Induction of intestinal Th17 cells by segmented filamentous bacteria.

The gastrointestinal tract of mammals is inhabited by hundreds of distinct species of commensal microorganisms that exist in a mutualistic relationship with the host. How commensal microbiota influence the host immune system is poorly understood. We show here that colonization of the small intestine of mice with a single commensal microbe, segmented filamentous bacterium (SFB), is sufficient to induce the appearance of CD4(+) T helper cells that produce IL-17 and IL-22 (Th17 cells) in the lamina propria. SFB adhere tightly to the surface of epithelial cells in the terminal ileum of mice with Th17 cells but are absent from mice that have few Th17 cells. Colonization with SFB was correlated with increased expression of genes associated with inflammation and antimicrobial defenses and resulted in enhanced resistance to the intestinal pathogen Citrobacter rodentium. Thus, manipulation of this commensal-regulated pathway may provide new opportunities for enhancing mucosal immunity and treating autoimmune disease.

To rarefy or not to rarefy: robustness and efficiency trade-offs of rarefying microbiome data.

MOTIVATION: Microbiome datasets provide rich information about microbial communities. However, vast library size variations across samples present great challenges for proper statistical comparisons. To deal with these challenges, rarefaction is often used in practice as a normalization technique, although there has been debate whether rarefaction should ever be used. Conventional wisdom and previous work suggested that rarefaction should never be used in practice, arguing that rarefying microbiome data is statistically inadmissible. These discussions, however, have been confined to particular parametric models and simulation studies. RESULTS: We develop a semiparametric graphical model framework for grouped microbiome data and analyze in the context of differential abundance testing the statistical trade-offs of the rarefaction procedure, accounting for latent variations and measurement errors. Under the framework, it can be shown rarefaction guarantees that subsequent permutation tests properly control the Type I error. In addition, the loss in sensitivity from rarefaction is solely due to increased measurement error; if the underlying variation in microbial composition is large among samples, rarefaction might not hurt subsequent statistical inference much. We develop the rarefaction efficiency index (REI) as an indicator for efficiency loss and illustrate it with a dataset on the effect of storage conditions for microbiome data. Simulation studies based on real data demonstrate that the impact of rarefaction on sensitivity is negligible when overdispersion is prominent, while low REI corresponds to scenarios in which rarefying might substantially lower the statistical power. Whether to rarefy or not ultimately depends on assumptions of the data generating process and characteristics of the data. AVAILABILITY AND IMPLEMENTATION: Source codes are publicly available at https://github.com/jcyhong/rarefaction. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Progressive dysbiosis of human orodigestive microbiota along the sequence of gastroesophageal reflux, Barrett's esophagus and esophageal adenocarcinoma.

The incidence of esophageal adenocarcinoma (EA) has drastically increased in the United States since 1970s for unclear reasons. We hypothesized that the widespread usage of antibiotics has increased the procarcinogenic potential of the orodigestive microbiota along the sequence of gastroesophageal reflux (GR), Barrett's esophagus (BE) and EA phenotypes. This case control study included normal controls (NC) and three disease phenotypes GR, BE and EA. Microbiota in the mouth, esophagus, and stomach, and rectum were analyzed using 16S rRNA gene sequencing. Overall, we discovered 44 significant pairwise differences in abundance of microbial taxa between the four phenotypes, with 12 differences in the mouth, 21 in the esophagus, two in the stomach, and nine in the rectum. Along the GR→BE→EA sequence, oral and esophageal microbiota were more diversified, the dominant genus Streptococcus was progressively depleted while six other genera Atopobium, Actinomyces, Veillonella, Ralstonia, Burkholderia and Lautropia progressively enriched. In NC, Streptococcus appeared to control populations of other genera in the foregut via numerous negative and positive connections, while in disease states, the rich network was markedly simplified. Inferred gene functional content showed a progressive enrichment through the stages of EA development in genes encoding antibiotic resistance, ligands of Toll-like and NOD-like receptors, nitrate-nitrite-nitric oxide pathway and acetaldehyde metabolism. The orodigestive microbiota is in a progressive dysbiotic state along the GR-BE-EA sequence. The increasing dysbiosis and antibiotic and procarcinogenic genes in the disease states warrants further study to define their roles in EA pathogenesis.

