Michael E. Sieracki

Eukaryotic plankton diversity in the sunlit ocean

Marine plankton support global biological and geochemical processes. Surveys of their biodiversity have hitherto been geographically restricted and have not accounted for the full range of plankton size. We assessed eukaryotic diversity from 334 size-fractionated photic-zone plankton communities collected across tropical and temperate oceans during the circumglobal Tara Oceans expedition. We analyzed 18S ribosomal DNA sequences across the intermediate plankton-size spectrum from the smallest unicellular eukaryotes (protists, >0.8 micrometers) to small animals of a few millimeters. Eukaryotic ribosomal diversity saturated at ~150,000 operational taxonomic units, about one-third of which could not be assigned to known eukaryotic groups. Diversity emerged at all taxonomic levels, both within the groups comprising the ~11,200 cataloged morphospecies of eukaryotic plankton and among twice as many other deep-branching lineages of unappreciated importance in plankton ecology studies. Most eukaryotic plankton biodiversity belonged to heterotrophic protistan groups, particularly those known to be parasites or symbiotic hosts.

Potential for Chemolithoautotrophy Among Ubiquitous Bacteria Lineages in the Dark Ocean

Bacteria isolated from a deep seawater mass seem to fix carbon using energy from the oxidation of inorganic sulfur. Recent studies suggest that unidentified prokaryotes fix inorganic carbon at globally significant rates in the immense dark ocean. Using single-cell sorting and whole-genome amplification of prokaryotes from two subtropical gyres, we obtained genomic DNA from 738 cells representing most cosmopolitan lineages. Multiple cells of Deltaproteobacteria cluster SAR324, Gammaproteobacteria clusters ARCTIC96BD-19 and Agg47, and some Oceanospirillales from the lower mesopelagic contained ribulose-1,5-bisphosphate carboxylase-oxygenase and sulfur oxidation genes. These results corroborated community DNA and RNA profiling from diverse geographic regions. The SAR324 genomes also suggested C1 metabolism and a particle-associated life-style. Microautoradiography and fluorescence in situ hybridization confirmed bicarbonate uptake and particle association of SAR324 cells. Our study suggests potential chemolithoautotrophy in several uncultured Proteobacteria lineages that are ubiquitous in the dark oxygenated ocean and provides new perspective on carbon cycling in the ocean’s largest habitat.

Assembling the Marine Metagenome, One Cell at a Time

The difficulty associated with the cultivation of most microorganisms and the complexity of natural microbial assemblages, such as marine plankton or human microbiome, hinder genome reconstruction of representative taxa using cultivation or metagenomic approaches. Here we used an alternative, single cell sequencing approach to obtain high-quality genome assemblies of two uncultured, numerically significant marine microorganisms. We employed fluorescence-activated cell sorting and multiple displacement amplification to obtain hundreds of micrograms of genomic DNA from individual, uncultured cells of two marine flavobacteria from the Gulf of Maine that were phylogenetically distant from existing cultured strains. Shotgun sequencing and genome finishing yielded 1.9 Mbp in 17 contigs and 1.5 Mbp in 21 contigs for the two flavobacteria, with estimated genome recoveries of about 91% and 78%, respectively. Only 0.24% of the assembling sequences were contaminants and were removed from further analysis using rigorous quality control. In contrast to all cultured strains of marine flavobacteria, the two single cell genomes were excellent Global Ocean Sampling (GOS) metagenome fragment recruiters, demonstrating their numerical significance in the ocean. The geographic distribution of GOS recruits along the Northwest Atlantic coast coincided with ocean surface currents. Metabolic reconstruction indicated diverse potential energy sources, including biopolymer degradation, proteorhodopsin photometabolism, and hydrogen oxidation. Compared to cultured relatives, the two uncultured flavobacteria have small genome sizes, few non-coding nucleotides, and few paralogous genes, suggesting adaptations to narrow ecological niches. These features may have contributed to the abundance of the two taxa in specific regions of the ocean, and may have hindered their cultivation. We demonstrate the power of single cell DNA sequencing to generate reference genomes of uncultured taxa from a complex microbial community of marine bacterioplankton. A combination of single cell genomics and metagenomics enabled us to analyze the genome content, metabolic adaptations, and biogeography of these taxa.

Matching phylogeny and metabolism in the uncultured marine bacteria, one cell at a time

