Sulfide (H₂S, HS⁻ and S²⁻) oxidation to sulfite and thiosulfate by heterotrophic bacteria, using sulfide:quinone oxidoreductase (SQR) and persulfide dioxygenase (PDO), has recently been reported as a possible detoxification mechanism for sulfide at high levels. Bioinformatic analysis revealed that the sqr and pdo genes were common in sequenced bacterial genomes, implying the sulfide oxidation may have other physiological functions. SQRs have previously been classified into six types. Here we grouped PDOs into three types and showed that some heterotrophic bacteria produced and released H₂S from organic sulfur into the headspace during aerobic growth, and others, for example, Pseudomonas aeruginosa PAO1, with sqr and pdo did not release H₂S. When the sqr and pdo genes were deleted, the mutants also released H₂S. Both sulfide-oxidizing and non-oxidizing heterotrophic bacteria were readily isolated from various environmental samples. The sqr and pdo genes were also common in the published marine metagenomic and metatranscriptomic data, indicating that the genes are present and expressed. Thus, heterotrophic bacteria actively produce and consume sulfide when growing on organic compounds under aerobic conditions. Given their abundance on Earth, their contribution to the sulfur cycle should not be overlooked.

Production of sulfide (H ₂ S, HS ⁻ , and S ²⁻ ) by heterotrophic bacteria during aerobic growth is a common phenomenon. Some bacteria with sulfide:quinone oxidoreductase (SQR) and persulfide dioxygenase (PDO) can oxidize self-produced sulfide to sulfite and thiosulfate, but other bacteria without these enzymes release sulfide into the medium, from which H ₂ S can volatilize into the gas phase. Here, we report that Cupriavidus necator H16, with the fccA and fccB genes encoding flavocytochrome c sulfide dehydrogenases (FCSDs), also oxidized self-produced H ₂ S. A mutant in which fccA and fccB were deleted accumulated and released H ₂ S. When fccA and fccB were expressed in Pseudomonas aeruginosa strain Pa3K with deletions of its sqr and pdo genes, the recombinant rapidly oxidized sulfide to sulfane sulfur. When PDO was also cloned into the recombinant, the recombinant with both FCSD and PDO oxidized sulfide to sulfite and thiosulfate. Thus, the proposed pathway is similar to the pathway catalyzed by SQR and PDO, in which FCSD oxidizes sulfide to polysulfide, polysulfide spontaneously reacts with reduced glutathione (GSH) to produce glutathione persulfide (GSSH), and PDO oxidizes GSSH to sulfite, which chemically reacts with polysulfide to produce thiosulfate. About 20.6% of sequenced bacterial genomes contain SQR, and only 3.9% contain FCSD. This is not a surprise, since SQR is more efficient in conserving energy because it passes electrons from sulfide oxidation into the electron transport chain at the quinone level, while FCSD passes electrons to cytochrome c . The transport of electrons from the latter to O ₂ conserves less energy. FCSDs are grouped into three subgroups, well conserved at the taxonomic level. Thus, our data show the diversity in sulfide oxidation by heterotrophic bacteria. IMPORTANCE Heterotrophic bacteria with SQR and PDO can oxidize self-produced sulfide and do not release H ₂ S into the gas phase. C. necator H16 has FCSD but not SQR, and it does not release H ₂ S. We confirmed that the bacterium used FCSD for the oxidation of self-produced sulfide. The bacterium also oxidized added sulfide. The common presence of SQRs, FCSDs, and PDOs in heterotrophic bacteria suggests the significant role of heterotrophic bacteria in sulfide oxidation, participating in sulfur biogeochemical cycling. Further, FCSDs have been identified in anaerobic photosynthetic bacteria and chemolithotrophic bacteria, but their physiological roles are unknown. We showed that heterotrophic bacteria use FCSDs to oxidize self-produced sulfide and extraneous sulfide, and they may be used for H ₂ S bioremediation.

