Dimethylsulfoniopropionate Cleavage Research Articles

Isolation and Assays of Bacterial Dimethylsulfoniopropionate Lyases.

The organosulfur metabolite dimethylsulfoniopropionate (DMSP) and its enzymatic breakdown product dimethyl sulfide (DMS) have important implications in the global sulfur cycle and in marine microbial food webs. Enormous amounts of DMSP are produced in marine environments where microbial communities import and catabolize it via either the demethylation or the cleavage pathways. The enzymes that cleave DMSP are termed "DMSP lyases" and generate acrylate or hydroxypropionate, and ~107tons of DMS annually. An important environmental factor affecting DMS generation by the DMSP lyases is the availability of metal ions as these enzymes use various cofactors for catalysis. This chapter summarizes advances on bacterial DMSP catabolism, with an emphasis on various biochemical methods employed for the isolation and characterization of bacterial DMSP lyases. Strategies are presented for the purification of DMSP lyases expressed in bacterial cells. Specific conditions for the efficient isolation of apoproteins in Escherichia coli are detailed. DMSP cleavage is effectively inferred, utilizing the described HPLC-based acrylate detection assay. Finally, substrate and metal binding interactions are examined using fluorescence and UV-visible assays. Together, these methods are rapid and well suited for the biochemical and structural characterization of DMSP lyases and in the assessment of uncharacterized DMSP catabolic enzymes, and new metalloenzymes in general.

Mechanistic Insights into Dimethylsulfoniopropionate Lyase DddY, a New Member of the Cupin Superfamily

The marine osmolyte dimethylsulfoniopropionate (DMSP) is one of Earth's most abundant organosulfur molecules. Bacterial DMSP lyases cleave DMSP, producing acrylate and dimethyl sulfide (DMS), a climate-active gas with roles in global sulfur cycling and atmospheric chemistry. DddY is the only known periplasmic DMSP lyase and is present in β-, γ-, δ- and ε-proteobacteria. Unlike other known DMSP lyases, DddY has not been classified into a protein superfamily, and its structure and catalytic mechanism are unknown. Here, we determined the crystal structure of DddY from the γ-proteobacterium Acinetobacter bereziniae originally isolated from human clinical specimens. This structure revealed that DddY contains a cap domain and a catalytic domain with a Zn2+ bound at its active site. We also observed that the DddY catalytic domain adopts a typical β-barrel fold and contains two conserved cupin motifs. Therefore, we concluded that DddY should belong to the cupin superfamily. Using structural and mutational analyses, we identified key residues involved in Zn2+ coordination, DMSP binding and the catalysis of DMSP cleavage, enabling elucidation of the catalytic mechanism, in which the residue Tyr271 of DddY acts as a general base to attack DMSP. Moreover, sequence analysis suggested that this proposed mechanism is common to DddY proteins from β-, γ-, δ- and ε-proteobacteria. The DddY structure and proposed catalytic mechanism provide a better understanding of how DMSP is catabolized to generate the important climate-active gas DMS.

Structural and Biochemical Insights into Dimethylsulfoniopropionate Cleavage by Cofactor-Bound DddK from the Prolific Marine Bacterium Pelagibacter.

Enormous amounts of the organic osmolyte dimethylsulfoniopropionate (DMSP) are produced in marine environments where bacterial DMSP lyases cleave it, yielding acrylate and the climate-active gas dimethyl sulfide (DMS). SAR11 bacteria are the most abundant clade of heterotrophic bacteria in the oceans and play a key role in DMSP catabolism. An important environmental factor affecting DMS generation via DMSP lyases is the availability of metal ions because they are essential cofactors for many of these enzymes. Here we examine the structure and activity of DddK in the presence of various metal ions. We have established that DddK containing a double-stranded β-helical motif utilizes various divalent metal ions as cofactors for catalytic activity. However, nickel, an abundant metal ion in marine environments, adopts a distorted octahedral coordination environment and conferred the highest DMSP lyase activity. Crystal structures of cofactor-bound DddK reveal key metal ion binding and catalytic residues and provide the first rationalization for varying activities with different metal ions. The structures of DddK along with site-directed mutagenesis and ultraviolet-visible studies are consistent with Tyr 64 acting as a base to initiate the β-elimination reaction of DMSP. Our biochemical and structural studies provide a detailed understanding of DMS generation by one of the ocean's most prolific bacteria.

