Molecular and physiological analysis of an OxyR-regulated ahpC promoter in Xanthomonas campestris pv. phaseoli.

Abstract

In Xanthomonas campestris pv. phaseoli, a gene for the alkyl hydroperoxide reductase subunit C (ahpC) had unique patterns of regulation by various forms of OxyR. Reduced OxyR repressed expression of the gene, whereas oxidized OxyR activated its expression. This dual regulation of ahpC is unique and unlike all other OxyR-regulated genes. The ahpC transcription start site was determined. Analysis of the region upstream of the site revealed promoter sequences that had high homology to the Xanthomonas consensus promoter sequence. Data from gel shift experiments indicated that both reduced and oxidized OxyR could bind to the ahpC regulatory region. Moreover, the reduced and the oxidized forms of OxyR gave different DNase I footprint patterns, indicating that they bound to different sites. The oxidized OxyR binding site overlapped the -35 region of the ahpC promoter by a few bases. This position is consistent with the role of the protein in activating transcription of the gene. Binding of reduced OxyR to the ahpC promoter showed an extended DNase I footprint and DNase I hypersensitive sites, suggesting that binding of the protein caused a shift in the binding site and bending of the target DNA. In addition, binding of reduced OxyR completely blocked the -35 region of the ahpC promoter and prevented binding of RNA polymerase, leading to repression of the gene. Monitoring of the ahpC promoter activity in vivo confirmed the location of the oxidized OxyR binding site required for activation of the promoter. A mutant that separated OxyR regulation from basal ahpC promoter activity was constructed. The mutant was unable to respond to oxidants by increasing ahpC expression. Physiologically, it had a slower aerobic growth rate and was more sensitive to organic peroxide killing. This indicated that oxidant induction of ahpC has important physiological roles in normal growth and during oxidative stress.