Search

ABSTRACT The photosynthetic and diazotrophic cyanobacterium Trichodesmium is a key contributor to marine biogeochemical cycles in the subtropical-oligotrophic oceans. Trichodesmium form colonies that harbor a distinct microbial community in comparison to the surrounding seawater. The presence of their associated bacteria can expand Trichodesmium’s functional potential and is predicted to influence the cycling of carbon, nitrogen, phosphorus, and iron (C, N, P, and Fe). To link the bacteria associated with Trichodesmium to key functional traits and elucidate how community structure can influence nutrient cycling, we characterized Red Sea Trichodesmium colonies using metagenomics and metaproteomics. Colonies harbored bacteria that typically associate with algae and particles, such as the ubiquitous Alteromonas macleodii, but also lineages specific to Trichodesmium, such as members from the order Balneolales. The majority of associated bacteria were auxotrophic for different vitamins, indicating their dependency on vitamin production by Trichodesmium. The associated bacteria carry functional traits including siderophore biosynthesis, reduced phosphorus metabolism, and denitrification pathways. The analysis supports Trichodesmium as an active hotspot for C, N, P, Fe, and vitamin exchange. In turn, Trichodesmium may rely on associated bacteria to meet its high Fe demand as several lineages synthesize photolabile siderophores (e.g., vibrioferrin, rhizoferrin, petrobactin) which can enhance the bioavailability of particulate Fe to the entire consortium. Collectively, the results indicate that Trichodesmium colonies provide a structure where these interactions can take place. While further studies are required to clarify the exact nature of these interactions, Trichodesmium’s reliance on particle and algae-associated bacteria and the observed redundancy of key functional traits likely underpins the resilience of Trichodesmium within an ever-changing global environment.IMPORTANCEColonies of the cyanobacteria Trichodesmium act as a biological hotspot for the usage and recycling of key resources such as C, N, P, and Fe within an otherwise oligotrophic environment. While Trichodesmium colonies are known to interact and support a unique community of algae and particle-associated microbes, our understanding of the taxa that populate these colonies and the gene functions they encode is still limited. Characterizing the taxa and adaptive strategies that influence consortium physiology and its concomitant biogeochemistry is critical in a future ocean predicted to have increasingly resource-depleted regions.

Inputs of new nitrogen by cyanobacterial diazotrophs are critical to ocean ecosystem structure and function. Relative to other ocean regions, there is a lack of data on the distribution of these microbes in the western South Atlantic. Here, the abundance of six diazotroph phylotypes: Trichodesmium, Crocosphaera, UCYN-A, Richelia associated with Rhizosolenia (Het-1) or Hemiaulus (Het-2), and Calothrix associated with Chaetoceros (Het-3) was measured by quantitative PCR (qPCR) of the nifH gene along a transect extending from the shelf-break to the open ocean along the Vitória-Trindade seamount chain (1200 km). Using nifH gene copies as a proxy for phylotype abundance, Crocosphaera signals were the most abundant, with a broad distribution throughout the study region. Trichodesmium signals were the second most abundant, with the greatest numbers confined to the warmer waters closer to the coast, and a significant positive correlation with temperature. The average signals for the host-associated diazotrophs (UCYN-A, Het-1, and Het-2) were consistently lower than for the other phylotypes. These findings expand measurements of cyanobacterial diazotroph distribution in the western South Atlantic, and provide a new resource to enhance modeling studies focused on patterns of nitrogen fixation in the global ocean.

