The Heterotrophic Bacterium Cupriavidus pinatubonensis JMP134 Oxidizes Sulfide to Sulfate with Thiosulfate as a Key Intermediate

Abstract

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 sulfur-oxidizing (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.

Keywords: heterotrophic bacteria; sulfane sulfur; sulfate; sulfate reduction; sulfide; sulfur oxidation pathway; thiosulfate.

FIG 1

Schematic overview of the sulfur-oxidizing genes in C. pinatubonensis JMP134. The genome of C. pinatubonensis JMP134 includes two chromosomes, A and B. Genes encoding the SOR, Sox, and FCSD systems are located on chromosome A, and the sqr-pdo operon is on chromosome B. The SOR system is encoded by sorA (GenBank accession number AAZ62443.1) and sorB (GenBank accession number AAZ62442.1). The Sox system genes include soxB (GenBank accession number AAZ62608.1), soxX (GenBank accession number AAZ62610.1), soxA (GenBank accession number AAZ62611.1), soxZ (GenBank accession number AAZ62613.1), soxY (GenBank accession number AAZ62614.1), soxD (GenBank accession number AAZ62616.1), and soxC (GenBank accession number AAZ62617.1). The FCSD system is encoded by fccB (GenBank accession number AAZ62620.1) and fccA (a possible cytochrome c; GenBank accession number AAZ62621.1). An operon coding for the SQR/PDO system genes consists of sqr (GenBank accession number AAZ62946.1), pdo2 (GenBank accession number AAZ62947.1), fisR (GenBank accession number AAZ62948.1), and tauE (GenBank accession number AAZ62949.1). The locus tag of each gene is given below the gene (e.g., Reut_A3252 is the tag for soxA).

FIG 2

The function of SQR, PDO, the Sox system, and FccAB in C. pinatubonensis JMP134. Cells were harvested, washed, and resuspended in 100 mM HEPES buffer, pH 7.4, at an OD₆₀₀ of 2.0. Sulfide was added to 500 μM to initiate the reaction. Sulfide (A), sulfane sulfur (B), thiosulfate (C), and sulfate (D) concentrations were determined. There was no apparent difference in the decrease in sulfide levels in HEPES buffer with or without heat-inactivated cells, but the heat-inactivated cells produced more sulfate (53 ± 13 μM) than the buffer (5 ± 3 μM) at 7 h (see Fig. S1 in the supplemental material). The apparent decrease in the buffer with heat-inactivated cells was likely due to evaporation via shaking and autoxidation. All data are averages for at least three samples with standard deviations (error bars).

FIG 3

Sulfide oxidation by the Δpdo1 Δpdo2 mutant and its derivatives. Cells were harvested, washed, and resuspended at an OD₆₀₀ of 2.0 in 100 mM HEPES buffer, pH 7.4. Sulfide was added to 500 μM to initiate the reaction. Sulfide (A), polysulfides (B), thiosulfate (C), and sulfate (D) levels were determined at different times. All data are averages for at least three samples with standard deviations (error bars).

FIG 4

Proposed pathways of sulfide oxidation in C. pinatubonensis JMP134 and its Δsqr Δpdo1 Δpdo2 mutant. (A) Sulfide oxidation in the wild type. Sulfide is oxidized to sulfane sulfur (S⁰) by SQR; PDO oxidizes S⁰ to sulfite, which spontaneously reacts with S⁰ to generate thiosulfate in the cytoplasm. Thiosulfate is transported to the periplasmic space and is oxidized by the Sox system to sulfate. (B) Sulfide oxidation by the Δsqr Δpdo1 Δpdo2 mutant without SQR and PDO. FCSD oxidizes sulfide to S⁰, which is then oxidized by the Sox system to sulfate. This pathway is marginal in the wild type.