Evolution of Dimethylsulfoniopropionate Metabolism in Marine Phytoplankton and Bacteria.

The elucidation of the pathways for dimethylsulfoniopropionate (DMSP) synthesis and metabolism and the ecological impact of DMSP have been studied for nearly 70 years. Much of this interest stems from the fact that DMSP metabolism produces the climatically active gas dimethyl sulfide (DMS), the primary natural source of sulfur to the atmosphere. DMSP plays many important roles for marine life, including use as an osmolyte, antioxidant, predator deterrent, and cryoprotectant for phytoplankton and as a reduced carbon and sulfur source for marine bacteria. DMSP is hypothesized to have become abundant in oceans approximately 250 million years ago with the diversification of the strong DMSP producers, the dinoflagellates. This event coincides with the first genome expansion of the Roseobacter clade, known DMSP degraders. Structural and mechanistic studies of the enzymes of the bacterial DMSP demethylation and cleavage pathways suggest that exposure to DMSP led to the recruitment of enzymes from preexisting metabolic pathways. In some cases, such as DmdA, DmdD, and DddP, these enzymes appear to have evolved to become more specific for DMSP metabolism. By contrast, many of the other enzymes, DmdB, DmdC, and the acrylate utilization hydratase AcuH, have maintained broad functionality and substrate specificities, allowing them to carry out a range of reactions within the cell. This review will cover the experimental evidence supporting the hypothesis that, as DMSP became more readily available in the marine environment, marine bacteria adapted enzymes already encoded in their genomes to utilize this new compound.

New Mechanistic Insight from Substrate- and Product-Bound Structures of the Metal-Dependent Dimethylsulfoniopropionate Lyase DddQ.

The marine microbial catabolism of dimethylsulfoniopropionate (DMSP) by the lyase pathway liberates ∼300 million tons of dimethyl sulfide (DMS) per year, which plays a major role in the biogeochemical cycling of sulfur. Recent biochemical and structural studies of some DMSP lyases, including DddQ, reveal the importance of divalent transition metal ions in assisting DMSP cleavage. While DddQ is believed to be zinc-dependent primarily on the basis of structural studies, excess zinc inhibits the enzyme. We examine the importance of iron in regulating the DMSP β-elimination reaction catalyzed by DddQ as our as-isolated purple-colored enzyme possesses ∼0.5 Fe/subunit. The UV-visible spectrum exhibited a feature at 550 nm, consistent with a tyrosinate-Fe(III) ligand-to-metal charge transfer transition. Incubation of as-isolated DddQ with added iron increases the intensity of the 550 nm peak, whereas addition of dithionite causes a bleaching as Fe(III) is reduced. Both the Fe(III) oxidized and Fe(II) reduced species are active, with similar kcat values and 2-fold differences in their Km values for DMSP. The slow turnover of Fe(III)-bound DddQ allowed us to capture a substrate-bound form of the enzyme. Our DMSP-Fe(III)-DddQ structure reveals conformational changes associated with substrate binding and shows that DMSP is positioned optimally to bind iron and is in the proximity of Tyr 120 that acts as a Lewis base to initiate catalysis. The structures of Tris-, DMSP-, and acrylate-bound forms of Fe(III)-DddQ reported here illustrate various states of the enzyme along the reaction pathway. These results provide new insights into DMSP lyase catalysis and have broader significance for understanding the mechanism of oceanic DMS production.

