Demethylation of Dimethylsulfoniopropionate and Production of Thiols in Anoxic Marine Sediments

Dimethylsulfoniopropionate (DMSP) is a natural product of algae and aquatic plants, particularly those from saline environments. We investigated whether DMSP could serve as a precursor of thiols in anoxic coastal marine sediments. The addition of 10 or 60 μM DMSP to anoxic sediment slurries caused the concentrations of 3-mercaptopropionate (3-MPA) and methanethiol (MSH) to increase. Antibiotics prevented the appearance of these thiols, indicating biological formation. Dimethyl sulfide (DMS) and acrylate also accumulated after the addition of DMSP, but these compounds were rapidly metabolized by microbes and did not reach high levels. Acrylate and DMS were probably generated by the enzymatic cleavage of DMSP. MSH arose from the microbial metabolism of DMS, since the direct addition of DMS greatly increased MSH production. Additions of 3-methiolpropionate gave rise to 3-MPA at rates similar to those with DMSP, suggesting that sequential demethylation of DMSP leads to 3-MPA formation. Only small amounts of MSH were liberated from 3-methiolpropionate, indicating that demethiolation was not a major transformation for 3-methiolpropionate. We conclude that DMSP was degraded in anoxic sediments by two different pathways. One involved the well-known enzymatic cleavage to acrylate and DMS, with DMS subsequently serving as a precursor of MSH. In the other pathway, successive demethylations of the sulfur atom proceeded via 3-methiolpropionate to 3-MPA.     

Rapid Microbial Production of Filamentous Sulfur Mats at Hydrothermal Vents

During recent oceanographic cruises to Pacific hydrothermal vent sites (9°N and the Guaymas Basin), the rapid microbial formation of filamentous sulfur mats by a new chemoautotrophic, hydrogen sulfide-oxidizing bacterium was documented in both in situ and shipboard experiments. Observations suggest that formation of these sulfur mats may be a factor in the initial colonization of hydrothermal surfaces by macrofaunal Alvinella worms. This novel metabolic capability, previously shown to be carried out by a coastal strain in H₂S continuous-flow reactors, may be an important, heretofore unconsidered, source of microbial organic matter production at deep-sea hydrothermal vents.     

Low-Molecular-Weight Sulfonates, a Major Substrate for Sulfate Reducers in Marine Microbial Mats

Several low-molecular-weight sulfonates were added to microbial mat slurries to investigate their effects on sulfate reduction. Instantaneous production of sulfide occurred after taurine and cysteate were added to all of the microbial mats tested. The rates of production in the presence of taurine and cysteate were 35 and 24 μM HS⁻ h⁻¹ in a stromatolite mat, 38 and 36 μM HS⁻ h⁻¹ in a salt pond mat, and 27 and 18 μM HS⁻ h⁻¹ in a salt marsh mat, respectively. The traditionally used substrates lactate and acetate stimulated the rate of sulfide production 3 to 10 times more than taurine and cysteate stimulated the rate of sulfide production in all mats, but when ethanol, glycolate, and glutamate were added to stromatolite mat slurries, the resulting increases were similar to the increases observed with taurine and cysteate. Isethionate, sulfosuccinate, and sulfobenzoate were tested only with the stromatolite mat slurry, and these compounds had much smaller effects on sulfide production. Addition of molybdate resulted in a greater inhibitory effect on acetate and lactate utilization than on sulfonate use, suggesting that different metabolic pathways were involved. In all of the mats tested taurine and cysteate were present in the pore water at nanomolar to micromolar concentrations. An enrichment culture from the stromatolite mat was obtained on cysteate in a medium lacking sulfate and incubated anaerobically. The rate of cysteate consumption by this enrichment culture was 1.6 pmol cell⁻¹ h⁻¹. Compared to the results of slurry studies, this rate suggests that organisms with properties similar to the properties of this enrichment culture are a major constituent of the sulfidogenic population. In addition, taurine was consumed at some of highest dilutions obtained from most-probable-number enrichment cultures obtained from stromatolite samples. Based on our comparison of the sulfide production rates found in various mats, low-molecular-weight sulfonates are important sources of C and S in these ecosystems.     