Feeding-Related Gut Microbial Composition Associates With Peripheral T-Cell Activation and Mucosal Gene Expression in African Infants.

Background: Exclusive breastfeeding reduces the rate of postnatal human immunodeficiency virus (HIV) transmission compared to nonexclusive breastfeeding; however, the mechanisms of this protection are unknown. Our study aimed to interrogate the mechanisms underlying the protective effect of exclusive breastfeeding. Methods: We performed a prospective, longitudinal study of infants from a high-HIV-prevalence, low-income setting in South Africa. We evaluated the role of any non-breast milk feeds, excluding prescribed medicines on stool microbial communities via 16S rRNA gene sequencing, peripheral T-cell activation via flow cytometry, and buccal mucosal gene expression via quantitative polymerase chain reaction assay. Results: A total of 155 infants were recruited at birth with mean gestational age of 38.9 weeks and mean birth weight of 3.2 kg. All infants were exclusively breastfed (EBF) at birth, but only 43.5% and 20% remained EBF at 6 or 14 weeks of age, respectively. We observed lower stool microbial diversity and distinct microbial composition in exclusively breastfed infants. These microbial communities, and the relative abundance of key taxa, were correlated with peripheral CD4+ T-cell activation, which was lower in EBF infants. In the oral mucosa, gene expression of chemokine and chemokine receptors involved in recruitment of HIV target cells to tissues, as well as epithelial cytoskeletal proteins, was lower in EBF infants. Conclusions: These data suggest that nonexclusive breastfeeding alters the gut microbiota, increasing T-cell activation and, potentially, mucosal recruitment of HIV target cells. Study findings highlight a biologically plausible mechanistic explanation for the reduced postnatal HIV transmission observed in EBF infants.

Human papillomavirus community in healthy persons, defined by metagenomics analysis of human microbiome project shotgun sequencing data sets.

UNLABELLED: Human papillomavirus (HPV) causes a number of neoplastic diseases in humans. Here, we show a complex normal HPV community in a cohort of 103 healthy human subjects, by metagenomics analysis of the shotgun sequencing data generated from the NIH Human Microbiome Project. The overall HPV prevalence was 68.9% and was highest in the skin (61.3%), followed by the vagina (41.5%), mouth (30%), and gut (17.3%). Of the 109 HPV types as well as additional unclassified types detected, most were undetectable by the widely used commercial kits targeting the vaginal/cervical HPV types. These HPVs likely represent true HPV infections rather than transitory exposure because of strong organ tropism and persistence of the same HPV types in repeat samples. Coexistence of multiple HPV types was found in 48.1% of the HPV-positive samples. Networking between HPV types, cooccurrence or exclusion, was detected in vaginal and skin samples. Large contigs assembled from short HPV reads were obtained from several samples, confirming their genuine HPV origin. This first large-scale survey of HPV using a shotgun sequencing approach yielded a comprehensive map of HPV infections among different body sites of healthy human subjects. IMPORTANCE: This nonbiased survey indicates that the HPV community in healthy humans is much more complex than previously defined by widely used kits that are target selective for only a few high- and low-risk HPV types for cervical cancer. The importance of nononcogenic viruses in a mixed HPV infection could be for stimulating or inhibiting a coexisting oncogenic virus via viral interference or immune cross-reaction. Knowledge gained from this study will be helpful to guide the designing of epidemiological and clinical studies in the future to determine the impact of nononcogenic HPV types on the outcome of HPV infections.

Divergent aquifer biogeochemical systems converge on similar and unexpected Cr(VI) reduction products.