Processes driving seasonal variability in DMS, DMSP, and DMSO concentrations and turnover in coastal Antarctic waters

AbstractThis study presents new measurements of the concentrations and turnover rates of dimethyl sulfide (DMS), dimethylsulfoniopropionate (DMSP), and dimethyl sulfoxide (DMSO) in coastal waters near Palmer Station, Antarctica, during the spring and summer of 2012–2013. Using several novel analytical and experimental techniques, we document variability in DMS, DMSP, and DMSO (DMS/P/O) concentrations and quantify dominant production and removal terms in the mixed layer DMS budget. Our results demonstrate considerable seasonal variability in the concentration of DMS (range 0–20 nM), total DMSP (8–160 nM), and total DMSO (4–160 nM). Over the seasonal cycle, dissolved DMSP concentrations were well correlated with total DMSP concentrations and the abundance of Phaeocystis antarctica, while DMSO concentrations (total and dissolved) were well correlated with DMS concentrations. DMSP cleavage from the dissolved pool (mean rate = 5.5 nM d−1) and release from microzooplankton grazing (mean 5.6 nM d−1) were the dominant sources of DMS, with smaller DMS production rates associated with DMSO reduction from the dissolved pool (mean 2.6 nM d−1) and krill grazing (mean 0.82 nM d−1). Specific rate constants for DMSP cleavage were inversely related to net primary production. Bacterial uptake was a primary contributor to DMS removal (mean −12 nM d−1), and we observed a significant correlation between bacterial production and gross DMS loss rate constants. Estimated sea‐air flux and photo‐oxidation constituted secondary DMS sinks. Our experimental and analytical methods provide insight into the DMS/P/O cycle at Palmer Station, and a starting point for future studies examining inter‐annual DMS/P/O variability in coastal Antarctic waters.

The abundant marine bacterium Pelagibacter simultaneously catabolizes dimethylsulfoniopropionate to the gases dimethyl sulfide and methanethiol.

Marine phytoplankton produce ∼10(9) tonnes of dimethylsulfoniopropionate (DMSP) per year(1,2), an estimated 10% of which is catabolized by bacteria through the DMSP cleavage pathway to the climatically active gas dimethyl sulfide(3,4). SAR11 Alphaproteobacteria (order Pelagibacterales), the most abundant chemo-organotrophic bacteria in the oceans, have been shown to assimilate DMSP into biomass, thereby supplying this cell's unusual requirement for reduced sulfur(5,6). Here, we report that Pelagibacter HTCC1062 produces the gas methanethiol, and that a second DMSP catabolic pathway, mediated by a cupin-like DMSP lyase, DddK, simultaneously shunts as much as 59% of DMSP uptake to dimethyl sulfide production. We propose a model in which the allocation of DMSP between these pathways is kinetically controlled to release increasing amounts of dimethyl sulfide as the supply of DMSP exceeds cellular sulfur demands for biosynthesis.

Effects of iron limitation and UV radiation on Phaeocystis antarctica growth and dimethylsulfoniopropionate, dimethylsulfoxide and acrylate concentrations

Environmental context Low iron concentrations and solar ultraviolet radiation can affect the growth of marine algae. We observed reduced growth and substantial increases in dissolved dimethylsulfoxide and cellular acrylate concentrations in low-iron cultures of a prevalent Southern Ocean algal species, Phaeocystis antarctica, with comparatively small increases observed for cellular dimethylsulfoniopropionate concentrations. Exposure of P. antarctica to high levels of ultraviolet and visible light had very little effect on concentrations of these compounds in culture, even under iron-limitation. Our results highlight the importance of iron to P. antarctica. Abstract Iron is a key nutrient regulating primary production in the Southern Ocean. We investigated the effect of iron limitation with and without exposure to ultraviolet radiation (UVR, 290–400nm) on concentrations of dimethylsulfoniopropionate (DMSP), dimethylsulfoxide (DMSO) and acrylate in axenic batch cultures of Phaeocystis antarctica, a dominant algal species in Antarctic waters. Cellular concentrations of DMSP and acrylate, and cell-number normalised dissolved DMSO concentrations were 1.4-, 11.5- and 6.9-fold higher in iron-limited cultures compared to iron-replete cultures, which we propose resulted from (1) increased reactions of DMSP and dimethylsulfide (DMS) with reactive oxygen species to produce DMSO and (2) increased DMSP cleavage under iron limitation to produce acrylate. Short-term exposure (4h) of iron-limited and iron-replete cultures to a range of photosynthetically active radiation (PAR) and UVR+PAR irradiances did not appreciably affect P. antarctica biomass or total DMSP, DMSO or acrylate concentrations, except at high UVR intensities, suggesting that iron limitation was the primary driver regulating growth and changes in concentrations of these compounds in P. antarctica. High millimolar cellular DMSP and acrylate concentrations under both iron-replete and iron-limited conditions indicated that these two compounds served as de facto antioxidants allowing P. antarctica to thrive under high UVR exposure and low iron concentrations. High dissolved acrylate concentrations indicate significant carbon removal possibly as part of an overflow mechanism during unbalanced growth.

