Duane C. Yoch

Dimethylsulfoniopropionate: Its Sources, Role in the Marine Food Web, and Biological Degradation to Dimethylsulfide

The massive quantities of phytoplankton in the North Atlantic and Antarctic oceans producing dimethylsulfoniopropionate (DMSP) as an osmoprotectant, much of which is degraded by marine bacteria to dimethylsulfide (DMS), ensures an important role for both compounds in the global sulfur cycle. The closest to a comprehensive review on this topic is a book of symposium proceedings edited by Kiene et al. (75); the more recent developments related specifically to DMSP degradation by microbial communities are found elsewhere (68). This article is more comprehensive, as it includes some of the earlier literature in describing the sources of DMSP, its release and linkage to the marine (primarily microbial) food web and subsequent degradation via cleavage to DMS and acrylic acid or demethylation and demethiolation to methanethiol. DMS production from DMSP has long been associated with marine algae according to the following reaction (20, 22): (1) DMSP is a tertiary sulfonium compound produced in high concentration by certain species of marine algae and plant halophytes for the regulation of their internal osmotic environment (1, 41, 47, 120), although its role in plants remains unclear. This alga-associated, i.e., particulate DMSP (DMSPp), when released into the marine environment as dissolved DMSP (DMSPd), can serve as a link between primary production and the microbial population, as it is readily degraded by chemoheterotrophic bacteria (59). DMSP turnover usually exceeds DMS production in natural waters (60) because DMSP is also demethylated to 3-methiolpropionate, which can be further demethylated to 3-mercaptopropionate or demethiolated, releasing methanethiol (72, 118). These reactions will be discussed in more detail below. The biogeochemical significance of DMSP cleavage was first suggested in 1972, when DMS was found to be universally present in seawater and emitted at a significant rate to the atmosphere (87). It was proposed that DMS, rather than H2S from coastal waters and mud flats, was the missing gaseous sulfur compound needed to enable the steady-state flow of sulfur between marine and terrestrial environments, making DMS emissions a key step in the global sulfur cycle (87). Atmospheric H2S, which arises primarily from dissimilatory sulfate reduction in organic matter-rich environments, could never be measured in sufficient quantity to be the vehicle for transferring large quantities of sulfur from sea to air to land. The total annual flux of biogenic DMS released to the atmosphere ranges from 28 to 45 Tg of S year−1, at least 10-fold higher than from all other sources (Table ​(Table1).1). Recent, more comprehensive calculations of global annual DMS flux from the oceans gave values that ranged from 13 to 37 Tg of S year−1 (57). This sea-to-air flux represents about 50% of the global biogenic sulfur flux to the atmosphere (3). However, anthropogenic sulfur emissions dominate the sulfur flux, representing 80 to 90% of the input to the global sulfur cycle (12, 23, 88). TABLE 1. Estimates of natural emissions of organosulfur compoundsa The magnitude of the marine DMS emissions is all the more remarkable considering that over half of the DMSP released is demethylated (68) and that a significant fraction of the DMS is oxidized by bacteria in the water column before it can be released to the atmosphere (13, 64). While most of the biogenic sulfur emissions (primarily DMS) come from the oceans, those coming from salt marshes and coastal wetlands are many times higher on a unit area basis (112). DMS flux per unit area from these marine wetlands is also much higher than from any known terrestrial soil (2). The biogeochemical cycling of DMSP and its biological degradation products are shown in Fig. ​Fig.11. FIG. 1. Scheme representing the mechanisms of DMSP and DMS cycling in the marine water column and atmosphere. DMSO, dimethyl sulfoxide; CCN, cloud-condensing nuclei; MMPA, 3-methiolpropionate; β-HP, β-hydroxypropionate; 3-MPA, 3-mercaptopropionate; ...

Phylogenetic analysis of culturable dimethyl sulfide-producing bacteria from a spartina-dominated salt marsh and estuarine water.

