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In this study, organic acids were demonstrated as a promising carbon source for bisabolene production by the non-conventional yeast, Rhodosporidium toruloides, at microscale with a maximum titre of 1055 ± 7 mg/L. A 125-fold scale-up of the optimal process, enhanced bisabolene titres 2.5-fold to 2606 mg/L. Implementation of a pH controlled organic acid feeding strategy at this scale lead to a further threefold improvement in bisabolene titre to 7758 mg/L, the highest reported microbial titre. Finally, a proof-of-concept sequential bioreactor approach was investigated. Firstly, the cellulolytic bacterium Ruminococcus flavefaciens was employed to ferment cellulose, yielding 4.2 g/L of organic acids. R. toruloides was subsequently cultivated in the resulting supernatant, producing 318 ± 22 mg/L of bisabolene. This highlights the feasibility of a sequential bioprocess for the bioconversion of cellulose, into biojet fuel candidates. Future work will focus on enhancing organic acid yields and the use of real lignocellulosic feedstocks to further enhance bisabolene production.

This study examines fermented soya bean (SBM) and sunflower meal (SFM) with three biological treatments: Aspergillus Oryza, Ruminococcus flavefaciens and Lactobacillus plantarum, as well as the effects of these treatments on chemical composition, cell wall constituents, in vitro dry matter disappearance (IVDMD), and enzyme activity of the fermented SBM and SFM. After 2 h of ruminal incubation, all biological treatments (fungi, Bacteria, fungi with Bacteria) improved the IVDMD for Fermented SBM compared to that of the control (19.47, 20.65 and 18.38%, respectively). The maximum improvement values of IVDMD for SBM were recorded after 4 h and 6 h of incubation for all treatments. In addition, SFM after 2 h of ruminal incubation with any examined biological treatment (fungi, Bacteria, fungi with Bacteria), the IVDMD values have improved compared to that of the control group. Moreover, enzyme activities (cellulase, amylase, xylanase, and protease) have increased in all treatments. It was determined that SSF with A. oryzae and Ruminococcus boosted the protein content and digestibility of soybean and sunflower meals. [ABSTRACT FROM AUTHOR]

[Objectives] XynD, one xylanase of Ruminococcus flavefaciens, possesses two independent catalytic domains; glucanase and xylanase. In this study, the xynD gene was expressed in Pichia pastoris, and its enzyme activity and enzymatic properties were studied to provide reference for its application. [Methods] According to the codon bias of P. pastor, the xynD gene was optimized(the optimized gene named xynDm). The recombinant plasmid pPIC9K-xynDm was constructed and transformed into P. pastoris GS115. The genetic engineering strain GS115-pPIC9K-xynDm was obtained by screening. The recombinant XynDm was obtained by induction, and its enzymatic properties were studied. [Results] The enzyme activity and specific activity of XynDm reached the maximum at 36 and 72 h for xylanase and glucanase by methanol induction, which were 265.32 and 468.36 IU mg-1, 2 260.87 and 3 208.31 IU mg-1, respectively. Xylanase activity of XynDm was stable at 37-40 °C and pH value 4.5-6.5. The optimum temperature and pH value were 37 °C and 6.0 respectively. XynDm showed good hydrolysis toward 4-0-methyl-D-glucurono-D-xylan, high viscosity arabinoxylan and birch xylan. The glucanase activity of XynDm was stable at 40-55 °C and pH value 5.0-9.0. The optimum temperature and pH value were 55 °C and 6.0 respectively. XynDm showed good hydrolysis toward barley B-glucan. Bifunctional enzyme activity of XynDm was stimulated by Cu2+, Zn2+ and Co2+, and was almost inhibited by Ag+ and SDS. [Conclusions] The optimum substrates of xylanase and glucanase of XynDm were 4-0-methyl-D-glucurono-D-xylan and barley low viscosity β-glucan, respectively. The optimum temperature of enzymatic reaction of xylanase and glucanase was 37 and 55 °C, and the optimum pH value was 6.0. [ABSTRACT FROM AUTHOR]

IntroductionThere are differences in the gut microbiome and metabolome when the host undergoes different physical or pathological conditions. However, the inter-relationship of microbiome and metabolome biomarkers to potentially promote the health of dairy cows needs to be studied. Further, the development of next-generation probiotics for dairy cattle health promotion has not been demonstrated.ObjectiveIn the present study, we identified the microbiome and metabolome biomarkers associated with healthy cows.MethodsWe analyzed the relationships of the ruminal microorganism profile and metabolites between healthy and mastitis lactating dairy cows. The roles of bacterial biomarker were further verified by in vitro fermentation and cow-to-mouse fecal microbiota transplantation (FMT).ResultsTwo species, Ruminococcus flavefaciens and Bifidobacterium longum subsp. longum, and six rumen metabolites were positively correlated with healthy cows by Spearman’s correlation analysis. Through in vitro ruminal fermentation, inoculating R. flavefaciens and B. longum subsp. longum showed the upregulation of the levels of putrescine, xanthurenic acid, and pyridoxal in the mastitis ruminal fluid, which confirmed the inter-relationships between these microbiota and metabolites associated with healthy cows. Further, we verified the role of R. flavefaciens and B. longum subsp. longum in promoting health by FMT. The administration of R. flavefaciens and B. longum subsp. longum reduced the death rate and recovered the bodyweight loss of germ-free mice caused by FMT mastitis feces.DiscussionWe provided evidence that the bacterial biomarkers alter downstream metabolites. This could indirectly indicate that the two bacterial biomarkers have the potential to be used as next-generation probiotics for dairy cattle, although it needs more evidence to support our hypothesis. Two species, R. flavefaciens and B. longum subsp. longum, with three metabolites, putrescine, xanthurenic acid, and pyridoxal, identified in the ruminal fluid, may point to a new health-promoting and disease-preventing approach for dairy cattle.