The identification of predominant microbial taxa with specific metabolic capabilities remains one the biggest challenges in environmental microbiology, because of the limits of current metagenomic and cell culturing methods. We report results from the direct analysis of multiple genes in individual marine bacteria cells, demonstrating the potential for high-throughput metabolic assignment of yet-uncultured taxa. The protocol uses high-speed fluorescence-activated cell sorting, whole-genome multiple displacement amplification (MDA), and subsequent PCR screening. A pilot library of 11 single amplified genomes (SAGs) was constructed from Gulf of Maine bacterioplankton as proof of concept. The library consisted of five flavobacteria, one sphingobacterium, four alphaproteobacteria, and one gammaproteobacterium. Most of the SAGs, apart from alphaproteobacteria, were phylogenetically distant from existing isolates, with 88–97% identity in the 16S rRNA gene sequence. Thus, single-cell MDA provided access to the genomic material of numerically dominant but yet-uncultured taxonomic groups. Two of five flavobacteria in the SAG library contained proteorhodopsin genes, suggesting that flavobacteria are among the major carriers of this photometabolic system. The pufM and nasA genes were detected in some 100-cell MDA products but not in SAGs, demonstrating that organisms containing bacteriochlorophyll and assimilative nitrate reductase constituted <1% of the sampled bacterioplankton. Compared with metagenomics, the power of our approach lies in the ability to detect metabolic genes in uncultured microorganisms directly, even when the metabolic and phylogenetic markers are located far apart on the chromosome.

Single-Cell Genomics Reveals Organismal Interactions in Uncultivated Marine Protists

Marine protist cells from the wild environment contain DNA from several viruses and bacteria, but apparently lack plastids. Whole-genome shotgun sequence data from three individual cells isolated from seawater, followed by analysis of ribosomal DNA, indicated that the cells represented three divergent clades of picobiliphytes. In contrast with the recent description of this phylum, we found no evidence of plastid DNA nor of nuclear-encoded plastid-targeted proteins, which suggests that these picobiliphytes are heterotrophs. Genome data from one cell were dominated by sequences from a widespread single-stranded DNA virus. This virus was absent from the other two cells, both of which contained non-eukaryote DNA derived from marine Bacteroidetes and large DNA viruses. By using shotgun sequencing of uncultured marine picobiliphytes, we revealed the distinct interactions of individual cells.

CELLULAR DNA CONTENT OF MARINE PHYTOPLANKTON USING TWO NEW FLUOROCHROMES: TAXONOMIC AND ECOLOGICAL IMPLICATIONS1

Two new fluorochromes, PicoGreen® and SYTOX Green™ stain (Molecular Probes, Inc.), are useful with flow cytometry for quantitative detection of cellular DNA in a variety of marina phytoplankton. The basic instrument configuration of modern low‐power flow cytometers (15 mW, 488 nm excitation) is sensitive enough to detect the DNA signal in nearly all of the 121 strains (from 12 taxonomic classes)examined. The major advantages of these dyes over others are 1)suitability for direct use in seawater, 2)green fluorescence emission of the DNA‐dye complex (wavelength 525 ± 15 nm) showing no overlap with the autofluorescence of the plankton pigments in the red band, 3) high fluorescence yield of the DNA‐dye complex with an increase in fluorescence > 100‐fold compared to the unstained cell, and 4)dyes can be used to quantify double‐stranded DNA. The high sensitivity allowed the quantification of the DNA of the smallest known phyto‐plankter (Prochlorococcus) as well as bacteria found in some of the algal cultures. Of the 12 taxonomic classes tested, only the 3 Nannochloropsis spp. (Eustagmatophyceae) stained poorly, and a few members of the Chlorophyceae and Pelagophyceae showed poor staining occasionally. In general, maximal fluorescence was achieved within 15 min after addition of the dye. Although the PicoGreen dye stained some living phytoplankton species, preservation is recommended for quantitation. SYTOX Green did not stain live cells. The combination of the dyes, therefore, allows the discrimination between live and dead cells in some algal groups (Prochlorococcus, diatoms, prasinophytes, and pelagophytes). Paraformaldehyde was preferred over glutaraldehyde for fixation to avoid (induced) green autofluorescence.

Plankton community response to sequential silicate and nitrate depletion during the 1989 North Atlantic spring bloom

Abstract The onset of the North Atlantic spring bloom is controlled by physical conditions and nutrient availability in the surface layer. The nutrient of interest is usually considered to be nitrate. In an intensive study of the 1989 bloom near 46°N, 18°W, dissolved silicate was rapidly depleted to a minimum before nitrate was depleted. This coincided with a shift in dominant phytoplankton from diatoms to small flagellates. The cyanobacteria, nanoplankton and some components of the microplankton community were followed over the course of two cruises through April and May. A true-color image analysis system, combined with epifluorescence microscopy, was used to yield rapid semi-automated measurements of cell abundances, population size spectra and trophic classification. Although few direct measurements of diatom abundance or biomass were made, the diatom bloom appeared rapidly and sank (or was grazed) over the course of 2–3 weeks. The diatoms were replaced by a community of abundant (10 4 ml −1 ), small (2–5 μm) phytoflagellates, presumed to be primarily prymnesiophytes, and chlorophyll levels remained high (2–4 μg l −1 ). Similarly abundant and small aplastidic (heterotrophic) flagellates coincided with the plastidic flagellates. Carbon standing stock budgets, as well as direct microscopic observations, indicated a large amount of detritus in the water column near the end of the diatom bloom that was not present 2 weeks later. The zooplankton community was dominated by protozoans and copepod nauplii, and mixotrophic ciliates increased significantly over the study period. The nutrient-controlled shift in phytoplankton community composition apparently reduced the flux of recently fixed carbon out of the surface and increased recycling and mixotrophy in the surface layer.

