Recipient Organization
MONTANA STATE UNIVERSITY
(N/A)
BOZEMAN,MT 59717
Performing Department
Land Resources & Environmental Sciences
Non Technical Summary
Yellowstone Lake WorkBiological methane (CH4) synthesis has long been viewed to be the final step in carbon cycling in nature. It is a strictly anaerobic process catalyzed by a phylogenetically confined group of microbes known as methanogens. Because methanogens are 02 sensitive, CH4 enrichment in 02- rich ocean and lake waters (2,000-3,000% oversaturation) is a conundrum for microbiologists and biogeochemists - termed the "methane paradox". Our research in Yellowstone Lake has demonstrated that methanogens are absent from CH4 enriched waters and that aerobic bacteria synthesize CH4 (Wang et al. 2017). Even more exciting, we identified methylamine (MeA) based CH4 synthesis, the relevant bacteria, enzyme and gene involved (manuscript in revision at Science). This transformative discovery breaks completely from established views of biogenic CH4 production in the biosphere, fills gaps in our understanding of global carbon cycling, and identifies previous unknowns important to climate modeling .Mouse Microbiome ResearchArsenic 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 (Objective 2) of the National Institute of Environmental Health Sciences (NIEHS).As an initial step toward defining the role of the human microbiome in arsenicosis, Objective 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 metabolism 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.Objective 2 will directly quantify the influence of microbially-produced, arsenic-active enzymes in the GIT on arsenical levels in gnotobiotic 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 with expected outcomes. 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.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
0%
Goals / Objectives
This project is composed of two research thrusts concerning:1) Aerobic Bacterial Mehtnae Synthesis in teh Environment.2) Arsenic - Microbe Interacitons in teh Gastrointestinal Tract.PROJECT 1) Aerobic Bacterial Mehtnae Synthesis in teh EnvironmentThe proposed research builds upon a productive research history by PI McDermott (microbial ecophysiologist) on Yellowstone Lake that includes 454-FLX and Titanium pyrosequencing characterization of the l 6S rRNA, culminating in the description of the Bacre,.;a (Clingenpeel et al. 2011), Archaea (Kan et al. 2011) and Nanoarchaea (Clingenpeel et al 2013), selected macrophytes and zooplankton (Lovalvo et al. 2010), reconstruction of three complete virophage genomes (Zhou et al. 2014), and extensive lake geochemistry characterization (Clingenpeel et al. 2011). McDermott has collaborated with Bothner (an expert in metabolomics, proteomics and biochemistry) for -15 years, spanning four NSF grants and together they began characterizing the MeA phenomenon and enzyme (Wang et al. 2019). Co-PI Hatzenpichler is an environmental microbiologist with strong expertise in single cell targeted studies, particularly organism organism interactions involving methane cycling. Co-PI Lawrence is a structural biochemist and expert in protein structure and molecular function. As a team, they are all experts covering their respective elements of the proposed research.The proposed project is comprised of hypothesis-driven experiments that target three integrated topics that seek to re-examine the rules governing microbial methane synthesis and how it integrates into ecosystem function. It spans from ecosystem -+ organism -+ enzyme -+ gene, launches with established momentum, and is exceptionally mature with respect to knowledge of the environment, model organisms and defined mutants, essential genes, as well as purified enzyme that catalyzes the underlying reaction. We approach the project at three levels:1)Ecosystem function: How does aerobic methane synthesis fit into aquatic carbon cycling?Using 13C isotopic finger prints derived from purified enzyme reactions and or ecologically relevant pure culture lake isolates, these experiments will examine the relative contribution of MeA -+ CH4 and MPn -+ CH4 to in situ CH4 synthesis, consumption, and efflux from a freshwater lake. 15N-based work will track the fate of primary amines generated as part of MeA demethylation..2. Microbial and metabolic interactions: What are the sources and fate (s) of MPn and MeA in the environment and are there specific organism-organism interactions at play? Preliminary data strongly suggests specific bacteria are involved and that algae are important sources of MPn and or MeA. We aim to define the organisms and metabolic pathway(s) that generate these metabolites (MeA in particular), and how different members of the microbial community might be interacting to lead to their release into the aquatic environment. We will employ in situ, pure culture and model co-culture-based metagenomics, transcriptomics, and metabolomics to understand the participants and their interactions as well as temporal community dynamics.3. Enzyme characterization: What is the biochemistry of this new methanogenesis enzyme and how many environmental organisms encode homologs also capable of this reaction? Efforts here will focus on mechanistic and structural characterizing the pyridoxal phosphate dependent aspartate aminotransferase we have characterized as converting MeA to CH4. BlastP searches of metagenomes illustrates literally thousands of homologs in virtually every environment, yet we know that not all will catalyze the MeA --> CH4 reaction. Structural studies will provide functional, mechanistic and evolutionary insight to synergistically complement the enzymology, and greatly aid in answering the central question of how this enzyme catalyzes this reaction. This will allow us to determine which homologs encode this activity and the importance of thisfunction in the context of distribution across taxonomic clades and ecosystem habitats.PROJECT 2) rsenic - Microbe Interacitons in teh Gastrointestinal Tract.Objective 1. Establish the baseline production of toxic arsenicals in germ free and humanized mice.This leverages the establishment of human GIT microbiomes in germ free mice to help differentiate between host vs. microbial metabolism. This approach has been powerful for differentiating between host vs. microbial metabolites in fecal samples (Marcobal et al. 2013). We will compare arsenical metabolism in germ free mice (no microbiome) to that of humanized mice (i.e. germ free mice gavaged with stool samples from humans). Organ samples (stool, brain, liver, and kidney) and body fluids (urine, blood) from the mice will be analyzed for arsenic biochemistries. Microbiome dynamics in response to As exposure will be tracked via Illumina sequencing of PCRs that target the V4 variable region.Objective 2. Quantify the influence of microbially-produced As-active enzymes in the GIT on arsenical levels in gnotobiotic mice.This project will directly quantify the effects of microbially-driven arsenic transformation on host mouse survival and arsenic biochemistry. These studies will be conducted in wild type and as3mt knockout (as3mt-/- that lack the liver enzyme for methylating As(III)) mice mono-associated with customized microbiomes comprised of E. coli AW3110 (?arsRBC) that will carry specified genes/functions. The customized operons are constructed and are awaiting recombination into AW3110 and encode the functions shown in Fig. 2. In each case, the effects on the host mouse are predicted.
