An Ortholog of OxyR in Legionella pneumophila Is Expressed Postexponentially and Negatively Regulates the Alkyl Hydroperoxide Reductase ( ahpC2D ) Operon

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 theahpC2D operon. The major protein captured was an ortholog of OxyR (OxyRLp ). Genetic studies indicated thatoxyRLp was an essential gene expressed postexponentially and only partially complemented anEscherichia coli oxyR mutant (GS077). Gel shift assays confirmed specific binding of OxyRLp toahpC2D promoter sequences, but not to promoters ofahpC1 oroxyRLp ; however, OxyRLp weakly bound toE. coli OxyR-regulated promoters (katG ,oxyR , andahpCF ). DNase I protection studies showed that the OxyRLp binding motif spanned the promoter and transcriptional start sequences ofahpC2 and that the protected region was unchanged by treatments with reducing agents or hydrogen peroxide (H2 O2 ). Moreover, the OxyRLp (pBADLpoxyR )-mediated repression of anahpC2 -gfp reporter construct inE. coli GS077 (theoxyR mutant) was not reversed by H2 O2 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.