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There are known associations between opioids, obesity, and the gut microbiome, but the molecular connection/mediation of these relationships is not understood. To better clarify the interplay of physiological, genetic, and microbial factors, this study investigated the microbiome and host inflammatory responses to chronic opioid administration in genetically obese, diet-induced obese, and lean mice. Samples of feces, urine, colon tissue, and plasma were analyzed using targeted LC-MS/MS quantification of metabolites, immunoassays of inflammatory cytokine levels, genome-resolved metagenomics, and metaproteomics. Genetic obesity, diet-induced obesity, and morphine treatment in lean mice each showed increases in distinct inflammatory cytokines. Metagenomic assembly and binning uncovered over 400 novel gut bacterial genomes and species. Morphine administration impacted the microbiome's composition and function, with the strongest effect observed in lean mice. This microbiome effect was less pronounced than either diet or genetically driven obesity. Based on inferred microbial physiology from the metaproteome datasets, a high-fat diet transitioned constituent microbes away from harvesting diet-derived nutrients and towards nutrients present in the host mucosal layer. Considered together, these results identified novel host-dependent phenotypes, differentiated the effects of genetic obesity versus diet induced obesity on gut microbiome composition and function, and showed that chronic morphine administration altered the gut microbiome., (© 2022. UT-Battele, LLC.)

Soil microbiomes are rapidly becoming known as an important driver of plant phenotypic variation and may mediate plant responses to environmental factors. However, integrating spatial scales relevant to climate change with plant intraspecific genetic variation and soil microbial ecology is difficult, making studies of broad inference rare. Here we hypothesize and show: 1) the degree to which tree genotypes condition their soil microbiomes varies by population across the geographic distribution of a widespread riparian tree, Populus angustifolia; 2) geographic dissimilarity in soil microbiomes among populations is influenced by both abiotic and biotic environmental variation; and 3) soil microbiomes that vary in response to abiotic and biotic factors can change plant foliar phenology. We show soil microbiomes respond to intraspecific variation at the tree genotype and population level, and geographic variation in soil characteristics and climate. Using a fully reciprocal plant population by soil location feedback experiment, we identified a climate-based soil microbiome effect that advanced and delayed bud break phenology by approximately 10 days. These results demonstrate a landscape-level feedback between tree populations and associated soil microbial communities and suggest soil microbes may play important roles in mediating and buffering bud break phenology with climate warming, with whole ecosystem implications.

Gas fermentation by autotrophic bacteria, such as clostridia, offers a sustainable path to numerous bioproducts from a range of local, highly abundant, waste and low-cost feedstocks, such as industrial flue gases or syngas generated from biomass or municipal waste. Unfortunately, designing and engineering clostridia remains laborious and slow. The ability to prototype individual genetic part function, gene expression patterns, and biosynthetic pathway performance in vitro before implementing designs in cells could help address these bottlenecks by speeding up design. Unfortunately, a high-yielding cell-free gene expression (CFE) system from clostridia has yet to be developed. Here, we report the development and optimization of a high-yielding (236 ± 24 μg/mL) batch CFE platform from the industrially relevant anaerobe, Clostridium autoethanogenum. A key feature of the platform is that both circular and linear DNA templates can be applied directly to the CFE reaction to program protein synthesis. We demonstrate the ability to prototype gene expression, and quantitatively map aerobic cell-free metabolism in lysates from this system. We anticipate that the C. autoethanogenum CFE platform will not only expand the protein synthesis toolkit for synthetic biology, but also serve as a platform in expediting the screening and prototyping of gene regulatory elements in non-model, industrially relevant microbes., (Copyright © 2020 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Drought stress negatively impacts microbial activity, but the magnitude of stress responses is likely dependent on a diversity of belowground interactions. Populus trichocarpa individuals and no-plant bulk soils were exposed to extended drought (∼0.03% gravimetric water content [GWC] after 12 days), rewet, and a 12-day "recovery" period to determine the effects of plant presence in mediating soil microbiome stability to water stress. Plant metabolomic analyses indicated that drought exposure increased host investment in C and N metabolic pathways (amino acids, fatty acids, phenolic glycosides) regardless of recovery. Several metabolites positively correlated with root-associated microbial alpha-diversity, but not those of soil communities. Soil bacterial community composition shifted with P. trichocarpa presence and with drought relative to irrigated controls, whereas soil fungal composition shifted only with plant presence. However, root fungal communities strongly shifted with drought, whereas root bacterial communities changed to a lesser degree. The proportion of bacterial water-stress opportunistic operational taxonomic units (OTUs) (enriched counts in drought) was high (∼11%) at the end of drying phases and maintained after rewet and recovery phases in bulk soils, but it declined over time in soils with plants present. For root fungi, opportunistic OTUs were high at the end of recovery in drought treatments (∼17% abundance), although relatively not responsive in soils, particularly planted soils (<0.5% abundance for sensitive or opportunistic). These data indicate that plants modulate soil and root-associated microbial drought responses via tight plant-microbe linkages during extreme drought scenarios, but trajectories after extreme drought vary with plant habitat and microbial functional groups. IMPORTANCE Climate change causes significant alterations in precipitation and temperature regimes that are predicted to become more extreme throughout the next century. Microorganisms are important members within ecosystems, and how they respond to these changing abiotic stressors has large implications for the functioning of ecosystems, the recycling of nutrients, and the health of the aboveground plant community. Drought stress negatively impacts microbial activity, but the magnitude of this stress response may be dependent on above- and belowground interactions. This study demonstrates that beneficial associations between plants and microbes can enhance tolerance to abiotic stress.

