Rumen metaproteomics: Closer to linking rumen microbial function to animal productivity traits.

The rumen microbiome constitutes a dense and complex mixture of anaerobic bacteria, archaea, protozoa, virus and fungi. Collectively, rumen microbial populations interact closely in order to degrade and ferment complex plant material into nutrients for host metabolism, a process which also produces other by-products, such as methane gas. Our understanding of the rumen microbiome and its functions are of both scientific and industrial interest, as the metabolic functions are connected to animal health and nutrition, but at the same time contribute significantly to global greenhouse gas emissions. While many of the major microbial members of the rumen microbiome are acknowledged, advances in modern culture-independent meta-omic techniques, such as metaproteomics, enable deep exploration into active microbial populations involved in essential rumen metabolic functions. Meaningful and accurate metaproteomic analyses are highly dependent on representative samples, precise protein extraction and fractionation, as well as a comprehensive and high-quality protein sequence database that enables precise protein identification and quantification. This review focuses on the application of rumen metaproteomics, and its potential towards understanding the complex rumen microbiome and its metabolic functions. We present and discuss current methods in sample handling, protein extraction and data analysis for rumen metaproteomics, and finally emphasize the potential of (meta)genome-integrated metaproteomics for accurate reconstruction of active microbial populations in the rumen.

IMG/VR: a database of cultured and uncultured DNA Viruses and retroviruses.

Viruses represent the most abundant life forms on the planet. Recent experimental and computational improvements have led to a dramatic increase in the number of viral genome sequences identified primarily from metagenomic samples. As a result of the expanding catalog of metagenomic viral sequences, there exists a need for a comprehensive computational platform integrating all these sequences with associated metadata and analytical tools. Here we present IMG/VR (https://img.jgi.doe.gov/vr/), the largest publicly available database of 3908 isolate reference DNA viruses with 264 413 computationally identified viral contigs from >6000 ecologically diverse metagenomic samples. Approximately half of the viral contigs are grouped into genetically distinct quasi-species clusters. Microbial hosts are predicted for 20 000 viral sequences, revealing nine microbial phyla previously unreported to be infected by viruses. Viral sequences can be queried using a variety of associated metadata, including habitat type and geographic location of the samples, or taxonomic classification according to hallmark viral genes. IMG/VR has a user-friendly interface that allows users to interrogate all integrated data and interact by comparing with external sequences, thus serving as an essential resource in the viral genomics community.

Stable Core Gut Microbiota across the Freshwater-to-Saltwater Transition for Farmed Atlantic Salmon.

Gut microbiota associations through habitat transitions are fundamentally important yet poorly understood. One such habitat transition is the migration from freshwater to saltwater for anadromous fish, such as salmon. The aim of the current work was therefore to determine the freshwater-to-saltwater transition impact on the gut microbiota in farmed Atlantic salmon, with dietary interventions resembling freshwater and saltwater diets with respect to fatty acid composition. Using deep 16S rRNA gene sequencing and quantitative PCR, we found that the freshwater-to-saltwater transition had a major association with the microbiota composition and quantity, while diet did not show significant associations with the microbiota. In saltwater there was a 100-fold increase in bacterial quantity, with a relative increase of Firmicutes and a relative decrease of both Actinobacteria and Proteobacteria Irrespective of an overall shift in microbiota composition from freshwater to saltwater, we identified three core clostridia and one Lactobacillus-affiliated phylotype with wide geographic distribution that were highly prevalent and co-occurring. Taken together, our results support the importance of the dominating bacteria in the salmon gut, with the freshwater microbiota being immature. Due to the low number of potentially host-associated bacterial species in the salmon gut, we believe that farmed salmon can represent an important model for future understanding of host-bacterium interactions in aquatic environments.IMPORTANCE Little is known about factors affecting the interindividual distribution of gut bacteria in aquatic environments. We have shown that there is a core of four highly prevalent and co-occurring bacteria irrespective of feed and freshwater-to-saltwater transition. The potential host interactions of the core bacteria, however, need to be elucidated further.

