Fibrobacter sp. HC4, a newly isolated strain, demonstrates a high cellulolytic activity as revealed by enzymatic measurements and in vitro assay.

Despite their low quantity and abundance, the cellulolytic bacteria that inhabit the equine large intestine are vital to their host, as they enable the crucial use of forage-based diets. Fibrobacter succinogenes is one of the most important intestinal cellulolytic bacteria. In this study, Fibrobacter sp. HC4, one cellulolytic strain newly isolated from the horse cecum, was characterized for its ability to utilize plant cell wall fibers. Fibrobacter sp. HC4 consumed only cellulose, cellobiose, and glucose and produced succinate and acetate in equal amounts. Among genes coding for CAZymes, 26% of the detected glycoside hydrolases (GHs) were involved in cellulolysis. These cellulases belong to the GH5, GH8, GH9, GH44, GH45, and GH51 families. Both carboxymethyl cellulase and xylanase activities of Fibrobacter sp. HC4 were detected using the Congo red method and were higher than those of F. succinogenes S85, the type strain. The in vitro addition of Fibrobacter sp. HC4 to a fecal microbial ecosystem of horses with large intestinal acidosis significantly enhanced fibrolytic activity as measured by the increase in gas and volatile fatty acids production during the first 48 h. According to this, the pH decreased and the disappearance of dry matter increased at a faster rate with Fibrobacter sp. HC4. Our data suggest a high specialization of the new strain in cellulose degradation. Such a strain could be of interest for future exploitation of its probiotic potential, which needs to be further determined by in vivo studies.IMPORTANCECellulose is the most abundant of plant cell wall fiber and can only be degraded by the large intestine microbiota, resulting in the production of volatile fatty acids that are essential for the host nutrition and health. Consequently, cellulolytic bacteria are of major importance to herbivores. However, these bacteria are challenged by various factors, such as high starch diets, which acidify the ecosystem and reduce their numbers and activity. This can lead to an imbalance in the gut microbiota and digestive problems such as colic, a major cause of mortality in horses. In this work, we characterized a newly isolated cellulolytic strain, Fibrobacter sp. HC4, from the equine intestinal microbiota. Due to its high cellulolytic capacity, reintroduction of this strain into an equine fecal ecosystem stimulates hay fermentation in vitro. Isolating and describing cellulolytic bacteria is a prerequisite for using them as probiotics to restore intestinal balance.

Quantitative qPCR Analysis of Ruminal Microorganisms in Beef Cattle Grazing in Pastures in the Rainy Season and Supplemented with Different Protein Levels.

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.

Fibrobacter communities in the gastrointestinal tracts of diverse hindgut-fermenting herbivores are distinct from those of the rumen.

The genus Fibrobacter contains cellulolytic bacteria originally isolated from the rumen. Culture-independent investigations have since identified Fibrobacter populations in the gastrointestinal tracts of numerous hindgut-fermenting herbivores, but their physiology is poorly characterized due to few representative axenic cultures. To test the hypothesis that novel Fibrobacter diversity exists in hindgut fermenters, we performed culturing and 16S rRNA gene amplicon sequencing on samples collected from phylogenetically diverse herbivorous hosts. Using a unique approach for recovering axenic Fibrobacter cultures, we isolated 45 novel strains from 11 different hosts. Full-length 16S rRNA gene sequencing of these isolates identified nine discrete phylotypes (cutoff = 0.03%) among them, including several that were only isolated from hindgut-fermenting hosts, and four previously unrepresented by axenic cultures. Our phylogenetic analysis indicated that six of the phylotypes are more closely related to previously described subspecies of Fibrobacter succinogenes, while the remaining three were more closely related to F. intestinalis. Culture-independent bacterial community profiling confirmed that most isolates were representative of numerically dominant phylotypes in their respective samples and strengthened the association of certain phylotypes with either ruminants or hindgut-fermenters. Despite considerable phylogenetic diversity observed among the Fibrobacter strains isolated here, phenotypic characterization suggests a conserved specialization for growth on cellulose.

