Aureococcus anophagefferens (Pelagophyceae) genomes improve evaluation of nutrient acquisition strategies involved in brown tide dynamics.

The pelagophyte Aureococcus anophagefferens causes harmful brown tide blooms in marine embayments on three continents. Aureococcus anophagefferens was the first harmful algal bloom species to have its genome sequenced, an advance that evidenced genes important for adaptation to environmental conditions that prevail during brown tides. To expand the genomic tools available for this species, genomes for four strains were assembled, including three newly sequenced strains and one assembled from publicly available data. These genomes ranged from 57.11 to 73.62 Mb, encoding 13,191-17,404 potential proteins. All strains shared ~90% of their encoded proteins as determined by homology searches and shared most functional orthologs as determined by KEGG, although each strain also possessed coding sequences with unique functions. Like the original reference genome, the genomes assembled in this study possessed genes hypothesized to be important in bloom proliferation, including genes involved in organic compound metabolism and growth at low light. Cross-strain informatics and culture experiments suggest that the utilization of purines is a potentially important source of organic nitrogen for brown tides. Analyses of metatranscriptomes from a brown tide event demonstrated that use of a single genome yielded a lower read mapping percentage (~30% of library reads) as compared to a database generated from all available genomes (~43%), suggesting novel information about bloom ecology can be gained from expanding genomic space. This work demonstrates the continued need to sequence ecologically relevant algae to understand the genomic potential and their ecology in the environment.

Strength in numbers: Collaborative science for new experimental model systems.

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

A comparative study of metatranscriptomic assessment methods to characterize Microcystis blooms.

Harmful algal blooms are increasing in duration and severity globally, resulting in increased research interest. The use of genetic sequencing technologies has provided a wealth of opportunity to advance knowledge, but also poses a risk to that knowledge if handled incorrectly. The vast numbers of sequence processing tools and protocols provide a method to test nearly every hypothesis, but each method has inherent strengths and weaknesses. Here, we tested six methods to classify and quantify metatranscriptomic activity from a harmful algal bloom dominated by Microcystis spp. Three online tools were evaluated (Kaiju, MG-RAST, and GhostKOALA) in addition to three local tools that included a command line BLASTx approach, recruitment of reads to individual Microcystis genomes, and recruitment to a combined Microcystis composite genome generated from sequenced isolates with complete, closed genomes. Based on the analysis of each tool presented in this study, two recommendations are made that are dependent on the hypothesis to be tested. For researchers only interested in the function and physiology of Microcystis spp., read recruitments to the composite genome, referred to as "Frankenstein's Microcystis", provided the highest total estimates of transcript expression. However, for researchers interested in the entire bloom microbiome, the online GhostKOALA annotation tool, followed by subsequent read recruitments, provided functional and taxonomic characterization, in addition to transcript expression estimates. This study highlights the critical need for careful evaluation of methods before data analysis.

Nitrogen flux into metabolites and microcystins changes in response to different nitrogen sources in Microcystis aeruginosa NIES-843.

The over-enrichment of nitrogen (N) in the environment has contributed to severe and recurring harmful cyanobacterial blooms, especially by the non-N2 -fixing Microcystis spp. N chemical speciation influences cyanobacterial growth, persistence and the production of the hepatotoxin microcystin, but the physiological mechanisms to explain these observations remain unresolved. Stable-labelled isotopes and metabolomics were employed to address the influence of nitrate, ammonium, and urea on cellular physiology and production of microcystins in Microcystis aeruginosa NIES-843. Global metabolic changes were driven by both N speciation and diel cycling. Tracing 15 N-labelled nitrate, ammonium, and urea through the metabolome revealed N uptake, regardless of species, was linked to C assimilation. The production of amino acids, like arginine, and other N-rich compounds corresponded with greater turnover of microcystins in cells grown on urea compared to nitrate and ammonium. However, 15 N was incorporated into microcystins from all N sources. The differences in N flux were attributed to the energetic efficiency of growth on each N source. While N in general plays an important role in sustaining biomass, these data show that N-speciation induces physiological changes that culminate in differences in global metabolism, cellular microcystin quotas and congener composition.