Outer membrane vesicles from Fibrobacter succinogenes S85 contain an array of carbohydrate-active enzymes with versatile polysaccharide-degrading capacity.

Fibrobacter succinogenes is an anaerobic bacterium naturally colonising the rumen and cecum of herbivores where it utilizes an enigmatic mechanism to deconstruct cellulose into cellobiose and glucose, which serve as carbon sources for growth. Here, we illustrate that outer membrane vesicles (OMVs) released by F. succinogenes are enriched with carbohydrate-active enzymes and that intact OMVs were able to depolymerize a broad range of linear and branched hemicelluloses and pectin, despite the inability of F. succinogenes to utilize non-cellulosic (pentose) sugars for growth. We hypothesize that the degradative versatility of F. succinogenes OMVs is used to prime hydrolysis by destabilising the tight networks of polysaccharides intertwining cellulose in the plant cell wall, thus increasing accessibility of the target substrate for the host cell. This is supported by observations that OMV-pretreatment of the natural complex substrate switchgrass increased the catalytic efficiency of a commercial cellulose-degrading enzyme cocktail by 2.4-fold. We also show that the OMVs contain a putative multiprotein complex, including the fibro-slime protein previously found to be important in binding to crystalline cellulose. We hypothesize that this complex has a function in plant cell wall degradation, either by catalysing polysaccharide degradation itself, or by targeting the vesicles to plant biomass.

Quantitative Metaproteomics Highlight the Metabolic Contributions of Uncultured Phylotypes in a Thermophilic Anaerobic Digester.

In this study, we used multiple meta-omic approaches to characterize the microbial community and the active metabolic pathways of a stable industrial biogas reactor with food waste as the dominant feedstock, operating at thermophilic temperatures (60°C) and elevated levels of free ammonia (367 mg/liter NH3-N). The microbial community was strongly dominated (76% of all 16S rRNA amplicon sequences) by populations closely related to the proteolytic bacterium Coprothermobacter proteolyticus. Multiple Coprothermobacter-affiliated strains were detected, introducing an additional level of complexity seldom explored in biogas studies. Genome reconstructions provided metabolic insight into the microbes that performed biomass deconstruction and fermentation, including the deeply branching phyla Dictyoglomi and Planctomycetes and the candidate phylum "Atribacteria" These biomass degraders were complemented by a synergistic network of microorganisms that convert key fermentation intermediates (fatty acids) via syntrophic interactions with hydrogenotrophic methanogens to ultimately produce methane. Interpretation of the proteomics data also suggested activity of a Methanosaeta phylotype acclimatized to high ammonia levels. In particular, we report multiple novel phylotypes proposed as syntrophic acetate oxidizers, which also exert expression of enzymes needed for both the Wood-Ljungdahl pathway and ß-oxidation of fatty acids to acetyl coenzyme A. Such an arrangement differs from known syntrophic oxidizing bacteria and presents an interesting hypothesis for future studies. Collectively, these findings provide increased insight into active metabolic roles of uncultured phylotypes and presents new synergistic relationships, both of which may contribute to the stability of the biogas reactor. IMPORTANCE: Biogas production through anaerobic digestion of organic waste provides an attractive source of renewable energy and a sustainable waste management strategy. A comprehensive understanding of the microbial community that drives anaerobic digesters is essential to ensure stable and efficient energy production. Here, we characterize the intricate microbial networks and metabolic pathways in a thermophilic biogas reactor. We discuss the impact of frequently encountered microbial populations as well as the metabolism of newly discovered novel phylotypes that seem to play distinct roles within key microbial stages of anaerobic digestion in this stable high-temperature system. In particular, we draft a metabolic scenario whereby multiple uncultured syntrophic acetate-oxidizing bacteria are capable of syntrophically oxidizing acetate as well as longer-chain fatty acids (via the ß-oxidation and Wood-Ljundahl pathways) to hydrogen and carbon dioxide, which methanogens subsequently convert to methane.

The effect of storage conditions on microbial community composition and biomethane potential in a biogas starter culture.

