Principles of calcium signaling. Salisbury Cove, Maine, June 28-30, 2007

A three-day International Symposium entitled "Principles of Calcium Signaling" organized by James N. Weiss, Yale E. Goldman, Stéphane Hatem, Lars Cleemann and Nikolai M. Soldatov in honor of the research contributions of Professor Martin Morad was held at the Mount Desert Island Biological Laboratory, Salisbury Cove, Maine. Support for this meeting was provided in part by GlaxoSmithKline, Leica Microsystems, Nikon Corp., St. Jude Medical, Inc., UCLA Cardiac Arrhythmia Center, Dr. Donald S. Orkand, Bob Hillis Family and OML, and Mount Desert Island Biological Laboratory. The symposium featured sessions on Cardiac physiology, Ion channels and Calcium signaling.     

The Fate of Nitrate in Intertidal Permeable Sediments

Coastal zones act as a sink for riverine and atmospheric nitrogen inputs and thereby buffer the open ocean from the effects of anthropogenic activity. Recently, microbial activity in sandy permeable sediments has been identified as a dominant source of N-loss in coastal zones, namely through denitrification. Some of the highest coastal denitrification rates measured so far occur within the intertidal permeable sediments of the eutrophied Wadden Sea. Still, denitrification alone can often account for only half of the substantial nitrate (NO₃ ⁻) consumption. Therefore, to investigate alternative NO₃ ⁻ sinks such as dissimilatory nitrate reduction to ammonium (DNRA), intracellular nitrate storage by eukaryotes and isotope equilibration effects we carried out ¹⁵NO₃ ⁻ amendment experiments. By considering all of these sinks in combination, we could quantify the fate of the ¹⁵NO₃ ⁻ added to the sediment. Denitrification was the dominant nitrate sink (50–75%), while DNRA, which recycles N to the environment accounted for 10–20% of NO₃ ⁻ consumption. Intriguingly, we also observed that between 20 and 40% of ¹⁵NO₃ ⁻ added to the incubations entered an intracellular pool of NO₃ ⁻ and was subsequently respired when nitrate became limiting. Eukaryotes were responsible for a large proportion of intracellular nitrate storage, and it could be shown through inhibition experiments that at least a third of the stored nitrate was subsequently also respired by eukaryotes. The environmental significance of the intracellular nitrate pool was confirmed by in situ measurements which revealed that intracellular storage can accumulate nitrate at concentrations six fold higher than the surrounding porewater. This intracellular pool is so far not considered when modeling N-loss from intertidal permeable sediments; however it can act as a reservoir for nitrate during low tide. Consequently, nitrate respiration supported by intracellular nitrate storage can add an additional 20% to previous nitrate reduction estimates in intertidal sediments, further increasing their contribution to N-loss.     

Characterization of beta-Glucosidase Activity in Intertidal Marine Sediments

Glycoside derivatives of 4-methylumbelliferone (MUF) were used to characterize the polysaccharidase enzyme systems present in sediments from an intertidal mud flat. The formation of highly fluorescent MUF on hydrolysis of the various glycosides was determined at low substrate concentrations (<1 muM) and with short incubation periods (>5 min). The hydrolysis of MUF-beta-d-glucose in sediments from depth intervals of 0 to 2 cm was insensitive to the presence of oxygen, dissolved sulfide, and iron; magnesium and calcium were stimulatory, however. A pronounced temperature optimum was observed at 40 degrees C, a salinity optimum at 30 per thousand, and a pH optimum at 8.5. Rates of hydrolysis were completely inhibited by the addition of mercuric chloride and sodium azide, but only partially inhibited by toluene and the specific beta-glucosidase inhibitor delta-1,5-gluconolactone. The response to delta-1,5-gluconolactone suggested that about 50% of the observed hydrolysis of MUF-beta-d-glucoside was due to exo- and endoglucanases. A wide variety of hydrolytic activities was observed, with at least some nonspecificity occurring in the case of MUF-beta-d-fucoside. Depth profiles indicated maximal activity in surface sediments with a rapid decline below 2 cm. MUF-glycosides provided a convenlent tool for initial analyses of the dynamics and controls of polymer hydrolysis in marine sediments.     

