Dimethylsulfoniopropionate and Methanethiol Are Important Precursors of Methionine and Protein-Sulfur in Marine Bacterioplankton

Organic sulfur compounds are present in all aquatic systems, but their use as sources of sulfur for bacteria is generally not considered important because of the high sulfate concentrations in natural waters. This study investigated whether dimethylsulfoniopropionate (DMSP), an algal osmolyte that is abundant and rapidly cycled in seawater, is used as a source of sulfur by bacterioplankton. Natural populations of bacterioplankton from subtropical and temperate marine waters rapidly incorporated 15 to 40% of the sulfur from tracer-level additions of [³⁵S]DMSP into a macromolecule fraction. Tests with proteinase K and chloramphenicol showed that the sulfur from DMSP was incorporated into proteins, and analysis of protein hydrolysis products by high-pressure liquid chromatography showed that methionine was the major labeled amino acid produced from [³⁵S]DMSP. Bacterial strains isolated from coastal seawater and belonging to the α-subdivision of the division Proteobacteria incorporated DMSP sulfur into protein only if they were capable of degrading DMSP to methanethiol (MeSH), whereas MeSH was rapidly incorporated into macromolecules by all tested strains and by natural bacterioplankton. These findings indicate that the demethylation/demethiolation pathway of DMSP degradation is important for sulfur assimilation and that MeSH is a key intermediate in the pathway leading to protein sulfur. Incorporation of sulfur from DMSP and MeSH by natural populations was inhibited by nanomolar levels of other reduced sulfur compounds including sulfide, methionine, homocysteine, cysteine, and cystathionine. In addition, propargylglycine and vinylglycine were potent inhibitors of incorporation of sulfur from DMSP and MeSH, suggesting involvement of the enzyme cystathionine γ-synthetase in sulfur assimilation by natural populations. Experiments with [methyl-³H]MeSH and [³⁵S]MeSH showed that the entire methiol group of MeSH was efficiently incorporated into methionine, a reaction consistent with activity of cystathionine γ-synthetase. Field data from the Gulf of Mexico indicated that natural turnover of DMSP supplied a major fraction of the sulfur required for bacterial growth in surface waters. Our study highlights a remarkable adaptation by marine bacteria: they exploit nanomolar levels of reduced sulfur in apparent preference to sulfate, which is present at 10⁶- to 10⁷-fold higher concentrations.     

Use of microautoradiography combined with fluorescence in situ hybridization to determine dimethylsulfoniopropionate incorporation by marine bacterioplankton taxa

The fraction of planktonic heterotrophic bacteria capable of incorporating dissolved dimethylsulfoniopropionate (DMSP) and leucine was determined at two coastal sites by microautoradioagraphy (AU). In Gulf of Mexico seawater microcosm experiments, the proportion of prokaryotes that incorporated sulfur from [(35)S]DMSP ranged between 27 and 51% of 4',6-diamidino-2-phenylindole (DAPI)-positive cells, similar to or slightly lower than the proportion incorporating [(3)H]leucine. In the northwest Mediterranean coast, the proportion of cells incorporating sulfur from [(35)S]DMSP increased from 5 to 42% from January to March, coinciding with the development of a phytoplankton bloom. At the same time, the proportion of cells incorporating [(3)H]leucine increased from 21 to 40%. The combination of AU and fluorescence in situ hybridization (FISH) revealed that the Roseobacter clade (alpha-proteobacteria) accounted for 13 to 43% of the microorganisms incorporating [(35)S]DMSP at both sampling sites. Significant uptake of sulfur from DMSP was also found among members of the gamma-proteobacteria and Cytophaga-Flavobacterium groups. Roseobacter and gamma-proteobacteria exhibited the highest percentage of DAPI-positive cells incorporating (35)S from DMSP (around 50%). Altogether, the application of AU with [(35)S]DMSP combined with FISH indicated that utilization of S from DMSP is a widespread feature among active marine bacteria, comparable to leucine utilization. These results point toward DMSP as an important substrate for a broad and diverse fraction of marine bacterioplankton.     

