Recent insights into oceanic dimethylsulfoniopropionate biosynthesis and catabolism

Dimethylsulfoniopropionate (DMSP), a globally important organosulfur compound is produced in prodigious amounts (2.0 Pg sulfur) annually in the marine environment by phytoplankton, macroalgae, heterotrophic bacteria, some corals and certain higher plants. It is an important marine osmolyte and a major precursor molecule for the production of climate-active volatile gas dimethyl sulfide (DMS). DMSP synthesis take place via three pathways: a transamination ‘pathway-’ in some marine bacteria and algae, a Met-methylation ‘pathway-’ in angiosperms and bacteria and a decarboxylation ‘pathway-’ in the dinoflagellate, Crypthecodinium. The enzymes DSYB and TpMMT are involved in the DMSP biosynthesis in eukaryotes while marine heterotrophic bacteria engage key enzymes such as DsyB and MmtN. Several marine bacterial communities import DMSP and degrade it via cleavage or demethylation pathways or oxidation pathway, thereby generating DMS, methanethiol, and dimethylsulfoxonium propionate, respectively. DMSP is cleaved through diverse DMSP lyase enzymes in bacteria and via Alma1 enzyme in phytoplankton. The demethylation pathway involves four different enzymes, namely DmdA, DmdB, DmdC and DmdD/AcuH. However, enzymes involved in the oxidation pathway have not been yet identified. We reviewed the recent advances on the synthesis and catabolism of DMSP and enzymes that are involved in these processes.     

Regulatory and Functional Diversity of Methylmercaptopropionate Coenzyme A Ligases from the Dimethylsulfoniopropionate Demethylation Pathway in Ruegeria pomeroyi DSS-3 and Other Proteobacteria

The organosulfur compound dimethylsulfoniopropionate (DMSP) is produced by phytoplankton and is ubiquitous in the surface ocean. Once released from phytoplankton, marine bacteria degrade DMSP by either the cleavage pathway to form the volatile gas dimethylsulfide (DMS) or the demethylation pathway, yielding methanethiol (MeSH), which is readily assimilated or oxidized. The enzyme DmdB, a methylmercaptopropionate (MMPA)-coenzyme A (CoA) ligase, catalyzes the second step in the demethylation pathway and is a major regulatory point. The two forms of DmdB present in the marine roseobacter Ruegeria pomeroyi DSS-3, RPO_DmdB1 and RPO_DmdB2, and the single form in the SAR11 clade bacterium “Candidatus Pelagibacter ubique” HTCC1062, PU_DmdB1, were characterized in detail. DmdB enzymes were also examined from Ruegeria lacuscaerulensis ITI-1157, Pseudomonas aeruginosa PAO1, and Burkholderia thailandensis E264. The DmdB enzymes separated into two phylogenetic clades. All enzymes had activity with MMPA and were sensitive to inhibition by salts, but there was no correlation between the clades and substrate specificity or salt sensitivity. All Ruegeria species enzymes were inhibited by physiological concentrations (70 mM) of DMSP. However, ADP reversed the inhibition of RPO_DmdB1, suggesting that this enzyme was responsive to cellular energy charge. MMPA reversed the inhibition of RPO_DmdB2 as well as both R. lacuscaerulensis ITI-1157 DmdB enzymes, suggesting that a complex regulatory system exists in marine bacteria. In contrast, the DmdBs of the non-DMSP-metabolizing P. aeruginosa PAO1 and B. thailandensis E264 were not inhibited by DMSP, suggesting that DMSP inhibition is a specific adaptation of DmdBs from marine bacteria.     

Towards a systematic understanding of structure–function relationship of dimethylsulfoniopropionate‐catabolizing enzymes

Each year, several million tons of dimethylsulfoniopropionate (DMSP) are produced by marine phytoplankton and bacteria as an important osmolyte to regulate their cellular osmosis. Microbial breakdown of DMSP to the volatile gas dimethylsulfide (DMS) plays an important role in global biogeochemical cycles of the sulphur element between land and the sea. Understanding the enzymes involved in the transformation of DMSP and DMS holds the key to a better understanding of oceanic DMSP cycles. Recent work by Shao et al. (2019) has resolved the crystal structure of two important enzymes, DmdB and DmdC, involved in DMSP transformation through the demethylation pathway. Their work represents an important step towards a systematic understanding of the structure–function relationships of DMSP‐catabolizing enzymes in marine microbes.     