Serendipitaceae represents a diverse fungal group in the Basidiomycota that includes endophytes and lineages that repeatedly evolved ericoid, orchid and ectomycorrhizal lifestyle. Plants rely upon both nitrogen and phosphorous, for essential growth processes, and are often provided by mycorrhizal fungi. In this study, we investigated the cellular proteome of Serendipita vermifera MAFF305830 and closely related Serendipita vermifera subsp. bescii NFPB0129 grown in vitro under (N) ammonium and (P) phosphate starvation conditions. Mycelial growth pattern was documented under these conditions to correlate growth-specific responses to nutrient starvation. We found that N-starvation accelerated hyphal radial growth, whereas P-starvation accelerated hyphal branching. Additionally, P-starvation triggers an integrated starvation response leading to remodelling of lipid metabolism. Higher abundance of an ammonium transporter known to serve as both an ammonium sensor and stimulator of hyphal growth was detected under N-starvation. Additionally, N-starvation led to strong up-regulation of nitrate, amino acid, peptide, and urea transporters, along with several proteins predicted to have peptidase activity. Taken together, our finding suggests S. bescii and S. vermifera have the metabolic capacity for nitrogen assimilation from organic forms of N compounds. We hypothesize that the nitrogen metabolite repression is a key regulator of such organic N assimilation., (© 2019 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Soil microbiome responses to short-term nitrogen (N) inputs remain uncertain when compared with previous research that has focused on long-term fertilization responses. Here, we examined soil bacterial/archaeal and fungal communities pre- and post-N fertilization in an 8 year-old switchgrass field, in which twenty-four plots received N fertilization at three levels (0, 100, and 200 kg N ha-1 as NH4NO3) for the first time since planting. Soils were collected at two depths, 0-5 and 5-15 cm, for DNA extraction and amplicon sequencing of 16S rRNA genes and ITS regions for assessment of microbial community composition. Baseline assessments prior to fertilization revealed no significant pre-existing divergence in either bacterial/archaeal or fungal communities across plots. The one-time N fertilizations increased switchgrass yields and tissue N content, and the added N was nearly completely removed from the soil of fertilized plots by the end of the growing season. Both bacterial/archaeal and fungal communities showed large spatial (by depth) and temporal variation (by season) within each plot, accounting for 17 and 12-22% of the variation as calculated from the Sq. root of PERMANOVA tests for bacterial/archaeal and fungal community composition, respectively. While N fertilization effects accounted for only ~4% of overall variation, some specific microbial groups, including the bacterial genus Pseudonocardia and the fungal genus Archaeorhizomyces, were notably repressed by fertilization at 200 kg N ha-1. Bacterial groups varied with both depth in the soil profile and time of sampling, while temporal variability shaped the fungal community more significantly than vertical heterogeneity in the soil. These results suggest that short-term effects of N fertilization are significant but subtle, and other sources of variation will need to be carefully accounted for study designs including multiple intra-annual sampling dates, rather than one-time "snapshot" analyses that are common in the literature. Continued analyses of these trends over time with fertilization and management are needed to understand how these effects may persist or change over time., Competing Interests: The authors of this manuscript have read the journal’s policy and have the following competing interest: DH is an Academic Editor for PLOS ONE. This manuscript has been authored by UT-Battelle, LLC under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Plants have developed defense strategies for phytopathogen and herbivore protection via coordinated metabolic mechanisms. Low-molecular weight metabolites produced within plant tissues, such as salicylic acid, represent one such mechanism which likely mediates plant - microbe interactions above and below ground. Salicylic acid is a ubiquitous phytohormone at low levels in most plants, yet are concentrated defense compounds in Populus, likely acting as a selective filter for rhizosphere microbiomes. We propagated twelve Populus trichocarpa genotypes which varied an order of magnitude in salicylic acid (SA)-related secondary metabolites, in contrasting soils from two different origins. After four months of growth, plant properties (leaf growth, chlorophyll content, and net photosynthetic rate) and plant root metabolomics specifically targeting SA metabolites were measured via GC-MS. In addition, rhizosphere microbiome composition was measured via Illumina MiSeq sequencing of 16S and ITS2 rRNA-genes., Results: Soil origin was the primary filter causing divergence in bacterial/archaeal and fungal communities with plant genotype secondarily influential. Both bacterial/archaeal and fungal evenness varied between soil origins and bacterial/archaeal diversity and evenness correlated with at least one SA metabolite (diversity: populin; evenness: total phenolics). The production of individual salicylic acid derivatives that varied by host genotype resulted in compositional differences for bacteria /archaea (tremuloidin) and fungi (salicylic acid) within one soil origin (Clatskanie) whereas soils from Corvallis did not illicit microbial compositional changes due to salicylic acid derivatives. Several dominant bacterial (e.g., Betaproteobacteria, Acidobacteria, Verrucomicrobia, Chloroflexi, Gemmatimonadete, Firmicutes) and one fungal phyla (Mortierellomycota) also correlated with specific SA secondary metabolites; bacterial phyla exhibited more negative interactions (declining abundance with increasing metabolite concentration) than positive interactions., Conclusions: These results indicate microbial communities diverge most among soil origin. However, within a soil origin, bacterial/archaeal communities are responsive to plant SA production within greenhouse-based rhizosphere microbiomes. Fungal microbiomes are impacted by root SA-metabolites, but overall to a lesser degree within this experimental context. These results suggest plant defense strategies, such as SA and its secondary metabolites, may partially drive patterns of both bacterial/archaeal and fungal taxa-specific colonization and assembly.