A new biogas process is initiated by adding a microbial community, typically in the form of a sample collected from a functional biogas plant. This inoculum has considerable impact on the initial performance of a biogas reactor, affecting parameters such as stability, biogas production yields and the overall efficiency of the anaerobic digestion process. In this study, we have analyzed changes in the microbial composition and performance of an inoculum during storage using barcoded pyrosequencing of bacterial and archaeal 16S ribosomal RNA (rRNA) genes, and determination of the biomethane potential, respectively. The inoculum was stored at room temperature, 4 and -20 °C for up to 11 months and cellulose was used as a standard substrate to test the biomethane potential. Storage up to 1 month resulted in similar final methane yields, but the rate of methane production was reduced by storage at -20 °C. Longer storage times resulted in reduced methane yields and slower production kinetics for all storage conditions, with room temperature and frozen samples consistently giving the best and worst performance, respectively. Both storage time and temperature affected the microbial community composition and methanogenic activity. In particular, fluctuations in the relative abundance of Bacteroidetes were observed. Interestingly, a shift from hydrogenotrophic methanogens to methanogens with the capacity to perform acetoclastic methanogensis was observed upon prolonged storage. In conclusion, this study suggests that biogas inocula may be stored up to 1 month with low loss of methanogenic activity, and identifies bacterial and archaeal species that are affected by the storage.

Analysis of the bovine rumen microbiome reveals a diversity of Sus-like polysaccharide utilization loci from the bacterial phylum Bacteroidetes.

Several unique Sus-like polysaccharide utilization loci (PULs) were identified from bacteria resident in bovine rumen microbiomes through functional screening of a fosmid library. The loci were phylogenetically assigned to the genus Prevotella within the phylum Bacteroidetes. These findings were augmented by a bioinformatic re-evaluation of ruminal Prevotella genomes, revealing additional loci not previously reported in the literature. Analysis of Bacteroidales-affiliated genomes reconstructed from a bovine rumen metagenome in a previous study further expanded the diversity of Sus-like PULs resident in this microbiome. Our findings suggest that Sus-like systems represent an important mechanism for degradation of a range of plant-derived glycans in ruminants.

Transcriptomic and proteomic changes associated with cobalamin-dependent propionate production by the rumen bacterium Xylanibacter ruminicola.

Xylanibacter ruminicola is an abundant rumen bacterium that produces propionate in a cobalamin (vitamin B12)-dependent manner via the succinate pathway. However, the extent to which this occurs across ruminal Xylanibacter and closely related bacteria, and the effect of cobalamin supplementation on the expression of propionate pathway genes and enzymes has yet to be investigated. To assess this, we screened 14 strains and found that almost all strains produced propionate when supplemented with cobalamin. X. ruminicola KHP1 was selected for further study, including complete genome sequencing, and comparative transcriptomics and proteomics of KHP1 cultures grown with and without supplemented cobalamin. The complete KHP1 genome was searched for cobalamin-binding riboswitches and four were predicted, though none were closely located to any of the succinate pathway genes, which were dispersed at numerous genomic loci. Cobalamin supplementation led to the differential expression of 17.5% of genes, including genes encoding the cobalamin-dependent methylmalonyl-CoA mutase and some methylmalonyl-CoA decarboxylase subunits, but most propionate biosynthesis pathway genes were not differentially expressed. The effect of cobalamin supplementation on the KHP1 proteome was much less pronounced, with the only differentially abundant propionate pathway enzyme being methylmalonyl-CoA mutase, which had greater abundance when supplemented with cobalamin. Our results demonstrate that cobalamin supplementation does not result in induction of the entire propionate biosynthesis pathway, but consistently increased expression of methylmalonyl-CoA mutase at transcriptome and proteome levels. The magnitude of the differential expression of propionate pathway genes observed was minor compared to that of genes proximate to predicted cobalamin riboswitches. IMPORTANCE: In ruminants, the rumen microbial community plays a critical role in nutrition through the fermentation of feed to provide vital energy substrates for the host animal. Propionate is a major rumen fermentation end-product and increasing its production is desirable given its importance in host glucose production and impact on greenhouse gas production. Vitamin B12 (cobalamin) can induce propionate production in the prominent rumen bacterium Xylanibacter ruminicola, but it is not fully understood how cobalamin regulates propionate pathway activity. Contrary to expectation, we found that cobalamin supplementation had little effect on propionate pathway expression at transcriptome and proteome levels, with minor upregulation of genes encoding the cobalamin-dependent enzyme of the pathway. These findings provide new insights into factors that regulate propionate production and suggest that cobalamin-dependent propionate production by X. ruminicola is controlled post-translationally.

