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2017
Zeigler Allen, L, McCrow JP, Ininbergs K, Dupont CL, Badger JH, Hoffman JM, Ekman M, Allen AE, Bergman B, Venter CJ.  2017.  The Baltic Sea virome: Diversity and transcriptional activity of DNA and RNA viruses. mSystems. 2( Weitz J, Ed.).   10.1128/mSystems.00125-16   Abstract

Metagenomic and metatranscriptomic data were generated from size-fractionated samples from 11 sites within the Baltic Sea and adjacent marine waters of Kattegat and freshwater Lake Torneträsk in order to investigate the diversity, distribution, and transcriptional activity of virioplankton. Such a transect, spanning a salinity gradient from freshwater to the open sea, facilitated a broad genome-enabled investigation of natural as well as impacted aspects of Baltic Sea viral communities. Taxonomic signatures representative of phages within the widely distributed order Caudovirales were identified with enrichments in lesser-known families such as Podoviridae and Siphoviridae. The distribution of phage reported to infect diverse and ubiquitous heterotrophic bacteria (SAR11 clades) and cyanobacteria (Synechococcus sp.) displayed population-level shifts in diversity. Samples from higher-salinity conditions (>14 practical salinity units [PSU]) had increased abundances of viruses for picoeukaryotes, i.e., Ostreococcus. These data, combined with host diversity estimates, suggest viral modulation of diversity on the whole-community scale, as well as in specific prokaryotic and eukaryotic lineages. RNA libraries revealed single-stranded DNA (ssDNA) and RNA viral populations throughout the Baltic Sea, with ssDNA phage highly represented in Lake Torneträsk. Further, our data suggest relatively high transcriptional activity of fish viruses within diverse families known to have broad host ranges, such as Nodoviridae (RNA), Iridoviridae (DNA), and predicted zoonotic viruses that can cause ecological and economic damage as well as impact human health.

Levering, J, Dupont CL, Allen AE, Palsson BO, Zengler K.  2017.  Integrated regulatory and metabolic networks of the marine diatom Phaeodactylum tricornutum predict the response to rising CO2 levels. mSystems. 2( Lin X, Ed.).   10.1128/mSystems.00142-16   Abstract

Diatoms are eukaryotic microalgae that are responsible for up to 40% of the ocean’s primary productivity. How diatoms respond to environmental perturbations such as elevated carbon concentrations in the atmosphere is currently poorly understood. We developed a transcriptional regulatory network based on various transcriptome sequencing expression libraries for different environmental responses to gain insight into the marine diatom’s metabolic and regulatory interactions and provide a comprehensive framework of responses to increasing atmospheric carbon levels. This transcriptional regulatory network was integrated with a recently published genome-scale metabolic model of Phaeodactylum tricornutum to explore the connectivity of the regulatory network and shared metabolites. The integrated regulatory and metabolic model revealed highly connected modules within carbon and nitrogen metabolism. P. tricornutum’s response to rising carbon levels was analyzed by using the recent genome-scale metabolic model with cross comparison to experimental manipulations of carbon dioxide.IMPORTANCE Using a systems biology approach, we studied the response of the marine diatom Phaeodactylum tricornutum to changing atmospheric carbon concentrations on an ocean-wide scale. By integrating an available genome-scale metabolic model and a newly developed transcriptional regulatory network inferred from transcriptome sequencing expression data, we demonstrate that carbon metabolism and nitrogen metabolism are strongly connected and the genes involved are coregulated in this model diatom. These tight regulatory constraints could play a major role during the adaptation of P. tricornutum to increasing carbon levels. The transcriptional regulatory network developed can be further used to study the effects of different environmental perturbations on P. tricornutum’s metabolism.

Mock, T, Otillar RP, Strauss J, McMullan M, Paajanen P, Schmutz J, Salamov A, Sanges R, Toseland A, Ward BJ, Allen AE, Dupont CL, Frickenhaus S, Maumus F, Veluchamy A, Wu T, Barry KW, Falciatore A, Ferrante MI, Fortunato AE, Glöckner G, Gruber A, Hipkin R, Janech MG, Kroth PG, Leese F, Lindquist EA, Lyon BR, Martin J, Mayer C, Parker M, Quesneville H, Raymond JA, Uhlig C, Valas RE, Valentin KU, Worden AZ, Armbrust VE, Clark MD, Bowler C, Green BR, Moulton V, van Oosterhout C, Grigoriev IV.  2017.  Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus. Nature. :1-5.: Macmillan Publishers Limited, part of Springer Nature. All rights reserved.   10.1038/nature20803   Abstract

The Southern Ocean houses a diverse and productive community of organisms. Unicellular eukaryotic diatoms are the main primary producers in this environment, where photosynthesis is limited by low concentrations of dissolved iron and large seasonal fluctuations in light, temperature and the extent of sea ice. How diatoms have adapted to this extreme environment is largely unknown. Here we present insights into the genome evolution of a cold-adapted diatom from the Southern Ocean, Fragilariopsis cylindrus, based on a comparison with temperate diatoms. We find that approximately 24.7 per cent of the diploid F. cylindrus genome consists of genetic loci with alleles that are highly divergent (15.1 megabases of the total genome size of 61.1 megabases). These divergent alleles were differentially expressed across environmental conditions, including darkness, low iron, freezing, elevated temperature and increased CO2. Alleles with the largest ratio of non-synonymous to synonymous nucleotide substitutions also show the most pronounced condition-dependent expression, suggesting a correlation between diversifying selection and allelic differentiation. Divergent alleles may be involved in adaptation to environmental fluctuations in the Southern Ocean.