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.

Biochemical, Kinetic, and Spectroscopic Characterization of Ruegeria pomeroyi DddW--A Mononuclear Iron-Dependent DMSP Lyase.

The osmolyte dimethylsulfoniopropionate (DMSP) is a key nutrient in marine environments and its catabolism by bacteria through enzymes known as DMSP lyases generates dimethylsulfide (DMS), a gas of importance in climate regulation, the sulfur cycle, and signaling to higher organisms. Despite the environmental significance of DMSP lyases, little is known about how they function at the mechanistic level. In this study we biochemically characterize DddW, a DMSP lyase from the model roseobacter Ruegeria pomeroyi DSS-3. DddW is a 16.9 kDa enzyme that contains a C-terminal cupin domain and liberates acrylate, a proton, and DMS from the DMSP substrate. Our studies show that as-purified DddW is a metalloenzyme, like the DddQ and DddP DMSP lyases, but contains an iron cofactor. The metal cofactor is essential for DddW DMSP lyase activity since addition of the metal chelator EDTA abolishes its enzymatic activity, as do substitution mutations of key metal-binding residues in the cupin motif (His81, His83, Glu87, and His121). Measurements of metal binding affinity and catalytic activity indicate that Fe(II) is most likely the preferred catalytic metal ion with a nanomolar binding affinity. Stoichiometry studies suggest DddW requires one Fe(II) per monomer. Electronic absorption and electron paramagnetic resonance (EPR) studies show an interaction between NO and Fe(II)-DddW, with NO binding to the EPR silent Fe(II) site giving rise to an EPR active species (g = 4.29, 3.95, 2.00). The change in the rhombicity of the EPR signal is observed in the presence of DMSP, indicating that substrate binds to the iron site without displacing bound NO. This work provides insight into the mechanism of DMSP cleavage catalyzed by DddW.

Molecular insight into bacterial cleavage of oceanic dimethylsulfoniopropionate into dimethyl sulfide

The microbial cleavage of dimethylsulfoniopropionate (DMSP) generates volatile DMS through the action of DMSP lyases and is important in the global sulfur and carbon cycles. When released into the atmosphere from the oceans, DMS is oxidized, forming cloud condensation nuclei that may influence weather and climate. Six different DMSP lyase genes are found in taxonomically diverse microorganisms, and dddQ is among the most abundant in marine metagenomes. Here, we examine the molecular mechanism of DMSP cleavage by the DMSP lyase, DddQ, from Ruegeria lacuscaerulensis ITI_1157. The structures of DddQ bound to an inhibitory molecule 2-(N-morpholino)ethanesulfonic acid and of DddQ inactivated by a Tyr131Ala mutation and bound to DMSP were solved. DddQ adopts a β-barrel fold structure and contains a Zn(2+) ion and six highly conserved hydrophilic residues (Tyr120, His123, His125, Glu129, Tyr131, and His163) in the active site. Mutational and biochemical analyses indicate that these hydrophilic residues are essential to catalysis. In particular, Tyr131 undergoes a conformational change during catalysis, acting as a base to initiate the β-elimination reaction in DMSP lysis. Moreover, structural analyses and molecular dynamics simulations indicate that two loops over the substrate-binding pocket of DddQ can alternate between "open" and "closed" states, serving as a gate for DMSP entry. We also propose a molecular mechanism for DMS production through DMSP cleavage. Our study provides important insight into the mechanism involved in the conversion of DMSP into DMS, which should lead to a better understanding of this globally important biogeochemical reaction.