Integrating host and microbiome biology using holo-omics.

Holo-omics is the use of omics data to study a host and its inherent microbiomes - a biological system known as a "holobiont". A microbiome that exists in such a space often encounters habitat stability and in return provides metabolic capacities that can benefit their host. Here we present an overview of beneficial host-microbiome systems and propose and discuss several methodological frameworks that can be used to investigate the intricacies of the many as yet undefined host-microbiome interactions that influence holobiont homeostasis. While this is an emerging field, we anticipate that ongoing methodological advancements will enhance the biological resolution that is necessary to improve our understanding of host-microbiome interplay to make meaningful interpretations and biotechnological applications.

Unraveling the phylogenomic diversity of Methanomassiliicoccales and implications for mitigating ruminant methane emissions.

BACKGROUND: Methanomassiliicoccales are a recently identified order of methanogens that are diverse across global environments particularly the gastrointestinal tracts of animals; however, their metabolic capacities are defined via a limited number of cultured strains. RESULTS: Here, we profile and analyze 243 Methanomassiliicoccales genomes assembled from cultured representatives and uncultured metagenomes recovered from various biomes, including the gastrointestinal tracts of different animal species. Our analyses reveal the presence of numerous undefined genera and genetic variability in metabolic capabilities within Methanomassiliicoccales lineages, which is essential for adaptation to their ecological niches. In particular, gastrointestinal tract Methanomassiliicoccales demonstrate the presence of co-diversified members with their hosts over evolutionary timescales and likely originated in the natural environment. We highlight the presence of diverse clades of vitamin transporter BtuC proteins that distinguish Methanomassiliicoccales from other archaeal orders and likely provide a competitive advantage in efficiently handling B12. Furthermore, genome-centric metatranscriptomic analysis of ruminants with varying methane yields reveal elevated expression of select Methanomassiliicoccales genera in low methane animals and suggest that B12 exchanges could enable them to occupy ecological niches that possibly alter the direction of H2 utilization. CONCLUSIONS: We provide a comprehensive and updated account of divergent Methanomassiliicoccales lineages, drawing from numerous uncultured genomes obtained from various habitats. We also highlight their unique metabolic capabilities involving B12, which could serve as promising targets for mitigating ruminant methane emissions by altering H2 flow.

Forecasting the dynamics of a complex microbial community using integrated meta-omics.

Predicting the behaviour of complex microbial communities is challenging. However, this is essential for complex biotechnological processes such as those in biological wastewater treatment plants (BWWTPs), which require sustainable operation. Here we summarize 14 months of longitudinal meta-omics data from a BWWTP anaerobic tank into 17 temporal signals, explaining 91.1% of the temporal variance, and link those signals to ecological events within the community. We forecast the signals over the subsequent five years and use 21 extra samples collected at defined time intervals for testing and validation. Our forecasts are correct for six signals and hint on phenomena such as predation cycles. Using all the 17 forecasts and the environmental variables, we predict gene abundance and expression, with a coefficient of determination ≥0.87 for the subsequent three years. Our study demonstrates the ability to forecast the dynamics of open microbial ecosystems using interactions between community cycles and environmental parameters.

Genomic and functional characterization of the Atlantic salmon gut microbiome in relation to nutrition and health.

