THE UNIVERSITY OF TENNESSEE

AQUATIC MICROBIAL ECOLOGY RESEARCH GROUP

 

 

Steven W. Wilhelm -  Professor OF Microbiology 

 

Office: 865-974-0665    Labs: 865-974-0682  or  865-974-4014

CURRENTLY FUNDED RESEARCH PROJECTS

 

Development of a tractable genetic system for Aureococcus anophagefferens.

Funding Source: The Gordon & Betty Moore Foundation

PI.  SW Wilhelm.  coPIs ER Zinser, T Sparer, T Reynolds and WH Wilson.

Aureococcus anophagefferens, the cause of Brown Tides, persists in coastal regions globally in what many considered hostile environments (i.e., turbid, enriched in nutrient and trace metal waste, etc.) at densities of greater than 109 cells L-1. This organism has a highly mosaic genome, indicative of frequent genomic modifications.  Along with suggestions that Aureococcus undergoes natural transformation, members of these populations are regularly infected by a giant virus (AaV) that is enriched with repeats believed to be active in genomic shuffling and frequent gene exchange between the virus and its host. Indeed, exchange has also occurred between AaV and other non-host organisms (i.e., bacteria and other algae) and implies a natural recombination mechanism with A. anophagefferens as the intermediate. We will develop a genetic system for Aureococcus that can serve to further our understanding of this algae’s ecology and evolution as well provide potential for biotechnological advances.  A series of approaches, including the use of the AaV and putative repeat regions that may encode promoters, will be employed in the transformation of Aureococcus. Along with direct transduction using modified AaV, we propose to take advantage of tools developed for the transformation of other algae (e.g., Chlorella, Nannochloropsis, Emiliania huxleyi) and other single-celled eukaryotes.

 

 R21AI113386-01: Effect of the gut microbiome on malaria. 

Funding Source: NIH Pathogenic eukaryotes panel

PI. N Schmidt, coPIs.  SW Wilhelm and SR Campagna.

Plasmodium infections, which cause malaria, have been a scourge to humanity for thousands of years. Unfortunately, we are still years away from an efficacious anti-malaria vaccine that is available to the >40% of the world's population that is at risk of malaria. Further complicating treatment and control of malaria is that both Plasmodium and the mosquito vector that transmits the parasite rapidly develop resistance to new drugs developed to treat malaria and control mosquito populations. Therefore, it is imperative novel approaches to control malaria are explored. We propose an exploratory project to determine the impact of the gut microbiota on regulating the severity of malaria, which has the potential to provide insights to novel and affordable approaches to treat malaria. The gut microbiota has been shown to shape susceptibility to obesity, diabetes, and regulate multiple aspects of host immunity. This is particularly true for components of the immune system that interface with the GI tract. Importantly, the gut microbiota also augments host immunity to bacterial and viral infections that occur outside of the GI tract. However, there are no reports as to how the gut microbiota influence host immune responses to non-GI tract parasitic infections, including Plasmodium. Our preliminary data demonstrate that C57BL/6 mice purchased from different vendors, which are known to have alterations in their gut microbiota, exhibit substantially different levels of parasite burden following infection with Plasmodium yoelii 17XNL. We have also demonstrated that C57BL/6 mice from different vendors treated with a cocktail of antibiotics exhibit dynamic responses following infection with Plasmodium compared to control treated mice. Collectively, our data suggest the gut microbiota regulates the severity of malaria. We hypothesize that specific gut bacteria and their metabolic by-products regulate susceptibility to malaria. We will address this hypothesis through the following specific aims: 1) Identify differences in gut microbiota populations that are associated with differential susceptibility to malaria, 2) Determine ability of the gut microbiota to transfer resistance or susceptibility to malaria to another mouse and 3) Identify gut metabolites that drive differential susceptibility to malaria.

