Dimethylsulfoniopropionate Catabolism Research Articles

Structural and molecular basis for the novel catalytic mechanism and evolution of DddP, an abundant peptidase‐like bacterial Dimethylsulfoniopropionate lyase: a new enzyme from an old fold

The microbial cleavage of dimethylsulfoniopropionate (DMSP) generates volatile dimethyl sulfide (DMS) and is an important step in global sulfur and carbon cycles. DddP is a DMSP lyase in marine bacteria, and the deduced dddP gene product is abundant in marine metagenomic data sets. However, DddP belongs to the M24 peptidase family according to sequence alignment. Peptidases hydrolyze C-N bonds, but DddP is deduced to cleave C-S bonds. Mechanisms responsible for this striking functional shift are currently unknown. We determined the structures of DMSP lyase RlDddP (the DddP from Ruegeria lacuscaerulensis ITI_1157) bound to inhibitory 2-(N-morpholino) ethanesulfonic acid or PO4 (3-) and of two mutants of RlDddP bound to acrylate. Based on structural, mutational and biochemical analyses, we characterized a new ion-shift catalytic mechanism of RlDddP for DMSP cleavage. Furthermore, we suggested the structural mechanism leading to the loss of peptidase activity and the subsequent development of DMSP lyase activity in DddP. This study sheds light on the catalytic mechanism and the divergent evolution of DddP, leading to a better understanding of marine bacterial DMSP catabolism and global DMS production.

Determination of 3-mercaptopropionic acid by HPLC: A sensitive method for environmental applications.

The organic sulfur compound 3-mercaptopropionic acid (3-MPA) is an important thiol intermediate in organic sulfur metabolism in natural environments. It is generated during degradation of sulfur-containing amino acids (e.g. methionine) and from demethylation of dimethylsulfoniopropionate (DMSP). This pathway is an alternative enzymatic process in the DMSP catabolism that routes sulfur away from the climatically-active dimethyl sulfide (DMS). 3-MPA detection and subsequent quantification in different matrices is difficult due to its extreme reactivity. We therefore developed a sensitive method for determination of 3-MPA based on pre-column derivatization with monobromobimane and analysis by high-performance liquid chromatography (HPLC) with fluorescence detection. This methodology was first tested with 3-MPA standards under low (0.005-0.2μmolL(-1)) and high (1-25μmolL(-1)) concentrations. For the optimization of the reaction, CHES and, alternatively, Tris-HCl buffers were evaluated in the derivatization step, with Tris-HCl showing more effective separation of thiol derivatives and a better 3-MPA peak shape. The detection limit was 4.3nmolL(-1) with a 10μL sample injection, and mean recoveries of 3-MPA ranged from 97 to 105% in estuarine waters with different salinities (0.17 and 35.9ppt). The linearity (r>0.99) and repeatability of detector response, with intra- and inter-day precision (% CV) of 2.68-7.01% and 4.86-12.5%, respectively, confirmed the reliability of the method. Previous 3-MPA analytical methods required immediate analysis due to unstable derivatives, but in this method we achieved high stability of the derivatized samples when stored at 4°C, with only a 3-5% loss after more than one year of storage. This method was successfully applied to measure 3-MPA concentrations and rates of 3-MPA production in a variety of intertidal estuarine sediment slurries. Dissolved 3-MPA concentrations in these sediment slurries varied between 2 and 237μmolL(-1) and, 3-MPA net fluxes ranged in wet sediments between -3.6±1.7 and 30±5μmolL(-1)g(-1)h(-1). Thus, the application of this optimized methodology showed an efficient performance for measuring 3-MPA in environmental samples, with a straightforward sample derivatization and a simple analysis of stable 3-MPA derivatives.

Salinity as a regulator of DMSP degradation in Ruegeria pomeroyi DSS-3.