In this study of reductive chromium immobilization, we found that flow-through columns constructed with homogenized aquifer sediment and continuously infused with lactate, chromate, and various native electron acceptors diverged to have very different Cr(VI)-reducing biogeochemical regimes characterized by either denitrifying or fermentative conditions (as indicated by effluent chemical data, 16S rRNA pyrotag data, and metatranscriptome data). Despite the two dramatically different biogeochemical environments that evolved in the columns, these regimes created similar Cr(III)-Fe(III) hydroxide precipitates as the predominant Cr(VI) reduction product, as characterized by micro-X-ray fluorescence and micro-X-ray absorption near-edge structure analysis. We discuss two conflicting scenarios of microbially mediated formation of Cr(III)-Fe(III) precipitates, each of which is both supported and contradicted by different lines of evidence: (1) enzymatic reduction of Cr(VI) to Cr(III) followed by coprecipitation of Cr(III) and Fe(III) and (2) both regimes generated at least small amounts of Fe(II), which abiotically reduced Cr(VI) to form a Cr-Fe precipitate. Evidence of zones with different levels of Cr(VI) reduction suggest that local heterogeneity may have confounded interpretation of processes based on bulk measurements. This study indicates that the bulk redox status and biogeochemical regime, as categorized by the dominant electron-accepting process, do not necessarily control the final product of Cr(VI) reduction.

Predictions of rhizosphere microbiome dynamics with a genome-informed and trait-based energy budget model.

Soil microbiomes are highly diverse, and to improve their representation in biogeochemical models, microbial genome data can be leveraged to infer key functional traits. By integrating genome-inferred traits into a theory-based hierarchical framework, emergent behaviour arising from interactions of individual traits can be predicted. Here we combine theory-driven predictions of substrate uptake kinetics with a genome-informed trait-based dynamic energy budget model to predict emergent life-history traits and trade-offs in soil bacteria. When applied to a plant microbiome system, the model accurately predicted distinct substrate-acquisition strategies that aligned with observations, uncovering resource-dependent trade-offs between microbial growth rate and efficiency. For instance, inherently slower-growing microorganisms, favoured by organic acid exudation at later plant growth stages, exhibited enhanced carbon use efficiency (yield) without sacrificing growth rate (power). This insight has implications for retaining plant root-derived carbon in soils and highlights the power of data-driven, trait-based approaches for improving microbial representation in biogeochemical models.

Genomic and physiological characterization of the chromate-reducing, aquifer-derived Firmicute Pelosinus sp. strain HCF1.

Pelosinus spp. are fermentative firmicutes that were recently reported to be prominent members of microbial communities at contaminated subsurface sites in multiple locations. Here we report metabolic characteristics and their putative genetic basis in Pelosinus sp. strain HCF1, an isolate that predominated anaerobic, Cr(VI)-reducing columns constructed with aquifer sediment. Strain HCF1 ferments lactate to propionate and acetate (the methylmalonyl-coenzyme A [CoA] pathway was identified in the genome), and its genome encodes two [NiFe]- and four [FeFe]-hydrogenases for H(2) cycling. The reduction of Cr(VI) and Fe(III) may be catalyzed by a flavoprotein with 42 to 51% sequence identity to both ChrR and FerB. This bacterium has unexpected capabilities and gene content associated with reduction of nitrogen oxides, including dissimilatory reduction of nitrate to ammonium (two copies of NrfH and NrfA were identified along with NarGHI) and a nitric oxide reductase (NorCB). In this strain, either H(2) or lactate can act as a sole electron donor for nitrate, Cr(VI), and Fe(III) reduction. Transcriptional studies demonstrated differential expression of hydrogenases and nitrate and nitrite reductases. Overall, the unexpected metabolic capabilities and gene content reported here broaden our perspective on what biogeochemical and ecological roles this species might play as a prominent member of microbial communities in subsurface environments.

Virus diversity and activity is driven by snowmelt and host dynamics in a high-altitude watershed soil ecosystem.