Formation of 30- to 40-Micrometer-Thick Laminations by High-Speed Marine Bacteria in Microbial Mats

The precision with which motile heterotrophic bacteria could position themselves in microbial mats was determined. This required the development of a technique to view motile bacteria in situ. This was successfully achieved by replacing a 1-cm-diameter minicore from the mat sediment with 210- to 300-(mu)m-diameter glass beads or sieved agar. After allowing 3 days for regrowth of the mat into the transparent medium, a cross section showed that bacteria formed a layer as thin as 30 to 40 (mu)m at a depth of 500 (mu)m below the surface. Bacterial concentrations in this microlamination were 20 times above background. Mean speeds were 200 (mu)m s(sup-1) inside and 60 (mu)m s(sup-1) outside the microlamination. The percentages of bacteria turning per 30 s were 93% inside and 10% outside the microlamination. Artificial chemical gradients were unsuccessful in stimulating microlamination formation or in eliciting the same extent of speed and turning responses. The significance of the results is that it is now possible to microscopically examine sedimentary bacteria in situ. Our first examination indicates that some bacteria form chemotactic microlaminations by increasing their turning frequency. This behavior is opposite that described in the enteric-based model of chemotactic movement, in which positive chemotaxis is achieved by decreasing the turning frequency.     

Microbial Communities and Exopolysaccharides from Polynesian Mats

Microbial mats present in two shallow atolls of French Polynesia were characterized by high amounts of exopolysaccharides associated with cyanobacteria as the predominating species. Cyanobacteria were found in the first centimeters of the gelatinous mats, whereas deeper layers showing the occurrence of the sulfate reducers Desulfovibrio and Desulfobacter species as determined by the presence of specific biomarkers. Exopolysaccharides were extracted from these mats and partially characterized. All fractions contained both neutral sugars and uronic acids with a predominance of the former. The large diversity in monosaccharides can be interpreted as the result of exopolymer biosynthesis by either different or unidentified cyanobacterial species. Received July 25, 2000; accepted October 21, 2000     

Consumption of Methane and CO2 by Methanotrophic Microbial Mats from Gas Seeps of the Anoxic Black Sea

The deep anoxic shelf of the northwestern Black Sea has numerous gas seeps, which are populated by methanotrophic microbial mats in and above the seafloor. Above the seafloor, the mats can form tall reef-like structures composed of porous carbonate and microbial biomass. Here, we investigated the spatial patterns of CH₄ and CO₂ assimilation in relation to the distribution of ANME groups and their associated bacteria in mat samples obtained from the surface of a large reef structure. A combination of different methods, including radiotracer incubation, beta microimaging, secondary ion mass spectrometry, and catalyzed reporter deposition fluorescence in situ hybridization, was applied to sections of mat obtained from the large reef structure to locate hot spots of methanotrophy and to identify the responsible microbial consortia. In addition, CO₂ reduction to methane was investigated in the presence or absence of methane, sulfate, and hydrogen. The mat had an average δ¹³C carbon isotopic signature of −67.1‰, indicating that methane was the main carbon source. Regions dominated by ANME-1 had isotope signatures that were significantly heavier (−66.4‰ ± 3.9 ‰ [mean ± standard deviation; n = 7]) than those of the more central regions dominated by ANME-2 (−72.9‰ ± 2.2 ‰; n = 7). Incorporation of ¹⁴C from radiolabeled CH₄ or CO₂ revealed one hot spot for methanotrophy and CO₂ fixation close to the surface of the mat and a low assimilation efficiency (1 to 2% of methane oxidized). Replicate incubations of the mat with ¹⁴CH₄ or ¹⁴CO₂ revealed that there was interconversion of CH₄ and CO_2. The level of CO₂ reduction was about 10% of the level of anaerobic oxidation of methane. However, since considerable methane formation was observed only in the presence of methane and sulfate, the process appeared to be a rereaction of anaerobic oxidation of methane rather than net methanogenesis.     

