Flavefaciens FD-1 Research Articles

Highly efficient, processive and multifunctional recombinant endoglucanase RfGH5_4 from Ruminococcus flavefaciens FD-1 v3 for recycling lignocellulosic plant biomasses

Gene encoding endoglucanase, RfGH5_4 from R. flavefaciens FD-1 v3 was cloned, expressed in Escherichia coli BL21(DE3) cells and purified. RfGH5_4 showed molecular size 41 kDa and maximum activity at pH 5.5 and 55 °C. It was stable between pH 5.0–8.0, retaining 85% activity and between 5 °C–45 °C, retaining 75% activity, after 60 min. RfGH5_4 displayed maximum activity (U/mg) against barley β-D-glucan (665) followed by carboxymethyl cellulose (450), xyloglucan (343), konjac glucomannan (285), phosphoric acid swollen cellulose (86), beechwood xylan (21.7) and carob galactomannan (16), thereby displaying the multi-functionality. Catalytic efficiency (mL.mg−1 s−1) of RfGH5_4 against carboxymethyl cellulose (146) and konjac glucomannan (529) was significantly high. TLC and MALDI-TOF-MS analyses of RfGH5_4 treated hydrolysates of cellulosic and hemicellulosic polysaccharides displayed oligosaccharides of degree of polymerization (DP) between DP2-DP11. TLC, HPLC and Processivity-Index analyses revealed RfGH5_4 to be a processive endoglucanase as initially, for 30 min it hydrolysed cellulose to cellotetraose followed by persistent release of cellotriose and cellobiose. RfGH5_4 yielded sufficiently high Total Reducing Sugar (TRS, mg/g) from saccharification of alkali pre-treated sorghum (72), finger millet (62), sugarcane bagasse (38) and cotton (27) in a 48 h saccharification reaction. Thus, RfGH5_4 can be considered as a potential endoglucanase for renewable energy applications.

Effect of oral dosing of live and autoclaved culture of Ruminococcus flavefaciens FD-1 on rumen bacterial and fungal populations in Murrah buffaloes

Effect of oral dosing of live and autoclaved culture of Ruminococcus flavefaciens FD-1 on rumen bacterial and fungal populations in Murrah buffaloes

Effects of yeast and yeast cell wall polysaccharides supplementation on beef cattle growth performance, rumen microbial populations and lipopolysaccharides production

This experiment was conducted to investigate the effects of live yeast and yeast cell wall polysaccharides on growth performance, rumen function and plasma lipopolysaccharides (LPS) content and immunity parameters of beef cattle. Forty Qinchuan cattle were randomly assigned to one of four treatments with 10 replicates in each treatment. The dietary treatments were: control diet (CTR), CTR supplemented with 1 g live yeast (2×1010 live cell g−1 per cattle per day (YST1), CTR supplemented with 2 g live yeast per cattle per day (YST2) and CTR supplemented with 20 g of yeast cell wall polysaccharides (30.0%≤β-glucan≤35.0%, and 28.0%≤mannanoligosaccharide≤32.0%) per cattle per day (YCW). The average daily gain was higher (P=0.023) and feed conversion ratio was lower (P=0.042) for the YST2 than the CTR. The digestibility of neutral detergent fiber (P=0.039) and acid detergent fiber (P=0.016) were higher in yeast supplemented groups. The acetic acid:propionic acid of the YST2 was lower compared with the CTR (P=0.033). Plasma LPS (P=0.032), acute phase protein haptoglobin (P=0.033), plasma amyloid A (P=0.015) and histamine (P=0.038) were lower in the YST2 compared with the CTR. The copies of fibrolytic microbial populations such as Fibrobacter succinogenes S85, Ruminococcus albus 7 and Ruminococcus flavefaciens FD-1 of the YST2 were higher (P<0.001), while the copies of typical lactate producing bacteria Streptococcus bovis JB1 was lower (P<0.001) compared with the CTR. Little differences were observed between the CTR, YST1 and YCW in growth performance, ruminal fermentation characteristics, microbial populations, immunity indices and total tract nutrient digestibility. It is concluded that the YST2 could promote fibrolytic microbial populations, decrease starch-utilizing bacteria, reduce LPS production in the rumen and LPS absorption into plasma and decrease inflammatory parameters, which can lead to an improvement in growth performance in beef cattle.