Microzooplankton grazing of primary production at 140°W in the equatorial Pacific

Abstract Phytoplankton growth rates and the grazing impact by microzooplankton were estimated from dilution experiments during spring and fall time-series cruises in the equatorial Pacific as part of the U.S. JGOFS program. The Time-series I (TS-I) cruise occurred during El Nino conditions, while Time-series II (TS-II) coincided with a relaxation event. Deck incubation experiments were conducted using samples from the upper mixed layer (15 m) and depths coinciding with subsurface peaks in chlorophyll a (30–60m). Initial chlorophyll a concentrations were similar at 15m (0.1-0.2 μg 1−1) and at 60 m (0.2-0.4 μg 1−1) in both cruises (experiments at 30 m were conducted only in TS-II). Phytoplankton growth rates were highest at 15 m and decreased with depth. Growth rates in the mixed layer were lower in TS-I (0.4-0.6 day−1) than TS-II (0.8-1.1 day−1). The same trend was observed in phytoplankton growth in the subsurface chlorophyll a maxima (0.2 vs 0.6-0.7 day−1). Grazing rates, which also declined with depth, were higher in TS-II than in TS-I at 60 m (0.6-0.7 vs 0.2-0.4 day−1), but lower at 15 m (0.5-0.8 vs 0.7-1.0 day−1). HPLC pigment analyses indicated that microzooplankton grazing generally balanced the daily production by prymnesiophytes, and consumed much of the daily production of picophytoplankton. However, microzooplankton apparently consumed only about half the potential production by diatoms, implying that other loss processes (macrozooplankton grazing, sinking) regulate diatom abundance in these waters. Herbivory by microzooplankton, primarily by small microflagellates and dinoflagellates, averaged 133 (15 m) to 123% (60 m) of phytoplankton growth in TS-I, and 70 (15–30 m) to 105% (60 m) in TS-II. Thus, grazing of phytoplankton by microzooplankton represented a major pathway of organic carbon transformation at the equator during El Nino and non-El Nino conditions.

Phylogenetic Diversity and Specificity of Bacteria Closely Associated with Alexandrium spp. and Other Phytoplankton

ABSTRACT While several studies have suggested that bacterium-phytoplankton interactions have the potential to dramatically influence harmful algal bloom dynamics, little is known about how bacteria and phytoplankton communities interact at the species composition level. The objective of the current study was to determine whether there are specific associations between diverse phytoplankton and the bacteria that co-occur with them. We determined the phylogenetic diversity of bacterial assemblages associated with 10 Alexandrium strains and representatives of the major taxonomic groups of phytoplankton in the Gulf of Maine. For this analysis we chose xenic phytoplankton cultures that (i) represented a broad taxonomic range, (ii) represented a broad geographic range for Alexandrium spp. isolates, (iii) grew under similar cultivation conditions, (iv) had a minimal length of time since the original isolation, and (v) had been isolated from a vegetative phytoplankton cell. 16S rRNA gene fragments of most Bacteria were amplified from DNA extracted from cultures and were analyzed by denaturing gradient gel electrophoresis and sequencing. A greater number of bacterial species were shared by different Alexandrium cultures, regardless of the geographic origin, than by Alexandrium species and nontoxic phytoplankton from the Gulf of Maine. In particular, members of the Roseobacter clade showed a higher degree of association with Alexandrium than with other bacterial groups, and many sequences matched sequences reported to be associated with other toxic dinoflagellates. These results provide evidence for specificity in bacterium-phytoplankton associations.

Grazing, growth and mortality of microzooplankton during the 1989 North Atlantic spring bloom at 47°N, 18°W

Grazing and growth rates of nano- and microzooplankton were measured as part of the 1989 North Atlantic Bloom Experiment, an interdisciplinary research program of the Joint Global Ocean Flux Study (JGOFS). Samples for shipboard experimental incubations were collected from the mixed layer of a drogued water mass (46°20′N, 17°50′W) over a 2 week period in May. Grazing and growth rates, measured using the size fractionation and dilution techniques, were calculated from changes in chlorophylls, accessory pigments, and cell abundances. The phytoplankton community was dominated by phytoflagellates, primarily prymnesiophytes, which passed 10 μm mesh. Chlorophyll a (Chl a) increased at an average rate of 0.9 doublings day−1 when incubated at 60 % I0. Grazing by nano- and microzooplankton removed 37–100% of estimated primary production in samples from 10 m, and 100% of that at 30 m. An attempt was made to budget estimated rates of community grazing to major groups of nano- and microzooplankton, using measured biomass and specific ingestion or growth rates from laboratory studies. Aplastidic microflagellates were apparently the most important herbivores. In addition to ciliates and heterotrophic dinoflagellates, various developmental stages of copepods were abundant in the <200 μm fraction. Predation within the microzooplankton community appeared to be substantial. Given the evidence of tight coupling between production and consumption within the upper water column, little material appeared to be available for direct export from the mixed layer to depth during this phase of the spring bloom.