Subsurface Microbial Methanotrophic Mats in the Black Sea

A nodule-shaped microbial mat was found subsurface in sediments of a gas seep in the anoxic Black Sea. This mat was dominated by ANME-1 archaea and consumed methane and sulfate simultaneously. We propose that such subsurface mats represent the initial stage of previously investigated microbial reefs.     

Microbial Iron Mats at the Mid-Atlantic Ridge and Evidence that Zetaproteobacteria May Be Restricted to Iron-Oxidizing Marine Systems

Chemolithoautotrophic iron-oxidizing bacteria play an essential role in the global iron cycle. Thus far, the majority of marine iron-oxidizing bacteria have been identified as Zetaproteobacteria, a novel class within the phylum Proteobacteria. Marine iron-oxidizing microbial communities have been found associated with volcanically active seamounts, crustal spreading centers, and coastal waters. However, little is known about the presence and diversity of iron-oxidizing communities at hydrothermal systems along the slow crustal spreading center of the Mid-Atlantic Ridge. From October to November 2012, samples were collected from rust-colored mats at three well-known hydrothermal vent systems on the Mid-Atlantic Ridge (Rainbow, Trans-Atlantic Geotraverse, and Snake Pit) using the ROV Jason II. The goal of these efforts was to determine if iron-oxidizing Zetaproteobacteria were present at sites proximal to black smoker vent fields. Small, diffuse flow venting areas with high iron(II) concentrations and rust-colored microbial mats were observed at all three sites proximal to black smoker chimneys. A novel, syringe-based precision sampler was used to collect discrete microbial iron mat samples at the three sites. The presence of Zetaproteobacteria was confirmed using a combination of 16S rRNA pyrosequencing and single-cell sorting, while light micros-copy revealed a variety of iron-oxyhydroxide structures, indicating that active iron-oxidizing communities exist along the Mid-Atlantic Ridge. Sequencing analysis suggests that these iron mats contain cosmopolitan representatives of Zetaproteobacteria, but also exhibit diversity that may be uncommon at other iron-rich marine sites studied to date. A meta-analysis of publically available data encompassing a variety of aquatic habitats indicates that Zetaproteobacteria are rare if an iron source is not readily available. This work adds to the growing understanding of Zetaproteobacteria ecology and suggests that this organism is likely locally restricted to iron-rich marine environments but may exhibit wide-scale geographic distribution, further underscoring the importance of Zetaproteobacteria in global iron cycling.     

Intact Phospholipid and Quinone Biomarkers to Assess Microbial Diversity and Redox State in Microbial Mats

Microbial mats are stratified microbial communities composed by highly inter-related populations and therefore are frequently chosen as model systems to study diversity and ecophysiological strategies. The present study describes an integrated approach to analyze microbial quinones and intact polar lipids (IPLs) in microbial mats within layers as thin as 500 μm by liquid chromatography–tandem mass spectrometry. Quinone profiles revealed important depth-related differences in community composition in two mat systems. The higher abundance of ubiquinones, compared to menaquinones, reflected the clear predominance of microorganisms belonging to aerobic α-, β-, and γ-Proteobacteria in Ebro delta estuarine mats. Hypersaline photosynthetic Camargue mats (France) showed a predominance of menaquinone-9 at the top of the mat, which is consistent with an important contribution of facultative aerobic or anaerobic bacteria in its photic zone. Quinone indices also indicated a higher diversity of non-phototrophs and a more anaerobic character in the hypersaline mats. Besides, the dissimilarity index suggested that the samples were greatly influenced by a depth-related redox state gradient. In the analysis of IPLs, there was a predominance of phosphatidylglycerols and sulfoquinovosyldiacylglycerols, the latter being an abundant biomarker of Cyanobacteria. This combined approach based on quinone and IPL analysis has proven to be a useful method to establish differences in the microbial diversity and redox state of highly structure microbial mat systems at a fine-scale level.     

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.     

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.     