Multifunctional endoglucanase, Rf GH5_4 from Ruminococcus flavefaciens showed (β/α) 8 -TIM barrel structure by homology modeling. Glu168 and Glu292 residues acted as general acid and base during catalysis. Circular Dichroism results, 40.83 % α-helices, 13.84 % β-strands and 45 % random turns-coils for Rf GH5_4 corroborated with predictions by PSIPRED and SOPMA. Molecular Dynamic simulation of Rf GH5_4 for 100 ns showed RMSD, 0.71 nm while for Rf GH5_4 - Cellopentaose complex was 0.55 nm, confirming that the binding of cellulosic ligand stabilizes its structural fold. Rf GH5_4 showed strong affinity towards cellulosic ligands having higher degree of polymerization such as cellohexaose (−11.70 kcal/mol) and cellodecaose (−12.64 kcal/mol). Interestingly, complex hemicellulosic ligands such as XLLG of xyloglucan also showed higher affinity (−13.2 kcal/mol) and accommodated at Rf GH5_4 active-site. Its catalytic cleft was broad enough to accommodate and hydrolyse various cellulosic and hemicellulosic ligands like XLLG of xyloglucan setting the basis of multifunctionality of Rf GH5_4. Loops L2, L3 and L4 having Trp58 formed barrier at active-site of Rf GH5_4 were responsible for processivity. Rf GH5_4 showed monodispersed state at 2.5 mg/mL and a rattle-toy shape by SAXS. Zeta potential, −16 mV of Rf GH5_4 indicated its higher stability. Multifunctional Rf GH5_4 endoglucanase could be beneficial for generation lignocellulosic bioethanol and in health, prebiotic and food sector. [Display omitted] • (β/α) 8 -TIM barrel structure of Rf GH5_4 contains 40.83 % α-helices, 13.84 % β-strands. • Active-site of Rf GH5_4 could fit large ligands, cellopentaose to cellodecaose. • Multifunctional endoglucanase, Rf GH5_4 also shows strong affinity for xyloglucan. • Loops, L2, L3 and L4 with Trp58 at active-site of Rf GH5_4 shape its processivity. • SAXS of Rf GH5_4 revealed monodispersed state and rattle-toy shape at 2.5 mg/mL. [ABSTRACT FROM AUTHOR]

Abstract Background The nature and amount of dietary medicinal plants are known to influence rumen fermentation and nutrient digestibility in ruminants. Nonetheless, changes in nutrient digestibility and rumen metabolism in response to dietary Andrographis paniculata (AP) in goats are unknown. This study examined the effects of dietary supplementation of leaves and whole plant of AP on nutrient digestibility, rumen fermentation, fatty acids and rumen microbial population in goats. Twenty-four Boer crossbred bucks (4 months old; average body weight of 20.18 ± 0.19 kg) were randomly assigned to three dietary groups of eight goats each. The dietary treatments included a control diet (Basal diet without additive), basal diet +1.5% (w/w) Andrographis paniculata leaf powder (APL) and basal diet +1.5% (w/w) Andrographis paniculata whole plant powder (APW). The trial lasted 100 d following 14 d of adjustment. Results The rumen pH and concentration of propionate were greater (P

In order to know the insights of a unique naturally existing trimodular licheninase from GH16 family, sub-family 21 ( Rf GH16_21) from Ruminococcus flavefaciens, its structure was modeled to understand its functional relations to reveal information regarding modifying the enzyme for improved properties with enhanced catalytic efficiency. Homology modeling revealed three tandem repeats of β-jelly roll like folds linked by natural linkers. Catalytic pockets and the catalytically important amino acids in each tandem repeat of Rf GH16_21 determined by multiple sequence alignment and structure superposition with its homologues indicated that two Glu residues are involved in a retaining-type of catalytic mechanism. Sequential molecular docking revealed maximum binding energy with mixed linked cellotriose showing that cellotriose is the lowest oligomeric hydrolysed product formed by the catalytic action of endo-β-1,3-1,4-glucanase. Molecular dynamic (MD) simulation of Rf GH16_21-cellotriose complex confirmed the structural specificity of catalytic residues and increased stability of enzyme in presence of ligand as compared to simulated Rf GH16_21 alone. The binding affinity of cellotriose towards the three tandem repeats of Rf GH16_21 was also confirmed by calculating total binding Gibbs free energy, i.e. -100.8 ± 2.6 KJ/mol, by using g_mmpbsa tool. The stability of the protein was determined by protein melting analysis that showed Ca 2+ and Mg 2+ ions imparted structural stability to Rf GH16_21. Dynamic light scattering analysis of Rf GH16_21 showed monodispersity and hydrodynamic radius of 4.0 nm at 2.0 mg/mL protein concentration, which was comparable with the radius of gyration of 3.2 nm determined by MD simulation showing the protein to be in monomeric form.Communicated by Ramaswamy H. Sarma.

The cellulosomal enzyme, RfGH51/2, of Ruminococcus flavefaciens contains an N-terminal module, a family 5 glycoside hydrolase GH5_4 with a putative endoglucanase activity, while C-terminal domain is a putative endo-mannanase (GH5_7). The two putative catalytic modules are separated by family 80 carbohydrate binding module (CBM80) having wide ligand specificity. The putative endo-mannanase module, GH5_7 (RfGH5_7), was cloned, expressed in Escherichia coli BL-21(DE3) cells and purified. SDS-PAGE analysis of purified RfGH5_7 showed molecular size ~ 35 kDa. Substrate specificity analysis of RfGH5_7 showed maximum activity against locust bean galactomannan (298.5 U/mg) followed by konjac glucomannan (256.2 U/mg) and carob galactomannan (177.2 U/mg). RfGH5_7 showed maximum activity at optimum pH 6.0 and temperature 60 °C. RfGH5_7 displayed stability in between pH 6.0 and 9.0 and thermostability till 50 °C. 10 mM Ca2+ ions increased the enzyme activity by 33%. The melting temperature of RfGH5_7 was 84 °C that was not affected by Ca2+ ions or chelating agents. RfGH5_7 showed, Vmax, 389 U/mg and Km, 0.92 mg/mL for locust bean galactomannan. TLC analysis revealed that RfGH5_7 hydrolysed locust bean galactomannan predominantly to mannose, mannobiose, mannotriose and higher degree of polymerization of manno-oligosaccharides indicating an endo-acting catalytic mechanism. This study revealed a highly active and thermostable endo-mannanase with considerable biotechnological potential. [ABSTRACT FROM AUTHOR]

This study assessed the influence of dietary blend of 20% palm oil and 80% canola oil on the population of rumen microbiota and nitrogen economy in goats. Twenty-four Boer bucks (4–5 months old; initial BW, 20.54 ± 0.474 kg) were randomly allotted to diets containing on a dry matter basis, 0, 4 or 8% oil blend, fed daily for 100 days and slaughtered. The rumen microbiota was examined by quantitative real-time polymerase chain reaction (PCR) using the 16S rRNA gene. The population of total protozoa and methanogens was lower (p 0.05) on microbial N yield, microbial protein synthesis, efficiency of microbial protein synthesis and the intake, retention and excretion of N. Dietary supplementation of blend of palm oil and canola oil altered rumen microbial profile but did not affect nitrogen metabolism in goats.

