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Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways., This collaborative effort was supported by the Gordon and Betty Moore Foundation EMS Program of the Marine Microbiology Initiative (grant nos. GBMF4972 and 4972.01 to F.-Y.B.; GBMF4970 and 4970.01 to M.A. and A.Z.W.; GBMF3788 to A.Z.W.; GBMF 4968 and 4968.01 to H.C.; GBMF4984 to V.H.; GBMF4974 and 4974.01 to C. Brownlee; GBMF4964 to Y. Hirakawa; GBMF4961 to T. Mock; GBMF4958 to P.S.; GBMF4957 to A.T.; GBMF4960 to G.J.S.; GBMF4979 to K.C.; GBMF4982 and 4982.01 to J.L.C.; GBMF4964 to P.J.K.; GBMF4981 to P.v.D.; GBMF5006 to A.E.A.; GBMF4986 to C.M.; GBMF4962 to J.A.F.R.; GBMF4980 and 4980.01 to S.L.; GBMF 4977 and 4977.01 to R.F.W.; GBMF4962.01 to C.H.S.; GBMF4985 to J.M.; GBMF4976 and 4976.01 to C.H.; GBMF4963 and 4963.01 to V.E.; GBMF5007 to C.L.D.; GBMF4983 and 4983.01 to J.L.; GBMF4975 and 4975.01 to A.D.T.; GBMF4973 and 4973.01 to I.R.-T. and GBMF4965 to N.K.), by The Leverhulme Trust (RPG-2017-364) to T. Mock and A. Hopes, and by ERD funds (16_019/0000759) from the Czech Ministry of Education to J.L.

Phaeocystis antarctica is an important phytoplankter of the Ross Sea where it dominates the early season bloom after sea ice retreat and is a major contributor to carbon export. The factors that influence Phaeocystis colony formation and the resultant Ross Sea bloom initiation have been of great scientific interest, yet there is little known about the underlying mechanisms responsible for these phenomena. Here, we present laboratory and field studies on Phaeocystis antarctica grown under multiple iron conditions using a coupled proteomic and transcriptomic approach. P. antarctica had a lower iron limitation threshold than a Ross Sea diatom Chaetoceros sp., and at increased iron nutrition (> 120 pM Fe') a shift from flagellate cells to a majority of colonial cells in P. antarctica was observed, implying a role for iron as a trigger for colony formation. Proteome analysis revealed an extensive and coordinated shift in proteome structure linked to iron availability and life cycle transitions with 327 and 436 proteins measured as significantly different between low and high iron in strains 1871 and 1374, respectively. The enzymes flavodoxin and plastocyanin that can functionally replace iron metalloenzymes were observed at low iron treatments consistent with cellular iron-sparing strategies, with plastocyanin having a larger dynamic range. The numerous isoforms of the putative iron-starvation-induced protein (ISIP) group (ISIP2A and ISIP3) had abundance patterns coinciding with that of either low or high iron (and coincident flagellate or the colonial cell types in strain 1871), implying that there may be specific iron acquisition systems for each life cycle type. The proteome analysis also revealed numerous structural proteins associated with each cell type: within flagellate cells actin and tubulin from flagella and haptonema structures as well as a suite of calcium-binding proteins with EF domains were observed. In the colony-dominated samples a variety of structural proteins were observed that are also often found in multicellular organisms including spondins, lectins, fibrillins, and glycoproteins with von Willebrand domains. A large number of proteins of unknown function were identified that became abundant at either high or low iron availability. These results were compared to the first metaproteomic analysis of a Ross Sea Phaeocystis bloom to connect the mechanistic information to the in situ ecology and biogeochemistry. Proteins associated with both flagellate and colonial cells were observed in the bloom sample consistent with the need for both cell types within a growing bloom. Bacterial iron storage and B12 biosynthesis proteins were also observed consistent with chemical synergies within the colony microbiome to cope with the biogeochemical conditions. Together these responses reveal a complex, highly coordinated effort by P. antarctica to regulate its phenotype at the molecular level in response to iron and provide a window into the biology, ecology, and biogeochemistry of this group.