Biotic Disturbance,Recolonization, and Early Succession of Bacterial Assemblages in Intertidal Sediments

The role of disturbance in structuring natural microbial communities has been largely unexplored. Disturbance associated with invertebrate ingestion can reduce bacterial biomass and alter metabolic activities and compositions of bacterial assemblages in marine sediments. The primary objectives of the research presented here were to test whether ingestion by a taxonomically diverse group of deposit feeders constituted a disturbance, and to determine the mechanisms by which bacterial assemblages recover following deposit-feeder ingestion. To test the question of disturbance, we compared fresh egesta vs surficial sediments with respect to bacterial assemblage structure. In emersed intertidal sediments, microbial recovery could be due to regrowth of bacterial populations surviving gut passage or to immigration from adjacent sediments. To differentiate between these modes of recolonization we used field manipulative experiments to exclude migration by isolating freshly extruded fecal coils of three deposit-feeding species from surrounding sediments. We then followed the quantitative and qualitative recovery in egesta and sediments through time using epifluorescence microscopy and PCR-DGGE analysis of 16S rDNA. Our findings indicate that (1) the degree and nature of the disturbance to bacterial assemblages from deposit feeding varies among invertebrate taxa, (2) recovery was significant but incomplete over 3 h, and (3) recolonization of biotically disturbed sediments is dominated by immigration.This revised version was published online in November 2004 with corrections to Volume 48.     

Microphytobenthos,their Extracellular Polymeric Substances,and the Morphogenesis of Intertidal Sediments

Intertidal sediments are important areas that separate the land from the sea and form natural coastal defenses. They are known as highly productive ecosystems, fueling the coastal food web. It is also conceived that microphytobenthos contribute to the stability of intertidal sediments by increasing the erosion threshold and that they are major players in coastal morphodynamics. Depending on the sedimentary composition of intertidal flats, different types of microphytobenthos colonize the sediment surface. Fine sand sediment is often colonized by cyanobacteria, prokaryotic algae, which form dense and rigid microbial mats. Mudflats on the other hand are characterized by the development of thin biofilms of epipelic diatoms. Both groups of phototrophic microorganisms excrete extracellular polymeric substances (EPS), but they do so in different ways and for different reasons. Two operationally defined fractions, water- and EDTA-extractable EPS, have been obtained from intertidal diatom biofilms and from cultures. They differ in composition and their production seems to be under different metabolic control. Water-extractable EPS are considered to be closely associated with the diatoms and are rich in neutral sugars, notably glucose. These EPS show a dynamic relationship with the microphytobenthic biomass. EDTA-extractable EPS are tightly bound to the sediment, probably through bridging by divalent ions. This material is rich in uronic acids and other acid sugars and is weakly related to chlorophyll. These EPS have been conceived to be a major factor in the structuring and diagenesis of coastal sediments and essential for increasing the sediment erosion threshold. However, this relationship is now questioned.     

Spatial and Temporal Variation of Phenanthrene-Degrading Bacteria in Intertidal Sediments

Phenanthrene-degrading bacteria were isolated from a 1-m² intertidal sediment site in Boston Harbor. Samples were taken six times over 2 years. A total of 432 bacteria were isolated and characterized by biochemical testing. When clustered on the basis of phenotypic characteristics, the isolates could be separated into 68 groups at a similarity level of approximately 70%. Several groups (a total of 200 isolates) corresponded to well-characterized species belonging the genera Vibrio and Pseudomonas. Only 51 of the 437 isolates (<11.7% of the total) hybridized to a DNA probe that encodes the upper pathway of naphthalene and phenanthrene degradation in Pseudomonas putida NCIB 9816. A cluster analysis indicated that the species composition of the phenanthrene-degrading community changed significantly from sampling date to sampling date. At one sampling time, 12 6-mm-diameter core subsamples were taken within the 1-m² site to determine the spatial variability of the degrading communities. An analysis of molecular variance, performed with the phenotypic characteristics, indicated that only 6% of the variation occurred among the 12 subsamples, suggesting that the subsamples were almost identical in composition. We concluded that the communities of phenanthrene-degrading bacteria in the sediments are very diverse, that the community structure undergoes significant change with time but does not vary significantly on a spatial scale of centimeters, and that the predominant genes that encode phenanthrene degradation in the communities are not well-characterized.     

