Export 90 results:
Sort by: Author Title Type [ Year  (Desc)]
Goodwin, KD, Muller-Karger FE, Djurhuus A, Zeigler Allen L, Allen AE, McCrow JP, Canonico Hyde G.  2019.  Chapter 32 - Molecular Approaches for an Operational Marine Biodiversity Observation Network. World Seas: an Environmental Evaluation (Second Edition). ( Sheppard C, Ed.).:613-631.: Academic Press   10.1016/B978-0-12-805052-1.00032-2   Abstract

Biological health underpins ecosystem services, including the production of food from the sea. Therefore, many policy frameworks rely on biological observations to guide the sustainable use of marine resources. For example, a marine biodiversity observation network can provide evidence of ecosystem degradation, remediation success, and progress toward goals of sustainable development. The utility of biodiversity observations is limited, however, if measurements are not standardized to allow comparisons across space and time or if they are too difficult or expensive to make routinely or at large scale. Molecular approaches (e.g., metagenomics, metabarcoding, eDNA, metatranscriptomics) offer efficiencies in sample collection and processing and provide analysis across trophic levels compared to traditional methods. Increased affordability and throughput of nucleic acid sequencing allow integration of molecular biological techniques into operational monitoring with the potential benefit of expanding the spatial and temporal scale of biological observations.

Brunson, JK, McKinnie SMK, Chekan JR, McCrow JP, Miles ZD, Bertrand EM, Bielinski VA, Luhavaya H, Oborník M, Smith JG, Hutchins DA, Allen AE, Moore BS.  2018.  Biosynthesis of the neurotoxin domoic acid in a bloom-forming diatom. Science. 361:1356-1358.   10.1126/science.aau0382   Abstract

Algal blooms can devastate marine mammal communities through the production of neurotoxins that accumulate within the food web. Brunson et al. identified a cluster of genes associated with biosynthesis of the neurotoxin domoic acid in a marine diatom (see the Perspective by Pohnert et al.). In vitro experiments established a series of enzymes that create the core structure of the toxin. Knowledge of the genes involved in domoic acid production will allow for genetic monitoring of algal blooms and aid in identifying conditions that trigger toxin production.Science, this issue p. 1356; see also p. 1308Oceanic harmful algal blooms of Pseudo-nitzschia diatoms produce the potent mammalian neurotoxin domoic acid (DA). Despite decades of research, the molecular basis for its biosynthesis is not known. By using growth conditions known to induce DA production in Pseudo-nitzschia multiseries, we implemented transcriptome sequencing in order to identify DA biosynthesis genes that colocalize in a genomic four-gene cluster. We biochemically investigated the recombinant DA biosynthetic enzymes and linked their mechanisms to the construction of DA’s diagnostic pyrrolidine skeleton, establishing a model for DA biosynthesis. Knowledge of the genetic basis for toxin production provides an orthogonal approach to bloom monitoring and enables study of environmental factors that drive oceanic DA production.

Murik, O, Tirichine L, Prihoda J, Thomas Y, Araújo WL, Allen AE, Fernie AR, Bowler C.  2018.  Downregulation of mitochondrial alternative oxidase affects chloroplast function, redox status and stress response in a marine diatom. New Phytologist.   10.1111/nph.15479   AbstractWebsite

Summary Diatoms dominance in contemporary aquatic environments indicates that they have developed unique and effective mechanisms to cope with the rapid and considerable fluctuations that characterize these environments. In view of their evolutionary history from a secondary endosymbiosis, inter-organellar regulation of biochemical activities may be of particular relevance. Diatom mitochondrial alternative oxidase (AOX) is believed to play a significant role in supplying chloroplasts with ATP produced in the mitochondria. Using the model diatom Phaeodactylum tricornutum we generated AOX knockdown lines, and followed sensitivity to stressors, photosynthesis and transcriptome and metabolome profiles of wild-type and knockdown lines. We show here that expression of the AOX gene is upregulated by various stresses including H2O2, heat, high-light illumination, and iron or nitrogen limitation. AOX knockdown results in hypersensitivity to stress. Knockdown lines also show significantly reduced photosynthetic rates and their chloroplasts are more oxidized. Comparisons of transcriptome and metabolome profiles suggest a strong impact of AOX activity on gene expression, which is carried through to the level of the metabolome. Our data provide evidence for the involvement of mitochondrial AOX in processes central to the cell biology of diatoms, revealing that cross-talk between mitochondria and chloroplasts is crucial for maintaining sensitivity to changing environments. This article is protected by copyright. All rights reserved.