Nutrient stoichiometry shapes microbial coevolution.

Coevolution is a force contributing to the generation and maintenance of biodiversity. It is influenced by environmental conditions including the scarcity of essential resources, which can drive the evolution of defence and virulence traits. We conducted a long-term chemostat experiment where the marine cyanobacterium Synechococcus was challenged with a lytic phage under nitrogen (N) or phosphorus (P) limitation. This manipulation of nutrient stoichiometry altered the stability of host-parasite interactions and the underlying mode of coevolution. By assessing the infectivity with > 18 000 pairwise challenges, we documented directional selection for increased phage resistance, consistent with arms-race dynamics while phage infectivity fluctuated through time, as expected when coevolution is driven by negative frequency-dependent selection. The resulting infection networks were 50% less modular under N- versus P-limitation reflecting host-range contraction and asymmetric coevolutionary trajectories. Nutrient stoichiometry affects eco-evolutionary feedbacks in ways that may alter the dynamics and functioning of environmental and host-associated microbial communities.

Viral and bacterial community responses to stimulated Fe(III)-bioreduction during simulated subsurface bioremediation.

The delivery of fermentable substrate(s) to subsurface environments stimulates Fe(III)-bioreduction and achieves detoxification of organic/inorganic contaminants. Although, much research has been conducted on the microbiology of such engineered systems at lab and field scales, little attention has been given to the phage-host interactions and virus community dynamics in these environments. The objective was to determine the responses of soil bacterial communities and viral assemblages to stimulated anaerobic Fe(III)-bioreduction following electron donor (e.g. acetate) addition. Microbial communities, including viral assemblages, were investigated after 60 days of Fe(III)-bioreduction in laboratory-scale columns continuously fed with acetate-amended artificial groundwater. Viral abundances were greatest in the influent section and decreased along the flow path. Acetate availability was important in influencing bacterial diversity, microbial interactions and viral abundance and community composition. The impact of acetate addition was most evident in the influent section of the columns. The increased relative abundance of Fe(III)-reducing bacteria coincided with an increase in viral abundance in areas of the columns exhibiting the most Fe(III) reduction. The genetic composition of viruses in these column sections also differed from the control column and distal sections of acetate-treated columns suggesting viral communities responded to biostimulated Fe(III)-bioreduction.

Cylindrospermopsis raciborskii Virus and host: genomic characterization and ecological relevance.

Cylindrospermopsis (Raphidiopsis) raciborskii is an invasive, filamentous, nitrogen-fixing cyanobacterium that forms frequent blooms in freshwater habitats. While viruses play key roles in regulating the abundance, production and diversity of their hosts in aquatic ecosystems, the role(s) of viruses in the ecology of C. raciborskii is almost unexplored. Progress in this field has been hindered by the absence of a characterized virus-host system in C. raciborskii. To bridge this gap, we sequenced the genome of CrV-01T, a previously isolated cyanosiphovirus, and its host, C. raciborskii strain Cr2010. Analyses suggest that CrV-01T represents a distinct clade of siphoviruses infecting, and perhaps lysogenizing, filamentous cyanobacteria. Its genome contains unique features that include an intact CRISPR array and a 12 kb inverted duplication. Evidence suggests CrV-01T recently gained the ability to infect Cr2010 and recently lost the ability to form lysogens. The cyanobacterial host contains a CRISPR-Cas system with CRISPR spacers matching protospacers within the inverted duplication of the CrV-01T genome. Examination of metagenomes demonstrates that viruses with high genetic identity to CrV-01T, but lacking the inverted duplication, are present in C. raciborskii blooms in Australia. The unique genomic features of the CrV/Cr2010 system offers opportunities to investigate in more detail virus-host interactions in an ecologically important bloom-forming cyanobacterium.

Composition of the gut microbiota modulates the severity of malaria.

