Effects of nutrient enrichment on surface microbial community gene expression in the oligotrophic North Pacific Subtropical Gyre.

Marine microbial communities are critical for biogeochemical cycles and the productivity of ocean ecosystems. Primary productivity in the surface ocean is constrained by nutrients which are supplied, in part, by mixing with deeper water. Little is known about the time scales, frequency, or impact of mixing on microbial communities. We combined in situ sampling using the Environmental Sample Processor and a small-scale mixing experiment with lower euphotic zone water to determine how individual populations respond to mixing. Transcriptional responses were measured using the MicroTOOLs (Microbiological Targets for Ocean Observing Laboratories) microarray, which targets all three domains of life and viruses. The experiment showed that mixing substantially affects photosynthetic taxa as expected, but surprisingly also showed that populations respond differently to unfiltered deep water which contains particles (organisms and detritus) compared to filtered deep water that only contains nutrients and viruses, pointing to the impact of biological interactions associated with these events. Comparison between experimental and in situ population transcription patterns indicated that manipulated populations can serve as analogs for natural populations, and that natural populations may be frequently or continuously responding to nutrients from deeper waters. Finally, this study also shows that the microarray approach, which is complementary to metatranscriptomic sequencing, is useful for determining the physiological status of in situ microbial communities.

Enigmatic relationship between chlorophyll a concentrations and photosynthetic rates at Station ALOHA.

An ordinary least squares (OLS) analysis of the relationship between chlorophyll a (chl a) concentrations and photosynthetic rates at depths of 5 and 25 m at Station ALOHA produced a slope that was only 28% of the mean productivity index at those depths and an intercept at zero chl a that equaled 70% of the mean photosynthetic rate. OLS regression lines are known to produce a slope and intercept that are biased estimates of the true slope and intercept when the explanatory variable, X, is uncontrolled, but in this case the measurement errors and natural variability of the chl a concentrations were much too small to explain the apparent bias. The bias was traceable to the fact that the photosynthetic rates were determined by more than one explanatory variable, a source of variability that is typically overlooked in discussions of OLS bias. Modeling the photosynthetic rates as a function of the product of chl a and surface irradiance produced a much more accurate and realistic description of the data, but the OLS continued to be biased, presumably because the photosynthetic rates were functions of factors in addition to chl a and surface irradiance (e.g., temperature, macronutrients, trace metals, and vitamins). The results underscore the need to recognize that the absence of bias in an OLS when X is not controlled implies that all scatter in the data about the OLS is due to errors in the dependent variable, an unlikely scenario. In most cases, resolution of the bias problem will require identification of the explanatory variables in addition to X that determine the dependent variable.

Clinical predictors of risk of hypoglycaemia during addition and titration of insulin glargine for type 2 diabetes mellitus.

AIM: Addition and titration of basal insulin is usually effective in improving glycaemic control in type 2 diabetes, but fear of hypoglycaemia remains a barrier. Ability to predict which patients might be at greatest risk of hypoglycaemia might facilitate individualization of treatment and improve safety. The aim of this study was to obtain information about clinical characteristics which might predict risk of hypoglycaemia during initiation of basal insulin. METHODS: Patient-level data from 2251 participants in 11 studies in which insulin glargine was started and titrated using similar treat-to-target methods was pooled and analysed with logistic regression models. RESULTS: Participants had mean age 58 years, diabetes duration 8.9 years, body mass index 31.0 and baseline A1c 8.8%. They attained mean A1c 7.1% during 6 months of treatment with final mean glargine dosage 0.44 units/kg. Symptomatic hypoglycaemia occurred in 52%, glucose-confirmed hypoglycaemia (blood glucose <50 mg/dl) in 17%, repeated glucose-confirmed events in 7% and severe hypoglycaemia in 1.5%. Independent predictors of glucose-confirmed hypoglycaemia were younger age, lower body mass index, use of a sulphonylurea in addition to metformin, lower attained A1c and lower dosage of glargine. CONCLUSIONS: These findings confirm low rates of clinically important hypoglycaemia using this method, and suggest that higher risk of hypoglycaemia may be suspected when patients needing insulin are younger, less obese and taking metformin and a sulphonylurea, and especially when A1c levels ≤7.0% are attained with glargine dosage ≤0.4 units/kg.

