Heritable Bovine Rumen Bacteria Are Phylogenetically Related and Correlated with the Cow's Capacity To Harvest Energy from Its Feed.

Ruminants sustain a long-lasting obligatory relationship with their rumen microbiome dating back 50 million years. In this unique host-microbiome relationship, the host's ability to digest its feed is completely dependent on its coevolved microbiome. This extraordinary alliance raises questions regarding the dependent relationship between ruminants' genetics and physiology and the rumen microbiome structure, composition, and metabolism. To elucidate this relationship, we examined the association of host genetics with the phylogenetic and functional composition of the rumen microbiome. We accomplished this by studying a population of 78 Holstein-Friesian dairy cows, using a combination of rumen microbiota data and other phenotypes from each animal with genotypic data from a subset of 47 animals. We identified 22 operational taxonomic units (OTUs) whose abundances were associated with rumen metabolic traits and host physiological traits and which showed measurable heritability. The abundance patterns of these microbes can explain high proportions of variance in rumen metabolism and many of the host physiological attributes such as its energy-harvesting efficiency. Interestingly, these OTUs shared higher phylogenetic similarity between themselves than expected by chance, suggesting occupation of a specific ecological niche within the rumen ecosystem. The findings presented here suggest that ruminant genetics and physiology are correlated with microbiome structure and that host genetics may shape the microbiome landscape by enriching for phylogenetically related taxa that may occupy a unique niche.IMPORTANCE Dairy cows are an essential nutritional source for the world's population; as such, they are extensively farmed throughout our planet and subsequently impact our environment. The microbial communities that reside in the upper digestive tract of these animals in a compartment named the rumen degrade and ferment the plant biomass that the animal ingests. Our recent efforts, as well as those of others, have shown that this microbial community's composition and functionality are tightly linked to the cow's capacity to harvest energy from its feed, as well as to other physiological traits. In this study, we identified microbial groups that are heritable and also linked to the cow's production parameters. This finding could potentially allow us to apply selection programs on specific rumen microbial components that are linked to the animal's physiology and beneficial to production. Hence, it is a steppingstone toward microbiome manipulation for increasing food availability while lowering environmental impacts such as methane emission.

Specific microbiome-dependent mechanisms underlie the energy harvest efficiency of ruminants.

Ruminants have the remarkable ability to convert human-indigestible plant biomass into human-digestible food products, due to a complex microbiome residing in the rumen compartment of their upper digestive tract. Here we report the discovery that rumen microbiome components are tightly linked to cows' ability to extract energy from their feed, termed feed efficiency. Feed efficiency was measured in 146 milking cows and analyses of the taxonomic composition, gene content, microbial activity and metabolomic composition was performed on the rumen microbiomes from the 78 most extreme animals. Lower richness of microbiome gene content and taxa was tightly linked to higher feed efficiency. Microbiome genes and species accurately predicted the animals' feed efficiency phenotype. Specific enrichment of microbes and metabolic pathways in each of these microbiome groups resulted in better energy and carbon channeling to the animal, while lowering methane emissions to the atmosphere. This ecological and mechanistic understanding of the rumen microbiome could lead to an increase in available food resources and environmentally friendly livestock agriculture.

Individualized medicine and the microbiome in reproductive tract.

Humans have evolved along with the millions of microorganisms that populate their bodies. These microbes (10(14)) outnumber human cells by 10 to 1 and account for 3 × 10(6) genes, more than ten times the 25,000 human genes. This microbial metagenome acts as our "other genome" and like our own genes, is unique to the individual. Recent international efforts such as the Human Microbiome Project (HMP) and the MetaHIT Project have helped catalog these microbial genomes using culture-independent, high-throughput, next-generation sequencing. This manuscript will describe recent efforts to define microbial diversity in the female reproductive tract because of the impact that microbial function has on reproductive efficiency. In this review, we will discuss current evidence that microbial communities are critical for maintaining reproductive health and how perturbations of microbial community structures can impact reproductive health from the aspect of infection, reproductive cyclicity, pregnancy, and disease states. Investigations of the human microbiome are propelling interventional strategies from treating medical populations to treating individual patients. In particular, we highlight how understanding and defining microbial community structures in different disease and physiological states have lead to the discovery of biomarkers and, more importantly, the development and implementation of microbial intervention strategies (probiotics) into modern day medicine. Finally this review will conclude with a literature summary of the effectiveness of microbial intervention strategies that have been implemented in animal and human models of disease and the potential for integrating these microbial intervention strategies into standard clinical practice.

