Methanethiol-dependent DMS production by Halomonas species

Halomonas alimentaria EF61 (isolated from Mariana Trench seawater22) produced MeSH and DMS when grown with l-Met (Fig. 1b). EF61 did not produce DMSP; thus its DMS production was not due to DMSP cleavage despite this bacterium exhibiting DMSP cleavage with exogenous DMSP (Fig. 1c). EF61 produced 11.3 ± 3.1 nmol DMS mg−1 total protein h−1 from MeSH (Fig. 1d). EF61 also produced MeSH and DMS from MMPA (Fig. 1e), the primary catabolite of the DMSP demethylase predicted to be in ~20% of marine bacteria15, and from H2S (Fig. 1f). DMS production from l-Met, MMPA, MeSH and H2S was also observed in 3 of 5 other tested Halomonas strains (SCS19, H33-56 and RT37) from diverse marine environments (Fig. 1 and Supplementary Table 1). In contrast, H. alimentaria H10-9-1 and H. saccharevitans H10-59 could not generate DMS from any of these four sulfur compounds but could produce MeSH from l-Met, MMPA and H2S (Fig. 1). These data implied that H10-9-1 and H10-59 lacked or did not express key MeSH S-methyltransferase enzyme(s) that were active in the four other Halomonas strains.

MddH, an H2S and MeSH S-methyltransferase in Halomonas

The genomes of all six Halomonas isolates contained megL encoding Met γ-lyase, consistent with their ability to liberate MeSH from l-Met (Fig. 1a,b). They contained dmdBC and acuH, encoding enzymes that catabolize MMPA and release MeSH15, consistent with their observed MMPA-dependent MeSH production phenotype (Fig. 1a,e). Similar to Halomonas HTNK1 (ref. 23), the strains contained the DMSP lyase gene dddD and generated DMS from exogenously added DMSP and acetyl CoA, probably produced intracellularly24 (Fig. 1a,c). None of the isolates contained mddA encoding the only known microbial H2S and MeSH S-methyltransferase, or proteins with >63% coverage and >34% amino acid identity to the human thiol S-methyltransferases TMT1A and TMT1B, indicating that the four Halomonas isolates with Mdd activity probably contained an unidentified Mdd enzyme.

There were 84 genes unique to the four Halomonas strains with Mdd activity compared with the two lacking this phenotype and only one encoded a candidate methyltransferase. This gene, termed mddH, was associated with multicopper oxidase genes and those predicted to be involved in metal transport and resistance, with no obvious link to sulfur metabolism (Supplementary Fig. 1). MddH shared no protein sequence identity with MddA. Instead, it encoded a ubiquinone methyltransferase UbiE family protein (COG2226) with only 37% amino acid identity to Escherichia coli UbiE, a SAM-dependent methyltransferase involved in menaquinone synthesis25. Notably, all six Halomonas strains contained a different UbiE homologue, with 74–75% protein identity to E. coli UbiE. EF61 MeSH S-methylation activity was cytosolic (2.46 ± 0.3 nmol DMS mg−1 total protein h−1) and not membranous, consistent with the EF61 MddH protein lacking a signal peptide. This differs from MddA which has 4–6 membrane-spanning helices and whose activity is enriched in Pseudomonas deceptionensis membrane fractions8. Indeed, MeSH S-methylation activity was detected in the cytosolic (2.46 ± 0.3 nmol DMS mg−1 total protein h−1) but not in membrane fractions of H. alimentaria EF61.

The MddH protein shared 31/50% and 34/53% amino acid sequence identity/similarity to the human thiol S-methyltransferases TMT1A and TMT1B, respectively, and their AlphaFold26 predicted structures were similar, particularly in their central and C-terminal regions, containing the conserved central GxGxG binding motif21 for SAM binding (Supplementary Fig. 2a). The predicted MddH structure was comparatively more compact than for TMT1A and TMT1B, lacked an extended N-terminal ‘hooked’ helical region and a conserved aspartate residue at position 98 previously implicated in SAM binding21, which was a glutamate at position 63 in MddH (Supplementary Fig. 2b). These observations support the hypothesis that MddH was a thiol S-methyltransferase similar to TMT1A and TMT1B.