Morphological Survey of Microbial Mats Near Deep-Sea Thermal Vents

A microscopic survey is presented of the most commonly observed and morphologically conspicuous microorganisms found attached to natural surfaces or to artificial materials deposited in the immediate vicinity of thermal submarine vents at the Galapagos Rift ocean spreading zone at a depth of 2,550 meters. Of special interest were the following findings: (i) all surfaces intermittently exposed to H₂S-containing hydrothermal fluid were covered by layers, ca. 5 to 10 μm thick, of procaryotic, gram-negative cells interspaced with amorphous metal (Mn-Fe) deposits; (ii) although some of the cells were encased by dense metal deposits, there was little apparent correlation between metal deposition and the occurrence of microbial mats, (iii) highly differentiated forms appeared to be analogues of certain cyanobacteria, (iv) isolates from massive mats of a prosthecate bacterium could be identified as Hyphomicrobium spp., (v) intracellular membrane systems similar to those found in methylotrophic and nitrifying bacteria were observed in approximately 20% of the cells composing the mats, (vi) thiosulfate enrichments made from mat material resulted in isolations of different types of sulfur-oxidizing bacteria including the obligately chemolithotrophic genus Thiomicrospira.     

Dimethyl Sulfide Production from Dimethylsulfoniopropionate in Coastal Seawater Samples and Bacterial Cultures

Dimethyl sulfide (DMS) was produced immediately after the addition of 0.1 to 2 μM β-dimethylsulfonio-propionate (DMSP) to coastal seawater samples. Azide had little effect on the initial rate of DMS production from 0.5 μM added DMSP, but decreased the rate of production after 6 h. Filtration of water samples through membrane filters (pore size, 0.2 μm) greatly reduced DMS production for approximately 10 h, after which time DMS production resumed at a high rate. Autoclaving completely eliminated the production of DMS. The antibiotics chloramphenicol, tetracycline, kanamycin, and vancomycin all had little effect on the accumulation of DMS over the first few hours of incubation, but produced significant inhibition thereafter. The effects of individual antibiotics were additive. Chloroform over a range of concentrations (0.25 to 1.25 mM) had no effects on DMS production. Similarly, organic amendments, including acrylate, glucose, protein, and starch, did not affect DMS accumulation from DMSP. Acrylate, a product of the enzymatic cleavage of DMSP, was metabolized in seawater samples, and two strains of bacteria were isolated with this compound as the growth substrate. These bacteria produced DMS from DMSP. The sensitivity to inhibitors with respect to growth and DMSP-lyase activity varied from strain to strain. These results illustrate the significant potential for microbial conversion of dissolved DMSP to DMS in coastal seawater.     

Dinitrogen-Fixing Cyanobacteria in Microbial Mats of Two Shallow Coral Reef Ecosystems

Dinitrogen-fixing organisms in cyanobacterial mats were studied in two shallow coral reef ecosystems: La Reunion Island, southwestern Indian Ocean, Sesoko (Okinawa) Island, and northwestern Pacific Ocean. Rapidly expanding benthic miniblooms, frequently dominated by a single cyanobacterial taxon, were identified by microscopy and molecular tools. In addition, nitrogenase activity by these blooms was measured in situ. Dinitrogen fixation and its contribution to mat primary production were calculated using ¹⁵N₂ and ¹³C methods. Dinitrogen-fixing cyanobacteria from mats in La Reunion and Sesoko showed few differences in taxonomic composition. Anabaena sp. among heterocystous and Hydrocoleum majus and Symploca hydnoides among nonheterocystous cyanobacteria occurred in microbial mats of both sites. Oscillatoria bonnemaisonii and Leptolyngbya spp. occurred only in La Reunion, whereas Hydrocoleum coccineum dominated in Sesoko. Other mats dominated by Hydrocoleum lyngbyaceum, Phormidium laysanense, and Trichocoleus tenerrimus occurred at lower frequencies. The 24-h nitrogenase activity, as measured by acetylene reduction, varied between 11 and 324 nmoles C₂H₂ reduced μg⁻¹ Chl a. The highest values were achieved by heterocystous Anabaena sp. performed mostly during the day. Highest values for nonheterocystous cyanobacteria were achieved by H. coccineum mostly during the night. Daily nitrogen fixation varied from nine (Leptolyngbya) to 238 nmoles N₂ μg⁻¹ Chl day⁻¹ (H. coccineum). Primary production rates ranged from 1,321 (S. hydnoides) to 9,933 nmoles C μg⁻¹ Chl day⁻¹ (H. coccineum). Dinitrogen fixation satisfied between 5% and 21% of the nitrogen required for primary production.     

