Hot springs viruses at Yellowstone National Park have ancient origins and are adapted to thermophilic hosts.

Geothermal springs house unicellular red algae in the class Cyanidiophyceae that dominate the microbial biomass at these sites. Little is known about host-virus interactions in these environments. We analyzed the virus community associated with red algal mats in three neighboring habitats (creek, endolithic, soil) at Lemonade Creek, Yellowstone National Park (YNP), USA. We find that despite proximity, each habitat houses a unique collection of viruses, with the giant viruses, Megaviricetes, dominant in all three. The early branching phylogenetic position of genes encoded on metagenome assembled virus genomes (vMAGs) suggests that the YNP lineages are of ancient origin and not due to multiple invasions from mesophilic habitats. The existence of genomic footprints of adaptation to thermophily in the vMAGs is consistent with this idea. The Cyanidiophyceae at geothermal sites originated ca. 1.5 Bya and are therefore relevant to understanding biotic interactions on the early Earth.

Aerobic methane synthesis and dynamics in a river water environment.

Reports of aerobic biogenic methane (CH4) have generated new views about CH4 sources in nature. We examine this phenomenon in the free-flowing Yellowstone river wherein CH4 concentrations were tracked as a function of environmental conditions, phototrophic microorganisms (using chlorophyll a, Chl a, as proxy), as well as targeted methylated amines known to be associated with this process. CH4 was positively correlated with temperature and Chl a, although diurnal measurements showed CH4 concentrations were greatest during the night and lowest during maximal solar irradiation. CH4 efflux from the river surface was greater in quiescent edge waters (71-94 µmol m-2 d) than from open flowing current (~ 57 µmol m-2 d). Attempts to increase flux by disturbing the benthic environment in the quiescent water directly below (~ 1.0 m deep) or at varying distances (0-5 m) upstream of the flux chamber failed to increase surface flux. Glycine betaine (GB), dimethylamine and methylamine (MMA) were observed throughout the summer-long study, increasing during a period coinciding with a marked decline in Chl a, suggesting a lytic event led to their release; however, this did not correspond to increased CH4 concentrations. Spiking river water with GB or MMA yielded significantly greater CH4 than nonspiked controls, illustrating the metabolic potential of the river microbiome. In summary, this study provides evidence that: (1) phototrophic microorganisms are involved in CH4 synthesis in a river environment; (2) the river microbiome possesses the metabolic potential to convert methylated amines to CH4; and (3) river CH4 concentrations are dynamic diurnally as well as during the summer active months.

Arsenic and Mercury Distribution in an Aquatic Food Chain: Importance of Femtoplankton and Picoplankton Filtration Fractions.

Arsenic (As) and mercury (Hg) were examined in the Yellowstone Lake food chain, focusing on two lake locations separated by approximately 20 km and differing in lake floor hydrothermal vent activity. Sampling spanned from femtoplankton to the main fish species, Yellowstone cutthroat trout and the apex predator lake trout. Mercury bioaccumulated in muscle and liver of both trout species, biomagnifying with age, whereas As decreased in older fish, which indicates differential exposure routes for these metal(loid)s. Mercury and As concentrations were higher in all food chain filter fractions (0.1-, 0.8-, and 3.0-µm filters) at the vent-associated Inflated Plain site, illustrating the impact of localized hydrothermal inputs. Femtoplankton and picoplankton size biomass (0.1- and 0.8-µm filters) accounted for 30%-70% of total Hg or As at both locations. By contrast, only approximately 4% of As and <1% of Hg were found in the 0.1-µm filtrate, indicating that comparatively little As or Hg actually exists as an ionic form or intercalated with humic compounds, a frequent assumption in freshwaters and marine waters. Ribosomal RNA (18S) gene sequencing of DNA derived from the 0.1-, 0.8-, and 3.0-µm filters showed significant eukaryote biomass in these fractions, providing a novel view of the femtoplankton and picoplankton size biomass, which assists in explaining why these fractions may contain such significant Hg and As. These results infer that femtoplankton and picoplankton metal(loid) loads represent aquatic food chain entry points that need to be accounted for and that are important for better understanding Hg and As biochemistry in aquatic systems. Environ Toxicol Chem 2023;42:225-241. © 2022 SETAC.

