Divergent functional isoforms drive niche specialisation for nutrient acquisition and use in rumen microbiome.
ABSTRACT: Many microbes in complex competitive environments share genes for acquiring and utilising nutrients, questioning whether niche specialisation exists and if so, how it is maintained. We investigated the genomic signatures of niche specialisation in the rumen microbiome, a highly competitive, anaerobic environment, with limited nutrient availability determined by the biomass consumed by the host. We generated individual metagenomic libraries from 14 cows fed an ad libitum diet of grass silage and calculated functional isoform diversity for each microbial gene identified. The animal replicates were used to calculate confidence intervals to test for differences in diversity of functional isoforms between microbes that may drive niche specialisation. We identified 153 genes with significant differences in functional isoform diversity between the two most abundant bacterial genera in the rumen (Prevotella and Clostridium). We found Prevotella possesses a more diverse range of isoforms capable of degrading hemicellulose, whereas Clostridium for cellulose. Furthermore, significant differences were observed in key metabolic processes indicating that isoform diversity plays an important role in maintaining their niche specialisation. The methods presented represent a novel approach for untangling complex interactions between microorganisms in natural environments and have resulted in an expanded catalogue of gene targets central to rumen cellulosic biomass degradation.
Project description:BACKGROUND: Sika deer (Cervus nippon) have different dietary preferences to other ruminants and are tolerant to tannin-rich plants. Because the rumen bacteria in domestic Sika deer have not been comprehensively studied, it is important to investigate its rumen bacterial population in order to understand its gut health and to improve the productivity of domestic Sika deer. RESULTS: The rumen bacterial diversity in domestic Sika deer (Cervus nippon) fed oak leaves- (OL group) and corn stalks-based diets (CS group) were elucidated using 16S rRNA gene libraries and denaturing gradient gel electrophoresis (DGGE). Overall, 239 sequences were examined from the two groups, 139 clones from the OL group were assigned to 57 operational taxonomic units (OTUs) and 100 sequences from the CS group were divided into 50 OTUs. Prevotella-like sequences belonging to the phylum Bacteroidetes were the dominant bacteria in both groups (97.2% OL and 77% CS), and sequences related to Prevotella brevis were present in both groups. However, Prevotella shahii-like, Prevotella veroralis-like, Prevotella albensis-like, and Prevotella salivae-like sequences were abundant in the OL group compared to those in the CS group, while Succinivibrio dextrinosolvens-like and Prevotella ruminicola-like sequences were prevalent in the CS group. PCR-DGGE showed that bacterial communities clustered with respect to diets and the genus Prevotella was the dominant bacteria in the rumen of domestic Sika deer. However, the distribution of genus Prevotella from two groups was apparent. In addition, other fibrolytic bacteria, such as Clostridium populeti and Eubacterium cellulosolvens were found in the rumen of domestic Sika deer. CONCLUSIONS: The rumen of domestic Sika deer harbored unique bacteria which may represent novel species. The bacterial composition appeared to be affected by diet, and sequences related to Prevotella spp. may represent new species that may be related to the degradation of fiber biomass or tannins. Moreover, the mechanism and biological functions of Prevotella spp. in the rumen ecosystem, and synergistic interactions with other microorganisms should be noticed.
Project description:Understanding the relationship between ingested plant material and the attached microbiome is essential for developing methodologies to improve ruminant nutrient use efficiency. We have previously shown that perennial ryegrass (PRG) rumen bacterial colonization events follow a primary (up to 4 h) and secondary (after 4 h) pattern based on the differences in diversity of the attached bacteria. In this study, we investigated temporal niche specialization of primary and secondary populations of attached rumen microbiota using metagenomic shotgun sequencing as well as monitoring changes in the plant chemistry using mid-infrared spectroscopy (FT-IR). Metagenomic Rapid Annotation using Subsystem Technology (MG-RAST) taxonomical analysis of shotgun metagenomic sequences showed that the genera <i>Butyrivibrio, Clostridium, Eubacterium, Prevotella</i>, and <i>Selenomonas</i> dominated the attached microbiome irrespective of time. MG-RAST also showed that <i>Acidaminococcus, Bacillus, Butyrivibrio</i>, and <i>Prevotella</i> rDNA increased in read abundance during secondary colonization, whilst <i>Blautia</i> decreased in read abundance. MG-RAST Clusters of Orthologous Groups (COG) functional analysis also showed that the primary function of the attached microbiome was categorized broadly within "metabolism;" predominantly amino acid, carbohydrate, and lipid metabolism and transport. Most sequence read abundances (51.6, 43.8, and 50.0% of COG families pertaining to amino acid, carbohydrate and lipid metabolism, respectively) within these categories were higher in abundance during secondary colonization. Kyoto encyclopedia of genes and genomes (KEGG) pathways analysis confirmed that the PRG-attached microbiota present at 1 and 4 h of rumen incubation possess a similar functional capacity, with only a few pathways being uniquely found in only one incubation time point only. FT-IR data for the plant residues also showed that the main changes in plant chemistry between primary and secondary colonization was due to increased carbohydrate, amino acid, and lipid metabolism. This study confirmed primary and secondary colonization events and supported the hypothesis that functional changes occurred as a consequence of taxonomical changes. Sequences within the carbohydrate metabolism COG families contained only 3.2% of cellulose activities, on average across both incubation times (1 and 4 h), suggesting that degradation of the plant cell walls may be a key rate-limiting factor in ensuring the bioavailability of intra-plant nutrients in a timely manner to the microbes and ultimately the animal. This suggests that a future focus for improving ruminant nutrient use efficiency should be altering the recalcitrant plant cell wall components and/or improving the cellulolytic capacity of the rumen microbiota.