E. coli cell extracts containing EF61 MddH protein showed in vitro SAM-dependent Mdd activity (46.4 ± 4.0 nmol DMS mg−1 total protein h−1) and H2S-dependent MeSH (14.4 ± 1.5 nmol mg−1 total protein h−1) and DMS (10.3 ± 0.2 nmol mg−1 total protein h−1) production (Table 1). Furthermore, an EF61 ΔmddH mutant (Supplementary Fig. 3a) overproduced MeSH when grown with l-Met or H2S compared with the wild-type strain and completely lacked Mdd activity (Supplementary Fig. 3b,c). These mutant phenotypes were restored to wild-type levels by cloned mddH, consistent with MddH being the Halomonas spp. SAM-dependent MeSH S-methyltransferase enzyme. These data and those in Fig. 1 indicated that there were also MddH-independent pathways converting H2S to MeSH in the tested Halomonas strains studied here, potentially through l-cysteine and l-Met as intermediates27,28.

Table 1 Activity of diverse MddH proteins expressed in E. coli

Importantly, when incubated in sterilized coastal seawater with 4 nM H2S or MeSH, EF61 but not the ΔmddH mutant showed DMS production (Supplementary Table 2) compared with the seawater control. The H2S or MeSH levels used were physiologically relevant for seawater and sediment samples29,30, implying that MddH yields DMS in marine environments.

MddH is widespread in diverse bacteria

Proteins with >45% amino acid identity to MddH were identified in diverse bacterial taxa, mainly Gammaproteobacteria and Alphaproteobacteria¸ but also some Betaproteobacteria, Deltaproteobacteria, Acidobacteria and Bacteroidetes (Fig. 2). When cloned, candidate mddH genes from marine and soil bacteria, but not Vibrio ubiE (negative control) conferred H2S and Mdd S-methylation activity to E. coli (Table 1). This included MlMddH from Marinobacter litoralis Sw-45, characterized below, which shared 60.3% amino acid identity to EF61 MddH. The diverse natural host bacteria containing mddH genes also had H2S and MeSH S-methylation activity (Supplementary Fig. 4). We predict that the candidate MddH enzymes in Fig. 2, which are distinct from the UbiE outgroup, constitute the ‘MddH’ family of H2S and MeSH S-methyltransferase enzymes. Several SAM-dependent methyltransferases were structurally similar to MddH (predicted by AlphaFold), but they had <21% amino acid identity to MddH and broad substrate specificity where characterized (Supplementary Table 3), and their activity on H2S/MeSH required further examination.

Fig. 2: Maximum-likelihood phylogenetic tree of MddH proteins.

The tree is drawn to scale, with branch lengths measured in number of substitutions per site. The scale bar indicates 0.5 amino acid substitutions per site. Different coloured circles at the end of each branch indicate bacterial taxonomy (see Taxonomy key). Different label colours indicate the source of the bacterial strain (see Source key). Proteins with experimentally ratified Mdd activity are marked with a yellow star near the labels. MddH from Halomonas alimentaria EF61 is highlighted by a red star. A putative UbiE protein from Vibrio sp. with no Mdd activity was used as the outgroup (shown in a black box).

Interestingly, M. litoralis Sw-45 mddH was adjacent to the cydABCD operon encoding a cytochrome bd oxidase complex (CydAB) and cysteine transporter (CydDC) involved in the regulation of intracellular cysteine and redox levels and H2S production31,32. However, mddH was not associated with any genes obviously linked to H2S or MeSH generation in other bacteria (Supplementary Fig. 1).

mddH and mddA were found in distinct but similarly diverse host bacteria. The mddA gene was mostly found in actinobacterial, alphaproteobacterial Rhizobiales and, except for Pseudomonas, was far less common in gammaproteobacteria than mddH8. The key difference between bacteria containing mddA and those with mddH was not in their host taxonomy, but more prominently in the environments they inhabit. Most bacteria with mddA were isolated from terrestrial soil or freshwater and not marine environments8. In contrast, mddH was predominantly in diverse bacteria from marine seawater or sediment such as Halomonas, Marinobacter, Novosphingobium and Erythrobacter (Fig. 2). mddH was also found but far less frequently in bacteria from soil, lake, spring and other sources such as wastewater plants, compost, fruits or animals (Fig. 2).

