Recipient Organization
MONTANA STATE UNIVERSITY
(N/A)
BOZEMAN,MT 59717
Performing Department
Land Resources & Environmental Sciences
Non Technical Summary
Project 1)In the western U.S., approximately 70% of carbon sink activity is located at elevations above 750 m, where 50-85% of land is dominated by hilly or mountainous topography. Given the extensive distribution of subalpine forests, a better global understanding of how these ecosystems contribute to C exchange with the atmosphere is critical. Surprisingly, there is little information regarding the soil microbial communities involved. This project is designed to illuminate the role and importance of soil methanotrophs in consuming atmospheric methane gas.Project 2)The United Nations designated 2005-2015 as an International Decade for Action: "Water for Life", with the intent of focusing attention of world governments on the declining availability of freshwater fit for human use. The severe arsenic contamination crisis in much of Asia is the most high profile example of this problem, though there are regions in the United States that also suffer from elevated arsenic in drinking water supplies. The fate, mobility, and ecotoxicology of arsenic in soil and water is highly dependent on the abiotic and biotic processes that regulate arsenic biogeochemical cycling in nature. It is now known that microbially catalyzed redox transformations are very important in controlling arsenic chemistry in most environments, and thus for reliable prediction of how and why arsenic moves within and across environments it is essential that microbe-arsenic interactions be understood in detail. The information generated from this study is expected to be of value to land and water resource managers in agriculture, mine reclamation, and municipal water treatment, finding application to bioremediation efforts aimed at manipulating microbe-As interactions in the environment and thus controlling As fate and transport. The project will include training opportunities for two Ph.D. graduate students that will include an international lab rotation in China, as well as undergraduate research internships targeting Native American students. In addition, the PIs will work with K-12 teachers to expand public and private school curricula by offering class period lectures and day-long microscopy based exercises that emphasize the importance of microorganisms to environmental health and function.Project 3)Arsenic poisoning, or arsenicosis, is a worldwide threat to public health, leading to a variety of human diseases, including cancer. The microbial community (microbiome) of the human GI tract (GIT) has been implicated as a significant influence on host exposure to toxic xenobiotics, including arsenic-containing compounds (arsenicals), but the individual roles of host vs. microbiome in arsenic biotransformation have not been clearly defined. The broad, long-term objective of this research is to better understand the functional components of the human microbiome that impact As-transformations in the GIT that can then be manipulated as prophylactic and/or detoxifying agents for use as novel treatment and prevention strategies against human arsenicosis. This research addresses the microbiome's role in human exposure to an environmental toxin and so specifically addresses a strategic theme ("Exposure Research") and a specific strategic goal (Goal 4, part b) of the National Institute of Environmental Health Sciences (NIEHS). As an initial step toward defining the role of the human microbiome in arsenicosis, Specific Aim 1 will establish the baseline production of arsenicals in germ free mice and germ free mice colonized with a human microbiome (humanized mice). Germ free mice are completely sterile and so arsenical production in these arsenic-exposed animals will be due to host cells alone. In contrast, arsenical production in humanized mice will reflect the net influence of host and microbe, thereby allowing a comparison of their individual roles. As the next step forward in defining the role of the human microbiome in arsenicosis, Specific Aim 2 will directly quantify the influence of microbially-produced, arsenic-active enzymes in the gastrointestinal tract on arsenical levels in genotobiotic mice. In this part of the project, germ free mice will be mono-associated with genetically defined strains of Escherichia coli that have been shown previously to metabolize arsenic. Arsenical production will be quantified from temporally collected mouse tissues and fluids by state-of-the-art methodology using high-performance liquid chromatography and inductively coupled plasma mass spectrometry (HPLC-ICPMS) and corresponding temporal microbiome dynamics will be tracked using 16S rRNA encoding gene metagenomic sequencing. These data will be analyzed together to provide statistical support to and experimental evidence for the in vivo transformation of arsenic by the human GIT microbiome.Project 4)New knowledge acquired from this study will serve as baseline data for long term monitoring of metal and metalloid accumulation in environments not directly affected by anthropogenic inputs but that accumulate these toxins as a result of non-source point contamination.Project 5)Our overall goal is to conduct a two year assessment of status and trends in bioaccumulation of mercury, a strong neurotoxin, in water, sediments, and aquatic species of depressional Prairie Potholes of Fort Belknap Indian Community. A survey of mercury levels in biotic and abiotic components of wetlands will support selection of a subset of pristine and impacted wetland types for more detailed characterization of hydrologic and biogeochemical controls of mercury fate and transport. These data are needed to 1) assess current risk of mercury bioaccumulation to wetland species and habitat, 2) establish a baseline for comparison against future levels of mercury, and 3) to evaluate changes of climate and/or mercury deposition on wetlands ecological functions and services for a prairie ecosystem.