Project description:The present study was aimed at understanding a shift in rumen microbiome of buffaloes fed various levels of total digestible nutrients. To understand the process, the metagenomics of rumen microbes, in vivo and in vitro rumen fermentation studies were carried out. Three rumen fistulated adult male Murrah buffaloes were fed three isonitrogenous diets varying in total digestible nutrients (70, 85 and 100% of TDN requirement) in 3X3 switch over design. On dry matter basis, wheat straw/ roughage content were 81, 63 and 51% and that of maize grain was 8, 16 and 21% in three diets respectively. After 20 d of feeding, rumen liquor and rumen contents were sampled just before (0h) and 4h post feeding. Ruminococcus flavefaciens and R. albus (estimated with real time PCR) were higher in high roughage diets. The predominant phyla in all the three groups were Bacteroidetes, Firmicutes followed by Proteobacteria, Actinobacteria and Fibrobacteres. A core group of more than fifty rumen bacteria was present in all the animals with very little variations due to level of TDN. The most predominant bacterial genera reported in order of decreasing abundance were: Prevotella, Bacteroides, Clostridium, Ruminococcus, Eubacterium, Parabacteroides, Fibrobacter, Butyrivibrio etc. The higher diversity of the enyzmes families GH 23, GH 28, GH 39, GH 97, GH 106, and GH 127 (the enzymes active in fibre and starch degradation) were significantly higher on 100%TDN diet while CE 14 (required for the hydrolysis of bond between carbohydrate and lignin) was higher on low TDN (70%) diet, indicating ester bond cleavage was better in animals fed high roughage (wheat straw) diet.
Project description:To identify differences in rumen function as a result of feeding monensin to beef cattle, rumen fluid metagenomics and metabolomics analyses were used to evaluate the functional attributes and metabolites of rumen microbiota in beef steers fed no or 200 mg/d of monensin. Eight rumen-fistulated steers were used in the study for a period of 53 days. Rumen fluid samples were collected on the last day of the experiment. Monensin increased the relative abundance of Selenomonas sp. ND2010, Prevotella dentalis, Hallella seregens, Parabacteroides distasonis, Propionispira raffinosivorans, and Prevotella brevis, but reduced the relative abundance of Robinsoniella sp. KNHs210, Butyrivibrio proteoclasticus, Clostridium botulinum, Clostridium symbiosum, Burkholderia sp. LMG29324, and Clostridium butyricum. Monensin increased the relative abundance of functional genes involved in amino acid metabolism and lipid metabolism. A total of 245 metabolites were identified. Thirty-one metabolites were found to be differentially expressed. Pathway analysis of the differentially expressed metabolites revealed upregulated metabolic pathways associated with metabolism of linoleic acid and some amino acids. These findings confirm that monensin affects rumen fermentation of forage-fed beef cattle by modulating the rumen microbiome, and by reducing amino acid degradation and biohydrogenation of linoleic acid in the rumen.