Metagenomic insights into strategies of carbon conservation and unusual sulfur biogeochemistry in a hypersaline Antarctic lake

Organic Lake is a shallow, marine-derived hypersaline lake in the Vestfold Hills, Antarctica that has the highest reported concentration of dimethylsulfide (DMS) in a natural body of water. To determine the composition and functional potential of the microbial community and learn about the unusual sulfur chemistry in Organic Lake, shotgun metagenomics was performed on size-fractionated samples collected along a depth profile. Eucaryal phytoflagellates were the main photosynthetic organisms. Bacteria were dominated by the globally distributed heterotrophic taxa Marinobacter, Roseovarius and Psychroflexus. The dominance of heterotrophic degradation, coupled with low fixation potential, indicates possible net carbon loss. However, abundant marker genes for aerobic anoxygenic phototrophy, sulfur oxidation, rhodopsins and CO oxidation were also linked to the dominant heterotrophic bacteria, and indicate the use of photo- and lithoheterotrophy as mechanisms for conserving organic carbon. Similarly, a high genetic potential for the recycling of nitrogen compounds likely functions to retain fixed nitrogen in the lake. Dimethylsulfoniopropionate (DMSP) lyase genes were abundant, indicating that DMSP is a significant carbon and energy source. Unlike marine environments, DMSP demethylases were less abundant, indicating that DMSP cleavage is the likely source of high DMS concentration. DMSP cleavage, carbon mixotrophy (photoheterotrophy and lithoheterotrophy) and nitrogen remineralization by dominant Organic Lake bacteria are potentially important adaptations to nutrient constraints. In particular, carbon mixotrophy relieves the extent of carbon oxidation for energy production, allowing more carbon to be used for biosynthetic processes. The study sheds light on how the microbial community has adapted to this unique Antarctic lake environment.

Genomic Insights into Bacterial DMSP Transformations

Genomic and functional genomic methods applied to both model organisms and natural communities have rapidly advanced understanding of bacterial dimethylsulfoniopropionate (DMSP) degradation in the ocean. The genes for the two main pathways in bacterial degradation, routing DMSP to distinctly different biogeochemical fates, have recently been identified. The genes dmdA, -B, -C, and -D mediate the demethylation of DMSP and facilitate retention of carbon and sulfur in the marine microbial food web. The genes dddD, -L, -P, -Q, -W, and -Y mediate the cleavage of DMSP to dimethylsulfide (DMS), with important consequences for ocean-atmosphere sulfur flux. In ocean metagenomes, sufficient copies of these genes are present for approximately 60% of surface ocean bacterial cells to directly participate in DMSP degradation. The factors that regulate these two competing pathways remain elusive, but gene transcription analyses of natural bacterioplankton communities are making headway in unraveling the intricacies of bacterial DMSP processing in the ocean.

A robust approach for the determination of dimethylsulfoxide in sea ice

The melting of sea ice samples is acknowledged to be deleterious to sympagic microorganisms due to the hypo‐osmotic shock undergone by the organism when released from high salinity brine inclusions into the sample melt. Because melting of sea ice samples was also anticipated to modify the initial proportions of dimethylsulfide (DMS), dimethylsulfoniopropionate (DMSP), and dimethylsulfoxide (DMSO), three sample treatments were tested on an Antarctic sea ice sample, with the aim of identifying an efficient procedure that could routinely be applied for the determination of DMSO in sea ice. Herein, it was demonstrated that purging the melted sample before the determination of DMSO in the sample via an enzyme‐linked method produced reliable DMSO results (215.8 ± 8.9 nmol L−1, precision 4.1%). However, analysis revealed that the unintentional enzymatic cleavage of DMSP through the subsequent production of interfering DMS during melting caused an overestimation of the DMSO content in the sample by more than 59% and concurrently an underestimation of the DMSP content by approximately 9%. The sequential determination of DMSP after the DMSO determination by the enzyme‐linked method was shown to be problematic. To circumvent all of these issues, we recommend an analytical procedure for the sequential determination of DMS, DMSP, and DMSO in sea ice. Ultimately, the first depth profile of DMSO at high resolution in sea ice was produced. The depth‐integrated DMSO concentration in sea ice was determined to be 718 µmol m−2, which indicated that Antarctic sea ice is a potentially important source of DMSO for the Southern Ocean.

