Steven Wilhelm is the Kenneth and Blaire Mossman Professor & Associate Head of the Department of Microbiology. In 2016 he became a fellow of the American Academy of Microbiology as well as a Sustaining Fellow of ASLO. In 2018 he was also named a James R. Cox Professor at the University of Tennessee. His group studies synergies between microbial communities and biogeochemical cycles in lakes and oceans. Lab members use biomolecular tools - DNA and RNA sequencing, metabolomics, and PCR-based quantitative analyses - to study viruses, bacteria, cyanobacteria and algae.
Skills and Expertise
EnvironmentWater QualityEcologyMarine EcologyMicrobiologyMarine BiologyAquatic EcologyMicrobial EcologyBiogeochemistryFreshwater EcologyLakesCommunity StructureBiological OceanographyMicrobial DiversityEutrophicationOceanographyAlgaeMetagenomicsPhytoplanktonLimnologySurface WaterPhytoplankton EcologyIronCyanobacteriaPlankton EcologyMolecular Microbial EcologyPlanktonDiatomsMarine MicrobiologyMicrobial GenomicsPyrosequencingAquatic Microbial EcologyMarine Microbial EcologyHarmful Algal BloomVirus Ecology
Awards & Achievements (2)
Award · Jun 2016
Named Fellow of the American Academy for Microbiology
Award · Feb 2016
Named Sustaining Fellow of ASLO
Microorganism plays a vital role in maintaining ecosystem function in lakes. For centuries, the patterns of plants and animals have been studied extensively at different temporal and spatial scales, while few similar studies have been carried out for microorganisms in the past due to the limitation of techniques. In this proposed study, five large shallow eutrophic lakes, i.e. Lake Taihu, Lake Chaohu, Lake Poyang, Lake Hongze and Lake Dianchi, were chosen to explore the biogeography patterns of prokaryotic microorganism (mainly bacteria and archaea). Using 16S rRNA gene based high-throughput sequencing, geographic information system and multivariate statistical analysis, we are going to elucidate the temporal and spatial patterns of prokaryotic microbial abundance, community composition and diversity. Meanwhile, the contribution of geography distance and modern environmental factors, such as cyanobacterial blooms, aquatic plant and riverine input, to the patterns were investigated quantitatively. The observed results would supply the clear evidence of whether the microbial pattern is similar to the macro-organism’s. And it would helpful to the development biogeography and provide important insights into the protection of eutrophic lake ecosystems.
Causes, consequences and controls of algal species outbreaks and 'harmful' blooms; delineation of the term 'harmful'; identification of harmful effects including toxins; molecular characterization of these events.
Research Items (184)
Establishing virus–host relationships has historically relied on culture-dependent approaches. Here we report on the use of marine metatranscriptomics to probe virus–host relationships. Statistical co-occurrence analyses of dsDNA, ssRNA and dsRNA viral markers of polyadenylation-selected RNA sequences from microbial communities dominated by Aureococcus anophagefferens (Quantuck Bay, NY), and diatoms (Narragansett Bay, RI) show active infections by diverse giant viruses (NCLDVs) associated with algal and nonalgal hosts. Ongoing infections of A. anophagefferens by a known Mimiviridae (AaV) occur during bloom peak and decline. Bloom decline is also accompanied by increased activity of viruses other than AaV, including ( þ ) ssRNA viruses. In Narragansett Bay, increased temporal resolution reveals active NCLDVs with both ‘boom-and-bust’ and ‘steady-state infection’-like ecologies that include known as well as novel virus–host interactions. Our approach offers a method for screening active viral infections and develops links between viruses and their potential hosts in situ. Our observations further demonstrate that previously unknown virus–host relationships in marine systems are abundant.
- May 2017
Annual cyanobacterial blooms dominated by Microcystis have occurred in western Lake Erie (USA/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 re-occurring Lake Erie blooms and thus may re-occur in the future.
Viruses are major pathogens in all biological systems. Virus propagation and downstream analysis remains a challenge, particularly in the ocean where the majority of their microbial hosts remain recalcitrant to current culturing techniques. We used a cultivation-independent approach to isolate and sequence individual viruses. The protocol uses high-speed fluorescence-activated virus sorting flow cytometry, multiple displacement amplification (MDA), and downstream genomic sequencing. We focused on 'giant viruses' that are readily distinguishable by flow cytometry. From a single-milliliter sample of seawater collected from off the dock at Boothbay Harbor, ME, USA, we sorted almost 700 single virus particles, and subsequently focused on a detailed genome analysis of 12. A wide diversity of viruses was identified that included Iridoviridae, extended Mimiviridae and even a taxonomically novel (unresolved) giant virus. We discovered a viral metacaspase homolog in one of our sorted virus particles and discussed its implications in rewiring host metabolism to enhance infection. In addition, we demonstrated that viral metacaspases are widespread in the ocean. We also discovered a virus that contains both a reverse transcriptase and a transposase; although highly speculative, we suggest such a genetic complement would potentially allow this virus to exploit a latency propagation mechanism. Application of single virus genomics provides a powerful opportunity to circumvent cultivation of viruses, moving directly to genomic investigation of naturally occurring viruses, with the assurance that the sequence data is virus-specific, non-chimeric and contains no cellular contamination.The ISME Journal advance online publication, 12 May 2017; doi:10.1038/ismej.2017.61.
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.
The North Pacific Ocean (between approximately 0°N and 50°N) contains the largest continuous ecosystem on Earth. This region plays a vital role in the cycling of globally important nutrients as well as carbon. Although the microbial communities in this region have been assessed, the dynamics of viruses (abundances and production rates) remains understudied. To address this gap, scientific cruises during the winter and summer seasons (2013) covered the North Pacific basin to determine factors that may drive virus abundances and production rates. Along with information on virus particle abundance and production, we collected a spectrum of oceanographic metrics as well as information on microbial diversity. The data suggest that both biotic and abiotic factors affect the distribution of virus particles. Factors influencing virus dynamics did not vary greatly between seasons, although the abundance of viruses was almost an order of magnitude greater in the summer. When considered in the context of microbial community structure, our observations suggest that members of the bacterial phyla Proteobacteria, Planctomycetes, and Bacteroidetes were correlated to both virus abundances and virus production rates: these phyla have been shown to be enriched in particle associated communities. The findings suggest that environmental factors influence virus community functions (e.g., virion particle degradation) and that particle-associated communities may be important drivers of virus activity.
Our current understanding of biology is heavily based on the contributions from 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 development of ecologically and taxonomically diverse marine protist genetic models. This multifaceted, complex but important challenge required a highly collaborative community-based approach. Herein we describe this approach, the advances achieved, and the lessons learned by participants in this novel community-based model for research.
While viruses with distinct phylogenetic origins and different nucleic acid types can infect and lyse eukaryotic phytoplankton, “giant” dsDNA viruses have been found to be associated with important ecological processes, including the collapse of algal blooms. However, the molecular aspects of giant virus–host interactions remain largely unknown. Aureococcus anophagefferens virus (AaV), a giant virus in the Mimiviridae clade, is known to play a critical role in regulating the fate of brown tide blooms caused by the pelagophyte Aureococcus anophagefferens. To understand the physiological response of A. anophagefferens CCMP1984 upon AaV infection, we studied the transcriptomic landscape of this host–virus pair over an entire infection cycle using a RNA-sequencing approach. A massive transcriptional response of the host was evident as early as 5 min post-infection, with modulation of specific processes likely related to both host defense mechanism(s) and viral takeover of the cell. Infected Aureococcus showed a relative suppression of host-cell transcripts associated with photosynthesis, cytoskeleton formation, fatty acid, and carbohydrate biosynthesis. In contrast, host cell processes related to protein synthesis, polyamine biosynthesis, cellular respiration, transcription, and RNA processing were overrepresented compared to the healthy cultures at different stages of the infection cycle. A large number of redox active host-selenoproteins were overexpressed, which suggested that viral replication and assembly progresses in a highly oxidative environment. The majority (99.2%) of annotated AaV genes were expressed at some point during the infection cycle and demonstrated a clear temporal–expression pattern and an increasing relative expression for the majority of the genes through the time course. We detected a putative early promoter motif for AaV, which was highly similar to the early promoter elements of two other Mimiviridae members, indicating some degree of evolutionary conservation of gene regulation within this clade. This large-scale transcriptome study provides insights into the Aureococcus cells infected by a giant virus and establishes a foundation to test hypotheses regarding metabolic and regulatory processes critical for AaV and other Mimiviridae members.
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 vs. 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.
Microcystins are secondary metabolites produced by several freshwater, bloom-forming cyanobacterial species. Microcystin-producing cyanobacteria co-occur with a complex community of heterotrophic bacteria. Though conflicting, studies suggest that microcystins affect the physiology of heterotrophic bacteria by inducing oxidative stress and increasing cell envelope permeability. Based on these observations, we hypothesized that exposure to microcystin should induce differential expression in genes responding to oxidative and envelope stress and trigger shifts in metabolite pools. We tested this hypothesis by exposing Escherichia coli MG1655 to 1 and 10 mg/L microcystin-LR and monitored global changes to gene expression, cellular metabolite pools, and lipid composition using RNA-sequencing and UPLC-MS. Contrary to reported studies, we observed no evidence that microcystin-LR induced oxidative or cell envelope stress in E. coli under the tested conditions. Our results suggest a potential difference in mechanism by which microcystin-LR interacts with heterotrophic bacteria vs. cyanobacteria.
The original publication of this Article included analysis of virus and microbial cell abundances and virus-to-microbial cell ratios. Data in the Article came from 25 studies intended to be exclusively from marine sites. However, 3 of the studies included in the original unified dataset were erroneously classified as marine sites during compilation. The records with mis-recorded longitude and latitude values were, in fact, taken from inland, freshwater sources. The three inland, freshwater datasets are ELA, TROUT and SWAT. The data from these three studies represent 163 of the 5,671 records in the original publication. In the updated version of the Article, all analyses have been recalculated using the same statistical analysis pipeline released via GitHub as part of the original publication. Removal of the three studies reduces the unified dataset to 5,508 records. Analyses involving all grouped datasets have been updated with changes noted in each figure. All key results remain qualitatively unchanged. All data and scripts used in this correction have been made available as a new, updated GitHub release to reflect the updated dataset and figures.
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 CyanoHAB. 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 co-limitation under warm, shallow conditions and support the need for dual nutrient (N and P) control.
The discovery of infectious particles that challenge conventional thoughts concerning “what is a virus” has led to the evolution a new field of study in the past decade. Here, we review knowledge and information concerning “giant viruses”, with a focus not only on some of the best studied systems, but also provide an effort to illuminate systems yet to be better resolved. We conclude by demonstrating that there is an abundance of new host–virus systems that fall into this “giant” category, demonstrating that this field of inquiry presents great opportunities for future research.