Monitoring of gene expression in Fibrobacter succinogenes S85 under the co-culture with non-fibrolytic ruminal bacteria.

Fibrobacter succinogenes is one of the most pivotal fibrolytic bacterial species in the rumen. In a previous study, we confirmed enhancement of fiber digestion in a co-culture of F. succinogenes S85 with non-fibrolytic ruminal strains R-25 and/or Selenomonas ruminantium S137. In the present study, mRNA expression level of selected functional genes in the genome of F. succinogenes S85 was monitored by real-time RT-PCR. Growth profile of F. succinogenes S85 was similar in both the monoculture and co-cultures with non-fibrolytics. However, expression of 16S rRNA gene of F. succinogenes S85 in the co-culture was higher (P < 0.01) than that of the monoculture. This finding suggests that metabolic activity of F. succinogenes S85 was enhanced by coexistence with strains R-25 and/or S. ruminantium S137. The mRNA expression of fumarate reductase and glycoside hydrolase genes was up-regulated (P < 0.01) when F. succinogenes S85 was co-cultured with non-fibrolytics. These results indicate the enhancement of succinate production and fiber hydrolysis by F. succinogenes S85 in co-cultures of S. ruminantium and R-25 strains.

Eremophila glabra reduces methane production and methanogen populations when fermented in a Rusitec.

Eremophila glabra Juss. (Scrophulariaceae), a native Australian shrub, has been demonstrated to have low methanogenic potential in a batch in vitro fermentation system. The present study aimed to test longer-term effects of E. glabra on rumen fermentation characteristics, particularly methane production and the methanogen population, when included as a component of a fermentation substrate in an in vitro continuous culture system (Rusitec). E. glabra was included at 150, 250, 400 g/kg DM (EG15, EG25, and EG40) with an oaten chaff and lupin-based substrate (control). Overall, the experiment lasted 33 days, with 12 days of acclimatization, followed by two periods during which fermentation characteristics (total gas, methane and VFA productions, dry matter disappearance, pH) were measured. The number of copies of genes specifically associated with total bacteria and cellulolytic bacteria (16S rRNA gene) and total ruminal methanogenic archaeal organisms (the methyl coenzyme M reductase A gene (mcrA)) was also measured during this time using quantitative real-time PCR. Total gas production, methane and volatile fatty acid concentrations were significantly reduced with addition of E. glabra. At the end of the experiment, the overall methane reduction was 32% and 45% for EG15 and EG25 respectively, compared to the control, and the reduction was in a dose-dependent manner. Total bacterial numbers did not change, but the total methanogen population decreased by up to 42.1% (EG40) when compared to the control substrate. The Fibrobacter succinogenes population was reduced at all levels of E. glabra, while Ruminococcus albus was reduced only by EG40. Our results indicate that replacing a portion of a fibrous substrate with E. glabra maintained a significant reduction in methane production and methanogen populations over three weeks in vitro, with some minor inhibition on overall fermentation at the lower inclusion levels.

Structural, Biochemical, and Phylogenetic Analysis of Bacterial and Fungal Carbohydrate Esterase Family 15 Glucuronoyl Esterases in the Rumen.

