嗜铁钩端螺旋菌Leptospirillum ferriphilum UBK03抗镍操纵子表达调控机理探究

【Abstract】wWw.shuoshilunwen.com A nickel resistance determinant ncrABCY was identified in Leptospirillum ferriphilum UBK03. There were four genes in this operon, ncrA, ncrB, ncrC and ncrY. NcrA, which was the base of the nickel resistance system, can constitute the nickel efflux pump together with the accessory protein NcrC. NcrY had a negative effect on nickel resistance. Moreover, NcrB can regulate the expression of ncrA as a transcriptional regulator. In this study, we aimed at the operon to elucidate its mechani of expression and regulation.By comparing the growth of E.coli NR21 either induced or not induced by NiCl2 in medium containing the NiCl2, we found that the growth of the uninduced group delayed about 2 h compared with the induced one. This result indicated that the nickel resistance determinant held an inducible property. RT-PCR confirmed the transcription of ncrA, ncrB, ncrC upregulated by Ni2+. Besides, qRT-PCR revealed that the presence of Ni2+ in culture medium resulted in a 10-fold increase in ncrA transcription.To further investigate the regulation, we determined the transcription start site of ncrA by the high resolution S1 nuclease mapping. A high GC content and inverted repeat sequence (p1p17) was identified at the downstream of the transcription start point. The results of Electrophoretic mobility shift (EMSAs) and DNase I footprinting assays showed that purified NcrB could specifically bind to the inverted repeat sequence in vitro.The interaction between the NcrB and pncrA was also confirmed by the bacterial one-hybrid system. Interestingly in vivo, this interaction was disrupted by added 1 mM Ni2+ to the screening medium plate. It demonstrated that NcrB was a nickel-responsive transcriptional regulator. Furthermore, pncrA was deleted to identify the region that was critical for NcrB bingding. We found that only the fragments contained intact p1p17 could bind with NcrB, revealing that NcrB bound directly to the inverted repeat sequence in the p1p17 region. Apo-NcrB might compete with RNA polymerase to bind to p1p17, which might repress transcription of ncrA.There were other two promoters (pncrB and pncrY) in the operon, with similar sequence to p1p17. However, they didn’t interact with NcrB in vivo. By comparing with this similar region, we found the five nucleotides between the two half-sites of p1p17 were critical for interaction. So we designed random library to alterative these five nucleotides. After screening, the conservative basic pair was the forth one, which might decide the interaction between the promoter and the NcrB.