Caron, DA, Alexander H, Allen AE, Archibald JM, Armbrust VE, Bachy C, Bell CJ, Bharti A, Dyhrman ST, Guida SM, Heidelberg KB, Kaye JZ, Metzner J, Smith SR, Worden AZ.  2017.  Probing the evolution, ecology and physiology of marine protists using transcriptomics. Nat Rev Micro. 15:6-20.: Nature Publishing Group   10.1038/nrmicro.2016.160   Abstract

Protists, which are single-celled eukaryotes, critically influence the ecology and chemistry of marine ecosystems, but genome-based studies of these organisms have lagged behind those of other microorganisms. However, recent transcriptomic studies of cultured species, complemented by meta-omics analyses of natural communities, have increased the amount of genetic information available for poorly represented branches on the tree of eukaryotic life. This information is providing insights into the adaptations and interactions between protists and other microorganisms and macroorganisms, but many of the genes sequenced show no similarity to sequences currently available in public databases. A better understanding of these newly discovered genes will lead to a deeper appreciation of the functional diversity and metabolic processes in the ocean. In this Review, we summarize recent developments in our understanding of the ecology, physiology and evolution of protists, derived from transcriptomic studies of cultured strains and natural communities, and discuss how these novel large-scale genetic datasets will be used in the future.

2016
Smith, SR, Gillard JTF, Kustka AB, McCrow JP, Badger JH, Zheng H, New AM, Dupont CL, Obata T, Fernie AR, Allen AE.  2016.  Transcriptional orchestration of the global cellular response of a model pennate diatom to diel light cycling under iron limitation. PLOS Genetics. 12:e1006490.: Public Library of Science   10.1371/journal.pgen.1006490   Abstract

Oceanic diatoms live in constantly fluctuating environments to which they must adapt in order to survive. During sunlit hours, photosynthesis occurs allowing diatoms to store energy used at night to sustain energy demands. Cellular and molecular mechanisms for regulation of phytoplankton growth are important to understand because of their environmental roles at the base of food webs and in regulating carbon flux out of the atmosphere. In ocean ecosystems, the availability of iron (Fe) commonly limits phytoplankton growth and diatoms typically outcompete other phytoplankton when Fe is added, indicating they have adaptations allowing them to both survive at low Fe and rapidly respond to Fe additions. These adaptations may be unique depending on isolation from coastal or oceanic locations. To identify adaptive strategies, we characterized the response of a model diatom, Phaeodactylum tricornutum, to limiting Fe conditions over day:night cycles using a combination of gene expression analyses, metabolite, and physiology measurements. Major coordinated shifts in metabolism and growth were documented over diel cycles, with peak expression of low Fe expressed genes in the dark phase. Diatoms respond to limiting Fe by increasing Fe acquisition, while decreasing growth rate through slowed cell cycle progression, reduced energy acquisition, and subtle metabolic remodeling.

Traller, JC, Cokus SJ, Lopez DA, Gaidarenko O, Smith SR, McCrow JP, Gallaher SD, Podell S, Thompson M, Cook O, Morselli M, Jaroszewicz A, Allen EE, Allen AE, Merchant SS, Pellegrini M, Hildebrand M.  2016.  Genome and methylome of the oleaginous diatom Cyclotella cryptica reveal genetic flexibility toward a high lipid phenotype. Biotechnology for Biofuels. 9:258.   10.1186/s13068-016-0670-3   AbstractWebsite

Improvement in the performance of eukaryotic microalgae for biofuel and bioproduct production is largely dependent on characterization of metabolic mechanisms within the cell. The marine diatom Cyclotella cryptica, which was originally identified in the Aquatic Species Program, is a promising strain of microalgae for large-scale production of biofuel and bioproducts, such as omega-3 fatty acids.

Kharbush, JJ, Allen AE, Moustafa A, Dorrestein PC, Aluwihare LI.  2016.  Intact polar diacylglycerol biomarker lipids isolated from suspended particulate organic matter accumulating in an ultraoligotrophic water column. Organic Geochemistry. 100:29-41.   10.1016/j.orggeochem.2016.07.008   Abstract

Intact polar diacylglycerols (IP-DAGs) are essential components of cell membranes. Because they are structurally diverse and hypothesized to represent primarily living cells, they are potential molecular markers for a recent contribution by microbial communities to various carbon reservoirs. This study employed a novel molecular networking approach to investigate the evolution of IP-DAG structural diversity with depth in an ultraoligotrophic environment of the western South Pacific Ocean to test the hypothesis that particle transport to depth is rapid enough to preserve the IP-DAG biomarker signature of the photosynthetic community. IP-DAG profiles of several cultured cyanobacteria and photosynthetic picoeukaryotes were used as templates for constructing molecular networks to compare and interpret IP-DAG signatures of suspended particles isolated from a water column depth profile. Analysis of corresponding genetic community composition data for the same field samples was used to connect IP-DAG structures with their likely biological sources. Our data show that, although most IP-DAG classes associated with photosynthetic organisms were not observed below the euphotic zone, several other IP-DAG classes in deep samples might provide interesting targets for future studies seeking to examine the in situ contribution of deep sea microbes to suspended particulate organic matter (POM). Overall, the results represent the deepest water column IP-DAG dataset to date and demonstrate the utility of molecular networking for analyzing and visualizing complex environmental datasets.

