Abstract
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.
Highlights
Metals are essential cofactors for many enzymes in bacterial cells
We identified a metal-resistant bacterium, L. ferriphilum UBK03, and cloned its nickel resistance determinant, including the ncrA, ncrB, ncrC, and ncrY genes
When non-induced E. coli NR21 was exposed to 4 mM NiCl2, there was a growth delay of 2 h compared with E. coli NR21 induced with 2 mM NiCl2, the growth yield was unaffected (Fig. 1)
Summary
Metals are essential cofactors for many enzymes in bacterial cells. The nickel ion, like other metal ions, is essential for bacterial metabolism and becomes toxic at high intracellular concentrations [2]. Escherichia coli can endure the presence of no more than 2 mM Ni2+ in culture media [3]. Nickel resistance in bacteria is accomplished principally by an operon-encoded and energy-dependent specific efflux system that pumps Ni2+ out of the cell, thereby lowering the intracellular concentration [4,5]. Several nickel-resistant bacteria have been isolated from heavy metal-contaminated locations, and their nickelresistance systems have been identified. A number of new efflux proteins have been identified; for example, RcnA of E. coli (Ni2+ and Co2+ resistance) [12,13], the cnr-like operon of Comamonas sp. A number of new efflux proteins have been identified; for example, RcnA of E. coli (Ni2+ and Co2+ resistance) [12,13], the cnr-like operon of Comamonas sp. [14], and mrdH of Pseudomonas putida (Ni2+ and Co2+)[15]
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