Viruses Occur Incorporated in Biogenic High-Mg Calcite from Hypersaline Microbial Mats

Using three different microscopy techniques (epifluorescence, electronic and atomic force microscopy), we showed that high-Mg calcite grains in calcifying microbial mats from the hypersaline lake “La Salada de Chiprana”, Spain, contain viruses with a diameter of 50–80 nm. Energy-dispersive X-ray spectrometer analysis revealed that they contain nitrogen and phosphorus in a molar ratio of ~9, which is typical for viruses. Nucleic acid staining revealed that they contain DNA or RNA. As characteristic for hypersaline environments, the concentrations of free and attached viruses were high (>10¹⁰ viruses per g of mat). In addition, we showed that acid treatment (dissolution of calcite) resulted in release of viruses into suspension and estimated that there were ~15 × 10⁹ viruses per g of calcite. We suggest that virus-mineral interactions are one of the possible ways for the formation of nano-sized structures often described as “nanobacteria” and that viruses may play a role in initiating calcification.     

Quantifying Microbial Diversity: Morphotypes, 16S rRNA Genes, and Carotenoids of Oxygenic Phototrophs in Microbial Mats

We quantified the diversity of oxygenic phototrophic microorganisms present in eight hypersaline microbial mats on the basis of three cultivation-independent approaches. Morphological diversity was studied by microscopy. The diversity of carotenoids was examined by extraction from mat samples and high-pressure liquid chromatography analysis. The diversity of 16S rRNA genes from oxygenic phototrophic microorganisms was investigated by extraction of total DNA from mat samples, amplification of 16S rRNA gene segments from cyanobacteria and plastids of eukaryotic algae by phylum-specific PCR, and sequence-dependent separation of amplification products by denaturing-gradient gel electrophoresis. A numerical approach was introduced to correct for crowding the results of chromatographic and electrophoretic analyses. Diversity estimates typically varied up to twofold among mats. The congruence of richness estimates and Shannon-Weaver indices based on numbers and proportional abundances of unique morphotypes, 16S rRNA genes, and carotenoids unveiled the underlying diversity of oxygenic phototrophic microorganisms in the eight mat communities studied.     

Diversity and Distribution in Hypersaline Microbial Mats of Bacteria Related to Chloroflexus spp.

Filamentous bacteria containing bacteriochlorophylls c and a were enriched from hypersaline microbial mats. Based on phylogenetic analyses of 16S rRNA gene sequences, these organisms form a previously undescribed lineage distantly related to Chloroflexus spp. We developed and tested a set of PCR primers for the specific amplification of 16S rRNA genes from filamentous phototrophic bacteria within the kingdom of “green nonsulfur bacteria.” PCR products recovered from microbial mats in a saltern in Guerrero Negro, Mexico, were subjected to cloning or denaturing gradient gel electrophoresis and then sequenced. We found evidence of a high diversity of bacteria related to Chloroflexus which exhibit different distributions along a gradient of salinity from 5.5 to 16%.     

Characterization of Exopolysaccharides Produced by Cyanobacteria Isolated from Polynesian Microbial Mats

Six cyanobacterial isolates recovered from Polynesian microbial mats, called “kopara,” were cultured using laboratory-closed photobioreactors and were shown to produce exopolymers as released and capsular exopolysaccharides (EPS). These polymers have been chemically characterized using colorimetric and elemental assays, infrared spectrometry, and gas chromatography. Both capsular and released EPS consisted of 7 to 10 different monosaccharides with neutral sugars predominating. Interestingly, four isolates exhibited sulfate contents ranging from 6% to 19%. On the basis of preliminary data, cyanobacteria from this unusual ecosystem appear to be an important source of novel EPS of a great interest in terms of their biological activities.