Dawn M. Moran

Defining DNA-Based Operational Taxonomic Units for Microbial-Eukaryote Ecology

ABSTRACT DNA sequence information has increasingly been used in ecological research on microbial eukaryotes. Sequence-based approaches have included studies of the total diversity of selected ecosystems, studies of the autecology of ecologically relevant species, and identification and enumeration of species of interest for human health. It is still uncommon, however, to delineate protistan species based on their genetic signatures. The reluctance to assign species-level designations based on DNA sequences is in part a consequence of the limited amount of sequence information presently available for many free-living microbial eukaryotes and in part a consequence of the problematic nature of and debate surrounding the microbial species concept. Despite the difficulties inherent in assigning species names to DNA sequences, there is a growing need to attach meaning to the burgeoning amount of sequence information entering the literature, and there is a growing desire to apply this information in ecological studies. We describe a computer-based tool that assigns DNA sequences from environmental databases to operational taxonomic units at approximately species-level distinctions. This approach provides a practical method for ecological studies of microbial eukaryotes (primarily protists) by enabling semiautomated analysis of large numbers of samples spanning great taxonomic breadth. Derivation of the algorithm was based on an analysis of complete small-subunit (18S) rRNA gene sequences and partial gene sequences obtained from the GenBank database for morphologically described protistan species. The program was tested using environmental 18S rRNA data sets for two oceanic ecosystems. A total of 388 operational taxonomic units were observed for 2,207 sequences obtained from samples collected in the western North Atlantic and eastern North Pacific oceans.

Iron conservation by reduction of metalloenzyme inventories in the marine diazotroph Crocosphaera watsonii

The marine nitrogen fixing microorganisms (diazotrophs) are a major source of nitrogen to open ocean ecosystems and are predicted to be limited by iron in most marine environments. Here we use global and targeted proteomic analyses on a key unicellular marine diazotroph Crocosphaera watsonii to reveal large scale diel changes in its proteome, including substantial variations in concentrations of iron metalloproteins involved in nitrogen fixation and photosynthesis, as well as nocturnal flavodoxin production. The daily synthesis and degradation of enzymes in coordination with their utilization results in a lowered cellular metalloenzyme inventory that requires ∼40% less iron than if these enzymes were maintained throughout the diel cycle. This strategy is energetically expensive, but appears to serve as an important adaptation for confronting the iron scarcity of the open oceans. A global numerical model of ocean circulation, biogeochemistry and ecosystems suggests that Crocosphaera’s ability to reduce its iron-metalloenzyme inventory provides two advantages: It allows Crocosphaera to inhabit regions lower in iron and allows the same iron supply to support higher Crocosphaera biomass and nitrogen fixation than if they did not have this reduced iron requirement.

Genomic and proteomic characterization of “Candidatus Nitrosopelagicus brevis”: An ammonia-oxidizing archaeon from the open ocean

Significance Thaumarchaeota are among the most abundant microbial cells in the ocean, but to date, complete genome sequences for marine Thaumarchaeota are lacking. Here, we report the 1.23-Mbp genome of the pelagic ammonia-oxidizing thaumarchaeon “Candidatus Nitrosopelagicus brevis” str. CN25. We present the first proteomic data, to our knowledge, from this phylum, which show a high proportion of proteins translated in oligotrophic conditions. Metagenomic fragment recruitment using data from the open ocean indicate the ubiquitous presence of Ca. N. brevis-like sequences in the surface ocean and suggest Ca. N. brevis as a model system for understanding the ecology and evolution of pelagic marine Thaumarchaeota. Thaumarchaeota are among the most abundant microbial cells in the ocean, but difficulty in cultivating marine Thaumarchaeota has hindered investigation into the physiological and evolutionary basis of their success. We report here a closed genome assembled from a highly enriched culture of the ammonia-oxidizing pelagic thaumarchaeon CN25, originating from the open ocean. The CN25 genome exhibits strong evidence of genome streamlining, including a 1.23-Mbp genome, a high coding density, and a low number of paralogous genes. Proteomic analysis recovered nearly 70% of the predicted proteins encoded by the genome, demonstrating that a high fraction of the genome is translated. In contrast to other minimal marine microbes that acquire, rather than synthesize, cofactors, CN25 encodes and expresses near-complete biosynthetic pathways for multiple vitamins. Metagenomic fragment recruitment indicated the presence of DNA sequences >90% identical to the CN25 genome throughout the oligotrophic ocean. We propose the provisional name “Candidatus Nitrosopelagicus brevis” str. CN25 for this minimalist marine thaumarchaeon and suggest it as a potential model system for understanding archaeal adaptation to the open ocean.