In eutrophic lakes, heterotrophic bacteria are closely associated with algal detritus and play a crucial role in nutrient cycling. However, the seasonal and spatial dynamics of free-living (FL) and particle-attached (PA) bacteria and the environmental factors shaping this relationship remain poorly understood. To address this issue, we explored the spatiotemporal patterns of bacterial community composition (BCC) in Lake Taihu, China, using terminal restriction fragment length polymorphism (T-RFLP) and 454-tag pyrosequencing of 16S rRNA gene. We generated a total of 218,027 high quality non-cyanobacterial sequence reads that resulted in 4940 OTUs (97% cutoff), with Actinobacteria, β- and α-proteobacteria being the predominant taxa. Although PA communities contained significantly higher alpha-diversity than FL ones, we found that 59% of OTUs, that accounted for 96% of the total reads, were shared by both communities. The high degree of overlap between FL and PA communities indicates a high rate of dispersal potential, highlighting an underestimated connectivity and potentially similar ecological role for these two components. Distinct seasonal trends were recorded in both FL and PA communities, while spatial differences in BCC were small. In addition, both FL and PA bacterial communities exhibited similar patterns and synchrony, correlated to water temperature, nitrate and total suspended solids (TSS). Accordingly, the effects of eutrophication and hydrodynamics on the phylogenetic overlap and diversity between FL and PA communities were discussed.
Harmful cyanobacterial blooms (cyanoHABs) are a major threat to freshwater ecosystems worldwide. Evidence suggests that both nitrogen and phosphorus are important nutrients in the development and proliferation of blooms, yet much less is known about nitrogen cycling dynamics in these systems. To assess the potential nitrogen cycling function of the cyanoHAB community, surface water samples were collected in Lake Tai (Taihu), China over a 5-month bloom event in 2014. The expression of six nitrogen cycling genes (nifH, hzsA, nxrB, nrfA, amoA, nosZ) was surveyed using a targeted microarray with probes designed to provide phylogenetic information. N-cycling gene expression varied spatially across Taihu, most notably near the mouth of the Dapu river. Expression of nifH was observed across the lake and attributable to both proteobacteria and cyanobacteria: proteobacteria were major contributors to nifH signal near shore. Other N transformations such as anaerobic ammonia oxidation and denitrification were evident in the surface waters as well. Observations in this study highlight the potential importance of heterotrophic bacteria in N-cycling associated with cyanoHABs.
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 over-generalized. 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 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.
Pyrodinium bahamense is a toxic, bioluminescent dinoflagellate with a record of intense bloom formation in both the Atlantic-Caribbean and Indo-Pacific regions. To date, limited genetic information exists for P. bahamense in comparison to other closely related harmful algal bloom taxa such as Alexandrium, or other bioluminescent taxa such as Pyrocystis. This study utilized single-cell PCR to explore the molecular diversity of P. bahamense within the Indian River Lagoon (IRL), Florida, USA, and a bioluminescent bay in Puerto Rico. Pyrodinium-specific primers targeting a ca.1.2-kb region of the 18S rRNA gene and degenerate primers targeting the conserved catalytic domain of the luciferase gene (lcf) were applied to single cells isolated from both geographic regions as well as single cells of clonal isolates from the IRL. Phylogenetic analysis revealed that while P. bahamense is more closely related to Alexandrium spp. at the 18S rRNA gene level, its lcf sequences are more closely related to Pyrocystis spp. than Alexandrium spp. Pyrodinium bahamense lcf sequences from the Western Atlantic formed 2 distinct clusters. These clusters were defined by a set of core amino acid substitutions, and the extent of variation was greater than that recorded between the established variants of Pyrocystis lcf. lcf sequences from an Indo-Pacific strain formed a third distinct cluster. Based on these results, the potential of lcf for use in tracking sub-populations of P. bahamense is discussed.
C57BL/6 mice are widely used for in vivo studies of immune function and metabolism in mammals. In a previous study, it was observed that when C57BL/6 mice purchased from different vendors were infected with Plasmodium yoelii, a causative agent of murine malaria, they exhibited both differential immune responses and significantly different parasite burdens: these patterns were reproducible when gut contents were transplanted into gnotobiotic mice. To gain insight into the mechanism of resistance, we removed whole ceca from mice purchased from two vendors, Taconic Biosciences (low parasitemia) and Charles River Laboratories (high parasitemia), to determine the combined host and microflora metabolome and metatranscriptome. With the exception of two Charles River samples, we observed ?90% similarity in overall bacterial gene expression within vendors and ?80% similarity between vendors. In total 33 bacterial genes were differentially expressed in Charles River mice (p-value < 0.05) relative to the mice purchased from Taconic. Included among these, fliC, ureABC, and six members of the nuo gene family were overrepresented in microbiomes susceptible to more severe malaria. Moreover, 38 mouse genes were differentially expressed in these purported genetically identical mice. Differentially expressed genes included basigin, a cell surface receptor required for P. falciparum invasion of red blood cells. Differences in metabolite pools were detected, though their relevance to malaria infection, microbial community activity, or host response is not yet understood. Our data have provided new targets that may connect gut microbial activity to malaria resistance and susceptibility phenotypes in the C57BL/6 model organism.
Viruses are generally considered to be amongst the smallest bioactive particles; dating back to the original observations, including those of luminaries such as Ivanosky and Beijerinck, size has always been at issue within the definition, a tradition that continued for many years . It was thus a surprise to the scientific community in the early 2000s when French scientists demonstrated that a particle, previously thought to be a bacterium, was indeed a virus . The discovery of the Mimivirus and the other “giants” that have followed, including Mamavirus, Pandoravirus, Faustovirus, and Mollivirus, has blurred the definition of what constitutes a virus and, indeed, the boundaries between viral particles and cellular life .
There is growing recognition that winter is an important season for growth of photoautotrophs in ice-covered freshwater environments. Exemplifying this are expansive under-ice blooms of filamentous diatoms in Lake Erie. Here we describe a metatranscriptome constructed from a phytoplankton community dominated by filamentous diatoms. As expected, a high percentage (> 73%) of the sequences with BLAST hits to nucleotides or proteins in National Center for Biotechnology Information databases were associated with photosynthetic algae of which the majority were diatoms, mainly Aulacoseira spp. and Stephanodiscus spp. which was confirmed by analysis of 18S rRNA gene transcripts and microscopy. Consistent with the winter growth environment, psychrophilic and low-light adaptations were observed. Prominent among adaptations to cold were transcripts for genes involved in biosynthesis of unsaturated fatty acids, which were consistent with expected increased membrane fluidity at low temperatures. Reflecting the combined effect of low winter insolation and high light attenuation were an abundant complement of reads for light-harvesting antennae, mainly genes encoding fucoxanthin chlorophyll a/c proteins. The presence of virulence factors originating from oomycetes offers support for new hypotheses into the eventual decline of Lake Erie's winter diatom bloom. Whereas fungi were identified both through the metatranscriptome and by microscopy, dsRNA viruses of fungi were detected that may indirectly counter against fungal infection. This study demonstrates the utility of an environmental omics approach to yield insights underlying phototrophic life as well as the interactions of the entire microbial community in an extreme environment.
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 >$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.
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 1942-2014. 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). This article is protected by copyright. All rights reserved.
In early August 2014, the municipality of Toledo, OH (USA) issued a ‘do not drink’ advisory on their water supply directly affecting over 400,000 residential customers and hundreds of businesses (Wilson, 2014). This order was attributable to levels of microcystin, a potent liver toxin, which rose to 2.5 μg L⁻¹ in finished drinking water. The Toledo crisis afforded an opportunity to bring together scientists from around the world to share ideas regarding factors that contribute to bloom formation and toxigenicity, bloom and toxin detection as well as prevention and remediation of bloom events. These discussions took place at an NSF- and NOAA-sponsored workshop at Bowling Green State University on April 13 and 14, 2015. In all, more than 100 attendees from six countries and 15 US states gathered together to share their perspectives. The purpose of this review is to present the consensus summary of these issues that emerged from discussions at the Workshop. As additional reports in this special issue provide detailed reviews on many major CHAB species, this paper focuses on the general themes common to all blooms, such as bloom detection, modeling, nutrient loading, and strategies to reduce nutrients.
This review summarizes the present state of knowledge regarding the toxic, bloom-forming cyanobacterium, Microcystis, with a specific focus on its geographic distribution, toxins, genomics, phylogeny, and ecology. A global analysis found documentation suggesting geographic expansion of Microcystis, with recorded blooms in at least 108 countries, 79 of which have also reported the hepatatoxin microcystin. The production of microcystins (originally “Fast-Death Factor”) by Microcystis and factors that control synthesis of this toxin are reviewed, as well as the putative ecophysiological roles of this metabolite. Molecular biological analyses have provided significant insight into the ecology and physiology of Microcystis, as well as revealed the highly dynamic, and potentially unstable, nature of its genome. A genetic sequence analysis of 27 Microcystis species, including 15 complete/draft genomes are presented. Using the strictest biological definition of what constitutes a bacterial species, these analyses indicate that all Microcystis species warrant placement into the same species complex since the average nucleotide identity values were above 95%, 16S rRNA nucleotide identity scores exceeded 99%, and DNA–DNA hybridization was consistently greater than 70%. The review further provides evidence from around the globe for the key role that both nitrogen and phosphorus play in controlling Microcystis bloom dynamics, and the effect of elevated temperature on bloom intensification. Finally, highlighted is the ability of Microcystis assemblages to minimize their mortality losses by resisting grazing by zooplankton and bivalves, as well as viral lysis, and discuss factors facilitating assemblage resilience.
Many giant dsDNA algal viruses share a common ancestor with Mimivirus – one of the largest viruses, in terms of genetic content. Together, these viruses form the proposed ‘Megaviridae’ clade of Nucleocytoplasmic Large DNA viruses (NCLDV). To gauge Megaviridae diversity we designed degenerate primers targeting the Major Capsid Protein (MCP) genes of algae-infecting viruses within this group and probed the clade's diversity during the course of a brown tide bloom caused by the harmful pelagophyte, Aureococcus anophagefferens. We amplified target sequences in water samples from two distinct locations (Weesuck Creek and Quantuck Bay, NY) covering twelve weeks concurrent with the proliferation and demise of a bloom. In total, 475 amplicons clustered into 145 Operational Taxonomic Units (OTUs) at 97% identity. One OTU contained 19 sequences with ≥ 97% identity to AaV, a member of the Megaviridae clade that infects A. anophagefferens, suggesting AaV was present during the bloom. Unifrac analysis showed clear temporal patterns in algal Megaviridae dynamics, with a shift in the virus community structure that corresponded to the Aureococcus bloom decline in both locations. Our data provide insights regarding the environmental relevance of algal Megaviridae members and raise important questions regarding their phylodynamics across different environmental gradients.