We tested the hypothesis that supplementation with three protein levels improves fermentation parameters without changing the rumen microbial population of grazing beef cattle in the rainy season. Four rumen-cannulated Nellore bulls (432 ± 21 kg of body weight) were used in a 4 × 4 Latin square design with four supplements and four experimental periods of 21 days each. The treatments were mineral supplement (ad libitum) and supplements with low, medium (MPS), and high protein supplement (HPS), supplying 106, 408, and 601 g/day of CP, respectively. The abundance of each target taxon was calculated as a fraction of the total 16S rRNA gene copies in the samples, using taxon-specific and domain bacteria primers. Supplemented animals showed lower (P < 0.05) proportions of Ruminococcus flavefaciens and greater (P < 0.05) proportions of Ruminococcus albus and Butyrivibrio fibrisolvens than animals that received only the mineral supplement. The HPS supplement resulted in higher (P < 0.05) proportions of Fibrobacter succinogenes, R. flavefaciens, and B. fibrisolvens and lower (P < 0.05) proportions of R. albus than the MPS supplement. Based on our results, high protein supplementation improves the ruminal conditions and facilitates the growth of cellulolytic bacteria in the rumen of bulls on pastures during the rainy season. [ABSTRACT FROM AUTHOR]

This study was conducted to evaluate the effects of exogenous fibrolytic enzymes (EFE) supplementation into diets with different non-fiber carbohydrates (NFC)/neutral detergent fiber (NDF) ratios on growth performance, nutrient digestibility, ruminal fermentation, and microbiome in Chinese domesticated Lezhi black goats. Treatments were 2 × 2 factorial arrangement of diets using low vs . high NFC/NDF ratios (1.06 or 1.66) and with or without EFE supplementation (0 or 0.4 g cellulase and xylanase/kg dry matter of diet). Sixty four Lezhi black goats (11 ± 0.8 kg of live weight) were randomly assigned to the four treatments. High NFC/NDF ratio and EFE supplementation achieved greater average daily gain and improve feed conversion ratio ( P <  .05). The same treatments also significantly increased digestibility of organic matter (OM), acid detergent fiber (ADF) and NDF. In addition, the high NFC/NDF ratio diet, but not the EFE supplement, improved digestibility of crude protein (CP) and reduced ruminal pH ( P <  .001). Both high NFC/NDF ratio and EFE supplement decreased ruminal NH 3 -N concentrations significantly ( P <  .001), and increased the production of rumen total volatile fatty acids (VFA, P <  .001), especially the molar proportion of propionate ( P <  .05). They reduced the molar proportion of acetate and thus the ratio of acetate to propionate (A: P) ( P <  .05). Both the high NFC/NDF ratio and the EFE supplement elevated the levels of microbial crude proteins ( P <  .01). Importantly, the combination of high NFC/NDF ratio and the EFE supplement diet raised the content of the microbial crude proteins to the highest levels among the 4 different treatment groups ( P  = .012). However, the high NFC/NDF ratio and the EFE supplementation had different impacts on fiber degrading activities. The high NFC/NDF ratio diet decreased, while EFE supplementation increased, the activities of cellulase and xylanase ( P <  .05). Among the three cellulytic bacteria detected ( Ruminococcus albus , Ruminococcus flavefaciens, Fibrobacter succinogenes ), only the abundance of Fibrobacter succinogenes was significantly decreased with the high NFC/NDF ratio ( P  = .004). These results illustrate that the diet with a high NFC/NDF ratio and EFE supplementation can improve ADG, feed conversion ratio and nutrient utilization in Lezhi black goats. These improvements are likely attributed to the elevated production of microbial crude proteins and a shift in the molar proportions of VFA in the rumen, rather than increase of cellulolytic bacteria in the rumen microbiota. [ABSTRACT FROM AUTHOR]

Abstract: The fibrolytic rumen bacterium Ruminococcus flavefaciensOS14 was isolated from swamp buffalo and its phylogenetic, ecological and digestive properties were partially characterized. Isolates from rumen contents of four swamp buffalo were screened for fibrolytic bacteria; one of the 40 isolates showed a distinctive feature of solubilizing cellulose powder in liquid culture and was identified as R. flavefaciens based on its 16S ribosomal DNA sequence. This isolate, OS14, was employed for detection and digestion studies, for which a quantitative PCR assay was developed and defined cultures were tested with representative forages in Thailand. OS14 was phylogenetically distant from other isolated and uncultured R. flavefaciens and showed limited distribution among Thai ruminants but was absent in Japanese cattle. OS14 digested rice straw and other tropical forage to a greater extent than the type strain C94 of R. flavefaciens. OS14 produced more lactate than C94, and digested para grass to produce propionate more extensively in co‐culture with lactate‐utilizing Selenomonas ruminantium S137 than a co‐culture of C94 with S137. These results indicate that phylogenetically distinct OS14 could digest Thai local forage more efficiently than the type strain, possibly forming a symbiotic cross‐feeding relationship with lactate‐utilizing bacteria. This strain might be useful for future animal and other industrial applications. [ABSTRACT FROM AUTHOR]

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.

The effects of different levels ofLeucaena leucocephalaandManihot esculentaleaves’ supplementation onin vitrogas production, rumen fermentation and microbial populations and urinary purine derivatives (PD) were investigated. Seven treatment groups – T1:C/rice straw (RS) (40:60) (Control); T2:C/RS/leucaena leaves (40:45:15); T3:C/RS/leucaena leaves (40:30:30); T4: C/RS/leucaena leaves (40:15:45); T5:C/RS/cassava leaves (40:45:15); T6:C/RS/cassava leaves (40:30:30) and T7:C/RS/cassava leaves (40:15:45) – were used in this experiment. In thein vitrostudy, acetate, propionate, butyrate and total VFA were found to increase significantly in T7. No significant difference was observed inin vitrogas production except the control diet althoughin vitrodry matter digestibility (IVDMD) was recorded significantly decreased. Determination of urinary PD, rumen fermentation and microbial population were done using 21 local Boer goats. Rumen NH₃-N, acetic and total VFA production were found to improve (P < .05) in all supplemented group. Significant (P < .05) reduction was noted in the urinary allantoin production and total PD at T7. The populations of total protozoa andRuminococcus flavefacienhad significantly increased (P < .05) whileRuminococcus albus,andFibrobacter succinogeneswere significantly reduced (P < .05) in supplemented group. In conclusion, the result from the present study suggested that 25% ofL. leucocephalaleaves diet and 50% ofM. esculentaleaves diet can be incorporated in the goat diet so as to improve the nutritive value of poor quality diet. [ABSTRACT FROM PUBLISHER]

Background: The nature and amount of dietary medicinal plants are known to influence rumen fermentation and nutrient digestibility in ruminants. Nonetheless, changes in nutrient digestibility and rumen metabolism in response to dietary Andrographis paniculata (AP) in goats are unknown. This study examined the effects of dietary supplementation of leaves and whole plant of AP on nutrient digestibility, rumen fermentation, fatty acids and rumen microbial population in goats. Twenty-four Boer crossbred bucks (4 months old; average body weight of 20. 18 ± 0.19 kg) were randomly assigned to three dietary groups of eight goats each. The dietary treatments included a control diet (Basal diet without additive), basal diet +1.5% (w/w) Andrographis paniculata leaf powder (APL) and basal diet +1.5% (w/w) Andrographis paniculata whole plant powder (APW). The trial lasted 100 d following 14 d of adjustment. Results: The rumen pH and concentration of propionate were greater (P < 0.05) in goats fed the APL and APW diets than those fed the control diet. The concentrations of ammonia nitrogen and acetate were greater (P < 0.05) in the control goats than the APL and APW goats. The digestibilities of crude protein, dry matter, acid detergent fibre and neutral detergent fibre were greater (P < 0.05) in the APL and APW goats compared to the control goats. Dietary APL and APW decreased (P < 0.05) the ruminal concentration of C18:0 and increased (P < 0.05) the ruminal concentration of C18:2n-6 and C18:3n-3. The APL goats had greater (P < 0. 05) ruminal concentration of C18:1 trans-11 and CLA cis-9 trans-11 than the APW and control goats. Dietary treatments had no significant effect on the population of protozoa and methanogens in the rumen of goats. The ruminal populations of Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes were greater (P < 0.05) in the APL and APW goats than the control goats. Conclusion: Dietary supplementation of leaves and whole plant of Andrographis paniculata can be used to manipulate rumen metabolism for improved nutrient digestibility in goats. [ABSTRACT FROM AUTHOR]