While recent reports demonstrate that the direct emission of methane from living tree trunks may be a significant terrestrial emission source, there has been debate whether tree emissions are due to transport from soils or produced in the wood environment itself. Reports of methanogens from wood of trees were prominent in the literature 40 years ago but have not been revisited with molecular ecology approaches. We examined communities associated with Populus deltoides using rRNA gene sequence analyses and how these vary with tree and wood properties. Our data indicate that wood environments are dominated by anaerobic microbiomes. Methanogens are prominent in heartwood (mean 34% relative abundance) compared to sapwood environments (13%), and dominant operational taxonomic units (OTUs) were classified as the Methanobacterium sp. Members of the Firmicutes phylum comprised 39% of total sequences and were in 42% greater abundance in sapwood over heartwood niches. Tree diameter was the strongest predictor of methanogen abundance, but wood moisture content and pH were also significant predictors of taxon abundance and overall community composition. Unlike microbiomes of the soil, rhizosphere and phyllosphere, wood associated communities are shaped by unique environmental conditions and may be prominent and overlooked sources of methane emissions in temperate forest systems., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Background: Lignin is a crucial molecule for terrestrial plants, as it offers structural support and permits the transport of water over long distances. The hardness of lignin reduces plant digestibility by cattle and sheep; it also makes inedible plant materials recalcitrant toward the enzymatic fermentation of cellulose, which is a potentially valuable substrate for sustainable biofuels. Targeted attempts to change the amount or composition of lignin in relevant plant species have been hampered by the fact that the lignin biosynthetic pathway is difficult to understand, because it uses several enzymes for the same substrates, is regulated in an ill-characterized manner, may operate in different locations within cells, and contains metabolic channels, which the plant may use to funnel initial substrates into specific monolignols., Results: We propose a dynamic mathematical model that integrates various datasets and other information regarding the lignin pathway in Brachypodium distachyon and permits explanations for some counterintuitive observations. The model predicts the lignin composition and label distribution in a BdPTAL knockdown strain, with results that are quite similar to experimental data., Conclusion: Given the present scarcity of available data, the model resulting from our analysis is presumably not final. However, it offers proof of concept for how one may design integrative pathway models of this type, which are necessary tools for predicting the consequences of genomic or other alterations toward plants with lignin features that are more desirable than in their wild-type counterparts.

Background: Anaerobic fermentation of lignocellulose occurs in both natural and managed environments, and is an essential part of the carbon cycle as well as a promising route to sustainable production of fuels and chemicals. Lignocellulose solubilization by mixed microbiomes is important in these contexts., Results: Here, we report the development of stable switchgrass-fermenting enrichment cultures maintained at various residence times and moderately high (55 °C) temperatures. Anaerobic microbiomes derived from a digester inoculum were incubated at 55 °C and fed semi-continuously with medium containing 30 g/L mid-season harvested switchgrass to achieve residence times (RT) of 20, 10, 5, and 3.3 days. Stable, time-invariant cellulolytic methanogenic cultures with minimal accumulation of organic acids were achieved for all RTs. Fractional carbohydrate solubilization was 0.711, 0.654, 0.581 and 0.538 at RT = 20, 10, 5 and 3.3 days, respectively, and glucan solubilization was proportional to xylan solubilization at all RTs. The rate of solubilization was described well by the equation r  =  k ( C  -  C 0 f r ), where C represents the concentration of unutilized carbohydrate, C 0 is the concentration of carbohydrate (cellulose and hemicellulose) entering the bioreactor and f r is the extrapolated fraction of entering carbohydrate that is recalcitrant at infinite residence time. The 3.3 day RT is among the shortest RT reported for stable thermophilic, methanogenic digestion of a lignocellulosic feedstock. 16S rDNA phylotyping and metagenomic analyses were conducted to characterize the effect of RT on community dynamics and to infer functional roles in the switchgrass to biogas conversion to the various microbial taxa. Firmicutes were the dominant phylum, increasing in relative abundance from 54 to 96% as RT decreased. A Clostridium clariflavum strain with genetic markers for xylose metabolism was the most abundant lignocellulose-solubilizing bacterium. A Thermotogae ( Defluviitoga tunisiensis ) was the most abundant bacterium in switchgrass digesters at RT = 20 days but decreased in abundance at lower RTs as did multiple Chloroflexi. Synergistetes and Euryarchaeota were present at roughly constant levels over the range of RTs examined., Conclusions: A system was developed in which stable methanogenic steady-states were readily obtained with a particulate biomass feedstock, mid-season switchgrass, at laboratory (1 L) scale. Characterization of the extent and rate of carbohydrate solubilization in combination with 16S rDNA and metagenomic sequencing provides a multi-dimensional view of performance, species composition, glycoside hydrolases, and metabolic function with varying residence time. These results provide a point of reference and guidance for future studies and organism development efforts involving defined cultures.

Background: Lignin is a natural polymer that is interwoven with cellulose and hemicellulose within plant cell walls. Due to this molecular arrangement, lignin is a major contributor to the recalcitrance of plant materials with respect to the extraction of sugars and their fermentation into ethanol, butanol, and other potential bioenergy crops. The lignin biosynthetic pathway is similar, but not identical in different plant species. It is in each case comprised of a moderate number of enzymatic steps, but its responses to manipulations, such as gene knock-downs, are complicated by the fact that several of the key enzymes are involved in several reaction steps. This feature poses a challenge to bioenergy production, as it renders it difficult to select the most promising combinations of genetic manipulations for the optimization of lignin composition and amount., Results: Here, we present several computational models than can aid in the analysis of data characterizing lignin biosynthesis. While minimizing technical details, we focus on the questions of what types of data are particularly useful for modeling and what genuine benefits the biofuel researcher may gain from the resulting models. We demonstrate our analysis with mathematical models for black cottonwood ( Populus trichocarpa ), alfalfa ( Medicago truncatula ), switchgrass ( Panicum virgatum ) and the grass Brachypodium distachyon ., Conclusions: Despite commonality in pathway structure, different plant species show different regulatory features and distinct spatial and topological characteristics. The putative lignin biosynthes pathway is not able to explain the plant specific laboratory data, and the necessity of plant specific modeling should be heeded.

Unlabelled: Bacterial endophytes that colonize Populus trees contribute to nutrient acquisition, prime immunity responses, and directly or indirectly increase both above- and below-ground biomasses. Endophytes are embedded within plant material, so physical separation and isolation are difficult tasks. Application of culture-independent methods, such as metagenome or bacterial transcriptome sequencing, has been limited due to the predominance of DNA from the plant biomass. Here, we describe a modified differential and density gradient centrifugation-based protocol for the separation of endophytic bacteria from Populus roots. This protocol achieved substantial reduction in contaminating plant DNA, allowed enrichment of endophytic bacteria away from the plant material, and enabled single-cell genomics analysis. Four single-cell genomes were selected for whole-genome amplification based on their rarity in the microbiome (potentially uncultured taxa) as well as their inferred abilities to form associations with plants. Bioinformatics analyses, including assembly, contamination removal, and completeness estimation, were performed to obtain single-amplified genomes (SAGs) of organisms from the phyla Armatimonadetes, Verrucomicrobia, and Planctomycetes, which were unrepresented in our previous cultivation efforts. Comparative genomic analysis revealed unique characteristics of each SAG that could facilitate future cultivation efforts for these bacteria., Importance: Plant roots harbor a diverse collection of microbes that live within host tissues. To gain a comprehensive understanding of microbial adaptations to this endophytic lifestyle from strains that cannot be cultivated, it is necessary to separate bacterial cells from the predominance of plant tissue. This study provides a valuable approach for the separation and isolation of endophytic bacteria from plant root tissue. Isolated live bacteria provide material for microbiome sequencing, single-cell genomics, and analyses of genomes of uncultured bacteria to provide genomics information that will facilitate future cultivation attempts., (Copyright © 2016 Utturkar et al.)