 

Award Abstract #1451528.  Collaborative Research: an integrated approach to understanding the function of the potent hepatotoxin microcystin in the growth & ecology of Microcystis

Funding source: NSF Division of Integrative Organismal Systems

PI  SW Wilhelm.  coPIs ER Zinser, SR Campagna, JM DeBruyn, EM Fozo and GL Boyer

Blooms of toxic photosynthetic bacteria (cyanobacteria) are occurring globally with expanding frequency, duration and intensity in lakes, reservoirs and river systems. Most recently blooms of the toxic cyanobacterium Microcystis shut down the water supply of the city of Toledo, OH for a weekend in August of 2014. While the scientific community has developed a solid understanding of the factors that contribute to the blooms of Microcystis, previous research has not explained why cells make the hepato- (liver) toxin microcystin. As a potent inhibitor of a key class of enzymes - protein phosphatases - microcystin might play important roles inside Microcystis cells, and once released, inside the cells of other (target) organisms. This project will use advanced tools in molecular biology (RNA sequencing), microbial genetics, the quantification of small metabolites (metabolomics) and enzyme analyses to understand how the presence of microcystin shapes the activity of both the cells that make the compound and the community of microorganisms around them. Experiments in the laboratory will be complemented by field surveys of bloom events across naturally occurring toxin gradients - areas of historically high and low concentrations of toxin during the summer bloom season. State-of-the-art statistical analyses combined with these advanced scientific approaches will transform the understanding of why these cyanobacteria make this toxic compound. Understanding of the biological functions of the microcystin, will lead to better stewardship of a valuable natural resource: potable water. The total research effort will train students, including those from underrepresented groups, and broadly disseminate information to the public, systems managers and the scientific community. A significant component will feed into state-associated, in-class 4H training that will expose as many as 200,000 students to cyanobacteria as a model system to examine complex biochemical questions.

The goal of this project is to develop a deeper understanding of the biochemical role of microcystins, a potent protein phosphatase inhibitor, within cells and communities, and address both ecological and evolutionary questions concerning the maintenance of this and other expensive biosynthetic pathways for non-ribosomally encoded secondary metabolites within a (sub)population of cells. To determine how microcystin shapes cellular biochemistry and physiology, controlled lab experiments with Microcystis isolates that make microcystin, engineered strains where the biosynthetic gene has been knocked out, and wild-type Microcystis cells that lack the biosynthetic pathway will be conducted. Other cyanobacterial pairs (Planktothrix and Anabaena spp.) that make or do not make the toxin, engineered bacteria that produce this compound and a set of microorganisms isolated from Lake Erie that co-occur with Microcystis and may be influenced by toxin will also be tested. Experiments in the presence and absence of exogenous toxin will be conducted with both producers and non-toxin producers. State-of-the-art techniques in metabolic (LC-MS and LC-MS/MS metabolomics and lipidomics), transcriptional (Illumina mRNA-sequencing), enzymatic (4:3:3-regulated processes) and physiological analyses (e.g., cellular growth rates, primary production, and photosynthetic efficiency) for these defined lab strains will be employed to develop "fingerprints" of cellular function and elucidate how microcystin shapes these biochemical pathways and the physiological ecology of these cells. Lab experiments will be complemented by field surveys of bloom events across naturally occurring and well documented toxin gradients. Relationships will be identified using univariate and multivariate techniques. This novel integration of sequencing, small molecule chemistry, physiological and enzymatic approaches will permit the mapping of the physiological biochemistry of cells and identify both isolated as well as synergistic effects: indeed this work may transform the study of secondary metabolites in complex microbial systems and provide insights into microbial evolutionary ecology.

Award Abstract #1240870.  Dimensions: Collaborative Research: Anthropogenic nutrient input drives genetic, functional and taxonomic biodiversity in hypereutrophic Lake Taihu, China.

Funding Source: NSF DEB (Dimensions of Biodiversity program).

PI HW Paerl. coPIs SW Wilhelm, W Gardner, F Hellweger.  Collaborator GL Boyer.