Aerobic bacterial methane synthesis.

Reports of biogenic methane (CH4) synthesis associated with a range of organisms have steadily accumulated in the literature. This has not happened without controversy and in most cases the process is poorly understood at the gene and enzyme levels. In marine and freshwater environments, CH4 supersaturation of oxic surface waters has been termed the "methane paradox" because biological CH4 synthesis is viewed to be a strictly anaerobic process carried out by O2-sensitive methanogens. Interest in this phenomenon has surged within the past decade because of the importance of understanding sources and sinks of this potent greenhouse gas. In our work on Yellowstone Lake in Yellowstone National Park, we demonstrate microbiological conversion of methylamine to CH4 and isolate and characterize an Acidovorax sp. capable of this activity. Furthermore, we identify and clone a gene critical to this process (encodes pyridoxylamine phosphate-dependent aspartate aminotransferase) and demonstrate that this property can be transferred to Escherichia coli with this gene and will occur as a purified enzyme. This previously unrecognized process sheds light on environmental cycling of CH4, suggesting that O2-insensitive, ecologically relevant aerobic CH4 synthesis is likely of widespread distribution in the environment and should be considered in CH4 modeling efforts.

Introducing the ArsR-Regulated Arsenic Stimulon.

The microbial ars operon encodes the primary bacterial defense response to the environmental toxicant, arsenic. An important component of this operon is the arsR gene, which encodes ArsR, a member of the family of proteins categorized as DNA-binding transcriptional repressors. As currently documented, ArsR regulates its own expression as well as other genes in the same ars operon. This study examined the roles of four ArsR proteins in the well-developed model Gram-negative bacterium Agrobacterium tumefaciens 5A. RNASeq was used to compare and characterize gene expression profiles in ± arsenite-treated cells of the wild-type strain and in four different arsR mutants. We report that ArsR-controlled transcription regulation is truly global, extending well beyond the current ars operon model, and includes both repression as well as apparent activation effects. Many cellular functions are significantly influenced, including arsenic resistance, phosphate acquisition/metabolism, sugar transport, chemotaxis, copper tolerance, iron homeostasis, and many others. While there is evidence of some regulatory overlap, each ArsR exhibits its own regulatory profile. Furthermore, evidence of a regulatory hierarchy was observed; i.e. ArsR1 represses arsR4, ArsR4 activates arsR2, and ArsR2 represses arsR3. Additionally and unexpectedly, aioB (arsenite oxidase small subunit) expression was shown to be under partial positive control by ArsR2 and ArsR4. Summarizing, this study demonstrates the regulatory portfolio of arsenite-activated ArsR proteins and includes essentially all major cellular functions. The broad bandwidth of arsenic effects on microbial metabolism assists in explaining and understanding the full impact of arsenic in natural ecosystems, including the mammalian gut.

A Single Microbiome Gene Alters Murine Susceptibility to Acute Arsenic Exposure.

Environmental toxicant exposure contributes to morbidity and mortality of many human diseases. With respect to arsenic, microbially driven chemical transformations dictate its toxicity and mobility in virtually every environment yet studied, so a general hypothesis is that the human gut microbiome determines disease outcome following exposure. However, the complex nature of the gut microbiome and the myriad of potential interactions with human cells/tissues make it challenging to quantify the influence of specific arsenic-active functions-a requisite step in developing effective disease prevention and/or clinical intervention strategies. To control both mammalian and microbial function during toxicant exposure, we genetically defined the gut microbiome of mice using only Escherichia coli strain, AW3110 (▵arsRBC), or the same strain carrying a single genome copy of the Fucus vesiculosus metallothionein gene (AW3110::fmt); a cysteine-rich peptide that complexes with arsenite, facilitating bioaccumulation and reducing its toxic effects. AW3110::fmt bioaccumulated significantly more arsenic and gnotobiotic mice colonized by this strain excreted significantly more arsenic in stool and accumulated significantly less arsenic in organs. Moreover, AW3110::fmt gnotobiotic mice were protected from acute toxicity exposure (20 ppm AsIII) relative to controls. This study demonstrates-in a highly controlled fashion-that a single microbiome function (arsenic bioaccumulation) encoded by a single gene in a single human gut microbiome bacterium significantly alters mammalian host arsenic exposure. The experimental model described herein allows for a highly controlled and directed assessment of microbiome functions, and is useful to quantify the influence of specific microbiome-arsenic interactions that help mitigate human disease.