To ensure sustainable aquaculture, it is essential to understand the path 'from feed to fish', whereby the gut microbiome plays an important role in digestion and metabolism, ultimately influencing host health and growth. Previous work has reported the taxonomic composition of the Atlantic salmon (Salmo salar) gut microbiome; however, functional insights are lacking. Here we present the Salmon Microbial Genome Atlas consisting of 211 high-quality bacterial genomes, recovered by cultivation (n = 131) and gut metagenomics (n = 80) from wild and farmed fish both in freshwater and seawater. Bacterial genomes were taxonomically assigned to 14 different orders, including 35 distinctive genera and 29 previously undescribed species. Using metatranscriptomics, we functionally characterized key bacterial populations, across five phyla, in the salmon gut. This included the ability to degrade diet-derived fibres and release vitamins and other exometabolites with known beneficial effects, which was supported by genome-scale metabolic modelling and in vitro cultivation of selected bacterial species coupled with untargeted metabolomic studies. Together, the Salmon Microbial Genome Atlas provides a genomic and functional resource to enable future studies on salmon nutrition and health.

The need for high-resolution gut microbiome characterization to design efficient strategies for sustainable aquaculture production.

Microbiome-directed dietary interventions such as microbiota-directed fibers (MDFs) have a proven track record in eliciting responses in beneficial gut microbes and are increasingly being promoted as an effective strategy to improve animal production systems. Here we used initial metataxonomic data on fish gut microbiomes as well as a wealth of a priori mammalian microbiome knowledge on α-mannooligosaccharides (MOS) and ß-mannan-derived MDFs to study effects of such feed supplements in Atlantic salmon (Salmo salar) and their impact on its gut microbiome composition and functionalities. Our multi-omic analysis revealed that the investigated MDFs (two α-mannans and an acetylated ß-galactoglucomannan), at a dose of 0.2% in the diet, had negligible effects on both host gene expression, and gut microbiome structure and function under the studied conditions. While a subsequent trial using a higher (4%) dietary inclusion of ß-mannan significantly shifted the gut microbiome composition, there were still no biologically relevant effects on salmon metabolism and physiology. Only a single Burkholderia-Caballeronia-Paraburkholderia (BCP) population demonstrated consistent and significant abundance shifts across both feeding trials, although with no evidence of ß-mannan utilization capabilities or changes in gene transcripts for producing metabolites beneficial to the host. In light of these findings, we revisited our omics data to predict and outline previously unreported and potentially beneficial endogenous lactic acid bacteria that should be targeted with future, conceivably more suitable, MDF strategies for salmon.

A concept for international societally relevant microbiology education and microbiology knowledge promulgation in society.