Background: Thermoanaerobacterium saccharolyticum is a hemicellulose-degrading thermophilic anaerobe that was previously engineered to produce ethanol at high yield. A major project was undertaken to develop this organism into an industrial biocatalyst, but the lack of genome information and resources were recognized early on as a key limitation., Results: Here we present a set of genome-scale resources to enable the systems level investigation and development of this potentially important industrial organism. Resources include a complete genome sequence for strain JW/SL-YS485, a genome-scale reconstruction of metabolism, tiled microarray data showing transcription units, mRNA expression data from 71 different growth conditions or timepoints and GC/MS-based metabolite analysis data from 42 different conditions or timepoints. Growth conditions include hemicellulose hydrolysate, the inhibitors HMF, furfural, diamide, and ethanol, as well as high levels of cellulose, xylose, cellobiose or maltodextrin. The genome consists of a 2.7 Mbp chromosome and a 110 Kbp megaplasmid. An active prophage was also detected, and the expression levels of CRISPR genes were observed to increase in association with those of the phage. Hemicellulose hydrolysate elicited a response of carbohydrate transport and catabolism genes, as well as poorly characterized genes suggesting a redox challenge. In some conditions, a time series of combined transcription and metabolite measurements were made to allow careful study of microbial physiology under process conditions. As a demonstration of the potential utility of the metabolic reconstruction, the OptKnock algorithm was used to predict a set of gene knockouts that maximize growth-coupled ethanol production. The predictions validated intuitive strain designs and matched previous experimental results., Conclusion: These data will be a useful asset for efforts to develop T. saccharolyticum for efficient industrial production of biofuels. The resources presented herein may also be useful on a comparative basis for development of other lignocellulose degrading microbes, such as Clostridium thermocellum.

This study investigated the abundance, distribution, and composition of microbial communities at the watershed scale in a boreal peatland within the Marcell Experimental Forest (MEF), Minnesota, USA. Through a close coupling of next-generation sequencing, biogeochemistry, and advanced analytical chemistry, a biogeochemical hot spot was revealed in the mesotelm (30- to 50-cm depth) as a pronounced shift in microbial community composition in parallel with elevated peat decomposition. The relative abundance of Acidobacteria and the Syntrophobacteraceae, including known hydrocarbon-utilizing genera, was positively correlated with carbohydrate and organic acid content, showing a maximum in the mesotelm. The abundance of Archaea (primarily crenarchaeal groups 1.1c and 1.3) increased with depth, reaching up to 60% of total small-subunit (SSU) rRNA gene sequences in the deep peat below the 75-cm depth. Stable isotope geochemistry and potential rates of methane production paralleled vertical changes in methanogen community composition to indicate a predominance of acetoclastic methanogenesis mediated by the Methanosarcinales in the mesotelm, while hydrogen-utilizing methanogens predominated in the deeper catotelm. RNA-derived pyrosequence libraries corroborated DNA sequence data to indicate that the above-mentioned microbial groups are metabolically active in the mid-depth zone. Fungi showed a maximum in rRNA gene abundance above the 30-cm depth, which comprised only an average of 0.1% of total bacterial and archaeal rRNA gene abundance, indicating prokaryotic dominance. Ratios of C to P enzyme activities approached 0.5 at the acrotelm and catotelm, indicating phosphorus limitation. In contrast, P limitation pressure appeared to be relieved in the mesotelm, likely due to P solubilization by microbial production of organic acids and C-P lyases. Based on path analysis and the modeling of community spatial turnover, we hypothesize that P limitation outweighs N limitation at MEF, and microbial communities are structured by the dominant shrub, Chamaedaphne calyculata, which may act as a carbon source for major consumers in the peatland.

Microbial reduction of toxic hexavalent chromium (Cr(VI)) in-situ is a plausible bioremediation strategy in electron-acceptor limited environments. However, higher [Cr(VI)] may impose stress on syntrophic communities and impact community structure and function. The study objectives were to understand the impacts of Cr(VI) concentrations on community structure and on the Cr(VI)-reduction potential of groundwater communities at Hanford, WA. Steady state continuous flow bioreactors were used to grow native communities enriched with lactate (30 mM) and continuously amended with Cr(VI) at 0.0 (No-Cr), 0.1 (Low-Cr) and 3.0 (High-Cr) mg/L. Microbial growth, metabolites, Cr(VI), 16S rRNA gene sequences and GeoChip based functional gene composition were monitored for 15 weeks. Temporal trends and differences in growth, metabolite profiles, and community composition were observed, largely between Low-Cr and High-Cr bioreactors. In both High-Cr and Low-Cr bioreactors, Cr(VI) levels were below detection from week 1 until week 15. With lactate enrichment, native bacterial diversity substantially decreased as Pelosinus spp., and Sporotalea spp., became the dominant groups, but did not significantly differ between Cr concentrations. The Archaea diversity also substantially decreased after lactate enrichment from Methanosaeta (35%), Methanosarcina (17%) and others, to mostly Methanosarcina spp. (95%). Methane production was lower in High-Cr reactors suggesting some inhibition of methanogens. Several key functional genes were distinct in Low-Cr bioreactors compared to High-Cr. Among the Cr resistant microbes, Burkholderia vietnamiensis, Comamonas testosterone and Ralstonia pickettii proliferated in Cr amended bioreactors. In-situ fermentative conditions facilitated Cr(VI) reduction, and as a result 3.0 mg/L Cr(VI) did not impact the overall bacterial community structure.

