Morgan M. Steffen

Comparative Metagenomics of Toxic Freshwater Cyanobacteria Bloom Communities on Two Continents

Toxic cyanobacterial blooms have persisted in freshwater systems around the world for centuries and appear to be globally increasing in frequency and severity. Toxins produced by bloom-associated cyanobacteria can have drastic impacts on the ecosystem and surrounding communities, and bloom biomass can disrupt aquatic food webs and act as a driver for hypoxia. Little is currently known regarding the genomic content of the Microcystis strains that form blooms or the companion heterotrophic community associated with bloom events. To address these issues, we examined the bloom-associated microbial communities in single samples from Lake Erie (North America), Lake Tai (Taihu, China), and Grand Lakes St. Marys (OH, USA) using comparative metagenomics. Together the Cyanobacteria and Proteobacteria comprised >90% of each bloom bacterial community sample, although the dominant phylum varied between systems. Relative to the existing Microcystis aeruginosa NIES 843 genome, sequences from Lake Erie and Taihu revealed a number of metagenomic islands that were absent in the environmental samples. Moreover, despite variation in the phylogenetic assignments of bloom-associated organisms, the functional potential of bloom members remained relatively constant between systems. This pattern was particularly noticeable in the genomic contribution of nitrogen assimilation genes. In Taihu, the genetic elements associated with the assimilation and metabolism of nitrogen were predominantly associated with Proteobacteria, while these functions in the North American lakes were primarily contributed to by the Cyanobacteria. Our observations build on an emerging body of metagenomic surveys describing the functional potential of microbial communities as more highly conserved than that of their phylogenetic makeup within natural systems.

The re-eutrophication of Lake Erie: Harmful algal blooms and hypoxia

Lake Erie supplies drinking water to more than 11 million consumers, processes millions of gallons of wastewater, provides important species habitat and supports a substantial industrial sector, with >

Nutrients drive transcriptional changes that maintain metabolic homeostasis but alter genome architecture in Microcystis

50 billion annual income to tourism, recreational boating, shipping, fisheries, and other industries. These and other key ecosystem services are currently threatened by an excess supply of nutrients, manifested in particular by increases in the magnitude and extent of harmful planktonic and benthic algal blooms (HABs) and hypoxia. Widespread concern for this important international waterbody has been manifested in a strong focus of scientific and public material on the subject, and commitments for Canada-US remedial actions in recent agreements among Federal, Provincial and State agencies. This review provides a retrospective synthesis of past and current nutrient inputs, impairments by planktonic and benthic HABs and hypoxia, modelling and Best Management Practices in the Lake Erie basin. The results demonstrate that phosphorus reduction is of primary importance, but the effects of climate, nitrogen and other factors should also be considered in the context of adaptive management. Actions to reduce nutrient levels by targeted Best Management Practices will likely need to be tailored for soil types, topography, and farming practices.

Metatranscriptomic Evidence for Co-Occurring Top-Down and Bottom-Up Controls on Toxic Cyanobacterial Communities

The cyanobacterium Microcystis aeruginosa is a globally distributed bloom-forming organism that degrades freshwater systems around the world. Factors that drive its dispersion, diversification and success remain, however, poorly understood. To develop insight into cellular-level responses to nutrient drivers of eutrophication, RNA sequencing was coupled to a comprehensive metabolomics survey of M. aeruginosa sp. NIES 843 grown in various nutrient-reduced conditions. Transcriptomes were generated for cultures grown in nutrient-replete (with nitrate as the nitrogen (N) source), nitrogen-reduced (with nitrate, urea or ammonium acting as the N sources) and phosphate-reduced conditions. Extensive expression differences (up to 696 genes for urea-grown cells) relative to the control treatment were observed, demonstrating that the chemical variant of nitrogen available to cells affected transcriptional activity. Of particular note, a high number of transposase genes (up to 81) were significantly and reproducibly up-regulated relative to the control when grown on urea. Conversely, phosphorus (P) reduction resulted in a significant cessation in transcription of transposase genes, indicating that variation in nutrient chemistry may influence transcription of transposases and may impact the highly mosaic genomic architecture of M. aeruginosa. Corresponding metabolomes showed comparably few differences between treatments, suggesting broad changes to gene transcription are required to maintain metabolic homeostasis under nutrient reduction. The combined observations provide novel and extensive insight into the complex cellular interactions that take place in this important bloom-forming organism during variable nutrient conditions and highlight a potential unknown molecular mechanism that may drive Microcystis blooms and evolution.

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

ABSTRACT 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.

Molecular prediction of lytic vs lysogenic states for Microcystis phage: Metatranscriptomic evidence of lysogeny during large bloom events

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.

A review of the global ecology, genomics, and biogeography of the toxic cyanobacterium, Microcystis spp.

Microcystis aeruginosa is a freshwater bloom-forming cyanobacterium capable of producing the potent hepatotoxin, microcystin. Despite increased interest in this organism, little is known about the viruses that infect it and drive nutrient mobilization and transfer of genetic material between organisms. The genomic complement of sequenced phage suggests these viruses are capable of integrating into the host genome, though this activity has not been observed in the laboratory. While analyzing RNA-sequence data obtained from Microcystis blooms in Lake Tai (Taihu, China), we observed that a series of lysogeny-associated genes were highly expressed when genes involved in lytic infection were down-regulated. This pattern was consistent, though not always statistically significant, across multiple spatial and temporally distinct samples. For example, samples from Lake Tai (2014) showed a predominance of lytic virus activity from late July through October, while genes associated with lysogeny were strongly expressed in the early months (June–July) and toward the end of bloom season (October). Analyses of whole phage genome expression shows that transcription patterns are shared across sampling locations and that genes consistently clustered by co-expression into lytic and lysogenic groups. Expression of lytic-cycle associated genes was positively correlated to total dissolved nitrogen, ammonium concentration, and salinity. Lysogeny-associated gene expression was positively correlated with pH and total dissolved phosphorous. Our results suggest that lysogeny may be prevalent in Microcystis blooms and support the hypothesis that environmental conditions drive switching between temperate and lytic life cycles during bloom proliferation.