Hydrogen peroxide (HOOH) is omnipresent in natural waters. Given that sunlight is the primary source of HOOH, we investigated the relationship between time of day and microbial HOOH degradation. Genes encoding HOOH-degrading enzymes were significantly more abundant during the day in ocean metatranscriptomes. While bacterial catalase-peroxidases were the most abundant transcripts, the abundance of algal peroxidases, along with the insensitivity of HOOH degradation rates to antibiotic treatment in our incubations, suggested that eukaryotic microorganisms were also important scavengers of HOOH. Phylogenetic placement of transcripts for HOOH degrading enzymes suggested that different taxa expressed these enzymes during the day than during the night. We also measured HOOH concentrations over a 24 h period in the South Pacific, and simultaneously conducted bottle incubations to measure HOOH dark degradation rates. Fitting these data to a dynamic model confirmed that the ability of the microbial community to degrade HOOH during the day increased, with peak HOOH removal rates occurring in late afternoon coincident with the highest HOOH concentrations. Collectively, these data suggest that even in dilute HOOH environments, there is a dynamic diel response to HOOH, and that the open ocean microbial community is complicit in its cross-protection of Prochlorococcus and other HOOH-sensitive taxa.
Significant evidence shows that nitrogen (N) supply may influence microbial community structure and, in some cases, the rate of primary productivity in fresh waters. To date, however, most focus has been on dissolved inorganic N (i.e., ammonia and nitrate), or dinitrogen gas. Far less is known about the effects of dissolved organic N such as urea on plankton activity, although this compound is both produced by in-lake processes and is a significant component of external loading. We evaluated the urea distribution and the activity of the major enzyme responsible for its assimilation (urease) in Lake Erie, which has a significant history of eutrophication. During 2012 and 2013, lake-wide surveys estimated surface urea concentrations and urease activity, along with phytoplankton composition and biomass, cyanobacterial toxins (microcystin), major nutrients and other physico-chemical parameters. In parallel, in situ 48-h microcosm experiments were executed to test whether different chemical forms of dissolved N could stimulate phytoplankton biomass. Results confirmed urea was a bioavailable form of N with in situ urea turnover times ranging from hours (for summer, i.e., Aug. 2012 and July 2013) to days (May 2013). Furthermore, we observed a positive correlation between urease activity and both microcystin concentrations and cyanobacterial dominance. Results also indicated a potential seasonal shift in the nutrient limiting phytoplankton biomass from phosphorus (P) to N. Our results reinforce the importance of both N and P in promoting phytoplankton growth and highlighted the need to consider organic nutrient sources as potentially important drivers of cyanobacterial blooms and toxin production.
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.
- Feb 2016
Viral lysis of phytoplankton constrains marine primary production, food web dynamics and biogeochemical cycles in the ocean. Yet, little is known about the biogeographical distribution of viral lysis rates across the global ocean. To address this, we investigated phytoplankton group-specific viral lysis rates along a latitudinal gradient within the North Atlantic Ocean. The data show large-scale distribution patterns of different virus groups across the North Atlantic that are associated with the biogeographical distributions of their potential microbial hosts. Average virus-mediated lysis rates of the picocyanobacteria Prochlorococcus and Synechococcus were lower than those of the picoeukaryotic and nanoeukaryotic phytoplankton (that is, 0.14 per day compared with 0.19 and 0.23 per day, respectively). Total phytoplankton mortality (virus plus grazer-mediated) was comparable to the gross growth rate, demonstrating high turnover rates of phytoplankton populations. Virus-induced mortality was an important loss process at low and mid latitudes, whereas phytoplankton mortality was dominated by microzooplankton grazing at higher latitudes (>56°N). This shift from a viral-lysis-dominated to a grazing-dominated phytoplankton community was associated with a decrease in temperature and salinity, and the decrease in viral lysis rates was also associated with increased vertical mixing at higher latitudes. Ocean-climate models predict that surface warming will lead to an expansion of the stratified and oligotrophic regions of the world’s oceans. Our findings suggest that these future shifts in the regional climate of the ocean surface layer are likely to increase the contribution of viral lysis to phytoplankton mortality in the higher-latitude waters of the North Atlantic, which may potentially reduce transfer of matter and energy up the food chain and thus affect the capacity of the northern North Atlantic to act as a long-term sink for CO2.
Marine viruses are critical drivers of ocean biogeochemistry, and their abundances vary spatiotemporally in the global oceans, with upper estimates exceeding 108 per ml. Over many years, a consensus has emerged that virus abundances are typically tenfold higher than microbial cell abundances. However, the true explanatory power of a linear relationship and its robustness across diverse ocean environments is unclear. Here, we compile 5,671 microbial cell and virus abundance estimates from 25 distinct marine surveys and find substantial variation in the virus-to-microbial cell ratio, in which a 10:1 model has either limited or no explanatory power. Instead, virus abundances are better described as nonlinear, power-law functions of microbial cell abundances. The fitted scaling exponents are typically less than 1, implying that the virus-to-microbial cell ratio decreases with microbial cell density, rather than remaining fixed. The observed scaling also implies that viral effect sizes derived from ‘representative’ abundances require substantial refinement to be extrapolated to regional or global scales.
- Jul 2015
Dissolved iron supply is pivotal in setting global phytoplankton productivity and pelagic ecosystem structure. However, most studies of the role of iron have focussed on carbon biogeochemistry within pelagic ecosystems, with less effort to quantify the iron biogeochemical cycle. Here we compare mixed‐layer biotic iron inventories from a low‐iron (~0.06 nmol L−1) subantarctic (FeCycle study) and a seasonally high‐iron (~0.6 nmol L−1) subtropical (FeCycle II study) site. Both studies were quasi‐Lagrangian, and had multi‐day occupation, common sampling protocols, and indirect estimates of biotic iron (from a limited range of available published biovolume/carbon/iron quotas). Biotic iron pools were comparable (~100 ± 30 pmol L−1) for low‐ and high‐iron waters, despite a tenfold difference in dissolved iron concentrations. Consistency in biotic iron inventories (~80 ± 24 pmol L−1, largely estimated using a limited range of available quotas) was also conspicuous for three Southern Ocean polar sites. Insights into the extent to which uniformity in biotic iron inventories was driven by the need to apply common iron quotas obtained from laboratory cultures were provided from FeCycle II. The observed twofold to threefold range of iron quotas during the evolution of FeCycle II subtropical bloom was much less than reported from laboratory monocultures. Furthermore, the iron recycling efficiency varied by fourfold during FeCycle II, increasing as stocks of new iron were depleted, suggesting that quotas and iron recycling efficiencies together set biotic iron pools. Hence, site‐specific differences in iron recycling efficiencies (which provide 20–50% and 90% of total iron supply in high‐ and low‐iron waters, respectively) help offset the differences in new iron inputs between low‐ and high‐iron sites. Future parameterization of iron in biogeochemical models must focus on the drivers of biotic iron inventories, including the differing iron requirements of the resident biota, and the subsequent fate (retention/export/recycling) of the biotic iron. Biotic iron pools are of similar magnitude in low‐ and high‐iron watersIron quotas and fe ratios together primarily set the size of biotic iron poolsIron‐rich microbes dominate biotic pools and iron recycling in HNLC waters
Here we examined the impact of a commonly employed method used to measure nitrogen fixation, the acetylene reduction assay (ARA), on a marine sediment community. Historically, the ARA technique has been broadly employed for its ease of use, in spite of numerous known artifacts. To gauge the severity of these effects in a natural environment, we employed high-throughput 16S rRNA gene sequencing to detect differences in acetylene-treated sediments vs. non-treated control sediments after a 7 h incubation. Within this short time period, significant differences were seen across all activity of microbes identified in the sediment, implying that the changes induced by acetylene occur quickly. The results have important implications for our understanding of marine nitrogen budgets. Moreover, because the ARA technique has been widely used in terrestrial and freshwater habitats, these results may be applicable to other ecosystems.
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 were examined from the western basin of Lake Erie collected during August 2012. 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 triplicate 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, revealed ongoing transcription of genes involved in 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 CO2aq-limitingconditions that favor cyanobacterial dominance. Additionally, the presence of Microcystis-specific cyanophage sequences provided preliminary evidence of possible top-down virus-mediated control of cHAB populations while the activity of Microcystis specific transposable elements suggest the potential for ongoing changes within this organism's genome. Overall, the 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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Viral lysis of microbial hosts releases organic matter that can then be assimilated by nontargeted microorganisms. Quantitative estimates of virus-mediated recycling of carbon in marine waters, first established in the late 1990s, were originally extrapolated from marine host and virus densities, host carbon content and inferred viral lysis rates. Yet, these estimates did not explicitly incorporate the cascade of complex feedbacks associated with virus-mediated lysis. To evaluate the role of viruses in shaping community structure and ecosystem functioning, we extend dynamic multitrophic ecosystem models to include a virus component, specifically parameterized for processes taking place in the ocean euphotic zone. Crucially, we are able to solve this model analytically, facilitating evaluation of model behavior under many alternative parameterizations. Analyses reveal that the addition of a virus component promotes the emergence of complex communities. In addition, biomass partitioning of the emergent multitrophic community is consistent with well-established empirical norms in the surface oceans. At steady state, ecosystem fluxes can be probed to characterize the effects that viruses have when compared with putative marine surface ecosystems without viruses. The model suggests that ecosystems with viruses will have (1) increased organic matter recycling, (2) reduced transfer to higher trophic levels and (3) increased net primary productivity. These model findings support hypotheses that viruses can have significant stimulatory effects across whole-ecosystem scales. We suggest that existing efforts to predict carbon and nutrient cycling without considering virus effects are likely to miss essential features of marine food webs that regulate global biogeochemical cycles.
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.
Microcystins are secondary metabolites produced by cyanobacteria that act as hepatotoxins in higher organisms. These toxins can be altered through abiotic processes, such as photodegradation and adsorption, as well as through biological processes via metabolism and bacterial degradation. Some species of bacteria can degrade microcystins, and many other organisms metabolize microcystins into a series of conjugated products. There are toxicokinetic models used to examine microcystin uptake and elimination, which can be difficult to compare due to differences in compartmentalization and speciation. Metabolites of microcystins are formed as a detoxification mechanism, and little is known about how quickly these metabolites are formed. In summary, microcystins can undergo abiotic and biotic processes that alter the toxicity and structure of the microcystin molecule. The environmental impact and toxicity of these alterations and the metabolism of microcystins remains uncertain, making it difficult to establish guidelines for human health. Here, we present the current state of knowledge regarding the alterations microcystins can undergo in the environment
Aureococcus anophagefferens causes economically and ecologically destructive “brown tides” in the United States, China and South Africa. Here we report the 370,920 bp genomic sequence of AaV, a virus capable of infecting and lysing A. anophagefferens. AaV is a member of the nucleocytoplasmic large DNA virus (NCLDV) group, harboring 377 putative coding sequences and 8 tRNAs. Despite being an algal virus, AaV shows no phylogenetic affinity to the Phycodnaviridae family, to which most algae-infecting viruses belong. Core gene phylogenies, shared gene content and genome-wide similarities suggest AaV is the smallest member of the emerging clade “Megaviridae”. The genomic architecture of AaV demonstrates that the ancestral virus had an even smaller genome, which expanded through gene duplication and assimilation of genes from diverse sources including the host itself – some of which probably modulate important host processes. AaV also harbors a number of genes exclusive to phycodnaviruses – reinforcing the hypothesis that Phycodna- and Mimiviridae share a common ancestor.