Dimethylsulfoniopropionate (DMSP) is an important carbon and sulfur source to marine bacterial communities and the main precursor of dimethylsulfide (DMS), a gas that influences atmospheric chemistry and potentially the global climate. In nature, bacterial DMSP catabolism can yield different proportions of DMS and methanethiol (MeSH), but relatively little is known about the factors controlling the pathways of bacterial degradation that select between their formation (cleavage vs. demethiolation). In this study, we carried out experiments to evaluate the influence of salinity on the routes of DMSP catabolism in Ruegeria pomeroyi DSS-3. We monitored DMS and MeSH accumulation in cell suspensions grown in a range of salinities (10, 20, 30 ppt) and with different DMSP amendments (0, 50, 500 µM). Significantly higher concentrations of DMS accumulated in low salinity treatments (10 ppt; P < 0.001), in both Marine Basal Medium (MBM) and half-strength Yeast Tryptone Sea Salts (1/2 YTSS) media. Results showed a 47.1% and 87.5% decrease of DMS accumulation, from salinity 10 to 20 ppt, in MBM and 1/2 YTSS media, respectively. On the other hand, MeSH showed enhanced accumulations at higher salinities (20, 30 ppt), with a 90.6% increase of MeSH accumulation from the 20 ppt to the 30 ppt salinity treatments. Our results with R. pomeroyi DSS-3 in culture are in agreement with previous results from estuarine sediments and demonstrate that salinity can modulate selection of the DMSP enzymatic degradation routes, with a consequent potential impact on DMS and MeSH liberation into the atmosphere.

The genetic potential for key biogeochemical processes in Arctic frost flowers and young sea ice revealed by metagenomic analysis

Newly formed sea ice is a vast and biogeochemically active environment. Recently, we reported an unusual microbial community dominated by members of the Rhizobiales in frost flowers at the surface of Arctic young sea ice based on the presence of 16S gene sequences related to these strains. Here, we use metagenomic analysis of two samples, from a field of frost flowers and the underlying young sea ice, to explore the metabolic potential of this surface ice community. The analysis links genes for key biogeochemical processes to the Rhizobiales, including dimethylsulfide uptake, betaine glycine turnover, and halocarbon production. Nodulation and nitrogen fixation genes characteristic of terrestrial root-nodulating Rhizobiales were generally lacking from these metagenomes. Non-Rhizobiales clades at the ice surface had genes that would enable additional biogeochemical processes, including mercury reduction and dimethylsulfoniopropionate catabolism. Although the ultimate source of the observed microbial community is not known, considerations of the possible role of eolian deposition or transport with particles entrained during ice formation favor a suspended particle source for this microbial community.

Marine bacteria from the Roseobacter clade produce sulfur volatiles via amino acid and dimethylsulfoniopropionate catabolism.

Dimethylsulfoniopropionate (DMSP) is a versatile sulfur source for the production of sulfur-containing secondary metabolites by marine bacteria from the Roseobacter clade. (34)S-labelled DMSP and cysteine, and several DMSP derivatives with modified S-alkyl groups were synthesised and used in feeding experiments that gave insights into the biosynthesis of sulfur volatiles from these bacteria.

The Ruegeria pomeroyi acuI Gene Has a Role in DMSP Catabolism and Resembles yhdH of E. coli and Other Bacteria in Conferring Resistance to Acrylate