Many industrial activities produce H₂S, which is toxic at high levels and odorous at even very low levels. Chemolithotrophic sulfur-oxidizing bacteria are often used in its remediation. Recently, we have reported that many heterotrophic bacteria can use sulfide:quinone oxidoreductase and persulfide dioxygenase to oxidize H₂S to thiosulfate and sulfite. These bacteria may also potentially be used in H₂S biotreatment. Here we report how various heterotrophic bacteria with these enzymes were cultured with organic compounds and the cells were able to rapidly oxidize H₂S to zero-valence sulfur and thiosulfate, causing no apparent acidification. Some also converted the produced thiosulfate to tetrathionate. The rates of sulfide oxidation by some of the tested bacteria in suspension, ranging from 8 to 50 µmol min⁻¹ g⁻¹ of cell dry weight at pH 7.4, sufficient for H₂S biotreatment. The immobilized bacteria removed H₂S as efficiently as the bacteria in suspension, and the inclusion of Fe₃O₄ nanoparticles during immobilization resulted in increased efficiency for sulfide removal, in part due to chemical oxidation H₂S by Fe₃O₄. Thus, heterotrophic bacteria may be used for H₂S biotreatment under aerobic conditions.

Elemental sulfur is a common fungicide, but its inhibition mechanism is unclear. Here, we investigated the effects of elemental sulfur on the single-celled fungus Saccharomyces cerevisiae and showed that the inhibition was due to its function as a strong oxidant. It rapidly entered S. cerevisiae. Inside the cytoplasm, it reacted with glutathione to generate glutathione persulfide that then reacted with another glutathione to produce H₂S and glutathione disulfide. H₂S reversibly inhibited the oxygen consumption by the mitochondrial electron transport chain, and the accumulation of glutathione disulfide caused disulfide stress and increased reactive oxygen species in S. cerevisiae. Elemental sulfur inhibited the growth of S. cerevisiae; however, it did not kill the yeast for up to 2 h exposure. The combined action of elemental sulfur and hosts’ immune responses may lead to the demise of fungal pathogens.

Heterotrophic bacteria have recently been reported to oxidize sulfide to sulfite and thiosulfate by using sulfide:quinone oxidoreductase (SQR) and persulfide dioxygenase (PDO). In chemolithotrophic bacteria, both SQR and PDO have been reported to function in the periplasmic space, with SQR as a peripheral membrane protein whose C terminus inserts into the cytoplasmic membrane and PDO as a soluble protein. Cupriavidus pinatubonensis JMP134, best known for its ability to degrade 2,4-dichlorophenoxyacetic acid and other aromatic pollutants, has a gene cluster of sqr and pdo encoding C. pinatubonensis SQR (CpSQR) and CpPDO2. When cloned in Escherichia coli , the enzymes are functional. Here we investigated whether they function in the periplasmic space or in the cytoplasm in heterotrophic bacteria. By using sequence analysis, biochemical detection, and green fluorescent protein (GFP)/PhoA fusion proteins, we found that CpSQR was located on the cytoplasmic side of the membrane and CpPDO2 was a soluble protein in the cytoplasm with a tendency to be peripherally located near the membrane. The location proximity of these proteins near the membrane in the cytoplasm may facilitate sulfide oxidation in heterotrophic bacteria. The information may guide the use of heterotrophic bacteria in bioremediation of organic pollutants as well as H ₂ S. IMPORTANCE Sulfide (H ₂ S, HS ⁻ , and S ²⁻ ), which is common in natural gas and wastewater, causes a serious malodor at low levels and is deadly at high levels. Microbial oxidation of sulfide is a valid bioremediation method, in which chemolithotrophic bacteria that use sulfide as the energy source are often used to remove sulfide. Heterotrophic bacteria with SQR and PDO have recently been reported to oxidize sulfide to sulfite and thiosulfate. Cupriavidus pinatubonensis JMP134 has been extensively characterized for its ability to degrade organic pollutants, and it also contains SQR and PDO. This paper shows the localization of SQR and PDO inside the cytoplasm in the vicinity of the membrane. The information may provide guidance for using heterotrophic bacteria in sulfide bioremediation.

Elemental sulfur and sulfite have been used to inhibit the growth of yeasts, but thiosulfate has not been reported to be toxic to yeasts. We observed that thiosulfate was more inhibitory than sulfite to Saccharomyces cerevisiae growing in a common yeast medium. At pH < 4, thiosulfate was a source of elemental sulfur and sulfurous acid, and both were highly toxic to the yeast. At pH 6, thiosulfate directly inhibited the electron transport chain in yeast mitochondria, leading to reductions in oxygen consumption, mitochondrial membrane potential and cellular ATP. Although thiosulfate was converted to sulfite and H₂S by the mitochondrial rhodanese Rdl1, its toxicity was not due to H₂S as the rdl1-deletion mutant that produced significantly less H₂S was more sensitive to thiosulfate than the wild type. Evidence suggests that thiosulfate inhibits cytochrome c oxidase of the electron transport chain in yeast mitochondria. Thus, thiosulfate is a potential agent against yeasts.