Mechanistic insight into acrylate metabolism and detoxification in marine dimethylsulfoniopropionate‐catabolizing bacteria

Dimethylsulfoniopropionate (DMSP) cleavage, yielding dimethyl sulfide (DMS) and acrylate, provides vital carbon sources to marine bacteria, is a key component of the global sulfur cycle and effects atmospheric chemistry and potentially climate. Acrylate and its metabolite acryloyl‐CoA are toxic if allowed to accumulate within cells. Thus, organisms cleaving DMSP require effective systems for both the utilization and detoxification of acrylate. Here, we examine the mechanism of acrylate utilization and detoxification in Roseobacters. We propose propionate‐CoA ligase (PrpE) and acryloyl‐CoA reductase (AcuI) as the key enzymes involved and through structural and mutagenesis analyses, provide explanations of their catalytic mechanisms. In most cases, DMSP lyases and DMSP demethylases (DmdAs) have low substrate affinities, but AcuIs have very high substrate affinities, suggesting that an effective detoxification system for acylate catabolism exists in DMSP‐catabolizing Roseobacters. This study provides insight on acrylate metabolism and detoxification and a possible explanation for the high K_m values that have been noted for some DMSP lyases. Since acrylate/acryloyl‐CoA is probably produced by other metabolism, and AcuI and PrpE are conserved in many organisms across all domains of life, the detoxification system is likely relevant to many metabolic processes and environments beyond DMSP catabolism.     

Metabolism of dimethylsulphoniopropionate by Ruegeria pomeroyi DSS‐3

Ruegeria pomeroyi DSS‐3 possesses two general pathways for metabolism of dimethylsulphoniopropionate (DMSP), an osmolyte of algae and abundant carbon source for marine bacteria. In the DMSP cleavage pathway, acrylate is transformed into acryloyl‐CoA by propionate‐CoA ligase (SPO2934) and other unidentified acyl‐CoA ligases. Acryloyl‐CoA is then reduced to propionyl‐CoA by AcuI or SPO1914. Acryloyl‐CoA is also rapidly hydrated to 3‐hydroxypropionyl‐CoA by acryloyl‐CoA hydratase (SPO0147). A SPO1914 mutant was unable to grow on acrylate as the sole carbon source, supporting its role in this pathway. Similarly, growth on methylmercaptopropionate, the first intermediate of the DMSP demethylation pathway, was severely inhibited by a mutation in the gene encoding crotonyl‐CoA carboxylase/reductase, demonstrating that acetate produced by this pathway was metabolized by the ethylmalonyl‐CoA pathway. Amino acids and nucleosides from cells grown on ¹³C‐enriched DMSP possessed labelling patterns that were consistent with carbon from DMSP being metabolized by both the ethylmalonyl‐CoA and acrylate pathways as well as a role for pyruvate dehydrogenase. This latter conclusion was supported by the phenotype of a pdh mutant, which grew poorly on electron‐rich substrates. Additionally, label from [¹³C‐methyl] DMSP only appeared in carbons derived from methyl‐tetrahydrofolate, and there was no evidence for a serine cycle of C‐1 assimilation.     

Structures of dimethylsulfoniopropionate-dependent demethylase from the marine organism Pelagabacter ubique

Dimethylsulfoniopropionate (DMSP) is a ubiquitous algal metabolite and common carbon and sulfur source for marine bacteria. DMSP is a precursor for the climatically active gas dimethylsulfide that is readily oxidized to sulfate, sulfur dioxide, methanesulfonic acid, and other products that act as cloud condensation nuclei. Although the environmental importance of DMSP metabolism has been known for some time, the enzyme responsible for DMSP demethylation by marine bacterioplankton, dimethylsufoniopropionate‐dependent demethylase A (DmdA, EC 2.1.1.B5), has only recently been identified and biochemically characterized. In this work, we report the structure for the apoenzyme DmdA from Pelagibacter ubique (2.1 Å), as well as for DmdA co‐crystals soaked with substrate DMSP (1.6 Å) or the cofactor tetrahydrofolate (THF) (1.6 Å). Surprisingly, the overall fold of the DmdA is not similar to other enzymes that typically utilize the reduced form of THF and in fact is a triple domain structure similar to what has been observed for the glycine cleavage T protein or sarcosine oxidase. Specifically, while the THF binding fold appears conserved, previous biochemical studies have shown that all enzymes with a similar fold produce 5,10‐methylene‐THF, while DmdA catalyzes a redox‐neutral methyl transfer reaction to produce 5‐methyl‐THF. On the basis of the findings presented herein and the available biochemical data, we outline a mechanism for a redox‐neutral methyl transfer reaction that is novel to this conserved THF binding domain.     