Bender, SJ, Moran DM, McIlvin MR, Zheng H, McCrow JP, Badger J, DiTullio GR, Allen AE, Saito MA.  2018.  Colony formation in Phaeocystis antarctica: connecting molecular mechanisms with iron biogeochemistry. Biogeosciences. 15:4923-4942.   10.5194/bg-15-4923-2018   AbstractWebsite

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 B-12 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.

Spackeen, JL, Bronk DA, Sipler RE, Bertrand EM, Hutchins DA, Allen AE.  2018.  Stoichiometric N:P Ratios, Temperature, and Iron Impact Carbon and Nitrogen Uptake by Ross Sea Microbial Communities. Journal of Geophysical Research: Biogeosciences.   10.1029/2017JG004316   Abstract

Abstract Phytoplankton growth in the seasonally productive Southern Ocean is typically limited by iron (Fe). In the next century, however, Fe inputs and temperature are predicted to increase. Dissolved concentrations of nitrogen (N) and phosphorus (P) may also change due to physical and biological drivers, altering the stoichiometric N:P ratio of the water column. Two separate experiments were conducted to study how these predicted changes will impact nutrient utilization by two natural microbial communities from the Ross Sea, Antarctica. The first investigation focused on a Terra Nova Bay community amended with Fe and grown under a wide range of seven different N:P ratios. Building on the results of the first study, a McMurdo Sound community was incubated under a factorial design at high, medium (ambient), and low N:P ratios, with and without Fe addition, and at ambient versus elevated temperature. In both experiments absolute uptake rates of bicarbonate, nitrate, and amino acids by two size fractions (0.7–5.0 μm and > 5.0 μm) of microorganisms were measured using stable isotopes. For Terra Nova Bay microorganisms, significant differences in nutrient uptake rates occurred when the N:P ratio was elevated, and nitrate uptake rates significantly increased early in the experiment but were not different at the end. For McMurdo Sound microorganisms, changing the N:P supply ratio did not have a clear effect, while temperature elevation and/or Fe addition significantly increased nutrient uptake. Results indicate that changing the dissolved N:P ratio can potentially alter nutrient uptake rates; however, the impact of temperature and Fe are greater.

Du, N, Gholami P, Kline DI, Dupont CL, Dickson AG, Mendola D, Martz T, Allen AE, Mitchell BG.  2018.  Simultaneous quantum yield measurements of carbon uptake and oxygen evolution in microalgal cultures. Plos One. 13   10.1371/journal.pone.0199125   AbstractWebsite

The photosynthetic quantum yield (F), defined as carbon fixed or oxygen evolved per unit of light absorbed, is a fundamental but rarely determined biophysical parameter. A method to estimate Phi for both net carbon uptake and net oxygen evolution simultaneously can provide important insights into energy and mass fluxes. Here we present details for a novel system that allows quantification of carbon fluxes using pH oscillation and simultaneous oxygen fluxes by integration with a membrane inlet mass spectrometer. The pHOS system was validated using Phaeodactylum tricornutum cultured with continuous illumination of 110 mu mole quanta m(-2) s(-1) at 25 degrees C. Furthermore, simultaneous measurements of carbon and oxygen flux using the pHOS-MIMS and photon flux based on spectral absorption were carried out to explore the kinetics of F in P. tricornutum during its acclimation from low to high light (110 to 750 mu mole quanta m(-2) s(-1)). Comparing results at 0 and 24 hours, we observed strong decreases in cellular chlorophyll a (0.58 to 0.21 pg cell(-1)), Fv/Fm (0.71 to 0.59) and maximum Phi(CO2) (0.019 to 0.004) and Phi(O2) (0.028 to 0.007), confirming the transition toward high light acclimation. The Phi time-series indicated a non-synchronized acclimation response between carbon uptake and oxygen evolution, which has been previously inferred based on transcriptomic changes for a similar experimental design with the same diatom that lacked physiological data. The integrated pHOS-MIMS system can provide simultaneous carbon and oxygen measurements accurately, and at the time-resolution required to resolve highresolution carbon and oxygen physiological dynamics.