We present the Aquatic Symbiosis Genomics Project, a global collaboration to generate high quality genome sequences for a wide range of eukaryotes and their microbial symbionts. Launched under the Symbiosis in Aquatic Systems Initiative of the Gordon and Betty Moore Foundation, the ASG Project brings together researchers from across the globe who hope to use these reference genomes to augment and extend their analyses of the dynamics, mechanisms and environmental importance of symbiosis. Applying large-scale, high-throughput sequencing and assembly technologies, the ASG collaboration will assemble and annotate the genomes of 500 symbiotic organisms – both the “hosts” and the microbial symbionts with which they associate. These data will be released openly to benefit all who work on symbiosis, from conservation geneticists to those interested in the origin of the eukaryotic cell.

Author(s): Faktorova, Drahomira; Nisbet, R Ellen R; Fernandez Robledo, Jose A; Casacuberta, Elena; Sudek, Lisa; Allen, Andrew E; Ares, Manuel; Areste, Cristina; Balestreri, Cecilia; Barbrook, Adrian C; Beardslee, Patrick; Bender, Sara; Booth, David S; Bouget, Francois-Yves; Bowler, Chris; Breglia, Susana A; Brownlee, Colin; Burger, Gertraud; Cerutti, Heriberto; Cesaroni, Rachele; Chiurillo, Miguel A; Clemente, Thomas; Coles, Duncan B; Collier, Jackie L; Cooney, Elizabeth C; Coyne, Kathryn; Docampo, Roberto; Dupont, Christopher L; Edgcomb, Virginia; Einarsson, Elin; Elustondo, Pia A; Federici, Fernan; Freire-Beneitez, Veronica; Freyria, Nastasia J; Fukuda, Kodai; Garcia, Paulo A; Girguis, Peter R; Gomaa, Fatma; Gornik, Sebastian G; Guo, Jian; Hampl, Vladimir; Hanawa, Yutaka; Haro-Contreras, Esteban R; Hehenberger, Elisabeth; Highfield, Andrea; Hirakawa, Yoshihisa; Hopes, Amanda; Howe, Christopher J; Hu, Ian; Ibanez, Jorge; Irwin, Nicholas AT; Ishii, Yuu; Janowicz, Natalia Ewa; Jones, Adam C; Kachale, Ambar; Fujimura-Kamada, Konomi; Kaur, Binnypreet; Kaye, Jonathan Z; Kazana, Eleanna; Keeling, Patrick J; King, Nicole; Klobutcher, Lawrence A; Lander, Noelia; Lassadi, Imen; Li, Zhuhong; Lin, Senjie; Lozano, Jean-Claude; Luan, Fulei; Maruyama, Shinichiro; Matute, Tamara; Miceli, Cristina; Minagawa, Jun; Moosburner, Mark; Najle, Sebastian R; Nanjappa, Deepak; Nimmo, Isabel C; Noble, Luke; Novak Vanclova, Anna MG; Nowacki, Mariusz; Nunez, Isaac; Pain, Arnab; Piersanti, Angela; Pucciarelli, Sandra; Pyrih, Jan; Rest, Joshua S | Abstract: An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Phaeocystis antarctica is an important phytoplankter of the Ross Sea where it dominates the early season bloom after sea ice retreat and is a major contributor to carbon export. The factors that influence Phaeocystis colony formation and the resultant Ross Sea bloom initiation have been of great scientific interest, yet there is little known about the underlying mechanisms responsible for these phenomena. Here, we present laboratory and field studies on Phaeocystis antarctica grown under multiple iron conditions using a coupled proteomic and transcriptomic approach. P. antarctica had a lower iron limitation threshold than a Ross Sea diatom Chaetoceros sp., and at increased iron nutrition (> 120 pM Fe') a shift from flagellate cells to a majority of colonial cells in P. antarctica was observed, implying a role for iron as a trigger for colony formation. Proteome analysis revealed an extensive and coordinated shift in proteome structure linked to iron availability and life cycle transitions with 327 and 436 proteins significantly different between low and high iron in strains 1871 and 1374, respectively. The enzymes flavodoxin and plastocyanin that can functionally replace iron metalloenzymes were observed at low iron treatments consistent with cellular iron sparing strategies, with plastocyanin being more dynamic in range. The numerous isoforms of the putative iron-starvation induced protein ISIP group (ISIP2A and ISIP3) had abundance patterns coincided with that of either low or high iron (and coincident flagellate or the colonial cell types in strain 1871), implying that there may be specific iron acquisition systems for each life cycle type. The proteome analysis also revealed numerous structural proteins associated with each cell type: within flagellate cells actin and tubulin from flagella and haptonema structures as well as a suite of calcium-binding proteins with EF domains were observed. In the colony-dominated samples a variety of structural proteins were observed that are also often found in multicellular organisms including spondins, lectins, fibrillins, and glycoproteins with von Willebrand domains. A large number of proteins of unknown function were identified that became abundant at either high and low iron availability. These results were compared to the first metaproteomic analysis of a Ross Sea Phaeocystis bloom to connect the mechanistic information to the in situ ecology and biogeochemistry. Proteins associated with both flagellate and colonial cells were observed in the bloom sample consistent with the need for both cell types within a growing bloom. Bacterial iron storage and B12 biosynthesis proteins were also observed consistent with chemical synergies within the colony microbiome to cope with the biogeochemical conditions. Together these responses reveal a complex, highly coordinated effort by P. antarctica to regulate its phenotype at the molecular level in response to iron and provide a window into the biology, ecology, and biogeochemistry of this group.

