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In the microbe-driven ecosystems of the open ocean, the small heterotrophic flagellates (sHF) are the chief microbial predators and recyclers of essential nutrients to phototrophic microbes. Even with intensive molecular phylogenetic studies of the sHF, the origins of their feeding success remain obscure because of limited understanding of their morphological adaptations to feeding. Here, we examined the sHF morphologies in the largest, most oligotrophic South Pacific and Atlantic (sub)tropical gyres and adjacent mesotrophic waters. On four research cruises, the sHF cells were flow cytometrically sorted from bacterioplankton and phytoplankton for electron microscopy. The sorted sHF comprised chiefly heterokont (HK) biflagellates and unikont choanoflagellates numerically at around 10-to-1 ratio. Of the four differentiated morphological types of HK omnipresent in the open ocean, the short-tinsel heterokont (stHK), whose tinsel flagellum is too short to propagate a complete wave, is predominant and a likely candidate to be the most abundant predator on Earth. Modeling shows that the described stHK propulsion is effective in feeding on bacterioplankton cells at low concentrations; however, owing to general prey scarcity in the oligotrophic ocean, selective feeding is unsustainable and omnivory is equally obligatory for the seven examined sHF types irrespective of their mode of propulsion. [ABSTRACT FROM AUTHOR]

Ubiquitous SAR11 Alphaproteobacteria numerically dominate marine planktonic communities. Because they are excruciatingly difficult to cultivate, there is comparatively little known about their physiology and metabolic responses to long- and short-term environmental changes. As surface oceans take up anthropogenic, atmospheric CO2, the consequential process of ocean acidification could affect the global biogeochemical significance of SAR11. Shipping accidents or inadvertent release of chemicals from industrial plants can have strong short-term local effects on oceanic SAR11. This study investigated the effect of 2.5-fold acidification of seawater on the metabolism of SAR11 and other heterotrophic bacterioplankton along a natural temperature gradient crossing the North Atlantic Ocean, Norwegian and Greenland Seas. Uptake rates of the amino acid leucine by SAR11 cells as well as other bacterioplankton remained similar to controls despite an instant ∼50% increase in leucine bioavailability upon acidification. This high physiological resilience to acidification even without acclimation, suggests that open ocean dominant bacterioplankton are able to cope even with sudden and therefore more likely with long-term acidification effects. [ABSTRACT FROM AUTHOR]

The dinoflagellate Neoceratium is commonly observed in oceanic waters, depleted in major inorganic nutrients such as nitrogen and phosphorus. Using culture isolates, we investigated whether two Neoceratium species (N. hexacanthum and N. candelabrum) can grow phototrophically at low nutrient concentrations found in surface waters of oligotrophic subtropical gyres (OSGs). No phototrophic growth (indicated by changes in cell numbers, the presence of dividing cells or cellular protein increase) was observed when N. hexacanthum and N. candelabrum were grown in low nutrient seawater. In separate experiments, to determine survival time under oligotrophic nutrient conditions, 68% of N. hexacanthum cells were able to re-establish growth after spending 1–10 days in North Atlantic gyre seawater; 40% recovered after 11–20 days and only 3% recovered after 21–30 days. The longest period any single cell survived, and then went on to divide, was 26 days. These findings demonstrate that Neoceratium cells could remain viable for >3 weeks in surface waters of OSGs, but to sustain their growth nutrients must be obtained periodically from an alternative source: via ephemeral upwelling of nutrient-rich waters, phagotrophy and/or movement to and from the nutricline. [ABSTRACT FROM PUBLISHER]

