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Ochrobactrum tritici  5bvl1 is able to resist to high concentrations of chromate through the expression of an inducible chromate-resistant determinant, found in a mobile element (Tn OtChr ), which carries the genes,  chrB ,  chrA ,  chrC  and  chrF . The regulation of  chr  operon present in Tn OtChr , which is controlled by a transcriptional regulator, ChrB, was characterized in the current work. Fusions of  chr  promoter, or  chr  promoter and  chrB  gene, upstream of a  gfp  reporter gene, identified the most probable promoter sequence within the  tnpR-chrB  intergenic region. This region contains an AT-rich imperfect inverted repeat sequence, which overlaps a part of the −10 sequence. The results of the  in vitro  DNA-binding assays with purified ChrB (His- or no-tagged) showed that the protein binds directly to the  chr  promoter region. In order to identify the ChrB functional domain for sensing chromate stress and for DNA-binding, site-directed mutagenesis of ChrB was performed. Among several single amino acid mutants, three mutants (R180; R187 and H229) prevented chromate induction without any modification to the protein’s stability. Interestingly, two ChrB mutants (R18 and R23) were constitutively active, regardless of chromate stress conditions, indicating that the residues most probably belong to the protein-DNA binding site. As such, the ChrB was classified as a transcriptional regulator that recognizes a specific DNA sequence, regulating the expression of a chromate resistance determinant.

Identification and Characterization of the Transcriptional Regulator ChrB in the Chromate Resistance Determinant of Ochrobactrum tritici 5bvl1