Animal Health Component
35%
Research Effort Categories
Basic
65%
Applied
35%
Developmental
(N/A)
Goals / Objectives
This is a multifaceted MAES project that covers considerable breadth in terms of topics investigated and collaborating scientists. All projects will require some time and effort on the part of project director, T.R. McDermott, and therefore are being listed and briefly characterized. All projects fall within the general topic of environmental microbiology and all are of relevance to food production and or human and environmental health; some have direct relevance to the state of Montana, some are more national in scope, while others are more general or are international in context.Goal 1) Characterize forest soil microbial community structure and diversity, with a specific interest in methane cycling.Goal 2) Study microbe interactions with arsenic and mercury, in environments that span from soil and aquatic environments to the human microbiome.
Project Methods
Project 1)Soil DNA will be used for PCR templates to generate barcoded 16S rRNA amplicons for paired-end Illumina sequencing to track relative abundances at the whole community level as well as of MB and methanogens, and for analyses using pmoA microarrays (6, 11, 44) that will allow us to track most known MB. We will also use qPCR to track the soluble methane monooxygenase, mmoX, of Methylocella (12). The microarrays will be calibrated with qPCR using MB group-specific pmoAs (five distinct groups including the forest clone group) (30). UniFrac (32) will be used for Illumina sequence analysis to provide relative community phyla changes over time and space (27). Soil SWC, pH, total carbon and selected solutes will be determined using standard methods. Geostatistical data analysis will use "geoR" (27, 37) to test for differences and correlate CH4 data with pmoA type and abundance as a function of location, depth, time and soil environmental variables. Ex situ soil mesocosm assays will be used to determine community CH4 consumption kinetics (Km and Vmax), following the methods of Knief et al. (29). 16S-based molecular analysis is blind to the USCα MB, so we will conduct 13C stable isotope probing (SIP) efforts in soil mesocosms to identify the 16S signature of this group (14). Repeated headspace replenishments over time with 13CH4 diluted into air at low (~10 ppm) concentrations will be used to focus the label into the DNA of the high affinity MB. The SIP approach will generate and separate 13C-enriched DNA for 16S-based PCR and metagenomic analysis (41).We seek to address a grand challenge in ecosystem ecology, namely the scaling gap between ecological processes observed at point or plot scales and their manifestation at landscape scales. Eddy covariance CO2 and water flux monitoring is ongoing at TCEF (16,39), and we have recently acquired two LI-7700 high-frequency open-path methane analyzers for our towers (2-m riparian and 40-m upland canopy) to provide top-down constraints on the magnitude/variability of ground-based CH4 sink/source observations. Due to time variance of driving variables and fluxes, and threshold behavior in rates of gas production and transport, our data analysis approaches take numerous forms. We will employ empirical and process model-based up-scaling of point and plot observations to the whole landscape while concurrently disaggregating (downscaling) tower-based measures of whole landscape and riparian component CH4 and CO2 fluxes, to understand how soil microbial and biogeochemical processes manifest at the landscape scale. We will also analyze flux time series across the landscape on diel (38) to seasonal scales to ascertain their relationships to one another and to driving variables, assessing potential error/uncertainty in sampling dynamic processes by conducting high-frequency sampling during selected field occupations and by making real-time measurements of key driving variables plus CO2 at benchmark sites. The variance and timing of CH4 fluxes and MB community structure dynamics across landscape positions/environmental conditions will be analyzed in simple and multiple regression frameworks, empirical modeling, and via parsimonious production and transport modeling (40) to better understand thresholds, nonlinearities, and feedbacks. While both our empirical and mechanistic