Ruminants have co-evolved with their gastrointestinal microbial communities that digest plant materials to provide energy for the host. Some arctic and boreal ruminants have already shown to be vulnerable to dietary shifts caused by changing climate, yet we know little about the metabolic capacity of the ruminant microbiome in these animals. Here, we use meta-omics approaches to sample rumen fluid microbial communities from Alaskan moose foraging along a seasonal lignocellulose gradient. Winter diets with increased hemicellulose and lignin strongly enriched for BS11, a Bacteroidetes family lacking cultivated or genomically sampled representatives. We show that BS11 are cosmopolitan host-associated bacteria prevalent in gastrointestinal tracts of ruminants and other mammals. Metagenomic reconstruction yielded the first four BS11 genomes; phylogenetically resolving two genera within this previously taxonomically undefined family. Genome-enabled metabolic analyses uncovered multiple pathways for fermenting hemicellulose monomeric sugars to short-chain fatty acids (SCFA), metabolites vital for ruminant energy. Active hemicellulosic sugar fermentation and SCFA production was validated by shotgun proteomics and rumen metabolites, illuminating the role BS11 have in carbon transformations within the rumen. Our results also highlight the currently unknown metabolic potential residing in the rumen that may be vital for sustaining host energy in response to a changing vegetative environment.

Our objectives were to evaluate potential signaling pathways regulating rumen protozoal chemotaxis using eukaryotic inhibitors potentially coordinated with phagocytosis as assessed by fluorescent bead uptake kinetics. Wortmannin (inhibitor of phosphoinositide 3-kinase), insulin, genistein (purported inhibitor of a receptor tyrosine kinase), U73122 (inhibitor of phospholipase C), and sodium nitroprusside (Snp, nitric oxide generator, activating protein kinase G) were preincubated with mixed ruminal protozoa for 3h before assessing uptake of fluorescent beads and chemosensory behavior to glucose, peptides, and their combination; peptides were also combined with guanosine triphosphate (GTP; a chemorepellent). Entodiniomorphids were chemoattracted to both glucose and peptides, but chemoattraction to glucose was increased by Snp and wortmannin without effect on chemoattraction to peptides. Rate of fluorescent bead uptake by an Entodinium caudatum culture decreased when beads were added simultaneously with feeding and incubated with wortmannin (statistical interaction). Wortmannin also decreased the proportion of mixed entodiniomorphids consuming beads. Isotrichid protozoa exhibited greater chemotaxis to glucose but, compared with entodiniomorphids, were chemorepelled to peptides. Wortmannin increased chemotaxis by entodiniomorphids but decreased chemotaxis to glucose by isotrichids. Motility assays documented that Snp and wortmannin decreased net swimming speed (distance among 2 points per second) but not total swimming speed (including turns) by entodiniomorphids. Wortmannin decreased both net and total swimming behavior in isotrichids. Results mechanistically explain the isotrichid migratory ecology to rapidly take up newly ingested sugars and subsequent sedimentation back to the ventral reticulorumen. In contrast, entodiniomorphids apparently integrate cellular motility with feeding behavior to consume small particulates and thereby stay associated and pass with the degradable fraction of rumen particulates. These results extend findings from aerobic ciliate models to explain how rumen protozoa have adapted physiology for their specific ecological niches.

BackgroundRumen ciliates play important roles in rumen function by digesting and fermenting feed and shaping the rumen microbiome. However, they remain poorly understood due to the lack of definitive direct evidence without influence by prokaryotes (including symbionts) in co-cultures or the rumen. In this study, we used RNA-Seq to characterize the transcriptome of Entodinium caudatum, the most predominant and representative rumen ciliate species.ResultsOf a large number of transcripts, > 12,000 were annotated to the curated genes in the NR, UniProt, and GO databases. Numerous CAZymes (including lysozyme and chitinase) and peptidases were represented in the transcriptome. This study revealed the ability of E. caudatum to depolymerize starch, hemicellulose, pectin, and the polysaccharides of the bacterial and fungal cell wall, and to degrade proteins. Many signaling pathways, including the ones that have been shown to function in E. caudatum, were represented by many transcripts. The transcriptome also revealed the expression of the genes involved in symbiosis, detoxification of reactive oxygen species, and the electron-transport chain. Overall, the transcriptomic evidence is consistent with some of the previous premises about E. caudatum. However, the identification of specific genes, such as those encoding lysozyme, peptidases, and other enzymes unique to rumen ciliates might be targeted to develop specific and effective inhibitors to improve nitrogen utilization efficiency by controlling the activity and growth of rumen ciliates. The transcriptomic data will also help the assembly and annotation in future genomic sequencing of E. caudatum.ConclusionAs the first transcriptome of a single species of rumen ciliates ever sequenced, it provides direct evidence for the substrate spectrum, fermentation pathways, ability to respond to various biotic and abiotic stimuli, and other physiological and ecological features of E. caudatum. The presence and expression of the genes involved in the lysis and degradation of microbial cells highlight the dependence of E. caudatum on engulfment of other rumen microbes for its survival and growth. These genes may be explored in future research to develop targeted control of Entodinium species in the rumen. The transcriptome can also facilitate future genomic studies of E. caudatum and other related rumen ciliates.