Ruminococcus flavefaciens is among the most important cellulolytic bacterial species in rumen and gastrointestinal tract of monogastric herbivorous animals. Its efficiency in degradation of (hemi)cellulosic substrates is associated with the production of remarkably intricate extracellular multienzyme complexes, named cellulosomes. In the present work we investigated the cellulolytic system of 007C. The bioinformatic analysis of the draft genome sequence revealed identical organization of sca gene cluster as has previously been found in four other strains of R. flavefaciens. The cluster consists of five genes in the following order: scaC-scaA-scaB-cttA-scaE. The cellulases of R. flavefaciens 007C belong to four families of glycoside hydrolases, namely GH48, GH44, GH9 in GH5. Majority of these enzymes are putative endoglucanases, belonging to families GH5 and GH9, whereas only one gene encoding GH44 and GH48 was found. Apart from catalytic domains, most of these proteins also contain dockerins – signature sequences, which indicate their attachement to cellulosomes. On the other hand, carbohydrate-binding modules were only found coupled to GH9 catalytic domains. Zymogram analysis showed that larger endoglucanases were mostly constitutively expressed, wheras smaller enzymes were only detected in later phases of Avicel-grown cultures.

The assembly of one of Nature's most elaborate multienzyme complexes, the cellulosome, results from the binding of enzymeborne dockerins to reiterated cohesin domains located in a noncatalytic primary scaffoldin. Generally, dockerins present two similar cohesin-binding interfaces that support a dual binding mode. The dynamic integration of enzymes in cellulosomes, afforded by the dual binding mode, is believed to incorporate additional flexibility in highly populated multienzyme complexes. Ruminococcus flavefaciens, the primary degrader of plant structural carbohydrates in the rumen of mammals, uses a portfolio of more than 220 different dockerins to assemble the most intricate cellulosome known to date. A sequence-based analysis organized R. flavefaciens dockerins into six groups. Strikingly, a subset of R. flavefaciens cellulosomal enzymes, comprising dockerins of groups 3 and 6, were shown to be indirectly incorporated into primary scaffoldins via an adaptor scaffoldin termed ScaC. Here, we report the crystal structure of a group 3 R. flavefaciens dockerin, Doc3, in complex with ScaC cohesin. Doc3 is unusual as it presents a large cohesin-interacting surface that lacks the structural symmetry required to support a dual binding mode. In addition, dockerins of groups 3 and 6, which bind exclusively to ScaC cohesin, display a conserved mechanism of protein recognition that is similar to Doc3. Groups 3 and 6 dockerins are predominantly appended to hemicellulose-degrading enzymes. Thus, single binding mode dockerins interacting with adaptor scaffoldins exemplify an evolutionary pathway developed by R. flavefaciens to recruit hemicellulases to the sophisticated cellulosomes acting in the gastrointestinal tract of mammals. [ABSTRACT FROM AUTHOR]

This study assessed the influence of dietary blend of 20% palm oil and 80% canola oil on the population of rumen microbiota and nitrogen economy in goats. Twenty-four Boer bucks (4–5 months old; initial BW, 20.54 ± 0.474 kg) were randomly allotted to diets containing on a dry matter basis, 0, 4 or 8% oil blend, fed daily for 100 days and slaughtered. The rumen microbiota was examined by quantitative real-time polymerase chain reaction (PCR) using the 16S rRNA gene. The population of total protozoa and methanogens was lower (p < .05) while the population ofFibrobacter succinogeneswas greater (p < .05) in goats fed oil blend compared with the control goats. The population of total bacteria was greater (p < .05) in goats fed 8% oil blend compared with those fed other diets. The population ofRuminococcus albusandRuminococcus flavefacienswas greater (p < .05) in goats fed 4% oil blend compared with those fed other diets. Diet had no effect (p> 0.05) on microbial N yield, microbial protein synthesis, efficiency of microbial protein synthesis and the intake, retention and excretion of N. Dietary supplementation of blend of palm oil and canola oil altered rumen microbial profile but did not affect nitrogen metabolism in goats. [ABSTRACT FROM AUTHOR]

The breakdown of plant cell wall (PCW) glycans is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Defining the portfolio of CBMs, the CBMome, of a PCW degrading system is central to understanding the mechanisms by which microbes depolymerize their target substrates. Ruminococcus flavefaciens, a major PCW degrading bacterium, assembles its catalytic apparatus into a large multienzyme complex, the cellulosome. Significantly, bioinformatic analyses of the R. flavefaciens cellulosome failed to identify a CBM predicted to bind to crystalline cellulose, a key feature of the CBMome of other PCW degrading systems. Here, high throughput screening of 177 protein modules of unknown function was used to determine the complete CBMome of R. flavefaciens. The data identified six previously unidentified CBMfamilies that targeted β-glucans, β-mannans, and the pectic polysaccharide homogalacturonan. The crystal structures of four CBMs, in conjunction with site-directed mutagenesis, provide insight into the mechanism of ligand recognition. In the CBMs that recognize β-glucans and β-mannans, differences in the conformation of conserved aromatic residues had a significant impact on the topology of the ligand binding cleft and thus ligand specificity. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan, indicating that the carboxylates of galacturonic acid are key specificity determinants. This report shows that the extended repertoire of proteins in the cellulosome of R. flavefaciens contributes to an extended CBMome that supports efficient PCW degradation in the absence of CBMs that specifically target crystalline cellulose. [ABSTRACT FROM AUTHOR]