BACKGROUND: Viruses impact nearly all organisms on Earth, including microbial communities and their associated biogeochemical processes. In soils, highly diverse viral communities have been identified, with a global distribution seemingly driven by multiple biotic and abiotic factors, especially soil temperature and moisture. However, our current understanding of the stability of soil viral communities across time and their response to strong seasonal changes in environmental parameters remains limited. Here, we investigated the diversity and activity of environmental soil DNA and RNA viruses, focusing especially on bacteriophages, across dynamics' seasonal changes in a snow-dominated mountainous watershed by examining paired metagenomes and metatranscriptomes. RESULTS: We identified a large number of DNA and RNA viruses taxonomically divergent from existing environmental viruses, including a significant proportion of fungal RNA viruses, and a large and unsuspected diversity of positive single-stranded RNA phages (Leviviricetes), highlighting the under-characterization of the global soil virosphere. Among these, we were able to distinguish subsets of active DNA and RNA phages that changed across seasons, consistent with a "seed-bank" viral community structure in which new phage activity, for example, replication and host lysis, is sequentially triggered by changes in environmental conditions. At the population level, we further identified virus-host dynamics matching two existing ecological models: "Kill-The-Winner" which proposes that lytic phages are actively infecting abundant bacteria, and "Piggyback-The-Persistent" which argues that when the host is growing slowly, it is more beneficial to remain in a dormant state. The former was associated with summer months of high and rapid microbial activity, and the latter with winter months of limited and slow host growth. CONCLUSION: Taken together, these results suggest that the high diversity of viruses in soils is likely associated with a broad range of host interaction types each adapted to specific host ecological strategies and environmental conditions. As our understanding of how environmental and host factors drive viral activity in soil ecosystems progresses, integrating these viral impacts in complex natural microbiome models will be key to accurately predict ecosystem biogeochemistry. Video Abstract.

microTrait: A Toolset for a Trait-Based Representation of Microbial Genomes.

Remote sensing approaches have revolutionized the study of macroorganisms, allowing theories of population and community ecology to be tested across increasingly larger scales without much compromise in resolution of biological complexity. In microbial ecology, our remote window into the ecology of microorganisms is through the lens of genome sequencing. For microbial organisms, recent evidence from genomes recovered from metagenomic samples corroborate a highly complex view of their metabolic diversity and other associated traits which map into high physiological complexity. Regardless, during the first decades of this omics era, microbial ecological research has primarily focused on taxa and functional genes as ecological units, favoring breadth of coverage over resolution of biological complexity manifested as physiological diversity. Recently, the rate at which provisional draft genomes are generated has increased substantially, giving new insights into ecological processes and interactions. From a genotype perspective, the wide availability of genome-centric data requires new data synthesis approaches that place organismal genomes center stage in the study of environmental roles and functional performance. Extraction of ecologically relevant traits from microbial genomes will be essential to the future of microbial ecological research. Here, we present microTrait, a computational pipeline that infers and distills ecologically relevant traits from microbial genome sequences. microTrait maps a genome sequence into a trait space, including discrete and continuous traits, as well as simple and composite. Traits are inferred from genes and pathways representing energetic, resource acquisition, and stress tolerance mechanisms, while genome-wide signatures are used to infer composite, or life history, traits of microorganisms. This approach is extensible to any microbial habitat, although we provide initial examples of this approach with reference to soil microbiomes.

METABOLIC: high-throughput profiling of microbial genomes for functional traits, metabolism, biogeochemistry, and community-scale functional networks.

