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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., (© The Author(s) 2022. Published by Oxford University Press.)

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., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 × 10(3) cells mL(-1)) and increased concentrations in the Southern temperate waters 8.3 × 10(3) 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., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

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., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

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., (© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

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 (35)S-methionine ((35)S-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 (35)S-Met uptake compared with controls, respectively). However, LNA bacterioplankton showed minor positive responses to dust leachate additions (7+/-4% increase in (35)S-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.

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 (3)H-leucine by phototrophic nanoflagellates (prasinophytes, pelagophytes and trebouxiophytes) and by associated bacteria using flow cytometric cell sorting. In contrast to (3)H-leucine-assimilating bacterial copopulations, none of the six studied nanoflagellates showed measurable direct uptake of (3)H-leucine, suggesting that the studied phototrophic protists were unable to utilize dissolved (3)H-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.

(35)S-Methionine and (3)H-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.

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.

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 [(35)S]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+G(2) cell cycle stages were constantly 1.5 times higher than those of cells at the G(1) stage.

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 nM d(-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 (35)S-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 amol cell(-1) d(-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.