Abstract The carbon isotope fractionation in algal organic matter (εp), including the long‐chain alkenones produced by the coccolithophorid family Noelaerhabdaceae, is used to reconstruct past atmospheric CO2 levels. The conventional proxy linearly relates εp to changes in cellular carbon demand relative to diffusive CO2 supply, with larger εp values occurring at lower carbon demand relative to supply (i.e., abundant CO2). However, the response of Gephyrocapsa oceanica, one of the dominant alkenone producers of the last few million years, has not been studied closely. Here, we subject G. oceanica to various CO2 levels by increasing pCO2 in the culture headspace, as opposed to increasing dissolved inorganic carbon (DIC) and alkalinity concentrations at constant pH. We note no substantial change in physiology, but observe an increase in εp as carbon demand relative to supply decreases, consistent with DIC manipulations. We compile existing Noelaerhabdaceae εp data and show that the diffusive model poorly describes the data. A meta‐analysis of individual treatments (unique combinations of lab, strain, and light conditions) shows that the slope of the εp response depends on the light conditions and range of carbon demand relative to CO2 supply in the treatment, which is incompatible with the diffusive model. We model εp as a multilinear function of key physiological and environmental variables and find that both photoperiod duration and light intensity are critical parameters, in addition to CO2 and cell size. While alkenone carbon isotope ratios indeed record CO2 information, irradiance and other factors are also necessary to properly describe alkenone εp.

There is growing interest in the use of metatranscriptomics to study virus community dynamics. We used RNA samples collected from harmful brown tides caused by the eukaryotic alga Aureococcus anophagefferens within New York (United States) estuaries and in the process observed how preprocessing of libraries by either selection for polyadenylation or reduction in ribosomal RNA (rRNA) influenced virus community analyses. As expected, more reads mapped to the A. anophagefferens genome in polyadenylation-selected libraries compared to the rRNA-reduced libraries, with reads mapped in each sample correlating to one another regardless of preprocessing of libraries. Yet, this trend was not seen for reads mapping to the Aureococcus anophagefferens Virus (AaV), where significantly more reads (approximately two orders of magnitude) were mapped to the AaV genome in the rRNA-reduced libraries. In the rRNA-reduced libraries, there was a strong and significant correlation between reads mappings to AaV and A. anophagefferens. Overall, polyadenylation-selected libraries produced fewer viral contigs, fewer reads mapped to viral contigs, and different proportions across viral realms and families, compared to their rRNA-reduced pairs. This study provides evidence that libraries generated by rRNA reduction and not selected for polyadenylation are more appropriate for quantitative characterization of viral communities in aquatic ecosystems by metatranscriptomics.

Pelagophytes are abundant picophytoplankton within open ocean ecosystems and the causative algae of harmful brown tide blooms in estuaries. The physiological capabilities facilitating the ecological success of pelagophytes in these diverse ecosystems remains poorly understood. Here, we investigated the transcriptional response of two coastal pelagophytes (Aureococcus anophagefferens and Aureoumbra lagunensis) and two open ocean pelagophytes (Pelagococcus subviridis and Pelagomonas calceolata) to conditions commonly found within the marine ecosystems where they thrive: low concentrations of nitrogen (N), phosphorus (P), or light. OrthoMCL was used to generate a total of 62,653 orthologous groups (OGs) with only a small fraction of these OGs (2,776 or 4.4%) being shared among all four pelagophytes. Of the commonly shared OGs, 8% were significantly differentially abundant under low N, P, or light with the majority associated with energy and lipid metabolism. Distinct responses among pelagophytes included increased abundance of transcripts encoding phosphate transporters (Aureococcus) and transcripts encoding a pyrophosphatase (Aureococcus and Pelagomonas) under low P, the expression of a suite of organic nitrogen-degrading enzymes under low N (Aureoumbra and Pelagomonas), increased abundance of transcripts encoding flavodoxins relative to ferredoxins (Pelagomonas) and transcripts encoding lysophospholipase (Pelagococcus) under low light, and both increases and decreases in abundance of transcripts encoding selenoproteins in all pelagophytes except Pelagococcus. Collectively, this study provides new information on the expressed gene compliment of these poorly characterized taxa and demonstrates that these pelagophytes possess a combination of shared and unique physiological features that likely facilitate their adaptation to distinct environmental conditions.

