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A flow-sorting technique was developed to determine unperturbed metabolic activities of phylogenetically characterized bacterioplankton groups with incorporation rates of [(35)S]methionine tracer. According to fluorescence in situ hybridization with rRNA targeted oligonucleotide probes, a clade of alpha-proteobacteria, related to Roseobacter spp., and a Cytophaga-Flavobacterium cluster dominated the different groups. Cytometric characterization revealed both these groups to have high DNA (HNA) content, while the alpha-proteobacteria exhibited high light scatter (hs) and the Cytophaga-Flavobacterium cluster exhibited low light scatter (ls). A third abundant group with low DNA (LNA) content contained cells from a SAR86 cluster of gamma-proteobacteria. Cellular specific activities of the HNA-hs group were 4- and 1.7-fold higher than the activities in the HNA-ls and LNA groups, respectively. However, the higher cellular protein synthesis by the HNA-hs could simply be explained by their maintenance of a larger cellular protein biomass. Similar biomass specific activities of the different groups strongly support the main assumption that underlies the determination of bacterial production: different bacteria in a complex community incorporate amino acids at a rate proportional to their protein synthesis. The fact that the highest growth-specific rates were determined for the smallest cells of the LNA group can explain the dominance of this group in nutrient-limited waters. The metabolic activities of the three groups accounted for almost the total bacterioplankton activity, indicating their key biogeochemical role in the planktonic ecosystem of the Celtic Sea.

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The algal osmolyte, dimethylsulphoniopropionate (DMSP), is abundant in the surface oceans and is the major precursor of dimethyl sulphide (DMS), a gas involved in global climate regulation. Here, we report results from an in situ Lagrangian study that suggests a link between the microbially driven fluxes of dissolved DMSP (DMSPd) and specific members of the bacterioplankton community in a North Sea coccolithophore bloom. The bacterial population in the bloom was dominated by a single species related to the genus Roseobacter, which accounted for 24% of the bacterioplankton numbers and up to 50% of the biomass. The abundance of the Roseobacter cells showed significant paired correlation with DMSPd consumption and bacterioplankton production, whereas abundances of other bacteria did not. Consumed DMSPd (28 nM day(-1)) contributed 95% of the sulphur and up to 15% of the carbon demand of the total bacterial populations, suggesting the importance of DMSP as a substrate for the Roseobacter-dominated bacterioplankton. In dominating DMSPd flux, the Roseobacter species may exert a major control on DMS production. DMSPd turnover rate was 10 times that of DMS (2.7 nM day(-1)), indicating that DMSPd was probably the major source of DMS, but that most of the DMSPd was metabolized without DMS production. Our study suggests that single species of bacterioplankton may at times be important in metabolizing DMSP and regulating the generation of DMS in the sea.

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Bacteria were deposited in tubes as compact pellets by centrifuging suspensions of cultured Vibrio at stationary phase. Numbers and protein biomass of flagellates added to these tubes and of the Vibrio, were followed and compared with the growth of the same and other protists on identical, uncentrifuged Vibrio. The flagellates Bodo saliens and Caecitellus parvulus, which could not be seen to multiply in tubes of suspended bacteria, grazed deposited bacteria actively as did the more versatile flagellate Cafeteria roenbergensis. The growth of these flagellates and their consumption of deposited bacteria were very similar to those of the flagellate Pteridomonas danica or the ciliate Uronema marinum fed with suspended bacteria, although deposit-feeders grew more slowly. Gross growth efficiencies (30-60%) of deposit-feeding flagellates were similar to those of the suspension-feeding protists. Caecitellus consumed 55 Vibrio to produce one flagellate, while 4,500 Vibrio were consumed to produce one Uronema. Surface-feeding flagellates are shown to be efficient bacterivores, capable of restricting the numbers of bacteria deposited on surfaces just as other protozoa control numbers of suspended bacteria.