Voluntary and forced exercise differentially alters the gut microbiome in C57BL/6J mice.

We have previously shown that voluntary wheel running (VWR) attenuates, whereas forced treadmill running (FTR) exacerbates, intestinal inflammation and clinical outcomes in a mouse model of colitis. As the gut microbiome is implicated in colitis, we hypothesized that VWR and FTR would differentially affect the gut microbiome. Mice (9-10/treatment) were randomly assigned to VWR, FTR, or sedentary home cage control (SED) for 6 wk. VWR were given running wheel access, whereas FTR ran on a treadmill for 40 min/day at 8-12 m/min, 5% grade. Forty-eight hours after the last exercise session, DNA was isolated from the fecal pellets and cecal contents, and the conserved bacterial 16S rRNA gene was amplified and sequenced using the Illumina Miseq platform. Permutational multivariate analysis of variance based on weighted UniFrac distance matrix revealed different bacterial clusters between feces and cecal contents in all groups (P < 0.01). Interestingly, the community structures of the three treatment groups clustered separately from each other in both gut regions (P < 0.05). Contrary to our hypothesis, the α-diversity metric, Chao1, indicated that VWR led to reduced bacterial richness compared with FTR or SED (P < 0.05). Taxonomic evaluation revealed that both VWR and FTR altered many individual bacterial taxa. Of particular interest, Turicibacter spp., which has been strongly associated with immune function and bowel disease, was significantly lower in VWR vs. SED/FTR. These data indicate that VWR and FTR differentially alter the intestinal microbiome of mice. These effects were observed in both the feces and cecum despite vastly different community structures between each intestinal region.

Functional phylotyping approach for assessing intraspecific diversity of Ruminococcus albus within the rumen microbiome.

Ruminococcus albus, a cellulolytic bacterium, is a critical member of the rumen community. Ruminococcus albus lacks a classical cellulosome complex, but it possesses a unique family 37 carbohydrate-binding module (CBM37), which is integrated into a variety of carbohydrate-active enzymes. We developed a potential molecular tool for functional phylotyping of the R. albus population in the rumen, based on a variable region in the cel48A gene. cel48A encodes a single copy of the CBM37-associated family 48 glycoside hydrolase in all known strains of this bacterium. A segment of the cel48A gene was amplified from rumen metagenomic samples of four bovines, and its abundance and diversity were evaluated. Analysis of the obtained sequences revealed the co-existence of multiple functional phylotypes of cel48A in all four animals. These included sequences identical or similar to those of R. albus isolates (reference strains), as well as several novel sequences. The dominant cel48A type varied among animals. This method can be used for detection of intraspecific diversity of R. albus in metagenomic samples. Together with scaC, a previously reported gene marker for R. flavefaciens, we present a set of two species-specific markers for phylotyping of Ruminococci in the herbivore rumen.

Pregnancy's stronghold on the vaginal microbiome.

OBJECTIVE: To assess the vaginal microbiome throughout full-term uncomplicated pregnancy. METHODS: Vaginal swabs were obtained from twelve pregnant women at 8-week intervals throughout their uncomplicated pregnancies. Patients with symptoms of vaginal infection or with recent antibiotic use were excluded. Swabs were obtained from the posterior fornix and cervix at 8-12, 17-21, 27-31, and 36-38 weeks of gestation. The microbial community was profiled using hypervariable tag sequencing of the V3-V5 region of the 16S rRNA gene, producing approximately 8 million reads on the Illumina MiSeq. RESULTS: Samples were dominated by a single genus, Lactobacillus, and exhibited low species diversity. For a majority of the patients (n = 8), the vaginal microbiome was dominated by Lactobacillus crispatus throughout pregnancy. Two patients showed Lactobacillus iners dominance during the course of pregnancy, and two showed a shift between the first and second trimester from L. crispatus to L. iners dominance. In all of the samples only these two species were identified, and were found at an abundance of higher than 1% in this study. Comparative analyses also showed that the vaginal microbiome during pregnancy is characterized by a marked dominance of Lactobacillus species in both Caucasian and African-American subjects. In addition, our Caucasian subject population clustered by trimester and progressed towards a common attractor while African-American women clustered by subject instead and did not progress towards a common attractor. CONCLUSION: Our analyses indicate normal pregnancy is characterized by a microbiome that has low diversity and high stability. While Lactobacillus species strongly dominate the vaginal environment during pregnancy across the two studied ethnicities, observed differences between the longitudinal dynamics of the analyzed populations may contribute to divergent risk for pregnancy complications. This helps establish a baseline for investigating the role of the microbiome in complications of pregnancy such as preterm labor and preterm delivery.