ABSTRACT Dimethylsulfoniopropionate (DMSP), an abundant osmoprotectant found in marine algae and salt marsh cordgrass, can be metabolized to dimethyl sulfide (DMS) and acrylate by microbes having the enzyme DMSP lyase. A suite of DMS-producing bacteria isolated from a salt marsh and adjacent estuarine water on DMSP agar plates differed markedly from the pelagic strains currently in culture. While many of the salt marsh and estuarine isolates produced DMS and methanethiol from methionine and dimethyl sulfoxide, none appeared to be capable of producing both methanethiol and DMS from DMSP. DMSP, and its degradation products acrylate and β-hydroxypropionate but not methyl-3-mecaptopropionate or 3-mercaptopropionate, served as a carbon source for the growth of all the α- and β- but only some of the γ-proteobacterium isolates. Phylogenetic analysis of 16S rRNA gene sequences showed that all of the isolates were in the group Proteobacteria, with most of them belonging to the α and γ subclasses. Only one isolate was identified as a β-proteobacterium, and it had >98% 16S rRNA sequence homology with a terrestrial species of Alcaligenes faecalis. Although bacterial population analysis based on culturability has its limitations, bacteria from the α and γ subclasses of the Proteobacteria were the dominant DMS producers isolated from salt marsh sediments and estuaries, with the γ subclass representing 80% of the isolates. The α-proteobacterium isolates were all in the Roseobactersubgroup, while many of the γ-proteobacteria were closely related to the pseudomonads; others were phylogenetically related toMarinomonas, Psychrobacter, or Vibrio species. These data suggest that DMSP cleavage to DMS and acrylate is a characteristic widely distributed among different phylotypes in the salt marsh-estuarine ecosystem.

Dimethylsulfoniopropionate lyase from the marine macroalga Ulva curvata: purification and characterization of the enzyme

This is a report on the purification and characterization of an algal dimethylsulfoniopropionate (DMSP) lyase. This enzyme, also found in bacteria, is responsible for producing most of the dimethylsulfide (DMS) in marine environments. It was purified from the green macroalga, Ulva curvata (Kützing) De Toni. Initial in-vivo experiments showed that DMSP lyase activity from endogenous DMSP in Ulva increased for 24 h and then decreased as the culture aged and endogenous DMSP levels were depleted. When amended with exogenous DMSP, rates of DMSP lyase activity remained high even when the culture was 5 d old. Following disruption of the DMSP-depleted U. curvata cells by grinding, a soluble DMSP lyase was purified. This enzyme is a monomer of 78 kDa which has a Km for DMSP of 0.52 mM. Soluble DMSP lyase had an optimum pH of 8 and an optimum osmotic strength of 75 mM NaCl. Following disruption of the algae by either grinding with sand or blending, and washing out the soluble enzyme, the green tissue, when treated with the non-ionic detergent, Triton X-100, solubilized additional DMSP lyase activity. Three hydrophobic variant forms of Ulva DMSP lyase were isolated and partially characterized from the detergent-solubilized activity. While the molecular and kinetic properties of the algal enzyme are different from the bacterial enzymes we purified earlier, both the soluble and membrane-bound forms did, nevertheless, cross-react with antibodies raised against the bacterial (Alcaligenes strain M3A) DMSP lyase.

Regulation of nitrogenase activity by covalent modification in Chromatium vinosum

Nitrogenase in Chromatium vinosum was rapidly, but reversibly inhibited by NH4+. Activity of the Fe protin component of nitrogenase required both Mn2+ and activating enzyme. Activating enzyme from Rhodospirillum rubrum could replace Chromatium chromatophores in activating the Chromatium Fe protein, and conversely, a protein fraction prepared from Chromatium chromatophores was effective in activating R. rubrum Fe protein. Inactive Chromatium Fe protein contained a peptide covalently modified by a phosphate-containing molecule, which migrated the same in SDS-polyacrylamide gels as the modified subunit of R. rubrum Fe protein. In sum, these observations suggest that Chromatium nitrogenase activity is regulated by a covalent modification of the Fe protein in a manner similar to that of R. rubrum.