EXECUTIVE SUMMARY: Microbes are all pervasive in their distribution and influence on the functioning and well-being of humans, life in general and the planet. Microbially-based technologies contribute hugely to the supply of important goods and services we depend upon, such as the provision of food, medicines and clean water. They also offer mechanisms and strategies to mitigate and solve a wide range of problems and crises facing humanity at all levels, including those encapsulated in the sustainable development goals (SDGs) formulated by the United Nations. For example, microbial technologies can contribute in multiple ways to decarbonisation and hence confronting global warming, provide sanitation and clean water to the billions of people lacking them, improve soil fertility and hence food production and develop vaccines and other medicines to reduce and in some cases eliminate deadly infections. They are the foundation of biotechnology, an increasingly important and growing business sector and source of employment, and the centre of the bioeconomy, Green Deal, etc. But, because microbes are largely invisible, they are not familiar to most people, so opportunities they offer to effectively prevent and solve problems are often missed by decision-makers, with the negative consequences this entrains. To correct this lack of vital knowledge, the International Microbiology Literacy Initiative-the IMiLI-is recruiting from the global microbiology community and making freely available, teaching resources for a curriculum in societally relevant microbiology that can be used at all levels of learning. Its goal is the development of a society that is literate in relevant microbiology and, as a consequence, able to take full advantage of the potential of microbes and minimise the consequences of their negative activities. In addition to teaching about microbes, almost every lesson discusses the influence they have on sustainability and the SDGs and their ability to solve pressing problems of societal inequalities. The curriculum thus teaches about sustainability, societal needs and global citizenship. The lessons also reveal the impacts microbes and their activities have on our daily lives at the personal, family, community, national and global levels and their relevance for decisions at all levels. And, because effective, evidence-based decisions require not only relevant information but also critical and systems thinking, the resources also teach about these key generic aspects of deliberation. The IMiLI teaching resources are learner-centric, not academic microbiology-centric and deal with the microbiology of everyday issues. These span topics as diverse as owning and caring for a companion animal, the vast range of everyday foods that are produced via microbial processes, impressive geological formations created by microbes, childhood illnesses and how they are managed and how to reduce waste and pollution. They also leverage the exceptional excitement of exploration and discovery that typifies much progress in microbiology to capture the interest, inspire and motivate educators and learners alike. The IMiLI is establishing Regional Centres to translate the teaching resources into regional languages and adapt them to regional cultures, and to promote their use and assist educators employing them. Two of these are now operational. The Regional Centres constitute the interface between resource creators and educators-learners. As such, they will collect and analyse feedback from the end-users and transmit this to the resource creators so that teaching materials can be improved and refined, and new resources added in response to demand: educators and learners will thereby be directly involved in evolution of the teaching resources. The interactions between educators-learners and resource creators mediated by the Regional Centres will establish dynamic and synergistic relationships-a global societally relevant microbiology education ecosystem-in which creators also become learners, teaching resources are optimised and all players/stakeholders are empowered and their motivation increased. The IMiLI concept thus embraces the principle of teaching societally relevant microbiology embedded in the wider context of societal, biosphere and planetary needs, inequalities, the range of crises that confront us and the need for improved decisioning, which should ultimately lead to better citizenship and a humanity that is more sustainable and resilient. ABSTRACT: The biosphere of planet Earth is a microbial world: a vast reactor of countless microbially driven chemical transformations and energy transfers that push and pull many planetary geochemical processes, including the cycling of the elements of life, mitigate or amplify climate change (e.g., Nature Reviews Microbiology, 2019, 17, 569) and impact the well-being and activities of all organisms, including humans. Microbes are both our ancestors and creators of the planetary chemistry that allowed us to evolve (e.g., Life's engines: How microbes made earth habitable, 2023). To understand how the biosphere functions, how humans can influence its development and live more sustainably with the other organisms sharing it, we need to understand the microbes. In a recent editorial (Environmental Microbiology, 2019, 21, 1513), we advocated for improved microbiology literacy in society. Our concept of microbiology literacy is not based on knowledge of the academic subject of microbiology, with its multitude of component topics, plus the growing number of additional topics from other disciplines that become vitally important elements of current microbiology. Rather it is focused on microbial activities that impact us-individuals/communities/nations/the human world-and the biosphere and that are key to reaching informed decisions on a multitude of issues that regularly confront us, ranging from personal issues to crises of global importance. In other words, it is knowledge and understanding essential for adulthood and the transition to it, knowledge and understanding that must be acquired early in life in school. The 2019 Editorial marked the launch of the International Microbiology Literacy Initiative, the IMiLI. HERE, WE PRESENT: our concept of how microbiology literacy may be achieved and the rationale underpinning it; the type of teaching resources being created to realise the concept and the framing of microbial activities treated in these resources in the context of sustainability, societal needs and responsibilities and decision-making; and the key role of Regional Centres that will translate the teaching resources into local languages, adapt them according to local cultural needs, interface with regional educators and develop and serve as hubs of microbiology literacy education networks. The topics featuring in teaching resources are learner-centric and have been selected for their inherent relevance, interest and ability to excite and engage. Importantly, the resources coherently integrate and emphasise the overarching issues of sustainability, stewardship and critical thinking and the pervasive interdependencies of processes. More broadly, the concept emphasises how the multifarious applications of microbial activities can be leveraged to promote human/animal, plant, environmental and planetary health, improve social equity, alleviate humanitarian deficits and causes of conflicts among peoples and increase understanding between peoples (Microbial Biotechnology, 2023, 16(6), 1091-1111). Importantly, although the primary target of the freely available (CC BY-NC 4.0) IMiLI teaching resources is schoolchildren and their educators, they and the teaching philosophy are intended for all ages, abilities and cultural spectra of learners worldwide: in university education, lifelong learning, curiosity-driven, web-based knowledge acquisition and public outreach. The IMiLI teaching resources aim to promote development of a global microbiology education ecosystem that democratises microbiology knowledge.