An annual cycle of dimethylsulfoniopropionate-sulfur and leucine assimilating bacterioplankton in the coastal NW Mediterranean

The contribution of major phylogenetic groups to heterotrophic bacteria assimilating sulfur from dissolved dimethylsulfoniopropionate (DMSP) and assimilating leucine was analysed in surface seawaters from Blanes Bay (NW Mediterranean) over an annual study between March 2003 and April 2004. The percentage of bacteria assimilating DMSP-S showed a strong seasonal pattern, with a steady increase from winter (8 +/- 5%) to summer (23 +/- 3%). The same seasonal pattern was observed for the rate of DMSP-S assimilation. The annual average percentage of DMSP-S-assimilating bacteria (16 +/- 8%) was lower than the corresponding percentage of leucine-assimilating cells (35 +/- 16%), suggesting that not all bacteria synthesizing protein incorporated DMSP-S. Smaller differences between both percentages were recorded in summer. Members of the Alphaproteobacteria (Roseobacter and SAR11) and Gammaproteobacteria groups accounted for most of bacterial DMSP-S-assimilating cells over the year. All major bacterial groups showed an increase of the percentage of cells assimilating DMSP-S during summer, and contributed to the increase of the DMSP-S assimilation rate in this period. In these primarily P-limited waters, enrichment with P + DMSP resulted in a stimulation of bacterial heterotrophic production comparable to, or higher than, that with P + glucose in summer, while during the rest of the year P + glucose induced a stronger response. This suggested that DMSP was more important a S and C source for bacteria in the warm stratified season. Overall, our results suggest that DMSP-S assimilation is controlled by the contribution of DMSP to S (and C) sources rather than by the phylogenetic composition of the bacterioplankton.     

Direct homocysteine biosynthesis from O-succinylhomoserine in Escherichia coli: an alternate pathway that bypasses cystathionine.

Mutations were found which enable Escherichia coli K-12 to form homocysteine in the absence of cystathionase. The formation of homocysteine in the mutant strains required cystathionine gamma-synthetase, the metB gene product, but bypassed the normal intermediate cystathionine. It is concluded that cystathionine gamma-synthetase catalyzes the formation of homocysteine directly from O-succinylhomoserine and an as-yet-unidentified sulfur donor. The mutation apparently causes the formation of this sulfur donor and has been named metQ. The expression of the metQ gene is under catabolite repression.     

Use of Microautoradiography Combined with Fluorescence In Situ Hybridization To Determine Dimethylsulfoniopropionate Incorporation by Marine Bacterioplankton Taxa

The fraction of planktonic heterotrophic bacteria capable of incorporating dissolved dimethylsulfoniopropionate (DMSP) and leucine was determined at two coastal sites by microautoradioagraphy (AU). In Gulf of Mexico seawater microcosm experiments, the proportion of prokaryotes that incorporated sulfur from [³⁵S]DMSP ranged between 27 and 51% of 4′,6-diamidino-2-phenylindole (DAPI)-positive cells, similar to or slightly lower than the proportion incorporating [³H]leucine. In the northwest Mediterranean coast, the proportion of cells incorporating sulfur from [³⁵S]DMSP increased from 5 to 42% from January to March, coinciding with the development of a phytoplankton bloom. At the same time, the proportion of cells incorporating [³H]leucine increased from 21 to 40%. The combination of AU and fluorescence in situ hybridization (FISH) revealed that the Roseobacter clade (α-proteobacteria) accounted for 13 to 43% of the microorganisms incorporating [³⁵S]DMSP at both sampling sites. Significant uptake of sulfur from DMSP was also found among members of the γ-proteobacteria and Cytophaga-Flavobacterium groups. Roseobacter and γ-proteobacteria exhibited the highest percentage of DAPI-positive cells incorporating ³⁵S from DMSP (around 50%). Altogether, the application of AU with [³⁵S]DMSP combined with FISH indicated that utilization of S from DMSP is a widespread feature among active marine bacteria, comparable to leucine utilization. These results point toward DMSP as an important substrate for a broad and diverse fraction of marine bacterioplankton.     