![Figure] Bloom of the toxic, non–N2-fixing cyanobacteria Microcystis spp. in eutrophic Lake Tai, China. CREDIT: H. PAERL, UNC-CHAPEL HILL, INSTITUTE OF MARINE SCIENCES Nutrient over-enrichment in lakes drives water-quality deterioration. The August 2014 water supply shutdown from Lake
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.
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.The ISME Journal advance online publication, 23 May 2014; doi:10.1038/ismej.2014.78.
To study the effect of urea nitrogen on the ecosystem of Lake Taihu, we conducted urea and various nitrogen analysis for the water samples collected from the lake and surrounding rivers during summer. The ecological index analysis of 82 sites in rivers and lake yielded the following results: (1) The urea nitrogen contents in Taihu ranged from 0.011 to 0.161 mg x L(-1), which was high in the northwest and low in the southeast, related to the main pollution sources distribution of its drainage basin. (2) The dissolved nitrogen was dominated by inorganic nitrogen and the ratio between ammonia nitrogen and nitrate nitrogen was 5: 1. The average percentage of urea nitrogen in total nitrogen, dissolved nitrogen, dissolved organic nitrogen and bioavailable nitrogen was respectively 2.28%, 5.91%, 15.86%, and 6.22%, which showed a significant ecological function in Taihu. (3) Urea nitrogen concentration in river was more than twice that in lake, and the lake river concentration was slightly higher than the river into the lake. (3) In Taihu, there was a transformation relationship between urea nitrogen and the nitrogen in other forms. It showed that urea nitrogen had a significant positive correlation with permanganate index and the other forms of nitrogen, and a significant negative correlation with dissolved oxygen. In addition, urea nitrogen was weakly and positively correlated with chlorophyll a, while closely related to the spatial distribution of benthos and zooplankton species. All the results above showed that urea nitrogen was the bridge of organic and inorganic nitrogen transformation, and was the sign of nitrogen cycle of Lake Taihu, which was controlled by the circulating rate. High nitrogen content (especially the organic nitrogen) and low dissolved oxygen content were the key contributors to the increased urea nitrogen content. In Taihu, the urea nitrogen content was affected by both exogenous input and endogenous release.
In marine environments, virus-mediated lysis of host cells leads to the release of cellular carbon and nutrients and is hypothesized to be a major driver of carbon recycling on a global scale. However, efforts to characterize the effects of viruses on nutrient cycles have overlooked the geochemical potential of the virus particles themselves, particularly with respect to their phosphorus content. In this Analysis article, we use a biophysical scaling model of intact virus particles that has been validated using sequence and structural information to quantify differences in the elemental stoichiometry of marine viruses compared with their microbial hosts. By extrapolating particle-scale estimates to the ecosystem scale, we propose that, under certain circumstances, marine virus populations could make an important contribution to the reservoir and cycling of oceanic phosphorus.
We announce the complete genome sequences of two temperate Podoviridae, Sulfitobacter phages ΦCB2047-A and ΦCB2047-C, which infect Sulfitobacter sp. strain 2047, a member of the Roseobacter clade. This is the first report of temperate podophage infecting members of the Sulfitobacter genus of the Roseobacter clade.
Viruses contribute to the mortality of marine microbes, consequentially altering biological species composition and system biogeochemistry. Although it is well established that host cells provide metabolic resources for virus replication, the extent to which infection reshapes host metabolism at a global level and the effect of this alteration on the cellular material released following viral lysis is less understood. To address this knowledge gap, the growth dynamics, metabolism and extracellular lysate of roseophage-infected Sulfitobacter sp. 2047 was studied using a variety of techniques, including liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics. Quantitative estimates of the total amount of carbon and nitrogen sequestered into particulate biomass indicate that phage infection redirects ∼75% of nutrients into virions. Intracellular concentrations for 82 metabolites were measured at seven time points over the infection cycle. By the end of this period, 71% of the detected metabolites were significantly elevated in infected populations, and stable isotope-based flux measurements showed that these cells had elevated metabolic activity. In contrast to simple hypothetical models that assume that extracellular compounds increase because of lysis, a profile of metabolites from infected cultures showed that >70% of the 56 quantified compounds had decreased concentrations in the lysate relative to uninfected controls, suggesting that these small, labile nutrients were being utilized by surviving cells. These results indicate that virus-infected cells are physiologically distinct from their uninfected counterparts, which has implications for microbial community ecology and biogeochemistry.The ISME Journal advance online publication, 5 December 2013; doi:10.1038/ismej.2013.216.
- May 2014
Macronutrients in sinking phytoplankton are typically remineralized at different rates, but less is known about the fate of micronutrient metals associated with sinking cells. Scavenging, the presence of co-occurring abiotic particles, and inadvertent contamination limit the utility of bulk analytical approaches to study remineralization of trace metals in sinking phytoplankton. We used synchrotron x-ray fluorescence mapping to measure macronutrients (P, S, and Si) and trace metals (Fe, Ni, and Zn) in individual cells of the diatom Asterionellopsis glacialis during a spring bloom in subtropical waters off New Zealand. P, S, Zn, and Ni were released significantly faster than Fe and Si from sinking cells in the upper 200 m. Bulk particulate element fluxes to sediment traps indicated similar trends, but biogenic silica flux was attenuated much faster than Si was lost from intact sinking cells collected in the traps. The metals were spatially co-located with P and S in upper ocean cells, but this association with P and S (based on a spatial resolution of 450 nm) was largely absent in sinking cells. In contrast, Fe retained a weak spatial association with Si, suggesting that remineralized Fe may be re-scavenged onto cell surfaces. As a result, dissolved Fe : macronutrient stoichiometries in the water column likely underestimate stoichiometries in sinking cells. We propose linkages between the selective loss of diatom cellular components (e.g., ribosomes or phospholipid membranes, Zn-finger proteins, and urease) and the observed recycling of specific elements (P, Zn, and Ni, respectively), which set the stoichiometry of macro-and micronutrient supply to surface waters.
Grand Lake St. Marys (Ohio, USA) is a hypereutrophic reservoir prone to persistent toxic cyanobacterial blooms fuelled by agricultural nutrient runoff. The pervasive and highly toxic 2010 bloom led to the collapse of the local tourism industry, with microcystin concentrations exceeding 2,000 μg mL-1 at some locations during the peak of the bloom. Sampling from the Celina Water Treatment Plant, chlorophyll a levels remained above 25 μg L-1 from June – September, reaching a maximum in excess of 100 μg L-1 in July. Ratios of dissolved inorganic nitrogen (DIN) to dissolved inorganic phosphorus (DIP) declined below 16 in July and August, suggesting the cyanobacterial population became N-limited during the bloom peak. Cyanobacterial biomass was dominated by Planktothrix spp. throughout late summer 2010, but phylogenetic analysis of mcyA sequences revealed the presence of toxic Microcystis spp. during July that coincided with the highest toxin measurements. August and September samples indicated the community shifted to yield Planktothrix spp. as the major toxic genus. Community shifts of this nature may impede targeted remediation efforts, and therefore a thorough understanding of the taxa involved is necessary prior to implementing strategies to limit bloom formation.
Upper ocean cycling of dissolved and particulate iron was investigated within an eddy during a study of the annual subtropical phytoplankton bloom east of New Zealand in the austral spring of 2008. During this GEOTRACES process study, dissolved iron surface mixed layer concentrations were initially high at ~ 0.6 nmol kg- 1 and declined to ~ 0.03 nmol kg- 1 due to biological consumption during a diatom-dominated bloom. The consequent iron limitation of the phytoplankton assemblage resulted in the decline and downward export of the bloom. Particulate iron concentrations were high in the surface mixed layer and varied between 3.9 nmol L- 1 and 12.1 nmol L- 1 during the onset and export of the bloom, respectively. The particulate iron and manganese results, along with a tracer model for the establishment of the eddy, suggest that the likely origin of the upper ocean particulate and dissolved iron pools within the eddy was from the continental margins along eastern New Zealand. Iron to aluminium (Fe:Al) ratios for suspended particulate material collected using McLane pumps deployed at 100 m and 200 m were significantly higher (range 0.16 to 2.90) than Fe:Al ratios for sinking particulate material intercepted using free-floating sediment traps (range 0.19 to 0.23) at corresponding deployment depths to the pumps. Based on the particulate Fe:Al ratios obtained for McLane pump collected samples, greater than > 70 % iron within the mixed layer was biogenic, indicating that either the resident biota are efficiently retaining iron or iron associated with organic detritus. Iron budgets, based on the dissolved, suspended and sinking particulate iron datasets, were constructed for the evolution and subsequent decline phases of the diatom bloom. The budgets reveal that the turnover time for dissolved iron within the mixed layer was on the order of days, suggesting that iron was rapidly exchanged from the dissolved pool to the particulate pools. In contrast, the residence time for iron in the particulate biogenic pool was in the order of 5–8 months indicating that iron was rapidly recycled but strongly retained by the biological community compared to the pelagic residence time for lithogenic iron, which was around 10–24 days. The regeneration of iron from biogenic particles was highest immediately below the euphotic zone (~ 50 m) and decreased with depth. Estimated regeneration fluxes for dissolved iron released from biogenic particles at 100 m depth were 0.34 ± 0.26 nmol m- 3 d- 1 during the development phase of the bloom and 7.1 ± 3.9 nmol m- 3 d- 1 at the peak of the bloom, when the overall export flux for particulate iron had increased 2-fold. Our results suggest a dynamic balance between iron regeneration from particulate organic material below the euphotic zone and the lability of this organic matter. Finally, the different Fe:Al ratios obtained for suspended and sinking particulate matter indicates that these pools are biogeochemically different. Future GEOTRACES process study work should focus on determining the biogechemical differences of iron with between these pools because this will assist with interpreting GEOTRACES sections and evaluating the impact of iron biogeochemistry on pelagic production and remineralisation within the global ocean.
- Feb 2014
Carbon and nutrient cycles in large temperate lakes such as Lake Erie are primarily driven by phototrophic and heterotrophic microorganisms, although our understanding of these is often constrained to late spring through summer due to logistical constraints. During periods of >90% ice-cover in February of 2008, 2009 and 2010, we collected samples from an icebreaker for an examination of bacterial production as well as microbial community structure. In comparison with summer months (August 2002 and 2010), we tested hypotheses concerning seasonal changes in microbial community diversity and production. Bacterial production estimates were ~ 2 orders of magnitude higher (volume normalised) in summer relative to winter. Our observations further demonstrate that the microbial community, including single celled phototrophs, varied in composition between August and February. Sediment traps deployed and collected over a three year period (2008 - 2011) confirmed that carbon export was ongoing and not limiting winter production. The results support the notion that active primary producers in winter months export carbon to the sediments that is not consumed until the warmer seasons. The establishment of this linkage is a critical observation in efforts to understand the extent and severity of annual summertime formation of a zone of regional hypoxia in Lake Erie. This article is protected by copyright. All rights reserved.