Zielinski, BL, Allen AE, Carpenter EJ, Coles VJ, Crump BC, Doherty M, Foster RA, Goes JI, Gomes HR, Hood RR, McCrow JP, Montoya JP, Moustafa A, Satinsky BM, Sharma S, Smith CB, Yager PL, Paul JH.  2016.  Patterns of transcript abundance of eukaryotic biogeochemically-relevant genes in the Amazon River plume. PLoS One. 11:e0160929.   10.1371/journal.pone.0160929   Abstract

The Amazon River has the largest discharge of all rivers on Earth, and its complex plume system fuels a wide array of biogeochemical processes, across a large area of the western tropical North Atlantic. The plume thus stimulates microbial processes affecting carbon sequestration and nutrient cycles at a global scale. Chromosomal gene expression patterns of the 2.0 to 156 mum size-fraction eukaryotic microbial community were investigated in the Amazon River Plume, generating a robust dataset (more than 100 million mRNA sequences) that depicts the metabolic capabilities and interactions among the eukaryotic microbes. Combining classical oceanographic field measurements with metatranscriptomics yielded characterization of the hydrographic conditions simultaneous with a quantification of transcriptional activity and identity of the community. We highlight the patterns of eukaryotic gene expression for 31 biogeochemically significant gene targets hypothesized to be valuable within forecasting models. An advantage to this targeted approach is that the database of reference sequences used to identify the target genes was selectively constructed and highly curated optimizing taxonomic coverage, throughput, and the accuracy of annotations. A coastal diatom bloom highly expressed nitrate transporters and carbonic anhydrase presumably to support high growth rates and enhance uptake of low levels of dissolved nitrate and CO2. Diatom-diazotroph association (DDA: diatoms with nitrogen fixing symbionts) blooms were common when surface salinity was mesohaline and dissolved nitrate concentrations were below detection, and hence did not show evidence of nitrate utilization, suggesting they relied on ammonium transporters to aquire recently fixed nitrogen. These DDA blooms in the outer plume had rapid turnover of the photosystem D1 protein presumably caused by photodegradation under increased light penetration in clearer waters, and increased expression of silicon transporters as silicon became limiting. Expression of these genes, including carbonic anhydrase and transporters for nitrate and phosphate, were found to reflect the physiological status and biogeochemistry of river plume environments. These relatively stable patterns of eukaryotic transcript abundance occurred over modest spatiotemporal scales, with similarity observed in sample duplicates collected up to 2.45 km in space and 120 minutes in time. These results confirm the use of metatranscriptomics as a valuable tool to understand and predict microbial community function.

Diner, RE, Bielinski VA, Dupont CL, Allen AE, Weyman PD.  2016.  Refinement of the diatom episome maintenance sequence and improvement of conjugation-based DNA delivery methods. Frontiers in Bioengineering and Biotechnology. 4   10.3389/fbioe.2016.00065   AbstractWebsite

Conjugation of episomal plasmids from bacteria to diatoms advances diatom genetic manipulation by simplifying transgene delivery and providing a stable and consistent gene expression platform. To reach its full potential, this nascent technology requires new optimized expression vectors and a deeper understanding of episome maintenance. Here we present the development of an additional diatom vector (pPtPBR1), based on the parent plasmid pBR322, to add a plasmid maintained at medium copy number in E. coli to the diatom genetic toolkit. Using this new vector, we evaluated the contribution of individual yeast DNA elements comprising the 1.4-kb tripartite CEN6-ARSH4-HIS3 sequence that enables episome maintenance in P. tricornutum. While various combinations of these individual elements enable efficient conjugation and high ex-conjugant yield in P. tricornutum, individual elements alone do not. Conjugation of episomes containing CEN6-ARSH4 and a small sequence from the low GC content 3’ end of HIS3 produced the highest number of diatom ex-conjugant colonies, resulting in a smaller and more efficient vector design. Our findings suggest that the CEN6 and ARSH4 sequences function differently in yeast and diatoms, and that low GC content regions of greater than ~500 bp are a potential indicator of a functional diatom episome maintenance sequence. Additionally, we have developed improvements to the conjugation protocol including a higher-throughput option utilizing 12-well plates, and plating methods that improve ex-conjugant yield and reduce time and materials required for the conjugation protocol. The data presented offer additional information regarding the mechanism by which the yeast-derived sequence enables diatom episome maintenance, and demonstrate options for flexible vector design.

Asplund-Samuelsson, J, Sundh J, Dupont CL, Allen AE, McCrow JP, Celepli NA, Bergman B, Ininbergs K, Ekman M.  2016.  Diversity and expression of bacterial metacaspases in an aquatic ecosystem. Frontiers in Microbiology. 7   10.3389/fmicb.2016.01043   AbstractWebsite

Metacaspases are distant homologs of metazoan caspase proteases, implicated in stress response and programmed cell death in bacteria and phytoplankton. While the few previous studies on metacaspases have relied on cultured organisms and sequenced genomes, no studies have focused on metacaspases in a natural setting. We here present data from the first microbial community-wide metacaspase survey; performed by querying metagenomic and metatranscriptomic datasets from the brackish Baltic Sea, a water body characterized by pronounced environmental gradients and periods of massive cyanobacterial blooms. Metacaspase genes were restricted to approximately 4% of the bacteria, taxonomically affiliated mainly to Bacteroidetes, Alpha- and Betaproteobacteria and Cyanobacteria. The gene abundance was significantly higher in larger or particle-associated bacteria (> 0.8 µm), and filamentous Cyanobacteria dominated metacaspase gene expression throughout the bloom season. Distinct seasonal expression patterns were detected for the three metacaspase genes in Nodularia spumigena, one of the main bloom-formers. Clustering of normalized gene expression in combination with analyses of genomic and assembly data suggest functional diversification of these genes, and possible roles of the metacaspase genes related to stress responses, i.e. sulfur metabolism in connection to oxidative stress, and nutrient stress induced cellular differentiation. Co-expression of genes encoding metacaspases and nodularin toxin synthesis enzymes was also observed in Nodularia spumigena. The study shows that metacaspases represent an adaptation of potentially high importance for several key organisms in the Baltic Sea, most prominently Cyanobacteria, and open up for further exploration of their physiological roles in microbes and assessment of their ecological impact in aquatic habitats.

