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Respiratory NADH oxidation in the rumen bacterium Prevotella bryantii is catalyzed by the Na+-translocating NADH:quinone oxidoreductase (NQR). A method for cell disruption and membrane isolation of P. bryantii under anoxic conditions using the EmulisFlex-C3 homogenizer is described. We compared NQR activity and protein yield after oxic and anoxic cell disruption by the EmulsiFlex, by ultrasonication, and by glass beads treatment. With an overall membrane protein yield of 50 mg L–1 culture and a NADH oxidation activity of 0.8 µmol min−1 mg−1, the EmulsiFlex was the most efficient method. Anoxic preparation yielded fourfold higher NQR activity compared to oxic preparation. P. bryantii lacks genes coding for superoxide dismutases and cell extracts do not exhibit superoxide dismutase activity. We propose that inactivation of NQR during oxic cell rupture is caused by superoxide, which accumulates in P. bryantii extracts exposed to air. Anoxic cell rupture is indispensable for the preparation of redox-active proteins and enzymes such as NQR from P. bryantii.

Monensin is an ionophore for monovalent cations, which is frequently used to prevent ketosis and to enhance performance in dairy cows. Studies have shown the rumen bacteria Prevotella bryantii B14 being less affected by monensin. The present study aimed to reveal more information about the respective molecular mechanisms in P.&nbsp, bryantii, as there is still a lack of knowledge about defense mechanisms against monensin. Cell growth experiments applying increasing concentrations of monensin and incubations up to 72 h were done. Harvested cells were used for label-free quantitative proteomics, enzyme activity measurements, quantification of intracellular sodium and extracellular glucose concentrations and fluorescence microscopy. Our findings confirmed an active cell growth and fermentation activity of P.&nbsp, bryantii B14 despite monensin concentrations up to 60 µM. An elevated abundance and activity of the Na+-translocating NADH:quinone oxidoreductase counteracted sodium influx caused by monensin. Cell membranes and extracellular polysaccharides were highly influenced by monensin indicated by a reduced number of outer membrane proteins, an increased number of certain glucoside hydrolases and an elevated concentration of extracellular glucose. Thus, a reconstruction of extracellular polysaccharides in P.&nbsp, bryantii in response to monensin is proposed, which is expected to have a negative impact on the substrate binding capacities of this rumen bacterium.

We investigated potential relationships between rumen microbiota and milk production in dairy cows during the transition period. Twelve dairy cows were divided into a low-yield (LY) or high-yield (HY) group based on their milk yield. Rumen samples were taken from dairy cows at 3 weeks before parturition, and at 4, 8, and 12 weeks after parturition. 16S rDNA-based metagenomic analysis showed that diversities of rumen microbiota in both groups were similar and the number of operational taxonomic units (OTUs) was lower in the postpartum than prepartum period in both groups. The abundance of Bacteroidetes and ratio of Bacteroidetes:Firmicutes was higher in the HY than the LY group. OTUs assigned to Prevotella bryantii, Fibrobacter succinogenes, Ruminococcus albus, Butyrivibrio fibrisolvens, and Succinivibrio sp. were abundant in the HY group. These OTUs were significantly related to the propionate molar proportion of rumen fluids in the HY group. OTUs assigned to Lachnospiraceae, Bifidobacterium sp. and Saccharofermentans were dominant in the LY group. Predictive functional profiling revealed that abundance of gene families involved in amino acid and vitamin metabolism was higher in the HY than the LY group. These results suggest that the community structure and fermentation products of rumen microbiota could be associated with milk production of dairy cows.

The effect of feeding a single TMR versus multiple rations across the day that differ in concentrations of neutral detergent fiber (NDF) and starch on the daily pattern of rumen microbial populations was characterized. Diets included a control total mixed ration (CON; 33.3% NDF), a low-fiber diet (LF; 29.6% NDF), and a high-fiber diet (HF; 34.8% NDF). Nine cannulated Holstein cows were assigned to 1 of 3 treatment sequences in a 3 × 3 Latin square design. Treatments included feeding CON ad libitum at 0900 h, feeding HF at 70% of daily offering at 0900 h and LF at 30% of daily offering at 2200 h (H/L), and feeding LF at 30% of daily offering at 0900 h and HF at 70% of daily offering at 1300 h (L/H). Rumen digesta was collected to represent every 3 h across the day, microbial DNA was extracted, and real-time quantitative PCR was used to determine the relative abundances of total bacteria, total fungi, total protozoa, Butyrivibrio fibrisolvens, Butyrivibrio hungatei, Fibrobacter succinogenes, Megasphaera elsdenii, Prevotella bryantii, Ruminococcus albus, Selenomonas ruminantium, and Streptococcus bovis. The relative abundances of total bacteria, total ciliated protozoa, F. succinogenes, P. bryantii, R. albus, S. ruminantium, and Strep. bovis were affected by time of day. Additionally, treatment affected the relative abundance of certain microbial groups at specific times of day. Notably, H/L treatment dramatically increased the relative abundances of B. fibrisolvens, B. hungatei, and Strep. bovis at 0900 h, by 2.5-, 5.4-, and 4.4-fold, respectively. Furthermore, the relative abundances of B. hungatei (3.9-fold), M. elsdenii (3.9-fold), R. albus (1.3-fold), S. ruminantium (1.3-fold), and Strep. bovis (4.5-fold) were greatly increased by L/H at 0900 h. At 0600 h, the relative abundance of F. succinogenes was 58% greater in L/H than H/L and the relative abundance of P. bryantii was 49% greater in H/L than L/H. Results suggest that there is a daily pattern of selected microbial populations that is altered by feeding rations that differ in NDF and starch within a day, with the greatest difference occurring before morning feeding.

Ruminants such as cattle and sheep depend on the breakdown of carbohydrates from plant-based feedstuff, which is accomplished by the microbial community in the rumen. Roughly 40% of the members of the rumen microbiota belong to the family Prevotellaceae, which ferments sugars to organic acids such as acetate, propionate, and succinate. These substrates are important nutrients for the ruminant. In a metaproteome analysis of the rumen of cattle, proteins that are homologous to the Na+-translocating NADH:quinone oxidoreductase (NQR) and the quinone:fumarate reductase (QFR) were identified in different Prevotella species. Here, we show that fumarate reduction to succinate in anaerobically growing Prevotella bryantii is coupled to chemiosmotic energy conservation by a supercomplex composed of NQR and QFR. This sodium-translocating NADH:fumarate oxidoreductase (SNFR) supercomplex was enriched by blue native PAGE (BN-PAGE) and characterized by in-gel enzyme activity staining and mass spectrometry. High NADH oxidation (850 nmol min−1 mg−1), quinone reduction (490 nmol min−1 mg−1), and fumarate reduction (1,200 nmol min−1 mg−1) activities, together with high expression levels, demonstrate that SNFR represents a charge-separating unit in P. bryantii. Absorption spectroscopy of SNFR exposed to different substrates revealed intramolecular electron transfer from the flavin adenine dinucleotide (FAD) cofactor in NQR to heme b cofactors in QFR. SNFR catalyzed the stoichiometric conversion of NADH and fumarate to NAD+ and succinate. We propose that the regeneration of NAD+ in P. bryantii is intimately linked to the buildup of an electrochemical gradient which powers ATP synthesis by electron transport phosphorylation. IMPORTANCE Feeding strategies for ruminants are designed to optimize nutrient efficiency for animals and to prevent energy losses like enhanced methane production. Key to this are the fermentative reactions of the rumen microbiota, dominated by Prevotella spp. We show that succinate formation by P. bryantii is coupled to NADH oxidation and sodium gradient formation by a newly described supercomplex consisting of Na+-translocating NADH:quinone oxidoreductase (NQR) and fumarate reductase (QFR), representing the sodium-translocating NADH:fumarate oxidoreductase (SNFR) supercomplex. SNFR is the major charge-separating module, generating an electrochemical sodium gradient in P. bryantii. Our findings offer clues to the observation that use of fumarate as feed additive does not significantly increase succinate production, or decrease methanogenesis, by the microbial community in the rumen.

Short-chain fatty acids (SCFAs) are bacterial products that are known to be used as energy sources in eukaryotic hosts, whereas their role in the metabolism of intestinal microbes is rarely explored. In the present study, acetic, propionic, butyric, isobutyric, valeric, and isovaleric acid, respectively, were added to a newly defined medium containing Prevotella bryantii B14 cells. After 8 h and 24 h, optical density, pH and SCFA concentrations were measured. Long-chain fatty acid (LCFA) profiles of the bacterial cells were analyzed via gas chromatography-time of flight-mass spectrometry (GC-ToF MS) and proteins were quantified using a mass spectrometry-based, label-free approach. Cultures supplemented with single SCFAs revealed different growth behavior. Structural features of the respective SCFAs were identified in the LCFA profiles, which suggests incorporation into the bacterial membranes. The proteomes of cultures supplemented with acetic and valeric acid differed by an increased abundance of outer membrane proteins. The proteome of the isovaleric acid supplementation showed an increase of proteins in the amino acid metabolism. Our findings indicate a possible interaction between SCFAs, the lipid membrane composition, the abundance of outer membrane proteins, and a modulation of branched chain amino acid biosynthesis by isovaleric acid.