Metabolism of methylated osmolytes by aerobic bacteria from Mono Lake, a moderately hypersaline, alkaline environment

Abstract: Three strains of aerobic bacteria were isolated from water and sediment samples of Mono Lake, a moderately hypersaline (90 ppt), alkaline (pH 9.7) lake in California. The organisms, Gram-negative rods, grew fastest at about pH 9.7 with no growth or much slower growth at pH 7.0. All three isolates grew on glycine betaine (GB) and respirometric experiments indicated that catabolism was by sequential demethylation with dimethyl glycine and sarcosine as intermediates. Two of the isolates also grew on dimethylsulfoniopropionate (DMSP), one with cleavage of the DMSP to yield dimethyl sulfide (DMS) and acrylate, and the other by demethylation with 3-methiolpropionate (MMPA) as an intermediate and the production of methanethiol from MMPA. The methylated osmolytes supported growth at salinities similar to those in Mono Lake, but, at higher salinities, catabolism was suppressed and GB and DMSP functioned as osmolytes. GB and DMSP probably originate from cyanobacteria and/or phytoplankton in Mono Lake and this report is the first indication of both the DMS and demethylation/methanethiol-producing pathways for DMSP degradation in a nonmarine environment.     

New Routes for Aerobic Biodegradation of Dimethylsulfoniopropionate

Dimethylsulfoniopropionate (DMSP), an osmolyte in marine plants, is biodegraded by cleavage of dimethyl sulfide (DMS) or by demethylation to 3-methiolpropionate (MMPA) and 3-mercaptopropionate (MPA). Sequential demethylation has been observed only with anoxic slurries of coastal sediments. Bacteria that grew aerobically on MMPA and DMSP were isolated from marine environments and phytoplankton cultures. Enrichments with DMSP selected for bacteria that generated DMS, whereas MMPA enrichments selected organisms that produced methanethiol (CH₃SH) from either DMSP or MMPA. A bacterium isolated on MMPA grew on MMPA and DMSP, but rapid production of CH₃SH from DMSP occurred only with DMSP-grown cells. Low levels of MPA accumulated during growth on MMPA, indicating demethylation as well as demethiolation of MMPA. The alternative routes for DMSP biodegradation via MMPA probably impact on net DMS fluxes to the marine atmosphere.     

Dimethylsulfoniopropionate metabolism by Pfiesteria-associated Roseobacter spp

The Roseobacter clade of marine bacteria is often found associated with dinoflagellates, one of the major producers of dimethylsulfoniopropionate (DMSP). In this study, we tested the hypothesis that Roseobacter species have developed a physiological relationship with DMSP-producing dinoflagellates mediated by the metabolism of DMSP. DMSP was measured in Pfiesteria and Pfiesteria-like (Cryptoperidiniopsis) dinoflagellates, and the identities and metabolic potentials of the associated Roseobacter species to degrade DMSP were determined. Both Pfiesteria piscicida and Pfiesteria shumwayae produce DMSP with an average intracellular concentration of 3.8 microM. Cultures of P. piscicida or Cryptoperidiniopsis sp. that included both the dinoflagellates and their associated bacteria rapidly catabolized 200 microM DMSP (within 30 h), and the rate of catabolism was much higher for P. piscicida cultures than for P. shumwayae cultures. The community of bacteria from P. piscicida and Cryptoperidiniopsis cultures degraded DMSP with the production of dimethylsulfide (DMS) and acrylate, followed by 3-methylmercaptopropionate (MMPA) and methanethiol (MeSH). Four DMSP-degrading bacteria were isolated from the P. piscicida cultures and found to be taxonomically related to Roseobacter species. All four isolates produced MMPA from DMSP. Two of the strains also produced MeSH and DMS, indicating that they are capable of utilizing both the lyase and demethylation pathways. The diverse metabolism of DMSP by the dinoflagellate-associated Roseobacter spp. offers evidence consistent with a hypothesis that these bacteria benefit from association with DMSP-producing dinoflagellates.     