BACKGROUND: Advances in microbiome science are being driven in large part due to our ability to study and infer microbial ecology from genomes reconstructed from mixed microbial communities using metagenomics and single-cell genomics. Such omics-based techniques allow us to read genomic blueprints of microorganisms, decipher their functional capacities and activities, and reconstruct their roles in biogeochemical processes. Currently available tools for analyses of genomic data can annotate and depict metabolic functions to some extent; however, no standardized approaches are currently available for the comprehensive characterization of metabolic predictions, metabolite exchanges, microbial interactions, and microbial contributions to biogeochemical cycling. RESULTS: We present METABOLIC (METabolic And BiogeOchemistry anaLyses In miCrobes), a scalable software to advance microbial ecology and biogeochemistry studies using genomes at the resolution of individual organisms and/or microbial communities. The genome-scale workflow includes annotation of microbial genomes, motif validation of biochemically validated conserved protein residues, metabolic pathway analyses, and calculation of contributions to individual biogeochemical transformations and cycles. The community-scale workflow supplements genome-scale analyses with determination of genome abundance in the microbiome, potential microbial metabolic handoffs and metabolite exchange, reconstruction of functional networks, and determination of microbial contributions to biogeochemical cycles. METABOLIC can take input genomes from isolates, metagenome-assembled genomes, or single-cell genomes. Results are presented in the form of tables for metabolism and a variety of visualizations including biogeochemical cycling potential, representation of sequential metabolic transformations, community-scale microbial functional networks using a newly defined metric "MW-score" (metabolic weight score), and metabolic Sankey diagrams. METABOLIC takes ~ 3 h with 40 CPU threads to process ~ 100 genomes and corresponding metagenomic reads within which the most compute-demanding part of hmmsearch takes ~ 45 min, while it takes ~ 5 h to complete hmmsearch for ~ 3600 genomes. Tests of accuracy, robustness, and consistency suggest METABOLIC provides better performance compared to other software and online servers. To highlight the utility and versatility of METABOLIC, we demonstrate its capabilities on diverse metagenomic datasets from the marine subsurface, terrestrial subsurface, meadow soil, deep sea, freshwater lakes, wastewater, and the human gut. CONCLUSION: METABOLIC enables the consistent and reproducible study of microbial community ecology and biogeochemistry using a foundation of genome-informed microbial metabolism, and will advance the integration of uncultivated organisms into metabolic and biogeochemical models. METABOLIC is written in Perl and R and is freely available under GPLv3 at https://github.com/AnantharamanLab/METABOLIC . Video abstract.

Simrank: Rapid and sensitive general-purpose k-mer search tool.

BACKGROUND: Terabyte-scale collections of string-encoded data are expected from consortia efforts such as the Human Microbiome Project http://nihroadmap.nih.gov/hmp. Intra- and inter-project data similarity searches are enabled by rapid k-mer matching strategies. Software applications for sequence database partitioning, guide tree estimation, molecular classification and alignment acceleration have benefited from embedded k-mer searches as sub-routines. However, a rapid, general-purpose, open-source, flexible, stand-alone k-mer tool has not been available. RESULTS: Here we present a stand-alone utility, Simrank, which allows users to rapidly identify database strings the most similar to query strings. Performance testing of Simrank and related tools against DNA, RNA, protein and human-languages found Simrank 10X to 928X faster depending on the dataset. CONCLUSIONS: Simrank provides molecular ecologists with a high-throughput, open source choice for comparing large sequence sets to find similarity.

A novel D-xylose isomerase from the gut of the wood feeding beetle Odontotaenius disjunctus efficiently expressed in Saccharomyces cerevisiae.

Carbohydrate rich substrates such as lignocellulosic hydrolysates remain one of the primary sources of potentially renewable fuel and bulk chemicals. The pentose sugar D-xylose is often present in significant amounts along with hexoses. Saccharomyces cerevisiae can acquire the ability to metabolize D-xylose through expression of heterologous D-xylose isomerase (XI). This enzyme is notoriously difficult to express in S. cerevisiae and only fourteen XIs have been reported to be active so far. We cloned a new D-xylose isomerase derived from microorganisms in the gut of the wood-feeding beetle Odontotaenius disjunctus. Although somewhat homologous to the XI from Piromyces sp. E2, the new gene was identified as bacterial in origin and the host as a Parabacteroides sp. Expression of the new XI in S. cerevisiae resulted in faster aerobic growth than the XI from Piromyces on D-xylose media. The D-xylose isomerization rate conferred by the new XI was also 72% higher, while absolute xylitol production was identical in both strains. Interestingly, increasing concentrations of xylitol (up to 8 g L-1) appeared not to inhibit D-xylose consumption. The newly described XI displayed 2.6 times higher specific activity, 37% lower KM for D-xylose, and exhibited higher activity over a broader temperature range, retaining 51% of maximal activity at 30 °C compared with only 29% activity for the Piromyces XI.

Microbial Phosphorus Mobilization Strategies Across a Natural Nutrient Limitation Gradient and Evidence for Linkage With Iron Solubilization Traits.