This study aimed to evaluate in vitro fermentation characteristics, microbial protein synthesis and microbial community composition when replacing maize silage (MS) with red clover silage (RCS) in total mixed rations (TMR) of dairy cows. Treatments included TMR containing forage (MS and RCS) and concentrates (0.75:0.25) with targeted proportions (dry matter (DM) basis) of RCS in TMR of 0.15 (RCS15 ), 0.30 (RCS30 ), 0.45 (RCS45 ), and 0.60 (RCS60 ), in substitution of MS. Samples of the TMR were incubated using the in vitro Ankom RF technique with a mixture of rumen fluid and buffer solution (1:2 v/v) for 8 and 24 hr. Gas production and total short-chain fatty acids concentration did not differ between diets, whereas ammonia-nitrogen concentration increased with increasing level of RCS. Acetate proportion was not affected by RCS level, but propionate showed a linear increase with increasing level of RCS at the expenses of butyrate. Branched fatty acids proportions linearly declined, reflecting a reduced deamination of true protein. Gene copy numbers of protozoa linearly decreased with increasing RCS levels, while total numbers of bacteria and methanogens were not affected by diet. The amylolytic bacteria Ruminobacter amylophillus and Prevotella bryantii showed evidence to increase with higher RCS levels after 8 hr and 24 hr, respectively, whereas no effects of diet where observed for the fibrolytic bacteria Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes. Concentrations of purine bases, and total N production in liquid-associated microbes declined with increasing RCS levels, suggesting a negative impact of this feed on microbial growth. The findings of this study suggest that in general, microbial protein synthesis might be impaired by the substitution of MS by RCS, therefore caution should be taken when formulating diets for dairy cows using high levels of RCS as ingredient.

In the Southern Hemisphere, ruminants are mostly raised in grazing systems where animals consume forage and are supplemented with low amounts of concentrates. Concentrates are usually given separately and are rapidly ingested. This practice leads to changing rumen environment conditions during the day, may alter the rumen microbial metabolism and could affect host performance. The native ruminal Prevotella bryantii strain 3C5 was administered every 48 h to wethers under experimental conditions simulating Southern-Hemisphere feeding to evaluate its potential as a rumen fermentation modulator. The inoculum potential was assessed on day 17. The ammonia nitrogen (NH3-N), volatile fatty acids and ruminal pH were monitored on a 24-h basis 19 days after the beginning of the experiment, and the microbial community structure was assessed by pyrosequencing. The administration of P. bryantii modified the fermentation products and daily pH values compared to the control. The NH3-N concentration in the rumen of treated animals was significantly higher than that of the untreated animals. Modification of the ruminal environment and fermentation pathways was achieved without altering the general structure of the microbial community or the potential methane production. P. bryantii 3C5 could be considered in potential probiotic formulations for ruminants in semi-intensive systems.

Respiratory NADH oxidation in the rumen bacterium Prevotella bryantii is catalyzed by the Na+-translocating NADH:quinone oxidoreductase (NQR). A method for cell disruption and membrane isolation of P. bryantii under anoxic conditions using the EmulisFlex-C3 homogenizer is described. We compared NQR activity and protein yield after oxic and anoxic cell disruption by the EmulsiFlex, by ultrasonication, and by glass beads treatment. With an overall membrane protein yield of 50 mg L–1 culture and a NADH oxidation activity of 0.8 µmol min−1 mg−1, the EmulsiFlex was the most efficient method. Anoxic preparation yielded fourfold higher NQR activity compared to oxic preparation. P. bryantii lacks genes coding for superoxide dismutases and cell extracts do not exhibit superoxide dismutase activity. We propose that inactivation of NQR during oxic cell rupture is caused by superoxide, which accumulates in P. bryantii extracts exposed to air. Anoxic cell rupture is indispensable for the preparation of redox-active proteins and enzymes such as NQR from P. bryantii. Electronic supplementary material The online version of this article (10.1007/s00203-019-01805-x) contains supplementary material, which is available to authorized users.

Transport stress syndrome often appears in beef cattle during ground transportation, leading to changes in their capacity to digest food due to changes in rumen microbiota. The present study aimed to analyze bacteria before and after cattle transport. Eight Xianan beef cattle were transported over 1000 km. Rumen fluid and blood were sampled before and after transport. Real-time PCR was used to quantify rumen bacteria. Cortisol and adrenocorticotrophic hormone (ACTH) were measured. Cortisol and ACTH were increased on day 1 after transportation and decreased by day 3. Cellulolytic bacteria (Fibrobacter succinogenes and Ruminococcus flavefaciens), Ruminococcus amylophilus and Prevotella albensis were increased at 6 h and declined by 15 days after transport. There was a significant reduction in Succinivibrio dextrinosolvens, Prevotella bryantii, Prevotella ruminicola and Anaerovibrio lipolytica after transport. Rumen concentration of acetic acid increased after transport, while rumen pH and concentrations of propionic and butyric acids were decreased. Body weight decreased by 3 days and increased by 15 days after transportation. Using real-time PCR analysis, we detected changes in bacteria in the rumen of beef cattle after transport, which might affect the growth of cattle after transport.

Vegetable oils are used to increase energy density of dairy cow diets, although they can provoke changes in rumen bacteria populations and have repercussions on the biohydrogenation process. The aim of this study was to evaluate the effect of two sources of dietary lipids: soybean oil (SO, an unsaturated source) and hydrogenated palm oil (HPO, a saturated source) on bacterial populations and the fatty acid profile of ruminal digesta. Three non-lactating Holstein cows fitted with ruminal cannulae were used in a 3x3 Latin square design with three periods consisting of 21 days. Dietary treatments consisted of a basal diet (Control, no fat supplement) and the basal diet supplemented with SO (2.7% of dry matter (DM)) or HPO (2.7% of DM). Ruminal digesta pH, NH3-N and volatile fatty acids were not affected by dietary treatments. Compared with control and HPO, total bacteria measured as copies of 16S ribosomal DNA/ml by quantitative PCR was decreased (P < 0.05) by SO. Fibrobacter succinogenes, Butyrivibrio proteoclasticus and Anaerovibrio lipolytica loads were not affected by dietary treatments. In contrast, compared with control, load of Prevotella bryantii was increased (P < 0.05) with HPO diet. Compared with control and SO, HPO decreased (P < 0.05) C18:2 cis n-6 in ruminal digesta. Contents of C15:0 iso, C18:11 trans-11 and C18:2 cis-9, trans-11 were increased (P < 0.05) in ruminal digesta by SO compared with control and HPO. In conclusion, supplementation of SO or HPO do not affect ruminal fermentation parameters, whereas HPO can increase load of ruminal P. bryantii. Also, results observed in our targeted bacteria may have depended on the saturation degree of dietary oils.

Current research indicates that changes in gut microbiota can impact the host, but it is not always clear how dietary and environmental factors alter gut microbiota. One potential factor is antimicrobial activity of compounds ingested by the host. The goal of this study was to determine the antimicrobial activity of common plant secondary metabolites against pure cultures of paired, structurally and phylogenetically distinct gastrointestinal bacteria of human or bovine origin: Prevotella bryantii B(1)4, Bacteroides fragilis 25285, Acetoanaerobium (Clostridium) sticklandii SR and Clostridioides difficile 9689. When growth media were amended with individual phytochemicals (the alkaloids: berberine, capsaicin, nicotine, piperine and quinine and the phenolic: curcumin), growth of each species was inhibited to varying degrees at the three greatest concentrations tested (0.10–10.00 mg mL(−1)). The viable cell numbers of all the cultures were reduced, ≥4-logs, by berberine at concentrations ≥1.00 mg mL(−1). Quinine performed similarly to berberine for B(1)4, 25285, and SR at the same concentrations. The other phytochemicals were inhibitory, but not as much as quinine or berberine. Nicotine had activity against all four species (≥2-log reduction in viable cell number at 10.00 mg mL(−1)), but had stronger activity against the Gram-positive bacteria, SR and 9689, (≥4-log reductions at 10.00 mg mL(−1)). In conclusion, the phytochemicals had varying spectra of antimicrobial activity. These results are consistent with the hypothesis that ingested phytochemicals have the ability to differentially impact gut microbiota through antimicrobial activity.

Background Residual feed intake (RFI) in dairy cattle typically calculated at peak lactation is a measure of feed efficiency independent of milk production level. The objective of this study was to evaluate differences in ruminal bacteria, biopolymer hydrolyzing enzyme activities, and overall performance between the most- and the least-efficient dairy cows during the peripartal period. Twenty multiparous Holstein dairy cows with daily ad libitum access to a total mixed ration from d − 10 to d 60 relative to the calving date were used. Cows were classified into most-efficient (i.e. with low RFI, n = 10) and least-efficient (i.e. with high RFI, n = 10) based on a linear regression model involving dry matter intake (DMI), fat-corrected milk (FCM), changes in body weight (BW), and metabolic BW. Results The most-efficient cows had ~ 2.6 kg/d lower DMI at wk 4, 6, 7, and 8 compared with the least-efficient cows. In addition, the most-efficient cows had greater relative abundance of total ruminal bacterial community during the peripartal period. Compared with the least-efficient cows, the most-efficient cows had 4-fold greater relative abundance of Succinivibrio dextrinosolvens at d − 10 and d 10 around parturition and tended to have greater abundance of Fibrobacter succinogenes and Megaspheara elsdenii. In contrast, the relative abundance of Butyrivibrio proteoclasticus and Streptococcus bovis was lower and Succinimonas amylolytica and Prevotella bryantii tended to be lower in the most-efficient cows around calving. During the peripartal period, the most-efficient cows had lower enzymatic activities of cellulase, amylase, and protease compared with the least-efficient cows. Conclusions The results suggest that shifts in ruminal bacteria and digestive enzyme activities during the peripartal period could, at least in part, be part of the mechanism associated with better feed efficiency in dairy cows. Electronic supplementary material The online version of this article (10.1186/s40104-018-0258-9) contains supplementary material, which is available to authorized users.