Glucuronoyl esterases (GEs) are carbohydrate active enzymes in carbohydrate esterase family 15 which are involved in the hydrolysis of lignin-carbohydrate complexes. They are encoded by a wide range of aerobic and anaerobic fungi and bacteria inhabiting diverse environments. The rumen microbiome is a complex microbial community with a wide array of enzymes that specialize in deconstructing plant cell wall carbohydrates. Enzymes from the rumen tend to show low similarity to homologues found in other environments, making the rumen microbiome a promising source for the discovery of novel enzymes. Using a combination of phylogenetic and structural analysis, we investigated the structure-function relationship of GEs from the rumen bacteria Fibrobacter succinogenes and Ruminococcus flavefaciens, and from the rumen fungus, Piromyces rhizinflata. All adopt a canonical α/ß hydrolase fold and possess a structurally conserved Ser-His-Glu/Asp catalytic triad. Structural variations in the enzymes are localized to loops surrounding the active site. Analysis of the active site structures in these enzymes emphasized the importance of structural plasticity in GEs with non-canonical active site conformations. We hypothesize that interkingdom HGT events may have contributed to the diversity of GEs in the rumen, and this is demonstrated by the phylogenetic and structural similarity observed between rumen bacterial and fungal GEs. This study advances our understanding of the structure-function relationship in glucuronoyl esterases and illuminates the evolutionary dynamics that contribute to enzyme diversity in the rumen microbiome.

Dynamic genome-based metabolic modeling of the predominant cellulolytic rumen bacterium Fibrobacter succinogenes S85.

Fibrobacter succinogenes is a cellulolytic bacterium that plays an essential role in the degradation of plant fibers in the rumen ecosystem. It converts cellulose polymers into intracellular glycogen and the fermentation metabolites succinate, acetate, and formate. We developed dynamic models of F. succinogenes S85 metabolism on glucose, cellobiose, and cellulose on the basis of a network reconstruction done with the automatic reconstruction of metabolic model workspace. The reconstruction was based on genome annotation, five template-based orthology methods, gap filling, and manual curation. The metabolic network of F. succinogenes S85 comprises 1,565 reactions with 77% linked to 1,317 genes, 1,586 unique metabolites, and 931 pathways. The network was reduced using the NetRed algorithm and analyzed for the computation of elementary flux modes. A yield analysis was further performed to select a minimal set of macroscopic reactions for each substrate. The accuracy of the models was acceptable in simulating F. succinogenes carbohydrate metabolism with an average coefficient of variation of the root mean squared error of 19%. The resulting models are useful resources for investigating the metabolic capabilities of F. succinogenes S85, including the dynamics of metabolite production. Such an approach is a key step toward the integration of omics microbial information into predictive models of rumen metabolism. IMPORTANCE F. succinogenes S85 is a cellulose-degrading and succinate-producing bacterium. Such functions are central for the rumen ecosystem and are of special interest for several industrial applications. This work illustrates how information of the genome of F. succinogenes can be translated to develop predictive dynamic models of rumen fermentation processes. We expect this approach can be applied to other rumen microbes for producing a model of rumen microbiome that can be used for studying microbial manipulation strategies aimed at enhancing feed utilization and mitigating enteric emissions.

The microbial ecology of anaerobic cellulose degradation in municipal waste landfill sites: evidence of a role for fibrobacters.

Cellulose is reputedly the most abundant organic polymer in the biosphere, yet despite the fundamental role of cellulolytic microorganisms in global carbon cycling and as potential sources of novel enzymes for biotechnology, their identity and ecology is not well established. Cellulose is a major component of landfill waste and its degradation is therefore a key feature of the anaerobic microbial decomposition process. Here, we targeted a number of taxa containing known cellulolytic anaerobes (members of the bacterial genus Fibrobacter, lineages of Clostridium clusters I, III, IV and XIV, and anaerobic fungi of the Neocallimastigales) in landfill leachate and colonized cellulose 'baits' via PCR and quantitative PCR (qPCR). Fibrobacter spp. and Clostridium clusters III, IV and XIV were detected in almost all leachate samples and cluster III and XIV clostridia were the most abundant (1-6% and 1-17% of total bacterial 16S rRNA gene copies respectively). Two landfill leachate microcosms were constructed to specifically assess those microbial communities that colonize and degrade cellulose substrates in situ. Scanning electron microscopy (SEM) of colonized cotton revealed extensive cellulose degradation in one microcosm, and Fibrobacter spp. and Clostridium cluster III represented 29% and 17%, respectively, of total bacterial 16S rRNA gene copies in the biofilm. Visible cellulose degradation was not observed in the second microcosm, and this correlated with negligible relative abundances of Clostridium cluster III and Fibrobacter spp. (≤ 0.1%), providing the first evidence that the novel fibrobacters recently detected in landfill sites and other non-gut environments colonize and degrade cellulose substrates in situ.