The Escherichia coli YhdH polypeptide is in the MDR012 sub-group of medium chain reductase/dehydrogenases, but its biological function was unknown and no phenotypes of YhdH− mutants had been described. We found that an E. coli strain with an insertional mutation in yhdH was hyper-sensitive to inhibitory effects of acrylate, and, to a lesser extent, to those of 3-hydroxypropionate. Close homologues of YhdH occur in many Bacterial taxa and at least two animals. The acrylate sensitivity of YhdH− mutants was corrected by the corresponding, cloned homologues from several bacteria. One such homologue is acuI, which has a role in acrylate degradation in marine bacteria that catabolise dimethylsulfoniopropionate (DMSP) an abundant anti-stress compound made by marine phytoplankton. The acuI genes of such bacteria are often linked to ddd genes that encode enzymes that cleave DMSP into acrylate plus dimethyl sulfide (DMS), even though these are in different polypeptide families, in unrelated bacteria. Furthermore, most strains of Roseobacters, a clade of abundant marine bacteria, cleave DMSP into acrylate plus DMS, and can also demethylate it, using DMSP demethylase. In most Roseobacters, the corresponding gene, dmdA, lies immediately upstream of acuI and in the model Roseobacter strain Ruegeria pomeroyi DSS-3, dmdA-acuI were co-regulated in response to the co-inducer, acrylate. These observations, together with findings by others that AcuI has acryloyl-CoA reductase activity, lead us to suggest that YdhH/AcuI enzymes protect cells against damaging effects of intracellular acryloyl-CoA, formed endogenously, and/or via catabolising exogenous acrylate. To provide “added protection” for bacteria that form acrylate from DMSP, acuI was recruited into clusters of genes involved in this conversion and, in the case of acuI and dmdA in the Roseobacters, their co-expression may underpin an interaction between the two routes of DMSP catabolism, whereby the acrylate product of DMSP lyases is a co-inducer for the demethylation pathway.

Diversity of DMSP transport in marine bacteria, revealed by genetic analyses

The enzyme product of the dddD gene, found in several different marine bacteria, acts on dimethylsulfoniopropionate (DMSP), liberating dimethyl sulfide (DMS) and generating 3-OH-propionate as the initially detected C3 product. In many bacteria, dddD is near genes whose sequence suggests that they encode a DMSP transporter. These are of two very different types, in the BCCT (betaine-carnitine-choline transporter) family or resembling members of the ABC super-family that import betaines. Even within these two families, the amino acid sequences of these putative transporters are not particularly similar to each other. Genes for the predicted DMSP transporters of Halomonas and Marinomonas (both BCCT type) and of Burkholderia ambifaria AMMD (ABC-type) were each cloned and introduced into an Escherichia coli mutant (MKH13) that is defective in betaine uptake, and so fails to catabolise DMSP even when a cloned dddD gene was present, due to the failure of the substrate to be imported. DMSP-dependent DMS production (Ddd+ phenotype) was restored by introducing any of these cloned transporters into MKH13 containing dddD. Other marine bacteria use a range of enzymes, called DddL, DddP, DddQ, DddW and DddY, to cleave DMSP, but the various ddd genes that encode them are usually unlinked to any that are predicted to encode betaine transporters. We identified one gene in Sulfitobacter sp. EE-36 and two in Roseovarius nubinhibens ISM, which, when cloned and introduced into E. coli MKH13, overcame its osmotic sensitivity when it was grown with DMSP or other exogenous betaines. These genes all encoded BCCT transporters, but were unlinked to any known genes involved in DMSP catabolism in these two strains of α-proteobacteria.

Multiple DMSP lyases in the γ-proteobacterium Oceanimonas doudoroffii

The marine γ-proteobacterium Oceanimonas doudoroffii was shown to have at least three different enzymes, each of which can cleave dimethylsulfoniopropionate (DMSP), an abundant compatible solute made by different classes of marine phytoplankton. These various DMSP lyases have similarities, but also some differences to those that had been identified in other bacteria. This was demonstrated by cloning each of the corresponding genes and transferring them into other species of bacteria in which backgrounds they conferred the ability to catabolise DMSP, releasing dimethyl sulfide (DMS) as one of the products (Ddd+ phenotype; DMSP-dependent DMS). One of these genes resembled dddD, which was in a cluster with other ddd genes variously involved in subsequent steps of DMSP catabolism, in DMSP import and in DMSP-dependent transcriptional regulation. The other two gene products both had sequence similarity to the previously identified DddP lyase. However, these two Oceanimonas DddP polypeptides were not particularly similar to each other and were in two different sub-branches compared to those that had been studied in strains of the Roseobacter clade of bacteria. One of these O. doudoroffii enzymes, DddP1, most closely resembled gene products in a disparate group of microbes that included two bacteria, Vibrio orientalis and Puniceispirillum marinum and, more strikingly, some Ascomycete fungi that can catabolise DMSP. Previously, the only bacteria known to have multiple ways to catabolise DMSP were in the Roseobacter clade, which were also the only bacteria that had been shown to have functional DddP DMSP lyases. Thus Oceanimonas doudoroffii is unusual on more than one count and likely acquired its dddD, dddP1 and dddP2 genes by independent horizontal gene transfer events.