Compared to grass silage (GS)-, corn silage (CS)-based diets appear to increase the efficiency of microbial protein synthesis (EMPS) in the rumen. Opposite results for the EMPS obtained in vitro have raised the question of whether an inadequate supply of amino N for microbes might explain the low EMPS. We examined the effects of supplementation with different N sources in CS on the EMPS and microbial populations in vitro. GS and CS were used as substrates for in vitro incubation. CS was non-supplemented or supplemented with urea, mixed amino acids (AA), peptone, or protein to adjust the N content to that of GS. Degradation of organic matter (OM) and crude protein (CP) revealed a positive effect of all N supplements, except protein. Additionally, N supplementation increased fiber degradation of CS. Peptone primarily stimulated hemicellulolytic activity and urea stimulated cellulolytic activity. The EMPS of CS was improved by all N supplements, with peptone and urea exhibiting the highest increase (57% and 54%, respectively), followed by AA mix (40%) and protein (11%) compared to that of CS alone (111 g microbial CP kg−1 fermented OM). However, the level of EMPS detected with GS (200 g microbial CP kg−1 fermented OM) was not achieved. Protozoal 18S rRNA gene copy numbers were negatively correlated with the EMPS, whereas no correlation was found between total bacteria and the EMPS. A stimulating effect of urea, AA mix, and peptone was detected for Ruminococcus albus and Prevotella bryantii, whereas Fibrobacter succinogenes was inhibited by N supplementation. This indicated that neither the amount of available N nor the N source was the only limiting factor in the low EMPS values of CS in vitro. Information is also provided on the stimulating effects of different N sources on several microbial species in mixed rumen culture.

Background Extensive degradation of amino acids in the rumen via microbial deamination decreases the post-ruminal availability of dietary indispensable amino acids. Together with the normal decrease in voluntary dry matter intake (DMI) around parturition in dairy cows, microbial metabolism contributes to a markedly negative balance of indispensable amino acids, including methionine which may be the first-limiting for milk production. The main objective of the current study was to profile changes in major bacterial species with key functions in cellulose and hemicellulose digestion, xylan breakdown, proteolytic action, propionic acid production, lactate utilization and ruminal biohydrogenation in cows supplemented with rumen-protected Methionine (SM; Smartamine M, Adisseo NA, Alpharetta, GA, USA) from −23 through 30 d relative to parturition. Because ~90% of the methionine in SM bypasses the rumen, ~10% of the methionine is released into the rumen and can be utilized by microbes. Results As expected, there was an increase in overall DMI after parturition (Day, P

It has become clear lately that many bacteria and even whole bacterial phyla do not use the classical Shine-Dalgarno sequence mediated pathway of protein translation initiation. The prominent phylum Bacteroidetes is one of them, and this was shown not only using bioinformatic but also functional reporter gene studies in its representative Prevotella bryantii. The latter studies revealed much higher sensitivity toward secondary structures in 5(') untranslated mRNA regions (5(') UTRs) during translation initiation compared to Escherichia coli. It was proposed that in the absence of Shine-Dalgarno sequence interaction the key elements enabling translation initiation are local absence of secondary structures in 5(') UTRs, and the ribosomal protein S1 which binds to mRNA. Here, we evaluate the 5(') UTRs devoid of secondary structures but containing divergent nucleotide compositions in P. bryantii reporter assay. We show that base composition profoundly affects the amount of the reporter synthesized, and further that these amounts were in agreement with S1 protein binding affinity for adenine/uracil bases in mRNA. This is the first, though indirect, clue that S1 is actually involved in translation initiation in Bacteroidetes and adds the second layer of control beside mRNA secondary structure affecting translation initiation in this phylum.

Eight rumen-cannulated Holstein heifers were used to explore the effect of trace mineral form and diet starch content on selected ruminal microbial populations under limit feeding conditions. Heifers were subjected to a split-plot, 4×4 Latin square design with 19-day periods. Trace mineral form [organic proteinates (OTM) or inorganic sulfates (ITM)] was the whole-plot factor, and starch content (3.5%, 12.9%, 22.3%, and 31.7% DM) was the sub-plot factor. Rumen samples were collected 3 h after feeding on day 18 of each period, and DNA was extracted. Relative abundances of 6 well-studied bacterial taxa, total anaerobic fungi, ciliate protozoa, methanogenic Archaea and bacteria were determined using validated primer sets by real-time quantitative PCR. Targeted populations had relative abundances comparable to those previously reported. Of the microbial populations measured, trace mineral form influenced only Prevotella bryantii, which was increased by OTM. Increasing dietary starch concentration linearly decreased methanogenic Archaea, total bacteria, Prevotella spp., and Prevotella bryantii, and tended to linearly decrease fungi and protozoa. In conclusion, contrary to the starch content, trace mineral form had limited impact on the abundance of selected microbial populations in limit fed heifers 3 h after feeding. The unexpected effect of starch content on bacterial populations and protozoa could be the result of different eating patterns of heifers fed diets varying in starch content.

U probavnom se sustavu biljojeda nalaze vrlo učinkoviti mikroorganizmi što razgrađuju celulolozu i hemicelulozu iz sažvakanog biljnog materijala, te ih opskrbljuju hranjivim tvarima. Osim protozoa i gljivica, razgradnji otporne (hemi)celulozne biomase u buragu znatno pridonose i bakterije. U fokusu je ovog preglednog rada opis enzimskog sustava triju predstavnika bakterija iz buraga što proizvode celulazu, i to: Ruminococcus flavefaciens, Prevotella bryantii i Pseudobutyrivibrio xylanivorans. R. flavefaciens je poznat po proizvodnji jedne od najkompleksnijih enzimskih struktura, pa bi se mogao upotrijebiti za dizajn enzimskog kompleksa za razgradnju stanične stijenke biljaka. S druge strane, bakterije Prevotella bryantii i Pseudobutyrivibrio xylanivorans proizvode jednostavne, slobodne i vrlo aktivne ksilanaze. Bakterija P. xylanivorans ima i probiotička svojstva, pa se može upotrijebiti za proizvodnju bioplina i kao dodatak krmivu. Ispitivanje je genoma i proteoma bakterija što razgrađuju celulozu i hemicelulozu usmjereno na identifikaciju novih enzima, koji se nakon kloniranja i ekspresije u odgovarajućim domaćinima mogu upotrijebiti za izradu vrlo aktivnih rekombinantnih hidrolitičkih mikroorganizama, te primijeniti u različitim biotehnološkim procesima., Herbivorous animals harbour potent cellulolytic and hemicellulolytic microorganisms that supply the host with nutrients acquired from degradation of ingested plant material. In addition to protozoa and fungi, rumen bacteria contribute a considerable part in the breakdown of recalcitrant (hemi)cellulosic biomass. The present review is focused on the enzymatic systems of three representative fibrolytic rumen bacteria, namely Ruminococcus flavefaciens, Prevotella bryantii and Pseudobutyrivibrio xylanivorans. R. flavefaciens is known for one of the most elaborated cellulosome architectures and might represent a promising candidate for the construction of designer cellulosomes. On the other hand, Prevotella bryantii and Pseudobutyrivibrio xylanivorans produce multiple free, but highly efficient xylanases. In addition, P. xylanivorans was also shown to have some probiotic traits, which makes it a promising candidate not only for biogas production, but also as an animal feed supplement. Genomic and proteomic analyses of cellulolytic and hemicellulolytic bacterial species aim to identify novel enzymes, which can then be cloned and expressed in adequate hosts to construct highly active recombinant hydrolytic microorganisms applicable for different biotechnological tasks.

This study determined the effects of aromatic amino acids (AA) and phenyl acids (phenylacetic acid and phenylpropionic acid) on fermentation of xylan by selected rumen bacteria. The ruminal bacteria were grown in a defined medium containing oat spelts xylan as the sole energy source, plus one of the following N sources: ammonia (no AA); ammonia plus a complete mixture of 20 AA commonly found in protein (CAA); ammonia plus complete AA mixture minus aromatic AA (MAR); ammonia plus phenyl acids (no AA+PA); ammonia plus complete AA mixture without aromatic AA plus phenyl acids (MAR+PA). There was varied growth response, with Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD1, and Butyrivibrio fibrisolvens JW11 not being able to grow. The supply of CAA mixture significantly (P

Summary Xylan is a major structural component of plant cell wall and the second most abundant plant polysaccharide in nature. Here, by combining genomic and functional analyses, we provide a comprehensive picture of xylan utilization by Xanthomonas campestris pv campestris (Xcc) and highlight its role in the adaptation of this epiphytic phytopathogen to the phyllosphere. The xylanolytic activity of Xcc depends on xylan-deconstruction enzymes but also on transporters, including two TonB-dependent outer membrane transporters (TBDTs) which belong to operons necessary for efficient growth in the presence of xylo-oligosaccharides and for optimal survival on plant leaves. Genes of this xylan utilization system are specifically induced by xylo-oligosaccharides and repressed by a LacI-family regulator named XylR. Part of the xylanolytic machinery of Xcc, including TBDT genes, displays a high degree of conservation with the xylose-regulon of the oligotrophic aquatic bacterium Caulobacter crescentus. Moreover, it shares common features, including the presence of TBDTs, with the xylan utilization systems of Bacteroides ovatus and Prevotella bryantii, two gut symbionts. These similarities and our results support an important role for TBDTs and xylan utilization systems for bacterial adaptation in the phyllosphere, oligotrophic environments and animal guts.