Multiple nutrient stresses at intersecting pacific ocean biomes detected by protein biomarkers

Protein markers of cyanobacterial stress Nutrients including iron, nitrogen, and phosphorus limit primary productivity in the oceans. Determining how abundant cyanobacteria such as Prochlorococcus adapt to nutrient stress across marine settings requires accurate molecular assays. Saito et al. developed a proteomic and metaproteomic approach capable of targeting specific metabolic biomarkers from mixed communities in seawater (see the Perspective by Moore). Prochlorococcus proteins are indicative of a major limiting nutrient across a wide transect in the Pacific Ocean; however, they also show that the limitation of multiple nutrients at overlapping biomes is an additional source of stress. Science, this issue p. 1173; see also p. 1120 The composition of cyanobacterial proteins reflects nutrient stress in central Pacific Ocean ecosystems. [Also see Perspective by Moore] Marine primary productivity is strongly influenced by the scarcity of required nutrients, yet our understanding of these nutrient limitations is informed by experimental observations with sparse geographical coverage and methodological limitations. We developed a quantitative proteomic method to directly assess nutrient stress in high-light ecotypes of the abundant cyanobacterium Prochlorococcus across a meridional transect in the central Pacific Ocean. Multiple peptide biomarkers detected widespread and overlapping regions of nutritional stress for nitrogen and phosphorus in the North Pacific Subtropical Gyre and iron in the equatorial Pacific. Quantitative protein analyses demonstrated simultaneous stress for these nutrients at biome interfaces. This application of proteomic biomarkers to diagnose ocean metabolism demonstrated Prochlorococcus actively and simultaneously deploying multiple biochemical strategies for low-nutrient conditions in the oceans.

Nutrient Acquisition and Population Growth of a Mixotrophic Alga in Axenic and Bacterized Cultures.

Axenic growth of a mixotrophic alga, Ochromonas sp., was compared in several inorganic and organic media, and in the presence of live bacteria under nutrient-replete and low-nutrient conditions. Axenic growth in the light was negligible in inorganic media with or without the addition of glucose. Addition of vitamins increased growth rate, but average cell size declined, resulting in no net increase in biomass. Supplementing axenic cultures with a more complex organic substrate resulted in moderate growth and higher maximal abundance (and biomass) than in the inorganic media with added vitamins. The absence of light did not greatly affect population growth rate in the presence of complex dissolved organic compounds, although cell size was significantly greater in the light than in the dark. The highest growth rates for the alga (up to 2.6 d?1) were measured in treatments containing live bacteria. Increases in cell number of Ochromonas sp. in the presence of bacterial prey were similar in the light and dark, although chloroplast and cell sizes differed. Bacterial abundance was reduced and dissolved phosphorus and ammonia were rapidly released in bacterized cultures in the light and dark, indicating high rates of bacterial ingestion and suggesting an inability of the alga to store or utilize N and P in excess of the quantities required for heterotrophic growth. Low-nutrient conditions in the presence of bacteria were promoted by adding glucose to stimulate bacterial growth and the uptake of N and P released by algal phagotrophy. Subsequent decreases in dissolved N and P following the addition of glucose corresponded to a second period of rapid growth of the alga in both light and dark. This result, combined with evidence for slow axenic growth of this strain, indicated that nutrient acquisition for this species in the presence of bacteria was accomplished primarily via ingestion of bacteria.

Effect of Temperature on Photosynthesis and Growth in Marine Synechococcus spp.

Marine Synechococcus regulates state transitions and photosynthetic protein content for temperature acclimation. In this study, we develop a mechanistic understanding of how temperature affects growth and photosynthesis in 10 geographically and physiologically diverse strains of Synechococcus spp. We found that Synechococcus spp. are able to regulate photochemistry over a range of temperatures by using state transitions and altering the abundance of photosynthetic proteins. These strategies minimize photosystem II (PSII) photodamage by keeping the photosynthetic electron transport chain (ETC), and hence PSII reaction centers, more oxidized. At temperatures that approach the optimal growth temperature of each strain when cellular demand for reduced nicotinamide adenine dinucleotide phosphate (NADPH) is greatest, the phycobilisome (PBS) antenna associates with PSII, increasing the flux of electrons into the ETC. By contrast, under low temperature, when slow growth lowers the demand for NADPH and linear ETC declines, the PBS associates with photosystem I. This favors oxidation of PSII and potential increase in cyclic electron flow. For Synechococcus sp. WH8102, growth at higher temperatures led to an increase in the abundance of PBS pigment proteins, as well as higher abundance of subunits of the PSII, photosystem I, and cytochrome b6f complexes. This would allow cells to increase photosynthetic electron flux to meet the metabolic requirement for NADPH during rapid growth. These PBS-based temperature acclimation strategies may underlie the larger geographic range of this group relative to Prochlorococcus spp., which lack a PBS.