Flow cytometric sorting, based on cellular optical properties and macromolecule content, has been successfully employed to taxonomically affiliate bacterioplankton. However, this approach has not been much used for eukaryotic plankton. To redress this imbalance, we identified a conspicuous group of red autofluorescent picoplankton in surface waters of the South Atlantic Ocean. Using catalysed reporter deposition fluorescence in situ hybridization, virtually, all cells sorted from that group were affiliated with the Mamiellales clade II (84 ± 4%, division Chlorophyta) with a size of 1.6 ± 0.03 μm. Based on electron microscopy, the Mamiellales clade II-sorted cells have a simple morphology with apparently no scales, flagella or surface features. Their latitudinal distribution resembled the distribution of Synechococcus with very low concentrations in the surface waters of the Southern subtropical gyre (0.6-1.6 × 103 cells mL−1) and increased concentrations in the Southern temperate waters 8.3 × 103 cells mL−1. Identification of the flow cytometric group as Mamiellales clade II allowed us to characterize the morphology of these enigmatic uncultured picoplanktonic cells by electron microscopy and to determine their apparent preference for temperate rather than subtropical oceanic photic waters. [ABSTRACT FROM AUTHOR]

The smallest phototrophic protists (<3 μm) are important primary producers in oligotrophic subtropical gyres - the Earth's largest ecosystems. In order to elucidate how these protists meet their inorganic nutrient requirements, we compared the phosphate uptake rates of plastidic and aplastidic protists in the phosphate-depleted subtropical and tropical North Atlantic (4-29°N) using a combination of radiotracers and flow cytometric sorting on two Atlantic Meridional Transect cruises. Plastidic protists were divided into two groups according to their size (<2 and 2-3 μm). Both groups of plastidic protists showed higher phosphate uptake rates per cell than the aplastidic protists. Although the phosphate uptake rates of protist cells were on average seven times ( P<0.001) higher than those of bacterioplankton, the biomass-specific phosphate uptake rates of protists were one fourth to one twentieth of an average bacterioplankton cell. The unsustainably low biomass-specific phosphate uptake by both plastidic and aplastidic protists suggests the existence of a common alternative means of phosphorus acquisition - predation on phosphorus-rich bacterioplankton cells. [ABSTRACT FROM AUTHOR]

A major obstacle in the molecular investigation of natural, especially oceanic, microbial cells is their adequate preservation for further land-based molecular analyses. Here, we examined the use of microwaves for cell fixation before high-speed flow cytometric sorting to define the metaproteomes and metagenomes of key microbial populations. The microwave fixation procedure was established using cultures of Synechococcus cyanobacteria, the photosynthetic eukaryote Micromonas pusilla and the gammaproteobacterium Halomonas variabilis. Shotgun proteomic analyses showed that the profile of microwave-fixed and -unfixed Synechococcus sp. WH8102 cells was the same, and hence proteome identification of microwave-fixed sorted cells by nanoLC-MS/MS is possible. Microwave-fixed flow-sorted Synechococcus cells can also be successfully used for whole-genome amplification and fosmid library construction. We then carried out successful metaproteomic and metagenomic analyses of microwave-fixed Synechococcus cells flow sorted from concentrates of microbial cells, collected in the North Atlantic Ocean. Thus, the microwave fixation procedure developed appears to be useful for molecular studies of microbial populations in aquatic ecosystems. [ABSTRACT FROM AUTHOR]

The metabolic responses of indigenous dominant bacterioplankton populations to additions of dust were examined in the tropical northeast Atlantic. Subsurface seawater samples were treated with dust, added directly or indirectly as a ‘leachate’ after its rapid dissolution in deionized water. Samples were incubated at ambient temperature and light for up to 24 h and microbial metabolic responses were assessed by 35S-methionine (35S-Met) uptake. Prochlorococcus and low nucleic acid (LNA) cells were sorted by flow cytometry to determine their group-specific responses. Sorted cells were also phylogenetically affiliated using FISH. The high-light-adapted ecotype II dominated the Prochlorococcus group and 73±14% of LNA prokaryotes belonged to the SAR11 clade of Alphaproteobacteria. Both Prochlorococcus and LNA cells were metabolically impaired by the addition of dust (40±28% and 37±22% decrease in 35S-Met uptake compared with controls, respectively). However, LNA bacterioplankton showed minor positive responses to dust leachate additions (7±4% increase in 35S-Met uptake), while the metabolic activity of Prochlorococcus cells decreased in the presence of dust leachate by 16±11%. Thus, dust dissolution in situ appears to be more deleterious to Prochlorococcus than SAR11-dominated LNA bacterioplankton and hence could initiate a compositional shift in the indigenous bacterioplankton. [ABSTRACT FROM AUTHOR]