Project description:Increasing feed efficiency is a key target in ruminant science which requires a better understanding of rumen microbiota. This study investigated the effect of a shift from a non-grazing to a grazing diet on the rumen bacterial, methanogenic archaea, fungal, and protozoal communities. A systems biology approach based on a description of the community structure, core microbiota, network analysis, and taxon abundance linked to the rumen fermentation was used to explore the benefits of increasing depth of the community analysis. A total of 24 sheep were fed ryegrass hay supplemented with concentrate (CON) and subsequently ryegrass pasture (PAS) following a straight through experimental design. Results showed that concentrate supplementation in CON-fed animals (mainly starch) promoted a simplified rumen microbiota in terms of network density and bacterial, methanogen and fungal species richness which favored the proliferation of amylolytic microbes and VFA production (+48%), but led to a lower (ca. 4-fold) ammonia concentration making the N availability a limiting factor certain microbes. The adaptation process from the CON to the PAS diet consisted on an increase in the microbial concentration (biomass of bacteria, methanogens, and protozoa), diversity (+221, +3, and +21 OTUs for bacteria, methanogens, and fungi, respectively), microbial network complexity (+18 nodes and +86 edges) and in the abundance of key microbes involved in cellulolysis (Ruminococcus, Butyrivibrio, and Orpinomyces), proteolysis (Prevotella and Entodiniinae), lactate production (Streptococcus and Selenomonas), as well as methylotrophic archaea (Methanomassiliicoccaceae). This microbial adaptation indicated that pasture degradation is a complex process which requires a diverse consortium of microbes working together. The correlations between the abundance of microbial taxa and rumen fermentation parameters were not consistent across diets suggesting a metabolic plasticity which allowed microbes to adapt to different substrates and to shift their fermentation products. The core microbiota was composed of 34, 9, and 13 genera for bacteria, methanogens, and fungi, respectively, which were shared by all sheep, independent of diet. This systems biology approach adds a new dimension to our understanding of the rumen microbial interactions and may provide new clues to describe the mode of action of future nutritional interventions.
Project description:Niche modification is a process whereby the activity of organisms modifies their local environment creating new niches for other organisms. This process can have a substantial role in community assembly of gut microbial ecosystems due to their vast and complex metabolic activities. We studied the postprandial diurnal community oscillatory patterns of the rumen microbiome and showed that metabolites produced by the rumen microbiome condition its environment and lead to dramatic diurnal changes in community composition and function. After feeding, microbiome composition undergoes considerable change in its phylogenetic breadth manifested as a significant 3-5-fold change in the relative abundance of methanogenic archaea and main bacterial taxa such as Prevotella, in a manner that was independent of individual host variation and diet. These changes in community composition were accompanied by changes in pH and methane partial pressure, suggesting a strong functional connection. Notably, cross-incubation experiments combining metabolites and organisms from different diurnal time points showed that the metabolites released by microbes are sufficient to reproduce changes in community function comparable to those observed in vivo. These findings highlight microbiome niche modification as a deterministic process that drives diurnal community assembly via environmental filtering.
Project description:The rumen microbiota plays an important role in animal functional attributes. These microbes are indispensable for the normal physiological development of the rumen, and may also convert the plant polysaccharides from grass into available milk and meat, making it highly valuable to humans. Exploring the microbial composition and metabolites of rumen across developmental stages is important for understanding ruminant nutrition and metabolism. However, relatively few reports have investigated the microbiome and metabolites across developmental stages in ruminants. Using 16S rRNA gene sequnecing, metabolomics and high-performance liquid chromatography techniques, we compared the rumen microbiota, metabolites and short chain fatty acids (SCFAs) between lambs and sub-adult Tibetan sheep (Ovis aries) from Qinghai-Tibetan Plateau. Bacteroidetes and Spirochaetae were enriched in sub-adult sheep, while Firmicutes and Tenericutes were more abundant in young individuals. The sub-adult individuals had higher alpha diversity values than those in young sheep. Metabolomics analysis showed that the content of essential amino acids and related gene functional pathways in rumen were different between the lambs and sub-adult population. L-Leucine that participates in valine, leucine and isoleucine biosynthesis was more abundant in the lambs, while phenylethylamine that takes part in phenylalanine metabolism was more enriched in the sub-adults. Both rumen microbial community structures and metabolite profiles were impacted by age, but rumen SCFA concentration was relatively stable between different age stages. Some specific microbes (e.g., Clostridium and Ruminococcaceae) were positively associated with L-Leucine but negatively correlated with phenylethylamine, implying that rumen microbes may play different roles for metabolite production at different ages. Mantel test analysis showed that rumen microbiota was significantly correlated with metabolomics and SCFA profiles. Our results indicates the close relationship between microbial composition and metabolites, and also reveal different nutritional requirement for different ages in ruminants, thus having important significance for regulating animal nutrition and metabolism by microbiome intervention.