Effect of phenotypic residual feed intake and dietary forage content on the rumen microbial community of beef cattle.

Feed-efficient animals have lower production costs and reduced environmental impact. Given that rumen microbial fermentation plays a pivotal role in host nutrition, the premise that rumen microbiota may contribute to host feed efficiency is gaining momentum. Since diet is a major factor in determining rumen community structure and fermentation patterns, we investigated the effect of divergence in phenotypic residual feed intake (RFI) on ruminal community structure of beef cattle across two contrasting diets. PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR) were performed to profile the rumen bacterial population and to quantify the ruminal populations of Entodinium spp., protozoa, Fibrobacter succinogenes, Ruminococcus flavefaciens, Ruminococcus albus, Prevotella brevis, the genus Prevotella, and fungi in 14 low (efficient)- and 14 high (inefficient)-RFI animals offered a low-energy, high-forage diet, followed by a high-energy, low-forage diet. Canonical correspondence and Spearman correlation analyses were used to investigate associations between physiological variables and rumen microbial structure and specific microbial populations, respectively. The effect of RFI on bacterial profiles was influenced by diet, with the association between RFI group and PCR-DGGE profiles stronger for the higher forage diet. qPCR showed that Prevotella abundance was higher (P < 0.0001) in inefficient animals. A higher (P < 0.0001) abundance of Entodinium and Prevotella spp. and a lower (P < 0.0001) abundance of Fibrobacter succinogenes were observed when animals were offered the low-forage diet. Thus, differences in the ruminal microflora may contribute to host feed efficiency, although this effect may also be modulated by the diet offered.

The Fibrobacteres: an important phylum of cellulose-degrading bacteria.

The phylum Fibrobacteres currently comprises one formal genus, Fibrobacter, and two cultured species, Fibrobacter succinogenes and Fibrobacter intestinalis, that are recognised as major bacterial degraders of lignocellulosic material in the herbivore gut. Historically, members of the genus Fibrobacter were thought to only occupy mammalian intestinal tracts. However, recent 16S rRNA gene-targeted molecular approaches have demonstrated that novel centres of variation within the genus Fibrobacter are present in landfill sites and freshwater lakes, and their relative abundance suggests a potential role for fibrobacters in cellulose degradation beyond the herbivore gut. Furthermore, a novel subphylum within the Fibrobacteres has been detected in the gut of wood-feeding termites, and proteomic analyses have confirmed their involvement in cellulose hydrolysis. The genome sequence of F. succinogenes rumen strain S85 has recently suggested that within this group of organisms a "third" way of attacking the most abundant form of organic carbon in the biosphere, cellulose, has evolved. This observation not only has evolutionary significance, but the superior efficiency of anaerobic cellulose hydrolysis by Fibrobacter spp., in comparison to other cellulolytic rumen bacteria that typically utilise membrane-bound enzyme complexes (cellulosomes), may be explained by this novel cellulase system. There are few bacterial phyla with potential functional importance for which there is such a paucity of phenotypic and functional data. In this review, we highlight current knowledge of the Fibrobacteres phylum, its taxonomy, phylogeny, ecology and potential as a source of novel glycosyl hydrolases of biotechnological importance.

Rational design to improve thermostability and specific activity of the truncated Fibrobacter succinogenes 1,3-1,4-ß-D-glucanase.

