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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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The interaction between NcrB and pncrA in vitro.Electromobility shift assays (A–B). (A) The pncrA fragment was incubated with His6-tagged NcrB or the His6-tag at the indicated concentrations. Lane 1, no protein; lane 2, 0.2 µM NcrB; lane 3, 0.4 µM NcrB; lane 4, 0.8 µM NcrB; and lane 5, 1.6 µM NcrB; lane 6, 0.2 µM His6-tag; lane 7, 0.4 µM His6-tag; lane 8, 0.8 µM His6-tag; and lane 9, 1.6 µM His6-tag. (B) The fragment was incubated with both 0.8 µM NcrB and unlabeled competitor at the fold-concentrations indicated above the lanes. (C) Sequence of the promoter pncrA. The GC-rich inverted repeat sequence (bold), the transcription start site of ncrA (bold and underlined) and the potential translation initiation codon (bold and italic) are indicated along the sequences. (D) The transcription start site of ncrA and Dnase I footprint of NcrB on pncrA. Lane 1, the arrowhead indicates the transcription start point. Lane G, A, T and C indicate the nucleotide sequence ladders of pncrA. Lane 2 and 6, DNase I digestions as a control (No NcrB). Lanes 3–5, purified NcrB protein was added to the final concentration from (0.1 µM, 0.2 µM and 0.4 µM).
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pone-0017367-g005: The interaction between NcrB and pncrA in vitro.Electromobility shift assays (A–B). (A) The pncrA fragment was incubated with His6-tagged NcrB or the His6-tag at the indicated concentrations. Lane 1, no protein; lane 2, 0.2 µM NcrB; lane 3, 0.4 µM NcrB; lane 4, 0.8 µM NcrB; and lane 5, 1.6 µM NcrB; lane 6, 0.2 µM His6-tag; lane 7, 0.4 µM His6-tag; lane 8, 0.8 µM His6-tag; and lane 9, 1.6 µM His6-tag. (B) The fragment was incubated with both 0.8 µM NcrB and unlabeled competitor at the fold-concentrations indicated above the lanes. (C) Sequence of the promoter pncrA. The GC-rich inverted repeat sequence (bold), the transcription start site of ncrA (bold and underlined) and the potential translation initiation codon (bold and italic) are indicated along the sequences. (D) The transcription start site of ncrA and Dnase I footprint of NcrB on pncrA. Lane 1, the arrowhead indicates the transcription start point. Lane G, A, T and C indicate the nucleotide sequence ladders of pncrA. Lane 2 and 6, DNase I digestions as a control (No NcrB). Lanes 3–5, purified NcrB protein was added to the final concentration from (0.1 µM, 0.2 µM and 0.4 µM).

Mentions: The transcription start point of ncrA was localized at position 44 nt upstream of the potential start codon (ATG) of ncrA by the high-resolution S1 nuclease mapping (Figure 5D). As shown in Fig. 5D, a high GC content and inverted repeat sequence (p1p17) was identified at the downstream of the transcription start point. The possibility of a direct interaction of NcrB with the putative operator in pncrA was assessed in vitro by EMSA. The ncrB gene was ligated into the expression plasmid pET30a(+), purified and assessed by SDS-PAGE. The pncrA fragment was labeled using infrared dye-labeled M13 oligos and purified [29]. The EMSA results showed that NcrB caused a slower movement of labeled pncrA, indicating that NcrB binds to ncrA (Fig. 5A). Moreover, given the large excess of competitor DNA [poly (dI-dC)] or M13 primer in the binding mix, NcrB–pncrA binding must be specific. Binding was significantly reduced in the presence of unlabeled pncrA or the 17-bp inverted repeat. Thus, NcrB could bind pncrA at the 17-bp inverted repeat region.


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)

The interaction between NcrB and pncrA in vitro.Electromobility shift assays (A–B). (A) The pncrA fragment was incubated with His6-tagged NcrB or the His6-tag at the indicated concentrations. Lane 1, no protein; lane 2, 0.2 µM NcrB; lane 3, 0.4 µM NcrB; lane 4, 0.8 µM NcrB; and lane 5, 1.6 µM NcrB; lane 6, 0.2 µM His6-tag; lane 7, 0.4 µM His6-tag; lane 8, 0.8 µM His6-tag; and lane 9, 1.6 µM His6-tag. (B) The fragment was incubated with both 0.8 µM NcrB and unlabeled competitor at the fold-concentrations indicated above the lanes. (C) Sequence of the promoter pncrA. The GC-rich inverted repeat sequence (bold), the transcription start site of ncrA (bold and underlined) and the potential translation initiation codon (bold and italic) are indicated along the sequences. (D) The transcription start site of ncrA and Dnase I footprint of NcrB on pncrA. Lane 1, the arrowhead indicates the transcription start point. Lane G, A, T and C indicate the nucleotide sequence ladders of pncrA. Lane 2 and 6, DNase I digestions as a control (No NcrB). Lanes 3–5, purified NcrB protein was added to the final concentration from (0.1 µM, 0.2 µM and 0.4 µM).
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Related In: Results  -  Collection

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pone-0017367-g005: The interaction between NcrB and pncrA in vitro.Electromobility shift assays (A–B). (A) The pncrA fragment was incubated with His6-tagged NcrB or the His6-tag at the indicated concentrations. Lane 1, no protein; lane 2, 0.2 µM NcrB; lane 3, 0.4 µM NcrB; lane 4, 0.8 µM NcrB; and lane 5, 1.6 µM NcrB; lane 6, 0.2 µM His6-tag; lane 7, 0.4 µM His6-tag; lane 8, 0.8 µM His6-tag; and lane 9, 1.6 µM His6-tag. (B) The fragment was incubated with both 0.8 µM NcrB and unlabeled competitor at the fold-concentrations indicated above the lanes. (C) Sequence of the promoter pncrA. The GC-rich inverted repeat sequence (bold), the transcription start site of ncrA (bold and underlined) and the potential translation initiation codon (bold and italic) are indicated along the sequences. (D) The transcription start site of ncrA and Dnase I footprint of NcrB on pncrA. Lane 1, the arrowhead indicates the transcription start point. Lane G, A, T and C indicate the nucleotide sequence ladders of pncrA. Lane 2 and 6, DNase I digestions as a control (No NcrB). Lanes 3–5, purified NcrB protein was added to the final concentration from (0.1 µM, 0.2 µM and 0.4 µM).
Mentions: The transcription start point of ncrA was localized at position 44 nt upstream of the potential start codon (ATG) of ncrA by the high-resolution S1 nuclease mapping (Figure 5D). As shown in Fig. 5D, a high GC content and inverted repeat sequence (p1p17) was identified at the downstream of the transcription start point. The possibility of a direct interaction of NcrB with the putative operator in pncrA was assessed in vitro by EMSA. The ncrB gene was ligated into the expression plasmid pET30a(+), purified and assessed by SDS-PAGE. The pncrA fragment was labeled using infrared dye-labeled M13 oligos and purified [29]. The EMSA results showed that NcrB caused a slower movement of labeled pncrA, indicating that NcrB binds to ncrA (Fig. 5A). Moreover, given the large excess of competitor DNA [poly (dI-dC)] or M13 primer in the binding mix, NcrB–pncrA binding must be specific. Binding was significantly reduced in the presence of unlabeled pncrA or the 17-bp inverted repeat. Thus, NcrB could bind pncrA at the 17-bp inverted repeat region.

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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