Many heterotrophic bacteria contain sulfide:quinone oxidoreductase (SQR) and persulfide dioxygenase (PDO) genes. It is unclear how these enzymes cooperate to oxidise sulfide in bacteria. Cupriavidus pinatubonensis JMP134 contains a gene cluster of sqr and pdo, and their functions were analysed in Escherichia coli. Recombinant E. coli cells with SQR and PDO rapidly oxidised sulfide to thiosulfate and sulfite. The SQR also contains a DUF442 domain that was shown to have rhodanese activities. E. coli cells with PDO and SQR-C94S, an active site mutant of the rhodanese domain, oxidised sulfide to thiosulfate with transitory accumulation of polysulfides. Cellular and enzymatic evidence showed that DUF442 speeds up the reaction of polysulfides with glutathione to produce glutathione persulfide (GSSH). Thus, SQR oxidises sulfide to polysulfides; rhodanese enhances the reaction of polysulfides with glutathione to produce GSSH; PDO oxidises GSSH to sulfite; sulfite spontaneously reacts with polysulfides to generate thiosulfate. The pathway is different from the proposed mitochondrial pathway because it has polysulfides, that is, disulfide and trisulfide, as intermediates. The data demonstrated that heterotrophic bacteria with SQR and PDO can rapidly oxidise sulfide to thiosulfate and sulfite, providing the foundation for using heterotrophic bacteria with SQR and PDO for sulfide bioremediation.

Heterotrophic bacteria actively participate in the biogeochemical cycle of sulfur on Earth. The heterotrophic bacterium Cupriavidus pinatubonensis JMP134 contains several enzymes involved in sulfur oxidation, but how these enzymes work together to oxidize sulfide in the bacterium has not been studied. Using gene-deletion and whole cell assays, we determined that the bacterium uses sulfide:quinone oxidoreductase to oxidize sulfide to polysulfide, which is further oxidized to sulfite by persulfide dioxygenase. Sulfite spontaneously reacts with polysulfide to produce thiosulfate. The Sox system oxidizes thiosulfate to sulfate. Flavocytochrome c sulfide dehydrogenase enhances thiosulfate oxidation by the Sox system, but couples with the Sox system for sulfide oxidation to sulfate in the absence of sulfide:quinone oxidoreductase. Thus, C. pinatubonensis JMP134 contains a main pathway and a contingent pathway for sulfide oxidation. Importance We establish a new pathway of sulfide oxidation with thiosulfate as a key intermediate in Cupriavidus pinatubonensis JMP134. The bacterium mainly oxidizes sulfide by using sulfide:quinone oxidoreductase, persulfide dioxygenase, and the Sox system with thiosulfate as a key intermediate. Although the purified and reconstituted Sox system oxidizes sulfide, its rate of sulfide oxidation in C. pinatubonensis JMP134 is too low to be physiologically relevant. The findings reveal how these sulfur oxidizing enzymes participate in sulfide oxidation in a single bacterium.