Probiotic dosing of Ruminococcus flavefaciens affects rumen microbiome structure and function in reindeer.

Highly cellulolytic bacterial species such as Ruminococcus flavefaciens are regarded essential for the microbial breakdown of cellulose in the rumen. We have investigated the effect of ruminal dosing of R. flavefaciens strain 8/94-32 during realimentation of starved reindeer (males, n = 3). Microbiome function measured as in situ digestion of cellulose and food pellets (percent DMD; dry matter disappearance) decreased after probiotic dosing. Microbial community analyses (>100,000 16S rDNA gene sequences for 27 samples) demonstrated that ruminal dosing influenced the microbiome structure; reflected by increased phylogenetic distances from background samples (unweighted UniFrac analysis) and reduced species diversity and evenness. Despite the inability to detect strain 8/94-32 post-dosing, the relative abundance of its affiliate family Ruminococcaceae remained consistent throughout the trial, whilst a dominant peak in the genus Prevotella and decline in uncharacterized Bacteroidetes (uBacNR) were observed in treatment samples. No clear relationships were observed between the relative abundance of Ruminococcaceae, Prevotella and uBacNR with cellulose DMD; however, Prevotella (negative) and uBacNR (positive) exhibited relationships with pellet DMD. These unexpected effects of ruminal dosing of a cellulolytic bacterium on digestibility are relevant for other studies on rumen manipulation.

Potential applications of gut microbiota to control human physiology.

The microorganisms living in our gut have been a black box to us for a long time. However, with the recent advances in high throughput DNA sequencing technologies, it is now possible to assess virtually all microorganisms in our gut including non-culturable ones. With the use of powerful bioinformatics tools to deal with multivariate analyses of huge amounts of data from metagenomics, metatranscriptomics, metabolomics, we now start to gain some important insights into these tiny gut inhabitants. Our knowledge is increasing about who they are, to some extent, what they do and how they affect our health. Gut microbiota have a broad spectrum of possible effects on health, from preventing serious diseases, improving immune system and gut health to stimulating the brain centers responsible for appetite and food intake control. Further, we may be on the verge of being capable of manipulating the gut microbiota by diet control to possibly improve our health. Diets consisting of different components that are fermentable by microbiota are substrates for different kinds of microbes in the gut. Thus, diet control can be used to favor the growth of some selected gut inhabitants. Nowadays, the gut microbiota is taken into account as a separate organ in human body and their activities and metabolites in gut have many physiological and neurological effects. In this mini-review, we discuss the diversity of gut microbiota, the technologies used to assess them, factors that affect microbial composition and metabolites that affect human physiology, and their potential applications in satiety control via the gut-brain axis.

Resin acids play key roles in shaping microbial communities during degradation of spruce bark.

The bark is the outermost defense of trees against microbial attack, largely thanks to toxicity and prevalence of extractive compounds. Nevertheless, bark decomposes in nature, though by which species and mechanisms remains unknown. Here, we have followed the development of microbial enrichments growing on spruce bark over six months, by monitoring both chemical changes in the material and performing community and metagenomic analyses. Carbohydrate metabolism was unexpectedly limited, and instead a key activity was metabolism of extractives. Resin acid degradation was principally linked to community diversification with specific bacteria revealed to dominate the process. Metagenome-guided isolation facilitated the recovery of the dominant enrichment strain in pure culture, which represents a new species (Pseudomonas abieticivorans sp. nov.), that can grow on resin acids as a sole carbon source. Our results illuminate key stages in degradation of an abundant renewable resource, and how defensive extractive compounds have major roles in shaping microbiomes.

Integrative meta-omics in Galaxy and beyond.