The Laurentian Great Lakes are among the most prominent sources of fresh water in the world. Lake Erie's infamous cyanobacterial blooms have, however, threatened the health of this valuable freshwater resource for decades. Toxic blooms dominated by the cyanobacterium Microcystis aeruginosa have most recently been one of primary ecological concerns for the lake. These toxic blooms impact the availability of potable water, as well as public health and revenues fromthe tourismand fishery industries. The socioeconomic effects of these blooms have spurred research efforts to pinpoint factors that drive bloom events. Despite decades of research and mitigation efforts, these blooms have expanded both in size and duration in recent years. However, through continued joint efforts between the Canadian and United States governments, scientists, and environmental managers, identification of the factors that drive bloom events is within reach. This review provides a summary of historical and contemporary research efforts in the realmof Lake Erie's harmful cyanobacterial blooms, both in terms of experimental and management achievements and insufficiencies, as well as future directions on the horizon for the lake's research community.
Abstract Biological processes are fundamentally driven by complex interactions between biomolecules. Integrated high-throughput omics studies enable multifaceted views of cells, organisms, or their communities. With the advent of new post-genomics technologies, omics studies are becoming increasingly prevalent; yet the full impact of these studies can only be realized through data harmonization, sharing, meta-analysis, and integrated research. These essential steps require consistent generation, capture, and distribution of metadata. To ensure transparency, facilitate data harmonization, and maximize reproducibility and usability of life sciences studies, we propose a simple common omics metadata checklist. The proposed checklist is built on the rich ontologies and standards already in use by the life sciences community. The checklist will serve as a common denominator to guide experimental design, capture important parameters, and be used as a standard format for stand-alone data publications. The omics metadata checklist and data publications will create efficient linkages between omics data and knowledge-based life sciences innovation and, importantly, allow for appropriate attribution to data generators and infrastructure science builders in the post-genomics era. We ask that the life sciences community test the proposed omics metadata checklist and data publications and provide feedback for their use and improvement.
Using marine sediment traps (named RESPIRE for REspiration of Sinking Particles In the subsuRface ocEan) designed to collect sinking particles and associated microbial communities in situ, we collected and incubated marine aggregates/particles in the southern Pacific Ocean from separate phytoplankton bloom events in-situ. We determined the phylogenetic affiliation for the microbes growing on aggregates by pyrosequencing partial 16S rRNA gene amplicons. Water column samples were also collected and sequenced for comparison between sinking-particle associated and planktonic bacterial communities. Statistically significant differences were found between the water column and sediment trap bacteria. Relative abundances of Pelagibacter sp. and multiple members of the Flavobacteria, Actinobacteria and α-Proteobacteria were elevated in water column samples, while trap samples contained members of the Roseobacter clade of α-Proteobacteria in high relative abundances. Our findings indicated that rapid changes -within 24 hours of collection - occurred to the microbial community associated with aggregates from either bloom type. There was little change to the bacterial assemblage after the initial 24 h incubation period. The most abundant early colonizer was a Sulfitobacter sp. This study provides further evidence that Roseobacters are rapid colonizers of marine aggregates and that colonization can occur on short time scales. This study further demonstrates that particle origin may be insignificant regarding the heterotrophic bacterial population that degrades them. This article is protected by copyright. All rights reserved.
We announce the complete genome sequence of a lytic podovirus, ΦCB2047-B, which infects the bacterium Sulfitobacter sp. strain 2047, a member of the Roseobacter clade. Genome analysis revealed ΦCB2047-B to be an N4-like phage, with its genome having high nucleotide similarity to other N4-like roseophage genomes.
We announce the complete genome sequence of a lytic podovirus,ΦCB2047-B, which infects the bacterium Sulfitobacter sp. strain 2047, a member of the Roseobacter clade. Genome analysis revealed ΦCB2047-B to be an N4-like phage, with its genome having high nucleotide similarity to other N4-like roseophage genomes.
Biological processes are fundamentally driven by complex interactions between biomolecules. Integrated high-throughput omics studies enable multifaceted views of cells, organisms, or their communities. With the advent of new post-genomics technologies, omics studies are becoming increasingly prevalent; yet the full impact of these studies can only be realized through data harmonization, sharing, meta-analysis, and integrated research. These essential steps require consistent generation, capture, and distribution of metadata. To ensure transparency, facilitate data harmonization, and maximize reproducibility and usability of life sciences studies, we propose a simple common omics metadata checklist. The proposed checklist is built on the rich ontologies and standards already in use by the life sciences community. The checklist will serve as a common denominator to guide experimental design, capture important parameters, and be used as a standard format for stand-alone data publications. The omics metadata checklist and data publications will create efficient linkages between omics data and knowledge-based life sciences innovation and, importantly, allow for appropriate attribution to data generators and infrastructure science builders in the post-genomics era. We ask that the life sciences community test the proposed omics metadata checklist and data publications and provide feedback for their use and improvement.
- Sep 2013
A multi-disciplinary examination of the drivers of dissolved methane was carried out during a phytoplankton bloom located in a subtropical mesoscale eddy. This investigation related temporal signals in methane concentrations with other biophysical and biogeochemical parameters in the upper waters (<300 m) of the southwest Pacific Ocean. In the surface mixed layer, methane supersaturation increased and δ13CCH4 became more depleted coincident with increases in particulate dimethylsulfoniopropionate (DMSPp) and succession from the diatom Asterionellopsis glacialis to the nanoflagellate Phaeocystis globosa and the cyanobacterium Synechococcus sp. In situ methane production was calculated in a surface mixed layer methane budget that incorporated sea-to-air exchange and vertical diffusion. Methane concentrations increased in and below the mixed layer when the export of biogenic particles increased. Increased grazing of microbes by microzooplankton may have contributed to particle recycling (rich in organic carbon and DMSP) and increased the potential for methanogenesis. Phytoplankton species composition and biomass in different bloom phases, and eddy dynamics, were important determinants of methane saturation and emission, and the potential implications for methane are considered for the future surface ocean.
We present the genome of a cyanosiphovirus (KBS2A) that infects a marine Synechococcus sp. (strain WH7803). Unique to this genome, relative to other sequenced cyanosiphoviruses, is the absence of elements associated with integration into the host chromosome, suggesting this virus may not be able to establish a lysogenic relationship.
- Jun 2013
Paralytic shellfish toxins are secondary metabolites produced by several species of dinoflagellates and cyanobacteria. Known targets of these toxins, which typically occur at detrimental concentrations during harmful algal blooms, include voltage-gated ion channels in humans and mammals. However, the effects of the toxins on the co-occurring phytoplankton community remain unknown. The present study examined the molecular mechanisms of the model photosynthetic alga Chlamydomonas reinhardtii in response to saxitoxin exposure as a means of gaining insights into the phytoplankton community response to a bloom. Previous work with yeast indicated that saxitoxin inhibited copper uptake, and so experiments were designed to examine whether saxitoxin exhibited a similar mode of action in algae. Expression profiling following exposure to saxitoxin or a copper chelator produced similar profiles in copper homeostasis genes, notably the induction of the cytochrome c6 (CYC6) and copper transporter (COPT1, CTR1) genes. Cytochrome c6 is used as an alternative to plastocyanin under conditions of copper deficiency, and immunofluorescence data showed this protein to be present in a significantly greater proportion of saxitoxin-exposed cells compared to controls. Live-cell imaging with a copper sensor probe for intracellular labile Cu(I) confirmed that saxitoxin blocked copper uptake. Extrapolations of these data to phytoplankton metabolic processes along with the copper transporter as a molecular target of saxitoxin based on existing structural models are discussed. Environ. Toxicol. Chem. © 2013 SETAC.
The rate of carbon fixation by phytoplank- ton in marine surface waters is often tied to the supply of growth-limiting nutrients such as iron (Fe). While average cellular requirements and ratios for various elements are well known in the literature, especially through laboratory culture work, the plasticity of these relationships in natural plankton communities has been less explored. To gauge how changes in the biological availability of dissolved Fe might influence cellular nutrient ratios of marine phytoplankton (and thus their physiology), we carried out incubation assays during a research expedition off the east coast of New Zealand. Trace-metal clean collection of plankton communities were amended with a continuum of concentrations of either Fe (as FeCl3) or desferroxamine B (to reduce bioavailable Fe) and then maintained for 72 h under in situ conditions. Along with standard assays (Fv/Fm, chlorophyll, nutrient drawdown), we measured ele- mental ratios in the bulk community by inductively coupled plasma mass spectrometry and within individ- ual plankton using synchrotron X-ray fluorescence. Our observations demonstrate that changes in the physiological ecology of the community (biomass, photosynthetic efficiency) were mirrored in changes in elemental ratios, including a 3-fold change in Fe stoi- chiometry and a 13-fold change in Zn stoichiometry when Fe-replete and Fe-depleted communities were compared. We present this information in consideration of the hypothesis that flexibility in elemental quotas influences the interactions between nutrient availabil- ity and planktonic physiological status, subsequently altering C flow through marine surface waters.
- Dec 2012
The distribution of cyanomyoviruses was estimated using a quantitative PCR (qPCR) approach that targeted the g20 gene as a proxy for phage. Samples were collected spatially during a >3,000 km transect through the Sargasso Sea and temporally during a gyre-constrained phytoplankton bloom within the southern Pacific Ocean. Cyanomyovirus abundances were lower in the Sargasso Sea than in the southern Pacific Ocean, ranging from 2.75 x 10(3) to 5.15 x 10(4) mL(-1) and correlating with the abundance of their potential hosts (Prochlorococcus and Synechococcus). Cyanomyovirus abundance in the southern Pacific Ocean (east of New Zealand) followed Synechococcus host populations in the system: this included a decrease in g20 gene copies (from 4.3 x 10(5) to 9.6 x 10(3) mL(-1) ) following the demise of a Synechococcus bloom. When compared with direct counts of viruses, observations suggest that the cyanomyoviruses comprised 0.5 to >25% of the total virus community. We estimated daily lysis rates of 0.2 - 46% of the standing stock of Synechococcus in the Pacific Ocean compared to ~ < 1.0 % in the Sargasso Sea. In total, our observations confirm this family of viruses is abundant in marine systems and that they are an important source of cyanobacterial mortality. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
Development and use of primer sets to amplify nucleic acid sequences of interest is fundamental to studies spanning many life science disciplines. As such, the validation of primer sets is essential. Several computer programs have been created to aid in the initial selection of primer sequences that may or may not require multiple nucleotide combinations (i.e., degeneracies). Conversely, validation of primer specificity has remained largely unchanged for several decades, and there are currently few available programs that allows for an evaluation of primers containing degenerate nucleotide bases. To alleviate this gap, we developed the program De-MetaST that performs an in silico amplification using user defined nucleotide sequence dataset(s) and primer sequences that may contain degenerate bases. The program returns an output file that contains the in silico amplicons. When De-MetaST is paired with NCBI's BLAST (De-MetaST-BLAST), the program also returns the top 10 nr NCBI database hits for each recovered in silico amplicon. While the original motivation for development of this search tool was degenerate primer validation using the wealth of nucleotide sequences available in environmental metagenome and metatranscriptome databases, this search tool has potential utility in many data mining applications.