This study investigated the effects of a 22:6 n-3 rich supplement on ruminal fermentation characteristics and microbial changes, and also the effects of a 22:6 n-3 rich supplement on plasma metabolites by using rumen cannulated weather goats in a 4×4 Latin square design. The 22:6 n-3 rich supplement was infused into the rumen of the goats twice daily in equal portions at a rate of 0.0, 2.5, 5.0 and 10.0 g/d [corresponding to 0, 0.4%, 0.8% and 1.5% of diets (dry material base), respectively]. The concentration of NH3-N and total volatile fatty acid (TVFA), and the molar proportions of acetate and butyrate were decreased by the supplement (P<0.001). The supplement decreased diversity of the rumen bacterial community (P<0.01), and reduced the abundance of Ruminococcus flavefaciens, R. albus, Fibrobacter succinogenes and protozoa (P<0.01). However, it increased the abundance of ruminal fungi (P<0.01). The supplement had no influence on the concentration of plasma glucose, HDL-cholesterol, LDL-cholesterol and urea but increased the concentration of triglycerides and total cholesterol (P<0.05). In conclusion, the 22:6 n-3 rich supplement inhibited ruminal fermentation and this was accompanied by the decrease of the abundance of ruminal microbes, and also affected plasma metabolites. [ABSTRACT FROM AUTHOR]

• Rf GH30 showed (β/α) 8 TIM barrel fold and an associated 'side-β structure'. • Circular dichroism displayed 28.09% α-helices and 21.02% β-strands in Rf GH30. • Molecular docking of Rf GH30 showed maximum binding affinity with xylobiose. • MD simulation of Rf GH30 xylobiose confirmed the structural specificity and stability. • Rf GH30 showed monomeric and monodispersed state at the lower concentration. The family 30 glycoside hydrolase (Rf GH30) from Ruminococcus flavefaciens expressing xylanase activity was homology modeled. The structure revealed a (β/α) 8 TIM barrel topology along with an associated 'side β-structure'. Secondary structure analysis by circular dichroism displayed 28.09% α-helices and 21.02% β-strands, which corroborated with the prediction results. Multiple Sequence Alignment and structure superposition of Rf GH30 with its homologue indicate that Glu200 and Glu302 are the catalytic residues displaying a retaining-type of catalysis. Molecular Dynamics (MD) simulation of Rf GH30 confirmed the structural compactness and steadiness of modeled structure. Molecular docking studies showed maximum binding affinity with xylobiose that was corroborated by the total binding Gibbs free energy of -160.5 kJ/mol calculated by g_mmpbsa tool. MD simulation of Rf GH30-xylobiose confirmed the structural specificity of catalytic residues and its increased stability in presence of ligand. Small angle X-ray scattering (SAXS) analysis of Rf GH30 confirmed its monodispersed, fully folded and elongated structure in solution form. Dummy atom model revealed monomeric form of Rf GH30 at 2.8 mg/ml, while at 4 mg/ml as dimer. Dynamic light scattering analysis of Rf GH30 showed monodispersity and increase in hydrodynamic radius (R h) from 2.0 to 4.0 mg/mL, displaying possible dimerization of Rf GH30 at 4.0 mg/mL as corroborated by SAXS analysis. The lowered zeta potential of Rf GH30 at 4 mg/mL also indicated the formation of a dimer. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • Sequential bioprocess demonstrated effective conversion of cellulose to bisabolene. • Maximum bisabolene titre of 7.8 g/L achieved, the highest reported microbial titre. • Organic acids are an effective carbon source for R. toruloides. • Bisabolene titres improved 7.4-fold through pH controlled organic acid feeding. • Fermentation of cellulose by R. flavefaciens yielded up to 5.1 g/L organic acids. In this study, organic acids were demonstrated as a promising carbon source for bisabolene production by the non-conventional yeast, Rhodosporidium toruloides, at microscale with a maximum titre of 1055 ± 7 mg/L. A 125-fold scale-up of the optimal process, enhanced bisabolene titres 2.5-fold to 2606 mg/L. Implementation of a pH controlled organic acid feeding strategy at this scale lead to a further threefold improvement in bisabolene titre to 7758 mg/L, the highest reported microbial titre. Finally, a proof-of-concept sequential bioreactor approach was investigated. Firstly, the cellulolytic bacterium Ruminococcus flavefaciens was employed to ferment cellulose, yielding 4.2 g/L of organic acids. R. toruloides was subsequently cultivated in the resulting supernatant, producing 318 ± 22 mg/L of bisabolene. This highlights the feasibility of a sequential bioprocess for the bioconversion of cellulose, into biojet fuel candidates. Future work will focus on enhancing organic acid yields and the use of real lignocellulosic feedstocks to further enhance bisabolene production. [ABSTRACT FROM AUTHOR]

Summary The discovery of new ribosomally synthesized and post-translationally modified peptide natural products (RiPPs) has greatly benefitted from the influx of genomic information. The lanthipeptides are a subset of this class of compounds. Adopting the genome-mining approach revealed a novel lanthipeptide gene cluster encoded in the genome of Ruminococcus flavefaciens FD-1, an anaerobic bacterium that is an important member of the rumen microbiota of livestock. The post-translationally modified peptides were produced via heterologous expression in Escherichia coli . Subsequent structural characterization and assessment of their bioactivity revealed features reminiscent of and distinct from previously reported lanthipeptides. The lanthipeptides of R. flavefaciens FD-1 represent a unique example within two-component lanthipeptides, consisting of a highly conserved α-peptide and a diverse set of eight β-peptides. [ABSTRACT FROM AUTHOR]

This study was conducted to evaluate the effects of Suaeda glauca crushed seed on rumen microbial populations, ruminal fermentation, methane emission, and growth performance in lambs. Thirty male Ujumqin lambs (a local breed, average body weight 25.5 ± 1.28 kg) were equally divided into three treatments for a 56 d feeding period. The treatments were control diet (CTR), 80 g of SG seed/kg of diet (SGS80, dry matter (DM) basis), and 160 g of SG seed/kg of diet (SGS160, DM basis). Ruminal samples were collected at 0, 3, 6 and 9 h after the morning feeding on d 28 and 56 to investigate treatment, sampling day, sampling hour, and their interactions effects on ruminal parameters and microorganism population. After the feeding trial, all lambs were moved to respiratory chambers to measure methane emission. Addition of SG seed decreased methanogen ( P < 0.05), protozoa ( P < 0.01), and Fibrobacter succinogenes populations ( P < 0.05). Methanogen and protozoa populations were also affected by an interaction between treatment and sampling day ( P < 0.01) and the effect of SG seed on methanogen and protozoa populations decreased with feeding time. Addition of SG seed did not change populations of fungi, Ruminococcus flavefaciens , and Ruminococcus albus . Lambs fed SG160 diet had lower rumen ammonia nitrogen concentration ( P < 0.01) compared with lambs fed CTR diet. Ammonia nitrogen concentration was affected by an interaction between treatment and sampling day ( P < 0.01). No SG seed effects were found in ruminal pH, total volatile fatty acid concentration and molar proportion of volatile fatty acid. Addition of SG seed increased average daily gain ( P < 0.05) and dry matter intake ( P < 0.05) of lambs but reduced methane emissions ( P < 0.05). No SG seed effects were found in feed efficiency. In conclusion, addition of SG seed seemed to be a feasible means of decreasing methane emissions from lambs by reducing methanogen and protozoa populations. [ABSTRACT FROM AUTHOR]