BACKGROUND: 'Omics methods have empowered scientists to tackle the complexity of microbial communities on a scale not attainable before. Individually, omics analyses can provide great insight; while combined as "meta-omics", they enhance the understanding of which organisms occupy specific metabolic niches, how they interact, and how they utilize environmental nutrients. Here we present three integrative meta-omics workflows, developed in Galaxy, for enhanced analysis and integration of metagenomics, metatranscriptomics, and metaproteomics, combined with our newly developed web-application, ViMO (Visualizer for Meta-Omics) to analyse metabolisms in complex microbial communities. RESULTS: In this study, we applied the workflows on a highly efficient cellulose-degrading minimal consortium enriched from a biogas reactor to analyse the key roles of uncultured microorganisms in complex biomass degradation processes. Metagenomic analysis recovered metagenome-assembled genomes (MAGs) for several constituent populations including Hungateiclostridium thermocellum, Thermoclostridium stercorarium and multiple heterogenic strains affiliated to Coprothermobacter proteolyticus. The metagenomics workflow was developed as two modules, one standard, and one optimized for improving the MAG quality in complex samples by implementing a combination of single- and co-assembly, and dereplication after binning. The exploration of the active pathways within the recovered MAGs can be visualized in ViMO, which also provides an overview of the MAG taxonomy and quality (contamination and completeness), and information about carbohydrate-active enzymes (CAZymes), as well as KEGG annotations and pathways, with counts and abundances at both mRNA and protein level. To achieve this, the metatranscriptomic reads and metaproteomic mass-spectrometry spectra are mapped onto predicted genes from the metagenome to analyse the functional potential of MAGs, as well as the actual expressed proteins and functions of the microbiome, all visualized in ViMO. CONCLUSION: Our three workflows for integrative meta-omics in combination with ViMO presents a progression in the analysis of 'omics data, particularly within Galaxy, but also beyond. The optimized metagenomics workflow allows for detailed reconstruction of microbial community consisting of MAGs with high quality, and thus improves analyses of the metabolism of the microbiome, using the metatranscriptomics and metaproteomics workflows.

Metabolic influence of core ciliates within the rumen microbiome.

Protozoa comprise a major fraction of the microbial biomass in the rumen microbiome, of which the entodiniomorphs (order: Entodiniomorphida) and holotrichs (order: Vestibuliferida) are consistently observed to be dominant across a diverse genetic and geographical range of ruminant hosts. Despite the apparent core role that protozoal species exert, their major biological and metabolic contributions to rumen function remain largely undescribed in vivo. Here, we have leveraged (meta)genome-centric metaproteomes from rumen fluid samples originating from both cattle and goats fed diets with varying inclusion levels of lipids and starch, to detail the specific metabolic niches that protozoa occupy in the context of their microbial co-habitants. Initial proteome estimations via total protein counts and label-free quantification highlight that entodiniomorph species Entodinium and Epidinium as well as the holotrichs Dasytricha and Isotricha comprise an extensive fraction of the total rumen metaproteome. Proteomic detection of protozoal metabolism such as hydrogenases (Dasytricha, Isotricha, Epidinium, Enoploplastron), carbohydrate-active enzymes (Epidinium, Diplodinium, Enoploplastron, Polyplastron), microbial predation (Entodinium) and volatile fatty acid production (Entodinium and Epidinium) was observed at increased levels in high methane-emitting animals. Despite certain protozoal species having well-established reputations for digesting starch, they were unexpectedly less detectable in low methane emitting-animals fed high starch diets, which were instead dominated by propionate/succinate-producing bacterial populations suspected of being resistant to predation irrespective of host. Finally, we reaffirmed our abovementioned observations in geographically independent datasets, thus illuminating the substantial metabolic influence that under-explored eukaryotic populations have in the rumen, with greater implications for both digestion and methane metabolism.

Draft genome sequence of the methane-oxidizing bacterium Methylococcus capsulatus (Texas).

Methanotrophic bacteria perform major roles in global carbon cycles via their unique enzymatic activities that enable the oxidation of one-carbon compounds, most notably methane. Here we describe the annotated draft genome sequence of the aerobic methanotroph Methylococcus capsulatus (Texas), a type strain originally isolated from sewer sludge.