- Oct 2012
- 2012 Society for Advancement of Hispanics/Chicanos and Native Americans in Science National Conference
It is well established that during phage infection, the host cell provides metabolic resources for virus replication. However, we know relatively little of how the infection process affects host metabolism at a global level. Our specific objective is to provide an understanding of how host metabolism is altered during phage infection and also to provide a framework for understanding how the infection process can alter the composition of cellular constituents that are released into the environment and are available for consumption by uninfected microbes. We hypothesize that although phage infection results in a global change in metabolite concentrations in a cell, pathways involved in nucleotide biosynthesis, nitrogen metabolism and intermediates in cell wall biosynthesis are especially affected by phage infection. We selected a representative member of the Roseobacter lineage of marine bacteria, Sulfitobacter sp. CB2047, as an environmentally relevant model for this work. Using a targeted tandem liquid chromatography and mass spectrometry approach, we determined the metabolite profile and the flux of selected metabolites of phage infected and uninfected CB2047 over an infection cycle. Our results indicate that phage infected cells are metabolically hyperactive compared to uninfected cells and that as a result of phage infection the host resources are redirected from energy production to the production of new phage particles. From our results we conclude that there is a metabolome remodeling during phage infection that affects nitrogen metabolism, amino acid metabolism, DNA synthesis and intermediates in call wall biosynthesis all of which are required for effective phage replication.
Viruses are the most abundant life forms on Earth, with an estimated 10(31) total viruses globally. The majority of these viruses infect microbes, whether bacteria, archaea or microeukaryotes. Given the importance of microbes in driving global biogeochemical cycles, it would seem, based on numerical abundances alone, that viruses also play an important role in the global cycling of carbon and nutrients. However, the importance of viruses in controlling host populations and ecosystem functions, such as the regeneration, storage and export of carbon and other nutrients, remains unresolved. Here, we report on advances in the study of ecological effects of viruses of microbes. In doing so, we focus on an area of increasing importance: the role that ocean viruses play in shaping microbial population sizes as well as in regenerating carbon and other nutrients.
1] Diatom blooms play a central role in supporting food-webs and sequestering biogenic carbon to depth. Oceanic conditions set bloom initiation, whereas both environmental and ecological factors determine bloom magnitude and lon-gevity. Our study reveals another fundamental determinant of bloom dynamics. A diatom spring bloom in offshore New Zealand waters was likely terminated by iron limitation, even though diatoms consumed <1/3 of the mixed-layer dis-solved iron inventory. Thus, bloom duration and magnitude were primarily set by competition for dissolved iron between microbes and small phytoplankton versus diatoms. Signifi-cantly, such a microbial mode of control probably relies both upon out-competing diatoms for iron (i.e., K-strategy), and having high iron requirements (i.e., r-strategy). Such resource competition for iron has implications for carbon biogeo-chemistry, as, blooming diatoms fixed three-fold more car-bon per unit iron than resident non-blooming microbes. Microbial sequestration of iron has major ramifications for determining the biogeochemical imprint of oceanic diatom blooms. Citation: Boyd, P. W., et al. (2012), Microbial control of diatom bloom dynamics in the open ocean, Geophys. Res. Lett., 39, L18601, doi:10.1029/2012GL053448.
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.
- Apr 2012
Recent investigations of Lake Erie in the winter have demonstrated the occurrence of substantial phytoplank-ton communities largely consisting of the diatom Aulacoseira islandica (O. Müller) Simonsen. To assess the ac-tivity of this diatom community, multiple measures of production, both general and diatom-specific, were undertaken. We measured oxygen (O 2) evolution as proxy for carbon (C)-fixation and 2-(4-pyridyl)-5-((4-(2-dimethylaminoethylaminocarbamoyl) methoxy)-phenyl)oxazole (PDMPO) incorporation as a measure of silica (Si) deposition. The latter demonstrated conclusively that diatoms were active during winter months and confirmed that diatoms are the primary drivers of winter productivity. The stoichiometric relationship between carbon and silica in the winter Lake Erie phytoplankton assemblage was further compared to the ac-tivity of the summer community. Although the winter phytoplankton community was observed to be active, it was less active than the summer community, with lower measured rates of O 2 evolution and Si deposition. These findings provide a new and expanded understanding of the biological carbon production in Lake Erie.
Saxitoxin is a secondary metabolite produced by several species of dinoflagellates and cyanobacteria which targets voltage-gated sodium and potassium channels in higher vertebrates. However, its molecular target in planktonic aquatic community members that co-occur with the toxin producers remains unknown. Previous microarray analysis with yeast identified copper and iron-homeostasis genes as being differentially regulated in response to saxitoxin. This study sought to identify the molecular target in microbial cells by comparing the transcriptional profiles of key copper and iron homeostasis genes (CTR1, FRE1, FET3, CUP1, CRS5) in cells exposed to saxitoxin, excess copper, excess iron, an extracellular Cu(I) chelator, or an intracellular Cu(I) chelator. Protein expression and localization of Ctr1p (copper transporter), Fet3p (multicopper oxidase involved in high-affinity iron uptake), and Aft1p (iron regulator) were also compared among treatments. Combined transcript and protein profiles suggested saxitoxin inhibited copper uptake. This hypothesis was confirmed by intracellular Cu(I) imaging with a selective fluorescent probe for labile copper. On the basis of the combined molecular and physiological results, a model is presented in which the copper transporter Ctr1p serves as a molecular target of saxitoxin and these observations are couched in the context of the eco-evolutionary role this toxin may serve for species that produce it.
For almost two decades, the western basin of Lake Erie has been plagued with recurring toxic algal blooms dominated by the colonial cyanobacterium, Microcystis spp. Since the Maumee River is a major source of nutrients and sediment inputs into the lake, and Microcystis spp. has been identified as a member of the upstream river algal assemblage, the possibility exists that the river Microcystis species serve as a seed population for the toxic blooms occurring in the lake. Genetic profiling of toxic cyanobacteria using the microcystin synthesis gene, mcyA, clearly indicates that the toxic cyanobacteria of the river are distinct from the toxic Microcystis spp. of Lake Erie. Indeed, mcyA sequences are almost exclusively from toxic Planktothrix spp., similar to what has been documented previously for Sandusky Bay. UniFrac statistical analysis of cyanobacterial community composition by comparison of 16S–23S ITS sequences also show that the Maumee River and Lake Erie communities are distinct. Overall, these data show that despite the importance of nutrient inputs and sediments from the river, the toxic cyanobacterial blooms of Lake Erie do not originate from toxic species endemic to the Maumee River and instead must originate elsewhere, most likely from the lake sediments.
- Mar 2012
Lake Erie is the most socioeconomically important and productive of the Laurentian (North American) Great Lakes. Since the mid-1990s cyanobacterial blooms dominated primarily by Microcystis have emerged to become annual, late summer events in the western basin of Lake Erie yet the effects of these blooms on food web dynamics and zooplankton grazing are unclear. From 2005 to 2007, grazing rates of cultured (Daphnia pulex) and natural assemblages of mesozooplankton and microzooplankton on five autotrophic populations were quantified during cyanobacterial blooms in western Lake Erie. While all groups of zooplankton grazed on all prey groups investigated, the grazing rates of natural and cultured mesozooplankton were inversely correlated with abundances of potentially toxic cyanobacteria (Microcystis, Anabaena, and Cylindrospermopsis; p < 0.05) while those of the in situ microzooplankton community were not. Microzooplankton grazed more rapidly and consistently on all groups of phytoplankton, including cyanobacteria, compared to both groups of mesozooplankton. Cyanobacteria displayed more rapid intrinsic cellular growth rates than other phytoplankton groups under enhanced nutrient concentrations suggesting that future nutrient loading to Lake Erie could exacerbate cyanobacterial blooms. In sum, while grazing rates of mesozooplankton are slowed by cyanobacterial blooms in the western basin of Lake Erie, microzooplankton are likely to play an important role in the top-down control of these blooms; this control could be weakened by any future increases in nutrient loads to Lake Erie.
The limnology of offshore Lake Erie during periods of extensive (> 70%) ice cover was examined from ship borne sampling efforts in 2007 to 2010, inclusive. Dense and discrete accumulations of the centric filamentous diatom Aulacoseria islandica (>10 μg Chl-a/L) were located in the isothermal (b1 °C) water column directly below the ice and only detectable in the ship wake; viable phytoplankton were also observed within ice. Evi-dence from these surveys supports the notions that winter blooms of diatoms occur annually prior to the onset of ice cover and that the phytoplankton from these blooms are maintained in the surface waters of Lake Erie and reduce silicate concentrations in the lake prior to spring. The mechanisms by which high phytoplankton biomass rise at this time of year requires further investigation, but these winter blooms probably have conse-quences for summer hypoxia and how the lake responds to climate change.
Phytoplankton interactions with iron (Fe) were examined in surface waters of Lake Erie during summer thermal stratification. Lake-wide sampling in June and September 2005 was conducted using a continuous surface water sampler (1 m sampling depth) and in July at 18 hydrographic stations (5 m sampling depth). In situ measurements of photosynthetic efficiency (maximum quantum yield of photosystem II) and phytoplankton community composition were measured using fast repetition rate fluorometry and a phytoplankton pigment-specific fluorometer, respectively, during June and September. High ratios (73%–85%) of intracellular Fe to particulate Fe coincident with increases in chlorophyll a (Chl a) concentrations in the western and central basins in June and July imply that the majority of Fe in these regions was associated with intracellular pools. Correlations between intracellular Fe and Chl a were frequently observed when Heterokontophyta and Pyrrophyta dominated the phytoplankton community. Assimilation of Fe by the phytoplankton strongly influenced its partitioning between the dissolved and particulate phase. Dissolved iron (<0.45 µm) concentrations were proportional to Chl a concentrations and both dissolved iron and Chl a were inversely proportional to nitrate concentrations in July and September, suggesting that dissolved iron influenced both nitrate drawdown and Chl a concentrations in Lake Erie surface waters in summer.
Biogenic Fe quotas were determined using three distinct techniques on samples collected concurrently in the subtropical Pacific Ocean east of New Zealand. Fe quotas were measured using radioisotope uptake experiments (24 h incubation), bulk filtration and analysis by inductively-coupled plasma mass spectrometer (ICPMS), and single-cell synchrotron x-ray fluorescence (SXRF) analysis over a sixteen-day period (year days 263 to 278 of 2008) during a quasi-Lagrangian drifter experiment that tracked the evolution of the annual spring diatom bloom within a counter-clockwise open-ocean eddy. Overall, radioisotope uptake-determined Fe quotas (washed with oxalate reagent to remove extracellular Fe) were the lowest (0.5-1.0 mmol Fe:mol P; 4-8 mumol Fe:mol C), followed by single-cell Fe quotas (2.3-7.5 mmol Fe:mol P; 17-57 mumol Fe:mol C), and the highest and most variable quotas were from the bulk filtration ICPMS approach that used the oxalate reagent wash, corrected for lithogenic Fe using Al (0.8-21 mmol Fe:mol P; 4-136 mumol Fe:mol C). During the evolution of the spring bloom within the eddy (year days 263 to 272), the surface mixed layer inventories of particulate organic elements (C, N, P, Si) and chlorophyll increased while Fe quotas estimated from all three approaches exhibited a general decline. After the onset of the bloom decline, the drogued buoys exited the eddy center (days 273 to 277). Fe quotas returned to pre-bloom values during this part of the study. Our standardized and coordinated sampling protocols reveal the general observed trend in Fe quotas: ICPMS > SXRF > radioisotope uptake. We discuss the inherent differences between the techniques and argue that each technique has its individual merits and uniquely contributes to the characterization of the oceanic particulate Fe pool.