Bacterial and fungal communities associated with plant roots are central to the host health, survival and growth. However, a robust understanding of the root-microbiome and the factors that drive host associated microbial community structure have remained elusive, especially in mature perennial plants from natural settings. Here, we investigated relationships of bacterial and fungal communities in the rhizosphere and root endosphere of the riparian tree species Populus deltoides, and the influence of soil parameters, environmental properties (host phenotype and aboveground environmental settings), host plant genotype (Simple Sequence Repeat (SSR) markers), season (Spring vs. Fall) and geographic setting (at scales from regional watersheds to local riparian zones) on microbial community structure. Each of the trees sampled displayed unique aspects to its associated community structure with high numbers of Operational Taxonomic Units (OTUs) specific to an individual trees (bacteria >90%, fungi >60%). Over the diverse conditions surveyed only a small number of OTUs were common to all samples within rhizosphere (35 bacterial and 4 fungal) and endosphere (1 bacterial and 1 fungal) microbiomes. As expected, Proteobacteria and Ascomycota were dominant in root communities (>50%) while other higher-level phylogenetic groups (Chytridiomycota, Acidobacteria) displayed greatly reduced abundance in endosphere compared to the rhizosphere. Variance partitioning partially explained differences in microbiome composition between all sampled roots on the basis of seasonal and soil properties (4% to 23%). While most variation remains unattributed, we observed significant differences in the microbiota between watersheds (Tennessee vs. North Carolina) and seasons (Spring vs. Fall). SSR markers clearly delineated two host populations associated with the samples taken in TN vs. NC, but overall host genotypic distances did not have a significant effect on corresponding communities that could be separated from other measured effects.

Next-generation sequencing has dramatically changed the landscape of microbial ecology, large-scale and in-depth diversity studies being now widely accessible. However, determining the accuracy of taxonomic and quantitative inferences and comparing results obtained with different approaches are complicated by incongruence of experimental and computational data types and also by lack of knowledge of the true ecological diversity. Here we used highly diverse bacterial and archaeal synthetic communities assembled from pure genomic DNAs to compare inferences from metagenomic and SSU rRNA amplicon sequencing. Both Illumina and 454 metagenomic data outperformed amplicon sequencing in quantifying the community composition, but the outcome was dependent on analysis parameters and platform. New approaches in processing and classifying amplicons can reconstruct the taxonomic composition of the community with high reproducibility within primer sets, but all tested primers sets lead to significant taxon-specific biases. Controlled synthetic communities assembled to broadly mimic the phylogenetic richness in target environments can provide important validation for fine-tuning experimental and computational parameters used to characterize natural communities., (© 2013 Society for Applied Microbiology and Blackwell Publishing Ltd.)

The mammalian gut harbors complex and variable microbial communities, across both host phylogenetic space and conspecific individuals. A synergy of host genetic and environmental factors shape these communities and account for their variability, but their individual contributions and the selective pressures involved are still not well understood. We employed barcoded pyrosequencing of V1-2 and V4 regions of bacterial small subunit ribosomal RNA genes to characterize the effects of host genetics and environment on cecum assemblages in 10 genetically distinct, inbred mouse strains. Eight of these strains are the foundation of the Collaborative Cross (CC), a panel of mice derived from a genetically diverse set of inbred founder strains, designed specifically for complex trait analysis. Diversity of gut microbiota was characterized by complementing phylogenetic and distance-based, sequence-clustering approaches. Significant correlations were found between the mouse strains and their gut microbiota, reflected by distinct bacterial communities. Cohabitation and litter had a reduced, although detectable effect, and the microbiota response to these factors varied by strain. We identified bacterial phylotypes that appear to be discriminative and strain-specific to each mouse line used. Cohabitation of different strains of mice revealed an interaction of host genetic and environmental factors in shaping gut bacterial consortia, in which bacterial communities became more similar but retained strain specificity. This study provides a baseline analysis of intestinal bacterial communities in the eight CC progenitor strains and will be linked to integrated host genotype, phenotype and microbiota research on the resulting CC panel.

Pelosinus fermentans 16S rRNA gene sequences have been reported from diverse geographical sites since the recent isolation of the type strain. We present the genome sequence of the P. fermentans type strain R7 (DSM 17108) and genome sequences for two new strains with different abilities to reduce iron, chromate, and uranium.

Background: Clostridium thermocellum is a candidate consolidated bioprocessing biocatalyst, which is a microorganism that expresses enzymes for both cellulose hydrolysis and its fermentation to produce fuels such as lignocellulosic ethanol. However, C. thermocellum is relatively sensitive to ethanol compared to ethanologenic microorganisms such as yeast and Zymomonas mobilis that are used in industrial fermentations but do not possess native enzymes for industrial cellulose hydrolysis., Results: In this study, C. thermocellum was grown to mid-exponential phase and then treated with ethanol to a final concentration of 3.9 g/L to investigate its physiological and regulatory responses to ethanol stress. Samples were taken pre-shock and 2, 12, 30, 60, 120, and 240 min post-shock, and from untreated control fermentations for systems biology analyses. Cell growth was arrested by ethanol supplementation with intracellular accumulation of carbon sources such as cellobiose, and sugar phosphates, including fructose-6-phosphate and glucose-6-phosphate. The largest response of C. thermocellum to ethanol shock treatment was in genes and proteins related to nitrogen uptake and metabolism, which is likely important for redirecting the cells physiology to overcome inhibition and allow growth to resume., Conclusion: This study suggests possible avenues for metabolic engineering and provides comprehensive, integrated systems biology datasets that will be useful for future metabolic modeling and strain development endeavors.