Biologically available concentrations of individual dissolved amino acids in the open ocean are generally <1 nM. Despite this, the microbial turnover of amino acids is usually measured in hours indicating high demand. It is thought that the majority of uptake is due to bacterioplankton, although protists, particularly phototrophic protists, are also expected to take up amino acids. In order to assess the ability of protists to compete with prokaryotes for amino acids at subnanomolar concentrations, we examined the direct uptake of 3H-leucine by phototrophic nanoflagellates (prasinophytes, pelagophytes and trebouxiophytes) and by associated bacteria using flow cytometric cell sorting. In contrast to 3H-leucine-assimilating bacterial copopulations, none of the six studied nanoflagellates showed measurable direct uptake of 3H-leucine, suggesting that the studied phototrophic protists were unable to utilize dissolved 3H-leucine at natural oceanic concentrations. More practically, the flow-sorting technique allowed rapid and unequivocal differentiation of organic nitrogen uptake between prokaryotic cells and eukaryotic cells in mixed microbial populations, reducing the need to establish and maintain axenic algal cultures. [ABSTRACT FROM AUTHOR]

Despite considerable advances in the understanding of the various microbial photoheterotrophic mechanisms, the role of solar radiation in the metabolism of bacterioplankton in the ocean is difficult to assess. It is already apparent that rates of CO2 fixation by prokaryotic cells may be only a part of the picture. Photophosphorylation is difficult to differentiate from respiratory phosphorylation and other types of ATP synthesis. Solar energy could by-pass ATP synthesis, instead being used to generate a proton-motive force, which in turn could be directly used for cell motility or even for importing molecules into cells. In addition, photoheterotrophic prokaryotes could actively regulate intake and use of solar energy for different metabolic functions depending on the energetic demands of the cell. The factors listed above hence require consideration when solar energy input into metabolism of oceanic photoheterotrophic prokaryotes is experimentally quantified and numerically modelled. [ABSTRACT FROM PUBLISHER]

Hydrophobic surfactants at the air–sea interface can retard evaporative and gaseous exchange between the atmosphere and the ocean. While numerous studies have examined the metabolic role of bacterioneuston at the air–sea interface, the interactions between hydrophobic surfactants and bacterioplankton are not well constrained. A novel experimental design was developed, using Vibrio natriegens and 3H-labelled hexadecanoic acid tracer, to determine how the bacterial metabolism of fatty acids affects evaporative fluxes. In abiotic systems, >92% of the added hexadecanoic acid remained at the air–water interface. In contrast, the presence of V. natriegens cells draws down insoluble hexadecanoic acid from the air–water interface as an exponential function of time. The exponents characterizing the removal of hexadecanoic acid from the interface co-vary with the concentration of V. natriegens cells in the underlying water, with the largest exponent corresponding to the highest cell abundance. Radiochemical budgets show that evaporative fluxes from the system are linearly proportional to the quantity of hexadecanoic acid at the interface. Thus, bacterioplankton could influence the rate of evaporation and gas transfer in the ocean through the metabolism of otherwise insoluble surfactants. [ABSTRACT FROM AUTHOR]

