Dimethylsulfoniopropionate Lyase Research Articles

Release of Dimethylsulfide from Dimethylsulfoniopropionate by Plant-Associated Salt Marsh Fungi

The range of types of microbes with dimethylsulfoniopropionate (DMSP) lyase capability (enzymatic release of dimethylsulfide [DMS] from DMSP) has recently been expanded from bacteria and eukaryotic algae to include fungi (a species of the genus Fusarium [M. K. Bacic and D. C. Yoch, Appl. Environ. Microbiol. 64:106-111, 1998]). Fungi (especially ascomycetes) are the predominant decomposers of shoots of smooth cordgrass, the principal grass of Atlantic salt marshes of the United States. Since the high rates of release of DMS from smooth cordgrass marshes have a temporal peak that coincides with peak shoot death, we hypothesized that cordgrass fungi were involved in this DMS release. We tested seven species of the known smooth cordgrass ascomycetes and discovered that six of them exhibited DMSP lyase activity. We also tested two species of ascomycetes from other DMSP-containing plants, and both were DMSP lyase competent. For comparison, we tested 11 species of ascomycetes and mitosporic fungi from halophytes that do not contain DMSP; of these 11, only 3 were positive for DMSP lyase. A third group tested, marine oomycotes (four species of the genera Halophytophthora and Pythium, mostly from mangroves), showed no DMSP lyase activity. Two of the strains of fungi found to be positive for DMSP lyase also exhibited uptake of DMS, an apparently rare combination of capabilities. In conclusion, a strong correlation exists between a fungal decomposer's ability to catabolize DMSP via the DMSP lyase pathway and the host plant's production of DMSP as a secondary product.

Partial characterisation of dimethylsulfoniopropionate (DMSP) lyase isozymes in 6 strains of Emiliania huxleyi

We characterised and compared dimethylsulfoniopropionate (DMSP) lyase isozymes in crude extracts of 6 axenic Emiliania huxleyi cultures (CCMP 370, 373, 374, 379, 1516, and strain L). This enzyme cleaves DMSP to form dimethyl sulfide (DMS), acrylate and a proton, but the function of this reaction in algae is still poorly understood. Most of the cultures produced high concentrations of intracellular DMSP, which was constant over the growth cycle and ranged from 157 to 242 mM, except for 1516 which had 50 mM DMSP cell-1. Extracts of all strains produced DMS from exogenous DMSP in vitro. DMSP lyases appeared constitutive, but enzyme activity and behaviour varied greatly among strains, and did not correlate with intracellular DMSP concentration. Strains 373 and 379 showed high DMSP lyase activities (12.5 and 6.1 fmol DMS cell-1 min-1, respectively), whereas DMS production was more than 100-fold lower in 370, 374, 1516 and L. This difference was intrinsic and the general pattern of high- and low-activity strains remained true over more than a 1 yr cultivation period. The cleavage reaction was optimal at pH 6 in the strains with high lyase activity and pH 5 was optimal for 374, 1516 and L. Strain 370 showed increasing activity with increasing pH. Experiments with additions of 0.125 to 2 M NaCl indicated halotolerant DMSP lyases in 373, 379 and 374. However, the halophilic DMSP lyases in 370 and L required 1 M NaCl addition for optimal DMSP cleavage, and 1516 showed optimal activity at 2 M NaCl. These results suggest that there are several structurally different DMSP lyase isozymes within E. huxleyi. However, it cannot be ruled out that varying concentrations of DMSP lyase per cell may have contributed to the differences in enzyme activity per cell. Comparison with other algal taxa indicates several families of DMSP lyases, hinting at possibly different cellular locations and functions, and varying DMS production under natural conditions.