Periodontitis has a polymicrobial etiology within the framework of a complex microbial ecosystem. With advances in sequencing technologies, comprehensive studies to elucidate bacterial community differences have recently become possible. We used 454 sequencing of 16S rRNA genes to compare subgingival bacterial communities from 29 periodontally healthy controls and 29 subjects with chronic periodontitis. Amplicons from both the V1-2 and V4 regions of the 16S gene were sequenced, yielding 1,393,579 sequences. They were identified by BLAST against a curated oral 16S database, and mapped to 16 phyla, 106 genera, and 596 species. 81% of sequences could be mapped to cultivated species. Differences between health- and periodontitis-associated bacterial communities were observed at all phylogenetic levels, and UniFrac and principal coordinates analysis showed distinct community profiles in health and disease. Community diversity was higher in disease, and 123 species were identified that were significantly more abundant in disease, and 53 in health. Spirochaetes, Synergistetes and Bacteroidetes were more abundant in disease, whereas the Proteobacteria were found at higher levels in healthy controls. Within the phylum Firmicutes, the class Bacilli was health-associated, whereas the Clostridia, Negativicutes and Erysipelotrichia were associated with disease. These results implicate a number of taxa that will be targets for future research. Some, such as Filifactor alocis and many Spirochetes were represented by a large fraction of sequences as compared with previously identified targets. Elucidation of these differences in community composition provides a basis for further understanding the pathogenesis of periodontitis.

To evaluate the effects of local fluid geochemistry on microbial communities associated with active hydrothermal vent deposits, we examined the archaeal and bacterial communities of 12 samples collected from two very different vent fields: the basalt-hosted Lucky Strike (37°17'N, 32°16.3'W, depth 1600-1750 m) and the ultramafic-hosted Rainbow (36°13'N, 33°54.1'W, depth 2270-2330 m) vent fields along the Mid-Atlantic Ridge (MAR). Using multiplexed barcoded pyrosequencing of the variable region 4 (V4) of the 16S rRNA genes, we show statistically significant differences between the archaeal and bacterial communities associated with the different vent fields. Quantitative polymerase chain reaction (qPCR) assays of the functional gene diagnostic for methanogenesis (mcrA), as well as geochemical modelling to predict pore fluid chemistries within the deposits, support the pyrosequencing observations. Collectively, these results show that the less reduced, hydrogen-poor fluids at Lucky Strike limit colonization by strict anaerobes such as methanogens, and allow for hyperthermophilic microaerophiles, like Aeropyrum. In contrast, the hydrogen-rich reducing vent fluids at the ultramafic-influenced Rainbow vent field support the prevalence of methanogens and other hydrogen-oxidizing thermophiles at this site. These results demonstrate that biogeographical patterns of hydrothermal vent microorganisms are shaped in part by large scale geological and geochemical processes., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

High concentrations of uranium, inorganic mercury [Hg(II)], and methylmercury (MeHg) have been detected in streams located in the Department of Energy reservation in Oak Ridge, TN. To determine the potential effects of the surface water contamination on the microbial community composition, surface stream sediments were collected 7 times during the year, from 5 contaminated locations and 1 control stream. Fifty-nine samples were analyzed for bacterial community composition and geochemistry. Community characterization was based on GS 454 FLX pyrosequencing with 235 Mb of 16S rRNA gene sequence targeting the V4 region. Sorting and filtering of the raw reads resulted in 588,699 high-quality sequences with lengths of >200 bp. The bacterial community consisted of 23 phyla, including Proteobacteria (ranging from 22.9 to 58.5% per sample), Cyanobacteria (0.2 to 32.0%), Acidobacteria (1.6 to 30.6%), Verrucomicrobia (3.4 to 31.0%), and unclassified bacteria. Redundancy analysis indicated no significant differences in the bacterial community structure between midchannel and near-bank samples. Significant correlations were found between the bacterial community and seasonal as well as geochemical factors. Furthermore, several community members within the Proteobacteria group that includes sulfate-reducing bacteria and within the Verrucomicrobia group appeared to be associated positively with Hg and MeHg. This study is the first to indicate an influence of MeHg on the in situ microbial community and suggests possible roles of these bacteria in the Hg/MeHg cycle.

In the genome of Shewanella oneidensis, genes encoding the global regulators ArcA, Crp, and EtrA have been identified. All these proteins deviate from their counterparts in E. coli significantly in terms of functionality and regulon. It is worth investigating the involvement and relationship of these global regulators in aerobic and anaerobic respiration in S. oneidensis. In this study, the impact of the transcriptional factors ArcA, Crp, and EtrA on aerobic and anaerobic respiration in S. oneidensis were assessed. While all these proteins appeared to be functional in vivo, the importance of individual proteins in these two major biological processes differed. The ArcA transcriptional factor was critical in aerobic respiration while the Crp protein was indispensible in anaerobic respiration. Using a newly developed reporter system, it was found that expression of arcA and etrA was not influenced by growth conditions but transcription of crp was induced by removal of oxygen. An analysis of the impact of each protein on transcription of the others revealed that Crp expression was independent of the other factors whereas ArcA repressed both etrA and its own transcription while EtrA also repressed arcA transcription. Transcriptional levels of arcA in the wild type, crp, and etrA strains under either aerobic or anaerobic conditions were further validated by quantitative immunoblotting with a polyclonal antibody against ArcA. This extensive survey demonstrated that all these three global regulators are functional in S. oneidensis. In addition, the reporter system constructed in this study will facilitate in vivo transcriptional analysis of targeted promoters.

Sulfate-reducing bacteria have been extensively studied for their potential in heavy-metal bioremediation. However, the occurrence of elevated nitrate in contaminated environments has been shown to inhibit sulfate reduction activity. Although the inhibition has been suggested to result from the competition with nitrate-reducing bacteria, the possibility of direct inhibition of sulfate reducers by elevated nitrate needs to be explored. Using Desulfovibrio vulgaris as a model sulfate-reducing bacterium, functional genomics analysis reveals that osmotic stress contributed to growth inhibition by nitrate as shown by the upregulation of the glycine/betaine transporter genes and the relief of nitrate inhibition by osmoprotectants. The observation that significant growth inhibition was effected by 70 mM NaNO(3) but not by 70 mM NaCl suggests the presence of inhibitory mechanisms in addition to osmotic stress. The differential expression of genes characteristic of nitrite stress responses, such as the hybrid cluster protein gene, under nitrate stress condition further indicates that nitrate stress response by D. vulgaris was linked to components of both osmotic and nitrite stress responses. The involvement of the oxidative stress response pathway, however, might be the result of a more general stress response. Given the low similarities between the response profiles to nitrate and other stresses, less-defined stress response pathways could also be important in nitrate stress, which might involve the shift in energy metabolism. The involvement of nitrite stress response upon exposure to nitrate may provide detoxification mechanisms for nitrite, which is inhibitory to sulfate-reducing bacteria, produced by microbial nitrate reduction as a metabolic intermediate and may enhance the survival of sulfate-reducing bacteria in environments with elevated nitrate level.