The formation of sulphur-containing amino acids in germinating seeds of rape (Brassica napus L.)

Sodium [(35)S]sulphide was fed to batches of germinating rapeseed, in some instances with the addition of unlabelled cysteine. Both the total radioactivity and specific radioactivity of the free sulphur-containing amino acids were examined. Cysteine and homocysteine were rapidly labelled; label subsequently appeared in cystathionine and methionine. The results obtained indicated that both the sulphydration and trans-sulphuration pathways were operating. This conclusion was reinforced by the results of experiments in which batches of rapeseed were incubated with l-[(14)C]homoserine. These showed the formation of labelled homocysteine, cystathione and methionine. It was thought the trans-sulphuration pathway was making the greater contribution to the biosynthesis of methionine in germinating rapeseed.     

A yeast with unusual sulphur amino acid metabolism

A yeast strain highly resistant to propargylglycine (an inhibitor of cystathionine gamma-lyase) was isolated from air. It was partially characterized, but it has not been identified with any known yeast species. Its sulphur amino acid metabolism differed from that of other fungi by the lack of the reverse transsulphuration pathway from methionine to cysteine, as no activity of cystathionine beta-synthase or cystathionine gamma-lyase was found. The functional lack of this pathway was confirmed by growth tests and by experiments with [35S]methionine. In contrast to Saccharomyces cerevisiae neither homocysteine synthase nor the sulphate assimilation pathway were repressible by methionine in the new strain; on the contrary, a regulatory effect of cysteine was observed.     

Biosynthesis of 3-dimethylsulfoniopropionate in Wollastonia biflora (L.) DC. Evidence that S-methylmethionine is an intermediate.

The compatible solute 3-dimethylsulfoniopropionate (DMSP) is accumulated by certain salt-tolerant flowering plants and marine algae. It is the major biogenic precursor of dimethylsulfide, an important sulfur-containing trace gas in the atmosphere. DMSP biosynthesis was investigated in Wollastonia biflora (L.) DC. [= Wedelia biflora (L.) DC., Melanthera biflora (L.) Wild, Asteraceae]. After characterizing DMSP and glycine betaine accumulation in three diverse genotypes, a glycine betaine-free genotype was chosen for radiotracer and stable isotope-labeling studies. In discs from young leaves, label from [U-14C]methionine was readily incorporated into the dimethylsulfide and acrylate moieties of DMSP. This establishes that DMSP is derived from methionine by deamination, decarboxylation, oxidation, and methylation steps, without indicating their order. Five lines of evidence indicated that methylation is the first step in the sequence, not the last. (a) In pulse-chase experiments with [14C]methionine, S-methylmethionine (SMM) had the labeling pattern expected of a pathway intermediate, whereas 3-methylthiopropionate (MTP) did not. (b) [14C]SMM was efficiently converted to DMSP but [14C]MTP was not. (c) The addition of unlabeled SMM, but not of MTP, reduced the synthesis of [14C]DMSP from [14C]methionine. (d) The dimethylsulfide group of [13CH3,C2H3]SMM was incorporated as a unit into DMSP. (e) When [C2H3,C2H3]SMM was given together with [13CH3]methionine, the main product was [C2H3,C2H3]DMSP, not [13CH3,C2H3]DMSP or [13CH3,13CH3]DMSP. The stable isotope labeling results also show that the SMM cycle does not operate at a high level in W. biflora leaves.     

A study of the sulphur amino acids of rat tissues

1. In a study of the metabolism of l-[(35)S]methionine in vivo, the labelled sulphur compounds of rat liver and brain were separated first by ion-exchange chromatography into two fractions containing (i) free sulphur amino acids such as methionine, cystathionine, cyst(e)ine and homocyst(e)ine and (ii) glutathione. 2. Two-dimensional paper chromatography with butan-1-ol-acetic acid or propionic acid-water in the first direction and 80% acetone or acetone-ethyl methyl ketone-water in the second direction was found superior to other solvent systems for separating the sulphur amino acids. 3. At 10min. after injection of [(35)S]methionine only a small part of the (35)S was found combined in free methionine or other free sulphur amino acids. 4. Evidence was obtained of the presence of adenosyl[(35)S]methionine and adenosyl[(35)S]homocysteine in perchloric acid extracts of rat liver and brain. 5. The trans-sulphuration pathway was active in brain as well as in liver.     