Plasmodium infections result in clinical presentations that range from asymptomatic to severe malaria, resulting in ∼1 million deaths annually. Despite this toll on humanity, the factors that determine disease severity remain poorly understood. Here, we show that the gut microbiota of mice influences the pathogenesis of malaria. Genetically similar mice from different commercial vendors, which exhibited differences in their gut bacterial community, had significant differences in parasite burden and mortality after infection with multiple Plasmodium species. Germfree mice that received cecal content transplants from "resistant" or "susceptible" mice had low and high parasite burdens, respectively, demonstrating the gut microbiota shaped the severity of malaria. Among differences in the gut flora were increased abundances of Lactobacillus and Bifidobacterium in resistant mice. Susceptible mice treated with antibiotics followed by yogurt made from these bacterial genera displayed a decreased parasite burden. Consistent with differences in parasite burden, resistant mice exhibited an elevated humoral immune response compared with susceptible mice. Collectively, these results identify the composition of the gut microbiota as a previously unidentified risk factor for severe malaria and modulation of the gut microbiota (e.g., probiotics) as a potential treatment to decrease parasite burden.

Diversity of Active Viral Infections within the Sphagnum Microbiome.

Sphagnum-dominated peatlands play an important role in global carbon storage and represent significant sources of economic and ecological value. While recent efforts to describe microbial diversity and metabolic potential of the Sphagnum microbiome have demonstrated the importance of its microbial community, little is known about the viral constituents. We used metatranscriptomics to describe the diversity and activity of viruses infecting microbes within the Sphagnum peat bog. The vegetative portions of six Sphagnum plants were obtained from a peatland in northern Minnesota, and the total RNA was extracted and sequenced. Metatranscriptomes were assembled and contigs were screened for the presence of conserved virus marker genes. Using bacteriophage capsid protein gp23 as a marker for phage diversity, we identified 33 contigs representing undocumented phages that were active in the community at the time of sampling. Similarly, RNA-dependent RNA polymerase and the nucleocytoplasmic large DNA virus (NCLDV) major capsid protein were used as markers for single-stranded RNA (ssRNA) viruses and NCLDV, respectively. In total, 114 contigs were identified as originating from undescribed ssRNA viruses, 22 of which represent nearly complete genomes. An additional 64 contigs were identified as being from NCLDVs. Finally, 7 contigs were identified as putative virophage or polinton-like viruses. We developed co-occurrence networks with these markers in relation to the expression of potential-host housekeeping gene rpb1 to predict virus-host relationships, identifying 13 groups. Together, our approach offers new tools for the identification of virus diversity and interactions in understudied clades and suggests that viruses may play a considerable role in the ecology of the Sphagnum microbiome.IMPORTANCESphagnum-dominated peatlands play an important role in maintaining atmospheric carbon dioxide levels by modifying conditions in the surrounding soil to favor the growth of Sphagnum over that of other plant species. This lowers the rate of decomposition and facilitates the accumulation of fixed carbon in the form of partially decomposed biomass. The unique environment produced by Sphagnum enriches for the growth of a diverse microbial consortia that benefit from and support the moss's growth, while also maintaining the hostile soil conditions. While a growing body of research has begun to characterize the microbial groups that colonize Sphagnum, little is currently known about the ecological factors that constrain community structure and define ecosystem function. Top-down population control by viruses is almost completely undescribed. This study provides insight into the significant viral influence on the Sphagnum microbiome and identifies new potential model systems to study virus-host interactions in the peatland ecosystem.

Seasonal Gene Expression and the Ecophysiological Implications of Toxic Microcystis aeruginosa Blooms in Lake Taihu.