1,3-1,4-ß-D-Glucanase has been widely used as a feed additive to help non-ruminant animals digest plant fibers, with potential in increasing nutrition turnover rate and reducing sanitary problems. Engineering of enzymes for better thermostability is of great importance because it not only can broaden their industrial applications, but also facilitate exploring the mechanism of enzyme stability from structural point of view. To obtain enzyme with higher thermostability and specific activity, structure-based rational design was carried out in this study. Eleven mutants of Fibrobacter succinogenes 1,3-1,4-ß-D-glucanase were constructed in attempt to improve the enzyme properties. In particular, the crude proteins expressed in Pichia pastoris were examined firstly to ensure that the protein productions meet the need for industrial fermentation. The crude protein of V18Y mutant showed a 2 °C increment of Tm and W203Y showed ∼30% increment of the specific activity. To further investigate the structure-function relationship, some mutants were expressed and purified from P. pastoris and Escherichia coli. Notably, the specific activity of purified W203Y which was expressed in E. coli was 63% higher than the wild-type protein. The double mutant V18Y/W203Y showed the same increments of Tm and specific activity as the single mutants did. When expressed and purified from E. coli, V18Y/W203Y showed similar pattern of thermostability increment and 75% higher specific activity. Furthermore, the apo-form and substrate complex structures of V18Y/W203Y were solved by X-ray crystallography. Analyzing protein structure of V18Y/W203Y helps elucidate how the mutations could enhance the protein stability and enzyme activity.

Screening of a xylanase clone from a fosmid library of rumen microbiota in Hu sheep.

The glycosyl hydrolase family 11, which is responsible for carbohydrate metabolism, was identified in the open reading frame (ORF) 6 of a xylanase positive clone from a fosmid library of rumen microbiota of Hu sheep. A BLASTP search of GenBank revealed that ORF6 encoded a 355-amino acid putative endoxylanase, having 61% similarity (e(-73)) to endo-1,4-ß-xylanase of Fibrobacter succinogenes S85 (YP_003250510.1). Predicted with the SWISS-MODEL, there were two separate ß-sandwich clusters linked with a high serine containing linker in ORF6. The N-terminal ß-sandwich is a novel endoxylanase of the glycosyl hydrolase family 11 with a specific activity of 1150.00 U/mg. The optimal pH and temperature for this enzyme were shown to be pH 5.0 and 50°C, respectively. The C-terminal helped increase the stability of the xylanase but decreased the activity to some degree. The C-terminal ß-sandwich could bind avicel, but no conserved domain could be found. It may be a novel carbohydrate-binding module.

In Vivo Competitions between Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminoccus albus in a Gnotobiotic Sheep Model Revealed by Multi-Omic Analyses.

Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens are the three predominant cellulolytic bacterial species found in the rumen. In vitro studies have shown that these species compete for adherence to, and growth upon, cellulosic biomass. Yet their molecular interactions in vivo have not heretofore been examined. Gnotobiotically raised lambs harboring a 17-h-old immature microbiota devoid of culturable cellulolytic bacteria and methanogens were inoculated first with F. succinogenes S85 and Methanobrevibacter sp. strain 87.7, and 5 months later, the lambs were inoculated with R. albus 8 and R. flavefaciens FD-1. Longitudinal samples were collected and profiled for population dynamics, gene expression, fibrolytic enzyme activity, in sacco fibrolysis, and metabolite profiling. Quantitative PCR, metagenome and metatranscriptome data show that F. succinogenes establishes at high levels initially but is gradually outcompeted following the introduction of the ruminococci. This shift resulted in an increase in carboxymethyl cellulase (CMCase) and xylanase activities but not in greater fibrolysis, suggesting that F. succinogenes and ruminococci deploy different but equally effective means to degrade plant cell walls. Expression profiles showed that F. succinogenes relied upon outer membrane vesicles and a diverse repertoire of CAZymes, while R. albus and R. flavefaciens preferred type IV pili and either CBM37-harboring or cellulosomal carbohydrate-active enzymes (CAZymes), respectively. The changes in cellulolytics also affected the rumen metabolome, including an increase in acetate and butyrate at the expense of propionate. In conclusion, this study provides the first demonstration of in vivo competition between the three predominant cellulolytic bacteria and provides insight on the influence of these ecological interactions on rumen fibrolytic function and metabolomic response.IMPORTANCE Ruminant animals, including cattle and sheep, depend on their rumen microbiota to digest plant biomass and convert it into absorbable energy. Considering that the extent of meat and milk production depends on the efficiency of the microbiota to deconstruct plant cell walls, the functionality of predominant rumen cellulolytic bacteria, Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens, has been extensively studied in vitro to obtain a better knowledge of how they operate to hydrolyze polysaccharides and ultimately find ways to enhance animal production. This study provides the first evidence of in vivo competitions between F. succinogenes and the two Ruminococcus species. It shows that a simple disequilibrium within the cellulolytic community has repercussions on the rumen metabolome and fermentation end products. This finding will have to be considered in the future when determining strategies aiming at directing rumen fermentations for animal production.

