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Identification of the transcriptional regulator NcrB in the nickel resistance determinant of Leptospirillum ferriphilum UBK03.

Zhu T, Tian J, Zhang S, Wu N, Fan Y - PLoS ONE (2011)

Bottom Line: The results revealed that ncrB encoded a transcriptional regulator that could regulate the expression of ncrA, ncrB, and ncrC.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.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China.

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.

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Inhibition of pncrA and NcrB binding in the presence of nickel.A bacterial one-hybrid system was used to detect n32p43 (pncrA)-protein and p1p17-protein interactions. Growth rates of cells containing different bait/prey combinations were examined under positive selection. Left panel: NM plate supplemented with 0.1% histamine and 1 mM NiCl2 as a control; Middle panel: NM plate supplemented with 2 mM 3-AT. Right panel: NM plate supplemented with 2.0 mM 3-AT and 1 mM NiCl2. Each population of cells was serially diluted in ten-fold steps (from left to right) and plated.
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pone-0017367-g007: Inhibition of pncrA and NcrB binding in the presence of nickel.A bacterial one-hybrid system was used to detect n32p43 (pncrA)-protein and p1p17-protein interactions. Growth rates of cells containing different bait/prey combinations were examined under positive selection. Left panel: NM plate supplemented with 0.1% histamine and 1 mM NiCl2 as a control; Middle panel: NM plate supplemented with 2 mM 3-AT. Right panel: NM plate supplemented with 2.0 mM 3-AT and 1 mM NiCl2. Each population of cells was serially diluted in ten-fold steps (from left to right) and plated.

Mentions: To test its effect on the binding between NcrB and the inverted repeat region, Ni2+ was added to the bacterial one-hybrid system by adding 1 mM Ni2+ to the 3-AT selective screening medium plate. As shown in Fig. 7, the strains containing the promoter (n32p43) or inverted repeat region (p1p17) in the prey plasmid grew on the 3-AT selective medium plates, but could not grow on the 3-AT plates containing 1 mM Ni2+. The presence of 1 mM Ni2+ in the medium did not affect the growth of E. coli [3], [9]. Thus, 1 mM Ni2+ could disrupt the binding between NcrB and the inverted repeat region (p1p17).


Identification of the transcriptional regulator NcrB in the nickel resistance determinant of Leptospirillum ferriphilum UBK03.

Zhu T, Tian J, Zhang S, Wu N, Fan Y - PLoS ONE (2011)

Inhibition of pncrA and NcrB binding in the presence of nickel.A bacterial one-hybrid system was used to detect n32p43 (pncrA)-protein and p1p17-protein interactions. Growth rates of cells containing different bait/prey combinations were examined under positive selection. Left panel: NM plate supplemented with 0.1% histamine and 1 mM NiCl2 as a control; Middle panel: NM plate supplemented with 2 mM 3-AT. Right panel: NM plate supplemented with 2.0 mM 3-AT and 1 mM NiCl2. Each population of cells was serially diluted in ten-fold steps (from left to right) and plated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046157&req=5

pone-0017367-g007: Inhibition of pncrA and NcrB binding in the presence of nickel.A bacterial one-hybrid system was used to detect n32p43 (pncrA)-protein and p1p17-protein interactions. Growth rates of cells containing different bait/prey combinations were examined under positive selection. Left panel: NM plate supplemented with 0.1% histamine and 1 mM NiCl2 as a control; Middle panel: NM plate supplemented with 2 mM 3-AT. Right panel: NM plate supplemented with 2.0 mM 3-AT and 1 mM NiCl2. Each population of cells was serially diluted in ten-fold steps (from left to right) and plated.
Mentions: To test its effect on the binding between NcrB and the inverted repeat region, Ni2+ was added to the bacterial one-hybrid system by adding 1 mM Ni2+ to the 3-AT selective screening medium plate. As shown in Fig. 7, the strains containing the promoter (n32p43) or inverted repeat region (p1p17) in the prey plasmid grew on the 3-AT selective medium plates, but could not grow on the 3-AT plates containing 1 mM Ni2+. The presence of 1 mM Ni2+ in the medium did not affect the growth of E. coli [3], [9]. Thus, 1 mM Ni2+ could disrupt the binding between NcrB and the inverted repeat region (p1p17).

Bottom Line: The results revealed that ncrB encoded a transcriptional regulator that could regulate the expression of ncrA, ncrB, and ncrC.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.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China.

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.

Show MeSH