modeling approaches will infer patterns and net landscape scale CH4 balances, the synergy gained in their combination represents yet unrealized potential to understand, quantify, and predict landscape scaleProject 2)We will use non-polar deletion mutants for all genes encoding the AioX-AioS-AioR system, the PhoR-PhoB system and four ArsR proteins in RNA-Seq-based transcriptomics of the wild type strain and selected regulatory mutants. These efforts will define the breadth of these regulatory systems as they pertain to AsIII, providing a comprehensive understanding of how the bacterial cell reacts to an environmental toxin at the transcriptional level.Other methods will focus on generating extensive metabolic maps of wild type and regulatory mutants used in Objective 1 as a function of exposure to AsIII. When combined with the RNA-Seq data, these experiments will generate in-depth insight into microbial cellular strategies for dealing with environmental toxins such as arsenic. These studies represent a bottom-up, functionally comprehensive approach to linking genes to whole cell behavior and performance. In our initial metabolomics efforts, we have found that the DphoR and DaioS mutants are essentially "blind" to AsIII.An ecological-economic (eco-economic) analysis will be performed on an in silico metabolic reconstruction of A. tumefaciens strain 5A. This will quantitatively define the limits of the genome-encoded physiological potential through convex basis analysis (extreme pathway analysis) with a focus on the two roles of AsIII: either an electron donor (energy source) or as a toxin. The mathematically defined physiological solution space will be data-mined using a combination of economic (resource investment) and ecological (competitiveness) tradeoff surfaces. Eco-economic-based model predictions will be correlated to the experimental data in an iterative manner to define and explain the metabolic acclimation of A. tumefaciens to AsIII stress.Project 3)We will compare arsenical metabolism in germ free mice (no microbiome) to that of humanized mice representing microbiomes from Chinese human volunteers (from Inner Mongolia, Hetao Basin; provided by collaborator Dr. Ping Li, Chinese University of Geosciences) that have had symptoms of arsenicosis and asymptomatic volunteers utilizing the same water source. We will quantify microbially-driven vs. host-driven AsV reduction to AsIII as well as microbially-driven As methylation in vivo.Project 4)Sampling will occur on Yellowstone Lake. Water samples will be size-fractionated filtered into mesoplankton, microplankton, nanoplankton, picoplankton and femtoplankton, respectively. Biomass is determined by weighing the collected zooplankton (i.e. meso- and microplankton) and by weighing the filters for the smaller size fractions. In addition to the plankton samples, we will be analyzing lake trout and cutthroat trout samples provided by Dr. Pat Bigelow, Yellowstone Fisheries. Each fish will be dissected into muscle, liver, and gut contents. Samples will be digested in 5% nitric acid and normalized based on mass for comparative purposes. ICP-MS will be used to detect and quantify both. ICP-MS can be combined with HPLC to speciate the As, using AsIII, AsV, MMAsV, DMAsV and trimethylarsineoxide as standards.Project 5)i) Collect water, soil, periphyton, and plant material from natural and constructed wetland microcosms of differing hydrology to quantify Hg species levels and to calculate bioconcentration factors. Samples for total Hg (HgT) and CH3Hg analysis will be collected using ultra-clean sampling techniques (Patterson and Settle,1976; Olson (1999). Duplicate field samples, field and laboratory blank samples of deionized water will be collected for QA/QC. All equipment used for sampling will be acid-washed and rinsed with Hg-free water, and cleaned between sampling sites.ii) Characterize microbial populations and key functional genes of the collected sediment. Total DNA will be extracted from sediments, periphyton and macroinvertebrate guts using the Power Soil DNA kit (Mo Bio Laboratories),shipped to Argonne National Laboratory for processing through their Illumina sequencing pipeline. The DNA will also be analyzed for the merA and hgcA genes by PCR using previously designed primers for the known diversity of these genes.