Nitrates have been fed to ruminants, including dairy cows, as an electron sink to mitigate CH 4 emissions. In the NO 3 − reduction process, NO 2 − can accumulate, which could directly inhibit methanogens and possibly other microbes in the rumen. Saccharomyces cerevisiae yeast was hypothesized to decrease NO 2 − through direct reduction or indirectly by stimulating the bacterium Selenomonas ruminantium, which is among the ruminal bacteria most well characterized to reduce both NO 3 − and NO 2 −. Ruminal fluid was incubated in continuous cultures fed diets without or with NaNO 3 (1.5% of diet dry matter; i.e., 1.09% NO 3 −) and without or with live yeast culture (LYC) fed at a recommended 0.010 g/d (scaled from cattle to fermentor intakes) in a 2 × 2 factorial arrangement of treatments. Treatments with LYC had increased NDF digestibility and acetate: propionate by increasing acetate molar proportion but tended to decrease total VFA production. The main effect of NO 3 − increased acetate: propionate by increasing acetate molar proportion; NO 3 − also decreased molar proportions of isobutyrate and butyrate. Both NO 3 − and LYC shifted bacterial community composition (based on relative sequence abundance of 16S rRNA genes). An interaction occurred such that NO 3 − decreased valerate molar proportion only when no LYC was added. Nitrate decreased daily CH 4 emissions by 29%. However, treatment × time interactions were present for both CH 4 and H 2 emission from the headspace; CH 4 was decreased by the main effect of NO 3 − until 6 h postfeeding, but NO 3 − and LYC decreased H 2 emission up to 4 h postfeeding. As expected, NO 3 − decreased methane emissions in continuous cultures; however, contrary to expectations, LYC did not attenuate NO 2 − accumulation.

In dairy rations, Met is often a limiting amino acid that is provided by rumen-undegradable protein and rumen-protected sources of Met. A Met precursor, 2-hydroxy-4-(methylthio) butanoic acid (HMB) has undergone considerable study for ruminal and postruminal metabolism, whereas its isopropyl ester (HMBi) has been evaluated primarily with respect to its supply of metabolizable Met rather than as a preformed source of Met for microbial metabolism. A control and 3 isomolar Met treatments-0.097% dl-Met, 0.048% dl-Met plus 0.055% HMBi (Met + HMBi treatment), and 0.11% HMBi-were pulse-dosed every 8h into continuous cultures simultaneously with feeding. Treatment had no effect on digestibilities of acid-detergent fiber or true organic matter. Digestibilities of neutral detergent fiber and hemicellulose were linearly decreased with increasing HMBi inclusion. Concentration of NH3-N tended to decrease linearly and quadratically, and NH3-N flow tended to decrease linearly, with increasing HMBi inclusion; in contrast, the proportion of bacterial N derived from NH3-N increased linearly. Peptide N increased linearly and tended to be affected quadratically (highest for the HMBi treatment). Acetate and propionate production both decreased with increasing HMBi, but acetate declined more such that acetate:propionate increased linearly. Isobutyrate production decreased, but isovalerate and valerate increased with increasing HMBi inclusion. Relative changes in population abundance were not detected by denaturing gradient gel electrophoresis. In the second study, which was done in batch culture, Met treatments consisted of control, 0.097% l-Met, 0.097% l-Met, 0.125% dl-HMBi, 0.098% dl-HMB, 0.250% dl-HMBi (2× HMBi), 0.049% dl-Met + 0.063% dl-HMBi (Met + HMBi), and 0.098% dl-HMB + 0.039% isopropanol. All of these Met treatments were unlabeled (i.e., at natural abundance of (13)C) but simultaneously dosed with equivalent dosages of [1-(13)C]-l-Met. All 8 treatments were inoculated with faunated or partially defaunated inocula. Protozoal abundance had minor effect on measurements. The unlabeled l-Met treatment had the lowest (13)C enrichment of Met in the microbial pellet followed by Met + HMBi and then d-Met or dl-HMB, which were lower than remaining treatments. The percentage of the [1-(13)C]-l-Met dose recovered in microbial Met was lowest for the l-Met treatment; intermediate for d-Met, dl-HMB (with or without isopropanol), and Met + HMBi treatments; and highest for HMBi, 2× HMBi, and control. Results suggest that racemization of d-Met lags behind l-Met. The similar conversions of the HMBi and 2× HMBi treatments compared with the control suggests a low degradation of HMBi to provide unlabeled Met to dilute the [1-(13)C]-l-Met dose for protein synthesis. The lack of treatment by time interaction suggests that these initial responses carried through during the 24h of incubation. The proportion of HMBi available to ruminal microbes can influence microbial metabolism, potentially through formation of l-Met.