by treatments with reducing agents or hydrogen peroxide (H₂O₂). Moreover, the LpOxyR (pBADLpoxyR) mediated repression of an ahpC2gfp reporter construct in E. coli GS077 (oxyR mutant) was not reversed by H₂O₂ challenge. Alignments with other OxyR proteins revealed several amino acid substitutions predicted to ablate thiol oxidation or conformational changes required for activation. We suggest these mutations have locked LpOxyR in an active DNA-binding conformation, which has permitted divergence of function from a regulator of oxidative stress to a cell cycle regulator, perhaps controlling gene expression during post-exponential differentiation.