Identification and functional analysis of a regulatory protein for the pertlI operon in Acidithiobacillus ferrooxidans ATCC23270
Some evidences indicated that the petll operon of Acidithiobacillus ferrooxidans ATCC23270 may encode the enzymes of the sulfur metabolism. In this study, the hypothetical protein gene AFE2726 located in the upstream region of the petll operon was cloned into the psv-β-galactosidase reporter gene vector together with the promoter of the petll operon and transformed into E. coli DH5α. Overnight cultured bacteria were inoculated into LB medium containing 0. lmol/L sodium thiosulfate ( Na2S2O3 ) , which acted as a potential inducer for the petll operon. The /3-galactosidase enzyme activity is increased by 35. 66％ compared with the control group (without sodium thiosulfate) ( P < 0. 05 ) . These data indicated that the promoter of the petll operon could respond to sodium thiosulfate, and the AFE2726 protein might play a potential role in the response process. Thus, the start codon( ATG) of the AFE2726 gene in the reporter vector was mutated to TTG for eliminating its expression. The β-galactosidase enzyme activity of the bacteria containing this mutated reporter vector remained unchanged no matter whether sodium thiosulfate was added or not. Furthermore, N-terminal six-his-tagged protein (his-AFE2726 from plasmid PLM1) was purified by affinity chromatography. Electrophoretic mobility shift assay ( EMSA) analysis showed that the recombinant AFE2726 protein did bind to the promoter sequence of the petll operon. Moreover, the AFE2726 protein was composed of 115 amino acids, containing two Cys residues (position 62 and 72) which were usually believed to be capable of interacting with sulfur atom. Missense mutations of the two Cys residues were performed in the reporter vector respectively. The result of the β-galactosidase reporter gene assay showed that the Cys72 residue rather than the Cys62 residue might be in the active site of the AFE2726 protein. Therefore, it appears that the petll operon should play an important role in the sulfur metabolism pathway of Acidithiobacillus ferrooxidans, and the AFE2726 protein should be the regulatory protein of the petll operon, and the Cys72 residue might play an important role in this protein interacting with sodium thiosulfate.
Acidithiobacillus ferrooxidans pefll operon sulfur metabolism regulatory protein
Yu Yang Jiangjiang Li Xiang Wang Li Yang Zhiying Huang Xueduan Liu Guanzhou Qiu
School of Minerals Processing and Bioengineering, Central South University, 410083, Changsha, China School of Minerals Processing and Bioengineering, Central South University, 410083, Changsha, China
国际会议
The 19th International Biohydrometallurgy Symposium(第19届国际生物湿法冶金大会 IBS2011)
长沙
英文
344-348
2011-09-18（万方平台首次上网日期，不代表论文的发表时间）