Dimethylsulfoniopropionate Metabolism by Pfiesteria-Associated Roseobacter spp.†

The Roseobacter clade of marine bacteria is often found associated with dinoflagellates, one of the major producers of dimethylsulfoniopropionate (DMSP). In this study, we tested the hypothesis that Roseobacter species have developed a physiological relationship with DMSP-producing dinoflagellates mediated by the metabolism of DMSP. DMSP was measured in Pfiesteria and Pfiesteria-like (Cryptoperidiniopsis) dinoflagellates, and the identities and metabolic potentials of the associated Roseobacter species to degrade DMSP were determined. Both Pfiesteria piscicida and Pfiesteria shumwayae produce DMSP with an average intracellular concentration of 3.8 μM. Cultures of P. piscicida or Cryptoperidiniopsis sp. that included both the dinoflagellates and their associated bacteria rapidly catabolized 200 μM DMSP (within 30 h), and the rate of catabolism was much higher for P. piscicida cultures than for P. shumwayae cultures. The community of bacteria from P. piscicida and Cryptoperidiniopsis cultures degraded DMSP with the production of dimethylsulfide (DMS) and acrylate, followed by 3-methylmercaptopropionate (MMPA) and methanethiol (MeSH). Four DMSP-degrading bacteria were isolated from the P. piscicida cultures and found to be taxonomically related to Roseobacter species. All four isolates produced MMPA from DMSP. Two of the strains also produced MeSH and DMS, indicating that they are capable of utilizing both the lyase and demethylation pathways. The diverse metabolism of DMSP by the dinoflagellate-associated Roseobacter spp. offers evidence consistent with a hypothesis that these bacteria benefit from association with DMSP-producing dinoflagellates.     

Dimethylsulfoniopropionate-dependent demethylase (DmdA) from Pelagibacter ubique and Silicibacter pomeroyi

The ubiquitous algal metabolite dimethylsulfoniopropionate (DMSP) is a major source of carbon and reduced sulfur for marine bacteria. Recently, the enzyme responsible for the demethylation of DMSP, designated DmdA, was identified, and homologs were found to be common in marine bacterioplankton cells. The recombinant DmdA proteins from the cultured marine bacteria Pelagibacter ubique HTCC1062 and Silicibacter pomeroyi DSS-3 were purified with a three-step procedure using anion-exchange, hydrophobic interaction, and hydroxyapatite chromatographies. The P. ubique enzyme possessed an M_r on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 38,500. Under nondenaturing conditions, the M_r was 68,000, suggesting that the enzyme was likely to be a dimer. The purified enzyme exhibited strict substrate specificity for DMSP, as DmdA from both S. pomeroyi and P. ubique possessed no detectable demethylase activity with glycine betaine, dimethyl glycine, methylmercaptopropionate, methionine, or dimethylsulfonioacetate. Less than 1% activity was found with dimethylsulfoniobutanoate and dimethylsulfoniopentanoate. The apparent K_ms for DMSP were 13.2 ± 2.0 and 5.4 ± 2.3 mM for the P. ubique and S. pomeroyi enzymes, respectively. In cell extracts of S. pomeroyi DSS-3, the apparent K_m for DMSP was 8.6 ± 1.2 mM, similar to that of purified recombinant DmdA. The intracellular concentration of DMSP in chemostat-grown S. pomeroyi DSS-3 was 70 mM. These results suggest that marine bacterioplankton may actively accumulate DMSP to osmotically significant concentrations that favor near-maximal rates of DMSP demethylation activity.     