A novel method for the measurement of dissolved adenosine and guanosine triphosphate in aquatic habitats: applications to marine microbial ecology.

A novel method for the measurement of dissolved adenosine-5'-triphosphate and guanosine-5'-triphosphate (D-ATP and D-GTP, respectively) in marine and freshwater habitats was developed and applied to samples collected from the oligotrophic North Pacific Ocean. Both D-ATP and D-GTP are co-precipitated by authigenically formed Mg(OH)(2) and can be concentrated by factors greater than 200-fold, for subsequent measurement by the firefly luciferin-luciferase bioluminescence reaction. The detection limit for this method was 2-3 pmol ml(-1) of concentrated sample (equivalent to an in situ concentration of 10 pM) with a 5% precision at concentrations of 10 pmol ml(-1) or above.A significant positive correlation (P<0.001) was observed between particulate ATP (P-ATP) and D-ATP in water samples collected from Station ALOHA (22.75 degrees N, 158 degrees W; depth profiles 0-1000 m). The highest concentrations of dissolved nucleotides were found in the euphotic zone (0-175 m) below which the concentrations were low and relatively invariant. The dissolved nucleotide pools generally exceeded their corresponding particulate pools.Using radioisotopic tracer techniques and the new concentration method, turnover times for both particulate and dissolved nucleotides can be determined. The ability to measure concentrations and follow nucleotide tracers accurately in a very dilute environment provides a unique opportunity to address questions on microbial community metabolism, nutrient dynamics and energy flux.

Unicellular cyanobacteria fix N2 in the subtropical North Pacific Ocean.

Fixed nitrogen (N) often limits the growth of organisms in terrestrial and aquatic biomes, and N availability has been important in controlling the CO2 balance of modern and ancient oceans. The fixation of atmospheric dinitrogen gas (N2) to ammonia is catalysed by nitrogenase and provides a fixed N for N-limited environments. The filamentous cyanobacterium Trichodesmium has been assumed to be the predominant oceanic N2-fixing microorganism since the discovery of N2 fixation in Trichodesmium in 1961 (ref. 6). Attention has recently focused on oceanic N2 fixation because nitrogen availability is generally limiting in many oceans, and attempts to constrain the global atmosphere-ocean fluxes of CO2 are based on basin-scale N balances. Biogeochemical studies and models have suggested that total N2-fixation rates may be substantially greater than previously believed but cannot be reconciled with observed Trichodesmium abundances. It is curious that there are so few known N2-fixing microorganisms in oligotrophic oceans when it is clearly ecologically advantageous. Here we show that there are unicellular cyanobacteria in the open ocean that are expressing nitrogenase, and are abundant enough to potentially have a significant role in N dynamics.

Archaeal dominance in the mesopelagic zone of the Pacific Ocean.

The ocean's interior is Earth's largest biome. Recently, cultivation-independent ribosomal RNA gene surveys have indicated a potential importance for archaea in the subsurface ocean. But quantitative data on the abundance of specific microbial groups in the deep sea are lacking. Here we report a year-long study of the abundance of two specific archaeal groups (pelagic euryarchaeota and pelagic crenarchaeota) in one of the ocean's largest habitats. Monthly sampling was conducted throughout the water column (surface to 4,750 m) at the Hawai'i Ocean Time-series station. Below the euphotic zone (> 150 m), pelagic crenarchaeota comprised a large fraction of total marine picoplankton, equivalent in cell numbers to bacteria at depths greater than 1,000 m. The fraction of crenarchaeota increased with depth, reaching 39% of total DNA-containing picoplankton detected. The average sum of archaea plus bacteria detected by rRNA-targeted fluorescent probes ranged from 63 to 90% of total cell numbers at all depths throughout our survey. The high proportion of cells containing significant amounts of rRNA suggests that most pelagic deep-sea microorganisms are metabolically active. Furthermore, our results suggest that the global oceans harbour approximately 1.3 x 10(28) archaeal cells, and 3.1 x 10(28) bacterial cells. Our data suggest that pelagic crenarchaeota represent one of the ocean's single most abundant cell types.

