CLONING AND CHARACTERIZATION OF SULFITE DEHYDROGENASE, 2 C-TYPE CYTOCHROMES, AND A FLAVOPROTEIN OF PARACOCCUS-DENITRIFICANS-GB17 - ESSENTIAL ROLE OF SULFITE DEHYDROGENASE IN LITHOTROPHIC SULFUR OXIDATION

A 13-kb genomic region of Paracoccus dentrificans GB17 is involved in lithotrophic thiosulfate oxidation, Adjacent to the previously reporte d soxB gene (C, Wodara, S, Kostka, M. Egert. D, P, Kelly, and C., G, F riedrich, J, Bacteriol, 176:6188-6191, 1994), 3.7 kb were sequenced, S equence analysis revealed four additional open reading frames, soxCDEF , soxC coded for a 430-amino-acid polypeptide with an M-r of 47,339 th at included a putative signal peptide of 40 amino acids (M-r of 3,599) with a RR motif present in periplasmic proteins with complex redox ce nters, The mature soxC gene product exhibited high amino acid sequence similarity to the eukaryotic molybdoenzyme sulfite oxidase and to nit rate reductase, We constructed a mutant, GBsoxC Delta, carrying an in- frame deletion in soxC which covered a region possibly coding for the molybdenum cofactor binding domain, GBsoxC Delta was unable to grow li thoautotrophically with thiosulfate but grew well with nitrate as a ni trogen source or as an electron acceptor, Whole cells and cell extract s of mutant GBsoxC Delta contained 10% of the thiosulfate-oxidizing ac tivity of the wild type, Only a marginal rate of sulfate-dependent cyt ochrome c reduction was observed from cell extracts of mutant GBsoxC D elta. These results demonstrated that sulfite dehydrogenase was essent ial for growth with thiosulfate of P. dentrificans GB17. soxD) coded f or a periplasmic diheme c-type cytochrome of 384 amino acids (M-r, of 39,983) containing a putative signal peptide with an M-r, of 2,363, so xE coded for a periplasmic monoheme c-type cytochrome of 236 amino aci ds (M-r of 25,926) containing a putative signal peptide with an M-r, o f 1,833, SoxD and SoxE were highly identical to c-type cytochromes of P. denitrificans and other organisms, soxF revealed an incomplete open reading frame coding for a peptide of 247 amino acids with a putative signal peptide (M-r of 2,629). The deduced amino acid sequence of sox F was 47% identical and 70% similar to the sequence of the flavoprotei n of flavocytochrome c of Chromatium vinosum, suggesting the involveme nt of the flavoprotein in thiosulfate oxidation of P, denitrificans GB 17.