Loss of sex and age driven differences in the gut microbiome characterize arthritis-susceptible 0401 mice but not arthritis-resistant 0402 mice.

BACKGROUND: HLA-DRB1 0401 is associated with susceptibility, while HLA-DRB1 0402 is associated with resistance to developing rheumatoid arthritis (RA) and collagen-induced arthritis in humans and transgenic mice respectively. The influence of gut-joint axis has been suggested in RA, though not yet proven. METHODOLOGY/PRINCIPAL FINDINGS: We have used HLA transgenic mice carrying arthritis susceptible and -resistant HLA-DR genes to explore if genetic factors and their interaction with gut flora gut can be used to predict susceptibility to develop arthritis. Pyrosequencing of the 16S rRNA gene from the fecal microbiomes of DRB1 0401 and DRB1 0402 transgenic mice revealed that the guts of 0401 mice is dominated by a Clostridium-like bacterium, whereas the guts of 0402 mice are enriched for members of the Porphyromonadaceae family and Bifidobacteria. DRB1 0402 mice harbor a dynamic sex and age-influenced gut microbiome while DRB1 0401 mice did not show age and sex differences in gut microbiome even though they had altered gut permeability. Cytokine transcripts, measured by rtPCR, in jejuna showed differential TH17 regulatory network gene transcripts in 0401 and 0402 mice. CONCLUSIONS/SIGNIFICANCE: We have demonstrated for the first time that HLA genes in association with the gut microbiome may determine the immune environment and that the gut microbiome might be a potential biomarker as well as contributor for susceptibility to arthritis. Identification of pathogenic commensal bacteria would provide new understanding of disease pathogenesis, thereby leading to novel approaches for therapy.

Phage-bacteria relationships and CRISPR elements revealed by a metagenomic survey of the rumen microbiome.

Viruses are the most abundant biological entities on the planet and play an important role in balancing microbes within an ecosystem and facilitating horizontal gene transfer. Although bacteriophages are abundant in rumen environments, little is known about the types of viruses present or their interaction with the rumen microbiome. We undertook random pyrosequencing of virus-enriched metagenomes (viromes) isolated from bovine rumen fluid and analysed the resulting data using comparative metagenomics. A high level of diversity was observed with up to 28,000 different viral genotypes obtained from each environment. The majority (~78%) of sequences did not match any previously described virus. Prophages outnumbered lytic phages approximately 2:1 with the most abundant bacteriophage and prophage types being associated with members of the dominant rumen phyla (Firmicutes and Proteobacteria). Metabolic profiling based on SEED subsystems revealed an enrichment of sequences with putative functional roles in DNA and protein metabolism, but a surprisingly low proportion of sequences assigned to carbohydrate and amino acid metabolism. We expanded our analysis to include previously described metagenomic data and 14 reference genomes. Clustered regularly interspaced short palindromic repeats (CRISPR) were detected in most of the microbial genomes, suggesting previous interactions between viral and microbial communities.

Cellulosomics, a gene-centric approach to investigating the intraspecific diversity and adaptation of Ruminococcus flavefaciens within the rumen.

BACKGROUND: The bovine rumen maintains a diverse microbial community that serves to break down indigestible plant substrates. However, those bacteria specifically adapted to degrade cellulose, the major structural component of plant biomass, represent a fraction of the rumen microbiome. Previously, we proposed scaC as a candidate for phylotyping Ruminococcus flavefaciens, one of three major cellulolytic bacterial species isolated from the rumen. In the present report we examine the dynamics and diversity of scaC-types both within and between cattle temporally, following a dietary switch from corn-silage to grass-legume hay. These results were placed in the context of the overall bacterial population dynamics measured using the 16S rRNA. PRINCIPAL FINDINGS: As many as 117 scaC-types were estimated, although just nineteen were detected in each of three rumens tested, and these collectively accounted for the majority of all types present. Variation in scaC populations was observed between cattle, between planktonic and fiber-associated fractions and temporally over the six-week survey, and appeared related to scaC phylogeny. However, by the sixth week no significant separation of scaC populations was seen between animals, suggesting enrichment of a constrained set of scaC-types. Comparing the amino-acid translation of each scaC-type revealed sequence variation within part of the predicted dockerin module but strong conservation in the N-terminus, where the cohesin module is located. CONCLUSIONS: The R. flavefaciens species comprises a multiplicity of scaC-types in-vivo. Enrichment of particular scaC-types temporally, following a dietary switch, and between fractions along with the phylogenetic congruence suggests that functional differences exist between types. Observed differences in dockerin modules suggest at least part of the functional heterogeneity may be conferred by scaC. The polymorphic nature of scaC enables the relative distribution of R. flavefaciens strains to be examined and represents a gene-centric approach to investigating the intraspecific adaptation of an important specialist population.