Phosphate depletion in the western North Atlantic Ocean.

Surface waters of the subtropical Sargasso Sea contain dissolved inorganic phosphate (DIP) concentrations of 0.2 to 1.0 nanomolar, which are sufficiently low to result in phosphorus control of primary production. The DIP concentrations in this area (which receives high inputs of iron-rich dust from arid regions of North Africa) are one to two orders of magnitude lower than surface levels in the North Pacific (where eolian iron inputs are much lower and water column denitrification is much more substantial). These data indicate a severe relative phosphorus depletion in the Atlantic. We hypothesize that nitrogen versus phosphorus limitation of primary production in the present-day ocean may be closely linked to iron supply through control of dinitrogen (N2) fixation, an iron-intensive metabolic process. Although the oceanic phosphorus inventory may set the upper limit for the total amount of organic matter produced in the ocean over geological time scales, at any instant in geological time, oceanic primary production may fall below this limit because of a persistent insufficient iron supply. By controlling N2 fixation, iron may control not only nitrogen versus phosphorus limitation but also carbon fixation and export stoichiometry and hence biological sequestration of atmospheric carbon dioxide.

Microorganisms in the accreted ice of Lake Vostok, Antarctica.

Analysis of a portion of Vostok ice core number 5G, which is thought to contain frozen water derived from Lake Vostok, Antarctica (a body of liquid water located beneath about 4 kilometers of glacial ice), revealed between 2 x 10(2) and 3 x 10(2) bacterial cells per milliliter and low concentrations of potential growth nutrients. Lipopolysaccharide (a Gram-negative bacterial cell biomarker) was also detected at concentrations consistent with the cell enumeration data, which suggests a predominance of Gram-negative bacteria. At least a portion of the microbial assemblage was viable, as determined by the respiration of carbon-14-labeled acetate and glucose substrates during incubations at 3 degrees C and 1 atmosphere. These accreted ice data suggest that Lake Vostok may contain viable microorganisms.

A computer-simulated restriction fragment length polymorphism analysis of bacterial small-subunit rRNA genes: efficacy of selected tetrameric restriction enzymes for studies of microbial diversity in nature.

An assessment of 10 tetrameric restriction enzymes (TREs) was conducted by using a computer-simulated restriction fragment length polymorphism (RFLP) analysis for over 100 proximally and distally related bacterial small-subunit (SSU) rRNA gene sequences. Screening SSU rDNA clone libraries with TREs has become an effective strategy because of logistic simplicity, commercial availability, and economy. However, the rationale for selecting the type and number of TREs has not been systematically evaluated. Our objective was to identify the optimal combination of TREs for RFLP screening of cloned SSU rRNA genes from undefined bacterial clone libraries. After computer-simulated TRE digestion, the resultant fragments were categorized on the basis of the frequency of different restriction fragment size classes. Three groups of distribution patterns for the TREs were determined and further examined via graphical exploratory data analysis. The RFLP size-frequency distribution data for each group of enzymes were then used to infer phylogenetic relationships via the neighbor-joining method. The resulting bootstrap values and the correct placement of node bifurcations were used as additional criteria to evaluate the efficacy of the selected TREs. These RFLP data were compared with known phylogenetic relationships based on SSU rRNA sequence analysis as defined by the Ribosomal Database Project. A heuristic approach testing random combinations of TREs showed that three or more TRE combinations detected > 99% of the operational taxonomic units (OTUs) within the model data set. OTUs that remained undetected after three TRE treatments had a median sequence similarity of 96.1%. Of the 10 restriction enzymes examined, HhaI, RsaI, and BstUI (group 3) were the most efficacious at detecting and differentiating bacterial SSU rRNA genes on the basis of their ability to correctly classify OTUs. Group 3 TREs are therefore recommended for screening in studies using bacterial SSU rRNA genes as descriptors of in situ microbial diversity.

The National Association of Marine Laboratories: a connected web for studying long-term changes in U.S. coastal and marine waters.