Domain analysis of a modular alpha-L-Arabinofuranosidase with a unique carbohydrate binding strategy from the fiber-degrading bacterium Fibrobacter succinogenes S85.

Family 43 glycoside hydrolases (GH43s) are known to exhibit various activities involved in hemicellulose hydrolysis. Thus, these enzymes contribute to efficient plant cell wall degradation, a topic of much interest for biofuel production. In this study, we characterized a unique GH43 protein from Fibrobacter succinogenes S85. The recombinant protein showed α-l-arabinofuranosidase activity, specifically with arabinoxylan. The enzyme is, therefore, an arabinoxylan arabinofuranohydrolase (AXH). The F. succinogenes AXH (FSUAXH1) is a modular protein that is composed of a signal peptide, a GH43 catalytic module, a unique ß-sandwich module (XX domain), a family 6 carbohydrate-binding module (CBM6), and F. succinogenes-specific paralogous module 1 (FPm-1). Truncational analysis and site-directed mutagenesis of the protein revealed that the GH43 domain/XX domain constitute a new form of carbohydrate-binding module and that residue Y484 in the XX domain is essential for binding to arabinoxylan, although protein structural analyses may be required to confirm some of the observations. Kinetic studies demonstrated that the Y484A mutation leads to a higher k(cat) for a truncated derivative of FSUAXH1 composed of only the GH43 catalytic module and the XX domain. However, an increase in the K(m) for arabinoxylan led to a 3-fold decrease in catalytic efficiency. Based on the knowledge that most XX domains are found only in GH43 proteins, the evolutionary relationships within the GH43 family were investigated. These analyses showed that in GH43 members with a XX domain, the two modules have coevolved and that the length of a loop within the XX domain may serve as an important determinant of substrate specificity.

Biochemical and domain analyses of FSUAxe6B, a modular acetyl xylan esterase, identify a unique carbohydrate binding module in Fibrobacter succinogenes S85.

Acetyl xylan esterase (EC 3.1.1.72) is a member of a set of enzymes required to depolymerize hemicellulose, especially xylan that is composed of a main chain of beta-1,4-linked xylopyranoside residues decorated with acetyl side groups. Fibrobacter succinogenes S85 Axe6B (FSUAxe6B) is an acetyl xylan esterase encoded in the genome of this rumen bacterium. The enzyme is a modular protein comprised of an esterase domain, a carbohydrate-binding module, and a region of unknown function. Sequences that are homologous to the region of unknown function are paralogously distributed, thus far, only in F. succinogenes. Therefore, the sequences were designated Fibrobacter succinogenes-specific paralogous module 1 (FPm-1). The FPm-1s are associated with at least 24 polypeptides in the genome of F. succinogenes S85. A bioinformatics search showed that most of the FPm-1-appended polypeptides are putative carbohydrate-active enzymes, suggesting a potential role in carbohydrate metabolism. Truncational analysis of FSUAxe6B, together with catalytic and substrate binding studies, has allowed us to delineate the functional modules in the polypeptide. The N-terminal half of FSUAxe6B harbors the activity that cleaves side chain acetyl groups from xylan-like substrates, and the binding of insoluble xylan was determined to originate from FPm-1. Site-directed mutagenesis studies of highly conserved active-site residues in the esterase domain suggested that the esterase activity is derived from a tetrad composed of Ser(44), His(273), Glu(194), and Asp(270), with both Glu(194) and Asp(270) functioning as helper acids, instead of a single carboxylate residue proposed to initiate catalysis.