Abundance and diversity of dockerin-containing proteins in the fiber-degrading rumen bacterium, Ruminococcus flavefaciens FD-1.

BACKGROUND: The cellulosome is a multi-enzyme machine, which plays a key role in the breakdown of plant cell walls in many anaerobic cellulose-degrading microorganisms. Ruminococcus flavefaciens FD-1, a major fiber-degrading bacterium present in the gut of herbivores, has the most intricate cellulosomal organization thus far described. Cellulosome complexes are assembled through high-affinity cohesin-dockerin interactions. More than two-hundred dockerin-containing proteins have been identified in the R. flavefaciens genome, yet the reason for the expansion of these crucial cellulosomal components is yet unknown. METHODOLOGY/PRINCIPAL FINDINGS: We have explored the full spectrum of 222 dockerin-containing proteins potentially involved in the assembly of cellulosome-like complexes of R. flavefaciens. Bioinformatic analysis of the various dockerin modules showed distinctive conservation patterns within their two Ca(2+)-binding repeats and their flanking regions. Thus, we established the conceptual framework for six major groups of dockerin types, according to their unique sequence features. Within this framework, the modular architecture of the parent proteins, some of which are multi-functional proteins, was evaluated together with their gene expression levels. Specific dockerin types were found to be associated with selected groups of functional components, such as carbohydrate-binding modules, numerous peptidases, and/or carbohydrate-active enzymes. In addition, members of other dockerin groups were linked to structural proteins, e.g., cohesin-containing proteins, belonging to the scaffoldins. CONCLUSIONS/SIGNIFICANCE: This report profiles the abundance and sequence diversity of the R. flavefaciens FD-1 dockerins, and provides the molecular basis for future understanding of the potential for a wide array of cohesin-dockerin specificities. Conserved differences between dockerins may be reflected in their stability, function or expression within the context of the parent protein, in response to their role in the rumen environment.

Diversity and strain specificity of plant cell wall degrading enzymes revealed by the draft genome of Ruminococcus flavefaciens FD-1.

BACKGROUND: Ruminococcus flavefaciens is a predominant cellulolytic rumen bacterium, which forms a multi-enzyme cellulosome complex that could play an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides. Identifying the major enzyme types involved in plant cell wall degradation is essential for gaining a better understanding of the cellulolytic capabilities of this organism as well as highlighting potential enzymes for application in improvement of livestock nutrition and for conversion of cellulosic biomass to liquid fuels. METHODOLOGY/PRINCIPAL FINDINGS: The R. flavefaciens FD-1 genome was sequenced to 29x-coverage, based on pulsed-field gel electrophoresis estimates (4.4 Mb), and assembled into 119 contigs providing 4,576,399 bp of unique sequence. As much as 87.1% of the genome encodes ORFs, tRNA, rRNAs, or repeats. The GC content was calculated at 45%. A total of 4,339 ORFs was detected with an average gene length of 918 bp. The cellulosome model for R. flavefaciens was further refined by sequence analysis, with at least 225 dockerin-containing ORFs, including previously characterized cohesin-containing scaffoldin molecules. These dockerin-containing ORFs encode a variety of catalytic modules including glycoside hydrolases (GHs), polysaccharide lyases, and carbohydrate esterases. Additionally, 56 ORFs encode proteins that contain carbohydrate-binding modules (CBMs). Functional microarray analysis of the genome revealed that 56 of the cellulosome-associated ORFs were up-regulated, 14 were down-regulated, 135 were unaffected, when R. flavefaciens FD-1 was grown on cellulose versus cellobiose. Three multi-modular xylanases (ORF01222, ORF03896, and ORF01315) exhibited the highest levels of up-regulation. CONCLUSIONS/SIGNIFICANCE: The genomic evidence indicates that R. flavefaciens FD-1 has the largest known number of fiber-degrading enzymes likely to be arranged in a cellulosome architecture. Functional analysis of the genome has revealed that the growth substrate drives expression of enzymes predicted to be involved in carbohydrate metabolism as well as expression and assembly of key cellulosomal enzyme components.