DddW, a third DMSP lyase in a model Roseobacter marine bacterium, Ruegeria pomeroyi DSS-3

Ruegeria pomeroyi DSS-3 is a model Roseobacter marine bacterium, particularly regarding its catabolism of dimethylsulfoniopropionate (DMSP), an abundant anti-stress molecule made by marine phytoplankton. We found a novel gene, dddW, which encodes a DMSP lyase that cleaves DMSP into acrylate plus the environmentally important volatile dimethyl sulfide (DMS). Mutations in dddW reduced, but did not abolish DMS production. Transcription of dddW was greatly enhanced by pre-growth of cells with DMSP, via a LysR-type regulator. Close DddW homologs occur in only one other Roseobacter species, and there are no close homologs and only a few related sequences in metagenomes of marine bacteria. In addition to DddW, R. pomeroyi DSS-3 had been shown to have two other, different, DMSP lyases, DddP and DddQ, plus an enzyme that demethylates DMSP, emphasizing the importance of this substrate for this model bacterium.

Unusual Regulation of a Leaderless Operon Involved in the Catabolism of Dimethylsulfoniopropionate in Rhodobacter sphaeroides

Rhodobacter sphaeroides strain 2.4.1 is a widely studied bacterium that has recently been shown to cleave the abundant marine anti-stress molecule dimethylsulfoniopropionate (DMSP) into acrylate plus gaseous dimethyl sulfide. It does so by using a lyase encoded by dddL, the promoter-distal gene of a three-gene operon, acuR-acuI-dddL. Transcription of the operon was enhanced when cells were pre-grown with the substrate DMSP, but this induction is indirect, and requires the conversion of DMSP to the product acrylate, the bona fide co-inducer. This regulation is mediated by the product of the promoter-proximal gene acuR, a transcriptional regulator in the TetR family. AcuR represses the operon in the absence of acrylate, but this is relieved by the presence of the co-inducer. Another unusual regulatory feature is that the acuR-acuI-dddL mRNA transcript is leaderless, such that acuR lacks a Shine-Dalgarno ribosomal binding site and 5′-UTR, and is translated at a lower level compared to the downstream genes. This regulatory unit may be quite widespread in bacteria, since several other taxonomically diverse lineages have adjacent acuR-like and acuI-like genes; these operons also have no 5′ leader sequences or ribosomal binding sites and their predicted cis-acting regulatory sequences resemble those of R. sphaeroides acuR-acuI-dddL.

Bacterial Catabolism of Dimethylsulfoniopropionate (DMSP)

Dimethylsulfoniopropionate (DMSP) is a metabolite produced primarily by marine phytoplankton and is the main precursor to the climatically important gas dimethylsulfide (DMS). DMS is released upon bacterial catabolism of DMSP, but it is not the only possible fate of DMSP sulfur. An alternative demethylation/demethiolation pathway results in the eventual release of methanethiol, a highly reactive volatile sulfur compound that contributes little to the atmospheric sulfur flux. The activity of these pathways control the natural flux of sulfur released to the atmosphere. Although these biochemical pathways and the factors that regulate them are of great interest, they are poorly understood. Only recently have some of the genes and pathways responsible for DMSP catabolism been elucidated. Thus far, six different enzymes have been identified that catalyze the cleavage of DMSP, resulting in the release of DMS. In addition, five of these enzymes appear to produce acrylate, while one produces 3-hydroxypropionate. In contrast, only one enzyme, designated DmdA, has been identified that catalyzes the demethylation reaction producing methylmercaptopropionate (MMPA). The metabolism of MMPA is performed by a series of three coenzyme-A mediated reactions catalyzed by DmdB, DmdC, and DmdD. Interestingly, Candidatus Pelagibacter ubique, a member of the SAR11 clade of Alphaproteobacteria that is highly abundant in marine surface waters, possessed functional DmdA, DmdB, and DmdC enzymes. Microbially mediated transformations of both DMS and methanethiol are also possible, although many of the biochemical and molecular genetic details are still unknown. This review will focus on the recent discoveries in the biochemical pathways that mineralize and assimilate DMSP carbon and sulfur, as well as the areas for which a comprehensive understanding is still lacking.