Bacterial Catabolism of Dimethylsulfoniopropionate (DMSP)

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

Effect of acidification on preservation of DMSP in seawater and phytoplankton cultures: Evidence for rapid loss and cleavage of DMSP in samples containing Phaeocystis sp.

The algal metabolite, dimethylsulfoniopropionate (DMSP), is known to be stable in acid solution and acidification of seawater samples to pH < 2 has been used previously to preserve samples for DMSP analysis. We, however, observed substantial (60–94%) DMSP losses in acidified seawater samples from a colonial Phaeocystis antarctica bloom in the Ross Sea, Antarctica, whereas little or no loss of DMSP occurred in other waters where Phaeocystis spp . were minor components of the phytoplankton community. Tests with cultures of colonial P. globosa showed that up to 68% of culture DMSP was lost during the first minutes after acid addition. After initial losses, DMSP remained constant, confirming the chemical stability of DMSP at pH < 2. Biological factors must therefore play a role in the initial acid-mediated DMSP loss in cultures or seawater. Dimethylsulfide (DMS) was produced immediately after acidification of P. globosa cultures suggesting that DMSP was degraded via lyase enzymes. Major acid-mediated DMSP losses were unique to colonial Phaeocystis spp., as no significant DMSP losses occurred with solitary P. globosa cells or other phytoplankton genera tested, although a small amount of DMS production (equivalent to < 3% of culture DMSP) was observed after acidification of a high DMSP lyase strain of Emiliania huxleyi (CCMP 373). Exogenous dissolved 35S-DMSP was not lost during acidification of colonial P. globosa cultures indicating that intracellular rather than dissolved DMSP was cleaved during the time between acid addition and lyase enzyme inactivation. Experiments with the differentially-permeable DMSP lyase inhibitors, p-CMB and p-CMBS, showed that only the membrane permeable p-CMB stopped DMSP loss under acidified conditions, confirming that DMSP loss occurred intracellularly. These results highlight the remarkable potential activity of DMSP lyases in Phaeocystis spp. and suggest that acidification alone is inadequate for preservation of samples containing colonial Phaeocystis spp.

Nutritional preferences override chemical defenses in determining food choice by a generalist herbivore, Littorina sitkana

In this study, we investigated why a littorinid snail, Littorina sitkana, preferentially consumes the ulvoid green algae Ulva lactuca and Ulva linza, even though these algae contain high concentrations of dimethylsulfoniopropionate (DMSP), which has been shown to be the precursor to an activated defense that deters feeding by sea urchins ( Strongylocentrotus droebachiensis and S. purpuratus). When urchins consume algae containing DMSP, the DMSP is rapidly cleaved into DMS and acrylic acid, which are feeding deterrents to the urchins. In multiple-choice feeding-preference assays, L. sitkana consumed U. lactuca and U. linza at a greater rate than six other algal species. In experiments to confirm that DMSP was being cleaved by the snails, DMS was generated when snails were fed either U. lactuca or U. linza, but was not present in containers with snails alone or algae alone. In laboratory bioassays, acrylic acid reduced feeding by snails when it was incorporated into agar-based diets at concentrations of 0.25% and 0.50% of the food's fresh mass, demonstrating that the animals are deterred by one of the products of DMSP cleavage. When feeding rates from multiple-choice feeding-preference assay were compared to the carbon to nitrogen ratio (C:N) of the macroalgae, there was a strong negative linear relationship between grazing rates and C:N until a C:N of ~ 8 was reached. Algae with a C:N higher than 8 were avoided by snails. When the snails were offered agar-based diets containing a range of nitrogen concentrations (1.96–6.85% of the dry mass), feeding rates were positively correlated with the diets' nitrogen concentrations and negatively correlated with their C:N. Because L. sitkana forages only during low tides, it may have a limited window of time to obtain sufficient nitrogen. Therefore, it may preferentially select foods with high nitrogen concentrations, even though these foods contain chemical defenses.