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Dilution cultures are a common technique for measuring the growth of bacterioplankton communities. In this study, the taxonomic composition of marine bacterioplankton dilution cultures was followed in water samples from Plymouth Sound and the English Channel (UK). Bacterial abundances as well as protein and DNA content were closely monitored by flow cytometry. Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragments and fluorescence in situ hybridization (FISH) were applied directly to the water samples and to cells sorted from the dilution cultures based on their protein and DNA content. As expected, a rapid activation of bacteria occurred. However, molecular techniques showed that the community developed in the dilution culture within 1 day was significantly different from that in the original water samples. Whereas in the original samples, cells detectable by FISH were dominated by members of the Cytophagal Flavobacterium (CF) cluster, in dilution cultures, gamma-proteobacteria accounted for the majority of cells detected, followed by alpha-proteobacteria. An actively growing and an apparently non-growing population with average cellular protein contents of 24 and 4.5 fg respectively, were sorted by flow cytometry. FISH indicated mostly gamma- (64%) and alpha-proteobacteria (33%) in the first active fraction and 78% members of the CF cluster in the second fraction. Sequencing of DGGE bands confirmed the FISH assignments of the latter two groups. The data presented clearly show that even relatively short-term dilution experiments do not measure in situ growth, but rather growth patterns of an enrichment. Furthermore, it was demonstrated that the combination of flow cytometric analysis and sorting combined with FISH and DGGE analysis presented a fairly rapid method of analysing the taxonomic composition of marine bacterioplankton.

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An assay has been developed for measuring protein biomass of marine planktonic bacteria by flow cytometry. The method was calibrated by using five species of Bacteria (an Arcobacter sp., a Cytophaga sp., an Oceanospirillum sp., a Pseudoalteromonas sp., and a Vibrio sp.) recently isolated from seawater samples and grown in culture at different temperatures. The intensity of SYPRO-protein fluorescence of these bacteria strongly correlated with their total protein content, measured by the bicinchoninic acid method to be in the range of 60 to 330 fg of protein cell-1 (r2 = 0.93, n = 34). According to the calibration, the mean biomass of planktonic bacteria from the North Sea in August 1998 was 24 fg of protein cell-1.

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Identification problems restrict quantitative ecological research on specific nanoflagellates. Identification by specific oligonucleotide probes permits use of flow cytometry for enumeration and measurement of size of nanoflagellates in statistically meaningful samples. Flow cytometry also permits measurement of intensity of probe binding by cells. Five fluorescent probes targeted to different regions of the small subunit rRNA of the common marine flagellate Paraphysomonas vestita all hybridized with cells of this flagellate. Cells fixed with trichloroacetic acid gave detectable signals at a probe concentration of 15 aM and specific fluorescence increased almost linearly to 1.5 fM, but at higher concentrations nonspecific binding increased sharply. Three flagellates, P. vestita, Paraphysomonas imperforata, and Pteridomonas danica, all bound a general eukaryotic probe approximately in proportion to their cell size, but the specific P. vestita probe gave 14 times more fluorescence with P. vestita than with either of the other flagellates. Cell fluorescence increased during the early growth of a batch culture and decreased toward the stationary phase; cell size changed in a comparable manner. Cell fluorescence intensity may allow inferences about growth rate, but whether fluorescence (assumed to reflect ribosome number) merely correlates with cell biomass or changes in a more complex manner remains unresolved.

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A procedure has been developed for preparing living bacteria, quantitatively labeled with (3)H-thymidine and (14)C-leucine, for short-term grazing experiments. The negligible rate of accumulation in protozoan macromolecules of moieties of bacterial macromolecules labeled with (3)H compared with moieties labeled with (14)C permits estimation of the consumption, digestion, and assimilation of prey biomass in protists without separating them from bacteria. The principles of this method are described, and the results of its application in examples of grazing by the ciliates Euplotes and Uronema and the flagellate Pteridomonas on the bacterium Vibrio are outlined.