35 S-Methionine and 3H-leucine bioassay tracer experiments were conducted on two meridional transatlantic cruises to assess whether dominant planktonic microorganisms use visible sunlight to enhance uptake of these organic molecules at ambient concentrations. The two numerically dominant groups of oceanic bacterioplankton were Prochlorococcus cyanobacteria and bacteria with low nucleic acid (LNA) content, comprising 60% SAR11-related cells. The results of flow cytometric sorting of labelled bacterioplankton cells showed that when incubated in the light, Prochlorococcus and LNA bacteria increased their uptake of amino acids on average by 50% and 23%, respectively, compared with those incubated in the dark. Amino acid uptake of Synechococcus cyanobacteria was also enhanced by visible light, but bacteria with high nucleic acid content showed no light stimulation. Additionally, differential uptake of the two amino acids by the Prochlorococcus and LNA cells was observed. The populations of these two types of cells on average completely accounted for the determined 22% light enhancement of amino acid uptake by the total bacterioplankton community, suggesting a plausible way of harnessing light energy for selectively transporting scarce nutrients that could explain the numerical dominance of these groups in situ. [ABSTRACT FROM AUTHOR]

Carbohydrate–protein interactions appear to play an important role in the phagocytosis of microbial prey by free-living protozoa. The present study utilizes FITC-labelled plant lectins to investigate the presence and localization of cell surface glycoconjugates on live and fixed planktonic protists ( Dunaliella primolecta, Oxyrrhis marina, Goniomonas amphinema, Paraphysomonas vestita and Euplotes vannus). With live flagellate preparations, lectins primarily bound to external cell surfaces, with minimal internal staining observed. In contrast, cell fixation permeabilized cell membranes, allowing lectins to bind to internal structures, such as nuclear membranes and food vacuoles, interfering with the characterization of cell surface glycoconjugates. The method developed to label cell surface sugar moieties of live planktonic protists successfully overcomes the problems associated with fixation, and thus provides a useful protocol for future studies on protistan cell surface carbohydrate characterization. [ABSTRACT FROM AUTHOR]

Abstract: A flow cytometric sorting technique for direct determination of bacterial biomass assimilation by phagotrophic flagellates was developed and tested in laboratory culture experiments. Living Vibrio bacteria were quantitatively pulse-chase labelled with [35S]methionine tracer and fed to Pteridomonas flagellates. Flow sorting revealed that the isotopically labelled material is in either bacterial prey or flagellate predators and the egested bacterial debris contained negligible amounts of tracer. These experimental results confirm an earlier hypothesis that flagellates release metabolised bacterial proteins primarily in a dissolved form. The assimilation efficiency of the Vibrio protein biomass by Pteridomonas was low, only about 20%, independently of the amount of consumed bacterial biomass, confirming our earlier indirect estimates. Additionally, against expectations that cells decrease their metabolic activity whilst preparing for and engaged in division, we found that the precursor uptake rates by flow sorted bacterial cells at the S+G2 cell cycle stages were constantly 1.5 times higher than those of cells at the G1 stage. [Copyright &y& Elsevier]

Abstract: Ambient concentrations and turnover rates of two amino acids, leucine and methionine, by total bacterioplankton and Prochlorococcus cyanobacteria were determined along a latitudinal transect across the Southern Atlantic gyre using a combined isotopic dilution and flow cytometric sorting technique. The ambient concentrations of methionine (0.2–0.65 nM) were about 2 times higher than the concentrations of leucine, while the turnover rates of the two amino acids were remarkably similar (0.1–0.7 nMd−1). The concentrations of both amino acids did not vary significantly with depth between 3 and 150 m but their turnover rates were 1.5–2 times higher in the top 3–80 m. Prochlorococcus took up amino acids in situ at high rates. Using a representative 35S-methionine precursor, about 25% of total bacterioplankton consumption of amino acids could be assigned to Prochlorococcus with low red fluorescence (Pro LRF) inhabiting the surface mixed layer down to 80 m and about 50% assigned to Prochlorococcus with high red fluorescence (Pro HRF) living below 100 m. In the same deep waters the cellular amino acid uptake of Pro LRF was less than 6% of that of the Pro HRF, indicating declining metabolic activity of the former. The mean cellular uptake rate of Pro HRF at depths below 120 m was 2.5 amolcell−1d−1, 4 times higher than the rates of Pro LRF in the top 80 m. The difference could be partially explained by Pro HRF cellular biomass being twice that of Pro LRF. The biomass specific rates of Prochlorococcus were comparable or higher (particular of the Pro HRF) than that of other bacterioplankton. The reported findings could explain ecological success of mixotrophic Prochlorococcus cyanobacteria over both strictly autotrophic algae and heterotrophic bacteria in oligotrophic regions sustained by nutrient remineralisation. [Copyright &y& Elsevier]