Arsenate-Induced Changes in Bacterial Metabolite and Lipid Pools during Phosphate Stress.

Agrobacterium tumefaciens GW4 is a heterotrophic arsenite-oxidizing bacterium with a high resistance to arsenic toxicity. It is now a model organism for studying the processes of arsenic detoxification and utilization. Previously, we demonstrated that under low-phosphate conditions, arsenate [As(V)] could enhance bacterial growth and be incorporated into biomolecules, including lipids. While the basic microbial As(V) resistance mechanisms have been characterized, global metabolic responses under low phosphate remain largely unknown. In the present work, the impacts of As(V) and low phosphate on intracellular metabolite and lipid profiles of GW4 were quantified using liquid chromatography-mass spectroscopy (LC-MS) in combination with transcriptional assays and the analysis of intracellular ATP and NADH levels. Metabolite profiling revealed that oxidative stress response pathways were altered and suggested an increase in DNA repair. Changes in metabolite levels in the tricarboxylic acid (TCA) cycle along with increased ATP are consistent with As(V)-enhanced growth of A. tumefaciens GW4. Lipidomics analysis revealed that most glycerophospholipids decreased in abundance when As(V) was available. However, several glycerolipid classes increased, an outcome that is consistent with maximizing growth via a phosphate-sparing phenotype. Differentially regulated lipids included phosphotidylcholine and lysophospholipids, which have not been previously reported in A. tumefaciens The metabolites and lipids identified in this study deepen our understanding of the interplay between phosphate and arsenate on chemical and metabolic levels.IMPORTANCE Arsenic is widespread in the environment and is one of the most ubiquitous environmental pollutants. Parodoxically, the growth of certain bacteria is enhanced by arsenic when phosphate is limited. Arsenate and phosphate are chemically similar, and this behavior is believed to represent a phosphate-sparing phenotype in which arsenate is used in place of phosphate in certain biomolecules. The research presented here uses a global approach to track metabolic changes in an environmentally relevant bacterium during exposure to arsenate when phosphate is low. Our findings are relevant for understanding the environmental fate of arsenic as well as how human-associated microbiomes respond to this common toxin.

Metabolic Responses to Arsenite Exposure Regulated through Histidine Kinases PhoR and AioS in Agrobacterium tumefaciens 5A.

Arsenite (AsIII) oxidation is a microbially-catalyzed transformation that directly impacts arsenic toxicity, bioaccumulation, and bioavailability in environmental systems. The genes for AsIII oxidation (aio) encode a periplasmic AsIII sensor AioX, transmembrane histidine kinase AioS, and cognate regulatory partner AioR, which control expression of the AsIII oxidase AioBA. The aio genes are under ultimate control of the phosphate stress response via histidine kinase PhoR. To better understand the cell-wide impacts exerted by these key histidine kinases, we employed 1H nuclear magnetic resonance (1H NMR) and liquid chromatography-coupled mass spectrometry (LC-MS) metabolomics to characterize the metabolic profiles of ΔphoR and ΔaioS mutants of Agrobacterium tumefaciens 5A during AsIII oxidation. The data reveals a smaller group of metabolites impacted by the ΔaioS mutation, including hypoxanthine and various maltose derivatives, while a larger impact is observed for the ΔphoR mutation, influencing betaine, glutamate, and different sugars. The metabolomics data were integrated with previously published transcriptomics analyses to detail pathways perturbed during AsIII oxidation and those modulated by PhoR and/or AioS. The results highlight considerable disruptions in central carbon metabolism in the ΔphoR mutant. These data provide a detailed map of the metabolic impacts of AsIII, PhoR, and/or AioS, and inform current paradigms concerning arsenic-microbe interactions and nutrient cycling in contaminated environments.