Microorganisms have evolved several mechanisms to mobilize and mineralize occluded and insoluble phosphorus (P), thereby promoting plant growth in terrestrial ecosystems. However, the linkages between microbial P-solubilization traits and the preponderance of insoluble P in natural ecosystems are not well known. We tested the P solubilization traits of hundreds of culturable bacteria representative of the rhizosphere from a natural gradient where P concentration and bioavailability decline as soil becomes progressively more weathered. Aluminum, iron phosphate and organic P (phytate) were expected to dominate in more weathered soils. A defined cultivation medium with these chemical forms of P was used for isolation. A combination of soil chemical, spectroscopic analyses and 16S rRNA gene sequencing were used to understand the in situ ability for solubilization of these predominant forms of P. Locations with more occluded and organic P harbored the greatest abundance of P-mobilizing microorganisms, especially Burkholderiaceae (Caballeronia and Paraburkholderia spp.). Nearly all bacteria utilized aluminum phosphate, however fewer could subsist on iron phosphate (FePO4) or phytate. Microorganisms isolated from phytic acid were also most effective at solubilizing FePO4, suggesting that phytate solubilization may be linked to the ability to solubilize Fe. Significantly, we observed Fe to be co-located with P in organic patches in soil. Siderophore addition in lab experiments reinstated phytase mediated P-solubilization from Fe-phytate complexes. Taken together, these results indicate that metal-organic-P complex formation may limit enzymatic P solubilization from phytate in soil. Additionally, the linked traits of phytase and siderophore production were mostly restricted to specific clades within the Burkholderiaceae. We propose that Fe complexation of organic P (e.g., phytate) represents a major constraint on P turnover and availability in acidic soils, as only a limited subset of bacteria appear to possess the traits required to access this persistent pool of soil P.

Presence and Persistence of Putative Lytic and Temperate Bacteriophages in Vaginal Metagenomes from South African Adolescents.

The interaction between gut bacterial and viral microbiota is thought to be important in human health. While fluctuations in female genital tract (FGT) bacterial microbiota similarly determine sexual health, little is known about the presence, persistence, and function of vaginal bacteriophages. We conducted shotgun metagenome sequencing of cervicovaginal samples from South African adolescents collected longitudinally, who received no antibiotics. We annotated viral reads and circular bacteriophages, identified CRISPR loci and putative prophages, and assessed their diversity, persistence, and associations with bacterial microbiota composition. Siphoviridae was the most prevalent bacteriophage family, followed by Myoviridae, Podoviridae, Herelleviridae, and Inoviridae. Full-length siphoviruses targeting bacterial vaginosis (BV)-associated bacteria were identified, suggesting their presence in vivo. CRISPR loci and prophage-like elements were common, and genomic analysis suggested higher diversity among Gardnerella than Lactobacillus prophages. We found that some prophages were highly persistent within participants, and identical prophages were present in cervicovaginal secretions of multiple participants, suggesting that prophages, and thus bacterial strains, are shared between adolescents. The number of CRISPR loci and prophages were associated with vaginal microbiota stability and absence of BV. Our analysis suggests that (pro)phages are common in the FGT and vaginal bacteria and (pro)phages may interact.

The Snowmelt Niche Differentiates Three Microbial Life Strategies That Influence Soil Nitrogen Availability During and After Winter.