Harmful cyanobacterial blooms represent an increasing threat to freshwater resources globally. Despite increased research, the physiological basis of how the dominant bloom-forming cyanobacteria, Microcystis spp., proliferate and then maintain high population densities through changing environmental conditions is poorly understood. In this study, we examined the transcriptional profiles of the microbial community in Lake Taihu, China at 9 stations sampled monthly from June to October in 2014. To target Microcystis populations, we collected metatranscriptomic data and mapped reads to the M. aeruginosa NIES 843 genome. Our results revealed significant temporal gene expression patterns, with many genes separating into either early or late bloom clusters. About one-third of genes observed from M. aeruginosa were differentially expressed between these two clusters. Conductivity and nutrient availability appeared to be the environmental factors most strongly associated with these temporal gene expression shifts. Compared with the early bloom season (June and July), genes involved in N and P transport, energy metabolism, translation, and amino acid biosynthesis were down-regulated during the later season (August to October). In parallel, genes involved in regulatory functions as well as transposases and the production of microcystin and extracellular polysaccharides were up-regulated in the later season. Our observation indicates an eco-physiological shift occurs within the Microcystis spp. transcriptome as cells move from the rapid growth of early summer to bloom maintenance in late summer and autumn.

Seasonally Relevant Cool Temperatures Interact with N Chemistry to Increase Microcystins Produced in Lab Cultures of Microcystis aeruginosa NIES-843.

Freshwater cyanobacterial blooms are regularly formed by Microcystis spp., which are well-known producers of the hepatotoxin microcystin. The environmental factors that regulate microcystin synthesis remain unclear. We used reverse transcription-quantitative PCR (RT-qPCR), metabolomics, and toxin profiling (both by LC-MS) to measure the response of Microcystis aeruginosa NIES-843 to nitrogen (N) concentration, N chemistry (nitrate versus urea), and a range of seasonally relevant temperatures. Growth rates at lower temperatures were slower but resulted in increased cellular microcystin content (quota), and at these lower temperatures, N concentration had no effect on toxin production. In contrast, at warmer temperatures, reduction in N concentration increased toxin production, especially when urea was supplied as the nitrogen source. Our culture results demonstrate how temperature may lead to physiological responses ranging from slow growing yet very toxic cells at cool temperatures, to faster growing but less-toxic cells at warmer temperatures. This response represents a key interaction in bloom dynamics. Capturing this phenomenon as a temperature-driven toxin phenotype incorporated into models might improve the ability to predict microcystin biosynthesis during cyanobacterial blooms.

Iron stable isotopes track pelagic iron cycling during a subtropical phytoplankton bloom.

The supply and bioavailability of dissolved iron sets the magnitude of surface productivity for ∼ 40% of the global ocean. The redox state, organic complexation, and phase (dissolved versus particulate) of iron are key determinants of iron bioavailability in the marine realm, although the mechanisms facilitating exchange between iron species (inorganic and organic) and phases are poorly constrained. Here we use the isotope fingerprint of dissolved and particulate iron to reveal distinct isotopic signatures for biological uptake of iron during a GEOTRACES process study focused on a temperate spring phytoplankton bloom in subtropical waters. At the onset of the bloom, dissolved iron within the mixed layer was isotopically light relative to particulate iron. The isotopically light dissolved iron pool likely results from the reduction of particulate iron via photochemical and (to a lesser extent) biologically mediated reduction processes. As the bloom develops, dissolved iron within the surface mixed layer becomes isotopically heavy, reflecting the dominance of biological processing of iron as it is removed from solution, while scavenging appears to play a minor role. As stable isotopes have shown for major elements like nitrogen, iron isotopes offer a new window into our understanding of the biogeochemical cycling of iron, thereby allowing us to disentangle a suite of concurrent biotic and abiotic transformations of this key biolimiting element.

Community Biological Ammonium Demand: A Conceptual Model for Cyanobacteria Blooms in Eutrophic Lakes.

Cyanobacterial harmful algal blooms (CyanoHABs) are enhanced by anthropogenic pressures, including excessive nutrient (nitrogen, N, and phosphorus, P) inputs and a warming climate. Severe eutrophication in aquatic systems is often manifested as non-N2-fixing CyanoHABs (e.g., Microcystis spp.), but the biogeochemical relationship between N inputs/dynamics and CyanoHABs needs definition. Community biological ammonium (NH4+) demand (CBAD) relates N dynamics to total microbial productivity and NH4+ deprivation in aquatic systems. A mechanistic conceptual model was constructed by combining nutrient cycling and CBAD observations from a spectrum of lakes to assess N cycling interactions with CyanoHABs. Model predictions were supported with CBAD data from a Microcystis bloom in Maumee Bay, Lake Erie, during summer 2015. Nitrogen compounds are transformed to reduced, more bioavailable forms (e.g., NH4+ and urea) favored by CyanoHABs. During blooms, algal biomass increases faster than internal NH4+ regeneration rates, causing high CBAD values. High turnover rates from cell death and remineralization of labile organic matter consume oxygen and enhance denitrification. These processes drive eutrophic systems to NH4+ limitation or colimitation under warm, shallow conditions and support the need for dual nutrient (N and P) control.