Archaeal communities from mercury and uranium-contaminated freshwater stream sediments were characterized and compared to archaeal communities present in an uncontaminated stream located in the vicinity of Oak Ridge, TN, USA. The distribution of the Archaea was determined by pyrosequencing analysis of the V4 region of 16S rRNA amplified from 12 streambed surface sediments. Crenarchaeota comprised 76% of the 1,670 archaeal sequences and the remaining 24% were from Euryarchaeota. Phylogenetic analysis further classified the Crenarchaeota as a Freshwater Group, Miscellaneous Crenarchaeota group, Group I3, Rice Cluster VI and IV, Marine Group I and Marine Benthic Group B; and the Euryarchaeota into Methanomicrobiales, Methanosarcinales, Methanobacteriales, Rice Cluster III, Marine Benthic Group D, Deep Sea Hydrothermal Vent Euryarchaeota 1 and Eury 5. All groups were previously described. Both hydrogen- and acetate-dependent methanogens were found in all samples. Most of the groups (with 60% of the sequences) described in this study were not similar to any cultivated isolates, making it difficult to discern their function in the freshwater microbial community. A significant decrease in the number of sequences, as well as in the diversity of archaeal communities was found in the contaminated sites. The Marine Group I, including the ammonia oxidizer Nitrosopumilus maritimus, was the dominant group in both mercury and uranium/nitrate-contaminated sites. The uranium-contaminated site also contained a high concentration of nitrate, thus Marine Group I may play a role in nitrogen cycle.

Background: Communities of microorganisms control the rates of key biogeochemical cycles, and are important for biotechnology, bioremediation, and industrial microbiological processes. For this reason, we constructed a model microbial community comprised of three species dependent on trophic interactions. The three species microbial community was comprised of Clostridium cellulolyticum, Desulfovibrio vulgaris Hildenborough, and Geobacter sulfurreducens and was grown under continuous culture conditions. Cellobiose served as the carbon and energy source for C. cellulolyticum, whereas D. vulgaris and G. sulfurreducens derived carbon and energy from the metabolic products of cellobiose fermentation and were provided with sulfate and fumarate respectively as electron acceptors., Results: qPCR monitoring of the culture revealed C. cellulolyticum to be dominant as expected and confirmed the presence of D. vulgaris and G. sulfurreducens. Proposed metabolic modeling of carbon and electron flow of the three-species community indicated that the growth of C. cellulolyticum and D. vulgaris were electron donor limited whereas G. sulfurreducens was electron acceptor limited., Conclusions: The results demonstrate that C. cellulolyticum, D. vulgaris, and G. sulfurreducens can be grown in coculture in a continuous culture system in which D. vulgaris and G. sulfurreducens are dependent upon the metabolic byproducts of C. cellulolyticum for nutrients. This represents a step towards developing a tractable model ecosystem comprised of members representing the functional groups of a trophic network.

Interspecies hydrogen transfer between organisms producing and consuming hydrogen promotes the decomposition of organic matter in most anoxic environments. Although syntrophic coupling between hydrogen producers and consumers is a major feature of the carbon cycle, mechanisms for energy recovery at the extremely low free energies of reactions typical of these anaerobic communities have not been established. In this study, comparative transcriptional analysis of a model sulfate-reducing microbe, Desulfovibrio vulgaris Hildenborough, suggested the use of alternative electron transfer systems dependent on growth modality. During syntrophic growth on lactate with a hydrogenotrophic methanogen, numerous genes involved in electron transfer and energy generation were upregulated in D. vulgaris compared with their expression in sulfate-limited monocultures. In particular, genes coding for the putative membrane-bound Coo hydrogenase, two periplasmic hydrogenases (Hyd and Hyn), and the well-characterized high-molecular-weight cytochrome (Hmc) were among the most highly expressed and upregulated genes. Additionally, a predicted operon containing genes involved in lactate transport and oxidation exhibited upregulation, further suggesting an alternative pathway for electrons derived from lactate oxidation during syntrophic growth. Mutations in a subset of genes coding for Coo, Hmc, Hyd, and Hyn impaired or severely limited syntrophic growth but had little effect on growth via sulfate respiration. These results demonstrate that syntrophic growth and sulfate respiration use largely independent energy generation pathways and imply that to understand microbial processes that sustain nutrient cycling, lifestyles not captured in pure culture must be considered.

In the genome of Shewanella oneidensis, a napDAGHB gene cluster encoding periplasmic nitrate reductase (NapA) and accessory proteins and an nrfA gene encoding periplasmic nitrite reductase (NrfA) have been identified. These two systems seem to be atypical because the genome lacks genes encoding cytoplasmic membrane electron transport proteins, NapC for NAP and NrfBCD/NrfH for NRF, respectively. Here, we present evidence that reduction of nitrate to ammonium in S. oneidensis is carried out by these atypical systems in a two-step manner. Transcriptional and mutational analyses suggest that CymA, a cytoplasmic membrane electron transport protein, is likely to be the functional replacement of both NapC and NrfH in S. oneidensis. Surprisingly, a strain devoid of napB encoding the small subunit of nitrate reductase exhibited the maximum cell density sooner than the wild type. Further characterization of this strain showed that nitrite was not detected as a free intermediate in its culture and NapB provides a fitness gain for S. oneidensis to compete for nitrate in the environments. On the basis results from mutational analyses of napA, napB, nrfA and napBnrfA in-frame deletion mutants, we propose that NapB is able to favor nitrate reduction by routing electrons to NapA exclusively.