In Vivo Characterization of Dimethylsulfoniopropionate Lyase in the Fungus Fusarium lateritium

A fungus, Fusarium lateritium, with dimethylsulfoniopropionate (DMSP) lyase activity was isolated from both seawater and a salt marsh due to its ability to grow on DMSP (with the evolution of dimethyl sulfide) as the sole source of carbon. This is the first reported case of DMSP lyase activity in a fungus. Several other common fungal genera tested did not have DMSP lyase activity. DMSP was taken up more rapidly by F. lateritium than it was utilized, leading to its intracellular accumulation. Inhibitor studies with nystatin and cyanide indicated that DMSP uptake was an energy-dependent process. The lyase was inducible by its substrate, DMSP (K(m), 1.2 mM), and by the substrate analogs choline and glycine betaine. During induction, DMSP lyase activity increased with time and then dropped rapidly. This loss of activity could be prevented by spiking the culture with fresh DMSP or choline. The V(max) for DMSP lyase was 34.7 mU . mg of protein. The inhibitory effects of nystatin, and p-chloromercuriphenylsulfonate on DMSP lyase activity suggested that the enzyme is cytosolic. Because plants like Spartina (a marsh grass) and marine algae contain high concentrations of DMSP, we speculate that DMSP-utilizing fungi may be involved in their decay.

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

Volume 63, no. 4, p. 1600: The caption to Table 2 should read as follows: "Inactivation of Clostridium perfringens spores by 5-mg/liter doses of mixed oxidants or free chlorine in buffer at pH 7 at 25(deg)C." [This corrects the article on p. 1598 in vol. 63.].

Comparison of selenium and sulfur volatilization by dimethylsulfoniopropionate lyase (DMSP) in two marine bacteria and estuarine sediments

The volatile forms of selenium emitted from soils and plants have been identified by others as dimethylselenide and dimethyldiselenide. Dimethylselenoniopropionate, a possible precursor of dimethylselenide, is the selenium analogue of the common marine osmoprotectant, dimethylsulfoniopropionate. Dimethylselenoniopropionate was organically synthesized from dimethylselenide and acrylate and used in a comparative study with dimethylsulfoniopropionate as a growth substrate, an inducer and substrate for dimethylsulfoniopropionate lyase in two marine bacterial isolates, Alcaligenes sp. strain M3A and Pseudomonas doudoroffii, and in salt marsh sediments and estuarine creek water samples. In P. doudoroffii, the rate of dimethylselenoniopropionate transport into cells was about half that observed with dimethylsulfoniopropionate; Alcaligenes does not take up these molecules. The rate and extent of induction of dimethylsulfoniopropionate lyase in P. doudoroffii at low concentrations of dimethylselenoniopropionate were similar to that of dimethylsulfoniopropionate. In Alcaligenes the low constitutive level of dimethylsulfoniopropionate lyase cleaved dimethylselenoniopropionate into dimethylselenide and acrylate, but induction of dimethylsulfoniopropionate lyase by dimethylselenoniopropionate did not follow. Dimethylsulfoniopropionate lyases from both strains, when induced by dimethylsulfoniopropionate, utilized dimethylselenoniopropionate as a substrate with emission of dimethylselenide. Rates of partially purified dimethylsulfoniopropionate lyase activity with dimethylselenoniopropionate were half those with dimethylsulfoniopropionate. Anoxic salt marsh sediments catalyzed dimethylsulfoniopropionate and dimethylselenoniopropionate degradation at equal rates. In sediments containing high organic matter, both dimethylsulfide and dimethylselenide served as substrates for methanogenesis. These data support the hypothesis that organic selenium molecules can be volatilized by the same biochemical pathways as organic sulfur molecules.

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.