Here, we examined diurnal changes in the ruminal microbial community and fermentation characteristics of dairy cows fed total mixed rations containing either corn silage (CS) or grass silage (GS) as forage. The rations, which consisted of 52% concentrate and 48% GS or CS, were offered for ad libitum intake over 20 days to three ruminal-fistulated lactating Jersey cows during three consecutive feeding periods. Feed intake, ruminal pH, concentrations of short chain fatty acids and ammonia in rumen liquid, as well as abundance change in the microbial populations in liquid and solid fractions, were monitored in 4-h intervals on days 18 and 20. The abundance of total bacteria and Fibrobacter succinogenes increased in solids in cows fed CS instead of GS, and that of protozoa increased in both solid and liquid fractions. Feeding GS favored numbers of F. succinogenes and Selenomonas ruminantium in the liquid fraction as well as the numbers of Ruminobacter amylophilus, Prevotella bryantii and ruminococci in both fractions. Minor effects of silage were detected on populations of methanogens. Despite quantitative changes in the composition of the microbial community, fermentation characteristics were less affected by forage source. These results suggest a functional adaptability of the ruminal microbiota to total mixed rations containing either GS or CS as the source of forage. Diurnal changes in microbial populations were primarily affected by feed intake and differed between species and fractions, with fewer temporal fluctuations evident in the solid than in the liquid fraction. Interactions between forage source and sampling time were of minor importance to most of the microbial species examined. Thus, diurnal changes of microbial populations and fermentative activity were less affected by the two silages.

The recent classification of glycoside hydrolase family 5 (GH5) members into subfamilies enhances the prediction of substrate specificity by phylogenetic analysis. However, the small number of well characterized members is a current limitation to understanding the molecular basis of the diverse specificity observed across individual GH5 subfamilies. GH5 subfamily 4 (GH5_4) is one of the largest, with known activities comprising (carboxymethyl) cellulases, mixedlinkage endo-glucanases, and endo-xyloglucanases. Through detailed structure-function analysis, we have revisited the characterization of a classic GH5_4 carboxymethylcellulase, PbGH5A (also known as Orf4, carboxymethylcellulase, and Cel5A), from the symbiotic rumen Bacteroidetes Prevotella bryantii B(1)4. We demonstrate that carboxymethylcellulose and phosphoric acid-swollen cellulose are in fact relatively poor substrates for PbGH5A, which instead exhibits clear primary specificity for the plant storage and cell wall polysaccharide, mixedlinkage beta-glucan. Significant activity toward the plant cell wall polysaccharide xyloglucan was also observed. Determination of PbGH5A crystal structures in the apo-form and in complex with (xylo) glucan oligosaccharides and an active-site affinity label, together with detailed kinetic analysis using a variety of well defined oligosaccharide substrates, revealed the structural determinants of polysaccharide substrate specificity. In particular, this analysis highlighted the PbGH5A active-site motifs that engender predominant mixed-linkage endo-glucanase activity vis a vis predominant endo-xyloglucanases in GH5_4. However the detailed phylogenetic analysis of GH5_4 members did not delineate particular clades of enzymes sharing these sequence motifs; the phylogeny was instead dominated by bacterial taxonomy. Nonetheless, our results provide key enzyme functional and structural reference data for future bioinformatics analyses of (meta) genomes to elucidate the biology of complex gut ecosystems.

BACKGROUND: There is a growing interest in the use of hops (Humulus lupulus) as an alternative to antibiotics to manipulate ruminal fermentation. However, the effects of different hop varieties on ruminal fermentation and bacterial populations have not been studied. Here the effects of three hop varieties, Cascade (CAS), Millennium (MIL) and Teamaker (TM), at a level of 800 µg mL−1 inoculum on ruminal fermentation and microbial populations in an artificial rumen system (rusitec) fed a barley silage-based total mixed ration were investigated. Bacterial populations were assessed using real-time polymerase chain reaction and expressed as a percentage of total bacterial 16S rRNA gene copies. RESULTS: All hops reduced (P < 0.001) total gas, methane and the acetate:propionate ratio. Liquid-associated Fibrobacter succinogenes, Ruminococcus albus and Streptococcus bovis were reduced (P < 0.05) by MIL and TM. Feed particle-associated S. bovis was reduced (P < 0.01) by MIL and TM, but TM and CAS increased (P < 0.01) Ruminobacter amylophilus and Prevotella bryantii respectively. Methanogens were decreased (P < 0.05) by MIL in both liquid and solid fractions and by CAS in the solid fraction. The total amount of α- and β-acids in hops affected the ruminal fermentation. CONCLUSION: Hop-induced changes in fermentation and microbial populations may improve energy efficiency use in the rumen. Further research is needed to determine the effects of hops on in vivo ruminal fermentation, microbial populations and animal performance. Copyright © 2012 Society of Chemical Industry

High-grain adaptation programs are widely used with feedlot cattle to balance enhanced growth performance against the risk of acidosis. This adaptation to a high-grain diet from a high-forage diet is known to change the rumen microbial population structure and help establish a stable microbial population within the rumen. Therefore, to evaluate bacterial population dynamics during adaptation to a high-grain diet, 4 ruminally cannulated beef steers were adapted to a high-grain diet using a step-up diet regimen containing grain and hay at ratios of 20:80, 40:60, 60:40, and 80:20. The rumen bacterial populations were evaluated at each stage of the step-up diet after 1 week of adaptation, before the steers were transitioned to the next stage of the diet, using terminal restriction fragment length polymorphism (T-RFLP) analysis, 16S rRNA gene libraries, and quantitative real-time PCR. The T-RFLP analysis displayed a shift in the rumen microbial population structure during the final two stages of the step-up diet. The 16S rRNA gene libraries demonstrated two distinct rumen microbial populations in hay-fed and high-grain-fed animals and detected only 24 common operational taxonomic units out of 398 and 315, respectively. The 16S rRNA gene libraries of hay-fed animals contained a significantly higher number of bacteria belonging to the phylum Fibrobacteres , whereas the 16S rRNA gene libraries of grain-fed animals contained a significantly higher number of bacteria belonging to the phylum Bacteroidetes . Real-time PCR analysis detected significant fold increases in the Megasphaera elsdenii , Streptococcus bovis , Selenomonas ruminantium , and Prevotella bryantii populations during adaptation to the high-concentrate (high-grain) diet, whereas the Butyrivibrio fibrisolvens and Fibrobacter succinogenes populations gradually decreased as the animals were adapted to the high-concentrate diet. This study evaluates the rumen microbial population using several molecular approaches and presents a broader picture of the rumen microbial population structure during adaptation to a high-grain diet from a forage diet.

Direct-fed microbials (DFM) are dietary supplements that inhibit gastrointestinal infection and provide optimally regulated microbial environments in the digestive tract. As the use of antibiotics in ruminant feeds has been banned, DFM have been emphasized as antimicrobial replacements. Microorganisms that are used in DFM for ruminants may be classified as lactic acid producing bacteria (LAB), lactic acid utilizing bacteria (LUB), or other microorganisms including species of Lactobacillus, Bifidobacterium, Enterococcus, Streptococcus, Bacillus and Propionibacterium, strains of Megasphaera elsdenii and Prevotella bryantii and yeast products containing Saccharomyces and Aspergillus. LAB may have beneficial effects in the intestinal tract and rumen. Both LAB and LUB potentially moderate rumen conditions and improve feed efficiency. Yeast DFM may reduce harmful oxygen, prevent excess lactate production, increase feed digestibility, and improve fermentation in the rumen. DFM may also compete with and inhibit the growth of pathogens, stimulate immune function, and modulate microbial balance in the gastrointestinal tract. LAB may regulate the incidence of diarrhea, and improve weight gain and feed efficiency. LUB improved weight gain in calves. DFM has been reported to improve dry matter intake, milk yield, fat corrected milk yield and milk fat content in mature animals. However, contradictory reports about the effects of DFM, dosages, feeding times and frequencies, strains of DFM, and effects on different animal conditions are available. Cultivation and preparation of ready-to-use strict anaerobes as DFM may be cost-prohibitive, and dosing methods, such as drenching, that are required for anaerobic DFM are unlikely to be acceptable as general on-farm practice. Aero-tolerant rumen microorganisms are limited to only few species, although the potential isolation and utilization of aero-tolerant ruminal strains as DFM has been reported. Spore forming bacteria are characterized by convenience of preparation and effectiveness of DFM delivery to target organs and therefore have been proposed as DFM strains. Recent studies have supported the positive effects of DFM on ruminant performance.