Molecular Cloning, Expression and Biochemical Characterization of a Family 5 Glycoside Hydrolase First Endo-Mannanase (RfGH5_7) from Ruminococcus flavefaciens FD-1 v3.

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 ~ 35kDa. 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. 10mM 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, 0.92mg/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.

Higher order scaffoldin assembly in Ruminococcus flavefaciens cellulosome is coordinated by a discrete cohesin-dockerin interaction

Cellulosomes are highly sophisticated molecular nanomachines that participate in the deconstruction of complex polysaccharides, notably cellulose and hemicellulose. Cellulosomal assembly is orchestrated by the interaction of enzyme-borne dockerin (Doc) modules to tandem cohesin (Coh) modules of a non-catalytic primary scaffoldin. In some cases, as exemplified by the cellulosome of the major cellulolytic ruminal bacterium Ruminococcus flavefaciens, primary scaffoldins bind to adaptor scaffoldins that further interact with the cell surface via anchoring scaffoldins, thereby increasing cellulosome complexity. Here we elucidate the structure of the unique Doc of R. flavefaciens FD-1 primary scaffoldin ScaA, bound to Coh 5 of the adaptor scaffoldin ScaB. The RfCohScaB5-DocScaA complex has an elliptical architecture similar to previously described complexes from a variety of ecological niches. ScaA Doc presents a single-binding mode, analogous to that described for the other two Coh-Doc specificities required for cellulosome assembly in R. flavefaciens. The exclusive reliance on a single-mode of Coh recognition contrasts with the majority of cellulosomes from other bacterial species described to date, where Docs contain two similar Coh-binding interfaces promoting a dual-binding mode. The discrete Coh-Doc interactions observed in ruminal cellulosomes suggest an adaptation to the exquisite properties of the rumen environment.

Complexity of the Ruminococcus flavefaciens FD-1 cellulosome reflects an expansion of family-related protein-protein interactions

Protein-protein interactions play a vital role in cellular processes as exemplified by assembly of the intricate multi-enzyme cellulosome complex. Cellulosomes are assembled by selective high-affinity binding of enzyme-borne dockerin modules to repeated cohesin modules of structural proteins termed scaffoldins. Recent sequencing of the fiber-degrading Ruminococcus flavefaciens FD-1 genome revealed a particularly elaborate cellulosome system. In total, 223 dockerin-bearing ORFs potentially involved in cellulosome assembly and a variety of multi-modular scaffoldins were identified, and the dockerins were classified into six major groups. Here, extensive screening employing three complementary medium- to high-throughput platforms was used to characterize the different cohesin-dockerin specificities. The platforms included (i) cellulose-coated microarray assay, (ii) enzyme-linked immunosorbent assay (ELISA) and (iii) in-vivo co-expression and screening in Escherichia coli. The data revealed a collection of unique cohesin-dockerin interactions and support the functional relevance of dockerin classification into groups. In contrast to observations reported previously, a dual-binding mode is involved in cellulosome cell-surface attachment, whereas single-binding interactions operate for cellulosome integration of enzymes. This sui generis cellulosome model enhances our understanding of the mechanisms governing the remarkable ability of R. flavefaciens to degrade carbohydrates in the bovine rumen and provides a basis for constructing efficient nano-machines applied to biological processes.

Structural Characterization and Bioactivity Analysis of the Two-Component Lantibiotic Flv System from a Ruminant Bacterium

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.

Rapid Evolution of Cellulosome Modules by Comparative Analyses of Five Clostridiales Genomes

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.