Diner, RE, Schwenck SM, McCrow JP, Zheng H, Allen AE.  2016.  Genetic manipulation of competition for nitrate between heterotrophic bacteria and diatoms. Frontiers in Microbiology. 7   10.3389/fmicb.2016.00880   Abstract

Diatoms are a dominant group of eukaryotic phytoplankton that contribute substantially to global primary production and the cycling of important elements such as carbon and nitrogen. Heterotrophic bacteria, including members of the gammaproteobacteria, are commonly associated with diatom populations and may rely on them for organic carbon while potentially competing with them for other essential nutrients. Considering that bacterioplankton drive oceanic release of CO2 (i.e., bacterial respiration) while diatoms drive ocean carbon sequestration vial the biological pump, the outcome of such competition could influence the direction and magnitude of carbon flux in the upper ocean. Nitrate availability is commonly a determining factor for the growth of diatom populations, particularly in coastal and upwelling regions. Diatoms as well as many bacterial species can utilize nitrate, however the ability of bacteria to compete for nitrate may be hindered by carbon limitation. Here we have developed a genetically tractable model system using the pennate diatom Phaeodactylum tricornutum and the widespread heterotrophic bacteria Alteromonas macleodii to examine carbon-nitrogen dynamics. While subsisting solely on P. tricornutum derived carbon, A. macleodii does not appear to be an effective competitor for nitrate, and may in fact benefit the diatom; particularly in stationary phase. However, allochthonous dissolved organic carbon addition in the form of pyruvate triggers A. macleodii proliferation and nitrate uptake, leading to reduced P. tricornutum growth. Nitrate reductase deficient mutants of A. macleodii (ΔnasA) do not exhibit such explosive growth and associated competitive ability in response to allochthonous carbon when nitrate is the sole nitrogen source, but could survive by utilizing solely P. tricornutum-derived nitrogen. Furthermore, allocthonous carbon addition enables wild-type A. macleodii to rescue nitrate reductase deficient P. tricornutum populations from nitrogen starvation, and RNA-seq transcriptomic evidence supports nitrogen-based interactions between diatoms and bacteria at the molecular level. This study provides key insights into the roles of carbon and nitrogen in phytoplankton-bacteria dynamics and lays the foundation for developing a mechanistic understanding of these interactions using co-culturing and genetic manipulation.

DÌez, B, Nylander JAA, Ininbergs K, Dupont CL, Allen AE, Yooseph S, Rusch DB, Bergman B.  2016.  Metagenomic analysis of the Indian Ocean picocyanobacterial community: Structure, potential function and evolution. PLoS ONE. 11:e0155757.: Public Library of Science   10.1371/journal.pone.0155757   Abstract

Unicellular cyanobacteria are ubiquitous photoautotrophic microbes that contribute substantially to global primary production. Picocyanobacteria such as Synechococcus and Prochlorococcus depend on chlorophyll a-binding protein complexes to capture light energy. In addition, Synechococcus has accessory pigments organized into phycobilisomes, and Prochlorococcus contains chlorophyll b. Across a surface water transect spanning the sparsely studied tropical Indian Ocean, we examined Synechococcus and Prochlorococcus occurrence, taxonomy and habitat preference in an evolutionary context. Shotgun sequencing of size fractionated microbial communities from 0.1 ?m to 20 ?m and subsequent phylogenetic analysis indicated that cyanobacteria account for up to 15% of annotated reads, with the genera Prochlorococcus and Synechococcus comprising 90% of the cyanobacterial reads, even in the largest size fraction (3.0?20 mm). Phylogenetic analyses of cyanobacterial light-harvesting genes (chl-binding pcb/isiA, allophycocyanin (apcAB), phycocyanin (cpcAB) and phycoerythin (cpeAB)) mostly identified picocyanobacteria clades comprised of overlapping sequences obtained from Indian Ocean, Atlantic and/or Pacific Oceans samples. Habitat reconstructions coupled with phylogenetic analysis of the Indian Ocean samples suggested that large Synechococcus-like ancestors in coastal waters expanded their ecological niche towards open oligotrophic waters in the Indian Ocean through lineage diversification and associated streamlining of genomes (e.g. loss of phycobilisomes and acquisition of Chl b); resulting in contemporary small celled Prochlorococcus. Comparative metagenomic analysis with picocyanobacteria populations in other oceans suggests that this evolutionary scenario may be globally important.