Structural and catalytic roles of amino acid residues located at substrate-binding pocket in Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase.

We created 12 mutant enzymes (E11L, F40I, Y42L, N44L, N44Q, E47I, L62G, K64A, K64M, R137M, R137Q, and N139A) from the truncated Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (TF-glucanase). The enzymes were used to investigate the structural and catalytic roles of specific amino acid residues located at the catalytic pocket and having direct interactions with glucose subsites of the product beta-1,3-1,4-cellotriose (CLTR). Fluorescence spectrometry showed no discernible changes in secondary structures among purified TF-glucanase and the mutants. Kinetic analyses showed E11L, F40I, Y42L, R137M, and R137Q with a >10-fold decrease of specific activity (11.2- to 67.4-fold), and E11L, N44Q, E47I, K64M, R137M, R137Q, and N139A with a 2.17- to 4.3-fold increase of K(m) value when compared with TF-glucanase. Notably, E11L, R137Q, R137M, F40I, and N139A showed the most significant decrease in catalytic efficiency relative to TF-glucanase, by 2155-, 84.9-, 48.5-, 41.1-, and 19.1-fold, respectively; the five mutants showed the greatest changes in comparative energy DeltaDeltaG(b), with values of 1.94 to 4.92 kcal/mol. Combined with results from kinetic and structure modeling analyses of all mutant enzymes and X-ray crystallography of F40I, we elucidate that Glu11, Phe40, Arg137, and Asn139 play a crucial role in the catalysis of TF-glucanase owing to their local and direct interaction through hydrogen bonds or van der Waals stacking interaction by aromatic rings onto the glucose subsites -3, -2, and -1 of CLTR/substrate. The overall globular structures in the wild-type and mutant F40I enzymes do not differ.

Detection of fiber-digesting bacteria in the ceca of ostrich using specific primer sets.

The purpose of this study was to detect three fibrolytic bacteria, Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminococcus albus, in the cecal digesta of the ostrich (Struthio camelus) by PCR using a species-specific primer set for each 16S ribosomal RNA gene (16S rDNA). Although amplified DNA fragments obtained from each primer set had the expected size, the clone library derived from the amplimer contained non-specific sequences. The F. succinogenes-specific primer set recovered a partial 16S rDNA sequence of an uncultivated Fibrobacter with low similarity (<95%) and distantly related phylogenetic positioning to Fibrobacter sequences deposited in the databases, indicating a novel species of Fibrobacter. The sequence was considered to be identical to a clone detected in our previous experiment. Thus, we confirm that the gastrointestinal tract of the ostrich is one of the habitats of Fibrobacter species. The clone library derived from the R. flavefaciens-specific primer set contained a 16S rDNA sequence with 97% similarity to R. flavefaciens, indicating it could be one of a major fibrolytic bacterium in the ostrich ceca. No R. albus 16S rDNA sequence was found in the clone library of the R. albus-specific primer set.

Bacterial community analysis on the different mucosal immune inductive sites of gastrointestinal tract in Bactrian camels.