Abstract Trichodesmium is a marine, diazotrophic cyanobacterium that plays a central role in the biogeochemical cycling of carbon and nitrogen. Colonies ubiquitously co‐occur with a diverse microbiome of heterotrophic bacteria. We show that manipulation of the microbiome with quorum sensing acyl homoserine lactones (AHLs) significantly modulated rates of N2 fixation by Trichodesmium collected from the western North Atlantic, with positive and negative effects of varied magnitude. Changes in Trichodesmium N2 fixation did not correlate with changes in microbiome composition or geochemistry. With AHL addition, a subset of the significantly differentially expressed genes was related to known quorum sensing responses in model bacteria. However, there was little overlap in specific microbiome transcriptional responses to AHL addition between stations. Overall, these host‐microbiome interactions reflect a complex interplay of biotic and environmental factors that together form an overlooked mechanism modulating Trichodesmium N2 fixation.

Abstract Marine eukaryotic phytoplankton adapt to low phosphorus (P) in the oceans through a variety of step‐wise mechanisms including lipid substitution and decreased nucleic acid content. Here, we examined the impact of low P concentrations on intracellular metabolites whose abundances can be quickly adjusted by cellular regulation within laboratory cultures of three model phytoplankton and in field samples from the Atlantic and Pacific Oceans. We quantified the relative abundances of monophosphate nucleotides and their corresponding nucleosides, using a combination of targeted and untargeted metabolomics methods. Under P‐deficient conditions, we observed a marked decrease in adenosine 5′‐monophosphate (AMP) with a concomitant increase in adenosine. This shift occurred within all detected pairs of monophosphate nucleotides and nucleosides, and was consistent with previous work showing transcriptional changes in nucleotide synthesis and salvage under P‐deficient conditions for model eukaryotes. In the field, we observed AMP‐to‐adenosine ratios that were similar to those in laboratory culture under P‐deficient conditions.

Viruses are partners in ecosystem ecology, yet their study has been primarily limited to laboratory models virus-host or derived from metagenomics. Here, Moniruzzamanet al. use metatranscriptomics to resolve interactions between giant viruses and single-celled eukaryotic hosts.

The N2 fixing cyanobacterium Trichodesmium is a critically important organism in oligotrophic marine ecosystems, supplying “new” nitrogen (N) to the otherwise N-poor tropical and subtropical regions where it occurs. Low concentrations of phosphorus (P) in these regions can constrain Trichodesmium distribution and N2 fixation rates. Physiological characterization of a single species in a mixed community can be challenging, and ‘omic approaches are increasingly important tools for tracking nutritional physiology in a taxon-specific manner. As such, studies examining the dynamics of gene and protein markers of physiology (e.g., nutrient stress) are critical for the application and interpretation of such ‘omic data in situ. Here we leveraged combined transcriptomics, proteomics, and enzyme activity assays to track the physiological response of Trichodesmium erythraeum IMS101 to P deficiency and subsequent P re-supply over 72 h of sampling. P deficiency resulted in differential gene expression, protein abundance, and enzyme activity that highlighted a synchronous shift in P physiology with increases in the transcripts and corresponding proteins for hydrolyzing organic phosphorus, taking up phosphate with higher affinity, and modulating intracellular P demand. After P deficiency was alleviated, gene expression of these biomarkers was reduced to replete levels within 4 h of P amendment. A number of these gene biomarkers were adjacent to putative pho boxes and their expression patterns were similar to a sphR response regulator. Protein products of the P deficiency biomarkers were slow to decline, with 84% of the original P deficient protein set still significantly differentially expressed after 72 h. Alkaline phosphatase activity tracked with proteins for this enzyme. With the rapid turnover time of transcripts, they appear to be good biomarkers of a P stress phenotype, whereas proteins, with a slower turnover time, may better reflect cellular activities. These results highlight the importance of validating and pairing transcriptome and proteome data that can be applied to physiological studies of key species in situ.