Substrates for Sulfate Reduction and Methane Production in Intertidal Sediments

The activity of and potential substrates for methane-producing bacteria and sulfate-reducing bacteria were examined in marsh, estuary, and beach intertidal sediments. Slow rates of methane production were detected in all sediments, although rates of sulfate reduction were 100- to 1,000-fold higher. After sulfate was depleted in sediments, the rates of methane production sharply increased. The addition of methylamine stimulated methanogenesis in the presence of sulfate, and [¹⁴C]methylamine was rapidly converted to ¹⁴CH₄ and ¹⁴CO₂ in freshly collected marsh sediment. Acetate, hydrogen, or methionine additions did not stimulate methanogenesis. [methyl-¹⁴C]methionine and [2-¹⁴C]acetate were converted to ¹⁴CO₂ and not to ¹⁴CH₄ in fresh sediment. No reduction of ¹⁴CO₂ to ¹⁴CH₄ occurred in fresh sediment. Molybdate, an inhibitor of sulfate reduction, inhibited [2-¹⁴C]acetate metabolism by 98.5%. Fluoracetate, an inhibitor of acetate metabolism, inhibited sulfate reduction by 61%. These results suggest that acetate is a major electron donor for sulfate reduction in marine sediments. In the presence of high concentrations of sulfate, methane may be derived from novel substrates such as methylamine.     

The Diverse Bacterial Community in Intertidal, Anaerobic Sediments at Sapelo Island, Georgia

The phylogenetic diversity and composition of the bacterial community in anaerobic sediments from Sapelo Island, GA, USA were examined using 16S rRNA gene libraries. The diversity of this community was comparable to that of soil, and 1,186 clones formed 817 OTUs at 99% sequence similarity. Chao1 estimators for the total richness were also high, at 3,290 OTUs at 99% sequence similarity. The program RDPquery was developed to assign clones to taxonomic groups based upon comparisons to the RDP database. While most clones could be assigned to describe phyla, fewer than 30% of the clones could be assigned to a described order. Similarly, nearly 25% of the clones were only distantly related (<90% sequence similarity) to other environmental clones, illustrating the unique composition of this community. One quarter of the clones were related to one or more undescribed orders within the γ-Proteobacteria. Other abundant groups included the δ-Proteobacteria, Bacteroidetes, and Cyanobacteria. While these phyla were abundant in other estuarine sediments, the specific members at Sapelo Island appeared to be different from those previously described in other locations, suggesting that great diversity exists between as well as within estuarine intertidal sediments. In spite of the large differences in pore water chemistry with season and depth, differences in the bacterial community were modest over the temporal and spatial scales examined and generally restricted to only certain taxa. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Calorimetric Studies of Behavior, Metabolism and Energetics of Sessile Intertidal Animals

SYNOPSIS: Behaviors to conserve water during intertidal exposureat the same time impair respiratory gas exchange, so that observedresponses to emersion may reflect compromises between theseincompatible needs. Behavioral isolation of the tissues fromair results in the complete or partial reliance on anoxic energymetabolism, which is most reliably measured directly as heatdissipation. Combined direct calorimetry and indirect calorimetry(respirometry) enable the partitioning of total metabolic heatdissipation into its aerobic and anoxic components, which mayvary according to physical and biological factors. The musselMytilus edulis is tolerant of anoxia and saves water and energyduring aerial exposure in its rocky intertidal habitat by closingits shell valves and becoming largely anoxic. Like most suspensionfeeders in this habitat, its compensation for reduced feedingtime involves energy conservation; there is little evidencefor energy supplementation such as increases in feeding rateor absorption efficiency. Ammonia production continues duringaerial exposure and is involved in acid-base balance in thehemolymph and mantle cavity fluid. Infaunal cockles (Cardiumedule) and mussels (Geukensia demissa) gape their shell valves,remain largely aerobic and have high rates of heat dissipationduring intertidal exposure, a response which appears relatedto the lower desiccation potential and exploitation of richertrophic resources in their soft-sediment habitats. The variableexpansion of the symbiotic sea anemone Anthopleura elegantissimareflects interaction among the responses to desiccation, irradianceand continued photosynthesis by its zooxanthellae during exposureto air.     

Quantitative Importance,Composition, and Seasonal Dynamics of Protozoan Communities in Polyhaline versus Freshwater Intertidal Sediments

The quantitative importance and composition of protozoan communities was investigated in sandy and silty intertidal sediments of a polyhaline and a freshwater site in the Schelde estuary. Total biomass of the protozoans studied, integrated over the upper 4 cm of the sediment, ranged from 41 to 597 mg C m^–2 and was in the same order of magnitude at the polyhaline and the freshwater intertidal site. Nanoheterotrophs were the dominant protozoans, in terms of both abundance and biomass. Ciliate abundances appeared to be largely determined by physical constraints, namely, the amount of interstitial space and hydrodynamic disturbances. It remains unclear which factors control nanoheterotrophic abundances and biomasses, which showed comparatively little seasonal and between-site fluctuations. Salinity differences were clearly reflected in the protozoan community composition. The dominant role of sessile ciliates is a unique feature of sediments in the freshwater tidal reaches, which can be attributed to the dynamic nature of sedimentation and resuspension processes associated with the maximum turbidity zone. Based on biomass ratios and estimated weight-specific metabolic rates, protozoa possibly accounted for ~29 to 96% of the estimated combined metabolic rate of protozoan and metazoan consumers at our sampling stations in late spring/early autumn. The contribution of protozoa to this combined metabolic rate was higher at the sandy than at the silty stations and was mainly accounted for by the nanoheterotrophs. These data emphasize the potential importance of small protozoa in sediments and suggest that protozoa are important components of benthic food webs.     