Alkali extracted beechwood glucuronoxylan methyl ester prepared by esterification of 4- O -methyl- d -glucuronic acid side residues by methanol was found to serve as substrate of microbial glucuronoyl esterases from Ruminococcus flavefaciens , Schizophyllum commune and Trichoderma reesei . The enzymatic deesterification was monitored by 1 H NMR spectroscopy and evaluated on the basis of the decrease of the signal of the ester methyl group and increase of the signal of methanol. The results show for the first time the action of enzymes on polymeric substrate, which imitates more closely the natural substrate in plant cell walls than the low molecular mass artificial substrates used up to present. [ABSTRACT FROM AUTHOR]

Ruminant herbivores meet their carbon and energy requirements from a symbiotic relationship with cellulosome-producing anaerobic bacteria that efficiently degrade plant cell-wall polysaccharides. The assembly of carbohydrate-active enzymes (CAZymes) into cellulosomes enhances protein stability and enzyme synergistic interactions. Cellulosomes comprise diverse CAZymes displaying a modular architecture in which a catalytic domain is connected, via linker sequences, to one or more noncatalytic carbohydrate-binding modules (CBMs). CBMs direct the appended catalytic modules to their target substrates, thus facilitating catalysis. The genome of the ruminal cellulolytic bacterium Ruminococcus flavefaciens strain FD-1 contains over 200 modular proteins containing the cellulosomal signature dockerin module. One of these is an endoglucanase Cel5A comprising two family 5 glycoside hydrolase catalytic modules (GH5) flanking an unclassified CBM (termed CBM-Rf2) and a C-terminal dockerin. This novel CBM-Rf2 has been purified and crystallized, and data from cacodylate-derivative crystals were processed to 1.02 and 1.29 Å resolution. The crystals belonged to the orthorhombic space group P212121. The CBM-Rf2 structure was solved by a single-wavelength anomalous dispersion experiment at the As edge. [ABSTRACT FROM AUTHOR]

The cellulolytic bacterium Ruminococcus flavefaciens of the herbivore rumen produces an elaborate cellulosome system, anchored to the bacterial cell wall via the covalently bound scaffoldin ScaE. Dockerin-bearing scaffoldins also bind to an autonomous cohesin of unknown function, called cohesin G (CohG). Here, we demonstrate that CohG binds to the scaffoldin-borne dockerin in opposite orientation on a distinct site, relative to that of ScaE. Based on these structural data, we propose that the complexed dockerin is still available to bind ScaE on the cell surface. CohG may thus serve as a molecular shuttle for delivery of scaffoldins to the bacterial cell surface. [ABSTRACT FROM AUTHOR]

Lignocellulosic substrates are widely available but not easily applied in biogas production due to their poor anaerobic degradation. The effect of bioaugmentation by anaerobic hydrolytic bacteria on biogas production was determined by the biochemical methane potential assay. Microbial biomass from full scale upflow anaerobic sludge blanket reactor treating brewery wastewater was a source of active microorganisms and brewery spent grain a model lignocellulosic substrate. Ruminococcus flavefaciens 007C, Pseudobutyrivibrio xylanivorans Mz5 T , Fibrobacter succinogenes S85 and Clostridium cellulovorans as pure and mixed cultures were used to enhance the lignocellulose degradation and elevate the biogas production. P. xylanivorans Mz5 T was the most successful in elevating methane production (+17.8%), followed by the coculture of P. xylanivorans Mz5 T and F. succinogenes S85 (+6.9%) and the coculture of C. cellulovorans and F. succinogenes S85 (+4.9%). Changes in microbial community structure were detected by fingerprinting techniques. [ABSTRACT FROM AUTHOR]

A number of anaerobic microorganisms produce multi-modular, multi-enzyme complexes termed cellulosomes. These extracellular macromolecular nanomachines are designed for the efficient degradation of plant cell-wall carbohydrates to smaller sugars that are subsequently used as a source of carbon and energy. Cellulolytic strains from the rumens of mammals, such as Ruminococcus flavefaciens, have been shown to have one of the most complex cellulosomal systems known. Cellulosome assembly requires the binding of dockerin modules located in cellulosomal enzymes to cohesin modules located in a macromolecular scaffolding protein. Over 220 genes encoding dockerin-containing proteins have been identified in the R. flavefaciens genome. The dockerin-containing enzymes can be incorporated into the primary scaffoldin (ScaA), which in turn can bind to adaptor scaffoldins (ScaB or ScaC) and subsequently to anchoring scaffoldin (ScaE), thereby attaching the whole complex to the cell surface. However, unlike other cellulosomes such as that from Clostridium thermocellum, the Ruminococcus species lack a specific carbohydrate-binding module (CBM) on ScaA which recruits the entire complex onto the surface of the substrate. Instead, a cellulose-binding protein, CttA, comprising two putative tandem novel carbohydrate-binding modules and a C-terminal X-dockerin module, which can bind to the cohesin of ScaE, may mediate the attachment of bacterial cells to cellulose. Here, the expression, purification and crystallization of the carbohydrate-binding modular part of the CttA from R. flavefaciens are described. X-ray data have been collected to resolutions of 3.23 and to 1.61 Å in space groups P3121 or P3221 and P21, respectively. The structure was phased using bound iodide from the crystallization buffer by SAD experiments. [ABSTRACT FROM AUTHOR]

Cellulosomes are large multicomponent cellulose-degrading assemblies found on the surfaces of cellulolytic microorganisms. Often containing hundreds of components, the self-assembly of cellulosomes is mediated by the ultra-high-affinity cohesin-dockerin interaction, which allows them to adopt the complex architectures necessary for degrading recalcitrant cellulose. Better understanding of how the cellulosome assembles and functions and what kinds of structures it adopts will further effort to develop industrial applications of cellulosome components, including their use in bioenergy production. Ruminococcus flavefaciens is a well-studied anaerobic cellulolytic bacteria found in the intestinal tracts of ruminants and other herbivores. Key to cellulosomal self-assembly in this bacterium is the dockerin ScaADoc, found on the non-catalytic structural subunit scaffoldin ScaA, which is responsible for assembling arrays of cellulose-degrading enzymes. This work expands on previous efforts by conducting a series of binding studies on ScaADoc constructs that contain mutations in their cohesin recognition interface, in order to identify which residues play important roles in binding. Molecular dynamics simulations were employed to gain insight into the structural basis for our findings. A specific residue pair in the first helix of ScaADoc, as well as a glutamate near the C-terminus, was identified to be essential for cohesin binding. By advancing our understanding of the cohesin binding of ScaADoc, this study serves as a foundation for future work to more fully understand the structural basis of cellulosome assembly in R. flavefaciens. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Microbial degradation of the plant cell wall is a fundamental biological process with considerable industrial importance. Hydrolysis of recalcitrant polysaccharides is orchestrated by a large repertoire of carbohydrate-active enzymes that display a modular architecture in which a catalytic domain is connected via linker sequences to one or more noncatalytic carbohydrate-binding modules (CBMs). CBMs direct the appended catalytic modules to their target substrates, thus potentiating catalysis. The genome of the most abundant ruminal cellulolytic bacterium, Ruminococcus flavefaciens strain FD-1, provides an opportunity to discover novel cellulosomal proteins involved in plant cell-wall deconstruction. It encodes a modular protein comprising a glycoside hydrolase family 9 catalytic module (GH9) linked to two unclassified tandemly repeated CBMs (termed CBM- Rf6A and CBM- Rf6B) and a C-terminal dockerin. The novel CBM- Rf6A from this protein has been crystallized and data were processed for the native and a selenomethionine derivative to 1.75 and 1.5 Å resolution, respectively. The crystals belonged to orthorhombic and cubic space groups, respectively. The structure was solved by a single-wavelength anomalous dispersion experiment using the CCP4 program suite and SHELXC/ D/ E. [ABSTRACT FROM AUTHOR]