Enzymatic depolymerization of lignocellulose by microbes in the bovine rumen and the human colon is critical to gut health and function within the host. Prevotella bryantii B(1)4 is a rumen bacterium that efficiently degrades soluble xylan. To identify the genes harnessed by this bacterium to degrade xylan, the transcriptomes of P. bryantii cultured on either wheat arabinoxylan or a mixture of its monosaccharide components were compared by DNA microarray and RNA sequencing approaches. The most highly induced genes formed a cluster that contained putative outer membrane proteins analogous to the starch utilization system identified in the prominent human gut symbiont Bacteroides thetaiotaomicron. The arrangement of genes in the cluster was highly conserved in other xylanolytic Bacteroidetes, suggesting that the mechanism employed by xylan utilizers in this phylum is conserved. A number of genes encoding proteins with unassigned function were also induced on wheat arabinoxylan. Among these proteins, a hypothetical protein with low similarity to glycoside hydrolases was shown to possess endoxylanase activity and subsequently assigned to glycoside hydrolase family 5. The enzyme was designated PbXyn5A. Two of the most similar proteins to PbXyn5A were hypothetical proteins from human colonic Bacteroides spp., and when expressed each protein exhibited endoxylanase activity. By using site-directed mutagenesis, we identified two amino acid residues that likely serve as the catalytic acid/base and nucleophile as in other GH5 proteins. This study therefore provides insights into capture of energy by xylanolytic Bacteroidetes and the application of their enzymes as a resource in the biofuel industry.

This study examined whether different corn silage to grass silage ratios in ruminant rations and different grinding levels of the feed affect the composition of the ruminal Bacteroides-Prevotella community in vitro. Three diets, composed of 10% soybean meal as well as of different corn silage and grass silage proportions, were ground through 1 mm or 4 mm screened sieves and incubated in a semi-continuous rumen simulation system. On day 14 of the incubation microbes were harvested by centrifugation from the liquid effluent of fermenter vessels. Microbial DNA was extracted for single strand conformation polymorphism (SSCP) analysis of 16S rRNA genes followed by sequencing of single SSCP bands. Fluorescence in situ hybridization (FISH) and real-time quantitative (q) PCR were used to quantify differences in the relative abundance of Bacteroides-Prevotella and Prevotella bryantii. SSCP profiles revealed a significant influence of the forage source as well as of the feed particle size on the community structure of the Bacteroides-Prevotella group. Different, phylogenetically distinct, so far uncultured Prevotella species were detected by sequence analysis of several treatment-dependent occurring SSCP bands indicating different nutritional requirements of these organisms for growth. No quantitative differences in the occurrence of Bacteroides-Prevotella-related species were detected between diets by FISH with probe BAC303. However, real-time qPCR data revealed a higher abundance of P. bryantii with increasing grass silage to corn silage ratio, thus again indicating changes within the community composition of the Bacteroides-Prevotella group. As P. bryantii possesses high proteolytic activity its higher abundance may have been caused by the higher contents of crude protein in the grass silage containing diets. To conclude, results of this study show an influence of the forage source on the ruminal community of Bacteroides-Prevotella. Furthermore, they suggest an effect of the feed particle size on this bacterial group.

16S rRNA gene-based analysis of rumen Prevotella was carried out to estimate the diversity and diet specificity of bacteria belonging to this genus. Total DNA was extracted from the rumen digesta of three sheep fed two diets with different hay-to-concentrate ratios (10 : 1 and 1 : 2). Real-time PCR quantification of Prevotella revealed that the relative abundance of this genus in the total rumen bacteria was up to 19.7%, while the representative species Prevotella bryantii and Prevotella ruminicola accounted for only 0.6% and 3.8%, respectively. Denaturing gradient gel electrophoresis analysis for Prevotella revealed shifts in the community composition with the diet. Analysis of 16S rRNA gene clone libraries showed significant differences (P=0.001) between clones detected from the sheep on the diets with different hay-to-concentrate ratios. The majority (87.8%) of Prevotella clones had

BACKGROUND: Use of brown algae (seaweed) in ruminant diets is increasing, but the effects of its phlorotannins (PT) on rumen microbial ecology have not been determined. Mixed forage (50:25:25 ground barley silage–alfalfa hay–grass hay) was used as substrate in a batch culture ruminal incubation that included PT extracted from Ascophyllum nodosum, with and without polyethylene glycol. Principal ruminal bacteria were quantified using real-time polymerase chain reaction. RESULTS: At 500 µg mL−1, PT reduced growth of Fibrobacter succinogenes by 78%, 83% and 65% at 6, 12 and 24 h (P < 0.001), Ruminococcus albus at 24 h only (P < 0.01) and did not affect R. flavefaciens. Non-cellulolytic bacteria Selenomonas ruminantium, Ruminobacter amylophilus and Prevotella bryantii were increased (P < 0.001) by PT at 12 and 24 h. Effects of PT on fermentation products (gas production, volatile fatty acid profiles and ammonia accumulation) were consistent with alterations in rumen microbial populations. CONCLUSION: The effects of PT on ruminal bacteria were species-dependent, which suggests that diet may mediate PT effects on animal performance. The variation in sensitivity of ruminal bacteria to PT reflects previously reported effects of condensed tannins from terrestrial plants on microbial populations. Copyright © 2009 Crown in the right of Canada. Published by John Wiley & Sons, Ltd

A xylanase-encoding gene, designated xynA19, was cloned from Sphingobacterium sp. TN19--a symbiotic bacterium isolated from the gut of Batocera horsfieldi larvae--and expressed in Escherichia coli BL21 (DE3). The full-length xynA19 (1,155 bp in length) encodes a 384-residue polypeptide (XynA19) containing a predicted signal peptide of 24 residues and a catalytic domain belonging to glycosyl hydrolase family 10 (GH 10). The deduced amino acid sequence of XynA19 is most similar (53.1% identity) to an endo-1,4-beta-xylanase from Prevotella bryantii B(1)4. Phylogenetic analysis of GH 10 Bacteroidia xylanases indicated that GH 10 xylanases from Sphingobacteria were separated into two clusters, and XynA19 is more closely related to the xylanases of Bacteroidia from gut or rumen than to those of Flavobacteria and Sphingobacteria from other sources. Recombinant XynA19 (r-XynA19) showed apparent optimal activity at pH 6.5 and 45 degrees C. Compared with thermophilic and mesophilic counterparts, r-XynA19 was more active at low temperatures, retaining65% of its maximum activity at 20-28 degrees C and approximately 40% even at 10 degrees C, and modeling indicated that XynA19 has fewer hydrogen bonds and salt bridges. These properties suggest that XynA19 has various potential applications, especially in aquaculture and the food industry.

Ingestion of high levels of rapidly fermented carbohydrates after parturition often leads to the production of excessive quantities of organic acids that may exceed the buffering capacity of the rumen and cause pH to drop. Ruminal acidosis results in animal discomfort, anorexia, depression, decreased digestibility, and decreased milk production. In the present study, we examined the effects of daily addition of cells of a newly isolated strain of Prevotella bryantii (25A) to the rumen of 12 ruminally cannulated cows in early lactation. This strain was selected based on earlier in vitro studies that indicated its ability to grow rapidly, compete for starch, and produce organic acids other than lactate. After calving, all cows received increasing amounts of an energy-dense diet containing barley grain, corn silage, and grass silage in a 40:60 forage-to-concentrate ratio. Animals were blocked according to milk production from their previous lactation. Treatments (control and P. bryantii) were distributed among cows within the same block. Cows were fed once a day. Six cows were given a daily dose of P. bryantii (2 x 10(11) cells/dose), administered directly with a syringe through the rumen cannula, from 3 wk prepartum up to 7 wk postpartum. Rumen fluid was sampled before feeding and at 2 and 3 h postfeeding on wk 1, 2, 3, 4, 6, and 7 postpartum. Feed intake and milk yield were recorded daily and milk composition was recorded 2 d/wk, up to wk 7 of lactation. Feed intake was similar between control and treated cows. Prevotella bryantii did not change milk production, but milk fat tended to be greater in treated cows compared with control cows (3.9 vs. 3.5%). Rumen pH was similar between the 2 groups and differed across sampling times, being higher before feeding (6.3) as opposed to 2 h (5.9) and 3 h (5.7) postfeeding. Rumen lactate concentration was similar before feeding between control and treated cows; however, 2 to 3 h after feeding, lactate concentration was lower in cows receiving P. bryantii compared with control cows (0.7 vs. 1.4 mM). This difference was maintained throughout the experimental period. Concentration of NH(3)-N was greater in treated cows than in control cows (174 vs. 142 mg/L). Acetate (65.5 vs. 57.8 mM), butyrate (12.7 vs. 10.5 mM), and branched-chain C4 fatty acid (0.90 vs. 0.75 mM) concentrations were greater in postfeeding samples of treated cows compared with control cows. Supplementing early-lactating cows with P. bryantii 25A increased ruminal fermentation products and milk fat concentration. Because signs of subacute ruminal acidosis were not observed in either treated or control cows, no conclusions can be made about possible protection against acidosis by P. bryantii.