Human activities have dramatically increased nitrogen inputs into many rivers and lakes, causing algal blooms that threaten economic and recreational uses of those waters. Lake Taihu, the third largest lake in China, experiences damaging blooms of toxic cyanobacteria as a result of excessive nutrient inputs. The identities, nitrogen processing capabilities, and activities of microbial communities in Lake Taihu will be examined to determine if nitrogen processing can be predicted from knowledge of the identity and genetic makeup of those communities. Various components of the nitrogen cycle will be measured and linked to representative molecular markers which, coupled with high throughput genetic sequencing, will provide a genetic database of nitrogen-cycling processes in freshwater ecosystems. A goal of the project will be to link microbial taxonomic, genetic, and functional data in a model that can predict how reduction of nutrient inputs will affect toxic cyanobacterial blooms.

This project will have broad applicability to the management of aquatic systems that are threatened by excessive nutrient inputs, a problem that is increasing worldwide. Additional broader impacts will include training of high school, undergraduate, and graduate students, international student exchanges, training of a postdoctoral researcher, and incorporation of science journalism students into field studies to promote adult scientific literacy.

Award Abstract 1230543. INSPIRE: An Ecologically-Driven Strategy for Ensuring Sustainability of Anthropogenically and Climatically Impacted Lakes

Funding Source: NSF  CBET / DEB

PI. HW Paerl. coPIs SW Wilhelm, JM DeBruyn.  Collaborator GL Boyer

This INSPIRE award is partially funded by the Environmental Sustainability Program of the Chemical, Bioengineering, Environmental, and Transport Systems Division (CBET) in the Engineering Directorate (ENG), the Ecosystems Studies Program of the Division of Environmental Biology (DEB) in the Biological Sciences Directorate (BIO), and the Office of International Science and Engineering (OISE).

INTELLECTUAL MERIT
Nutrient enrichment (eutrophication) in freshwaters has promoted global proliferation of harmful (toxic) cyanobacterial blooms (CyanoHABs), and this problem is exacerbated by global warming. This award is based on a new hypothesis that challenges the prevailing paradigm that CyanoHABs can be countered by control of the single nutirent, phosphorus (P). The new paradigm-challenging hypothesis is that nitrogen (N) loading in large lakes controls phytoplankton community structure, and that magnitude, spatial extent, and duration of CyanoHABs in large lakes are strongly dependent on both N and P inputs. This award is appropriate for the INSPIRE program due to the proposed transformational paradigm shift as well as the proposed problem-driven research that requires a comprehensive and integrative approach (ecosystem science as well as engineering analysis) to the grand challenge issue of ensuring availability of adequate quality freshwater resources. The fundamental questions raised are at the interface of biological science and engineering research. An additional challenge is addressing this new paradigm in the context of evolving climatic conditions. While it is crucial to understand how input reductions of N and P substrates can beneficially shape phytoplankton communities, this needs to be accomplished while accounting for climatic variations that are known to favor CyanoHABs. Managing nutrients requires an engineering solution, but implementation can only be successful if it is ecologically-constrained, so that the resulting biological species are desirable (e.g., non-toxin producing genera). Moreover, given the time frame required for traditional surveys of remediation success (i.e., years), the development of more rapid indicators is needed that will show more quickly whether an ecosystem is on a trajectory towards desired sustainability. How future climatic conditions will favor CyanoHAB formation must and will be considered. As more external pressure is placed on aquatic systems to provide ecosystems services, interdisciplinary (molecular, toxicological, biogeochemical, geohydrologic) efforts are needed to address the problem and ensure long term sustainability. In this research, in situ bioassays will be employed as well as quantitative mapping of gene expression to determine how various amounts of anthropogenic N (including ammonium and nitrate) and P (as phosphate) shape phytoplankton community structure and function and also CyanoHAB potential under ambient and elevated temperatures consistent with warming trends in a large lake basin. The particular place-based study will done on Lake Taihu, the third largest lake in China. The Lake Taihu study will be accomplished using mesocosms in a recently constructed flow-thru pond system, located at the Chinese Academy of Sciences Taihu Lake Laboratory for Environmental Research field station. In collaboration with Chinese researchers, data from sites in Taihu will be collected to compare experimental conditions to seasonal in situ bloom dynamics. Results will be used to calibrate and verify eutrophication-CyanoHAB modeling.

