Bacterial SBP56 identified as a Cu-dependent methanethiol oxidase widely distributed in the biosphere

Abstract

Oxidation of methanethiol (MT) is a significant step in the sulfur cycle. MT is an intermediate of metabolism of globally significant organosulfur compounds including dimethylsulfoniopropionate (DMSP) and dimethylsulfide (DMS), which have key roles in marine carbon and sulfur cycling. In aerobic bacteria, MT is degraded by a MT oxidase (MTO). The enzymatic and genetic basis of MT oxidation have remained poorly characterized. Here, we identify for the first time the MTO enzyme and its encoding gene (mtoX) in the DMS-degrading bacterium Hyphomicrobium sp. VS. We show that MTO is a homotetrameric metalloenzyme that requires Cu for enzyme activity. MTO is predicted to be a soluble periplasmic enzyme and a member of a distinct clade of the Selenium-binding protein (SBP56) family for which no function has been reported. Genes orthologous to mtoX exist in many bacteria able to degrade DMS, other one-carbon compounds or DMSP, notably in the marine model organism Ruegeria pomeroyi DSS-3, a member of the Rhodobacteraceae family that is abundant in marine environments. Marker exchange mutagenesis of mtoX disrupted the ability of R. pomeroyi to metabolize MT confirming its function in this DMSP-degrading bacterium. In R. pomeroyi, transcription of mtoX was enhanced by DMSP, methylmercaptopropionate and MT. Rates of MT degradation increased after pre-incubation of the wild-type strain with MT. The detection of mtoX orthologs in diverse bacteria, environmental samples and its abundance in a range of metagenomic data sets point to this enzyme being widely distributed in the environment and having a key role in global sulfur cycling.

Figure 1

Simplified schematic showing the role of MT as an intermediate in the metabolism of sulfur compounds. A single arrow does not imply a single biotransformation step. DMDS, dimethyldisulfide; DMSO, dimethylsulfoxide; DMSO₂, dimethylsulfone; DOM, dissolved organic matter; HS⁻, sulfide ion; MTA, 5′-methylthioadenosine; SO₃²⁻, sulfite ion; S⁰, elemental sulfur; S₂O₃²⁻, thiosulfate; S₄O₆²⁻, tetrathionate; SO₄²⁻, sulfate.

Figure 2

Genomic context of mtoX genes in selected bacteria showing the clustering of mtoX with genes encoding proteins containing SCO1/SenC and/or MauG domains, see inset for definition of coloring and patterns to particular gene annotation. As discussed in the text, in some instances, genes are encoding fusion proteins of SCO1 and mauG domains. Further information about the presence of SCO1 and MauG domain encoding genes in the vicinity of mtoX genes is given in Supplementary Table S7.

Figure 3

Transcriptional regulation of Ruegeria pomeroyi DSS-3 SPOA0268 and the methanethiol oxidase gene encoded by SPOA0269, assessed by beta galactosidase transcriptional fusion assay using various potential inducers. Values are reported in Miller units.

Figure 4

Phylogenetic analysis of translated methanethiol oxidase genes obtained from public databases, selected bacterial isolates by PCR, clone libraries of enrichment cultures and DNA extracted from surface sediments of Stiffkey saltmarsh. The tree was based on an alignment of full length and partial MtoX sequences in Arb and was derived using the neighbor joining algorithm and PAM correction implemented in Arb from a region comprising amino acid positions 85–300 of the Hyphomicrobium VS MtoX polypeptide. Bootstrap values (100 iterations) were derived in Mega 5, only those supporting terminal nodes with a confidence of 75% or higher are shown. Taxa shown in bold tested positive for MT oxidation.