Samples were collected from the top 200 m of the water column at 50 stations during two cruises in different, near equinoctial seasons on an Atlantic transect near the 20°W meridian between 50°N and 50°S. These samples were analysed to determine characteristics of the heterotrophic bacterial populations. Flow cytometry was used to enumerate these bacteria and determine their average size so as to calculate their biomass. Heterotrophic bacterial production, and the rate of grazing of these bacteria by heterotrophic nanoplankton in the main depth layers, were determined using 3H thymidine and 14C leucine techniques. The biomass of heterotrophic nanoplankton in these layers was determined using a glucosaminidase assay. Five provinces were distinguished along the transect and characterized by average values of all measured parameters. The relative composition and activity of the microbial community in the water columns within each province changed little between the two cruises. Lowest heterotrophic bacterial biomass of 1–2 mg C m–3 and production of 0.1–0.2 mg C m–3 day–1 were found in the northern and southern Atlantic gyres, and were relatively similar in both seasons. Biomass and production were 2–4 times higher in the northern and southern temperate waters, and in equatorial waters, than in the gyres and tended to show more seasonal variation. Production and biomass in the layer below the pycnocline were lower by 10–30% and about 50%, respectively, than values determined in the surface mixed layer, and varied less with latitude. Depth-integrated values of these two parameters were generally of similar size in the mixed water layer and the layer of the chlorophyll maximum and pycnocline, and tended to vary with season. The specific growth rate of heterotrophic bacteria was in the range 0.05 to 0.12 day–1 in the top mixed layer at all latitudes. In spite of the elevated temperatures, bacterial growth appears to be restricted by a shortage of nutrients so that the microbial community cycles very slowly, with a turnover time of the order of 1 week or more. The depth-integrated biomass of heterotrophic nanoplankton was generally about 100% of the heterotrophic bacterial biomass in the same water. Grazing by these nanoplankton at the rate measured could consume all of the new production of heterotrophic bacteria in all waters, and they probably control the populations of both heterotrophic and phototrophic bacteria. [ABSTRACT FROM PUBLISHER]

A dual radioactive-labelled bacteria technique using Vibrio (DRLV), developed for laboratory studies on bacterivory, has been refined for use at the concentrations of prey and predators typcially found at sea. Experiments with estuarine water collected in spring and in autumn showed that bacterivorous nanoflagellates (HNF) (concentration 1.38±0.35×103 HNF ml−1) ingested 2.7±0.96 DRLV flagellate1−1 h−1 at concentrations of 0.8–2.2×106 DRLV ml−1 in the presence of 2.04±0.68×106 natural bacteria ml−1. The method was also applied to samples collected in October in the Celtic Sea, when on average 1 ml of water from the surface layer contained 1.41±0.16×106 natural bacteria, 14.6×103 cyanobacteria, 530±170 HNF, 7.3±3.0×103 phototrophic nanoflagellates (1.5–4 μm), 49.0±26.5 phototrophic dinoflagellates, 36.3±12.6 heterotrophic dinoflagellates and 21.3±9.5 Leucocryptos marina. Under these conditions the grazing rate in most samples did not exceed the coefficient of variation of the method (2%), although we estimate the grazing rate was -1.6 DRLV HNF−1 h−1 and on one occasion a rate of 2.45 was recorded. The gross growth efficiency for protein of -30% displayed by natural HNF means that they could release about ∼70% of inorganic nutrients derived from their food. [ABSTRACT FROM PUBLISHER]