Characterization of the MddH enzyme

The purified MlMddH enzyme (Fig. 3a) showed SAM-dependent S-methylation of H2S and MeSH, producing both MeSH and slightly lower amounts of DMS from H2S (Fig. 3b). MlMddH had an optimal pH of ~9.0 (Supplementary Fig. 5a) and temperature of 45 °C (Supplementary Fig. 5b) for MeSH and H2S, and showed high activities at ~pH 8 and at 10–20 °C, physiologically relevant seawater pH and temperature values, respectively (Supplementary Fig. 5). MlMddH had Km (the Michaelis constant) and kcat (enzyme turnover number) values of 0.23 mM and 0.08 s−1, respectively, for MeSH, and 0.07 mM and 0.06 s−1, respectively, for the SAM co-substrate. The Km of MlMddH for H2S (0.22 mM) was similar to that for MeSH (0.23 mM), while the kcat value of 0.16 s−1 measured for H2S was ~2-fold higher than that for MeSH (Fig. 3e,f). Overall, MlMddH was ~2-fold more efficient when using H2S (kcat/Km ≈ 727 M−1 s−1) over MeSH (kcat/Km ≈ 347 M−1 s−1) as substrate.

Fig. 3: Characterization of the MddH enzyme.

a, SDS–PAGE of purified MlMddH. b, In vitro DMS and/or MeSH production by purified MlMddH with MeSH or H2S as substrates. The units (nmol mg−1 h−1) represent the nanomolar amount of DMS or MeSH produced by MlMddH per milligram per hour. c, The ability of MlMddH to S-methylate a range of substrates (as detailed) as monitored by the formation of S-adenosyl homocysteine (SAH) from S-adenosyl methionine (SAM). d, The effect of EDTA addition on MlMddH activity. e, Michaelis–Menten curves of purified MlMddH for H2S S-methylation and SAM. f, Michaelis–Menten curves of purified MlMddH for MeSH S-methylation and SAM. Initial rates were determined with 0.27 µM MlMddH (molecular weight: 24.24 kDa) and 0–2 mM SAM (1 mM MeSH/H2S), or 0–2 mM MeSH/H2S (1 mM SAM) at 45 °C and pH 9 for 30 min. Kinetic parameters for MddH were determined by nonlinear fitting using the Michaelis–Menten equation in the form v/[E] = kcat×[S]/(Km + [S]) based on the initial rates of DMS production (or DMS and MeSH production) in triplicate experiments. Data are shown as mean ± s.d.

Source data

MlMddH turnover rates for H2S (0.16 s−1) and MeSH (0.08 s−1) were consistent with enzymes involved in secondary metabolism33 and the SAM-dependent S-methyltransferase MddA with MeSH (~0.09 s−1) and H2S (~0.01 s−1)9. Compared with MddA, the specificity constants for MlMddH with H2S and MeSH were substantially higher by around an order of magnitude, indicating higher catalytic efficiency. The modestly lower kcat/Km values observed for MlMddH relative to the lower limit expected for most enzymes33 may be due to the reactive nature of these gaseous substrates and/or substrate diffusion limitation in assays. MlMddH showed no S-methylation activity towards most other tested sulfur compounds including glutathione (GSH), cysteine (l-Cys), coenzyme A (CoA), 2-mercaptoethanesulfonate (Coenzyme M) or the DMSP synthesis intermediates l-Met and 4-methylthio-2-hydroxybutyrate (MTHB) (Fig. 3c). However, S-adenosyl homocysteine was formed from SAM when MlMddH was incubated with ethanethiol and 1-propanethiol at levels ~23 and ~40% less, respectively, compared with MeSH (Supplementary Fig. 5e). This is consistent with MddH being able to S-methylate other short-chain low molecular weight alkyl thiols.

The purified MlMddH protein contained only up to 0.15 Zn and 0.038 Ca metals per protein and addition of mM levels of the metal-chelator EDTA only slightly reduced its activity (Fig. 3d). Furthermore, MddH activity was not enhanced by the addition of various metals and was even reduced by mM levels of Mn2+, Zn2+ and Co2+ (Supplementary Fig. 5c,d). Thus, despite Halomonas mddH being linked to candidate metal transporters and metalloenzymes (Supplementary Fig. 1), MddH does not likely require a metal co-factor for activity.