Background: The Arc two-component system is a global regulator controlling many genes involved in aerobic/anaerobic respiration and fermentative metabolism in Escherichia coli. Shewanella oneidensis MR-1 contains a gene encoding a putative ArcA homolog with ~81% amino acid sequence identity to the E. coli ArcA protein but not a full-length arcB gene., Results: To understand the role of ArcA in S. oneidensis, an arcA deletion strain was constructed and subjected to both physiological characterization and microarray analysis. Compared to the wild-type MR-1, the mutant exhibited impaired aerobic growth and a defect in utilizing DMSO in the absence of O2. Microarray analyses on cells grown aerobically and anaerobically on fumarate revealed that expression of 1009 genes was significantly affected (p < 0.05) by the mutation. In contrast to E. coli ArcA, the protein appears to be dispensable in regulation of the TCA cycle in S. oneidensis. To further determine genes regulated by the Arc system, an ArcA recognition weight matrix from DNA-binding data and bioinformatics analysis was generated and used to produce an ArcA sequence affinity map. By combining both techniques, we identified an ArcA regulon of at least 50 operons, of which only 6 were found to be directly controlled by ArcA in E. coli., Conclusion: These results indicate that the Arc system in S. oneidensis differs from that in E. coli substantially in terms of its physiological function and regulon while their binding motif are strikingly similar.

We investigated the efficiency of RNA interference (RNAi) in Arabidopsis using transitive and homologous inverted repeat (hIR) vectors. hIR constructs carry self-complementary intron-spliced fragments of the target gene whereas transitive vectors have the target sequence fragment adjacent to an intron-spliced, inverted repeat of heterologous origin. Both transitive and hIR constructs facilitated specific and heritable silencing in the three genes studied (AP1, ETTIN and TTG1). Both types of vectors produced a phenotypic series that phenocopied reduction of function mutants for the respective target gene. The hIR yielded up to fourfold higher proportions of events with strongly manifested reduction of function phenotypes compared to transitive RNAi. We further investigated the efficiency and potential off-target effects of AP1 silencing by both types of vectors using genome-scale microarrays and quantitative RT-PCR. The depletion of AP1 transcripts coincided with reduction of function phenotypic changes among both hIR and transitive lines and also showed similar expression patterns among differentially regulated genes. We did not detect significant silencing directed against homologous potential off-target genes when constructs were designed with minimal sequence similarity. Both hIR and transitive methods are useful tools in plant biotechnology and genomics. The choice of vector will depend on specific objectives such as cloning throughput, number of events and degree of suppression required.

A new approach, termed whole-community RNA amplification (WCRA), was developed to provide sufficient amounts of mRNAs from environmental samples for microarray analysis. This method employs fusion primers (six to nine random nucleotides with an attached T7 promoter) for the first-strand synthesis. The shortest primer (T7N6S) gave the best results in terms of the yield and representativeness of amplification. About 1,200- to 1,800-fold amplification was obtained with amounts of the RNA templates ranging from 10 to 100 ng, and very representative detection was obtained with 50 to 100 ng total RNA. Evaluation with a Shewanella oneidensis Deltafur strain revealed that the amplification method which we developed could preserve the original abundance relationships of mRNAs. In addition, to determine whether representative detection of RNAs can be achieved with mixed community samples, amplification biases were evaluated with a mixture containing equal quantities of RNAs (100 ng each) from four bacterial species, and representative amplification was also obtained. Finally, the method which we developed was applied to the active microbial populations in a denitrifying fluidized bed reactor used for denitrification of contaminated groundwater and ethanol-stimulated groundwater samples for uranium reduction. The genes expressed were consistent with the expected functions of the bioreactor and groundwater system, suggesting that this approach is useful for analyzing the functional activities of microbial communities. This is one of the first demonstrations that microarray-based technology can be used to successfully detect the activities of microbial communities from real environmental samples in a high-throughput fashion.

This study presents a global transcriptional analysis of the cold shock response of Shewanella oneidensis MR-1 after a temperature downshift from 30 degrees C to 8 or 15 degrees C based on time series microarray experiments. More than 700 genes were found to be significantly affected (P < or = 0.05) upon cold shock challenge, especially at 8 degrees C. The temporal gene expression patterns of the classical cold shock genes varied, and only some of them, most notably so1648 and so2787, were differentially regulated in response to a temperature downshift. The global response of S. oneidensis to cold shock was also characterized by the up-regulation of genes encoding membrane proteins, DNA metabolism and translation apparatus components, metabolic proteins, regulatory proteins, and hypothetical proteins. Most of the metabolic proteins affected are involved in catalytic processes that generate NADH or NADPH. Mutational analyses confirmed that the small cold shock proteins, So1648 and So2787, are involved in the cold shock response of S. oneidensis. The analyses also indicated that So1648 may function only at very low temperatures.

A marine psychrotolerant, dissimilatory Fe(III)-reducing bacterium, Shewanella sp. strain PV-4, from the microbial mat at a hydrothermal vent of Loihi Seamount in the Pacific Ocean has been further characterized, with emphases on metal reduction and iron biomineralization. The strain is able to reduce metals such as Fe(III), Co(III), Cr(VI), Mn(IV), and U(VI) as electron acceptors while using lactate, formate, pyruvate, or hydrogen as an electron donor. Growth during iron reduction occurred over the pH range of 7.0 to 8.9, a sodium chloride range of 0.05 to 5%, and a temperature range of 0 to 37 degrees C, with an optimum growth temperature of 18 degrees C. Unlike mesophilic dissimilatory Fe(III)-reducing bacteria, which produce mostly superparamagnetic magnetite (<35 nm), this psychrotolerant bacterium produces well-formed single-domain magnetite (>35 nm) at temperatures from 18 to 37 degrees C. The genome size of this strain is about 4.5 Mb. Strain PV-4 is sensitive to a variety of commonly used antibiotics except ampicillin and can acquire exogenous DNA (plasmid pCM157) through conjugation.