A novel soxO gene, encoding a glutathione disulfide reductase, is essential for tetrathionate oxidation in Advenella kashmirensis

Abstract

Molecular mechanisms of chemolithotrophic tetrathionate oxidation are not clearly understood. Here we used transposon(Tn5-mob)-insertion mutagenesis to search for novel tetrathionate oxidation genes in the facultatively chemolithoautotrophic betaproteobacterium Advenella kashmirensis that not only oxidizes tetrathionate, but also produces the same as an intermediate during thiosulfate oxidation. Genome-wide random insertion of Tn5-mob occurred at a frequency of one per 104 donor E. coli cells. A library of 8000 transconjugants yielded five tetrathionate-oxidation-impaired mutants, of which, the one named Ak_Tn_16 was studied here in detail. When grown chemolithoautotrophically on thiosulfate, Ak_Tn_16 converted the total thiosulfate supplied to equivalent amount of tetrathionate, exactly in the same way as the wild type. It could not, however, oxidize the intermediary tetrathionate to sulfate; Ak_Tn_16 could not also oxidize tetrathionate when it was supplied as the starting chemolithotrophic substrate. In the Ak_Tn_16 genome, Tn5-mob was found to have transposed in a novel soxO gene, located just-upstream of soxB, within the sox gene cluster. SoxO was predicted, via iterative threading assembly simulation, to be a glutathione-disulfide (GSSG) reductase. When Ak_Tn_16 was grown in tetrathionate-based chemolithoautotrophic medium supplemented with reduced glutathione (GSH) its tetrathionate-oxidation deficiency, remarkably, was ameliorated. Implications for a key role of GSH in tetrathionate oxidation are discussed in the light of other molecular evidences available for A. kashmirensis.