As graduate students, we learn how to condense manuscripts into abstracts packed with jargon and dense nuggets of information. These abstracts are efficient, effective ways of communicating with colleagues and collaborators. However, they are not likely to attract a broad readership. How often is your 250word gold mine of information intelligible to your non-scientist friends and neighbors? Or even to scientists outside your narrow subdiscipline? Training in—and, more importantly, practicing—science communication can help researchers connect across science disciplines as well as with the general public (e.g., National Research Council [NRC] 2000; Diaz-Munoz 2014; Besley et al. 2015). This particular science communication project entailed students and scientists translating their research abstracts into articles that could be easily understood by the general public. Our approach was to provide science communication training, practice writing for nonspecialists, and a carrot: an opportunity to create a peer-reviewed science communication article ready to submit for publication. Eight graduate students and early-career researchers from the Center for Microbial Oceanography: Research and Education (C-MORE) were recruited to participate. Each was asked to translate a recent science abstract into an ∼800–1000 word article that would be accessible to nonspecialists having some college education. Erin Loury, a science communication professional, peer-reviewed the translations, and provided feedback to authors. Over the next few paragraphs, Loury and several authors describe the main challenges they faced during this project. These communication case studies give us insight (highlighted in italics below) into the process behind translating science into stories. All of the translated articles are available online as Supporting Information.

About half the carbon fixed by phytoplankton in the ocean is taken up and metabolized by marine bacteria, a transfer that is mediated through the seawater dissolved organic carbon (DOC) pool. The chemical complexity of marine DOC, along with a poor understanding of which compounds form the basis of trophic interactions between bacteria and phytoplankton, have impeded efforts to identify key currencies of this carbon cycle link. Here, we used transcriptional patterns in a bacterial-diatom model system based on vitamin B12 auxotrophy as a sensitive assay for metabolite exchange between marine plankton. The most highly up-regulated genes (up to 374-fold) by a marine Roseobacter clade bacterium when cocultured with the diatom Thalassiosira pseudonana were those encoding the transport and catabolism of 2,3-dihydroxypropane-1-sulfonate (DHPS). This compound has no currently recognized role in the marine microbial food web. As the genes for DHPS catabolism have limited distribution among bacterial taxa, T. pseudonana may use this sulfonate for targeted feeding of beneficial associates. Indeed, DHPS was both a major component of the T. pseudonana cytosol and an abundant microbial metabolite in a diatom bloom in the eastern North Pacific Ocean. Moreover, transcript analysis of the North Pacific samples provided evidence of DHPS catabolism by Roseobacter populations. Other such biogeochemically important metabolites may be common in the ocean but difficult to discriminate against the complex chemical background of seawater. Bacterial transformation of this diatom-derived sulfonate represents a previously unidentified and likely sizeable link in both the marine carbon and sulfur cycles.