Soil microbial biomass can reach its annual maximum pool size beneath the winter snowpack and is known to decline abruptly following snowmelt in seasonally snow-covered ecosystems. Observed differences in winter versus summer microbial taxonomic composition also suggests that phylogenetically conserved traits may permit winter- versus summer-adapted microorganisms to occupy distinct niches. In this study, we sought to identify archaea, bacteria, and fungi that are associated with the soil microbial bloom overwinter and the subsequent biomass collapse following snowmelt at a high-altitude watershed in central Colorado, United States. Archaea, bacteria, and fungi were categorized into three life strategies (Winter-Adapted, Snowmelt-Specialist, Spring-Adapted) based upon changes in abundance during winter, the snowmelt period, and after snowmelt in spring. We calculated indices of phylogenetic relatedness (archaea and bacteria) or assigned functional attributes (fungi) to organisms within life strategies to infer whether phylogenetically conserved traits differentiate Winter-Adapted, Snowmelt-Specialist, and Spring-Adapted groups. We observed that the soil microbial bloom was correlated in time with a pulse of snowmelt infiltration, which commenced 65 days prior to soils becoming snow-free. A pulse of nitrogen (N, as nitrate) occurred after snowmelt, along with a collapse in the microbial biomass pool size, and an increased abundance of nitrifying archaea and bacteria (e.g., Thaumarchaeota, Nitrospirae). Winter- and Spring-Adapted archaea and bacteria were phylogenetically clustered, suggesting that phylogenetically conserved traits allow Winter- and Spring-Adapted archaea and bacteria to occupy distinct niches. In contrast, Snowmelt-Specialist archaea and bacteria were phylogenetically overdispersed, suggesting that the key mechanism(s) of the microbial biomass crash are likely to be density-dependent (e.g., trophic interactions, competitive exclusion) and affect organisms across a broad phylogenetic spectrum. Saprotrophic fungi were the dominant functional group across fungal life strategies, however, ectomycorrhizal fungi experienced a large increase in abundance in spring. If well-coupled plant-mycorrhizal phenology currently buffers ecosystem N losses in spring, then changes in snowmelt timing may alter ecosystem N retention potential. Overall, we observed that snowmelt separates three distinct soil niches that are occupied by ecologically distinct groups of microorganisms. This ecological differentiation is of biogeochemical importance, particularly with respect to the mobilization of nitrogen during winter, before and after snowmelt.

Niche differentiation is spatially and temporally regulated in the rhizosphere.

The rhizosphere is a hotspot for microbial carbon transformations, and is the entry point for root polysaccharides and polymeric carbohydrates that are important precursors to soil organic matter (SOM). However, the ecological mechanisms that underpin rhizosphere carbohydrate depolymerization are poorly understood. Using Avena fatua, a common annual grass, we analyzed time-resolved metatranscriptomes to compare microbial functions in rhizosphere, detritusphere, and combined rhizosphere-detritusphere habitats. Transcripts were binned using a unique reference database generated from soil isolate genomes, single-cell amplified genomes, metagenomes, and stable isotope probing metagenomes. While soil habitat significantly affected both community composition and overall gene expression, the succession of microbial functions occurred at a faster time scale than compositional changes. Using hierarchical clustering of upregulated decomposition genes, we identified four distinct microbial guilds populated by taxa whose functional succession patterns suggest specialization for substrates provided by fresh growing roots, decaying root detritus, the combination of live and decaying root biomass, or aging root material. Carbohydrate depolymerization genes were consistently upregulated in the rhizosphere, and both taxonomic and functional diversity were highest in the combined rhizosphere-detritusphere, suggesting coexistence of rhizosphere guilds is facilitated by niche differentiation. Metatranscriptome-defined guilds provide a framework to model rhizosphere succession and its consequences for soil carbon cycling.

Hormonal contraception alters vaginal microbiota and cytokines in South African adolescents in a randomized trial.

Young women in sub-Saharan Africa are disproportionally affected by HIV infection and unintended pregnancies. However, hormonal contraceptive (HC) use may influence HIV risk through changes in genital tract microbiota and inflammatory cytokines. To investigate this, 130 HIV negative adolescent females aged 15-19 years were enrolled into a substudy of UChoose, an open-label randomized crossover study (NCT02404038), comparing acceptability and contraceptive product preference as a proxy for HIV prevention delivery methods. Participants were randomized to injectable norethisterone enanthate (Net-En), combined oral contraceptives (COC) or etonorgesterol/ethinyl estradiol combined contraceptive vaginal ring (CCVR) for 16 weeks, then crossed over to another HC for 16 weeks. Cervicovaginal samples were collected at baseline, crossover and exit for characterization of the microbiota and measurement of cytokine levels; primary endpoints were cervical T cell activation, vaginal microbial diversity and cytokine concentrations. Adolescents randomized to COCs had lower vaginal microbial diversity and relative abundance of HIV risk-associated taxa compared to Net-En or CCVR. Cervicovaginal inflammatory cytokine concentrations were significantly higher in adolescents randomized to CCVR compared to COC and Net-En. This suggests that COC use may induce an optimal vaginal ecosystem by decreasing bacterial diversity and inflammatory taxa, while CCVR use is associated with genital inflammation.