English

This study determined the effects of aromatic amino acids (AA) and phenyl acids (phenylacetic acid and phenylpropionic acid) on fermentation of xylan by selected rumen bacteria. The ruminal bacteria were grown in a defined medium containing oat spelts xylan as the sole energy source, plus one of the following N sources: ammonia (no AA); ammonia plus a complete mixture of 20 AA commonly found in protein (CAA); ammonia plus complete AA mixture minus aromatic AA (MAR); ammonia plus phenyl acids (no AA+PA); ammonia plus complete AA mixture without aromatic AA plus phenyl acids (MAR+PA). There was varied growth response, with Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD1, and Butyrivibrio fibrisolvens JW11 not being able to grow. The supply of CAA mixture significantly (P&lt;0.05) increased acetate production in all species, in comparison with no AA treatment, by 54, 30, 97 and 33% in Prevotella bryantii B14, Ruminococcus albus SY3, R. flavefaciens 17 and Pseudobutyrivibrio xylanivorans JK170 cultures, respectively. The addition of phenyl acids significantly (P&lt;0.05) increased acetate yields by 34, 8 and 5% in P. bryantii B14, R. albus SY3 and P. xylanivorans JK170 cultures, respectively. Sugar utilization by all the species significantly (P&lt;0.05) increased by 42 and 77% when CAA mixture was provided. The deletion of aromatic AA from the complete AA mixture caused a significant (P&lt;0.05) reduction in sugar utilization in P. bryantii B14 and P. xylanivorans JK170 by 25 and 10%, respectively. Phenyl acids addition increased sugar utilization by 4.8 and 5.1% in R. albus SY3 and R. flavefaciens 17, respectively. The supply of CAA mixture significantly (P&lt;0.05) increased microbial yield in P. bryantii B14, R. albus SY3, R. flavefaciens 17 and P. xylanivorans JK170 species. It is concluded that supplementation of complete mixture of AA is beneficial in stimulating bacterial growth in the rumen. &nbsp; Key words: Aromatic amino acids, phenylacetic acid, phenylpropionic acid, rumen bacteria, xylan.

Antimicrobial activity of Brazilian propolis extracts against rumen bacteria in vitro

The antimicrobial activity of three Brazilian propolis extracts was evaluated on bacterial strains representing major rumen functional groups. The extracts were prepared using different concentrations of propolis and alcohol, resulting in different phenolic compositions. The propolis extracts inhibited the growth of Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, Ruminococcus albus 7, Butyrivibrio fibrisolvens D1, Prevotella albensis M384, Peptostreptococcus sp. D1, Clostridium aminophilum F and Streptococcus bovis Pearl11, while R. albus 20, Prevotella bryantii B₁4 and Ruminobacter amylophilus H18 were resistant to all the extracts. The inhibited strains showed also different sensitivity to propolis; the hyper-ammonia-producing bacteria (C. aminophilum F and Peptostreptococcus sp. D1) being the most sensitive. Inhibition of hyper-ammonia-producing bacteria by propolis would be beneficial to the animal. The extract containing the lowest amount of phenolic compounds (LLOS C3) showed the lowest antimicrobial activity against all the bacteria. The major phenolic compounds identified in the propolis extracts (naringenin, chrysin, caffeic acid, p-coumaric acid and Artepillin C) were also evaluated on four sensitive strains. Only naringenin showed inhibitory effect against all strains, suggesting that naringenin is one of the components participating to the antibacterial activity of propolis.

Expression patterns of Ruminococcus flavefaciens 007S cellulases as revealed by zymogram approach

The expression of Ruminococcus flavefaciens 007S cellulases in different incubation time points (growth stages) and their substrate inducibility were analyzed by comparing the zymogram expression profiles of cultures grown on insoluble cellulose (Avicel) with cellobiose-grown cultures. The molecular weights of the enzymes were compared to (putative) cellulases encoded in the R. flavefaciens FD-1 genome.

Effect of Exogenous Fibrolytic Enzyme Application on the Microbial Attachment and Digestion of Barley Straw In vitro.

The effects of exogenous fibrolytic enzymes (EFE; a mixture of two preparations from Trichoderma spp., with predominant xylanase and β-glucanase activities, respectively) on colonization and digestion of ground barley straw and alfalfa hay by Fibrobacter succinogenes S85 and Ruminococcus flavefaciens FD1 were studied in vitro. The two levels (28 and 280 μg/ml) of EFE tested and both bacteria were effective at digesting NDF of hay and straw. With both substrates, more NDF hydrolysis (p<0.01) was achieved with EFE alone at 280 than at 28 μg/ml. A synergistic effect (p<0.01) of F. succinogenes S85 and EFE on straw digestion was observed at 28 but not 280 μg/ml of EFE. Strain R. flavefaciens FD1 digested more (p<0.01) hay and straw with higher EFE than with lower or no EFE, but the effect was additive rather than synergistic. Included in the incubation medium, EFE showed potential to improve fibre digestion by cellulolytic ruminal bacteria. In a second batch culture experiment using mixed rumen microbes, DM disappearance (DMD), gas production and incorporation of 15N into particle-associated microbial N (15N-PAMN) were higher (p<0.001) with ammoniated (5% w/w; AS) than with native (S) ground barley straw. Application of EFE to the straws increased (p<0.001) DMD and gas production at 4 and 12 h, but not at 48 h of the incubation. EFE applied onto S increased (p<0.01) 15N-PAMN at 4 h only, but EFE on AS increased (p<0.001) 15N-PAMN at all time points. Prehydrolysis increased (p<0.01) DMD from both S and AS at 4 and 12 h, but reduced (p<0.01) 15N-PAMN in the early stage (4 h) of the incubation, as compared to non-prehydrolyzed samples. Application of EFE to barley straw increased rumen bacterial colonization of the substrate, but excessive hydrolytic action of EFE prior to incubation decreased it.