McQuaid, JB, Kustka AB, Oborník M, Horák A, McCrow JP, Karas BJ, Zheng H, Kindeberg T, Andersson AJ, Barbeau KA, Allen AE.  2018.  Carbonate-sensitive phytotransferrin controls high-affinity iron uptake in diatoms. Nature. 555:534.: Macmillan Publishers Limited, part of Springer Nature. All rights reserved.   10.1038/nature25982   AbstractWebsite

In vast areas of the ocean, the scarcity of iron controls the growth and productivity of phytoplankton1,2. Although most dissolved iron in the marine environment is complexed with organic molecules3, picomolar amounts of labile inorganic iron species (labile iron) are maintained within the euphotic zone4 and serve as an important source of iron for eukaryotic phytoplankton and particularly for diatoms5. Genome-enabled studies of labile iron utilization by diatoms have previously revealed novel iron-responsive transcripts6,7, including the ferric iron-concentrating protein ISIP2A8, but the mechanism behind the acquisition of picomolar labile iron remains unknown. Here we show that ISIP2A is a phytotransferrin that independently and convergently evolved carbonate ion-coordinated ferric iron binding. Deletion of ISIP2A disrupts high-affinity iron uptake in the diatom Phaeodactylum tricornutum, and uptake is restored by complementation with human transferrin. ISIP2A is internalized by endocytosis, and manipulation of the seawater carbonic acid system reveals a second-order dependence on the concentrations of labile iron and carbonate ions. In P. tricornutum, the synergistic interaction of labile iron and carbonate ions occurs at environmentally relevant concentrations, revealing that carbonate availability co-limits iron uptake. Phytotransferrin sequences have a broad taxonomic distribution8 and are abundant in marine environmental genomic datasets9,10, suggesting that acidification-driven declines in the concentration of seawater carbonate ions will have a negative effect on this globally important eukaryotic iron acquisition mechanism.

Rastogi, A, Maheswari U, Dorrell RG, Vieira FRJ, Maumus F, Kustka A, McCarthy J, Allen AE, Kersey P, Bowler C, Tirichine L.  2018.  Integrative analysis of large scale transcriptome data draws a comprehensive landscape of Phaeodactylum tricornutum genome and evolutionary origin of diatoms. Scientific Reports. 8:4834.   10.1038/s41598-018-23106-x   AbstractWebsite

Diatoms are one of the most successful and ecologically important groups of eukaryotic phytoplankton in the modern ocean. Deciphering their genomes is a key step towards better understanding of their biological innovations, evolutionary origins, and ecological underpinnings. Here, we have used 90 RNA-Seq datasets from different growth conditions combined with published expressed sequence tags and protein sequences from multiple taxa to explore the genome of the model diatom Phaeodactylum tricornutum, and introduce 1,489 novel genes. The new annotation additionally permitted the discovery of extensive alternative splicing in diatoms, including intron retention and exon skipping, which increase the diversity of transcripts generated in changing environments. In addition, we have used up-to-date reference sequence libraries to dissect the taxonomic origins of diatom genes. We show that the P. tricornutum genome is enriched in lineage-specific genes, with up to 47% of the gene models present only possessing orthologues in other stramenopile groups. Finally, we have performed a comprehensive de novo annotation of repetitive elements showing novel classes of transposable elements such as SINE, MITE and TRIM/LARD. This work provides a solid foundation for future studies of diatom gene function, evolution and ecology.