Uncovering multiple molecular targets for caffeine using a drug target validation strategy combining A 2A receptor knockout mice with microarray profiling.

Caffeine is the most widely consumed psychoactive substance and has complex pharmacological actions in brain. In this study, we employed a novel drug target validation strategy to uncover the multiple molecular targets of caffeine using combined A(2A) receptor (A(2A)R) knockouts (KO) and microarray profiling. Caffeine (10 mg/kg) elicited a distinct profile of striatal gene expression in WT mice compared with that by A(2A)R gene deletion or by administering caffeine into A(2A)R KO mice. Thus, A(2A)Rs are required but not sufficient to elicit the striatal gene expression by caffeine (10 mg/kg). Caffeine (50 mg/kg) induced complex expression patterns with three distinct sets of striatal genes: 1) one subset overlapped with those elicited by genetic deletion of A(2A)Rs; 2) the second subset elicited by caffeine in WT as well as A(2A)R KO mice; and 3) the third subset elicited by caffeine only in A(2A)R KO mice. Furthermore, striatal gene sets elicited by the phosphodiesterase (PDE) inhibitor rolipram and the GABA(A) receptor antagonist bicucullin, overlapped with the distinct subsets of striatal genes elicited by caffeine (50 mg/kg) administered to A(2A)R KO mice. Finally, Gene Set Enrichment Analysis reveals that adipocyte differentiation/insulin signaling is highly enriched in the striatal gene sets elicited by both low and high doses of caffeine. The identification of these distinct striatal gene populations and their corresponding multiple molecular targets, including A(2A)R, non-A(2A)R (possibly A(1)Rs and pathways associated with PDE and GABA(A)R) and their interactions, and the cellular pathways affected by low and high doses of caffeine, provides molecular insights into the acute pharmacological effects of caffeine in the brain.

Maintenance of Sympatric and Allopatric Populations in Free-Living Terrestrial Bacteria.

For free-living bacteria and archaea, the equivalent of the biological species concept does not exist, creating several obstacles to the study of the processes contributing to microbial diversification. These obstacles are particularly high in soil, where high bacterial diversity inhibits the study of closely related genotypes and therefore the factors structuring microbial populations. Here, we isolated strains within a single Curtobacterium ecotype from surface soil (leaf litter) across a regional climate gradient and investigated the phylogenetic structure, recombination, and flexible gene content of this genomic diversity to infer patterns of gene flow. Our results indicate that microbial populations are delineated by gene flow discontinuities, with distinct populations cooccurring at multiple sites. Bacterial population structure was further delineated by genomic features allowing for the identification of candidate genes possibly contributing to local adaptation. These results suggest that the genetic structure within this bacterium is maintained both by ecological specialization in localized microenvironments (isolation by environment) and by dispersal limitation between geographic locations (isolation by distance).IMPORTANCE Due to the promiscuous exchange of genetic material and asexual reproduction, delineating microbial species (and, by extension, populations) remains challenging. Because of this, the vast majority of microbial studies assessing population structure often compare divergent strains from disparate environments under varied selective pressures. Here, we investigated the population structure within a single bacterial ecotype, a unit equivalent to a eukaryotic species, defined as highly clustered genotypic and phenotypic strains with the same ecological niche. Using a combination of genomic and computational analyses, we assessed the phylogenetic structure, extent of recombination, and flexible gene content of this genomic diversity to infer patterns of gene flow. To our knowledge, this study is the first to do so for a dominant soil bacterium. Our results indicate that bacterial soil populations, similarly to those in other environments, are structured by gene flow discontinuities and exhibit distributional patterns consistent with both isolation by distance and isolation by environment. Thus, both dispersal limitation and local environments contribute to the divergence among closely related soil bacteria as observed in macroorganisms.