Arsenic and the gastrointestinal tract microbiome.

Arsenic is a toxin, ranking first on the Agency for Toxic Substances and Disease Registry and the Environmental Protection Agency Priority List of Hazardous Substances. Chronic exposure increases the risk of a broad range of human illnesses, most notably cancer; however, there is significant variability in arsenic-induced disease among exposed individuals. Human genetics is a known component, but it alone cannot account for the large inter-individual variability in the presentation of arsenicosis symptoms. Each part of the gastrointestinal tract (GIT) may be considered as a unique environment with characteristic pH, oxygen concentration, and microbiome. Given the well-established arsenic redox transformation activities of microorganisms, it is reasonable to imagine how the GIT microbiome composition variability among individuals could play a significant role in determining the fate, mobility and toxicity of arsenic, whether inhaled or ingested. This is a relatively new field of research that would benefit from early dialogue aimed at summarizing what is known and identifying reasonable research targets and concepts. Herein, we strive to initiate this dialogue by reviewing known aspects of microbe-arsenic interactions and placing it in the context of potential for influencing host exposure and health risks. We finish by considering future experimental approaches that might be of value.

Transcriptomics analysis defines global cellular response of Agrobacterium tumefaciens 5A to arsenite exposure regulated through the histidine kinases PhoR and AioS.

In environments where arsenic and microbes coexist, microbes are the principal drivers of arsenic speciation, which directly affects bioavailability, toxicity and bioaccumulation. Speciation reactions influence arsenic behaviour in environmental systems, directly affecting human and agricultural exposures. Arsenite oxidation decreases arsenic toxicity and mobility in the environment, and therefore understanding its regulation and overall influence on cellular metabolism is of significant interest. The arsenite oxidase (AioBA) is regulated by a three-component signal transduction system AioXSR, which is in turn regulated by the phosphate stress response, with PhoR acting as the master regulator. Using RNA-sequencing, we characterized the global effects of arsenite on gene expression in Agrobacterium tumefaciens 5A. To further elucidate regulatory controls, mutant strains for histidine kinases PhoR and AioS were employed, and illustrate that in addition to arsenic metabolism, a host of other functional responses are regulated in parallel. Impacted functions include arsenic and phosphate metabolism, carbohydrate metabolism, solute transport systems and iron metabolism, in addition to others. These findings contribute significantly to the current understanding of the metabolic impact and genetic circuitry involved during arsenite exposure in bacteria. This informs how arsenic contamination will impact microbial activities involving several biogeochemical cycles in nature.

The gut microbiome is required for full protection against acute arsenic toxicity in mouse models.

Arsenic poisons an estimated 200 million people worldwide through contaminated food and drinking water. Confusingly, the gut microbiome has been suggested to both mitigate and exacerbate arsenic toxicity. Here, we show that the microbiome protects mice from arsenic-induced mortality. Both antibiotic-treated and germ-free mice excrete less arsenic in stool and accumulate more arsenic in organs compared to control mice. Mice lacking the primary arsenic detoxification enzyme (As3mt) are hypersensitive to arsenic after antibiotic treatment or when derived germ-free, compared to wild-type and/or conventional counterparts. Human microbiome (stool) transplants protect germ-free As3mt-KO mice from arsenic-induced mortality, but protection depends on microbiome stability and the presence of specific bacteria, including Faecalibacterium. Our results demonstrate that both a functional As3mt and specific microbiome members are required for protection against acute arsenic toxicity in mouse models. We anticipate that the gut microbiome will become an important explanatory factor of disease (arsenicosis) penetrance in humans, and a novel target for prevention and treatment strategies.

Phosphate starvation response controls genes required to synthesize the phosphate analog arsenate.