Nuclear magnetic resonance analysis of [1-13C]dimethylsulfoniopropionate (DMSP) and [1-13C]acrylate metabolism by a DMSP lyase-producing marine isolate of the α-subclass of Proteobacteria

ABSTRACT The prominence of the α-subclass of Proteobacteria in the marine bacterioplankton community and their role in dimethylsulfide (DMS) production has prompted a detailed examination of dimethylsulfoniopropionate (DMSP) metabolism in a representative isolate of this phylotype, strain LFR. [1-13C]DMSP was synthesized, and its metabolism and that of its cleavage product, [1-13C]acrylate, were studied using nuclear magnetic resonance (NMR) spectroscopy. [1-13C]DMSP additions resulted in the intracellular accumulation and then disappearance of both [1-13C]DMSP and [1-13C]β-hydroxypropionate ([1-13C]β-HP), a degradation product. Acrylate, the immediate product of DMSP cleavage, apparently did not accumulate to high enough levels to be detected, suggesting that it was rapidly β-hydroxylated upon formation. When [1-13C]acrylate was added to cell suspensions of strain LFR it was metabolized to [1-13C]β-HP extracellularly, where it first accumulated and was then taken up in the cytosol where it subsequently disappeared, indicating that it was directly decarboxylated. These results were interpreted to mean that DMSP was taken up and metabolized by an intracellular DMSP lyase and acrylase, while added acrylate was β-hydroxylated on (or near) the cell surface to β-HP, which accumulated briefly and was then taken up by cells. Growth on acrylate (versus that on glucose) stimulated the rate of acrylate metabolism eightfold, indicating that it acted as an inducer of acrylase activity. DMSP, acrylate, and β-HP all induced DMSP lyase activity. A putative model is presented that best fits the experimental data regarding the pathway of DMSP and acrylate metabolism in the α-proteobacterium, strain LFR.

N-Terminal Amino Acid Sequences and Comparison of DMSP Lyases from Pseudomonas Doudoroffii and Alcagenes Strain M3A

A comparative in vitro study of the DMSP lyases from an estuarine isolate, Alcaligenes strain M3A and a marine organism, Pseudomonas doudoroffii has been carried out. The enzymes in both organisms were induced to high levels by aeration, but unlike Alcaligenes, the P. doudoroffii enzyme was easily inactivated in vivo by processes not yet understood. The enzymes from both organisms bound to phenyl-Sepharose CL-4B and could then be purified by hydrophobic, anion exchange and gel filtration chromatography. A minor form (isozyme) of DMSP lyase (DL-2) was detected in Alcaligenes; its size and subunit composition were similar to the major isoform (DL-1) as was its K m for DMSP. Polyclonal antibodies raised against the Alcaligenes DMSP lyase were equally reactive against both the Alcaligenes and P. doudoroffii enzymes. Western blots of SDS-polyacrylamide gels together with gel filtration analysis showed that both enzymes were monomers of 48 kDa. The purified DMSP lyases had a similar K m (1 to 2 mM) and v max (ca. 500 units/mg protein) for DMSP. Methyl-3-mercaptopropionate (MMPA) was inhibitory to DMSP lyase activity in vivo and in vitro in both organisms. Cyanide and p-chloromercuri-benzoate inhibited P. doudoroffii DMSP lyase activity in vivo but not in vitro; activity in Alcaligenes was unaffected. Based on results of kinetic and inhibitor studies, a working model of DMS production, which includes a postulated DMSP-binding protein in P. doudoroffii, but not Alcaligenes, has been presented. The N-terminal amino acid sequences of the DMSP lyases purified from Alcaligenes and P. doudoroffii had 75% homology to each other in the first 20 amino acid residues, with 90% homology in the first 10 residues suggesting that the N-terminal region of DMSP lyase in these two marine bacteria is highly conserved. The N-terminal amino acid sequences of these enzymes showed no significant degree of homology with any existing protein in the database.