Development of a signature probe targeting the 16S-23S rRNA internal transcribed spacer of a ruminal Ruminococcus flavefaciens isolate from reindeer

The cellulolytic Ruminococcus flavefaciens has previously been introduced into the ruminant rumen to increase microbial degradation of plant cell wall carbohydrates. The functional effect of an introduced bacterium depends on its ability to establish in the digestive tract, and signature probes can be used as a tool to track and quantify introduced strains. The purpose of this current study was to develop an oligonucleotide signature probe targeting the 16S-23S rRNA internal transcribed spacer (ITS) of a putative probiotic cellulolytic isolate (R. flavefaciens strain 8/94-32) from the rumen of reindeer (Rangifer tarandus tarandus). The 16S-23S rRNA gene ITS of three Ruminococcus strains; R. flavefaciens strain 8/94-32, R. flavefaciens FD-1 and Ruminococcus albus Ra-8, was investigated. The ITS region has been reported to vary more between closely related bacteria compared to the widely used 16S rRNA gene, and a high degree of sequence polymorphism was indeed detected between the three Ruminococcus strains studied. Based on observed sequence differences, two oligonucloetide probes, ITSRumi1 and ITSRumi2, targeting the ITS region of the R. flavefaciens isolate 8/94-32 were developed. Probe specificity was evaluated in dot blot hybridisations with R. flavefaciens isolate 8/94-32 and four other Ruminococcus-strains tested. The probe ITSRumi1 gave positive signals for the R. flavefaciens isolate 8/94-32 only, while probe ITSRumi2 gave positive signals for R. flavefaciens isolate 8/94-32 as well as for R. albus Ra-8. The result of hybridisations with the probe ITSRumi1 indicates that the probe is specific for the R. flavefaciens strain 8/94-32 amongst the four Ruminococcus-strains tested, and is promising for further studies using it as a signature probe for tracking this strain when re-introduced to the reindeer rumen.

Kinetic characterization of a glycoside hydrolase family 44 xyloglucanase/endoglucanase from Ruminococcus flavefaciens FD-1

Two forms of Ruminococcus flavefaciens FD-1 endoglucanase B, a member of glycoside hydrolase family 44, one with only a catalytic domain and the other with a catalytic domain and a carbohydrate binding domain (CBM), were produced. Both forms hydrolyzed cellotetraose, cellopentaose, cellohexaose, carboxymethylcellulose (CMC), birchwood and larchwood xylan, xyloglucan, lichenan, and Avicel but not cellobiose, cellotriose, mannan, or pullulan. Addition of the CBM increased catalytic efficiencies on both CMC and birchwood xylan but not on xyloglucan, and it decreased rates of cellopentaose and cellohexaose hydrolysis. Catalytic efficiencies were much higher on xyloglucan than on other polysaccharides. Hydrolysis rates increased with increasing cellooligosaccharide chain length. Cellotetraose hydrolysis yielded only cellotriose and glucose. Hydrolysis of cellopentaose gave large amounts of cellotetraose and glucose, somewhat more of the former than of the latter, and much smaller amounts of cellobiose and cellotriose. Cellohexaose hydrolysis yielded much more cellotetraose than cellobiose and small amounts of glucose and cellotriose, along with a low and transient amount of cellopentaose.