Project description:High throughput sequencing was used to examine the rumen microbiota of sika deer fed high (OLH) and low concentration (OLL) of tannin rich oak leaves. The results showed that Prevotella spp. were the most dominant bacteria. The most predominant methanogens were the members of the order Methanoplasmatales. The dominant rumen protozoa were Entodinium longinucleatum, Eudiplodinium maggii, and Epidinium caudatum, and the fungal communities were mostly represented by Piromyces spp. Moreover, the relative abundance of Pseudobutyrivibrio spp. (P=0.026), unidentified bacteria (P=0.028), and Prevotella spp. (P=0.022) was lower in the OLH group than in the OLL group. The concentration of propionate in the OLH group was greater than in the OLL group (P=0.006). Patterns of relationships showed that methanogens belonging to the order Methanoplasmatales were negatively correlated with Treponema spp., Ent. Longinucleatum, and acetate. Methanosphaera stadtmanae was positively correlated to propionate, while Methanobrevibacter ruminantium was negatively associated with Methanobrevibacter thaueri and Methanobrevibacter millerae. Tannins altered the rumen microbes and fermentation patterns. However, the response of the entire rumen microbiota and the relationship between rumen microorganisms and the fermentation parameters were not fully understood.
Project description:BACKGROUND:The rumen contains a myriad of microbes whose primary role is to degrade and ferment dietary nutrients, which then provide the host with energy and nutrients. Rumen microbes commonly attach to ingested plant materials and form biofilms for effective plant degradation. Quorum sensing (QS) is a well-recognised form of bacterial communication in most biofilm communities, with homoserine lactone (AHL)-based QS commonly being used by Gram-negative bacteria alone and AI-2 Lux-based QS communication being used to communicate across Gram-negative and Gram-positive bacteria. However, bacterial cell to cell communication in the rumen is poorly understood. In this study, rumen bacterial genomes from the Hungate collection and Genbank were prospected for QS-related genes. To check that the discovered QS genes are actually expressed in the rumen, we investigated expression levels in rumen metatranscriptome datasets. RESULTS:A total of 448 rumen bacterial genomes from the Hungate collection and Genbank, comprised of 311 Gram-positive, 136 Gram-negative and 1 Gram stain variable bacterium, were analysed. Abundance and distribution of AHL and AI-2 signalling genes showed that only one species (Citrobacter sp. NLAE-zl-C269) of a Gram-negative bacteria appeared to possess an AHL synthase gene, while the Lux-based genes (AI-2 QS) were identified in both Gram-positive and Gram-positive bacteria (191 genomes representing 38.2% of total genomes). Of these 192 genomes, 139 are from Gram-positive bactreetteria and 53 from Gram-negative bacteria. We also found that the genera Butyrivibrio, Prevotella, Ruminococcus and Pseudobutyrivibrio, which are well known as the most abundant bacterial genera in the rumen, possessed the most lux-based AI-2 QS genes. Gene expression levels within the metatranscriptome dataset showed that Prevotella, in particular, expressed high levels of LuxS synthase suggesting that this genus plays an important role in QS within the rumen. CONCLUSION:This is the most comprehensive study of QS in the rumen microbiome to date. This study shows that AI-2-based QS is rife in the rumen. These results allow a greater understanding on plant-microbe interactions in the rumen.
Project description:Rumen microbiota plays an important role in animal productivity, methane production and health. Several different locations have been used to obtain rumen samples (i.e., liquid-phase samples, solid-phase samples, buccal swabs) in previous studies. Here we assess differences in the rumen microbiota between solid- and liquid-phases of the rumen under differing dietary conditions (white clover vs. perennial ryegrass); there were 4 sample types: liquid-associated/grass (LG), solid-associated/grass (SG), liquid-associated/clover (LC), and solid-associated/clover (SC). Four Holstein-Friesian cows were strip grazed on pure stands of perennial ryegrass or white clover in a change-over design experiment with 3 periods (each lasting for 3 weeks). Solid- and liquid- phase microbes were obtained following total rumen evacuation on the penultimate day of each period. DNA was extracted and multiplexed libraries sequenced using 16S next generation sequencing (Illumina MiSeq). Demultiplexed sequences underwent quality control and taxonomic profiles were generated for each sample. Statistical analysis for the effects of diet and phase was conducted both overall [using non-metric multidimensional scaling (NMDS) and diversity indices] and for individual taxa. Separation of both diet and phase was observed NMDS, with significant effects of diet (P < 0.001) and phase (P < 0.001) being observed. Regardless of diet, Prevotella was most abundant in the liquid samples. When assessing differences between phases, the majority of statistically significant taxa (predominantly from Archaea and the order Clostridiales) were found at higher relative abundances in solid-phase samples. Diversity (Shannon Index) was lower in the liquid-phase samples, possibly because of the higher relative abundance of Prevotella. A presence vs. absence approach, followed by Chi-squared testing, was adopted. Differences between phases (LG vs. LC, LC vs. LG, SG vs. SC, and SC vs. SG) and differences between phases for the clover diet (LC vs. SC and SC vs. LC) were significant (P < 0.001); differences between phases for the grass diet were non-significant. Sampling technique has a profound impact on reported microbial communities, which must be taken into consideration, particularly as archaea may be underestimated in the liquid-phase.