Pathways and Substrate Specificity of DMSP Catabolism in Marine Bacteria of the Roseobacter Clade

The volatiles released by Phaeobacter gallaeciensis, Oceanibulbus indolifex and Dinoroseobacter shibae have been investigated by GC-MS, and several MeSH-derived sulfur volatiles have been identified. An important sulfur source in the oceans is the algal metabolite dimethylsulfoniopropionate (DMSP). Labelled [2H6]DMSP was fed to the bacteria to investigate the production of volatiles from this compound through the lysis pathway to [2H6]dimethylsulfide or the demethylation pathway to [2H3]-3-(methylmercapto)propionic acid and lysis to [2H3]MeSH. [2H6]DMSP was efficiently converted to [2H3]MeSH by all three species. Several DMSP derivatives were synthesised and used in feeding experiments. Strong dealkylation activity was observed for the methylated ethyl methyl sulfoniopropionate and dimethylseleniopropionate, as indicated by the formation of EtSH- and MeSeH-derived volatiles, whereas no volatiles were formed from dimethyltelluriopropionate. In contrast, the dealkylation activity for diethylsulfoniopropionate was strongly reduced, resulting in only small amounts of EtSH-derived volatiles accompanied by diethyl sulfide in P. gallaeciensis and O. indolifex, while D. shibae produced the related oxidation product diethyl sulfone. The formation of diethyl sulfide and diethyl sulfone requires the lysis pathway, which is not active for [2H6]DMSP. These observations can be explained by a shifted distribution between the two competing pathways due to a blocked dealkylation of ethylated substrates.

Molecular dissection of bacterial acrylate catabolism – unexpected links with dimethylsulfoniopropionate catabolism and dimethyl sulfide production

A bacterium in the genus Halomonas that grew on dimethylsulfoniopropionate (DMSP) or acrylate as sole carbon sources and that liberated the climate-changing gas dimethyl sulfide in media containing DMSP was obtained from the phylloplane of the macroalga Ulva. We identified a cluster that contains genes specifically involved in DMSP catabolism (dddD, dddT) or in degrading acrylate (acuN, acuK) or that are required to break down both substrates (dddC, dddA). Using NMR and HPLC analyses to trace 13C- or 14C-labelled acrylate and DMSP in strains of Escherichia coli with various combinations of cloned ddd and/or acu genes, we deduced that DMSP is imported by the BCCT-type transporter DddT, then converted by DddD to 3-OH-propionate (3HP), liberating dimethyl sulfide in the process. As DddD is a predicted acyl CoA transferase, there may be an earlier, unidentified catabolite of DMSP. Acrylate is also converted to 3HP, via a CoA transferase (AcuN) and a hydratase (AcuK). The 3HP is predicted to be catabolized by an alcohol dehydrogenase, DddA, to malonate semialdehyde, thence by an aldehyde dehydrogenase, DddC, to acyl CoA plus CO2. The regulation of the ddd and acu genes is unusual, as a catabolite, 3HP, was a co-inducer of their transcription. This first description of genes involved in acrylate catabolism in any organism shows that the relationship between the catabolic pathways of acrylate and DMSP differs from that which had been suggested in other bacteria.