Biogenic Fe quotas were determined using three distinct techniques on samples collected concurrently in the subtropical Pacific Ocean east of New Zealand. Fe quotas were measured using radioisotope uptake experiments (24 h incubation), bulk filtration and analysis by inductively-coupled plasma mass spectrometer (ICPMS), and single-cell synchrotron x-ray fluorescence (SXRF) analysis over a sixteen-day period (year days 263 to 278 of 2008) during a quasi-Lagrangian drifter experiment that tracked the evolution of the annual spring diatom bloom within a counter-clockwise open-ocean eddy. Overall, radioisotope uptake-determined Fe quotas (washed with oxalate reagent to remove extracellular Fe) were the lowest (0.5–1.0 mmol Fe:mol P; 4–8 μmol Fe:mol C), followed by single-cell Fe quotas (2.3–7.5 mmol Fe:mol P; 17–57 μmol Fe:mol C), and the highest and most variable quotas were from the bulk filtration ICPMS approach that used the oxalate reagent wash, corrected for lithogenic Fe using Al (0.8–21 mmol Fe:mol P; 4–136 μmol Fe:mol C). During the evolution of the spring bloom within the eddy (year days 263 to 272), the surface mixed layer inventories of particulate biogenic elements (C, N, P, Si) and chlorophyll increased while Fe quotas estimated from all three approaches exhibited a general decline. After the onset of the bloom decline, the drogued buoys exited the eddy center (days 273 to 277). Fe quotas returned to pre-bloom values during this part of the study. Our standardized and coordinated sampling protocols reveal the general observed trend in Fe quotas: ICPMS > SXRF > radioisotope uptake. We discuss the inherent differences between the techniques and argue that each technique has its individual merits and uniquely contributes to the characterization of the oceanic particulate Fe pool.
Blooms of the potentially toxic cyanobacterium Microcystis are common events globally, and as a result significant resources continue to be dedicated to monitoring and controlling these events. Recent studies have shown that a significant proportion of total cell-associated phosphorus (P) in marine phytoplankton can be surface adsorbed; as a result studies completed to date do not accurately report the P demands of these organisms. In this study we measure the total cell-associated and intracellular P as well as growth rates of two toxic strains of Microcystis aeruginosa Kütz grown under a range of P concentrations. The results show that the intracellular P pool in Microcystis represents a percentage of total cell-associated P (50-90%) similar to what has been reported for actively growing algae in marine systems. Intracellular P concentrations (39-147 fg cell(-1)) generally increased with increasing P concentrations in the growth medium, but growth rate and the ratio of total cell-associated to intracellular P remained generally stable. Intracellular P quotas and growth rates in cells grown under the different P treatments illustrate the ability of this organism to successfully respond to changes in ambient P loads, and thus have implications for ecosystem scale productivity models employing P concentrations to predict algal bloom events.
- Dec 2011
In this study we investigated the effect of the phosphonate herbicide glyphosate (N-(phosphonomethyl)glycine) on the phytoplankton community structure in Lake Erie using lake water incubations, laboratory growth experiments and phylogenetic analysis of phosphonate metabolism genes. In microcosms, addition of glyphosate to Sandusky Bay water resulted in a significant increase in phytoplankton abundance, specifically causing an increase in the abundance of Planktothrix spp. In microcosms using Maumee Bay water, glyphosate did not stimulate phytoplankton growth but caused a decrease in Microcystis spp. abundance. The difference in the ability of Planktothrix spp. and Microcystis spp. to grow in the presence of glyphosate was confirmed in laboratory growth experiments. Further, an examination of the molecular pathways involved in phosphonate metabolism demonstrated that heterotrophic bacteria may be critical in allowing this proliferation. The results indicate that glyphosate has both positive and negative influences on phytoplankton community structure, serving as a nutrient source to microbes able to tolerate the herbicidal effects of the compound while killing those less tolerant. Moreover, this work highlights that in natural environments microorganisms function as communities, and the metabolic abilities of individual species are often less important than the collective ability of the community.
- Nov 2011
Lagrangian studies of virus activity in pelagic environments over extended temporal scales are rare. To address this, viruses and bacteria were examined during the course of a natural phytoplankton bloom in the pelagic South Pacific Ocean east of New Zealand. Daily samples were collected in a mesoscale eddy from year days 263-278 (September 19th-October 4th, 2008). The productive bloom transitioned from a diatom to a pico- and nanoplankton-dominated system, resulting in chlorophyll a concentrations up to 2.43 μg L(-1) . Virus abundances fluctuated c. 10-fold (1.8 × 10(10) -1.3 × 10(11) L(-1) ) over 16 days. The production rates of virus particles were high compared with those reported in other marine systems, ranging from 1.4 × 10(10) to 2.1 × 10(11) L(-1) day(-1) . Our observations suggest viruses contributed significantly to the mortality of bacteria throughout the bloom, with 19-216% of the bacterial standing stock being lysed daily. This mortality released nutrient elements (N, Fe) that likely helped sustain the bloom through the sampling period. Parametric analyses found significant correlations with both biotic (e.g. potential host abundances) and abiotic parameters (e.g. nutrient concentrations, temperature). These observations demonstrate that viruses may be critical in the extended maintenance of regeneration-driven biological production.
- Oct 2011
We completed a transect through the Western Pacific Warm Pool to examine how environmental variables may influence viral and bacterial abundance and production rates in this globally important oceanic region. Of the variables analyzed, viral abundance and production had the most significant relationship to bacterial cell abundance: viral parameters were not significantly correlated to the measured environmental variables, including temperature. Bacterial production rates were significantly correlated to temperature in open ocean waters, but not in waters close to land masses. Analyses of 16S rRNA gene by pyrosequencing indicated only minor changes in eubacterial community structure across the transect, with α-proteobacteria dominating all sampled populations. Diversity within the prokaryotic community did not correlate directly with viral abundance or activity. Comparisons to two other ocean-scale transects (> 8000 km of open ocean in total) in the Atlantic Ocean indicated that correlations between viral and bacterial abundance and production relative to environmental variables are regime dependent. In particular, correlations to temperature showed remarkable differences across the three transects. Collectively, our observations suggest that seemingly similar oceanic regions may have very different microbial community responses to environmental variables. Our observations and analyses demonstrate that ocean-scale generalizations may not apply in the case of viral ecology.
Picocyanobacteria represented by Prochlorococcus and Synechococcus have an important role in oceanic carbon fixation and nutrient cycling. In this study, we compared the community composition of picocyanobacteria from diverse marine ecosystems ranging from estuary to open oceans, tropical to polar oceans and surface to deep water, based on the sequences of 16S-23S rRNA internal transcribed spacer (ITS). A total of 1339 ITS sequences recovered from 20 samples unveiled diverse and several previously unknown clades of Prochlorococcus and Synechococcus. Six high-light (HL)-adapted Prochlorococcus clades were identified, among which clade HLVI had not been described previously. Prochlorococcus clades HLIII, HLIV and HLV, detected in the Equatorial Pacific samples, could be related to the HNLC clades recently found in the high-nutrient, low-chlorophyll (HNLC), iron-depleted tropical oceans. At least four novel Synechococcus clades (out of six clades in total) in subcluster 5.3 were found in subtropical open oceans and the South China Sea. A niche partitioning with depth was observed in the Synechococcus subcluster 5.3. Members of Synechococcus subcluster 5.2 were dominant in the high-latitude waters (northern Bering Sea and Chukchi Sea), suggesting a possible cold-adaptation of some marine Synechococcus in this subcluster. A distinct shift of the picocyanobacterial community was observed from the Bering Sea to the Chukchi Sea, which reflected the change of water temperature. Our study demonstrates that oceanic systems contain a large pool of diverse picocyanobacteria, and further suggest that new genotypes or ecotypes of picocyanobacteria will continue to emerge, as microbial consortia are explored with advanced sequencing technology.
In experiments designed primarily to investigate viral lysis, we found that the presence of viruses had a positive effect on the growth of Synechococcus. A Landry-Hassett-type stepwise dilution experiment conducted during a Synechococcus bloom in the Gulf of Mexico used both (i) 0.2-mu m filtered seawater in which the abundance of bacteria and grazers were reduced but the majority of viruses were retained, and (ii) ultrafiltered (30 000 MW cutoff) virus-free seawater in which the abundance of viruses, bacteria and grazers were reduced. High growth rates and frequency of dividing cells (FDCs) were recorded in 0.2-mm filtered treatments while growth was inhibited in incubations with a high proportion of virus-free ultrafiltered water. In two subsequent experiments using Mediterranean Sea populations, a two-point dilution approach in which viral abundance was reduced by 80-90% yielded similar results, and showed that Synechococcus only grew well in the presence of viruses, bacteria and grazers. In four further Mediterranean experiments viruses removed via ultrafiltration were added back, either untreated, or inactivated by a heat treatment. Growth rates and FDCs were higher in the presence of untreated viruses than with viruses inactivated by heat, suggesting that it was not organic matter in the virus-size fraction but rather the presence of infectious viruses which sustained growth. While Synechococcus was also infected by viruses during these experiments, our data imply that growth of Synechococcus may depend upon viral lysis of heterotrophic bacteria. This finding is consistent with the view that nutrient cycling by viral lysis of heterotrophic bacteria may control phytoplankton growth and ecosystem scale carbon production.
Noxious cyanobacteria pose a considerable health threat to freshwater ecosystems. Research now suggests that toxic strains may be outcompeted by their non-toxic counterparts as surface concentrations of carbon dioxide increase.