Levering, J, Broddrick J, Dupont CL, Peers G, Beeri K, Mayers J, Gallina AA, Allen AE, Palsson BO, Zengler K.  2016.  Genome-scale model reveals metabolic basis of biomass partitioning in a model diatom. Plos One. 11   10.1371/journal.pone.0155038   AbstractWebsite

Diatoms are eukaryotic microalgae that contain genes from various sources, including bacteria and the secondary endosymbiotic host. Due to this unique combination of genes, diatoms are taxonomically and functionally distinct from other algae and vascular plants and confer novel metabolic capabilities. Based on the genome annotation, we performed a genome-scale metabolic network reconstruction for the marine diatom Phaeodactylum tricornutum. Due to their endosymbiotic origin, diatoms possess a complex chloroplast structure which complicates the prediction of subcellular protein localization. Based on previous work we implemented a pipeline that exploits a series of bioinformatics tools to predict protein localization. The manually curated reconstructed metabolic network iLB1027_lipid accounts for 1,027 genes associated with 4,456 reactions and 2,172 metabolites distributed across six compartments. To constrain the genome-scale model, we determined the organism specific biomass composition in terms of lipids, carbohydrates, and proteins using Fourier transform infrared spectrometry. Our simulations indicate the presence of a yet unknown glutamine-ornithine shunt that could be used to transfer reducing equivalents generated by photosynthesis to the mitochondria. The model reflects the known biochemical composition of P. tricornutum in defined culture conditions and enables metabolic engineering strategies to improve the use of P. tricornutum for biotechnological applications.

Smith, SR, Glé C, Abbriano RM, Traller JC, Davis A, Trentacoste E, Vernet M, Allen AE, Hildebrand M.  2016.  Transcript level coordination of carbon pathways during silicon starvation-induced lipid accumulation in the diatom Thalassiosira pseudonana. New Phytologist.   10.1111/nph.13843   Abstract

* Diatoms are one of the most productive and successful photosynthetic taxa on Earth and possess attributes such as rapid growth rates and production of lipids, making them candidate sources of renewable fuels. Despite their significance, few details of the mechanisms used to regulate growth and carbon metabolism are currently known, hindering metabolic engineering approaches to enhance productivity. * To characterize the transcript level component of metabolic regulation, genome-wide changes in transcript abundance were documented in the model diatom Thalassiosira pseudonana on a time-course of silicon starvation. Growth, cell cycle progression, chloroplast replication, fatty acid composition, pigmentation, and photosynthetic parameters were characterized alongside lipid accumulation. * Extensive coordination of large suites of genes was observed, highlighting the existence of clusters of coregulated genes as a key feature of global gene regulation in T. pseudonana. The identity of key enzymes for carbon metabolic pathway inputs (photosynthesis) and outputs (growth and storage) reveals these clusters are organized to synchronize these processes. * Coordinated transcript level responses to silicon starvation are probably driven by signals linked to cell cycle progression and shifts in photophysiology. A mechanistic understanding of how this is accomplished will aid efforts to engineer metabolism for development of algal-derived biofuels.

2015
Bertrand, EM, McCrow JP, Moustafa A, Zheng H, McQuaid JB, Delmont TO, Post AF, Sipler RE, Spackeen JL, Xu K, Bronk DA, Hutchins DA, Allen AE.  2015.  Phytoplankton–bacterial interactions mediate micronutrient colimitation at the coastal Antarctic sea ice edge. Proceedings of the National Academy of Sciences.   10.1073/pnas.1501615112   Abstract

Southern Ocean primary productivity plays a key role in global ocean biogeochemistry and climate. At the Southern Ocean sea ice edge in coastal McMurdo Sound, we observed simultaneous cobalamin and iron limitation of surface water phytoplankton communities in late Austral summer. Cobalamin is produced only by bacteria and archaea, suggesting phytoplankton–bacterial interactions must play a role in this limitation. To characterize these interactions and investigate the molecular basis of multiple nutrient limitation, we examined transitions in global gene expression over short time scales, induced by shifts in micronutrient availability. Diatoms, the dominant primary producers, exhibited transcriptional patterns indicative of co-occurring iron and cobalamin deprivation. The major contributor to cobalamin biosynthesis gene expression was a gammaproteobacterial population, Oceanospirillaceae ASP10-02a. This group also contributed significantly to metagenomic cobalamin biosynthesis gene abundance throughout Southern Ocean surface waters. Oceanospirillaceae ASP10-02a displayed elevated expression of organic matter acquisition and cell surface attachment-related genes, consistent with a mutualistic relationship in which they are dependent on phytoplankton growth to fuel cobalamin production. Separate bacterial groups, including Methylophaga, appeared to rely on phytoplankton for carbon and energy sources, but displayed gene expression patterns consistent with iron and cobalamin deprivation. This suggests they also compete with phytoplankton and are important cobalamin consumers. Expression patterns of siderophore- related genes offer evidence for bacterial influences on iron availability as well. The nature and degree of this episodic colimitation appear to be mediated by a series of phytoplankton–bacterial interactions in both positive and negative feedback loops.

Llewellyn, CA, Sommer U, Dupont CL, Allen AE, Viant MR.  2015.  Using community metabolomics as a new approach to discriminate marine microbial particulate organic matter in the western English Channel. Progress in Oceanography.   10.1016/j.pocean.2015.04.022   AbstractWebsite

Metabolomics provides an unbiased assessment of a wide range of metabolites and is an emerging ‘omics technique in the marine sciences. We use ‘non-targeted’ community metabolomics to determine patterns in metabolite profiles associated with particulate organic matter (POM) at four locations from two long-term monitoring stations (L4 and E1) in the western English Channel. The polar metabolite fractions were measured using ultra-high performance liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (UHPLC-FT-ICR-MS), and the lipid fractions by direct infusion Fourier transform ion cyclotron resonance mass spectrometry (DI-FT-ICR-MS); these were then analysed to statistically compare the metabolite distributions. Results show significantly different profiles of metabolites across the four locations with the largest differences for both the polar and lipid fractions found between the two stations relative to the smaller differences associated with depth. We putatively annotate the most discriminant metabolites revealing a range of amino-acid derivatives, diacylglyceryltrimethylhomoserine (DGTS) lipids, oxidised fatty acids (oxylipins), glycosylated compounds, oligohexoses, phospholipids, triacylglycerides (TAGs) and oxidised TAGs. The majority of the polar metabolites were most abundant in the surface waters at L4 and least abundant in the deep waters at E1 (E1-70m). In contrast, the oxidised TAGs were more abundant at E1 and most abundant at E1-70m. The differentiated metabolites are discussed in relation to the health of the phytoplankton as indicated by nutrients, carbon and chlorophyll, and to the dominance (determined from metatranscript data) of the picoeukaryote Ostreococcus. Our results show proof of concept for community metabolomics in discriminating and characterising polar and lipid metabolite patterns associated with marine POM.