Role of Methionine in Cephalosporin Synthesis

Methionine has an almost unique stimulatory effect on biosynthesis of cephalosporins (by Cephalosporium acremonium). No other sulfur-containing compound tested, except dl-methionine-dl-sulfoxide, replaced methionine. dl-Methionine stimulated the synthesis of cephalosporins when added after the growth phase. The utilization of inorganic sulfate was repressed by methionine. Experiments with l-methionine-S(35) showed that essentially all the sulfur in the cephalosporins was derived from methionine. Sulfur-labeled compounds found in the soluble pool from cells grown with methionine-S(35) were methionine, homocysteine, taurine, cystathionine, cysteic acid, glutathionine, and cysteine. dl-Serine-3-C(14) was incorporated into the antibiotics, and its utilization was stimulated by methionine. l-Cysteine had a sparing effect on the incorporation of methionine-S(35) and serine-C(14) into the antibiotics. The data are consistent with the hypothesis that a cystathionine-mediated pathway is operative in the transfer of sulfur between methionine and cysteine.     

Sulfur assimilation by Oxyrrhis marina feeding on a 35S‐DMSP‐labelled prey

A laboratory grazing experiment was conducted with the aim of quantifying the sulfur assimilation by a herbivore protist feeding on a dimethylsulfoniopropionate (DMSP)‐containing phytoplankter. When supplied with dissolved ³⁵S‐DMSP, cultures of an axenic strain of the diatom Thalassiosira pseudonana took up 60–95% of the added radioisotope and accumulated it untransformed in the cytoplasm. Radiolabelled diatom cells were offered as prey to the heterotrophic dinoflagellate Oxyrrhis marina. After 32 h in the dark, all the prey had been grazed and digested, leaving only radiolabelled O. marina in the grazing bottles and thus providing an estimate of the percentage of DMSP‐sulfur retained by the predator. Subsequent precipitation with cold trichloroacetic acid (TCA) provided the fraction of retained DMSP‐S that had been assimilated into the micrograzer macromolecules. In parallel incubations with predator and dissolved ³⁵S‐DMSP only (no prey), O. marina (and their closely associated bacteria) took up the radiolabelled substrate osmotrophically to an activity of 0.04 dpm cell^?1 and assimilated it all into macromolecules. By correcting grazing ³⁵S‐DMSP assimilation for osmotrophic ³⁵S‐DMSP assimilation, and comparing it with the ingested radioisotope, the percentage of ingested DMSP‐sulfur retained and assimilated by the predator was determined to be 32 ± 4%. This is the first study that provides direct evidence that ingestion of a DMSP‐containing prey supplies structural sulfur to a herbivore protist and that quantifies this assimilative supply at one‐third of ingested DMSP.     

Functional demonstration of reverse transsulfuration in the Mycobacterium tuberculosis complex reveals that methionine is the preferred sulfur source for pathogenic Mycobacteria

Methionine can be used as the sole sulfur source by the Mycobacterium tuberculosis complex although it is not obvious from examination of the genome annotation how these bacteria utilize methionine. Given that genome annotation is a largely predictive process, key challenges are to validate these predictions and to fill in gaps for known functions for which genes have not been annotated. We have addressed these issues by functional analysis of methionine metabolism. Transport, followed by metabolism of (35)S methionine into the cysteine adduct mycothiol, demonstrated the conversion of exogenous methionine to cysteine. Mutational analysis and cloning of the Rv1079 gene showed it to encode the key enzyme required for this conversion, cystathionine gamma-lyase (CGL). Rv1079, annotated metB, was predicted to encode cystathionine gamma-synthase (CGS), but demonstration of a gamma-elimination reaction with cystathionine as well as the gamma-replacement reaction yielding cystathionine showed it encodes a bifunctional CGL/CGS enzyme. Consistent with this, a Rv1079 mutant could not incorporate sulfur from methionine into cysteine, while a cysA mutant lacking sulfate transport and a methionine auxotroph was hypersensitive to the CGL inhibitor propargylglycine. Thus, reverse transsulfuration alone, without any sulfur recycling reactions, allows M. tuberculosis to use methionine as the sole sulfur source. Intracellular cysteine was undetectable so only the CGL reaction occurs in intact mycobacteria. Cysteine desulfhydrase, an activity we showed to be separable from CGL/CGS, may have a role in removing excess cysteine and could explain the ability of M. tuberculosis to recycle sulfur from cysteine, but not methionine.     