Considerable research has shown that cyanobacteria and the viruses that infect them (cyanophage) are pervasive and diverse in global lake populations. Few studies have seasonally analyzed freshwater systems, and little is known about the bacterial and viral communities that coexist during the harsh winters of the Laurentian Great Lakes. Here, we employed quantitative PCR to estimate the abundance of cyanomyoviruses in this system, using the portal vertex g20 gene as a proxy for cyanophage abundance and to determine the potential ecological relevance of these viruses. Cyanomyoviruses were abundant in both the summer and the winter observations, with up to 3.1 × 106 copies of g20 genes ml−1 found at several stations and depths in both seasons, representing up to 4.6% of the total virus community. Lake Erie was productive during both our observations, with high chlorophyll a concentrations in the summer (up to 10.3 μg liter−1) and winter (up to 5.2 μg liter−1). Both bacterial and viral abundances were significantly higher during the summer than during the winter (P < 0.05). Summer bacterial abundances ranged from 3.3 × 106 to 1.6 × 107 ml−1 while winter abundances ranged between ∼3.4 × 105 and 1.2 × 106 ml−1. Total virus abundances were high during both months, with summer abundances significantly higher at most stations, ranging from 6.5 × 107 to 8.8 × 107 ml−1, and with winter abundances ranging from 3.4 × 107 to 6.6 × 107 ml−1. This work confirms that putative cyanomyoviruses are ubiquitous in both summer and winter months in this large freshwater lake system and that they are an abundant component of the virioplankton group.
Epifluorescence microscopy is a common method used to enumerate virus-like particles (VLP) from environmental samples and relies on the use of filter membranes with pore sizes < 0.02 μm; the most commonly used protocols employ 25 mm Anodisc™ membranes with a built-in support ring. Other filters with small pore sizes exist, including the 13 mm Anodisc™ membranes without a support ring. However, the use of these membranes for viral enumeration has not been previously reported. Here we describe a modified protocol for 13 mm Anodisc membranes that uses a custom filter holder that can be readily constructed in individual investigators' laboratories from commercially available Swinnex® filter holders. We compared VLP concentrations obtained from phage lysates and seawater samples using both Anodisc membranes, as well as Nuclepore™ small pore-size membranes (0.015 or 0.030 μm). The 13 mm Anodisc membranes gave comparable estimates of VLP abundance to those obtained with the 25 mm Anodisc membranes when similar staining methods were employed. Both Nuclepore membranes typically gave an order of magnitude lower VLP abundance values for environmental samples. The 13 mm Anodisc membranes are less costly and require smaller sample volumes than their 25 mm counterpart making them ideal for large-scale studies and sample replication. This method increases the options of reliable approaches available for quantifying VLP from environmental samples.
We discuss below the correspondence relating to our Opinion article about the 'microbial carbon pump' (MCP) (Microbial production of recalcitrant dissolved organic matter: long-term carbon storage in the global ocean. Nature Rev. Microbiol. 8, 593–599 (2010)
- May 2011
Studies of the Phycodnaviridae have traditionally relied on the DNA polymerase (pol) gene as a biomarker. However, recent investigations have suggested that the major capsid protein (MCP) gene may be a reliable phylogenetic biomarker. We used MCP gene amplicons gathered across the North Atlantic to assess the diversity of Emiliania huxleyi-infecting Phycodnaviridae. Nucleotide sequences were examined across >6000 km of open ocean, with comparisons between concentrates of the virus-size fraction of seawater and of lysates generated by exposing host strains to these same virus concentrates. Analyses revealed that many sequences were only sampled once, while several were over-represented. Analyses also revealed nucleotide sequences distinct from previous coastal isolates. Examination of lysed cultures revealed a new richness in phylogeny, as MCP sequences previously unrepresented within the existing collection of E. huxleyi viruses (EhV) were associated with viruses lysing cultures. Sequences were compared with previously described EhV MCP sequences from the North Sea and a Norwegian Fjord, as well as from the Gulf of Maine. Principal component analysis indicates that location-specific distinctions exist despite the presence of sequences common across these environments. Overall, this investigation provides new sequence data and an assessment on the use of the MCP gene.
- Mar 2011
We have examined the bioavailability of Fe complexed to a siderophore produced by the heterotrophic marine bacterium Vibrio alginolyticus isolate PWH3a and Fe from ligand-complexes present in virus-mediated lysates (using phage PWH3a-P1) of this same bacterium. Fe-binding functional groups, stability constants and the bioavailability of Fe associated with these two separate ligand pools were determined and contrasted to previous work. Under low-Fe growth conditions, axenic cultures of V. alginolyticus PWH3a were shown to produce catecholate siderophores, while neither catecholate nor hydroxamate-type Fe-binding moieties were detected in virus-generated cell lysates. Analysis of the overall binding strength using electrochemical techniques revealed that the siderophore-containing organic extract and the organics in the virus-mediated lysates had Fe-binding constants comparable to the weaker L-2-type ligands found throughout the water column in seawater. A further purification of the siderophore-containing extract revealed a ligand with a stability constant of logK'(FeL,Fe3+) = 22.3, typical for siderophores and L-1-type of ligands found in marine surface waters. In assimilation studies, the Fe in the lysate was found to be more bioavailable to our model heterotrophic bacterium, autotrophic cyanobacterium and eukaryotic diatom cultures than the catecholate siderophore produced by the Vibrio sp. This work demonstrates that the Fe-containing components of virus-mediated cell lysates are different than siderophores secreted by these same cells, and as such continues to build the argument supporting the importance of virus-mediated Fe regeneration in marine surface waters.
Microcystis blooms occur worldwide and threaten aquatic ecosystems and human health. Sublethal effects on early developmental stages of fish are largely unknown, and research has mainly focused on microcystin toxins (such as MC-LR) rather than Microcystis cells. We exposed (96 h) zebrafish larvae to purified MC-LR (0-1000 μg/L) or lyophilized Microcystis aeruginosa containing 4.5 μg/L MC-LR and evaluated changes in global gene expression (Affymetrix GeneChip zebrafish genome arrays). Significant changes in gene expression (≥ 1.7-fold change, p < 0.0001) were determined with Rosetta Resolver 7.0, and ontology analysis was conducted with the DAVID bioinformatics tool. The number of differentially expressed genes relative to control increased with MC-LR concentration and included genes related to known mechanisms of action for MC-LR in mammals and older life stages of fish, as well as genes unique to larval zebrafish. Up-regulation of vitellogenin genes (vtg) (19.2-fold to >100-fold on arrays; 619.3-fold confirmed by quantitative PCR) was observed in Microcystis-exposed larvae but not in larvae exposed to MC-LR. Up-regulation of vtg indicates exposure to estrogenic substance(s) and suggests that Microcystis may be a natural source of environmental estrogens. Concerns about effects of Microcystis blooms may extend beyond those associated with the microcystin toxin.
The field of viral ecology has long endeavored to devise and adapt methodologies to peer beyond the visible and elucidate the roles of viruses in the environment. Much has been learned regarding the dynamics of viral assemblages and the significant role viruses have in biogeochemical cycles (Brussaard et al., 2008). Despite these advances, detailed under- standing of biological processes behind ecosystem- scale effects of viral infection has remained largely obscured, with research relegated to a handful of available host–virus culture systems. Increasingly affordable DNA sequencing has provided a route to assess the genetic diversity of viruses in the environ- ment. Current research seeks to apply genomic technologies to address knowledge gaps in environ- mental virology, but obstacles presented by the unique biology of viruses must be addressed to understand the context and significance of viral genome and metagenome sequence data. Coincident with the twentieth anniversary of the publication that launched the field (Bergh et al., 1989), viral ecologists from around the world met in 2009 for a workshop of the Scientific Committee for Oceano- graphic Research (SCOR) Working Group on the Role ofVirusesinMarine viral-ecology.dbi.udel.edu) and at a session entitled ‘From Direct Counts to Metagenomics: Two Decades of Discovery in Aquatic Viral Ecology’ at the 109th General Meeting of the American Society for Micro- biology (ASM; http://www.asm.org). These meetings covered a broad range of topics relevant to environ- mental virology, however, the impact of metagenomics emerged as a major topic. Highlighted are important issues for viral metagenomics raised during a round- table discussion (SCOR) and through various abstracts presented at both forums.
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.
- Jan 2011
In recent years, excessive anthropogenic nutrient loading in Taihu (Lake Tai), China's third largest freshwater lake, has promoted the growth of harmful cyanobacterial blooms. Often composed of toxic species, these blooms threaten the supply of drinking water and fisheries-related food supplies for more than 8 million people. During a spatial survey in May 2009, surface water samples were collected from 14 stations located throughout this 2338km2 lake. Physicochemical and toxin measurements were compared to results from PCR based analyses of extracted DNA to examine microbial community diversity. Maximum concentrations of the hepatotoxin microcystin in this region were >600μgg−1 dry weight. Statistical comparisons of lake chemistry demonstrate relationships between toxin concentrations and physicochemistry (e.g., temperature, nutrients) within the water column. Pyrosequencing of bacterial 16S rRNA gene fragments revealed a diverse community that includes potential fecal bacteria. However, subsequent source tracking of specific fecal bacteria (by quantitative PCR) indicated that fecal bacteria concentrations in the lake water bacteria were relatively low and likely not of human origin. In total the data suggest that the proliferation of cyanobacteria, but not the production of microcystin, is influenced (and perhaps regulated) by both nitrogen and phosphorus concentrations. Our observations further suggest that microcystin concentrations are correlated to the diversity of the eubacterial community, implying that specific bacteria may associate with bloom events and/or be associated with nutrient sources loading into this system.
In experiments designed primarily to investigate viral lysis, we found that the presence of viruses had a positive effect on the growth of Synechococcus. A Landry-Hassett-type stepwise dilution experiment conducted during a Synechococcus bloom in the Gulf of Mexico used both (i) 0.2-µm filtered seawater in which the abundance of bacteria and grazers were reduced but the majority of viruses were retained, and (ii) ultrafiltered (30 000 MW cutoff) virus-free seawater in which the abundance of viruses, bacteria and grazers were reduced. High growth rates and frequency of dividing cells (FDCs) were recorded in 0.2-µm filtered treatments while growth was inhibited in incubations with a high proportion of virus-free ultrafiltered water. In two subsequent experiments using Mediterranean Sea populations, a two-point dilution approach in which viral abundance was reduced by 80–90% yielded similar results, and showed that Synechococcus only grew well in the presence of viruses, bacteria and grazers. In four further Mediterranean experiments viruses removed via ultrafiltration were added back, either untreated, or inactivated by a heat treatment. Growth rates and FDCs were higher in the presence of untreated viruses than with viruses inactivated by heat, suggesting that it was not organic matter in the virus-size fraction but rather the presence of infectious viruses which sustained growth. While Synechococcus was also infected by viruses during these experiments, our data imply that growth of Synechococcus may depend upon viral lysis of heterotrophic bacteria. This finding is consistent with the view that nutrient cycling by viral lysis of heterotrophic bacteria may control phytoplankton growth and ecosystem scale carbon production.
Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative estimates of this process is critical as we can then develop better models of microbial community structure and function as well as advance our understanding of how viruses work to alter aquatic biogeochemical cycles. The virus reduction technique allows researchers to estimate the rate at which virus particles are released from the endemic microbial community. In brief, the abundance of free (extracellular) viruses is reduced in a sample while the microbial community is maintained at near ambient concentration. The microbial community is then incubated in the absence of free viruses and the rate at which viruses re-occur in the sample (through the lysis of already infected members of the community) can be quantified by epifluorescence microscopy or, in the case of specific viruses, quantitative PCR. These rates can then be used to estimate the rate of microbial mortality due to virus-mediated cell lysis.