Veluchamy, A, Rastogi A, Lin X, Lombard B, Murik O, Thomas Y, Dingli F, Rivarola M, Ott S, Liu X, Sun Y, Rabinowicz PD, McCarthy J, Allen AE, Loew D, Bowler C, Tirichine L.  2015.  An integrative analysis of post-translational histone modifications in the marine diatom Phaeodactylum tricornutum. Genome Biol. 16:102.   10.1186/s13059-015-0671-8   Abstract

BACKGROUND: Nucleosomes are the building blocks of chromatin where gene regulation takes place. Chromatin landscapes have been profiled for several species, providing insights into the understanding of fundamental mechanisms of chromatin-mediated transcriptional regulation of gene expression. However, knowledge is missing from several major and deep-branching eukaryotic groups, such as the marine model diatom Phaeodactylum tricornutum. Diatoms are highly diverse and ubiquitous species of phytoplankton that play a key role in global biogeochemical cycles. Dissecting chromatin-mediated regulation of genes in diatoms will help understand the ecological success of these organisms in contemporary oceans. RESULTS: Here, we use high resolution mass spectrometry to identify a full repertoire of post-translational modifications on P. tricornutum histones, including eight novel modifications. We map five histone marks coupled with expression data and show that P. tricornutum displays both unique and broadly conserved chromatin features, reflecting the chimeric nature of its genome. Combinatorial analysis of histone marks and DNA methylation demonstrates the presence of an epigenetic code defining active or repressive chromatin states. We further profile three specific histone marks under conditions of nitrate depletion and show that the histone code is dynamic and targets specific sets of genes. CONCLUSIONS: This study is the first genome-wide characterization of the histone code from a Stramenopile and a marine phytoplankton. The work represents an important initial step for understanding the evolutionary history of chromatin and how epigenetic modifications affect gene expression in response to environmental cues in marine environments.

Karas, BJ, Diner RE, Lefebvre SC, McQuaid J, Phillips APR, Noddings CM, Brunson JK, Valas RE, Deerinck TJ, Jablanovic J, Gillard JTF, Beeri K, Ellisman MH, Glass JI, Hutchison Iii CA, Smith HO, Venter CJ, Allen AE, Dupont CL, Weyman PD.  2015.  Designer diatom episomes delivered by bacterial conjugation. Nat Commun. 6: Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.   10.1038/ncomms7925   Abstract

Eukaryotic microalgae hold great promise for the bioproduction of fuels and higher value chemicals. However, compared with model genetic organisms such as Escherichia coli and Saccharomyces cerevisiae, characterization of the complex biology and biochemistry of algae and strain improvement has been hampered by the inefficient genetic tools. To date, many algal species are transformable only via particle bombardment, and the introduced DNA is integrated randomly into the nuclear genome. Here we describe the first nuclear episomal vector for diatoms and a plasmid delivery method via conjugation from Escherichia coli to the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. We identify a yeast-derived sequence that enables stable episome replication in these diatoms even in the absence of antibiotic selection and show that episomes are maintained as closed circles at copy number equivalent to native chromosomes. This highly efficient genetic system facilitates high-throughput functional characterization of algal genes and accelerates molecular phytoplankton research.

Morrissey, J, Sutak R, Paz-Yepes J, Tanaka A, Moustafa A, Veluchamy A, Thomas Y, Botebol H, Bouget F-Y, McQuaid J B, Tirichine L, Allen A E, Lesuisse E, Bowler C.  2015.  A novel protein, ubiquitous in marine phytoplankton, concentrates iron at the cell surface and facilitates uptake. Current Biology. 25:364-371.   10.1016/j.cub.2014.12.004   Abstract

Summary Numerous cellular functions including respiration require iron. Plants and phytoplankton must also maintain the iron-rich photosynthetic electron transport chain, which most likely evolved in the iron-replete reducing environments of the Proterozoic ocean [1]. Iron bioavailability has drastically decreased in the contemporary ocean [1], most likely selecting for the evolution of efficient iron acquisition mechanisms among modern phytoplankton. Mesoscale iron fertilization experiments often result in blooms dominated by diatoms [2], indicating that diatoms have adaptations that allow survival in iron-limited waters and rapid multiplication when iron becomes available. Yet the genetic and molecular bases are unclear, as very few iron uptake genes have been functionally characterized from marine eukaryotic phytoplankton, and large portions of diatom iron starvation transcriptomes are genes encoding unknown functions [3–5]. Here we show that the marine diatom Phaeodactylum tricornutum utilizes ISIP2a to concentrate Fe(III) at the cell surface as part of a novel, copper-independent and thermodynamically controlled iron uptake system. ISIP2a is expressed in response to iron limitation several days prior to the induction of ferrireductase activity, and it facilitates significant Fe(III) uptake during the initial response to Fe limitation. ISIP2a is able to directly bind Fe(III) and increase iron uptake when heterologously expressed, whereas knockdown of ISIP2a in P. tricornutum decreases iron uptake, resulting in impaired growth and chlorosis during iron limitation. ISIP2a is expressed by diverse marine phytoplankton, indicating that it is an ecologically significant adaptation to the unique nutrient composition of marine environments.