The microbial communities colonize the mucosal immune inductive sites could be captured by hosts, which could initiate the mucosal immune responses. The aggregated lymphoid nodule area (ALNA) and the ileal Payer's patches (PPs) in Bactrian camels are both the mucosal immune inductive sites of the gastrointestinal tract. Here, the bacteria community associated with the ALNA and ileal PPs were analyzed using of 16S rDNA-Illumina Miseq sequencing. The mutual dominant bacterial phyla at the two sites were the Bacteroidetes, Firmicutes, Verrucomicrobia and Proteobacteria, and the mutual dominant genus in both sits was Prevotella. The abundances of the Fibrobacter, Campylobacter and RFP12 were all higher in ALNA than in ileal PPs. While, the abundances of the 5-7N15, Clostridium, and Escherichia were all higher in ileal PPs than in ALNA. The results suggested that the host's intestinal microenvironment is selective for the symbiotic bacteria colonizing the corresponding sites, on the contrary, the symbiotic bacteria could impact on the physiological functions of this local site. In ALNA and ileal PPs of Bactrian camel, the bacteria which colonized different immune inductive sites have the potential to stimulate different immune responses, which is the result of the mutual selection and adaptation between microbial communities and their host.

Molecular biological detection and quantification of novel Fibrobacter populations in freshwater lakes.

PCR and quantitative PCR (qPCR) primers targeting the 16S rRNA gene were used to detect and quantify members of the genus Fibrobacter in lake water, sediment and colonized cotton taken from two freshwater lakes. Phylogenetic analysis identified two groups of sequences; those clustered with Fibrobacter succinogenes, the type species, and a defined cluster of clones loosely associated with several Fibrobacter sequences observed previously in clone libraries from freshwater environments. 16S rRNA gene sequences recovered in the same way from soil samples and ovine feces in the surrounding land were all F. succinogenes and did not include any from this group of the "freshwater" Fibrobacteres. In all cases, nested PCR was required to detect Fibrobacter 16S rRNA genes, and qPCR analysis of reverse transcribed bacterial community RNA confirmed their very low relative abundance on colonized cotton baits in the water column (at 0, 3, 7, 11, and 13 m) and on the sediment surface (<0.02% of total bacterial rRNA). However, in Esthwaite Water sediment itself, the relative abundance of fibrobacters was 2 orders of magnitude higher (ca. 1% of total bacterial rRNA). The presence of fibrobacters, including the cellulolytic rumen species F. succinogenes, on colonized cellulose samples and in lake sediment suggests that these organisms may contribute to the primary degradation of plant and algal biomass in freshwater lake ecosystems.

Use of real-time PCR technique in studying rumen cellulolytic bacteria population as affected by level of roughage in swamp buffalo.

A real-time polymerase chain reaction approach was used in this study to determine the population of major ruminal bacterial species (Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens) in digesta and rumen fluid of swamp buffalo (Bubalus bubalis). Four rumen-fistulated, male swamp buffalo were randomly assigned according to a 4 x 4 Latin square design to evaluate the effect of the urea-treated rice straw (roughage source)-to-concentrate ratio on cellulolytic bacterial distribution. Animals were fed roughage-to-concentrate (R:C) ratios of 100:0, 75:25, 50:50, and 25:75, respectively. At the end of each period, rumen fluid and digesta were collected at 0 h and 4 h post-morning-feeding. It was found that feeding urea-treated rice straw solely increased these three cellulolytic bacteria numbers up to 2.65 x 10(9) and 3.54 x 10(9) copies per milliliter for F. succinogenes, 5.10 x 10(7) and 7.40 x 10(7) copies per milliliter for R. flavefaciens, and 4.00 x 10(6) and 6.00 x 10(6) copies per milliliter for R. albus in rumen fluid and digesta, respectively. The distribution of the three cellulolytic bacteria species in digesta were highest at 3.21 x 10(9), 4.55 x 10(7), and 4.56 x 10(6) copies per milliliter for F. succinogenes, R. flavefaciens, and R. albus, respectively. Moreover, at 4 h post-morning-feeding, the populations of the three cellulolytic bacteria were higher than found at 0 h post-morning-feeding. It is most notable that F. succinogenes were the highest in population in the rumen of swamp buffalo and cellulolytic bacteria mostly adhered to feed digesta in the rumen.