Abundance and Diversity of Dockerin-Containing Proteins in the Fiber-Degrading Rumen Bacterium, Ruminococcus flavefaciens FD-1

BackgroundThe cellulosome is a multi-enzyme machine, which plays a key role in the breakdown of plant cell walls in many anaerobic cellulose-degrading microorganisms. Ruminococcus flavefaciens FD-1, a major fiber-degrading bacterium present in the gut of herbivores, has the most intricate cellulosomal organization thus far described. Cellulosome complexes are assembled through high-affinity cohesin-dockerin interactions. More than two-hundred dockerin-containing proteins have been identified in the R. flavefaciens genome, yet the reason for the expansion of these crucial cellulosomal components is yet unknown.Methodology/Principal FindingsWe have explored the full spectrum of 222 dockerin-containing proteins potentially involved in the assembly of cellulosome-like complexes of R. flavefaciens. Bioinformatic analysis of the various dockerin modules showed distinctive conservation patterns within their two Ca2+-binding repeats and their flanking regions. Thus, we established the conceptual framework for six major groups of dockerin types, according to their unique sequence features. Within this framework, the modular architecture of the parent proteins, some of which are multi-functional proteins, was evaluated together with their gene expression levels. Specific dockerin types were found to be associated with selected groups of functional components, such as carbohydrate-binding modules, numerous peptidases, and/or carbohydrate-active enzymes. In addition, members of other dockerin groups were linked to structural proteins, e.g., cohesin-containing proteins, belonging to the scaffoldins.Conclusions/SignificanceThis report profiles the abundance and sequence diversity of the R. flavefaciens FD-1 dockerins, and provides the molecular basis for future understanding of the potential for a wide array of cohesin-dockerin specificities. Conserved differences between dockerins may be reflected in their stability, function or expression within the context of the parent protein, in response to their role in the rumen environment.

Diversity and Strain Specificity of Plant Cell Wall Degrading Enzymes Revealed by the Draft Genome of Ruminococcus flavefaciens FD-1

Background Ruminococcus flavefaciens is a predominant cellulolytic rumen bacterium, which forms a multi-enzyme cellulosome complex that could play an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides. Identifying the major enzyme types involved in plant cell wall degradation is essential for gaining a better understanding of the cellulolytic capabilities of this organism as well as highlighting potential enzymes for application in improvement of livestock nutrition and for conversion of cellulosic biomass to liquid fuels.Methodology/Principal FindingsThe R. flavefaciens FD-1 genome was sequenced to 29x-coverage, based on pulsed-field gel electrophoresis estimates (4.4 Mb), and assembled into 119 contigs providing 4,576,399 bp of unique sequence. As much as 87.1% of the genome encodes ORFs, tRNA, rRNAs, or repeats. The GC content was calculated at 45%. A total of 4,339 ORFs was detected with an average gene length of 918 bp. The cellulosome model for R. flavefaciens was further refined by sequence analysis, with at least 225 dockerin-containing ORFs, including previously characterized cohesin-containing scaffoldin molecules. These dockerin-containing ORFs encode a variety of catalytic modules including glycoside hydrolases (GHs), polysaccharide lyases, and carbohydrate esterases. Additionally, 56 ORFs encode proteins that contain carbohydrate-binding modules (CBMs). Functional microarray analysis of the genome revealed that 56 of the cellulosome-associated ORFs were up-regulated, 14 were down-regulated, 135 were unaffected, when R. flavefaciens FD-1 was grown on cellulose versus cellobiose. Three multi-modular xylanases (ORF01222, ORF03896, and ORF01315) exhibited the highest levels of up-regulation.Conclusions/SignificanceThe genomic evidence indicates that R. flavefaciens FD-1 has the largest known number of fiber-degrading enzymes likely to be arranged in a cellulosome architecture. Functional analysis of the genome has revealed that the growth substrate drives expression of enzymes predicted to be involved in carbohydrate metabolism as well as expression and assembly of key cellulosomal enzyme components.

Efeito dos taninos condensados de forrageiras nativas do semi-árido nordestino sobre o crescimento e atividade celulolítica de Ruminococcus flavefaciens FD1

Avaliaram-se o crescimento da população, a atividade in vitro da enzima 1,4-&lt;FONT FACE=Symbol&gt;b-&lt;/FONT&gt;endoglucanase e a taxa de digestão de celulose em culturas de Ruminococcus flavefaciens FD1 na presença de 50, 100, 200 e 400µg/ml de taninos purificado das leguminosas Mimosa hostilis (Jurema Preta), Mimosa caesalpinifolia (Sabiá) e Bauhinia cheilantha (Mororó). O crescimento bacteriano, a atividade da endoglucanase e a digestão de celulose foram fortemente inibidos pela presença dos taninos condensados purificados das três espécies, entretanto, a intensidade da inibição foi variável em função da espécie da leguminosa e da concentração de tanino.