The role of MddH in bacteria

The wild-type EF61, ΔmddH mutant and genetically complemented strains were assessed for their ability to grow with mM l-Met, H2S, MeSH, cysteine, H2O2, cobalt or zinc levels. These compounds and metals can be cytotoxic if allowed to accumulate, cause oxidative stress31,32,34 and/or were associated with the action of gene products situated near mddH in microbial genomes (Supplementary Fig. 1). Except for MeSH, none of these compounds or metals affected the growth or yield of the ΔmddH compared with the wild-type strain (Supplementary Fig. 6). In contrast, despite having a similar initial growth rate to the wild-type and complemented strains, the ΔmddH mutant had reduced final biomass when grown with 2 mM MeSH compared with the wild-type and complemented strains (Supplementary Fig. 6). Furthermore, mddH transcription was significantly 2.5-fold upregulated by growth with MeSH but not with l-Met or H2S (Supplementary Fig. 6e). These data are consistent with MddH having a role to detoxify MeSH when it reaches high environmental levels, through generation of non-toxic DMS, as was recently shown for MddA9. Although MeSH is potentially abundant in Earth’s oceans due to the prominence of DMSP demethylation, it is rarely likely to reach mM levels35. Thus, if MddH does have a role in MeSH detoxification, it is probably minor under physiologically relevant marine conditions. Alternatively, we hypothesize that there are other detoxification strategies for the MeSH and/or the other tested stress-inducing molecules in EF61 that compensate for the loss of MddH in the EF61/ΔmddH mutant. This hypothesis was supported by the MddH-independent S-methylation of H2S observed in all Halomonas strains tested here (Fig. 1).

MddH is abundant in marine environments

mddH was found in 242 out of 243 Tara Oceans samples in the OM-RCG marine metagenome database36, comprising 68 sampling locations in epipelagic and mesopelagic waters across the globe. In the 178 prokaryote-enriched samples (>0.22 μm size fractionated), the percentage of mddH normalized by cell numbers ranged between 0.09 and 5.2% (with an average of 2.19 ± 0.93%) (Fig. 4 and Supplementary Table 4). Marine samples with abundant mddH-containing bacteria (>4%) were from the South/North Atlantic Ocean, South/North Pacific Ocean, Indian Ocean and Mediterranean Sea. The relative abundance of mddH in surface water (SRF, median: 2.26%) and the deep chlorophyll maximum layers (DCM, median: 2.21%) were similar but significantly higher than in the mesopelagic zone (MES, median: 1.60%) (Kruskal–Wallis test, Chi square = 16.0, d.f. = 2, P < 0.05) (Supplementary Fig. 7). Surprisingly, many copies of mddH were also identified in virus-enriched samples (<0.22 μm) (5.79 × 10−8 to 4.06 × 10−5 per mapped read). Indeed, 32 distinct MddH homologues were identified from marine viruses in Tara Oceans Viromes37 data. Many of these were highly homologous to bacterial mddH genes (Supplementary Table 5 and Supplementary Fig. 8), supporting the hypothesis of mddH horizontal gene transfer between viruses and bacteria. In contrast, mddA was detected in far less Tara Oceans (190 of the 243) and marine prokaryote-enriched samples (169 of 178) than mddH. mddA was significantly less abundant than mddH in these samples (~50-fold lower, Mann–Whitney test, P < 0.05) with on average only 0.04 ± 0.07% of bacteria predicted to contain mddA (Supplementary Table 4). In addition, unlike mddH, the percentage of bacteria with mddA was highest in the MES samples (Kruskal–Wallis test, Chi square = 34.8, d.f. = 2, P < 0.05). The 216 mddH sequences retrieved from Tara Oceans metagenomes were all from Proteobacteria (73.1% Gammaproteobacteria, 12.0% Alphaproteobacteria, 0.5% Betaproteobacteria and 14.4% others) (Fig. 4b). In contrast, the 25 mddA sequences were distributed in more diverse bacterial taxa, including Bacteroidetes, Cyanobacteria, Planctomycetes and Alpha-, Gamma- and Epsilon-proteobacteria (Fig. 4b).