Paerl, RW, Bertrand EM, Allen AE, Palenik B, Azam F.  2015.  Vitamin B1 ecophysiology of marine picoeukaryotic algae: Strain-specific differences and a new role for bacteria in vitamin cycling. Limnology and Oceanography. 60:215-228.   10.1002/lno.10009   AbstractWebsite

We confirmed multiple picoeukaryotic algae, Ostreococcus, Micromonas, and Pelagomonas spp., as thiamine (vitamin B1) auxotrophs in laboratory experiments with axenic cultures. Examined strains have half saturation growth constants (K-s) for B1 between 1.26 and 6.22 pmol B1 L-1, which is higher than reported seawater concentrations. Minimum B1 cell quotas for Ostreococcus and Micromonas spp. are high (2.20 x 10(-8)-4.46 x 10(-8) pmol B1 cell(-1)) relative to other B1 auxotrophic phytoplankton, potentially making them B1 rich prey for zooplankton and significant B1 reservoirs in oligotrophic marine habitats. Ostreococcus and Micromonas genomes are nonuniformly missing portions of the B1 biosynthesis pathway. Given their gene repertoires, Ostreococcus lucimarinus CCE9901 and Ostreococcus tauri OTH95 are expected to salvage B1 from externally provided 4-methyl-5-thiazoleethanol (HET) and 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP). However, in culture, neither could use HET plus HMP instead of B1, highlighting current limitations of genome-based prediction of B1 salvaging by picoeukaryotic algae. HMP and phosphorylated B1 use varied amongst tested strains and notably all Prasinophytes tested could not use HMP. B1-limited O. lucimarinus CCE9901 could not grow on added thiamine diphosphate (TDP), a phosophorylated B1 form. However, in co-culture with Pseudoalteromonas sp. TW7, a bacterium known to exhibit phosphatase activity, O. lucimarinus CCE9901 exhibited increased growth following TDP additions. This demonstrates that bacteria influence vitamin B1 availability beyond de novo synthesis and consumption; they can also serve as conduits that chemically alter, but not completely degrade or retain B1 analogs (e.g., TDP), and make them accessible to a broader range of microbes.

Dupont, CL, McCrow JP, Valas R, Moustafa A, Walworth N, Goodenough U, Roth R, Hogle SL, Bai J, Johnson ZI, Mann E, Palenik B, Barbeau KA, Craig Venter J, Allen AE.  2015.  Genomes and gene expression across light and productivity gradients in eastern subtropical Pacific microbial communities. ISME J. 9:1076-1092.: International Society for Microbial Ecology   10.1038/ismej.2014.198   Abstract

Transitions in community genomic features and biogeochemical processes were examined in surface and subsurface chlorophyll maximum (SCM) microbial communities across a trophic gradient from mesotrophic waters near San Diego, California to the oligotrophic Pacific. Transect end points contrasted in thermocline depth, rates of nitrogen and CO2 uptake, new production and SCM light intensity. Relative to surface waters, bacterial SCM communities displayed greater genetic diversity and enrichment in putative sulfur oxidizers, multiple actinomycetes, low-light-adapted Prochlorococcus and cell-associated viruses. Metagenomic coverage was not correlated with transcriptional activity for several key taxa within Bacteria. Low-light-adapted Prochlorococcus, Synechococcus, and low abundance gamma-proteobacteria enriched in the>3.0-[mu]m size fraction contributed disproportionally to global transcription. The abundance of these groups also correlated with community functions, such as primary production or nitrate uptake. In contrast, many of the most abundant bacterioplankton, including SAR11, SAR86, SAR112 and high-light-adapted Prochlorococcus, exhibited low levels of transcriptional activity and were uncorrelated with rate processes. Eukaryotes such as Haptophytes and non-photosynthetic Aveolates were prevalent in surface samples while Mamielles and Pelagophytes dominated the SCM. Metatranscriptomes generated with ribosomal RNA-depleted mRNA (total mRNA) coupled to in vitro polyadenylation compared with polyA-enriched mRNA revealed a trade-off in detection eukaryotic organelle and eukaryotic nuclear origin transcripts, respectively. Gene expression profiles of SCM eukaryote populations, highly similar in sequence identity to the model pelagophyte Pelagomonas sp. CCMP1756, suggest that pelagophytes are responsible for a majority of nitrate assimilation within the SCM.

Weyman, PD, Beeri K, Lefebvre SC, Rivera J, McCarthy JK, Heuberger AL, Peers G, Allen AE, Dupont CL.  2015.  Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis. Plant Biotechnology Journal. 13:460-470.   10.1111/pbi.12254   AbstractWebsite

Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow on urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle.

