A regulation mechanism for the promoter region of the pet II operon in Acidithiobacillus ferrooxidans ATCC23270

Acidithiobacillus ferrooxidans ATCC23270 is a gram-negative and autotrophic bacillus acquiring energy via the oxidation of iron and sulfur. The pet II operon is involved in the sulfur metabolism of A. ferrooxidans. However, the mechanisms that control the expression of the pet II operon are poorly understood. We previously described that the AFE2726 protein is associated with the expression of the pet II operon. Here, we attempted to analyze the involvement of AFE2726 in the regulation of pet II operon expression. First, pEGF recombinant vectors driven by the promotor of the pet II operon, denoted pEGF-pet II, were constructed. Then, DH5α E. coli cultures containing the vector mentioned above were cultivated in Na₂S₂O₃, as this medium substantially enhances the expression of green fluorescent proteins. To examine the regulatory effect of AFE2726 on the pet II operon, the C62/V and C72/V mutants for AFE2726 were constructed in pEGF-pet II vectors using the site-directed deletion method. Compared to pEFG-pet II and pEFG-pet II-Δ-C62/V, pEFG-pet II-Δ-C72/V reduced the expression of green fluorescent proteins dramatically when transformed into DH5α E.coli in Na₂S₂O₃ medium. This suggested that the 72nd cysteine was a crucial residue of the AFE2726 protein, affecting the response of the pet II operon to sodium thiosulfate. Furthermore, the binding site of AFE2726 on the promotor of the pet II operon was identified using the electrophoretic mobility shift assay (EMSA), and it was found to be a 34bp inverted repeat sequence (named IR4), which ranged from -65 to -32. In summary, our results indicated that the AFE2726 protein regulates the pet II operon by binding to the IR4 sequence in its promotor region, whose function is likely affected by Na₂S₂O₃ binding to its Cys72 residue counterpart.