Harmful algal blooms (HABs) threaten ecosystems and human health worldwide. Controlling nitrogen inputs to coastal waters is a common HAB management strategy, as nutrient concentrations often suggest coastal blooms are nitrogen-limited. However, defining best nutrient management practices is a long-standing challenge: in part, because of difficulties in directly tracking the nutritional physiology of harmful species in mixed communities. Using metatranscriptome sequencing and incubation experiments, we addressed this challenge by assaying the in situ physiological ecology of the ecosystem destructive alga, Aureococcus anophagefferens. Here we show that gene markers of phosphorus deficiency were expressed in situ, and modulated by the enrichment of phosphorus, which was consistent with the observed growth rate responses. These data demonstrate the importance of phosphorus in controlling brown-tide dynamics, suggesting that phosphorus, in addition to nitrogen, should be evaluated in the management and mitigation of these blooms. Given that nutrient concentrations alone were suggestive of a nitrogen-limited ecosystem, this study underscores the value of directly assaying harmful algae in situ for the development of management strategies.

With rising atmospheric CO2, phytoplankton face shifts in ocean chemistry including increased dissolved CO2 and acidification that will likely influence the relative competitive fitness of different phytoplankton taxa. Here we compared the physiological and gene expression responses of six species of phytoplankton including a diatom, a raphidophyte, two haptophytes, and two dinoflagellates to ambient (~400 ppm) and elevated (~800 ppm) CO2. Dinoflagellates had significantly slower growth rates and higher, yet variable, chlorophyll a per cell under elevated CO2. The other phytoplankton tended to have increased growth rates and/or decreased chlorophyll a per cell. Carbon and nitrogen partitioning of cells shifted under elevated CO2 in some species, indicating potential changes in energy fluxes due to changes in carbon concentrating mechanisms (CCM) or photorespiration. Consistent with these phenotypic changes, gene set enrichment analyses revealed shifts in energy, carbon and nitrogen metabolic pathways, though with limited overlap between species in the genes and pathways involved. Similarly, gene expression responses across species revealed few conserved CO2-responsive genes within CCM and photorespiration categories, and a survey of available transcriptomes found high diversity in biophysical CCM and photorespiration expressed gene complements between and within the four phyla represented by these species. The few genes that displayed similar responses to CO2 across phyla were from understudied gene families, making them targets for further research to uncover the mechanisms of phytoplankton acclimation to elevated CO2. These results underscore that eukaryotic phytoplankton have diverse gene complements and gene expression responses to CO2 perturbations and highlight the value of cross-phyla comparisons for identifying gene families that respond to environmental change.

The concentration and composition of bioavailable nitrogen (N) and phosphorus (P) in the upper ocean shape eukaryotic phytoplankton communities and influence their physiological responses. Phytoplankton are known to exhibit similar physiological responses to limiting N and P conditions such as decreased growth rates, chlorosis, and increased assimilation of N and P. Are these responses similar at the molecular level across multiple species? To interrogate this question, five species from biogeochemically important, bloom-forming taxa (Bacillariophyta, Dinophyta, and Haptophyta) were grown under similar low N, low P, and replete nutrient conditions to identify transcriptional patterns and associated changes in biochemical pools related to N and P stress. Metabolic profiles, revealed through the transcriptomes of these taxa, clustered together based on species rather than nutrient stressor, suggesting that the global metabolic response to nutrient stresses was largely, but not exclusively, species-specific. Nutrient stress led to few transcriptional changes in the two dinoflagellates, consistent with other research. An orthologous group analysis examined functionally conserved (i.e., similarly changed) responses to nutrient stress and therefore focused on the diatom and haptophytes. Most conserved ortholog changes were specific to a single nutrient treatment, but a small number of orthologs were similarly changed under both N and P stress in 2 or more species. Many of these orthologs were related to photosynthesis and may represent generalized stress responses. A greater number of orthologs were conserved across more than one species under low P compared to low N. Screening the conserved orthologs for functions related to N and P metabolism revealed increased relative abundance of orthologs for nitrate, nitrite, ammonium, and amino acid transporters under N stress, and increased relative abundance of orthologs related to acquisition of inorganic and organic P substrates under P stress. Although the global transcriptional responses were dominated by species-specific changes, the analysis of conserved responses revealed functional similarities in resource acquisition pathways among different phytoplankton taxa. This overlap in nutrient stress responses observed among species may be useful for tracking the physiological ecology of phytoplankton field populations.