BROADER IMPACTS
An immediate societal product will be a science-based nutrient input reduction strategy for Chinese provincial/central governments that will bring and maintain Taihu below the CyanoHAB threshold and be transferrable to shallow hypereutrophic lakes worldwide that are experiencing both N and P over-enrichment. Student journalists and a U.S. (UNC/UTK) - China (Hohai, Nanjing Universities, NIGLAS, Chinese Academy of Sciences) student exchange will disseminate successes and the emerging scientific results to the general public. This project will train graduate, undergraduate, and secondary school students in interdisciplinary, cross-cutting research combining ecological analysis and environmental remediation within the context of socioeconomic policy issues concerning the sustainability of ecosystem services. While the lead U.S. institution is UNC-CH, the U.S. partner institution (UTK) is an EPSCoR institution, so this study will support students that are historically under-supported and typically limited with hands-on research experience. Teacher-training workshops will demonstrate the utility of land-water interface research in developing STEM knowledge for students, with a focus on making projects tractable with limited resources. This project will employ an interdisciplinary (ecology, molecular biology, toxicology, ecohydrology, water management) and international team of researchers (including a new female faculty member) and students to assess, control and mitigate CyanoHABs. This will enhance conceptual and technical understanding of interactive environmental drivers of long-term change in large lake ecosystems, information that will help ensure their sustainability in a warming world.

Award Abstract NA11NOS4780021. Biological degradation of microcystins: a first step towards biofilters for high efficiency toxin removal

Funding Source: NOAA PCM Program

PI. SW Wilhelm.  coPI GL Boyer

Research over the last decade has demonstrated bacteria that co-occur in the environment with toxic cyanobacteria blooms have the ability to break down cyanobacterial toxins: sometimes using these toxins as sole carbon sources. The University of Tennessee has isolated such organisms from Lake Erie, and acquired the original Australian isolate to degrade microcystins. This proposal will identify new microcystin-degrading species and characterize their decomposition of microcystins, a class of cyanotoxins that have been detected annually in the Laurentian Great Lakes since 1995. Field samples will be collected from, western Lake Erie, embayments of Lake Ontario, and obtained from international locations via ongoing research programs (e.g., Lake Tai (aka Taihu) in eastern China).  The rates of biological microcystin decomposition will be determined along with the identity of end products and factors constraining this process (temperature, inorganic nutrient availability). Biological characterizations (growth rate, growth efficiency, ability to use as sole carbon source vs. biological “doping” with stimulatory organic and inorganics, and genetic identity) will be coupled with chemical measurements of toxin and breakdown product concentrations. These are the essential first steps for development of a biological filter for toxin removal.  Specific objectives of this proposal include: 

(1) Isolate microcystin-degrading bacteria from blooms in Lakes Erie, Ontario and Tai.

(2) Identify bacteria (from 1) capable of using microcystins as a sole carbon source.

(3) Characterize bacteria and their growth rates under different conditions of temperature, nutrients and available carbon.

(4) Characterize degradation rates of microcystins under different physiological conditions.

(5) Identify the degradation products formed by these bacteria, including estimates of toxicity.

(6) Identifying a physical infrastructure (filter support, type, etc) that can be used in a bioreactor for the degradation of microcystins.

(7) Identify and address the NEPA requirements for application of a biofilter to remove microcystins from an external body of water.

The information on their rates of toxin degradation, byproducts and other metabolites produced by the degrading organism as well as microbial growth (both in lab or on the potential platform) will be used select a subset of bacteria for further study and testing in the bioreator.  The end-goal of this phase I study is to develop and deploy a biological “digester” under “pilot plant” conditions, similar to those currently used for the removal of other persistent organic pollutants. This process is now constrained from moving forward due to the lack basic information on the growth of the organisms, process rates and the nature of the resulting end products. This effort is an essential first step for preparation of a scalable biological filtration system to mitigate and prevent microcystins from passing into water distribution systems.  Potential end users include resource managers and water providers, but may also include aquaculture, recreational water body managers or agricultural providers. To help address the engineering and regulatory challenges for incorporation of this technology into existing water infrastructure, this project has established an advisory team that includes a representative from a large water engineering firm and NEPA experts to help guide the early development of the technology.