Assessment of biases associated with profiling simple, model communities using terminal-restriction fragment length polymorphism-based analyses

Community profiles based on terminal-restriction fragment length polymorphism (T-RFLP) analyses of amplified ribosomal RNA genes are used to monitor changes in microbial community structure and are sometimes employed for semi-quantitative estimates of species richness and abundance in environmental samples. To assess the accuracy of T-RFLP community profiles representing the relative abundance of bacteria in a sample, five species of ruminal bacteria were used to construct simple “communities”. Template DNA for PCR amplification was generated either by mixing equal quantities of genomic DNA from pure cultures or by mixing equal numbers of cells prior to DNA extraction. Pairwise mixtures of Fibrobacter succinogenes S85 with Ruminococcus albus 8, Ruminococcus flavefaciens FD-1, Butyrivibrio fibrisolvens 49 and Streptococcus bovis JB1 were created and a 5-member community was constructed. With genomic DNA mixes, relative abundance calculations based on T-RFLP patterns did not reflect input ratios. These discrepancies could not be accounted for by differences in genome size and rRNA operon copy number. In cell mixing experiments, easily lysed cells were overrepresented. To determine if a numerical correction factor could be used to compensate for observed discrepancies, we attempted to quantify biases attributed to DNA extraction and PCR amplification. Biases attributable to these factors led to deviations from expected PCR product ratios by 6% to 38%. We found that interactions were so complex that a suitable factor could not be derived. The unsystematic dependence of T-RFLP peak ratios on variability of DNA extraction and PCR amplification prevents accurate quantification of the relative abundance of microorganisms designed to represent simplified natural populations.

Degradation of lucerne stem cell walls by five rumen bacterial species

The rumen bacterial strains Butyrivibrio fibrisolvens H17c, Fibrobacter succinogenes S85, Lachnospira multiparas 40, Ruminococcus albus 7 and R. flavefaciens FD-1 were compared individually and as a five-species mixture with a rumen inoculum for their ability to degrade lucerne ( Medicago sativa L.) stem cell wall polysaccharides. Two maturity stages of lucerne (bud and full flower) were utilized as substrates and incubation periods of 24 and 96 h were employed. Lucerne stem substrates and fermentation residues were analysed for cell wall content and composition. Degradation of total cell wall polysaccharides and all polysaccharide fractions was greater for immature than for mature lucerne stems. The 96-h incubation resulted in significantly more degradation of all cell wall polysaccharides than did the 24-h incubation. The R. albus culture was able to degrade all cell wall polysaccharide fractions as well or better than any other culture, including the rumen inoculum. Co-culture of R. albus with the four other ruminal species did not improve degradation of any wall polysaccharides compared with R. albus alone. Microscopic examination indicated that R. albus and the five-species mixture appeared to degrade lucerne tissues in thin sections to almost the same extent as did rumen fluid.

Strain-specific genomic regions of Ruminococcus flavefaciens FD-1 as revealed by combinatorial random-phase genome sequencing and suppressive subtractive hybridization.

Two closely related strains of the Gram-positive, cellulolytic ruminal bacterium Ruminococcus flavefaciens were compared at the genomic level by suppressive subtractive hybridization. The two strains investigated in this study differ by 1.94% in their respective 16S rDNA genes. Three hundred and eighty-four PCR-amplified products were cloned and then screened for their strain identity by dot blot hybridization. Based on redundancy percentages of the clones sequenced, 9.5% of the genome of the R. flavefaciens FD-1 strain is not present in the JM1 strain. The majority of identities of individual cloned subtracted products (642 bp average length) bore no relation to deposited sequences in GenBank (42% of the subtracted library), whereas of those with putative assigned functions 7% are loosely associated with fibre-degradation, 6% with insertion elements, transposons and phage-like ORFs, 5% with cell membrane associated proteins and 3% with signal transduction. Subtracted sequences were then supplemented with the draft (2 x coverage) genome sequence of R. flavefaciens FD-1 to indicate potential regions of rearrangement within the genome, including a novel insertion sequence.