Long-term time-series measurements provide data that test specific hypotheses or suggest new avenues of study. Such studies are widely acknowledged as important for differentiating the influence of human activities from natural background variability. Several long-term research or monitoring programs are active in coastal and marine environments around the world and serve as models for development of new studies. The spatial array of U.S. coastal laboratories is suitable for resolving latitudinal trends and for many types of comparative studies. However, establishing a network of coastal laboratories focused on long-term monitoring and research problems presents special challenges in setting research priorities at appropriate scales, in data management, and in coordination of the scientific effort. The National Association of Marine Laboratories (NAML) is uniquely positioned to promote long-term studies among networks of its member institutions. The NAML can play an effective role in publicizing the importance of long-term studies, in providing access to expertise in this type of research, and in promoting its continuance for periods longer than the length of individual scientific careers.

Phylogenetic diversity of the bacterial community from a microbial mat at an active, hydrothermal vent system, Loihi Seamount, Hawaii.

The phylogenetic diversity of small-subunit rRNA genes associated with the domain Bacteria was examined (by using previously defined operational taxonomic units [C. L. Moyer, F.C. Dobbs, and D. M. Karl, Appl. Environ. Microbiol. 60:871-879, 1994]; those for Pele's Vents Bacteria are hereafter abbreviated PVB OTUs) with samples from a microbial mat at an active, deep-sea hydrothermal vent system. A cluster of phylogenetically related PVB OTUs (OTUs 2, 3, 6, and 8) was closely affiliated with Thiovulum sp. contained within the epsilon subclass of the class Proteobacteria and accounted for 60.5% of the small-subunit rRNA bacterial clone library from Pele's Vents. A second, smaller cluster of PVB OTUs (OTUs 1 and 11) was closely affiliated with Xanthomonas sp., contained within the gamma subclass of the Proteobacteria and accounted for a total of 27.1% of the bacterial clone library. The remaining five PVB OTUs each accounted for 2.1% of the clones recovered and were affiliated with the following phylogenetic groups: PVB OTU 5 was a member of the Alteromonas group; PVB OTU 12 was a member of the Colwellia assemblage; PVB OTU 4 was loosely determined to be a member of the Thiothrix group, with the endosymbiotic bacteria from Bathymodiolus thermophilus and Calyptogena magnifica as the nearest relatives; PVB OTU 10B was a member of the Myxobacterium group; and PVB OTU 9A was a member of the Paraphyletic assemblage, with the Octopus Spring microbial mat type K clone as the closest known relative.(ABSTRACT TRUNCATED AT 250 WORDS)

Estimation of diversity and community structure through restriction fragment length polymorphism distribution analysis of bacterial 16S rRNA genes from a microbial mat at an active, hydrothermal vent system, Loihi Seamount, Hawaii.

PCR was used to amplify (eu)bacterial small-subunit (16S) rRNA genes from total-community genomic DNA. The source of total-community genomic DNA used for this culture-independent analysis was the microbial mats from a deep-sea, hydrothermal vent system, Pele's Vents, located at Loihi Seamount, Hawaii. Oligonucleotides complementary to conserved regions in the 16S rRNA-encoding DNA (rDNA) of bacteria were used to direct the synthesis of PCR products, which were then subcloned by blunt-end ligation into phagemid vector pBluescript II. Restriction fragment length polymorphism patterns, created by using tandem tetrameric restriction endonucleases, revealed the presence of 12 groups of 16S rRNA genes representing discrete operational taxonomic units (OTUs). The rank order abundance of these putative OTUs was measured, and the two most abundant OTUs accounted for 72.9% of all of the 16S rDNA clones. Among the remaining 27.1% of the 16S rDNA clones, none of the 10 OTUs was represented by more than three individual clones. The cumulative OTU distribution for 48 bacterial 16S rDNA clones demonstrated that the majority of taxa represented in the clone library were detected, a result which we assume to be an estimate of the diversity of bacteria in the native hydrothermal vent habitat. 16S rDNA fingerprinting of individual clones belonging to particular OTUs by using an oligonucleotide probe that binds to a universally conserved region of the 16S rDNA fragments was conducted to confirm OTU specificity and 16S rDNA identity.

