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.
Journal of bacteriology (Impact Factor: 2.81). 06/2008; 190(10):3444-55. DOI: 10.1128/JB.00141-08
Source: PubMed


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 (OxyRLp). Genetic studies indicated that oxyRLp was an essential gene expressed postexponentially and only partially complemented an Escherichia coli oxyR mutant (GS077). Gel shift assays confirmed specific binding of OxyRLp to ahpC2D promoter sequences, but not to promoters of ahpC1 or oxyRLp; however, OxyRLp weakly bound to E. coli OxyR-regulated promoters (katG, oxyR, and ahpCF). DNase I protection studies showed that the OxyRLp 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 OxyRLp (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 OxyRLp 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.

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