Pathways of Assimilative Sulfur Metabolism in Pseudomonas putida

Cysteine and methionine biosynthesis was studied in Pseudomonas putida S-313 and Pseudomonas aeruginosa PAO1. Both these organisms used direct sulfhydrylation of O-succinylhomoserine for the synthesis of methionine but also contained substantial levels of O-acetylserine sulfhydrylase (cysteine synthase) activity. The enzymes of the transsulfuration pathway (cystathionine gamma-synthase and cystathionine beta-lyase) were expressed at low levels in both pseudomonads but were strongly upregulated during growth with cysteine as the sole sulfur source. In P. aeruginosa, the reverse transsulfuration pathway between homocysteine and cysteine, with cystathionine as the intermediate, allows P. aeruginosa to grow rapidly with methionine as the sole sulfur source. P. putida S-313 also grew well with methionine as the sulfur source, but no cystathionine gamma-lyase, the key enzyme of the reverse transsulfuration pathway, was found in this species. In the absence of the reverse transsulfuration pathway, P. putida desulfurized methionine by the conversion of methionine to methanethiol, catalyzed by methionine gamma-lyase, which was upregulated under these conditions. A transposon mutant of P. putida that was defective in the alkanesulfonatase locus (ssuD) was unable to grow with either methanesulfonate or methionine as the sulfur source. We therefore propose that in P. putida methionine is converted to methanethiol and then oxidized to methanesulfonate. The sulfonate is then desulfonated by alkanesulfonatase to release sulfite for reassimilation into cysteine.     

Influence of salinity on dimethyl sulfide and methanethiol formation in estuarine sediments and its side effect on nitrous oxide emissions

We investigated the regulatory effect of salinity on the production of dimethylsulfide (DMS) and methanethiol (MeSH) in estuarine sediments and the potential interactions with the nitrous oxide (N₂O) reductase step of the denitrification pathway. This was achieved by monitoring DMS, MeSH and N₂O accumulation in sediment slurries retrieved from a temperate estuary (Ave, NW Portugal). Treatments were performed with and without amendments of potential sulfur gas precursors, DMSP (0–50?μM) or methionine (0–500?μM) at different salinities (0, 15 and 30?ppt). Experimental increases of salinity inhibited DMS accumulation under both oxic and anoxic incubation conditions, and the pattern was observed whether DMSP or methionine was added or not, i.e. lower salinities stimulated DMS net production. In contrast, MeSH tended to accumulate to higher concentrations in higher salinity treatments (15 and 30?ppt). Our results also suggest that while salinity had a direct influence on N₂O accumulation, it also may modulated N₂O production through its regulatory effect on the formation of MeSH, a compound previously shown to inhibit N₂O reduction activity. Overall, our results suggest that changes in salinity may have an important regulatory role in net production of DMS, MeSH and N₂O and their potential emissions to the atmosphere.     

Cysteine and methionine content of the Escherichia coli ribonucleic acid polymerase subunits.

We describe a procedure that allows cysteine and methionine content to be determined on microgram amounts of partially purified protein. The only requirements are that the protein can be obtained as a pure band after electrophoresis on a polyacrylamide gel and that some data on amino acid content be available. This method involves double labeling by growing bacterial cells with [3H]leucine and [35S]SO4 and determining the ratio of these radioisotopes incorporated into the ribonucleic acid polymerase subunits. The relative specific activities of [3H]leucine and [35S]cysteine and methionine are determined from the ratio of these isotopes incorporated into beta-galactosidase, the leucine, cysteine, and methionine contents of which are known. We have used this procedure to determine the sulfur content of the subunits of Escherichia coli ribonucleic acid polymerase. These new data are necessary to quantitate the rates of synthesis of these subunits by in vivo labeling with [35S]SO4.     