Microcosm experiments were conducted along the Washington and Oregon coasts in May 2009, May 2010, and July 2010 to determine whether variation in the supply of silicic acid from the Columbia River could influence the silicification and sinking potential of coastal diatom blooms. The chlorophyll a concentration increased similarly in communities incubated with added nitrate or both nitrate and silicic acid, indicating that growth was limited by nitrate availability. Communities that grew in the treatment with added silicic acid and nitrate were more silicified than communities in the treatment with only nitrate added. No difference in community composition was detected between these treatments in three out of four experiments. Isolates of Minutocellus, Cylindrotheca, Thalassiosira, and Odontella were obtained from the microcosm experiment conducted in May 2010 and were maintained in the laboratory in 20 µmol L−1 silicic acid. All four diatom isolates contained ∼ 2.5 times more silica per cell when silicic acid concentration in the media was increased to 80 µmol L−1. The intensity of a fluorescent cellular stain of newly precipitated silica (2-(4-pyridyl)-5{[4-dimethylaminoethyl-aminocarbamoyl)-methoxy]phenyl}oxazole) strongly correlated with silica content among species, but was a less sensitive indicator of changing silicification within a single species. Changes in silicification were not correlated with changes in the transcript abundance of silicic acid transporters. Sinking rates increased roughly 2-fold for cells that contained ∼ 2.5 times more silica. Variation in silicic acid supply alters the silicification of nitrate-fueled coastal diatom blooms and the potential sink of carbon from coastal zones.

A microcosm study in iron-limited waters of the northeast subarctic Pacific Ocean was conducted to examine how iron availability affects the frustule-related response of individual diatoms and thus the total quantity of silica precipitated by the community. New silica precipitated per cell was estimated using the fluorescent cell stain 2-(4-pyridyl)-5{[4-dimethylaminoethyl-aminocarbamoyl)-methoxy]phenyl}oxazole (PDMPO). Differences in new silica precipitation within a particular genus before and after iron enrichment were small compared to differences among genera, indicating that the quantity of total silica precipitated is particularly sensitive to community composition. Transcriptional patterns of genes encoding silicon transporters, aminopropyltransferases, chitin synthases, and a protein with uncharacterized function were measured in natural populations to identify indicators of the frustule-related responses of different genera to iron limitation. Transcripts associated with silicon transporters were the most readily detectable in three metatranscriptome datasets and were capable of resolving species composition shifts and physiological responses. Silicon transporter transcripts from a distinct phylogenetic clade were most abundant in the iron-limited community, and transcripts from a separate clade were more abundant in the community that bloomed after iron enrichment. Transcripts of the gene present in the iron-limited community were also more abundant in iron-limited laboratory cultures of Pseudo-nitzschia multiseries, suggesting that this gene plays a role in silicon uptake during iron limitation. The responses of individual cells, as detected in this study, determine how the community influences silicon cycling in iron-limited environments.

Diatoms are photoautotrophic organisms capable of growing on a variety of inorganic and organic nitrogen sources. Discovery of a complete urea cycle in diatoms was surprising, as this pathway commonly functions in heterotrophic organisms to rid cells of waste nitrogen. To determine how the urea cycle is integrated into cellular nitrogen metabolism and energy management, the centric diatom Thalassiosira pseudonana was maintained in semi-continuous batch cultures on nitrate, ammonium, or urea as the sole nitrogen source, under a 16: 8 light: dark cycle and at light intensities that were low, saturating, or high for growth. Steady-state transcript levels were determined for genes encoding enzymes linked to the urea cycle, urea hydrolysis, glutamine synthesis, pyrimidine synthesis, photorespiration, and energy storage. Transcript abundances were significantly affected by nitrogen source, light intensity and a diel cycle. The impact of N source on differential transcript accumulation was most apparent under the highest light intensity. Models of cellular metabolism under high light were developed based on changes in transcript abundance and predicted enzyme localizations. We hypothesize that the urea cycle is integrated into nitrogen metabolism through its connection to glutamine and in the eventual production of urea. These findings have important implications for nitrogen flow in the cell over diel cycles at surface ocean irradiances.

Our current understanding of biology is heavily based on a small number of genetically tractable model organisms. Most eukaryotic phyla lack such experimental models, and this limits our ability to explore the molecular mechanisms that ultimately define their biology, ecology, and diversity. In particular, marine protists suffer from a paucity of model organisms despite playing critical roles in global nutrient cycles, food webs, and climate. To address this deficit, an initiative was launched in 2015 to foster the development of ecologically and taxonomically diverse marine protist genetic models. The development of new models faces many barriers, some technical and others institutional, and this often discourages the risky, long-term effort that may be required. To lower these barriers and tackle the complexity of this effort, a highly collaborative community-based approach was taken. Herein, we describe this approach, the advances achieved, and the lessons learned by participants in this novel community-based model for research.

