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An Ortholog of OxyR in Legionella pneumophila Is Expressed Postexponentially 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,¹ Department of Microbiology and Immunology,² Medicine, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada,³ Department of Medicine, Division of Infectious Diseases and International Health,⁴ Department of Microbiology, University of Virginia School of Medicine, Charlottesville, Virginia 22908-1340⁵

Received 28 January 2008/ Accepted 11 March 2008

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 postexponentially 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 (OxyR_Lp). Genetic studies indicated that oxyR_Lp was an essential gene expressed postexponentially and only partially complemented an Escherichia coli oxyR mutant (GS077). Gel shift assays confirmed specific binding of OxyR_Lp to ahpC2D promoter sequences, but not to promoters of ahpC1 or oxyR_Lp; however, OxyR_Lp weakly bound to E. coli OxyR-regulated promoters (katG, oxyR, and ahpCF). DNase I protection studies showed that the OxyR_Lp 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 OxyR_Lp (pBADLpoxyR)-mediated repression of an ahpC2-gfp reporter construct in E. coli GS077 (the 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 OxyR_Lp in an active DNA-binding conformation, which has permitted a divergence of function from a regulator of oxidative stress to a cell cycle regulator, perhaps controlling gene expression during postexponential differentiation.

Published ahead of print on 21 March 2008.