Purification and characterization of a dimethylsulfoniopropionate cleaving enzyme from Desulfovibrio acrylicus

An enzyme that cleaves the algal osmolyte dimethylsulfoniopropionate to dimethylsulfide and acrylate was purified almost 400-fold from the marine sulfate- and acrylate-reducing bacterium Desulfovibrio acrylicus DSM 10141. Dimethylsulfoniopropionate lyase activity was induced by acrylate and dimethylsulfoniopropionate. At 30 °C, the enzyme had a K m for dimethylsulfoniopropionate of 0.45 mM and a V max of 2590 μmol min −1 mg protein −1. Dimethylsulfoniopropionate was the only substrate of the enzyme. Among the compounds tested, dimethylsulfoniobutyrate was the most potent inhibitor ( kK i = 0.25 mM). On a denaturing polyacrylamide gel, the protein migrated as a single band of 49 kDa.

Purification and characterization of a dimethylsulfoniopropionate cleaving enzyme from Desulfovibrio acrylicus

Abstract An enzyme that cleaves the algal osmolyte dimethylsulfoniopropionate to dimethylsulfide and acrylate was purified almost 400-fold from the marine sulfate- and acrylate-reducing bacterium Desulfovibrio acrylicus DSM 10141. Dimethylsulfoniopropionate lyase activity was induced by acrylate and dimethylsulfoniopropionate. At 30 °C, the enzyme had a K m for dimethylsulfoniopropionate of 0.45 mM and a V max of 2590 μmol min −1 mg protein −1 . Dimethylsulfoniopropionate was the only substrate of the enzyme. Among the compounds tested, dimethylsulfoniobutyrate was the most potent inhibitor ( kK i = 0.25 mM). On a denaturing polyacrylamide gel, the protein migrated as a single band of 49 kDa.

Enzymatic cleavage of dimethylsulfoniopropionate (DMSP) in cell-free extracts of the marine macroalga Enteromorpha clathrata (Roth) Grev. (Ulvales, Chlorophyta)

The enzymatic cleavage of dimethylsulfoniopropionate (DMSP) to dimethylsulfide (DMS) in the marine macroalga Enteromorpha clathrata (Roth) Grev. (Ulvales, Chlorophyta) showed a maximum activity of 3.1 μmol DMS min −1 (mg protein) −1 at 25 °C and a pH optimum of 6.2 to 6.4. The activity was more sensitive to temperature than to pH changes. With ammonium sulfate precipitation, approximately 90% of the activity was found in the 0–35% fraction, indicating a fairly hydrophobic enzyme. Activity was increased by addition of detergents during extraction. These results suggest that the DMSP lyase enzyme is probably membrane-bound in vivo. The enzyme activity is relatively insensitive to chloride salt concentrations. Addition of nucleosides and nucleotides did not increase enzyme activity. After hyperosmotic shock for 72 h, enzyme activity increased 1.7-fold and intracellular DMSP concentration was enhanced from initially 80 mmol DMSP (kg freshweight) −1 to 107 mmol DMSP (kg freshweight) −1. The results suggest that DMSP cleavage within the cell by algal DMSP lyase may contribute to the production of oceanic and atmospheric DMS.

Grazing‐activated production of dimethyl sulfide (DMS) by two clones of Emiliania huxleyi

Emiliania huxleyi clones CCMP 370 and CCMP 373 produced similar amounts of dimethylsulfoniopropionate (DMSP) during axenic exponential growth, averaging 109 mM internal DMSP. Both clones had detectable DMSP lyase activity, as measured by production of dimethyl sulfide (DMS) during in vitro assays of crude cell preparations, but activities and conditions differed considerably between clones. Clone 373 had high activity; clone 370 had low activity and required chloride. For both strains, enzyme activity per cell was constant during exponential growth, but little DMS was produced by healthy cells. Rather, DMS production was activated when cells were subjected to physical or chemical stresses that caused cell lysis. We propose that DMSP lyase and DMSP are segregated within these cells and reaction only under conditions that result in cell stress or damage. Such activation occurs during microzooplankton grazing. When these clones were grazed by the dinoflagellate Oxyrrhis marina, DMS was produced; ungrazed cells, as well as those exposed to grazer exudates and associated bacteria, generated no DMS. Grazing of clone 373 produced much more DMS than grazing of clone 370, consistent with their relative in vitro DMSP lyase activities. DMS was only generated when cells were actually being grazed, indicating that ingested cells were responsible for the DMS formation. We suggest that even low levels of grazing can greatly accelerate DMS production.