2014
Dupont, CL, Larsson J, Yooseph S, Ininbergs K, Goll J, Asplund-Samuelsson J, McCrow JP, Celepli N, Allen LZ, Ekman M, Lucas AJ, Hagstrom A, Thiagarajan M, Brindefalk B, Richter AR, Andersson AF, Tenney A, Lundin D, Tovchigrechko A, Nylander JAA, Brami D, Badger JH, Allen AE, Rusch DB, Hoffman J, Norrby E, Friedman R, Pinhassi J, Venter JC, Bergman B.  2014.  Functional tradeoffs underpin salinity-driven divergence in microbial community composition. Plos One. 9   10.1371/journal.pone.0089549   AbstractWebsite

Bacterial community composition and functional potential change subtly across gradients in the surface ocean. In contrast, while there are significant phylogenetic divergences between communities from freshwater and marine habitats, the underlying mechanisms to this phylogenetic structuring yet remain unknown. We hypothesized that the functional potential of natural bacterial communities is linked to this striking divide between microbiomes. To test this hypothesis, metagenomic sequencing of microbial communities along a 1,800 km transect in the Baltic Sea area, encompassing a continuous natural salinity gradient from limnic to fully marine conditions, was explored. Multivariate statistical analyses showed that salinity is the main determinant of dramatic changes in microbial community composition, but also of large scale changes in core metabolic functions of bacteria. Strikingly, genetically and metabolically different pathways for key metabolic processes, such as respiration, biosynthesis of quinones and isoprenoids, glycolysis and osmolyte transport, were differentially abundant at high and low salinities. These shifts in functional capacities were observed at multiple taxonomic levels and within dominant bacterial phyla, while bacteria, such as SAR11, were able to adapt to the entire salinity gradient. We propose that the large differences in central metabolism required at high and low salinities dictate the striking divide between freshwater and marine microbiomes, and that the ability to inhabit different salinity regimes evolved early during bacterial phylogenetic differentiation. These findings significantly advance our understanding of microbial distributions and stress the need to incorporate salinity in future climate change models that predict increased levels of precipitation and a reduction in salinity.

Smith, DR, Arrigo KR, Alderkamp A-C, Allen AE.  2014.  Massive difference in synonymous substitution rates among mitochondrial, plastid, and nuclear genes of Phaeocystis algae. Molecular Phylogenetics and Evolution. 71:36-40.   10.1016/j.ympev.2013.10.018   AbstractWebsite

We are just beginning to understand how mutation rates differ among mitochondrial, plastid, and nuclear genomes. In most seed plants the mitochondrial mutation rate is estimated to be lower than those of the plastid and nucleus, whereas in the red alga Porphyra the opposite is true, and in certain green algae all three genomes appear to have similar rates of mutation. Relative rate statistics of organelle vs nuclear genes, however, are lacking for lineages that acquired their plastids through secondary endosymbiosis, but recent organelle DNA analyses suggest that they may differ drastically from what is observed in lineages with primary plastids, such as green plants and red algae. Here, by measuring synonymous nucleotide substitutions, we approximate the relative mutation rates within the haptophyte genus Phaeocystis, which has a red-algal-derived, secondary plastid. Synonymous-site divergence data indicate that for Phaeocystis antarctica and P. globosa the mitochondrial mutation rate is 10 and 3 times that of the plastid and nucleus, respectively. This differs drastically from relative rate estimates for primary-plastid-bearing lineages and presents a much more dynamic view of organelle vs nuclear mutation rates across the eukaryotic domain.

2013
Read, BA, Kegel J, Klute MJ, Kuo A, Lefebvre SC, Maumus F, Mayer C, Miller J, Monier A, Salamov A, Young J, Aguilar M, Claverie JM, Frickenhaus S, Gonzalez K, Herman EK, Lin YC, Napier J, Ogata H, Sarno AF, Shmutz J, Schroeder D, de Vargas C, Verret F, von Dassow P, Valentin K, Van de Peer Y, Wheeler G, Dacks JB, Delwiche CF, Dyhrman ST, Glockner G, John U, Richards T, Worden AZ, Zhang XY, Grigoriev IV, Allen AE, Bidle K, Borodovsky M, Bowler C, Brownlee C, Cock JM, Elias M, Gladyshev VN, Groth M, Guda C, Hadaegh A, Iglesias-Rodriguez MD, Jenkins J, Jones BM, Lawson T, Leese F, Lindquist E, Lobanov A, Lomsadze A, Malik SB, Marsh ME, Mackinder L, Mock T, Mueller-Roeber B, Pagarete A, Parker M, Probert I, Quesneville H, Raines C, Rensing SA, Riano-Pachon DM, Richier S, Rokitta S, Shiraiwa Y, Soanes DM, van der Giezen M, Wahlund TM, Williams B, Wilson W, Wolfe G, Wurch LL, Annotation EH.  2013.  Pan genome of the phytoplankton Emiliania underpins its global distribution. Nature. 499:209-213.   10.1038/Nature12221   AbstractWebsite

Coccolithophores have influenced the global climate for over 200 million years(1). These marine phytoplankton can account for 20 per cent of total carbon fixation in some systems(2). They form blooms that can occupy hundreds of thousands of square kilometres and are distinguished by their elegantly sculpted calcium carbonate exoskeletons (coccoliths), rendering them visible from space(3). Although coccolithophores export carbon in the form of organic matter and calcite to the sea floor, they also release CO2 in the calcification process. Hence, they have a complex influence on the carbon cycle, driving either CO2 production or uptake, sequestration and export to the deep ocean(4). Here we report the first haptophyte reference genome, from the coccolithophore Emiliania huxleyi strain CCMP1516, and sequences from 13 additional isolates. Our analyses reveal a pan genome (core genes plus genes distributed variably between strains) probably supported by an atypical complement of repetitive sequence in the genome. Comparisons across strains demonstrate that E. huxleyi, which has long been considered a single species, harbours extensive genome variability reflected in different metabolic repertoires. Genome variability within this species complex seems to underpin its capacity both to thrive in habitats ranging from the equator to the subarctic and to form large-scale episodic blooms under a wide variety of environmental conditions.