Cohesins and dockerins are two groups of structural modules critical to the assembly of cellulosomes, the protein complexes for biomass degradation in cellulolytic microorganisms. To understand their sequence diversity and evolution, we re-mined five well-studied cellulosome-producing genomes in the order of Clostridiales for both domain modules and performed a comparative study. We found many putatively new cohesins and dockerins (designated as NEW) besides those identified previously (PUB). We showed that a significant number of NEW domain-containing proteins have signatures to be cellulosome-related, such as having CAZyme (carbohydrate-active enzymes) domains or other cellulosome-related domains, differentially expressed in cellulose degradation, having similar GC content and codon adaptation index as PUB domains. Evolutionary analyses suggest that NEW domains are more divergent in sequence than PUB domains, and PUB domains evolve more rapidly than CAZyme domains. Phylogenetic and functional domain analyses revealed that duplications play a major role in the expansion of dockerins and cohesins. For example, orthologous proteins of closely related strains have different numbers of cohesins, suggesting duplications occurred in a genome-specific manner after the strains diverged. High abundance of cohesin- and dockerin-containing proteins in Ruminococcus flavefaciens FD-1 and Acetivibrio cellulolyticus CD2, relative to Clostridium thermocellum, may be a result of gene duplication and horizontal gene transfer under relaxed purifying selection in environments with high species diversity. Overall, this study suggests that dockerins and cohesins evolve with a high rate of domain birth and death in cellulolytic microorganisms. [ABSTRACT FROM AUTHOR]

Anaerobic bacteria organize carbohydrate-active enzymes into a multi-component complex, the cellulosome, which degrades cellulose and hemicellulose highly efficiently. Genome sequencing of Ruminococcus flavefaciens FD-1 offers extensive information on the range and diversity of the enzymatic and structural components of the cellulosome. The R. flavefaciens FD-1 genome encodes over 200 dockerin-containing proteins, most of which are of unknown function. One of these modular proteins comprises a glycoside hydrolase family 5 catalytic module (GH5) linked to an unclassified carbohydrate-binding module (CBM- Rf1) and a dockerin. The novel CBM- Rf1 has been purified and crystallized. The crystals belonged to the trigonal space group R32: H. The CBM- Rf1 structure was determined by a multiple-wavelength anomalous dispersion experiment using AutoSol from the PHENIX suite using both selenomethionyl-derivative and native data to resolutions of 2.28 and 2.0 Å, respectively. [ABSTRACT FROM AUTHOR]

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Cellulosomes are massive cell-bound multienzyme complexes tethered by macromolecular scaffolds that coordinate the efforts of many anaerobic bacteria to hydrolyze plant cell-wall polysaccharides, which are a major untapped source of carbon and energy. Integration of cellulosomal components occurs via highly ordered protein-protein interactions between cohesin modules, located in the scaffold, and dockerin modules, found in the enzymes and other cellulosomal proteins. The proposed cellulosomal architecture for Ruminococcus flavefaciens strain FD-1 consists of a major scaffoldin (ScaB) that acts as the backbone to which other components attach. It has nine cohesins and a dockerin with a fused X-module that binds to the cohesin on ScaE, which in turn is covalently attached to the cell wall. The ScaA dockerin binds to ScaB cohesins allowing more carbohydrate-active modules to be assembled. ScaC acts as an adaptor that binds to both ScaA and selected ScaB cohesins, thereby increasing the repertoire of dockerin-bearing proteins that integrate into the complex. In previous studies, a screen for novel cohesin-dockerin complexes was performed which led to the identification of a total of 58 probable cohesin-dockerin pairs. Four were selected for subsequent structural and biochemical characterization based on the quality of their expression and the diversity in their specificities. One of these is C12D22, which comprises the cohesin from the adaptor ScaC protein bound to the dockerin of a CBM-containing protein. This complex has been purified and crystallized, and data were collected to resolutions of 2.5 Å (hexagonal, P65), 2.16 Å (orthorhombic, P212121) and 2.4 Å (orthorhombic, P21212) from three different crystalline forms. [ABSTRACT FROM AUTHOR]

Ruminococcus flavefaciens is a cellulolytic bacterium found in the rumen of herbivores and produces one of the most elaborate and variable cellulosome systems. The structure of an R. flavefaciens protein ( RfCohG, ZP_06142108), representing a freestanding (non-cellulosomal) type III cohesin module, has been determined. A selenomethionine derivative with a C-terminal histidine tag was crystallized and diffraction data were measured to 2.44 Å resolution. Its structure was determined by single-wavelength anomalous dispersion, revealing eight molecules in the asymmetric unit. RfCohG exhibits the most complex among all known cohesin structures, possessing four α-helical elements and a topographical protuberance on the putative dockerin-binding surface. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The study addressed the importance of yeast (Saccharomyces cerevisiae) viability for reduc-ing the incidence of subacute ruminal acidosis (SARA) and improving total tract nutrient digestibility in beef heifers. Six ruminally cannulated beef heifers (680 ± 50 kg BW) were used in a replicated 3x3 Latin square design and were fed a diet consisting of 40% barley silage, 10% chopped grass hay, and 50% barley grain-based concentrate (DM basis). Treatments were 1) no yeast (Control), 2) active dried yeast (ADY; 4 g pro-viding 1010 cfu/g; AB Vista, Marlborough, UK), and 3) killed dried yeast (KDY; 4 g autoclaved ADY). The treatments were directly dosed via the ruminal cannula daily at the time of feeding. The periods consisted of 2 wk of adaptation (d 1 to 14) and 7 d of measurements (d 15 to 21). Ruminal pH was continuously measured (d 15 to 21) using an indwelling system. Ruminal con-tents were sampled on d 15 and 17 at 0, 3,6, 9, and 12 h after feeding. Total tract nutrient digestibility was mea-sured using an external marker (YbCl3) from d 15 to 19. No treatment difference was observed for DMI (P = 0.86). Yeast supplementation (ADY and KDY) tended to increase total tract digestibility of starch (P = 0.07) whereas no effects were observed on digestibility of other nutrients. Both ADY and KDY elevated minimum (P < 0.01) and mean ruminal pH (P = 0.02) whereas no effects were observed on maximum pH (P = 0.12). Irrespective of its viability, yeast supplementation was effective in reducing time that ruminal pH was below 5.8 (P < 0.01) and 5.6 (P < 0.01). No treatment differ-ences were observed for the ruminal VFA profile and lactate concentration. No treatment differences were observed on the relative population size of Streptococ-cus bovis, Fibrobacter succinogenes, and Megasphaera elsdenii (P > 0.10); however, the proportion of Rumi-nococcus flavefaciens in solid fraction of digesta was greater with KDY (P = 0.05). The study demonstrates the positive effects of yeast, irrespective of its viabil-ity, in reducing the severity of SARA. However, further studies are required to evaluate the importance of yeast viability for other dietary conditions, particularly when the risk of acidosis is high. [ABSTRACT FROM AUTHOR]