Environmental arsenic poisoning affects roughly 200 million people worldwide. The toxicity and mobility of arsenic in the environment is significantly influenced by microbial redox reactions, with arsenite (AsIII ) being more toxic than arsenate (AsV ). Microbial oxidation of AsIII to AsV is known to be regulated by the AioXSR signal transduction system and viewed to function for detoxification or energy generation. Here, we show that AsIII oxidation is ultimately regulated by the phosphate starvation response (PSR), requiring the sensor kinase PhoR for expression of the AsIII oxidase structural genes aioBA. The PhoRB and AioSR signal transduction systems are capable of transphosphorylation cross-talk, closely integrating AsIII oxidation with the PSR. Further, under PSR conditions, AsV significantly extends bacterial growth and accumulates in the lipid fraction to the apparent exclusion of phosphorus. This could spare phosphorus for nucleic acid synthesis or triphosphate metabolism wherein unstable arsenic esters are not tolerated, thereby enhancing cell survival potential. We conclude that AsIII oxidation is logically part of the bacterial PSR, enabling the synthesis of the phosphate analog AsV to replace phosphorus in specific biomolecules or to synthesize other molecules capable of a similar function, although not for total replacement of cellular phosphate.

Methylphosphonate metabolism by Pseudomonas sp. populations contributes to the methane oversaturation paradox in an oxic freshwater lake.

The 'CH4 oversaturation paradox' has been observed in oxygen-rich marine and lake waters, and viewed to significantly contribute to biosphere cycling of methane, a potent greenhouse gas. Our study focused on the intriguing well-defined pelagic methane enriched zone (PMEZ) in freshwater lakes. Spiking Yellowstone Lake PMEZ samples with 13 C-labeled potential methanogenesis substrates found only 13 C-methylphosphonate (MPn) resulted in 13 CH4 generation. In 16S rRNA gene Illumina libraries, four Pseudomonas sp. operational taxonomic units surprisingly accounted for ∼11% abundance in the PMEZ community. Pseudomonas sp. isolates were also obtained from MPn enrichments with PMEZ water; they were most aggressive in MPn metabolism and their 16S rRNA gene sequences matched 35% of the Illumina PMEZ Pseudomonas reads. Further, two key genes encoding C-P lyase (phnJL, an important enzyme for dealkylation of MPn), were only amplifiable from PMEZ DNA and all PCR generated phnJL clones matched those of the Pseudomonas sp. isolates. Notably, methanogen 16S rRNA signatures were absent in all Illumina libraries and mcrA was not detected via PCR. Collectively, these observations are consistent with the conclusion that MPn metabolism contributes significantly to CH4 oversaturation in Yellowstone Lake and likely other oxic freshwater lake environments, and that Pseudomonas sp. populations are critical participants.

Arsenite oxidation regulator AioR regulates bacterial chemotaxis towards arsenite in Agrobacterium tumefaciens GW4.

Some arsenite [As(III)]-oxidizing bacteria exhibit positive chemotaxis towards As(III), however, the related As(III) chemoreceptor and regulatory mechanism remain unknown. The As(III)-oxidizing bacterium Agrobacterium tumefaciens GW4 displays positive chemotaxis towards 0.5-2 mM As(III). Genomic analyses revealed a putative chemoreceptor-encoding gene, mcp, located in the arsenic gene island and having a predicted promoter binding site for the As(III) oxidation regulator AioR. Expression of mcp and other chemotaxis related genes (cheA, cheY2 and fliG) was inducible by As(III), but not in the aioR mutant. Using capillary assays and intrinsic tryptophan fluorescence spectra analysis, Mcp was confirmed to be responsible for chemotaxis towards As(III) and to bind As(III) (but not As(V) nor phosphate) as part of the sensing mechanism. A bacterial one-hybrid system technique and electrophoretic mobility shift assays showed that AioR interacts with the mcp regulatory region in vivo and in vitro, and the precise AioR binding site was confirmed using DNase I foot-printing. Taken together, these results indicate that this Mcp is responsible for the chemotactic response towards As(III) and is regulated by AioR. Additionally, disrupting the mcp gene affected bacterial As(III) oxidation and growth, inferring that Mcp may exert some sort of functional connection between As(III) oxidation and As(III) chemotaxis.

Metabolic response of Agrobacterium tumefaciens 5A to arsenite.