Berg, C, Dupont CL, Asplund-Samuelsson J, Celepli NA, Eiler A, Allen AE, Ekman M, Bergman B, Ininbergs K.  2018.  Dissection of microbial community functions during a cyanobacterial bloom in the Baltic Sea via metatranscriptomics. Frontiers in Marine Science. 5   10.3389/fmars.2018.00055   AbstractWebsite

Marine and brackish surface waters are highly dynamic habitats that undergo repeated seasonal variations in microbial community composition and function throughout time. While succession of the various microbial groups has been well investigated, little is known about the underlying gene-expression of the microbial community. We investigated microbial interactions via metatranscriptomics over a spring to fall seasonal cycle in the brackish Baltic Sea surface waters, a temperate brackish water ecosystem periodically promoting massive cyanobacterial blooms, which have implications for e.g. primary production, nutrient cycling, and expansion of hypoxic zones. Network analysis of the gene expression of all microbes from 0.22 - 200 µm in size and of the major taxonomic groups dissected the seasonal cycle into four components that comprised genes peaking during different periods of the bloom. Photoautotrophic nitrogen-fixing Cyanobacteria displayed the highest connectivity among the microbes, in contrast to chemoautotrophic ammonia-oxidizing Thaumarchaeota, while heterotrophs dominated connectivity among pre- and post-bloom peaking genes. The network was also composed of distinct functional connectivities, with an early season balance between carbon metabolism and ATP synthesis shifting to a dominance of ATP synthesis during the bloom, while carbon degradation, specifically through the glyoxylate shunt, characterized the post-bloom period, driven by Alphaproteobacteria as well as by Gammaproteobacteria of the SAR86 and SAR92 clusters. Our study stresses the exceptionally strong biotic driving force executed by cyanobacterial blooms on associated microbial communities in the Baltic Sea and highlights the impact cyanobacterial blooms have on functional microbial community composition.

Tatters, AO, Schnetzer A, Xu K, Walworth NG, Fu F, Spackeen JL, Sipler RE, Bertrand EM, McQuaid JB, Allen AE, Bronk DA, Gao K, Sun J, Caron DA, Hutchins DA.  2018.  Interactive effects of temperature, CO2 and nitrogen source on a coastal California diatom assemblage. Journal of Plankton Research. 40:151-164.   10.1093/plankt/fbx074   AbstractWebsite

Diatoms are often considered to be a single functional group, yet there is a great deal of morphological, genetic and ecological diversity within the class. How these differences will translate into species-specific responses to rapid changes in the ocean environment resulting from climate change and eutrophication is currently poorly understood. We investigated the response of a natural diatom-dominated assemblage in coastal California waters to interactions between the variables nitrogen source (nitrate and urea), temperature (19 and 23°C) and CO2 (380 and 800 ppm) in a factorial experimental matrix using continuous culture (ecostat) methods. The community included diatoms of the cosmopolitan genera Pseudo-nitzschia and Chaetoceros, as well as Leptocylindrus and Cylindrotheca. Our results demonstrate strong interactive effects of these variables on community composition; notably, nitrogen source alone and nitrogen and CO2 together had a much greater influence on diatom community structure at 23°C compared with 19°C. In addition, warming and acidification interactions significantly increased cellular quotas of the neurotoxin domoic acid produced by Pseudo-nitzschia multiseries. In general, the effects observed for the factors tested differed significantly between the various diatom genera in this assemblage, suggesting potentially divergent responses of some of these ecologically and biogeochemically important phytoplankton taxa to interactions between global-scale and local-scale anthropogenic stressors in a changing ocean.

Hippmann, AA, Schuback N, Moon K-M, McCrow JP, Allen AE, Foster LJ, Green BR, Maldonado MT.  2017.  Contrasting effects of copper limitation on the photosynthetic apparatus in two strains of the open ocean diatom Thalassiosira oceanica. PLOS ONE. 12:e0181753.: Public Library of Science   10.1371/journal.pone.0181753   AbstractWebsite