Abstract: The cellulosome is a large extracellular multi-enzyme complex that facilitates the efficient hydrolysis and degradation of crystalline cellulosic substrates. During the course of our studies on the cellulosome of the rumen bacterium Ruminococcus flavefaciens, we focused on the critical ScaA dockerin (ScaADoc), the unique dockerin that incorporates the primary enzyme-integrating ScaA scaffoldin into the cohesin-bearing ScaB adaptor scaffoldin. In the absence of a high-resolution structure of the ScaADoc module, we generated a computational model, and, upon its analysis, we were surprised to discover a putative stacking interaction between an N-terminal Trp and a C-terminal Pro, which we termed intramolecular clasp. In order to verify the existence of such an interaction, these residues were mutated to alanine. Circular dichroism spectroscopy, intrinsic tryptophan and ANS fluorescence, and NMR spectroscopy indicated that mutation of these residues has a destabilizing effect on the functional integrity of the Ca2+-bound form of ScaADoc. Analysis of recently determined dockerin structures from other species revealed the presence of other well-defined intramolecular clasps, which consist of different types of interactions between selected residues at the dockerin termini. We propose that this thematic interaction may represent a major distinctive structural feature of the dockerin module. [Copyright &y& Elsevier]

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

The rumen bacterium Ruminococcus flavefaciens produces a highly organized multienzyme cellulosome complex that plays a key role in the degradation of plant cell wall polysaccharides, notably cellulose. The R. flavefaciens cellulosomal system is anchored to the bacterial cell wall through a relatively small ScaE scaffoldin subunit, which bears a single type IIIe cohesin responsible for the attachment of two major dockerin-containing scaffoldin proteins, ScaB and the cellulose-binding protein CttA. Although ScaB recruits the catalytic machinery onto the complex, CttA mediates attachment of the bacterial substrate via its two putative carbohydrate-binding modules. In an effort to understand the structural basis for assembly and cell surface attachment of the cellulosome in R. flavefaciens, we determined the crystal structure of the high affinity complex (Kd=20.83 nM) between the cohesin module of ScaE (CohE) and its cognate X-dockerin (XDoc) modular dyad from CttA at 1.97-Å resolution. The structure reveals an atypical calcium-binding loop containing a 13-residue insert. The results further pinpoint two charged specificity-related residues on the surface of the cohesin module that are responsible for specific versus promiscuous cross-strain binding of the dockerin module. In addition, a combined functional role for the three enigmatic dockerin inserts was established whereby these extraneous segments serve as structural buttresses that reinforce the stalklike conformation of the X-module, thus segregating its tethered complement of cellulosomal components from the cell surface. The novel structure of the RfCohE-XDoc complex sheds light on divergent dockerin structure and function and provides insight into the specificity features of the type IIIe cohesin-dockerin interaction. [ABSTRACT FROM AUTHOR]

Four, rumen fistulated swamp buffalo bulls were used to study microbial populations in the rumen when supplemented with coconut oil and mangosteen peel. Animals were randomly assigned to a 4 × 4 Latin square design. Four treatments were un-supplemented (Control), supplementation with coconut oil at 50 g/kg (CO5), supplementation with mangosteen peel at 30 g/kg (MP3) and supplementation with CO5 and MP3 (COM), of total DM intake. Animals received concentrate at 10 g/kg of BW, and rice straw was given ad libitum. Abundance of total bacteria was increased by CO5 supplementation, whereas populations of protozoa and Fibrobacter succinogenes were reduced by CO5 and COM supplementation. Dietary supplementation did not affect methanogen, Ruminococcus flavefaciens or Ruminococcus albus abundances. Dietary treatments changed denaturing gradient gel electrophoresis (DGGE) band patterns of methanogens and protozoa when compared with the control group, especially when supplemented with MP3. Supplementation of COM resulted in the greatest difference in pattern of DGGE bands for total bacteria compared with the control. Coconut oil and mangosteen peel supplementation resulted in changing of rumen microbial abundances and communities; however, combination of them could be more benefit to improve rumen fermentation of swamp buffalo fed on rice straw. [ABSTRACT FROM AUTHOR]

Background: Ruminococcus flavefaciens is an important fibre-degrading bacterium found in the mammalian gut. Cellulolytic strains from the bovine rumen have been shown to produce complex cellulosome structures that are associated with the cell surface. R. flavefaciens 007 is a highly cellulolytic strain whose ability to degrade dewaxed cotton, but not Avicel cellulose, was lost following initial isolation in the variant 007S. The ability was recovered after serial subculture to give the cotton-degrading strain 007C. This has allowed us to investigate the factors required for degradation of this particularly recalcitrant form of cellulose. Methodology/Principal Findings: The major proteins associated with the bacterial cell surface and with the culture supernatant were analyzed for R. flavefaciens 007S and 007C grown with cellobiose, xylan or Avicel cellulose as energy sources. Identification of the proteins was enabled by a draft genome sequence obtained for 007C. Among supernatant proteins a cellulosomal GH48 hydrolase, a rubrerthyrin-like protein and a protein with type IV pili N-terminal domain were the most strongly up-regulated in 007C cultures grown on Avicel compared with cellobiose. Strain 007S also showed substrate-related changes, but supernatant expression of the Pil protein and rubrerythrin in particular were markedly lower in 007S than in 007C during growth on Avicel. Conclusions/Significance: This study provides new information on the extracellular proteome of R. flavefaciens and its regulation in response to different growth substrates. Furthermore it suggests that the cotton cellulose non-degrading strain (007S) has altered regulation of multiple proteins that may be required for breakdown of cotton cellulose. One of these, the type IV pilus was previously shown to play a role in adhesion to cellulose in R. albus, and a related pilin protein was identified here for the first time as a major extracellular protein in R. flavefaciens. [ABSTRACT FROM AUTHOR]