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The nickel resistance determinant  ncrABCY  was identified in  Leptospirillum ferriphilum  UBK03. Within this operon,  ncrA  and  ncrC  encode two membrane proteins that form an efflux system, and  ncrB  encodes NcrB, which belongs to an uncharacterized family (DUF156) of proteins. How this determinant is regulated remains unknown. Our data indicate that expression of the nickel resistance determinant is induced by nickel. The promoter of  ncrA , designated  pncrA , was cloned into the promoter probe vector pPR9TT, and co-transformed with either a wild-type or mutant nickel resistance determinant. The results revealed that  ncrB  encoded a transcriptional regulator that could regulate the expression of  ncrA ,  ncrB , and  ncrC . A GC-rich inverted repeat sequence was identified in the promoter  pncrA . Electrophoretic mobility shift assays (EMSAs) and footprinting assays showed that purified NcrB could specifically bind to the inverted repeat sequence of  pncrA in vitro ; this was confirmed by bacterial one-hybrid analysis. Moreover, this binding was inhibited in the presence of nickel ions. Thus, we classified NcrB as a transcriptional regulator that recognizes the inverted repeat sequence binding motif to regulate the expression of the key nickel resistance gene,  ncrA .

Identification of the Transcriptional Regulator NcrB in the Nickel Resistance Determinant of Leptospirillum ferriphilum UBK03