The recent EU ban of growth-promoting antibiotics in animal production was based on fears concerning antibiotic resistance being transmitted to human pathogens. This paper explores the adaptation mechanism of a common ruminal bacterium, Prevotella bryantii, to one of the banned compounds, flavomycin (flavophospholipol).Growth in the presence of flavomycin (2 and 20 microg ml(-1)) was characterized by a concentration-dependent increase in the length of the lag phase, which decreased after previous flavomycin exposure. From growth patterns on solid medium, decreased sensitivity appeared to be due to a whole-population adaptation. Proteomic analysis indicated upregulation of three native proteins occurred following flavomycin adaptation. Further analysis of two of these proteins resulted in no database matches, suggesting that they may be species-specific. Flavomycin adaptation also resulted in co-adaptation to bacitracin and vancomycin.Adaptation of P. bryantii to flavomycin, which also resulted in co-adaptation to bacitracin and vancomycin, may involve an increased availability of undecaprenyl pyrophosphate.The use of flavomycin, and similar growth-promoting antibiotics, in animal production may prompt adaptive responses in ruminal bacteria which can significantly change their antibiotic sensitivity.

Aims: To identify and characterize the major lactic acid bacteria in the rumen of dairy cattle grazing improved pasture of rye grass and white clover and receiving a maize silage and grain supplement with and without virginiamycin. Methods and Results: Eighty-five bacterial isolates were obtained from the rumen of 16 Holstein-Friesian dairy cows. The isolates were initially grouped on the basis of their Gram morphology and by restriction fragment length polymorphism analysis of the PCR amplified 16S rDNA. A more definitive analysis was undertaken by comparing the 16S rDNA sequences. Many of the isolates were closely related to other previously characterized rumen bacteria, including Streptococcus bovis, Lactobacillus vitulinus, Butyrivibrio fibrisolvens, Prevotella bryantii and Selenomonas ruminantium. The in vitro production of l- and ⁄or d-lactate was seen with all but five of the isolates examined, many of which were also resistant to virginiamycin. Conclusion: Supplementation of grain with virginiamycin may reduce the risk of acidosis but does not prevent its occurrence in dairy cattle grazing improved pasture. Significance and Impact of the Study: This study shows that lactic acid production is caused, not only by various thoroughly researched types of bacteria, but also by others previously identified in the rumen but not further characterized.

Aims: To investigate the mode of action of a blend of essential oil compounds on the colonization of starch-rich substrates by rumen bacteria. Methods and Results: Starch-rich substrates were incubated, in nylon bags, in the rumen of sheep organized in a 4 × 4 latin square design and receiving a 60 : 40 silage : concentrate diet. The concentrate was either high or low in crude protein, and the diet was supplemented or not with a commercial blend of essential oil compounds (110 mg per day). The total genomic DNA was extracted from the residues in the bags. The total eubacterial DNA was quantified by real-time PCR and the proportion of Ruminobacter amylophilus, Streptococcus bovis and Prevotella bryantii was determined. Neither the supplementation with essential oil compounds nor the amount of crude protein affected the colonization of the substrates by the bacteria quantified. However, colonization was significantly affected by the substrate colonized. Conclusions: The effect of essential oils on the colonization of starch-rich substrates is not mediated through the selective inhibition of R. amylophilus. Significance and Impact of the Study: This study enhances our understanding of the colonization of starch-rich substrates, as well as of the mode of action of the essential oils as rumen manipulating agents.

Available tools for genetic analysis in the anaerobic rumen bacterium Prevotella bryantii are limited to only two known systems for gene delivery, and no genes, with the exception of plasmid maintenance and selection genes, have been successfully expressed from plasmids in any species of the genus Prevotella until now. It is shown here that nucB, a newly cloned nuclease gene from P. bryantii, can be controllably expressed from shuttle vector pRH3 in P. bryantii strain TC1-1, depending on the tetracycline concentration in the growth medium. nucB expression is also growth-medium dependent and this regulation presumably takes place at the translational level. His-tagged NucB was purified from P. bryantii TC1-1 culture supernatant and was shown to degrade DNA as well as RNA; it is most likely a minor 36 kDa P. bryantii non-specific nuclease.

The ruminant provides a powerful model for understanding the temporal dynamics of gastrointestinal microbial communities. Diet-induced milk fat depression (MFD) in the dairy cow is caused by rumen-derived bioactive fatty acids, and is commonly attributed to the changes in the microbial population. The aim of the present study was to determine the changes occurring in nine ruminal bacterial taxa with well-characterised functions, and abundance of total fungi, ciliate protozoa and bacteria during the induction of and recovery from MFD. Interactions between treatment and time were observed for ten of the twelve populations. The total number of both fungi and ciliate protozoa decreased rapidly (days 4 and 8, respectively) by more than 90 % during the induction period and increased during the recovery period. The abundance ofStreptococcus bovis(amylolytic) peaked at 350 % of control levels on day 4 of induction and rapidly decreased during the recovery period. The abundance ofPrevotella bryantii(amylolytic) decreased by 66 % from day 8 to 20 of the induction period and increased to the control levels on day 12 of the recovery period. The abundance ofMegasphaera elsdeniiandSelenomonas ruminantium(lactate-utilising bacteria) increased progressively until day 12 of induction (>170 %) and decreased during the recovery period. The abundance ofFibrobacter succinogenes(fibrolytic) decreased by 97 % on day 4 of induction and increased progressively to an equal extent during the recovery period, although smaller changes were observed for other fibrolytic bacteria. The abundance of theButyrivibrio fibrisolvens/Pseudobutyrivibriogroup decreased progressively during the induction period and increased during the recovery period, whereas the abundance ofButyrivibrio hungateiwas not affected by treatment. Responsive taxa were modified rapidly, with the majority of changes occurring within 8 d and their time course was similar to the time course of the induction of MFD, demonstrating a strong correlation between changes in ruminal microbial populations and MFD.

Seven multiparous Holstein cows with a ruminal fistula were used to investigate the changes in rumen microbiota, gene expression of the ruminal epithelium, and blood biomarkers of metabolism and inflammation during the transition period. Samples of ruminal digesta, biopsies of ruminal epithelium, and blood were obtained during −14 through 28 d in milk (DIM). A total of 35 genes associated with metabolism, transport, inflammation, and signaling were evaluated by quantitative reverse transcription-PCR. Among metabolic-related genes, expression of HMGCS2 increased gradually from −14 to a peak at 28 DIM, underscoring its central role in epithelial ketogenesis. The decrease of glucose and the increase of nonesterified fatty acids and β-hydroxybutyrate in the blood after calving confirmed the state of negative energy balance. Similarly, increases in bilirubin and decreases in albumin concentrations after calving were indicative of alterations in liver function and inflammation. Despite those systemic signs, lower postpartal expression of TLR2, TLR4, CD45, and NFKB1 indicated the absence of inflammation within the epithelium. Alternatively, these could reflect an adaptation to react against inducers of the immune system arising in the rumen (e.g., bacterial endotoxins). The downregulation of RXRA, INSR, and RPS6KB1 between −14 and 10 DIM indicated a possible increase in insulin resistance. However, the upregulation of IRS1 during the same time frame could serve to restore sensitivity to insulin of the epithelium as a way to preserve its proliferative capacity. The upregulation of TGFB1 from −14 and 10 DIM coupled with upregulation of both EGFR and EREG from 10 to 28 DIM indicated the existence of 2 waves of epithelial proliferation. However, the downregulation of TGFBR1 from −14 through 28 DIM indicated some degree of cell proliferation arrest. The downregulation of OCLN and TJP1 from −14 to 10 DIM indicated a loss of tight-junction integrity. The gradual upregulation of membrane transporters MCT1 and UTB to peak levels at 28 DIM reflected the higher intake and fermentability of the lactation diet. In addition, those changes in the diet after calving resulted in an increase of butyrate and a decrease of ruminal pH and acetate, which partly explain the increase of Anaerovibrio lipolytica, Prevotella bryantii, and Megasphaera elsdenii and the decrease of fibrolytic bacteria (Fibrobacter succinogenes, Butyrivibrio proteoclasticus). Overall, these multitier changes revealed important features associated with the transition into lactation. Alterations in ruminal epithelium gene expression could be driven by nutrient intake–induced changes in microbes; microbial metabolism; and the systemic metabolic, hormonal, and immune changes. Understanding causes and mechanisms driving the interaction among ruminal bacteria and host immunometabolic responses merits further study. Fil: Minuti, A.. Universita Cattolica del Sacro Cuore; Italia Fil: Palladino, Rafael Alejandro. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Khan, M. J.. University of Illinois at Urbana; Estados Unidos Fil: Alqarni, S.. University of Illinois at Urbana; Estados Unidos Fil: Agrawal, A.. University of Illinois at Urbana; Estados Unidos Fil: Piccioli Capelli, F.. Universita Cattolica del Sacro Cuore; Italia Fil: Hidalgo, F.. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal; Argentina Fil: Cardoso, F. C.. University of Illinois at Urbana; Estados Unidos Fil: Trevisi, E.. Universita Cattolica del Sacro Cuore; Italia Fil: Loor, J. J.. University of Illinois at Urbana; Estados Unidos

The restriction endonucleases PbrTI and Pru2I, isoschizomers of Sau3AI and HaeIII, were partially purified and characterized from anaerobic rumen bacteria Prevotella bryantii TC1-1 and Prevotella ruminicola 23, respectively. These are the first type II restriction endonucleases discovered in strains of the genus Prevotella, and they represent one of the barriers hindering gene transfer in these microorganisms. Heterologous DNA was protected against the action of the PbrTI or Pru2I by incubation in a cell-free extract of the respective strain which contained 20 mM EDTA. This led to the development of a protocol enabling successful electrotransformation of the P. bryantii TC1-1 strain with a pRH3 Bacteroides--Escherichia coli shuttle vector containing up to 7-kb long DNA inserts. Plasmid DNA isolated from the transformed strain facilitated the transfer with further increased efficiency and made possible the introduction of ligation reaction products directly to P. bryantii TC1-1 without passing them first through E. coli.