Ammonia switch-off of nitrogenase from Rhodobacter sphaeroides and Methylosinus trichosporium: no evidence for Fe protein modification

In vivo switch-off of nitrogenase activity by NH4+is a reversible process in Rhodobacter sphaeroides and Methylosinus trichosporium OB3b. The same pattern of switch-off in Rhodospirillum rubrum is explained by ADP-ribosylation of one of the Fe protein subunits, however, no evidence of covalent modification could be found in the subunits from either R. sphaeroides or M. trichosporium. Fe protein subunits from these organisms showed no variant behaviour on SDS-PAGE, nor were they 32P-labeled following switch-off. These observations suggest either that the attachment of the modifying group to the Fe protein in these organisms is quite labile and does not survive in vitro manipulation, or that the mechanism of switch-off is different than that seen in Rhodospirillum.

Methylamine metabolism and its role in nitrogenase “Switch off” in Rhodopseudomonas capsulata

In the photosynthetic bacterium Rhodopseudomonas capsulata, NH4+switch-off of nitrogenase activity can be mimicked by its analog, methylamine. Like NH4+, methylamine appeared to require processing by glutamine synthetase (GS) before it was effective; γ-glutamylmethylamide was shown to be the product of this reaction. Evidence that this glutamine analog functioned directly to initiate nitrogenase inactivation was suggested first by the fact that it was a poor substrate for glutamate synthase (i.e., it was not further metabolized by this pathway) and secondly, azaserine which blocks the transfer of the glutamine amide group had no effect on CH3NH3+(or NH4+) switch-off. These observations are taken as preliminary evidence to suggest that when NH4+inhibits nitrogenase activity, inactivation is initiated by glutamine itself, and not a molecule derived from it. Finally, evidence was presented that R. capsulata would use CH3NH3+as a nitrogen substrate, but lag periods and generation times increased with subsequent passages.

Differential Metabolism of Dimethylsulfoniopropionate and Acrylate in Saline and Brackish Intertidal Sediments

In anoxic Spartina altemiflora—dominated sediments along a naturally occuring salinity gradient (the Cooper River estuary, South Carolina, U.S.A.), dimethylsulfoniopropionate (DMSP) was metabolized to dimethyl sulfide (DMS) and acrylate by sediment microbes. The rate of DMSP degradation and acrylate mineralization by sediment microbes was similar at all sites along this 25-km transect. However, sediments amended with acrylate (or DMSP) showed significantly higher rates of N2 fixation (measured as acetylene reduction activity) (ARA) in the saline sediments downstream than brackish sediments. These results are consistent with the fact that acrylate stimulated the rates of both denitrification and CO2 production in the saline sediments at the mouth of the river more than tenfold over rates in brackish sediments. Enrichment experiments indicate that microbes capable of using DMSP or acrylate were not present in upstream sediments despite the fact that microbial biomass, percent organic matter, and both glucose-stimulated ARA and denitrification were highest upstream. It appears that acrylate utilizing, N2 fixing, and denitrifying populations are insignificant in the lower salinity sediments of the estuary. These results may reflect the availability of DMSP, which averaged 10.3 nmol g wet wt−1 of saline sediments and levels less than our detection limit (1 μm) in brackish sediments.

Molecular cloning and sequencing of the ferredoxin I fdxN gene of the photosynthetic bacterium Rhodospirillum rubrum

Using an oligonucleotide probe derived from the amino acid sequence of Rhodospirillum rubrum ferredoxin I, the gene (fdxN) was identified, cloned and sequenced. The FdxN coding region is 183 nucleotides which codes for a 61 amino acid (7267 Da) protein. Phylogenetic comparisons between the R. rubrum FdI and other 8Fe-8S nif-coupled ferredoxins showed only moderate degrees of similarity between the amino acid sequences. R. rubrum FdI synthesis was stimulated by nif derepressing conditions, but was not completely repressed by nif repression. Previous reports of an extracellular clostridial-type ferredoxin in R. rubrum could not be confirmed.