Transcriptome profiling was performed on four treatments of the harmful algal bloom-forming pelagophyte Aureococcus anophagefferens strain CCMP 1850 to assess responses to common stressors for dense phytoplankton blooms: low inorganic nitrogen concentrations, low inorganic phosphorus concentrations, low light levels, and a replete control. The de novo assemblies of pooled reads from all treatments reconstructed ~ 54,000 transcripts using Trinity, and ~ 31,000 transcripts using ABySS. Comparison to the strain CCMP 1984 genome showed that the majority of the gene models were present in both de novo assemblies and that roughly 95% of contigs from both assemblies mapped to the genome, with Trinity capturing slightly more genome content. Sequence reads were mapped back to the de novo assemblies as well as the gene models and differential expression was analyzed using a Bayesian approach called Analysis of Sequence Counts (ASC). On average, 93% of significantly upregulated transcripts recovered by genome mapping were present in the significantly upregulated pool from both de novo assembly methods. Transcripts related to the transport and metabolism of nitrogen were upregulated in the low nitrogen treatment, transcripts encoding enzymes that hydrolyze organic phosphorus or relieve arsenic toxicity were upregulated in the low phosphorus treatment, and transcripts for enzymes that catabolize organic compounds, restructure lipid membranes, or are involved in sulfolipid biosynthesis were upregulated in the low light treatment. A comparison of this transcriptome to the nutrient regulated transcriptional response of CCMP 1984 identified conserved responses between these two strains. These analyses reveal the transcriptional underpinnings of physiological shifts that could contribute to the ecological success of this species in situ: organic matter processing, metal detoxification, lipid restructuring, and photosynthetic apparatus turnover.

Three phosphate-regulated proteins in the coccolithophorid Emiliania huxleyi were detected by the biotinylation of cell-surface proteins. Two of these phosphate-regulated proteins have reduced denatured molecular weights near 110 000 Da (118 078 and 110 541, respectively), while the third, and most abundant, is 69 087 Da. Induction of the three proteins and the common marker of phosphate stress, alkaline phosphatase activity, occur in the presence of <0.25 μM inorganic phosphate in batch culture. Phosphate-regulated proteins and enzyme activity differed among E. huxleyi strains. Alkaline phosphatase is an enzyme commonly induced by phytoplankton in response to phosphate stress in order for cells to scavenge inorganic phosphate from organic sources. In E. huxleyi, this enzyme activity and the phosphate-regulated proteins are rapidly lost when phosphate is added back to phosphate-stressed cultures. This contrasts with the slower loss of alkaline phosphatase activity in the dinoflagellate Prorocentrum minimum. The presence of the three phosphate-regulated proteins and enzyme activity appear to differ somewhat among E. huxleyi strains. Based on these differences between strains, kinetic data, growth experiments and enzyme activities, the 69 087 Da protein may be a phosphatase with a high specificity for 5′-nucleotides. [ABSTRACT FROM AUTHOR]

Genes that are constitutively expressed across multiple environmental stimuli are crucial to quantifying differentially expressed genes, particularly when employing quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) assays. However, the identification of these potential reference genes in non-model organisms is challenging and is often guided by expression patterns in distantly related organisms. Here, transcriptome datasets from the diatom Thalassiosira pseudonana grown under replete, phosphorus-limited, iron-limited, and phosphorus and iron co-limited nutrient regimes were analyzed through literature-based searches for homologous reference genes, k-means clustering, and Analysis of Sequence Counts (ASC) to identify putative reference genes. A total of 9759 genes were identified and screened for stable expression. Literature-based searches surveyed 18 generally accepted reference genes, revealing 101 homologs in T. pseudonana with variable expression and a wide range of mean tags per million. K-means analysis parsed the whole transcriptome into 15 clusters. The two most stable clusters contained 709 genes but still had distinct patterns in expression. ASC analyses identified 179 genes that were stably expressed (p < 0.1 for 1.25 fold change). Genes known to have a stable expression pattern across the test treatments, like actin, were identified in this pool of 179 candidate genes. ASC can be employed on data without biological replicates and was more robust than the k-means approach in isolating genes with stable expression. The intersection of the genes identified through ASC with commonly used reference genes from the literature suggests that actin and ubiquitin ligase may be useful reference genes for T. pseudonana and potentially other diatoms. With the wealth of transcriptome sequence data becoming available, ASC can be easily applied to transcriptome datasets from other phytoplankton to identify reference genes.