Ecophysiological Examination of the Lake Erie Microcystis Bloom in 2014: Linkages between Biology and the Water Supply Shutdown of Toledo, OH.

Annual cyanobacterial blooms dominated by Microcystis have occurred in western Lake Erie (U.S./Canada) during summer months since 1995. The production of toxins by bloom-forming cyanobacteria can lead to drinking water crises, such as the one experienced by the city of Toledo in August of 2014, when the city was rendered without drinking water for >2 days. It is important to understand the conditions and environmental cues that were driving this specific bloom to provide a scientific framework for management of future bloom events. To this end, samples were collected and metatranscriptomes generated coincident with the collection of environmental metrics for eight sites located in the western basin of Lake Erie, including a station proximal to the water intake for the city of Toledo. These data were used to generate a basin-wide ecophysiological fingerprint of Lake Erie Microcystis populations in August 2014 for comparison to previous bloom communities. Our observations and analyses indicate that, at the time of sample collection, Microcystis populations were under dual nitrogen (N) and phosphorus (P) stress, as genes involved in scavenging of these nutrients were being actively transcribed. Targeted analysis of urea transport and hydrolysis suggests a potentially important role for exogenous urea as a nitrogen source during the 2014 event. Finally, simulation data suggest a wind event caused microcystin-rich water from Maumee Bay to be transported east along the southern shoreline past the Toledo water intake. Coupled with a significant cyanophage infection, these results reveal that a combination of biological and environmental factors led to the disruption of the Toledo water supply. This scenario was not atypical of reoccurring Lake Erie blooms and thus may reoccur in the future.

Dynamic, mechanistic, molecular-level modelling of cyanobacteria: Anabaena and nitrogen interaction.

Phytoplankton (eutrophication, biogeochemical) models are important tools for ecosystem research and management, but they generally have not been updated to include modern biology. Here, we present a dynamic, mechanistic, molecular-level (i.e. gene, transcript, protein, metabolite) model of Anabaena - nitrogen interaction. The model was developed using the pattern-oriented approach to model definition and parameterization of complex agent-based models. It simulates individual filaments, each with individual cells, each with genes that are expressed to yield transcripts and proteins. Cells metabolize various forms of N, grow and divide, and differentiate heterocysts when fixed N is depleted. The model is informed by observations from 269 laboratory experiments from 55 papers published from 1942 to 2014. Within this database, we identified 331 emerging patterns, and, excluding inconsistencies in observations, the model reproduces 94% of them. To explore a practical application, we used the model to simulate nutrient reduction scenarios for a hypothetical lake. For a 50% N only loading reduction, the model predicts that N fixation increases, but this fixed N does not compensate for the loading reduction, and the chlorophyll a concentration decreases substantially (by 33%). When N is reduced along with P, the model predicts an additional 8% reduction (compared to P only).

It Takes Two to Tango: When and Where Dual Nutrient (N & P) Reductions Are Needed to Protect Lakes and Downstream Ecosystems.