Demethylation and cleavage of dimethylsulfoniopropionate in marine intertidal sediments

Abstract Demethylation and cleavage of dimethylsulfoniopropionate (DMSP) was measured in three different types of intertidal marine sediments: a cyanobacterial mat, a diatom-covered tidal flat and a carbonate sediment. Consumption rates of added DMSP were highest in cyanobacterial mat slurries (59 μmol DMSP 1⁻¹) and lower in slurries from a diatom mat and a carbonate tidal sediment (24 and 9 μmol DMSP 1⁻¹ h⁻¹, respectively). Dimethyl sulfide (DMS) and 3-mercaptopropionate (MPA) were produced simultaneously during DMSP consumption, indicating that cleavage and demethylation occurred at the same time. Viable counts of DMSP-utilizing bacteria revealed a population of 2 × 10⁷ cells cm⁻³ sediment (90% of these cleaved DMSP to DMS, 10% demethylated DMSP to MPA) in the cyanobacterial mat, 7 × 10⁵ cells cm⁻³ in the diatom mat (23% cleavers, 77% demethylators), and 9 × 10⁴ cells cm⁻³ (20% cleavers and 80% demethylators) in the carbonate sediment. In slurries of the diatom mat, the rate of MPA production from added 3-methiolpropionate (MMPA) was 50% of the rate of MPA formation from DMSP. The presence of a large population of demethylating bacteria and the production of MPA from DMSP suggest that the demethylation pathway, in addition to cleavage, contributes significantly to DMSP consumption in coastal sediments.     

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.     

Biogenic production of DMSP and its degradation to DMS—their roles in the global sulfur cycle

Dimethyl sulfide(DMS) is the most abundant form of volatile sulfur in Earth's oceans, and is mainly produced by the enzymatic clevage of dimethylsulfoniopropionate(DMSP). DMS and DMSP play important roles in driving the global sulfur cycle and may affect climate. DMSP is proposed to serve as an osmolyte, a grazing deterrent, a signaling molecule, an antioxidant, a cryoprotectant and/or as a sink for excess sulfur. It was long believed that only marine eukaryotes such as phytoplankton produce DMSP. However, we recently discovered that marine heterotrophic bacteria can also produce DMSP, making them a potentially important source of DMSP. At present, one prokaryotic and two eukaryotic DMSP synthesis enzymes have been identified.Marine heterotrophic bacteria are likely the major degraders of DMSP, using two known pathways: demethylation and cleavage.Many phytoplankton and some fungi can also cleave DMSP. So far seven different prokaryotic and one eukaryotic DMSP lyases have been identified. This review describes the global distribution pattern of DMSP and DMS, the known genes for biosynthesis and cleavage of DMSP, and the physiological and ecological functions of these important organosulfur molecules, which will improve understanding of the mechanisms of DMSP and DMS production and their roles in the environment.     

Metabolic reconstructions identify plant 3‐methylglutaconyl‐CoA hydratase that is crucial for branched‐chain amino acid catabolism in mitochondria

The proteinogenic branched‐chain amino acids (BCAAs) leucine, isoleucine and valine are essential nutrients for mammals. In plants, BCAAs double as alternative energy sources when carbohydrates become limiting, the catabolism of BCAAs providing electrons to the respiratory chain and intermediates to the tricarboxylic acid cycle. Yet, the actual architecture of the degradation pathways of BCAAs is not well understood. In this study, gene network modeling in Arabidopsis and rice, and plant‐prokaryote comparative genomics detected candidates for 3‐methylglutaconyl‐CoA hydratase (4.2.1.18), one of the missing plant enzymes of leucine catabolism. Alignments of these protein candidates sampled from various spermatophytes revealed non‐homologous N‐terminal extensions that are lacking in their bacterial counterparts, and green fluorescent protein‐fusion experiments demonstrated that the Arabidopsis protein, product of gene At4g16800, is targeted to mitochondria. Recombinant At4g16800 catalyzed the dehydration of 3‐hydroxymethylglutaryl‐CoA into 3‐methylglutaconyl‐CoA, and displayed kinetic features similar to those of its prokaryotic homolog. When at4g16800 knockout plants were subjected to dark‐induced carbon starvation, their rosette leaves displayed accelerated senescence as compared with control plants, and this phenotype was paralleled by a marked increase in the accumulation of free and total leucine, isoleucine and valine. The seeds of the at4g16800 mutant showed a similar accumulation of free BCAAs. These data suggest that 3‐methylglutaconyl‐CoA hydratase is not solely involved in the degradation of leucine, but is also a significant contributor to that of isoleucine and valine. Furthermore, evidence is shown that unlike the situation observed in Trypanosomatidae, leucine catabolism does not contribute to the formation of the terpenoid precursor mevalonate.     