Dissolved organic phosphorus (DOP) is increasingly recognized as an important phosphorus source that can support primary production in a variety of marine systems. Despite its importance, the production rate and fate of DOP is poorly understood. In this study, four shipboard nutrient addition experiments were conducted during the upwelling summer season off the coast of Oregon (USA) to track DOP production. Nitrogen (N) and phosphorus (P) additions were used to decouple DOP production and hydrolysis by inducing or repressing, respectively, community alkaline phosphatase activity. In order to examine the progression of nutrient uptake and DOP production under upwelling versus relaxation conditions, two experiments were initiated with waters collected during upwelling events, and two with waters collected during relaxation events. Increases in DOP concentration occurred by day-5 in control treatments in all experiments. Nitrogen treatments had increased chlorophyll a, and yielded lower net DOP production rates relative to controls, as well as increased alkaline phosphatase activity, suggesting that DOP levels were depressed as a consequence of increased hydrolysis of bioavailable DOP substrates. Phosphorus additions resulted in a significant net production of DOP at all stations, but no increase in chlorophyll a relative to control treatments. Taken together these data suggest that changes in DIN:DIP will influence DOP production, which in turn may impact the potential export of DOP to offshore ecosystems. A comparison of experimental data from the four sites also suggests that geographic/physiographic conditions exert greater control on DOP production than physical (upwelling versus relaxation) conditions.

Phosphorus (P) is a critical driver of phytoplankton growth and ecosystem function in the ocean. Diatoms are an abundant class of marine phytoplankton that are responsible for significant amounts of primary production. With the control they exert on the oceanic carbon cycle, there have been a number of studies focused on how diatoms respond to limiting macro and micronutrients such as iron and nitrogen. However, diatom physiological responses to P deficiency are poorly understood. Here, we couple deep sequencing of transcript tags and quantitative proteomics to analyze the diatom Thalassiosira pseudonana grown under P-replete and P-deficient conditions. A total of 318 transcripts were differentially regulated with a false discovery rate of

Shotgun mass spectrometry was used to detect proteins in the harmful alga, Aureococcus anophagefferens, and monitor their relative abundance across nutrient replete (control), phosphate-deficient (-P) and -P refed with phosphate (P-refed) conditions. Spectral counting techniques identified differentially abundant proteins and demonstrated that under phosphate deficiency, A. anophagefferens increases proteins involved in both inorganic and organic phosphorus (P) scavenging, including a phosphate transporter, 5'-nucleotidase, and alkaline phosphatase. Additionally, an increase in abundance of a sulfolipid biosynthesis protein was detected in -P and P-refed conditions. Analysis of the polar membrane lipids showed that cellular concentrations of the sulfolipid sulphoquinovosyldiacylglycerol (SQDG) were nearly two-fold greater in the -P condition versus the control condition, while cellular phospholipids were approximately 8-fold less. Transcript and protein abundances were more tightly coupled for gene products involved in P metabolism compared to those involved in a range of other metabolic functions. Comparison of protein abundances between the -P and P-refed conditions identified differences in the timing of protein degradation and turnover. This suggests that culture studies examining nutrient starvation responses will be valuable in interpreting protein abundance patterns for cellular nutritional status and history in metaproteomic datasets.