Prevotella spp. are believed to play a central role in ruminal nitrogen metabolism, but little is understood about the genetics and biochemistry of nitrogen assimilation and regulation in these bacteria. The gene encoding a family III glutamine synthetase (GSIII, glnN ) in Prevotella bryantii B 1 4 was cloned by Escherichia coli mutant complementation, and enzyme assays as well as Northern blot analysis showed that maximal enzyme activity and glnN transcription occurred in cells grown under nitrogen-limiting conditions. Addition of methionine sulfoximine (MSX), a GS inhibitor, terminated bacterial growth when ammonium was provided as the sole nitrogen source, but the inhibitory effect could be overcome by the inclusion of either L -glutamine or trypticase in the growth medium. A P. bryantii mutant lacking glutamate dehydrogenase (GdhA) activity was isolated by ethylmethylsulfonate mutagenesis. Growth studies with different nitrogen sources showed that the mutant strain was still capable of growth with ammonium as the sole nitrogen source, albeit at a decreased growth rate. The mutant strain could also grow with L -glutamine as a nitrogen source in the presence of MSX. These data suggest that GlnN provides an effective route of ammonium assimilation for P. bryantii, in addition to that afforded by the glutamate dehydrogenase pathway.

Prevotella species are important in catabolic protein metabolism by the mixed ruminal microbial population. This study was conducted to purify and investigate properties of one of the enzymes involved in amino acid metabolism by Prevotella bryantii B 1 4, glutamate-phenylpyruvate aminotransferase (GPA; EC 2.6.1.64). GPA was purified 51-fold from a cell-free extract by ammonium sulfate precipitation and column chromatography with Phenyl-superose, DEAE-Toyopearl 650 M, Sephacryl S-100 HR and Sephadex G-100. The molecular mass of GPA was estimated to be 28.0 kDa by SDS-PAGE. The optimum pH was 6.5 and the activity declined above pH 9.0. GPA was reactive over a wide range of pH from 5.0 to 10.5. Maximum activity of GPA occurred at 45°C and the activity declined at temperatures over 55°C. GPA was stable below 60°C. Aminooxyacetate and phenylhydrazine were highly inhibitory, while SDS, EDTA and some heavy metal ions also inhibited activity. The purification and characterization of enzyme will help to isolate the gene and ultimately to understand the role of GPA in both anabolic and catabolic amino acid metabolism by P. bryantii B 1 4.

The glucomannokinase gene of Prevotella bryantii B 1 4, a strictly anaerobic ruminal bacterium, was amplified with degenerate PCR primers. The degenerate PCR primers were based on the N-terminal amino acid residues of the purified glucomannokinase and the C-terminus of other bacterial glucokinases. The PCR product had a molecular weight of approximately 550 bp. Because the purified glucomannokinase was composed of dimers with a monomer molecular weight of 34.5 kDa, the PCR product accounted for approximately 60% of the glucomannokinase DNA sequence. The B 1 4 sequence had significant similarity with the glucokinases of several aerobic, Gram-positive bacteria ( Streptomyces coelicolor , Staphylococcus xylosus , Bacillus subtilis , Bacillus megaterium ). Previous work indicated that the Gram-positive glucokinases and the B 1 4 glucomannokinase have a role in catabolite repression. These presumptive regulatory kinases formed a cluster that was distinct from other bacterial glucokinases. Amino acid sequence analyses indicated that the regulatory glucokinase genes (including the P. bryantii B 1 4 glucomannokinase gene) had a mean sequence similarity of 42 ± 2.4%. The regulatory kinases had some highly conserved regions with other bacterial glucokinases, but the mean similarity was lower (17 ± 2.0%). The role of regulatory kinases in catabolite repression has not been precisely defined, but sequence comparisons suggest a common theme may exist. Because P. bryantii B 1 4 is a Gram-negative bacterium, it appears that regulatory glucokinases may be widely distributed in eubacteria and not restricted to Gram-positive species.

Enzymes that degrade dietary and host-derived glycans represent the most abundant functional activities encoded by genes unique to the human gut microbiome. However, the biochemical activities of a vast majority of the glycan-degrading enzymes are poorly understood. Here, we use transcriptome sequencing to understand the diversity of genes expressed by the human gut bacteria Bacteroides intestinalis and Bacteroides ovatus grown in monoculture with the abundant dietary polysaccharide xylan. The most highly induced carbohydrate active genes encode a unique glycoside hydrolase (GH) family 10 endoxylanase (BiXyn10A or BACINT_04215 and BACOVA_04390) that is highly conserved in the Bacteroidetes xylan utilization system. The BiXyn10A modular architecture consists of a GH10 catalytic module disrupted by a 250 amino acid sequence of unknown function. Biochemical analysis of BiXyn10A demonstrated that such insertion sequences encode a new family of carbohydrate-binding modules (CBMs) that binds to xylose-configured oligosaccharide/polysaccharide ligands, the substrate of the BiXyn10A enzymatic activity. The crystal structures of CBM1 from BiXyn10A (1.8 Å), a cocomplex of BiXyn10A CBM1 with xylohexaose (1.14 Å), and the CBM from its homolog in the Prevotella bryantii B14 Xyn10C (1.68 Å) reveal an unanticipated mode for ligand binding. A minimal enzyme mix, composed of the gene products of four of the most highly up-regulated genes during growth on wheat arabinoxylan, depolymerizes the polysaccharide into its component sugars. The combined biochemical and biophysical studies presented here provide a framework for understanding fiber metabolism by an important group within the commensal bacterial population known to influence human health.

Prevotella bryantii B14 has a transport system for glucose and mannose, but β-glucanase expression is only catabolite-repressed by glucose. P. bryantii B14 cell extracts had ATP-dependent gluco- and mannokinase activities, and significant phosphoenolpyruvate- or GTP-dependent hexose phosphorylation was not observed. Mannose inhibited glucose phosphorylation (and vice versa), and activity gels indicated that a single protein was responsible for both activities. Glucose was phosphorylated at a faster rate than was mannose [V max 280 nmol hexose (mg protein)−1 min−1 versus 60 nmol hexose (mg protein)−1 min−1, respectively] and glucose was a better substrate for the kinase (K m 0·12 mM versus 1·2 mM, respectively). The purified glucomannokinase (1250-fold) had a molecular mass of 68 kDa, but SDS-PAGE gels indicated that it was a dimer (monomer 34·5 kDa). The N-terminus (25 residues) had an 8 amino acid segment that was homologous to other bacterial glucokinases. The glucomannokinase was competitively inhibited by the nonmetabolizable glucose analogue 2-deoxyglucose (2DG), and cells grown with glucose and 2DG had lower rates of glucose consumption than did cells given only glucose. When the ratio of 2DG to glucose was increased, the glucose consumption rate decreased and the β-glucanase activity increased. The glucose consumption rate and the glucomannokinase activity of cells treated with 2DG were highly correlated (r 2=0·98). This result suggested that glucomannokinase activity was either directly or indirectly regulating β-glucanase expression.

Gram-negative, ruminal Prevotella strains (n = 15) differed greatly in their sensitivity to the feed additive monensin. Strains that were repeatedly transferred with sublethal doses tolerated more monensin than those that were unadapted, but growth experiments indicated that the sensitivity range was as great as 2000-fold. Prevotella bryantii B(1)4 grew with monensin concentrations as high as 20 microM, but P. ruminicola H15a, D31d, 20-63, E40a, and D42f never initiated growth if monensin was greater than 0.01 microM. Washed cell preparations that were energized with glucose lost intracellular potassium when monensin was added, and potassium depletion could also be used as an index of monensin sensitivity. Adapted cells of P. bryantii B(1)4 had a half-maximal potassium depletion constant (K(d)) of 3.2 microM, but the K(d) values of P. ruminicola strains H15a, D31d, 20-63, E40a, and D42f were less than 0.04 microM. Maximal potassium depletion (K(max)) values range from 90% to 40%, and monensin-adapted cells always had lower K(max) values than unadapted cells. A linear regression of log K(d)/K(max) versus percentage decrease in optical density divided by monensin concentration had an r(2) of 0.75, and this regression indicated that potassium depletion from washed cells closely correlated with growth inhibition. P. bryantii B(1)4 had a K(d)/K(max) ratio that was sevenfold greater than other Prevotella strains, and this result indicated that P. bryantii may be unusual in its ability to grow with very high concentrations of monensin.