Project Methods
Yellowstone Lake ResearchMethods to be used have all been published or are described in submitted manuscripts. Text directly below is taken from these manuscripts and provides detailed descriptions of the methods the McDermott and Bothner groups use for this work. Enzyme structural studies will be conducted in the Lawrence laboratory and are not provided here. For in situ CH4 analysis, duplicate lake water samples for each depth were collected in 250 mL serum bottles, filling from the bottom and overflowing with at least one volume to expel bubbles. Vials are immediately sealed and secured with aluminum crimps, and killed by injecting 200 µL of a saturated HgCl2 solution and stored on ice for transport to the laboratory where they were stored at 4°C. CH4 analysis is by gas chromatography of an introduced headspace (using ultra-high purity N2) on a Hewlett-Packard HP5890A (for 2016 work) or a Varian CP-3800 (for 2017 work) gas chromatograph with flame ionization detection. The original CH4 concentration in solution will be calculated using Henry's Law and solubility equations (Wiesenburg & Guinasso, 1979).Enrichments with 13C-labeled substrates will identify and qualitatively assess potential methanogenesis substrates yielding CH4. Vials are incubated in the dark at 12°C for 15 days. The isotopic signature (δ13C) of the CH4 of the headspace is measured using a modification of the protocols described by Wang et al. (Wang et al. 2017) on a Picarro G2201-i cavity ring-down spectrometer (CRDS) equipped with a Small Sample Introduction Module 2 (SSIM2). For CH4 analysis , headspace gas is injected directly into an Agilent 7890 GC-MS equipped with Carboxen-1010 PLOT capillary GC column in splitless mode. The mass spectrometer was operated in scan/SIM mode; scan range (10 to 100 m/z), for SIM m/z 14, 15, 16, 17 were used. Chromatography experiments are done on an Agilent 6538 Q-TOF mass spectrometry, positive mode, equipped with a reversed-phase Agilent Zorbax Eclepse Plus C18 column (2.1x150mm). Solvent A was 0.1% formic acid in HPLC water, and solvent B was 0.1% formic acid in acetonitrile. The 15 min binary gradient elution profile was as follows: t 1) 1min, 0% B; t 2) 11 min, 55% B; t 3) 14 min, 100% B; and t 4) 15 min, 0% B. The wavelength was 320nm, the flow rate was 600 µL/min, and the sample injection volume was 10 µL. Limit of detection was determined to be 20nM. For detection of TMA, ethyl bromoacetate was used to label the compound of interest prior to analysis by LCMS. The labelling procedure was performed as described by Johnson (2008).DNA is extracted from all water column depths for use as PCR templates to determine whether the methanogen indicator gene, mcrA, was detectable and for Illumina sequencing of the community 16S rRNA genes. In each case, biomass from triplicate one liter samples were separately collected on Sterivex-GV 0.22 μm filter cartridges (Millipore Sigma, USA), and then DNA extracted using the PowerWater DNA isolation kit (MO BIO Laboratories, INC.) following the manufacturer's instructions. Both mcrA PCRs and Illumina community analysis were conducted as previously described (Wang et al. 2017).Enzyme assaysFor assessing aspartate amino transferase activity, enzyme reactions contained purified his-tag Aat, 20 mM aspartate, 2 mM -ketoglutarate, and 2 mM 5' pyridoxal phosphate in a 100 mM phosphate, pH 7.0. Activity was measured as described by Reitman and Frankel ( MeA transferase activity assays were the same except 2 mM MeA replaced aspartate and were carried out in 15 mL sealed serum bottles for tracking CH4 synthesis as described above. MeA transferase assays conducted with E. coli cytoplasmic extract were of the same composition except the phosphate buffer was replaced by the cytoplasmic extract, which was prepared by growing E. coli BL21(pET28a) to mid log phase in M9 minimal salts broth, collecting the cells via centrifugation (6,000 x g, 5 minutes), washing (3x) and resuspending the pellet in 100 mM phosphate buffer, and then lysing by sonication. Following centrifugation to pellet unbroken cells, the supernatant was filtered through 3 KDa cut-off filters to remove large molecular weight proteins with the intent of removing extraneous enzymes. For all of the above reaction types, control reactions consisted of the same compositions except the Aat enzyme preparation was inactivated by boiling for 20 minutes, or where only MeA was added without a-ketoglutarate.Mouse Microbiome ResearchSynthetic operons are being (or have already been built) that will examine specific microbial x arsenic interactions that are viewed to be relevant to influencing arsenic mobility and or toxicity. Specifially, we will clone different combinations of arsH, arsM, arsP, arsI, acr3, arsJ and gapdh.Mouse husbandry, experimetnal treatments and care will be as previously described by Coryelle et al. (Coryell M, McAlpine M, Pinkham NV, McDermott TR, Walk ST. (2018) The gut microbiome is required for full protection against acute arsenic toxicity in mouse models. Nat Commun. 9:5424)