In the modern ocean, phytoplankton maintain extremely high primary production/biomass ratios, indicating that they bloom, die, and are replaced weekly. The molecular mechanisms regulating cellular mortality and turnover are largely unknown, even though they effectively short-circuit carbon export to the deep ocean and channel primary productivity to microbial food webs. Here, we present morphological, biochemical, and molecular evidence of caspase-mediated, autocatalytic programmed cell death (PCD) in the diatom Thalassiosira pseudonana in response to iron starvation. Transmission electron microscopy revealed internal degradation of nuclear, chloroplastic, and mitochondrial organelles, all while the plasma membranes remained intact. Cellular degradation was concomitant with dramatic decreases in photosynthetic efficiency, externalization of phosphatidylserine, and significantly elevated caspase-specific activity, with the addition of a broad-spectrum caspase inhibitor rescuing cells from death. A search of the T. pseudonana genome identified six distinct putative metacaspases containing a conserved caspase domain structure. Quantitative reverse transcription-PCR and Western blot analysis revealed differential gene and protein expression of T. pseudonana metacaspases, some of which correlated with physiological stress and caspase activity. Taken together with the recent discovery of the metacaspase-mediated viral infection of phytoplankton (K. D. Bidle, L. Haramaty, J. Barcelos-Ramos, and P. G. Falkowski, Proc. Natl. Acad. Sci. USA 104: 6049-6054, 2007), our findings reveal a key role for metacaspases in the turnover of phytoplankton biomass in the oceans. Furthermore, given that Fe is required for photosynthetic electron transfer and is chronically limiting in a variety of oceanic systems, including high-nutrient low-chlorophyll regions, our findings provide a potential ecological context for PCD in these unicellular photoautotrophs.

Diatoms are among the most diverse groups of phytoplankton in the ocean. Despite their widely recognized influence on ocean ecosystems and global biogeochemistry, little is known about the impact of this diversity on large-scale processes. Here, we examined the ramifications of between-species diversity by documenting the transcriptional response of three diatoms —Thalassiosira pseudonana, Fragilariopsis cylindrus, and Pseudo-nitzschia multiseries —to the onset of nitrate limitation of growth, a common limiting nutrient in the ocean. The three species shared 5583 clusters of orthologous genes based on OrthoMCL clustering of publically available diatom genomes. These clusters represent 30–54% of the predicted genes in each diatom genome. Less than 5% of genes within these core clusters displayed the same transcriptional responses across species when growth was limited by nitrate availability. Orthologs, such as those involved in nitrogen uptake and assimilation, as well as carbon metabolism, were differently expressed across the three species. The two pennate diatoms, F. cylindrus and P. multiseries, shared 3839 clusters without orthologs in the genome of the centric diatom T. pseudonana. A majority of these pennate-clustered genes, as well as the non-orthologous genes in each species, had minimal annotation information, but were often significantly differentially expressed under nitrate limitation, indicating their potential importance in the response to nitrogen availability. Despite these variations in the specific transcriptional response of each diatom, overall transcriptional patterns suggested that all three diatoms displayed a common physiological response to nitrate limitation that consisted of a general reduction in carbon fixation and carbohydrate and fatty acid metabolism and an increase in nitrogen recycling. Characterization of these finely tuned responses will help to better predict which types of diatoms will bloom under which sets of environmental factors.

Strain HIMB11 is a planktonic marine bacterium isolated from coastal seawater in Kaneohe Bay, Oahu, Hawaii belonging to the ubiquitous and versatile Roseobacter clade of the alphaproteobacterial family Rhodobacteraceae. Here we describe the preliminary characteristics of strain HIMB11, including annotation of the draft genome sequence and comparative genomic analysis with other members of the Roseobacter lineage. The 3,098,747 bp draft genome is arranged in 34 contigs and contains 3,183 protein-coding genes and 54 RNA genes. Phylogenomic and 16S rRNA gene analyses indicate that HIMB11 represents a unique sublineage within the Roseobacter clade. Comparison with other publicly available genome sequences from members of the Roseobacter lineage reveals that strain HIMB11 has the genomic potential to utilize a wide variety of energy sources (e.g. organic matter, reduced inorganic sulfur, light, carbon monoxide), while possessing a reduced number of substrate transporters., Gordon and Betty Moore Foundation, Agouron Institute, University of Hawaii and Manoa School of Ocean and Earth Science and Technology, National Science Foundation (U.S.). Science and Technology Center (Award EF0424599)