Bacterial chemotaxis and its potential role in marine dimethylsulfide production and biogeochemical sulfur cycling

Dimethylsulfide (DMS) gas constitutes 90% of biogenic sulfur emissions from oceans and is an important agent in climate regulation. DMS in seawater arises primarily via lyase degradation of dimethylsulfoniopropionate (DMSP), an osmolyte produced in high concentrations by some marine phytoplankton. After being induced to synthesize DMSP lyase, cells of a marine bacterium (Alcaligenes strain M3A) significantly reduce their rate of change in direction and tumbling frequency, and they are attracted to DMSP at levels similar to those occurring near senescing phytoplankton cells (10‒7–10‒6 M). In contrast, genetically identical bacteria without lyase induction are not attracted to DMSP. Combined with lyase activity, bacterial chemotaxis to DMSP could increase the rate of DMS production and therefore play a critical role in biogeochemical sulfur cycling between dissolved organic matter in seawater and the atmosphere.

Cleavage of dimethylsulfoniopropionate and reduction of acrylate by Desulfovibrio acrylicus sp. nov.

From anoxic intertidal sediment, a dimethylsulfoniopropionate (DMSP)-cleaving anaerobe (strain W218) was isolated that reduced the acrylate formed to propionate. The bacterium was vibrio- to rod-shaped and motile by means of multiple polar flagella. It reduced sulfate, thiosulfate, and acrylate, and used lactate, fumarate, succinate, malate, pyruvate, ethanol, propanol, glycerol, glycine, serine, alanine, cysteine, hydrogen, and formate as electron donors. Sulfate and acrylate were reduced simultaneously; growth with sulfate was faster than with acrylate. Extracts of cells grown in the presence of DMSP contained high DMSP lyase activities (9.8 U/mg protein). The DNA mol% G+C was 45.1. On the basis of its characteristics and the 16S rRNA gene sequence, strain W218 was assigned to a new Desulfovibrio species for which the name Desulfovibrio acrylicus is proposed. A variety of other sulfate-reducing bacteria (eight of them originating from a marine or saline environment and five from other environments) did not reduce acrylate.

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 (Kutzing) 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.

Comparative Physiology of Dimethyl Sulfide Production by Dimethylsulfoniopropionate Lyase in Pseudomonas doudoroffii and Alcaligenes sp. Strain M3A

Dimethylsulfoniopropionate (DMSP) lyase enzymatically cleaves DMSP, an algal metabolite, to produce acrylate, a proton, and dimethyl sulfide (DMS), the most abundant volatile sulfur compound emitted from oceans. The physiology of DMS production by DMSP lyase was studied in vivo in an Alcaligenes-like organism, strain M3A, a salt marsh bacterial isolate, and in a marine strain, Pseudomonas doudoroffii. Enzymes from both strains were induced at optimum rates by 1 mM DMSP and vigorous aeration. P. doudoroffii was very sensitive to continued aeration and lost activity rapidly; the enzyme was more stable when aeration ceased. In addition to DMSP, acrylate and several of its analogs acted as inducers of DMSP lyase in Alcaligenes sp. strain M3A but not in P. doudoroffii. Turnover of DMSP by P. doudoroffii was enhanced by 3.5% NaCl or seawater, whereas the Alcaligenes sp. strain M3A enzyme was not salt dependent and salt did not greatly affect its activity. The pH profile showed two peaks of DMSP lyase activity (6.5 and 8.8) for Alcaligenes sp. strain M3A and a single peak at pH 8 for P. doudoroffii. Enzyme activity in both organisms was inhibited by methyl-3-mercaptopropionate and homocysteine. Cyanide, azide and p-chloromercuribenzoate inhibited only the P. doudoroffii DMSP lyase. The apparent K(infm) values for DMSP for cell cultures of Alcaligenes sp. strain M3A and P. doudoroffii were ca. 2 mM and <20 (mu)M, respectively. The differences in the physiology of DMSP metabolism in these two bacterial isolates may enable them to exist in diverse ecological niches.