There is an intricate interaction between iron (Fe) and copper (Cu) physiology in diatoms. However, strategies to cope with low Cu are largely unknown. This study unveils the comprehensive restructuring of the photosynthetic apparatus in the diatom Thalassiosira oceanica (CCMP1003) in response to low Cu, at the physiological and proteomic level. The restructuring results in a shift from light harvesting for photochemistry—and ultimately for carbon fixation—to photoprotection, reducing carbon fixation and oxygen evolution. The observed decreases in the physiological parameters Fv/Fm, carbon fixation, and oxygen evolution, concomitant with increases in the antennae absorption cross section (σPSII), non-photochemical quenching (NPQ) and the conversion factor (φe:C/ηPSII) are in agreement with well documented cellular responses to low Fe. However, the underlying proteomic changes due to low Cu are very different from those elicited by low Fe. Low Cu induces a significant four-fold reduction in the Cu-containing photosynthetic electron carrier plastocyanin. The decrease in plastocyanin causes a bottleneck within the photosynthetic electron transport chain (ETC), ultimately leading to substantial stoichiometric changes. Namely, 2-fold reduction in both cytochrome b6f complex (cytb6f) and photosystem II (PSII), no change in the Fe-rich PSI and a 40- and 2-fold increase in proteins potentially involved in detoxification of reactive oxygen species (ferredoxin and ferredoxin:NADP+ reductase, respectively). Furthermore, we identify 48 light harvesting complex (LHC) proteins in the publicly available genome of T. oceanica and provide proteomic evidence for 33 of these. The change in the LHC composition within the antennae in response to low Cu underlines the shift from photochemistry to photoprotection in T. oceanica (CCMP1003). Interestingly, we also reveal very significant intra-specific strain differences. Another strain of T. oceanica (CCMP 1005) requires significantly higher Cu concentrations to sustain both its maximal and minimal growth rate compared to CCMP 1003. Under low Cu, CCMP 1005 decreases its growth rate, cell size, Chla and total protein per cell. We argue that the reduction in protein per cell is the main strategy to decrease its cellular Cu requirement, as none of the other parameters tested are affected. Differences between the two strains, as well as differences between the well documented responses to low Fe and those presented here in response to low Cu are discussed.

Spackeen, JL, Sipler RE, Xu K, Tatters AO, Walworth NG, Bertrand EM, McQuaid JB, Hutchins DA, Allen AA, Bronk DA.  2017.  Interactive effects of elevated temperature and CO2 on nitrate, urea, and dissolved inorganic carbon uptake by a coastal California, USA, microbial community. Marine Ecology Progress Series. 577:49-65.   10.3354/meps12243   Abstract

Average global temperatures and carbon dioxide (CO2) levels are expected to increase in the coming decades. Implications for ocean ecosystems include shifts in microbial community structure and subsequent modifications to nutrient pathways. Studying how predicted future temperature and CO2 conditions will impact the biogeochemistry of the ocean is important because of the ocean’s role in regulating global climate. We determined how elevated temperature and CO2 affect uptake rates of nitrate, urea, and dissolved inorganic carbon (DIC) by 2 size classes (0.7-5.0 and >5.0 µm) of a microbial assemblage collected from coastal California, USA. This microbial community was incubated for 10 d using an ecostat continuous culture system that supplied the microorganisms with either nitrate or urea as the dominant nitrogen source. Biomass parameters, nutrient concentrations, and uptake rates were measured throughout the experiment. In all treatments, urea uptake rates were greater than nitrate, and larger microorganisms had higher uptake rates than smaller microorganisms. Uptake rates of urea and DIC within both size fractions were higher at elevated temperature, and uptake rates of nitrate by smaller microorganisms increased with elevated CO2. These findings suggest that the rate at which nutrients cycle in temperate coastal waters will increase as temperature and CO2 levels rise and that the effect will vary between nitrogen substrates and different microorganisms.

McCarthy, JK, Smith SR, McCrow JP, Tan M, Zheng H, Beeri K, Roth RA, Lichtle C, Goodenough U, Bowler C, Dupont CL, Allen AE.  2017.  Nitrate reductase knockout uncouples nitrate transport from nitrate assimilation and drives repartitioning of carbon flux in a model pennate diatom. The Plant Cell. Abstract