The effect of nitrogen limitation on cellular DMSP and DMS release in marine phytoplankton: climate feedback implications

The effect of nitrogen limitation on intracellular dimethylsulfoniopropionate (DMSP) and its enzymatic cleavage to dimethylsulfide (DMS) was investigated in semi-continuous cultures of the coccolithophore Emiliania huxleyi and the chain-forming coastal diatom Skeletonema costatum, both ecologically important species. The diatom had a DMSP/C molar ratio of 0.0020 (1% of cell carbon) under nutrient sufficient conditions, and increased its DMSP/C ratio by 70% under nitrogen limitation. The coccolithophore had a constitutively high intracellular DMSP concentration (~200 mmol per liter of cell volume, an estimated 5% of cell carbon) and N-limitation of this species caused no measurable increase in DMSP. Instead, N-limitation of this species increased the activity of the DMSP cleavage enzyme, DMSPlyase, which resulted in a 20-fold increase in the moles of DMS in the culture per unit of cell volume and a 40- to 80-fold increase in the ratio of DMS to chl a. Our results and previously published findings suggest that N-limitation is likely a contributing factor to the extremely high DMS:chl a ratios observed in nutrient limited surface ocean waters under thermally stratified conditions. Thus, N-limitation may play a role in climate feedback mechanisms involving biologically derived DMS.

INTRASPECIFIC VARIATION IN STRESS‐INDUCED HYDROGEN PEROXIDE SCAVENGING BY THE ULVOID MACROALGA ULVA LACTUCA1

We investigated the presence and kinetics of the oxidative stress response in intertidal and subtidal individuals of the ulvoid macroalga Ulva lactuca L. Stress responses, as measured with both enzymatic and fluorescent‐based antioxidant assays, differed between individuals collected from a subtidal and an intertidal habitat. Subtidal individuals secreted significantly more hydrogen peroxide (H2O2) than intertidal individuals when subjected to osmotic stress or desiccation. The activity of reactive‐oxygen‐scavenging enzymes and the ability to scavenge exogenous H2O2 were lower in subtidal than in intertidal individuals, suggesting that subtidal individuals are less stress tolerant. In vitro experimentation demonstrated that millimolar concentrations of dimethylsulfoniopropionate (DMSP) and its breakdown products could efficiently scavenge H2O2, with DMSP being a less‐effective scavenger than dimethyl sulfide (DMS), acrylic acid, and acrylate. The addition of H2O2 at concentrations of 2.5 mM or greater induced the cleavage of DMSP into DMS and acrylic acid in subtidal individuals. Intertidal individuals were affected in the same manner with the addition of 5 mM H2O2. There were no differences in the amounts of DMSP cleavage in subtidal and intertidal algae when the algae were subjected to hyposaline conditions. Our data suggest that the oxidative‐stress‐induced cleavage of DMSP affords products with efficient H2O2‐scavenging abilities. In addition, U. lactuca individuals growing in intertidal habitats are better acclimatized to changing environments and thus have a higher threshold for oxidative stress than conspecifics in subtidal habitats.

The relative significance of viral lysis and microzooplankton grazing as pathways of dimethylsulfoniopropionate (DMSP) cleavage: An Emiliania huxleyi culture study

Dimethylsulfoniopropionate (DMSP) cleavage was investigated during culture studies of grazing by the microzooplankter Oxyrrhis marina and viral lysis by Emiliania huxleyi virus 86 (EhV‐86) on two axenic strains of E. huxleyi. The cleavage products of DMSP, dimethyl sulfide (DMS) and acrylic acid (AA), accumulated during viral infection of both strains, confirming that viral lysis of algae can lead directly to DMSP cleavage. AA and DMS accumulated in parallel with compromised E. huxleyi cells, indicating that DMSP cleavage occurred during the physical disruption of the infected cells. This is in agreement with the hypothesis that DMSP and DMSP lyase ([DL] the enzyme responsible for cleaving DMSP) are segregated in healthy or undamaged cells. During grazing, the concentrations of DMS and AA produced per eaten cell were an order of magnitude higher than the concentrations resulting from cell death caused by viral infection, suggesting that grazing is the quantitatively more significant pathway of DMS production in E. huxleyi. Levels of DL activity decreased in infected cultures to a minimum of 0.00065 fmol cell−1 min−1 as compared with an average of 0.09 fmol cell−1 min−1 in the control cultures, indicating that reduced DL activity in virally infected cells was responsible for the lower levels of DMSP cleavage observed during viral lysis.