Accurate estimation of microbial loop processes and rates.

Our conceptual models of microbial food webs in aquatic ecosystems provide a unifying hypothesis for the design and conduct of field studies. Our ability to provide a rigorous test of these hypotheses, however, relies to a large extent on the availability of precise and accurate methods. Although considerable progress has been made over the past two decades, unambiguous resolution of in situ microbial rates and processes will probably require improved or novel methodologies.

Quantification of chemiluminescent DNA probes using liquid scintillation counting.

A novel method for quantifying chemiluminescent DNA probes is described. The method uses liquid scintillation counting to measure light emission from the alkaline phosphatase-catalyzed breakdown of the substrate PPD (3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo[3.3.1.1 (3.7[decan]- 4-yl)phenyl phosphate) on dot blot preparations. Serial dilutions of either pUC18 DNA or lambda DNA were hybridized with digoxigenin-labeled probes and detected using the method described. Light flux (luminescence) was linearly related to DNA concentration, typically with a coefficient of determination (r2) of 0.9 or better. Due to the stability of alkaline phosphatase and the long-lived luminescence of PPD in the Lumi-phos formulation, repetitive analyses of a given sample can be made for up to 20 h. The method can reliably detect 17 amol of DNA (30 pg pUC18DNA) with a coefficient of variation on replicate samples of 14%. Optimization experiments showed that 7% sodium dodecyl sulfate in the prehybridization and hybridization buffers resulted in the lowest background; the best combination of signal-to-noise ratio and reproducibility was obtained using Bio-Rad Zeta-Probe GT nylon membranes. Direct immersion of samples into a solution of substrate was found to give the most precise results and ensured that substrate limitation at high concentrations of alkaline phosphatase (i.e., higher DNA amounts) did not occur.

The grounding of the Bahia Paraiso: Microbial ecology of the 1989 Antarctic oil spill.

On 28 January 1989 the Bahia Paraiso ran aground and sank near Palmer Station, Antarctica. At least 6.8 × 10(5) liters of diesel fuel arctic (DFA) were released into semi-enclosed Arthur Harbor and deposited in the nearby intertidal regions. Approximately 6 weeks later, a group of scientists was deployed to evaluate the impact of the oil spill on the surrounding coastal marine ecosystem.Microbial hydrocarbon oxidation potential ((14)CO2 evolved from (14)C-labeled hexadecane) was detected throughout both the oil-impacted and control regions. Hexadecane was mineralized at extremely low rates (0.13-1.21 pmol g(-1) sediment dry weight day(-1)); microbiological turnover time exceeded 2 years. The acute effects of DFA (measured over exposure periods of 3-7 days) on the metabolic activities of sedimentary microorganisms appear to be negligible even at seawater saturation concentrations of DFA. Long-term exposure (120 days) to varying concentrations of DFA resulted in significant decreases (>90%) in total ATP, but had either no effect or a slight stimulatory effect on metabolic activity and production. In contrast to planktonic microbial communities, increasing incubation temperatures of between 0 and 30°C had a positive effect on rates of metabolism and production of sedimentary assemblages. These results may influence the overall weathering rates of hydrocarbons deposited in the intertidal and supratidal regions of Arthur Harbor and other polar regions.

Top predators in the Southern ocean: a major leak in the biological carbon pump.

Primary productivity in the Southern Ocean is approximately 3.5 gigatons of carbon per year, which accounts for nearly 15 percent of the global total. The presence of high concentrations of nitrate in Antarctic waters suggests that it might be possible to increase primary production significantly and thereby alleviate the net accumulation of atmospheric carbon dioxide. An analysis of the food web for these waters implies that the Southern Ocean may be remarkably inefficient as a carbon sink. This inefficiency is caused by the large flux of carbon respired to the atmosphere by air-breathing birds and mammals, dominant predators in the unusually simple food web of Antarctic waters. These top predators may transfer into the atmosphere as much as 20 to 25 percent of photosynthetically fixed carbon.