ISOLATION, PURIFICATION, AND CHARACTERIZATION OF DMSP LYASE (DIMETHYLPROPIOTHETIN DETHIOMETHYLASE (4.4.1.3)) FROM THE RED ALGA POLYSIPHONIA PANICULATA1

ABSTRACTPolysiphonia paniculata Montagne is an intertidal red alga known to produce large amounts of the compound dimethylsulfoniopropionate (DMSP). Conversion of this substrate into dimethylsulfide is accomplished in P, paniculata by an enzyme called DMSP lyase (dimethylpropiothetin dethiomethyla.se (4.4.1.3)). DMSP lyase has been purified and characterized from P. paniculata. Enzymie activity is found in two different proteins: the larger with a molecular weight of 9.26 ± 104 daltons and the smaller with a molecular weight of 3.65 ± 104 daltons. Specific activity of the enzyme is 526 μmols min−1mg−1 for the smaller protein a nd 263 μmols min −1 mg−1 for the la rger protein. The Michaelis‐Menten constant (Km) is 72.8 μM ± 17.15 and the vmax is 1.62 μmols min−1± 0.928 for the 92.6‐kDa protein. The p1 of the larger protein is 5.8 and 5.9 for the smaller protein. Interaction with cysteine protease inhibitors L‐trans‐epoxysuccinyl‐leucylamido (4‐guanidino)‐butane, dithiobis‐(2‐nitrobenzoate), or N‐ethylmaleimide inactivated enzyme activity. The presence of either magnesium or calcium with DMSP lyase enhanced activity al concentrations between 20 and 40 μM but had little effect above these levels. Addition of the divalent chelators ethylenebis(oxyethylenenitrilo) tetraacetic acid and ethylenediaminetetraacetate decreased activity of the enzyme, but activity was restored when either chelator was removed and magnesium or calcium was added to the enzyme.

Purification and characterization of dimethylsulfoniopropionate lyase from an alcaligenes-like dimethyl sulfide-producing marine isolate

Dimethyl sulfide (DMS) is quantitatively the most important biogenic sulfur compound emitted from oceans and salt marshes. It is formed primarily by the action of dimethylsulfoniopropionate (DMSP) lyase which cleaves DMSP, an algal osmolyte, to equimolar amounts of DMS and acrylate. This report is the first to describe the isolation and purification of DMSP lyase. The soluble enzyme was purified to electrophoretic homogeneity from a facultatively anaerobic gram-negative rod-shaped marine bacterium identified as an Alcaligenes species by the Vitek gram-negative identification method. The key to successful purification of the enzyme was its binding to, and hydrophobic chromatography on, a phenyl-Sepharose CL-4B column. DMSP lyase biosynthesis was induced by its substrate, DMSP; its product, acrylate; and also by acrylamide. The relative effectivenesses of the inducers were 100, 90, and 204%, respectively. DMSP lyase is a 48-kDa monomer with a Michaelis-Menten constant (K(infm)) for DMSP of 1.4 mM and a V(infmax) of 408 (mu)mol/min/mg of protein. It converted DMSP to DMS and acrylate stoichiometrically. The similar K(infm) values measured for pure DMSP lyase and the axenic culture, seawater, and surface marsh sediment suggest that the microbes in these ecosystems must have enzymes similar to the one purified from our marine isolate. Anoxic sediment populations, however, have a 40-fold-lower K(infm) for this enzyme (30 (mu)M), possibly giving them the capability to metabolize much lower levels of DMSP than the aerobes.