Analysis of mutations that alter HO sensing and transcription regulation properties of a global peroxide regulator OxyR in Xanthomonas campestris pv. phaseoli

OxyR5, from a Xanthomonas campestris pv. phaseoli H(2)O(2)-resistant mutant, contains the two mutations G197D and L301R. The protein exists in its oxidized-like form in the absence of oxidants as judged by the protein's ability to activate the ahpC promoter. Analysis of DNase I footprint patterns indicates that under reducing conditions OxyR5 and OxyRG197D bind to the target site in the ahpC promoter in a manner similar to oxidized wild-type OxyR. Site-directed mutagenesis showed that OxyR5 behaves like oxidized OxyR, independent of the highly conserved C residues at positions 199 and 208 where, in normal OxyR, a disulfide bond between these residues converts the protein from its reduced to the oxidized form. The presence of aspartic acid or valine residue at position 197 caused OxyR to behave like the oxidized form in uninduced cells. Changing D197 to A or T in OxyR5 resulted in proteins with similar properties to native OxyR. In vivo, OxyR5 probably locked in an oxidized-like conformation, resulting in continuous high-level activation of target genes in the OxyR regulon.