Flow-cytometric cell sorting and subsequent molecular analyses for culture-independent identification of bacterioplankton involved in dimethylsulfoniopropionate transformations

Marine bacterioplankton transform dimethylsulfoniopropionate (DMSP) into the biogeochemically important and climatically active gas dimethylsulfide. In order to identify specific bacterial taxa mediating DMSP processing in a natural marine ecosystem, we amended water samples from a southeastern U.S. salt marsh with 20 microM DMSP and tracked community shifts with flow cytometry (FCM) coupled to 16S rRNA gene analyses. In two out of four seasons studied, DMSP amendments induced the formation of distinct bacterioplankton populations with elevated nucleic acid (NA) content within 24 h, indicative of cells actively utilizing DMSP. The 16S rRNA genes of the cells with and without elevated NA content were analyzed following cell sorting and PCR amplification with sequencing and terminal restriction fragment length polymorphism approaches. Compared to cells in the control FCM populations, bacteria with elevated NA content in the presence of DMSP were relatively enriched in taxa related to Loktanella, Oceanicola, and Sulfitobacter (Roseobacter lineage, alpha-Proteobacteria); Caulobacter (alpha-Proteobacteria); and Brachymonas and Xenophilus (beta-Proteobacteria) in the May-02 sample and to Ketogulonicigenium (Roseobacter lineage, alpha-Proteobacteria) and novel gamma-Proteobacteria in the Sept-02 sample. Our study suggests that diverse bacterioplankton participate in the metabolism of DMSP in coastal marine systems and that their relative importance varies temporally.     

In vivo regulation of de novo methionine biosynthesis in a higher plant (lemna)

Administration of methionine to growing Lemna had essentially no effect on accumulation of sulfate sulfur in protein cysteine, but decreased accumulation into cystathionine and its products (homocysteine, methionine, S-methylmethioninesulfonium salt, S-adenosylmethionine, and S-adenosylhomocysteine) to as low as 21% that of control plants, suggesting that methionine regulates its own de novo synthesis at cystathionine synthesis. Methionine caused only a slight reduction (to 80% that of control plants) in the accumulation of sucrose carbon into the 4-carbon moieties of cystathionine and products. This observation was puzzling since cystathionine synthesis proceeds by incorporation of equivalent amounts of sulfur (from cysteine) and 4-carbon moieties (from O-phosphohomoserine). The apparent inconsistency was resolved by the demonstration in Lemna (Giovanelli, Datko, Mudd, Thompson 1983 Plant Physiol 71: 319-326) that de novo synthesis of the methionine 4-carbon moiety occurs not only via the established transsulfuration route from O-phosphohomoserine, but also via the ribose moiety of 5′-methylthioadenosine. It is now clear that the more accurate assessment of the flux of sulfur (and 4-carbon moieties) through transsulfuration is provided by the amount of ³⁵S from ³⁵SO₄²⁻ that accumulates in cystathionine and its products, rather than by the corresponding measurements with ¹⁴C. These studies therefore unequivocally demonstrate in higher plants that methionine does indeed feedback regulate it own de novo synthesis in vivo, and that cystathionine synthesis is a locus for this regulation.     

Abundant and diverse bacteria involved in DMSP degradation in marine surface waters