Irreversibly increased nitrogen fixation in Trichodesmium experimentally adapted to elevated carbon dioxide

Nitrogen fixation rates of the globally distributed, biogeochemically important marine cyanobacterium Trichodesmium increase under high carbon dioxide (CO2) levels in short-term studies due to physiological plasticity. However, its long-term adaptive responses to ongoing anthropogenic CO2 increases are unknown. Here we show that experimental evolution under extended selection at projected future elevated CO2 levels results in irreversible, large increases in nitrogen fixation and growth rates, even after being moved back to lower present day CO2 levels for hundreds of generations. This represents an unprecedented microbial evolutionary response, as reproductive fitness increases acquired in the selection environment are maintained after returning to the ancestral environment. Constitutive rate increases are accompanied by irreversible shifts in diel nitrogen fixation patterns, and increased activity of a potentially regulatory DNA methyltransferase enzyme. High CO2-selected cell lines also exhibit increased phosphorus-limited growth rates, suggesting a potential advantage for this keystone organism in a more nutrient-limited, acidified future ocean.

Development and Application of a Monoclonal-Antibody Technique for Counting Aureococcus anophagefferens, an Alga Causing Recurrent Brown Tides in the Mid-Atlantic United States

ABSTRACT A method was developed for the rapid detection and enumeration of Aureococcus anophagefferens, the cause of harmful algal blooms called “brown tides” in estuaries of the Mid-Atlantic United States. The method employs a monoclonal antibody (MAb) and a colorimetric, enzyme-linked immunosorbent assay format. The MAb obtained exhibits high reactivity with A. anophagefferens and very low cross-reactivities with a phylogenetically diverse array of other protists and bacteria. Standard curves are constructed for each 96-well microtiter plate by using known amounts of a preserved culture of A. anophagefferens. This approach allows estimation of the abundance of the alga in natural samples. The MAb method was compared to an existing method that employs polyclonal antibodies and epifluorescence microscopy and to direct microscopic counts of A. anophagefferens in samples with high abundances of the alga. The MAb method provided increased quantitative accuracy and greatly reduced sample processing time. A spatial survey of several Long Island estuaries in May 2000 using this new approach documented a range of abundances of A. anophagefferens in these bays spanning nearly 3 orders of magnitude.

ABUNDANCE OF A NOVEL DINOFLAGELLATE PHYLOTYPE IN THE ROSS SEA, ANTARCTICA1

The biodiversity of protistan assemblages present in microhabitats of the Ross Sea, Antarctica, was examined using molecular biological methods to obtain a greater understanding of the genetic diversity present. Sequencing of 18S clone libraries indicated genetically diverse collections of organisms in the water column, ice, and meltwater layer (slush), but a single small subunit ribosomal DNA (srDNA) sequence type dominated clone libraries (>30%) from seawater and slush samples taken within the ice pack of this ecosystem. The BLAST searches indicated that this dominant clone was derived from a dinoflagellate, and that it shared sequence similarity (97.6%–98.3%) with both Karenia and Karlodinium species. Phylogenetic analyses based on small subunit ribosomal gene sequences supported its placement as a sister group to these taxa, and suggested that it represented a novel genus. The dinoflagellate was successfully recovered in culture, and morphological analyses have shown that it contains chloroplasts, is gymnodinoid, appears not to have thecal plates, and has an apical groove and sulcal structure that confirm its placement as a relative of the Karenia/Karlodinium group. The abundance of this phylotype in natural samples was confirmed by quantitative PCR analyses of water and slush communities, and suggests that this dinoflagellate can be a major constituent of the protistan assemblages of some Antarctic microhabitats of the Ross Sea.

A Description of Seven Antarctic Marine Gymnamoebae Including a New Subspecies, Two New Species and a New Genus: Neoparamoeba aestuarina antarctica n. subsp., Platyamoeba oblongata n. sp., Platyamoeba contorta n. sp. and Vermistella antarctica n. gen. n. sp.

ABSTRACT. Seven marine gymnamoebae were isolated from different environments of seawater, slush (pack ice meltwater), and sediment in the Ross Sea area of Antarctica. All amoebae were isolated and maintained at temperatures below 4°C. Growth, rate of locomotion, and general morphology were observed at an environmentally appropriate temperature (1°C) and at room temperature (∼25°C). Molecular (srDNA sequences) and microscopical techniques were used to identify the gymnamoebae and establish their phylogenetic affinities. Three isolates (S‐131‐2, SL‐200, and W4‐3) were assigned to a psychrophilic subspecies of Neoparamoeba aestuarina, N. aestuarina antarctica n. subsp., one isolate (S‐205) was assigned to a new species of Platyamoeba, P. oblongata n. sp., two isolates (W51C#4 & W51C#5) were also assigned to a new species of Platyamoeba, P. contorta n. sp., and one isolate (S‐241) was a novel psychrophilic gymnamoeba Vermistella antarctica n. gen. n. sp. Molecular and morphological results revealed that V. antarctica was not related to any described family of gymnamoebae. Strains S‐205, W51C#4, and W51C#5 were capable of locomotion at room temperature, while strains SL‐200, S‐131‐2, W4‐3, and S‐241 exhibited locomotion only below ∼10°C. Our results imply that the Antarctic environment is host both to cosmopolitan gymnamoebae that have acquired adaptations for existence at low environmental temperature and to apparently novel psychrophilic amoebae described here for the first time.