Prevotella bryantii B14 grew faster on glucose than mannose (0.70 versus 0.45 h−1), but these sugars were used simultaneously rather than diauxically. 2-deoxy-glucose (2DG) decreased the growth rate of cells that were provided with either glucose or mannose, but 2DG did not completely prevent growth. Cells grown on glucose or mannose transported both 14C-glucose and 14C-mannose, but cells grown on glucose had over three-fold higher rates of 14C-glucose transport than cells grown on mannose. The 14C-mannose transport rates of glucose- and mannose-grown cells were similar. Woolf-Augustinsson-Hofstee plots were not linear, and it appeared that the glucose/mannose/2DG carrier acted as a facilitated diffusion system at high substrate concentrations. When cultures were grown on nitrogen-deficient (excess sugar) medium, isolates had three-fold lower 14C-glucose transport, but the 14C-mannose transport did not change significantly. 14C-glucose and 14C-mannose transport rates could be inhibited by 2DG and either mannose or glucose, respectively. The 14C-glucose transport of mannose-grown cells was inhibited more strongly by mannose and 2DG than those grown on glucose. Cells grown on glucose or mannose had similar ATP-dependent glucokinase activity, and 2DG was a competitive inhibitor (Ki=0.75 mM). Thin layer chromatography indicated that cell extracts also had ATP-dependent mannose phosphorylation, but only a small amount of phosphorylated 2DG was detected. Glucose, mannose or 2DG were not phosphorylated in the presence of PEP. Based on these results, it appeared that P. bryantii B14 had: (1) two mechanisms of glucose transport, a constitutive glucose/mannose/2DG carrier and an alternative glucose carrier that was regulated by glucose availability, (2) an ATP-dependent glucokinase that was competitively inhibited by 2DG but was unable to phosphorylate 2DG at a rapid rate, and (3) virtually no PEP-dependent glucose, mannose or 2DG phosphorylation activities.

The current research was aimed at comparing proteolytic activities among ruminal Prevotella spp. Growth rates of Prevotella sp. 2202, Prevotella ruminicola D31d, P. brevis GA33, P. albensis M384, and P. bryantii B(1)4 varied with N source, and no one N source produced the fastest growth in all species. Proteolytic activity was greatest with casein compared with peptides, AA, and NH(4)Cl in all species. Proteolytic activity of Prevotella sp. 2202, P. brevis GA33, and P. bryantii B(1)4 was modulated by N source. With gelatin co-polymerized SDS-PAGE, the extracellular activities of the Prevotella spp. showed wide variation in number, size, and type of proteases. Prevotella sp. 2202 and P. albensis M384 produced metalloproteases of low molecular weight (40 kDa). P. ruminicola D31d produced one cysteine protease (100-200 kDa) and two metalloproteases (90-100 kDa). P. brevis GA33 generated a diffuse clearing zone (95-160 kDa) containing serine, cysteine, and metalloproteases. P. bryantii B(1)4 produced a metalloprotease greater than 200 kDa in size. The molecular sizes provided are estimations and served only to differentiate among the bacterial species in this study. Large variations in proteolytic activities among species and the known genetic diversity of the Prevotella taxon suggested that targeting this bacterial assemblage for genetic manipulation in order to alter the bacterial impact on ruminal protein degradation would be difficult.

The non-cellulolytic ruminal bacterium, Prevotella bryantii, grew rapidly on water soluble β-glucans, but a mutant deficient in carboxymethylcellulase (CMCase) activity could not. Native activity gels prepared with CMC and β-glucan indicated that the CMCase and β-glucanase activities migrated at similar rates. When a plasmid carrying the P. bryantii CMCase was transferred to Escherichia coli, the clone had CMCase and β-glucanase activities. P. bryantii grew on mixed β-1,3-1,4 glucans, but not on β-1,3 glucan, and similar results were obtained with the cellulolytic ruminal bacteria (Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD1 and Ruminococcus albus B199). Mixed ruminal bacteria from cattle fed hay had twice as much CMCase activity as bacteria from cattle fed 90% cereal grain (P

We describe an approach for determining the genetic composition of Bacteroides and Prevotella populations in gut contents based on selective amplification of 16S rRNA gene sequences (rDNA) followed by cleavage of the amplified material with restriction enzymes. The relative contributions of different ribotypes to total Bacteroides and Prevotella 16S rDNA are estimated after end labelling of one of the PCR primers, and the contribution of Bacteroides and Prevotella sequences to total eubacterial 16S rDNA is estimated by measuring the binding of oligonucleotide probes to amplified DNA. Bacteroides and Prevotella 16S rDNA accounted for between 12 and 62% of total eubacterial 16S rDNA in samples of ruminal contents from six sheep and a cow. Ribotypes 4, 5, 6, and 7, which include most cultivated rumen Prevotella strains, together accounted for between 20 and 86% of the total amplified Bacteroides and Prevotella rDNA in these samples. The most abundant Bacteroides or Prevotella ribotype in four animals, however, was ribotype 8, for which there is only one known cultured isolate, while ribotypes 1 and 2, which include many colonic Bacteroides spp., were the most abundant in two animals. This indicates that some abundant Bacteroides and Prevotella groups in the rumen are underrepresented among cultured rumen Prevotella isolates. The approach described here provides a rapid, convenient, and widely applicable method for comparing the genotypic composition of bacterial populations in gut samples. Methods for enumerating gut bacteria that are based on cultivation, isolation, and biochemical testing are generally laborious and do not guarantee recovery of the less easily cultivated species. This is a particular problem for obligately anaerobic bacteria, which make up the great majority of organisms present in densely populated gut habitats such as the rumen and hind gut (13, 31). For this reason, there has been increasing interest in the rapid enumeration of microbial groupings by analysis of nucleic acids extracted from gut samples. Probing of extracted RNA with radiolabelled or fluorescently labelled oligonucleotide probes has been used in several studies (6, 14, 20, 30) but relies on developing panels of probes for different groups from available sequence data. Sequencing of random PCR-amplified 16S rRNA gene (rDNA) clones has provided valuable information on total eubacterial diversity for human fecal microflora (37). However, more rapid approaches to the study of diversity that allow the examination of large numbers of samples are required, and a semiquantitative PCR detection approach based on serial dilution has been reported for some of the predominant gut anaerobes (35). The approach we take here is to perform selective PCR amplification of 16S rRNA genes from the gram-negative anaerobic genera Bacteroides and Prevotella by using DNA extracted from gut samples and then to estimate the genotypic composition of samples from restriction enzyme cleavage patterns (restriction fragment length polymorphism [RFLP]) of the amplified DNA (PCR-RFLP). 16S rDNA PCR-RFLP approaches have proved valuable for typing isolated bacterial strains (see, e.g., refer

The influence of peptides and amino acids on ammonia assimilation and de novo synthesis of amino acids by three predominant noncellulolytic species of ruminal bacteria, Prevotella bryantii B 1 4, Selenomonas ruminantium HD4, and Streptococcus bovis ES1, was determined by growing these bacteria in media containing 15 NH 4 Cl and various additions of pancreatic hydrolysates of casein (peptides) or amino acids. The proportion of cell N and amino acids formed de novo decreased as the concentration of peptides increased. At high concentrations of peptides (10 and 30 g/liter), the incorporation of ammonia accounted for less than 0.16 of bacterial amino acid N and less than 0.30 of total N. At 1 g/liter, which is more similar to peptide concentrations found in the rumen, 0.68, 0.87, and 0.46 of bacterial amino acid N and 0.83, 0.89, and 0.64 of total N were derived from ammonia by P. bryantii , S. ruminantium , and S. bovis , respectively. Concentration-dependent responses were also obtained with amino acids. No individual amino acid was exhausted in any incubation medium. For cultures of P. bryantii , peptides were incorporated and stimulated growth more effectively than amino acids, while cultures of the other species showed no preference for peptides or amino acids. Apparent growth yields increased by between 8 and 57%, depending on the species, when 1 g of peptides or amino acids per liter was added to the medium. Proline synthesis was greatly decreased when peptides or amino acids were added to the medium, while glutamate and aspartate were enriched to a greater extent than other amino acids under all conditions. Thus, the proportion of bacterial protein formed de novo in noncellulolytic ruminal bacteria varies according to species and the form and identity of the amino acid and in a concentration-dependent manner.

Selected phenotypic characteristics of isolates of Prevotella ruminicola (formerly Bacteroides ruminicola) were studied in order to establish whether the characteristics of genotypic strain groups established previously on the basis of 16S ribosomal DNA sequences differed systematically. Among strains formerly considered P. ruminicola subsp. brevis, strains related to strain GA33T (T = type strain) typically failed to produce carboxymethyl cellulase (CMCase) activity detectable by plate assays and failed to ferment xylose, while strains related to strain B14T produced abundant CMCase and fermented xylose. We propose that strains related to GA33T, which have DNA G + C contents between 45 and 51 mol%, should be assigned to a new species, Prevotella brevis, and that strains related to B14T, which have DNA G + C contents between 39 and 43 mol%, should be assigned to another new species, Prevotella bryantii. Most of the isolates formerly classified as P. ruminicola subsp. ruminicola strains produced CMCase and had DNA G + C contents between 45 and 51 mol%, and we propose that these organisms should be placed in the redefined species P. ruminicola. A small group of isolates that have lower G + C contents are assigned to another new species, Prevotella albensis. Most P. brevis and P. bryantii strains produced abundatn extracellular DNase activity. Proteinase activities (as determined by [14C]casein hydrolysis) varied widely between strains, and P. brevis strains exhibited the highest mean activity. All strains produced dipeptidyl peptidase activity, but the relative activities against different peptide substrates exhibited by P. bryantii, P. albensis, and P. brevis differed systematically. The phenotypic differences among the newly defined species suggest that they may occupy distinct niches within the rumen ecosystem.