Wide-spread abundance in soil and water, coupled with high toxicity have put arsenic at the top of the list of environmental contaminants. Early studies demonstrated that both concentration and the valence state of inorganic arsenic (arsenite, As(III) vs. arsenate As(V)) can be modulated by microbes. Using genetics, transcriptomic and proteomic techniques, microbe-arsenic detoxification, respiratory As(V) reduction and As(III) oxidation have since been examined. The effect of arsenic exposure on whole-cell intracellular microbial metabolism, however, has not been extensively studied. We combined LC-MS and 1 H NMR to quantify metabolic changes in Agrobacterium tumefaciens (strain 5A) upon exposure to sub-lethal concentrations of As(III). Metabolomics analysis reveals global differences in metabolite concentrations between control and As(III) exposure groups, with significant perturbations to intermediates shuttling into and cycling within the TCA cycle. These data are most consistent with the disruption of two key TCA cycle enzymes, pyruvate dehydrogenase and α-ketoglutarate dehydrogenase. Glycolysis also appeared altered following As(III) stress, with carbon accumulating as complex saccharides. These observations suggest that an important consequence of As(III) contamination in nature will be to alter microbial carbon metabolism at the microbial community level and thus has the potential to foundationally impact all biogeochemical cycles in the environment.

Regulatory Activities of Four ArsR Proteins in Agrobacterium tumefaciens 5A.

UNLABELLED: ArsR is a well-studied transcriptional repressor that regulates microbe-arsenic interactions. Most microorganisms have an arsR gene, but in cases where multiple copies exist, the respective roles or potential functional overlap have not been explored. We examined the repressors encoded by arsR1 and arsR2 (ars1 operon) and by arsR3 and arsR4 (ars2 operon) in Agrobacterium tumefaciens 5A. ArsR1 and ArsR4 are very similar in their primary sequences and diverge phylogenetically from ArsR2 and ArsR3, which are also quite similar to one another. Reporter constructs (lacZ) for arsR1, arsR2, and arsR4 were all inducible by As(III), but expression of arsR3 (monitored by reverse transcriptase PCR) was not influenced by As(III) and appeared to be linked transcriptionally to an upstream lysR-type gene. Experiments using a combination of deletion mutations and additional reporter assays illustrated that the encoded repressors (i) are not all autoregulatory as is typically known for ArsR proteins, (ii) exhibit variable control of each other's encoding genes, and (iii) exert variable control of other genes previously shown to be under the control of ArsR1. Furthermore, ArsR2, ArsR3, and ArsR4 appear to have an activator-like function for some genes otherwise repressed by ArsR1, which deviates from the well-studied repressor role of ArsR proteins. The differential regulatory activities suggest a complex regulatory network not previously observed in ArsR studies. The results indicate that fine-scale ArsR sequence deviations of the reiterated regulatory proteins apparently translate to different regulatory roles. IMPORTANCE: Given the significance of the ArsR repressor in regulating various aspects of microbe-arsenic interactions, it is important to assess potential regulatory overlap and/or interference when a microorganism carries multiple copies of arsR This study explores this issue and shows that the four arsR genes in A. tumefaciens 5A, associated with two separate ars operons, encode proteins exhibiting various degrees of functional overlap with respect to autoregulation and cross-regulation, as well as control of other functional genes. In some cases, differences in regulatory activity are associated with only limited differences in protein primary structure. The experiments summarized herein also present evidence that ArsR proteins appear to have activator functions, representing novel regulatory activities for ArsR, previously known only to be a repressor.

Geochemistry and Mixing Drive the Spatial Distribution of Free-Living Archaea and Bacteria in Yellowstone Lake.