The structure of S. lividans acetoacetyl‐CoA synthetase shows a novel interaction between the C‐terminal extension and the N‐terminal domain

The adenosine monoposphate‐forming acyl‐CoA synthetase enzymes catalyze a two‐step reaction that involves the initial formation of an acyl adenylate that reacts in a second partial reaction to form a thioester between the acyl substrate and CoA. These enzymes utilize a Domain Alternation catalytic mechanism, whereby a ～110 residue C‐terminal domain rotates by 140° to form distinct catalytic conformations for the two partial reactions. The structure of an acetoacetyl‐CoA synthetase (AacS) is presented that illustrates a novel aspect of this C‐terminal domain. Specifically, several acetyl‐ and acetoacetyl‐CoA synthetases contain a 30‐residue extension on the C‐terminus compared to other members of this family. Whereas residues from this extension are disordered in prior structures, the AacS structure shows that residues from this extension may interact with key catalytic residues from the N‐terminal domain. Proteins 2015; 83:575–581. © 2014 Wiley Periodicals, Inc.     

Single-taxon field measurements of bacterial gene regulation controlling DMSP fate

The ‘bacterial switch'' is a proposed regulatory point in the global sulfur cycle that routes dimethylsulfoniopropionate (DMSP) to two fundamentally different fates in seawater through genes encoding either the cleavage or demethylation pathway, and affects the flux of volatile sulfur from ocean surface waters to the atmosphere. Yet which ecological or physiological factors might control the bacterial switch remains a topic of considerable debate. Here we report the first field observations of dynamic changes in expression of DMSP pathway genes by a single marine bacterial species in its natural environment. Detection of taxon-specific gene expression in Roseobacter species HTCC2255 during a month-long deployment of an autonomous ocean sensor in Monterey Bay, CA captured in situ regulation of the first gene in each DMSP pathway (dddP and dmdA) that corresponded with shifts in the taxonomy of the phytoplankton community. Expression of the cleavage pathway was relatively greater during a high-DMSP-producing dinoflagellate bloom, and expression of the demethylation pathway was greater in the presence of a mixed diatom and dinoflagellate community. These field data fit the prevailing hypothesis for bacterial DMSP gene regulation based on bacterial sulfur demand, but also suggest a modification involving oxidative stress response, evidenced as upregulation of catalase via katG, when DMSP is demethylated.     

Evidence for Intracellular and Extracellular Dimethylsulfoniopropionate (DMSP) Lyases and DMSP Uptake Sites in Two Species of Marine Bacteria

The kinetics of dimethylsulfoniopropionate (DMSP) uptake and dimethylsulfide (DMS) production from DMSP in two bacterial species, Alcaligenes sp. strain M3A, an isolate from estuarine surface sediments, and Pseudomonas doudoroffii, from seawater, were investigated. In Alcaligenes cells induced for DMSP lyase (DL) activity, DMS production occurred without DMSP uptake. In DL-induced suspensions of P. doudoroffii, uptake of DMSP preceded the production of DMS, indicating an intracellular location of DL; intracellular DMSP levels reached ca. 7 mM. DMSP uptake rates in noninduced cells showed saturation at three concentrations (K(inft) [transport] values, 3.4, 127, and 500 (mu)M). In DL-induced cells of P. doudoroffii, DMSP uptake rates increased ca. threefold (V(infmax), 0.022 versus 0.065 (mu)mol of DMSP taken up min(sup-1) mg of cell protein(sup-1)), suggesting that the uptake binding proteins were inducible. DMSP uptake and DL activity in P. doudoroffii were both inhibited by CN(sup-), 2,4-dinitrophenol, and membrane-impermeable thiol-binding reagents, further indicating active uptake of DMSP by cell surface components. The respiratory inhibitors had limited or no effect on DL activity by the Alcaligenes sp. Of the structural analogs of DMSP tested for their effect on DMSP metabolism, glycine betaine (GBT), but not methyl-3-mercaptopropionic acid (MMPA), inhibited DMSP uptake by P. doudoroffii, suggesting that GBT shares a binding protein with DMSP and that MMPA is taken up at a separate site. Two models of DMSP uptake, induction, and DL location found in marine bacteria are presented.     

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.