Identification And Functional Analysis Of A Regulatory Protein For The PetⅡ Operon In Acidithiobacillus Ferrooxidans

Evidences gradually came to show that the six proteins encoded by pet II operon might be involved in sulfur metabolism of Acidithiobacillus ferrooxidans(A. ferrooxidans). We have identified the-35 and-10 box core element of pet II operon promoter. According to the genome sequences of A. ferrooxidans ATCC23270 in Genbank, the AFE2726 gene, which encode a putative protein, was concerned.Theβ-galactosidase reporter gene plasmid (pSV72) which contained the AFE2726 gene and pet II operon promoter region was transformed into E.coli DH5a andβ-galactosidase activity was tested. Theβ-galactosidase activity would increase 35.66% if sodium thiosulfate was added into LB medium. This result indicated that sodium thiosulfate could enhance the activity of the regulatory unit of pet II operon.In order to confirming if protein AFE2726 was involved in the induction of sodium thiosulfate to the regulatory unit of pet II operon, the site-directed mutagenesis was used to mutate the start codon of protein AFE2726 for eliminating its expression. The mutated reporter plasmid (pSV72m) was transformed into E.coli DH5a, and the (3-galactosidase activity was not influenced by sodium thiosulfate any more. This result indicated that protein AFE2726 should be involved in the response of petⅡoperon promoter to sodium thiosulfate.Furthermore, the gene AFE2726 was subcloned into plasmid pLM1 and transformed into E.coli BL21, and recombinant His-AFE2726 protein was expressed and purified subsequently. The result of electrophoretic mobility shift assay (EMSA) showed that the recombinant AFE2726 protein did bind to the promoter sequence of the pet II operon, and the protein AF2726 was really the regulatory protein for pet II operon.Bioinformatic analysis of the protein AFE2726 showed that it contained two cysteine residues (Cys62 and Cys72). Cysteine residue in protein was usually believed to be capable of binding with sodium thiosulfate, which might change the structure of protein and affect the binding capacity of protein to promoter. Thus, missense mutations of these two cysteine residues were performed in the reporter vector pSV72 respectively. The result of theβ-galactosidase reporter gene assay showed that the Cys72 residue rather than the Cys62 residue might be in the active site of the AFE2726 protein.In conclusion, petⅡoperon should be involved in sulfur metabolism of A. ferrooxidans, and the protein AFE2726 should be the regulatory protein for petⅡoperon, and the Cys72 residue should be the key binding site of the regulatory protein to sodium thiosulfate.