Sulfate-Dependent Interspecies H2 Transfer between Methanosarcina barkeri and Desulfovibrio vulgaris during Coculture Metabolism of Acetate or Methanol

We compared the metabolism of methanol and acetate when Methanosarcina barkeri was grown in the presence and absence of Desulfovibrio vulgaris. The sulfate reducer was not able to utilize methanol or acetate as the electron donor for energy metabolism in pure culture, but was able to grow in coculture. Pure cultures of M. barkeri produced up to 10 μmol of H₂ per liter in the culture headspace during growth on acetate or methanol. In coculture with D. vulgaris, the gaseous H₂ concentration was ≤2 μmol/liter. The fractions of ¹⁴CO₂ produced from [¹⁴C]methanol and 2-[¹⁴C]acetate increased from 0.26 and 0.16, respectively, in pure culture to 0.59 and 0.33, respectively, in coculture. Under these conditions, approximately 42% of the available electron equivalents derived from methanol or acetate were transferred and were utilized by D. vulgaris to reduce approximately 33 μmol of sulfate per 100 μmol of substrate consumed. As a direct consequence, methane formation in cocultures was two-thirds that observed in pure cultures. The addition of 5.0 mM sodium molybdate or exogenous H₂ decreased the effects of D. vulgaris on the metabolism of M. barkeri. An analysis of growth and carbon and electron flow patterns demonstrated that sulfate-dependent interspecies H₂ transfer from M. barkeri to D. vulgaris resulted in less methane production, increased CO₂ formation, and sulfide formation from substrates not directly utilized by the sulfate reducer as electron donors for energy metabolism and growth.     

Interactions between Benthic Copepods,Bacteria and Diatoms Promote Nitrogen Retention in Intertidal Marine Sediments

The present study aims at evaluating the impact of diatoms and copepods on microbial processes mediating nitrate removal in fine-grained intertidal sediments. More specifically, we studied the interactions between copepods, diatoms and bacteria in relation to their effects on nitrate reduction and denitrification. Microcosms containing defaunated marine sediments were subjected to different treatments: an excess of nitrate, copepods, diatoms (Navicula sp.), a combination of copepods and diatoms, and spent medium from copepods. The microcosms were incubated for seven and a half days, after which nutrient concentrations and denitrification potential were measured. Ammonium concentrations were highest in the treatments with copepods or their spent medium, whilst denitrification potential was lowest in these treatments, suggesting that copepods enhance dissimilatory nitrate reduction to ammonium over denitrification. We hypothesize that this is an indirect effect, by providing extra carbon for the bacterial community through the copepods'' excretion products, thus changing the C/N ratio in favour of dissimilatory nitrate reduction. Diatoms alone had no effect on the nitrogen fluxes, but they did enhance the effect of copepods, possibly by influencing the quantity and quality of the copepods'' excretion products. Our results show that small-scale biological interactions between bacteria, copepods and diatoms can have an important impact on denitrification and hence sediment nitrogen fluxes.     

Carbohydrate Metabolism in Leukocytes VII. Metabolism of Glucose, Acetate, and Propionate by Human Plasma Cells

Plasma cells obtained from the peripheral blood of a patient with multiple myeloma was incubated in serum and Krebs-Ringer bicarbonate buffer with (14)C-labeled glucose, acetate, and propionate. Glucose utilization by these cells amounted to 0.5 mumole per hr per 10(8) cells and was mainly via the Embden-Meyerhof pathway, and only 6% or less traversed the hexose monophosphate shunt. The presence of Krebs cycle activity was demonstrated by direct isolation of several labeled intermediates after incubation with either (14)C-acetate or (14)C-propionate. The distribution of (14)C in lactate, succinate, fumarate, malate, aspartate, and glutamate indicate a complete Krebs cycle. Acetate was metabolized via the Krebs cycle to the extent of 0.15 mumoles per hr per 10(8) cells, and the rate of propionate utilization was 0.17 mumoles per hr per 10(8) cells.     