The ecological prominence of diatoms in the ocean environment largely results from their superior competitive ability for dissolved nitrate (NO3-). To investigate the cellular and genetic basis of diatom NO3− assimilation, we generated a knockout in the nitrate reductase gene (NR-KO) of the model pennate diatom Phaeodactylum tricornutum. In NR-KO cells, N assimilation was abolished although NO3−transport remained intact. Unassimilated NO3− accumulated in NR-KO cells, resulting in swelling and associated changes in biochemical composition and physiology. Elevated expression of genes encoding putative vacuolar NO3− chloride channel (ClC) transporters plus electron micrographs indicating enlarged vacuoles suggested vacuolar storage of NO3−. Triacylglycerol concentrations in the NR-KO cells increased immediately following the addition of NO3−. and these increases coincided with elevated gene expression of key TAG biosynthesis components. Simultaneously, induction of transcripts encoding proteins involved in thylakoid membrane lipid recycling suggested more abrupt repartitioning of carbon resources in NR-KO cells compared to WT. Conversely, ribosomal structure and photosystems genes were immediately deactivated in NR-KO cells following NO3− addition, followed within hours by deactivation of genes encoding enzymes for chlorophyll biosynthesis and carbon fixation and metabolism. N-assimilation pathway genes respond uniquely, apparently induced simultaneously by both NO3− replete and deplete conditions.

Diner, RE, Noddings CM, Lian NC, Kang AK, McQuaid JB, Jablanovic J, Espinoza JL, Nguyen NA, Anzelmatti MA, Jansson J, Bielinski VA, Karas BJ, Dupont CL, Allen AE, Weyman PD.  2017.  Diatom centromeres suggest a mechanism for nuclear DNA acquisition. Proceedings of the National Academy of Sciences. 114:E6015-E6024.   10.1073/pnas.1700764114   Abstract

Centromeres are essential for cell division and growth in all eukaryotes, and knowledge of their sequence and structure guides the development of artificial chromosomes for functional cellular biology studies. Centromeric proteins are conserved among eukaryotes; however, centromeric DNA sequences are highly variable. We combined forward and reverse genetic approaches with chromatin immunoprecipitation to identify centromeres of the model diatom Phaeodactylum tricornutum. We observed 25 unique centromere sequences typically occurring once per chromosome, a finding that helps to resolve nuclear genome organization and indicates monocentric regional centromeres. Diatom centromere sequences contain low-GC content regions but lack repeats or other conserved sequence features. Native and foreign sequences with similar GC content to P. tricornutum centromeres can maintain episomes and recruit the diatom centromeric histone protein CENH3, suggesting nonnative sequences can also function as diatom centromeres. Thus, simple sequence requirements may enable DNA from foreign sources to persist in the nucleus as extrachromosomal episomes, revealing a potential mechanism for organellar and foreign DNA acquisition.

Lee, ZM-P, Poret-Peterson AT, Siefert JL, Kaul D, Moustafa A, Allen AE, Dupont CL, Eguiarte LE, Souza V, Elser JJ.  2017.  Nutrient stoichiometry shapes microbial community structure in an evaporitic shallow pond. Frontiers in Microbiology. 8   10.3389/fmicb.2017.00949   Abstract

Nutrient availability and ratios can play an important role in shaping microbial communities of freshwater ecosystems. The Cuatro Ciénegas Basin (CCB) in Mexico is a desert oasis where, perhaps paradoxically, high microbial diversity coincides with extreme oligotrophy. To better understand the effects of nutrients on microbial communities in CCB, a mesocosm experiment was implemented in a stoichiometrically imbalanced pond, Lagunita, which has an average TN:TP ratio of 122 (atomic). The experiment had four treatments, each with five spatial replicates - unamended controls and three fertilization treatments with different N:P regimes (P only, N:P = 16 and N:P = 75 by atoms). In the water column, quantitative PCR of the 16S rRNA gene indicated that P enrichment alone favored proliferation of bacterial taxa with high rRNA gene copy number, consistent with a previously hypothesized but untested connection between rRNA gene copy number and P requirement. Bacterial and microbial eukaryotic community structure was investigated by pyrosequencing of 16S and 18S rRNA genes from the planktonic and surficial sediment samples. Nutrient enrichment shifted the composition of the planktonic community in a treatment-specific manner and promoted the growth of previously rare bacterial taxa at the expense of the more abundant, potentially endemic, taxa. The eukaryotic community was highly enriched with phototrophic populations in the fertilized treatment. The sediment microbial community exhibited high beta diversity among replicates within treatments, which obscured any changes due to fertilization. Overall, these results showed that nutrient stoichiometry can be an important factor in shaping microbial community structure.

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.

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.