Preventing harmful algal blooms (HABs) is needed to protect lakes and downstream ecosystems. Traditionally, reducing phosphorus (P) inputs was the prescribed solution for lakes, based on the assumption that P universally limits HAB formation. Reduction of P inputs has decreased HABs in many lakes, but was not successful in others. Thus, the "P-only" paradigm is overgeneralized. Whole-lake experiments indicate that HABs are often stimulated more by combined P and nitrogen (N) enrichment rather than N or P alone, indicating that the dynamics of both nutrients are important for HAB control. The changing paradigm from P-only to consideration of dual nutrient control is supported by studies indicating that (1) biological N fixation cannot always meet lake ecosystem N needs, and (2) that anthropogenic N and P loading has increased dramatically in recent decades. Sediment P accumulation supports long-term internal loading, while N may escape via denitrification, leading to perpetual N deficits. Hence, controlling both N and P inputs will help control HABs in some lakes and also reduce N export to downstream N-sensitive ecosystems. Managers should consider whether balanced control of N and P will most effectively reduce HABs along the freshwater-marine continuum.

Metatranscriptomic evidence for co-occurring top-down and bottom-up controls on toxic cyanobacterial communities.

Little is known about the molecular and physiological function of co-occurring microbes within freshwater cyanobacterial harmful algal blooms (cHABs). To address this, community metatranscriptomes collected from the western basin of Lake Erie during August 2012 were examined. Using sequence data, we tested the hypothesis that the activity of the microbial community members is independent of community structure. Predicted metabolic and physiological functional profiles from spatially distinct metatranscriptomes were determined to be ≥90% similar between sites. Targeted analysis of Microcystis aeruginosa, the historical causative agent of cyanobacterial harmful algal blooms over the past ∼20 years, as well as analysis of Planktothrix agardhii and Anabaena cylindrica, revealed ongoing transcription of genes involved in microcystin toxin synthesis as well as the acquisition of both nitrogen and phosphorus, nutrients often implicated as independent bottom-up drivers of eutrophication in aquatic systems. Transcription of genes involved in carbon dioxide (CO2) concentration and metabolism also provided support for the alternate hypothesis that high-pH conditions and dense algal biomass result in CO2-limiting conditions that further favor cyanobacterial dominance. Additionally, the presence of Microcystis-specific cyanophage sequences provided preliminary evidence of possible top-down virus-mediated control of cHAB populations. Overall, these data provide insight into the complex series of constraints associated with Microcystis blooms that dominate the western basin of Lake Erie during summer months, demonstrating that multiple environmental factors work to shape the microbial community.

Niche of harmful alga Aureococcus anophagefferens revealed through ecogenomics.

Harmful algal blooms (HABs) cause significant economic and ecological damage worldwide. Despite considerable efforts, a comprehensive understanding of the factors that promote these blooms has been lacking, because the biochemical pathways that facilitate their dominance relative to other phytoplankton within specific environments have not been identified. Here, biogeochemical measurements showed that the harmful alga Aureococcus anophagefferens outcompeted co-occurring phytoplankton in estuaries with elevated levels of dissolved organic matter and turbidity and low levels of dissolved inorganic nitrogen. We subsequently sequenced the genome of A. anophagefferens and compared its gene complement with those of six competing phytoplankton species identified through metaproteomics. Using an ecogenomic approach, we specifically focused on gene sets that may facilitate dominance within the environmental conditions present during blooms. A. anophagefferens possesses a larger genome (56 Mbp) and has more genes involved in light harvesting, organic carbon and nitrogen use, and encoding selenium- and metal-requiring enzymes than competing phytoplankton. Genes for the synthesis of microbial deterrents likely permit the proliferation of this species, with reduced mortality losses during blooms. Collectively, these findings suggest that anthropogenic activities resulting in elevated levels of turbidity, organic matter, and metals have opened a niche within coastal ecosystems that ideally suits the unique genetic capacity of A. anophagefferens and thus, has facilitated the proliferation of this and potentially other HABs.

Aureococcus anophagefferens strain CCMP1851: draft genome of a second Kratosvirus quantuckense-susceptible host strain for an emerging host-giant virus model system.

Here, we report the draft genome of Aureococcus anophagefferens strain CCMP1851, which is susceptible to the virus Kratosvirus quantuckense. CCMP1851 complements an available genome for a virus-resistant strain (CCMP1850) isolated from the same bloom. Future studies can now use this genome to examine genetic hints of virus resistance and susceptibility.