Chemoautotrophic Carbon Fixation Rates and Active Bacterial Communities in Intertidal Marine Sediments

Chemoautotrophy has been little studied in typical coastal marine sediments, but may be an important component of carbon recycling as intense anaerobic mineralization processes in these sediments lead to accumulation of high amounts of reduced compounds, such as sulfides and ammonium. We studied chemoautotrophy by measuring dark-fixation of ¹³C-bicarbonate into phospholipid derived fatty acid (PLFA) biomarkers at two coastal sediment sites with contrasting sulfur chemistry in the Eastern Scheldt estuary, the Netherlands. At one site where free sulfide accumulated in the pore water right to the top of the sediment, PLFA labeling was restricted to compounds typically found in sulfur and ammonium oxidizing bacteria. At the other site, with no detectable free sulfide in the pore water, a very different PLFA labeling pattern was found with high amounts of label in branched i- and a-PLFA besides the typical compounds for sulfur and ammonium oxidizing bacteria. This suggests that other types of chemoautotrophic bacteria were also active, most likely Deltaproteobacteria related to sulfate reducers. Maximum rates of chemoautotrophy were detected in first 1 to 2 centimeters of both sediments and chemosynthetic biomass production was high ranging from 3 to 36 mmol C m⁻² d⁻¹. Average dark carbon fixation to sediment oxygen uptake ratios were 0.22±0.07 mol C (mol O₂)⁻¹, which is in the range of the maximum growth yields reported for sulfur oxidizing bacteria indicating highly efficient growth. Chemoautotrophic biomass production was similar to carbon mineralization rates in the top of the free sulfide site, suggesting that chemoautotrophic bacteria could play a crucial role in the microbial food web and labeling in eukaryotic poly-unsaturated PLFA was indeed detectable. Our study shows that dark carbon fixation by chemoautotrophic bacteria is a major process in the carbon cycle of coastal sediments, and should therefore receive more attention in future studies on sediment biogeochemistry and microbial ecology.     

Carbon and Electron Flow in Mud and Sandflat Intertidal Sediments at Delaware Inlet, Nelson, New Zealand

An investigation of carbon and electron flow in mud and sandflat intertidal sediments showed that the terminal electron acceptor was principally sulfate and that the carbon flow was mainly to CO₂. Studies with thin layers of sediment exposed to H₂ showed that methane production accounted for virtually none of the H₂ utilized, whereas sulfate reduction accounted for a major proportion of the gas uptake. At all sampling sites except one (site B7), rates of methanogenesis were low but sulfate concentrations in the interstitial water were high (>18 mM). At site B7, the sulfate concentrations declined with depth from 32 mM at 2 cm to <1 mM at 10 cm or below, and active methanogenesis occurred in the low-sulfate zone. Sulfate-reducing activity at this site initially decreased and then increased with depth so that elevated rates occurred in both the active and nonactive methanogenic zones. The respiratory index (RI) [RI = ¹⁴CO₂/(¹⁴CO₂ + ¹⁴CH₄)] for [2-¹⁴C]acetate catabolism at site B7 ranged from 0.98 to 0.2 in the depth range of 2 to 14 cm. Addition of sulfate to sediment from the low-sulfate zone resulted in an increase in RI and a decrease in methanogenesis. At all other sites examined, RI ranged from 0.97 to 0.99 and was constant with depth. The results suggested that although methanogenesis was inhibited by sulfate (presumably through the activity of sulfate-reducing bacteria), it was not always limited by sulfate reduction.     

Competition for Sulfate and Ethanol Among Desulfobacter, Desulfobulbus, and Desulfovibrio Species Isolated from Intertidal Sediments

Competition for sulfate and ethanol among Desulfobacter, Desulfobulbus, and Desulfovibrio species isolated from estuarine sediments was studied in energy-limited chemostats. Desulfovibrio baculatus was the most successful competitor for limiting amounts of sulfate and ethanol, followed by Desulfobulbus propionicus. The success of Desulfovibrio baculatus was dependent on the availability of sufficient iron. Of the three species studied, Desulfobacter postgatei was the least successful competitor for limiting amounts of sulfate. Although stimulating the growth of Desulfobacter postgatei, addition of Ca-saturated illite particles to culture media did not affect the outcome of competition for sulfate. Thus, under sulfate limitation acetate accumulated. This phenomenon was briefly discussed in relation to the flow of electrons during anaerobic mineralization in marine and estuarine sulfate-limited sediments.