Yellowstone Lake, the largest subalpine lake in the United States, harbors great novelty and diversity of Bacteria and Archaea. Size-fractionated water samples (0.1-0.8, 0.8-3.0, and 3.0-20 µm) were collected from surface photic zone, deep mixing zone, and vent fluids at different locations in the lake by using a remotely operated vehicle (ROV). Quantification with real-time PCR indicated that Bacteria dominated free-living microorganisms with Bacteria/Archaea ratios ranging from 4037:1 (surface water) to 25:1 (vent water). Microbial population structures (both Bacteria and Archaea) were assessed using 454-FLX sequencing with a total of 662,302 pyrosequencing reads for V1 and V2 regions of 16S rRNA genes. Non-metric multidimensional scaling (NMDS) analyses indicated that strong spatial distribution patterns existed from surface to deep vents for free-living Archaea and Bacteria in the lake. Along with pH, major vent-associated geochemical constituents including CH4, CO2, H2, DIC (dissolved inorganic carbon), DOC (dissolved organic carbon), SO4 (2-), O2 and metals were likely the major drivers for microbial population structures, however, mixing events occurring in the lake also impacted the distribution patterns. Distinct Bacteria and Archaea were present among size fractions, and bigger size fractions included particle-associated microbes (> 3 µm) and contained higher predicted operational taxonomic unit richness and microbial diversities (genus level) than free-living ones (<0.8 µm). Our study represents the first attempt at addressing the spatial distribution of Bacteria and Archaea in Yellowstone Lake, and our results highlight the variable contribution of Archaea and Bacteria to the hydrogeochemical-relevant metabolism of hydrogen, carbon, nitrogen, and sulfur.

Landscape position influences microbial composition and function via redistribution of soil water across a watershed.

Subalpine forest ecosystems influence global carbon cycling. However, little is known about the compositions of their soil microbial communities and how these may vary with soil environmental conditions. The goal of this study was to characterize the soil microbial communities in a subalpine forest watershed in central Montana (Stringer Creek Watershed within the Tenderfoot Creek Experimental Forest) and to investigate their relationships with environmental conditions and soil carbonaceous gases. As assessed by tagged Illumina sequencing of the 16S rRNA gene, community composition and structure differed significantly among three landscape positions: high upland zones (HUZ), low upland zones (LUZ), and riparian zones (RZ). Soil depth effects on phylogenetic diversity and ß-diversity varied across landscape positions, being more evident in RZ than in HUZ. Mantel tests revealed significant correlations between microbial community assembly patterns and the soil environmental factors tested (water content, temperature, oxygen, and pH) and soil carbonaceous gases (carbon dioxide concentration and efflux and methane concentration). With one exception, methanogens were detected only in RZ soils. In contrast, methanotrophs were detected in all three landscape positions. Type I methanotrophs dominated RZ soils, while type II methanotrophs dominated LUZ and HUZ soils. The relative abundances of methanotroph populations correlated positively with soil water content (R = 0.72, P < 0.001) and negatively with soil oxygen (R = -0.53, P = 0.008). Our results suggest the coherence of soil microbial communities within and differences in communities between landscape positions in a subalpine forested watershed that reflect historical and contemporary environmental conditions.

Proteomics and Genetics for Identification of a Bacterial Antimonite Oxidase in Agrobacterium tumefaciens.

Antimony (Sb) and its compounds are listed by the United States Environmental Protection Agency (USEPA, 1979) and the European Union (CEC, 1976) as a priority pollutant. Microbial redox transformations are presumed to be an important part of antimony cycling in nature; however, regulation of these processes and the enzymology involved are unknown. In this study, comparative proteomics and reverse transcriptase-PCR analysis of Sb(III)-oxidizing bacterium Agrobacterium tumefaciens GW4 revealed an oxidoreductase (anoA) is widely distributed in microorganisms, including at least some documented to be able to oxidize Sb(III). Deletion of the anoA gene reduced Sb(III) resistance and decreased Sb(III) oxidation by ∼27%, whereas the anoA complemented strain was similar to the wild type GW4 and a GW4 anoA overexpressing strain increased Sb(III) oxidation by ∼34%. Addition of Sb(III) up-regulated anoA expression and cloning anoA to Escherichia coli demonstrated direct transferability of this activity. A His-tag purified AnoA was found to require NADP(+) as cofactor, and exhibited a K(m) for Sb(III) of 64 ± 10 µM and a V(max) of 150 ± 7 nmol min(-1) mg(-1). This study contributes important initial steps toward a mechanistic understanding of microbe-antimony interactions and enhances our understanding of how microorganisms participate in antimony biogeochemical cycling in nature.