An expanded analysis of oceanic metagenomic data indicates that the majority of prokaryotic cells in marine surface waters have the genetic capability to demethylate dimethylsulfoniopropionate (DMSP). The 1701 homologues of the DMSP demethylase gene, dmdA , identified in the (2007) Global Ocean Sampling (GOS) metagenome, are sufficient for 58% (±9%) of sampled cells to participate in this critical step in the marine sulfur cycle. This remarkable frequency of DMSP-demethylating cells is in accordance with biogeochemical data indicating that marine phytoplankton direct up to 10% of fixed carbon to DMSP synthesis, and that most of this DMSP is subsequently degraded by bacteria via demethylation. The GOS metagenomic data also revealed a new cluster of dmdA sequences (designated Clade E) that implicates marine gammaproteobacteria in DMSP demethylation, along with previously recognized alphaproteobacterial groups Roseobacter and SAR11. Analyses of G+C content and gene order indicate that lateral gene transfer is likely responsible for the wide distribution of dmdA among diverse taxa, contributing to the homogenization of biogeochemical roles among heterotrophic marine bacterioplankton. Candidate genes for the competing bacterial degradation process that converts DMSP to the climate-active gas dimethylsulfide (DMS) ( dddD and dddL ) occur infrequently in the (2007) GOS metagenome, suggesting either that the key DMS-producing bacterial genes are yet to be identified or that DMS formation by free-living bacterioplankton is insignificant relative to their demethylation activity.     

Production and fate of methylated sulfur compounds from methionine and dimethylsulfoniopropionate in anoxic salt marsh sediments

Anoxic salt marsh sediments were amended with dl-methionine and dimethylsulfoniopropionate (DMSP). Microbial metabolism of methionine yielded methane thiol (MSH) as the major volatile organosulfur product, with the formation of lesser amounts of dimethylsulfide (DMS). Biological transformation of DMSP resulted in the rapid release of DMS and only small amounts of MSH. Experiments with microbial inhibitors indicated that production of MSH from methionine was carried out by procaryotic organisms, probably sulfate-reducing bacteria. Methane-producing bacteria did not metabolize methionine. The involvement of specific groups of organisms in DMSP hydrolysis could not be determined with the inhibitors used, because DMSP was hydrolyzed in all samples except those which were autoclaved. Unamended sediment slurries, prepared from Spartina alterniflora sediments, contained significant (1 to 10 muM) concentrations of DMS. Endogenous methylated sulfur compounds and those produced from added methionine and DMSP were consumed by sediment microbes. Both sulfate-reducing and methane-producing bacteria were involved in DMS and MSH consumption. Methanogenesis was stimulated by the volatile organosulfur compounds released from methionine and DMSP. However, apparent competition for these compounds exists between methanogens and sulfate reducers. At low (1 muM) concentrations of methionine, the terminal S-methyl group was metabolized almost exclusively to CO(2) and only small amounts of CH(4). At higher (>100 muM) concentrations of methionine, the proportion of the methyl-sulfur group converted to CH(4) increased. The results of this study demonstrate that methionine and DMSP are potential precursors of methylated sulfur compounds in anoxic sediments and that the microbial community is capable of metabolizing volatile methylated sulfur compounds.     

REGULATION OF BIOSYNTHESIS OF DIMETHYLSULFONIOPROPIONATE AND ITS UPTAKE IN STERILE MUTANT OF ULVA PERTUSA (CHLOROPHYTA)1

It has been shown that marine algae produce the compatible solute dimethylsulfoniopropionate (DMSP) from methionine (Met) via four enzymatic reactions in which the third step, synthesis of 4‐dimethylsulfonio‐2‐hydroxy‐butyrate (DMSHB) from 4‐methylthio‐2‐hydroxybutyrate (MTHB), is the committing step. However, regulation of the biosynthetic pathways and transport properties of DMSP is largely unknown. Here, the effects of sulfur and sodium concentrations on the uptake and synthesis of DMSHB and DMSP were examined in a sterile mutant of Ulva pertusa Kjellm. Sulfur deficiency increased the activity of the sulfur assimilation enzyme O‐acetyl serine sulfhydrylase but decreased the MTHB S‐methyltransferase activity, suggesting the preferential utilization of sulfur atoms for Met metabolites other than DMSP. Uptake of DMSP and DMSHB was enhanced by S deficiency. High salinity enhanced the MTHB S‐methyltransferase activity as well as the uptake of DMSHB. The MTHB S‐methyltransferase activity was inhibited by its product DMSP. These data demonstrate the importance of MTHB S‐methyltransferase activity and uptake of DMSHB for the regulation of DMSP.