Fig. 4: The abundance of mdd genes and/or transcripts in global seawaters and coastal sediments.

a, The relative abundance of mddH and mddA in Tara Oceans metagenome samples from the OM-RGCv1 database (normalized by cell numbers). b, Taxonomic assignment of MddH and MddA sequences in Tara Oceans metagenome samples from the OM-RGCv1 database. c, The relative abundance of mddH and mddA transcripts in Tara Oceans metatranscriptome samples from the OM-RGCv2 database (normalized by per cent of mapped reads). d, The relative abundance of mddH and mddA in sediment metagenome samples from the Yellow Sea and the Bohai Sea (normalized by cell numbers). e, Taxonomy assignment of MddH and MddA sequences from sediment metagenome samples from the Yellow Sea and the Bohai Sea.

Source data

mddH and mddA transcripts were respectively found in 186 and 63 of the 187 metatranscriptomes in the OM-RGCv2 database38 (Fig. 4c). Consistent with their gene abundance, the abundance of mddH transcripts (2.80 × 10−7 to 5.33 × 10−5 per mapped read) was far higher than for mddA (4.84 × 10−9 to 8.03 × 10−7 per mapped read). These data are consistent with MddH being an important enzyme in Earth’s oceans and marine H2S and MeSH S-methylation, being more crucial than previously predicted8.

In contrast, the most abundant DMSP lyase gene dddP was predicted to be in 12.4 ± 6.7% (0.4%–29.3%) of bacteria in Tara Oceans metagenomes, which was ~5-fold more than those with MddH (Supplementary Table 4). dddP transcript levels (2.78 × 10−7 to 9.98 × 10−5 per mapped read) were also slightly higher than those of mddH (1.86 × 10−7 to 5.32 × 10−5 per mapped read). These data imply that the Mdd pathway is probably a less important source of DMS than DMSP-dependent DMS production in marine systems.

As much as ~15% of bacteria (0.88–14.74%, normalized by cell number) in surface sediments from the Bohai and Yellow Seas near China39 were predicted to contain mddH (Fig. 4d). Indeed, mddH was far more abundant than mddA (predicted in 0.39–3.34% of bacteria) in most samples. These omics data again suggest that bacteria with mddH and mddA are generally more abundant in sediment than in aquatic marine samples, and that mddH is the dominant gene in these marine settings (Fig. 4d). Sediment mddH genes were mainly gammaproteobacterial but were also in Acidobacteria and some sulfate-reducing Deltaproteobacteria, whereas mddA was found in more diverse phyla including Ignavibacteriae, Nitrospirae, Planctomycetes and Bacteroidetes (Fig. 4e). These sediment environments probably contain higher physiological levels of l-Met, MeSH and, more prominently, H2S (that can be present at mM levels) than seawater environments22,39. However, the genetic potential to cleave DMSP was still higher in these sediments, with 10.0–29.5% of bacteria predicted to contain dddP. Once again, this implies DMSP cleavage as the likely dominant DMS-producing pathway. Nevertheless, considering the large number of bacteria in marine sediment40 and the often-high substrate availability22,39, H2S- and MeSH-dependent DMS production pathways are probably important sources of DMS in marine sediment environments.

Given that Carrión et al.8 predicted from metagenomic analysis that 5–76% of soil bacteria contained mddA, we also examined the abundance of mddH in these soil metagenomes (Supplementary Table 6)8. No reliable mddH sequence was identified in these soil metagenomes possibly due to their sequence depth. Thus, we also investigated the abundance of mddA and mddH in larger metagenome datasets from rhizosphere soil samples of different plants (Glycine soja, Sesbania cannabina and Sorghum bicolor)41. Only 0.1–1.67% of bacteria in these soil samples were predicted to contain mddH, whereas mddA was far more abundant (8.74–13.11%) (Supplementary Fig. 9). These data are consistent with MddA being the major H2S and MeSH S-methylation enzyme in terrestrial soils, while MddH probably dominates in marine settings.