 

Award Abstract #1046042. Biogeochemical implications of marine phage: Roseophage as a relevant and tractable model

Funding Source: NSF Biological Oceanography

PI. A Buchan.  coPIs SW Wilhelm and SR Campagna

Intellectual Merit: Prokaryotic viruses (phage) have long been hypothesized to influence microbial community composition, nutrient biogeochemistry and both the flux and character of carbon in the world’s oceans. Model estimates indicate that > 25% of the daily carbon production in marine surface waters is shunted to the dissolved organic matter (DOM) pool by viral activity. Through this process viruses redistribute nutrient elements from large biological particles (i.e., bacteria, algae) into biologically inactive (dead) particulate and dissolved pools of organic compounds. Many of these compounds contain macro- and micronutrients (e.g., P, N, Fe) that can be rapidly recycled back into the food web. While we now have a better (yet far from complete) appreciation of the role of viruses in the regeneration of nutrient elements, we remain almost completely ignorant to, and have almost no data for, the role of viruses in the regeneration of organic carbon, the subsequent partitioning of this carbon by size (e.g. dissolved vs particulate) and bioavailability (labile vs recalcitrant). Understanding the contribution of virus activity to

the various carbon pools and the rates associated with this process is an absolute necessity if we are to develop accurate marine and global carbon models. There is little doubt that the global-scale influence of viruses is determined by host-phage interactions, yet our understanding of these interactions and their quantitative effects on system processes is in its infancy. To address these questions, it is imperative that we examine ecologically relevant model systems. To that end, our proposal focuses on phage that infect the Roseobacter clade, a numerically

abundant and biogeochemically active group of heterotrophic marine bacteria. Despite the recognized importance of lineage members to the global cycling of elements (particularly carbon), we know little of the viruses (“roseophage”) that infect them, the influence viruses have on host processes and the effects of this interaction on other members of the marine microbial community. As such this proposal is transformative in that it will exploit recently characterized virus-host models for biogeochemical and molecular studies of a major heterotrophic bacterioplankton lineage that is truly ecologically relevant. The overall objectives of this proposal are to: (i) examine the distribution, diversity and production of roseophage, (ii) assess the composition and bioavailability of Roseobacter cell lysis-derived DOM and (iii) to track the subsequent uptake and metabolism of Roseobacter-derived carbon and nitrogen by marine surface water microbial communities. These objectives will be achieved through a combination of lab and field-based experiments: we will develop molecular tools to quantify specific rates of roseophage production and Roseobacter mortality under three different environmental regimes (a naturally productive open ocean regime, a near shore to off-shore gradient and induced phytoplankton blooms from mesocosms). We will specifically determine the character and biological availability of carbon from lysates of Roseobacter in lab trials with model microbes. We will examine the rates of assimilation of radiolabeled Roseobacter lysate by natural communities which, when coupled with data on the composition, bioavailability and fate of the DOM released, will form the baseline for a model of Roseobacter-phage C-cycling through the microbial foodweb. Finally, complementary metabolomics studies of lysate consuming populations (in the lab and field) will provide unprecedented insight into how microbes perceive and process viral-lysed material. Collectively, these data will provide critical information on the interplay of phage with a major marine bacterial lineage and the ensuing influence these interactions have on microbial food webs.

 

Broader impacts: The broader impacts of this study include training of graduate, undergraduate and precollegiate students as well as facilitation of outreach and diversification at an EPSCoR (Experimental Program to Stimulate Competitive Research) institution. At least 3 graduate, 14 undergraduate, and 4 precollegiate students will be supported. The training aspects of the proposed work are high: many of the scientific activities are ideally suited for undergraduates and upper-level high school students research experiences. Two undergraduate journalism students will receive hand-on experiences in the lab and document the scientific efforts as articles and electronic media for dissemination to the general public and K-12 educators. Finally, the PIs are actively engaged in programs designed to recruit and retain underrepresented groups into graduate programs in biological and chemical sciences.


                                                     
updated 10/07/2015