Flavefaciens FD-1 Research Articles

Nucleotide sequence of thecelA gene encoding a cellodextrinase ofRuminococcus flavefaciens FD-1

The nucleotide sequence of a 3.6 kb DNA fragment containing a cellodextrinase gene (celA) from Ruminococcus flavefaciens FD-1 was determined. The gene was expressed from its own regulatory region in Escherichia coli and a putative consensus promoter sequence was identified upstream of a ribosome binding site and a TTG start codon. The complete amino acid sequence of the CelA enzyme (352 residues) was deduced and showed no significant homology to cellulases from other organisms. Two lysozyme-type active sites were found in the amino-terminal third of the enzyme. In E. coli the cloned Cel A protein was translocated into the periplasm. The lack of a typical signal sequence, and the results of transposon phoA mutagenesis experiments indicated that CelA is secreted by a mechanism other than a leader peptide.

Assessment of the endo-1,4-beta-glucanase components of Ruminococcus flavefaciens FD-1

The extracellular endo-1,4-beta-glucanase components of Ruminococcus flavefaciens FD-1 were analyzed by high-performance liquid chromatography (HPLC) by using DEAE ion-exchange, hydroxylapatite, and gel filtration chromatography and polyacrylamide gel electrophoresis (PAGE). Two endo-1,4-beta-glucanase peaks were resolved by DEAE-HPLC and termed endoglucanases A and B. Carboxymethyl cellulose (CMC) zymograms were achieved by enzyme separation using nondenaturing PAGE followed by incubation of the gel on top of a CMC-agarose gel. This revealed no less than 13 and 5 endo-1,4-beta-glucanase components present in endoglucanases A and B, respectively. Hydroxylapatite chromatography of endoglucanases A and B revealed one activity peak for each preparation, which contained 4 and 5 endo-1,4-beta-glucanase components, respectively. Gel filtration chromatography of endoglucanase A following hydroxylapatite chromatography resolved the most active carboxymethylcellulase (CMCase) component from other endo-1,4-beta-glucanase activities. Gel filtration of endoglucanase B following hydroxylapatite chromatography showed one CMCase activity peak. Protein stains of sodium dodecyl sulfate-PAGE and nondenaturing PAGE gels of endoglucanases A and B from hydroxylapatite and gel filtration chromatography revealed multiple protein components. When xylan was substituted for CMC in zymograms, identical separation patterns for CMCase and xylanase activities were observed for both endoglucanases A and B. These data suggest that both 1,4-beta linkage-hydrolyzing activities reside on the same polypeptide or protein complex. The highest endo-1,4-beta-glucanase-specific activities were observed following DEAE-HPLC chromatography, with 16.2 and 7.5 mumol of glucose equivalents per min per mg of protein for endoglucanases A and B, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial cell surface structures involved in lucerne cell wall degradation by pure cultures of cellulolytic rumen bacteria

Pure cultures of the cellulolytic rumen bacterial strains Bacteroides succinogenes S85, Ruminococcus flavefaciens FD1 and Ruminococcus albus 7 were grown on lucerne cell walls (CW) or on cellobiose as the sole added carbohydrate substrate. Scanning electron microscopy visualization using cationized-feritin pretreatment have shown that cell surface topology of these strains grown on and attached to CW particles was specified by a dense coat of characteristic protuberant structures. In contrast, when grown on cellobiose, the surface topology of these bacterial strains was smoother, and contained fewer protuberant structures. The ability of these bacterial strains to attach to cellulose was higher for bacteria previously adapted to lucerne CW compared to cellobiose adaptation. Bacteroides succinogenes S85 was the best digester of lucerne CW (46.5%) and also had the best adhesion capability (65.6%) after adaption to grow on CW.

Improved assay for quantitating adherence of ruminal bacteria to cellulose.

A quantitative technique suitable for the determination of adherence of ruminal bacteria to cellulose was developed. This technique employs adherence of cells to cellulose disks and alleviates the problem of nonspecific cell entrapment within cellulose particles. By using this technique, it was demonstrated that the adherence of Ruminococcus flavefaciens FD1 to cellulose was inhibited by formaldehyde, methylcellulose, and carboxymethyl cellulose. Adherence was unaffected by acid hydrolysates of methylcellulose, glucose, and cellobiose.

Analysis of antibiotic susceptibility and extrachromosomal DNA content of Ruminococcus albus and Ruminococcus flavefaciens.

Seventeen Ruminococcus albus and Ruminococcus flavefaciens strains have been screened for naturally occurring antibiotic resistance, as determined by zones of inhibition from antibiotic disks. These strains were also examined for extrachromosomal DNA content. All strains screened are resistant to low levels (10-200 micrograms/mL) of streptomycin. In contrast to the previously reported data, we have found that R. flavefaciens C-94 is now susceptible to both kanamycin and tetracycline. However, R. flavefaciens FD-1 is not susceptible to kanamycin (minimum inhibitory concentration (MIC) = 40 micrograms/mL). Furthermore, R. albus 8 is resistant to tetracycline (MIC = 40 micrograms/mL), and erythromycin (MIC = 100 micrograms/mL). Six freshly isolated strains showed resistance to tetracycline (35-70 micrograms/mL), and all tetracycline-resistant strains also showed resistance to minocycline. None of these Ruminococcus determinants share homology with the streptococcal tetL, tetM, or tetN determinants. All 17 strains were screened for extrachromosomal DNA content. Nine different techniques for the detection and isolation of extrachromosomal DNA were tested. However, owing to difficulties in demonstrating or isolating plasmid DNA, it has not been possible to determine if these antibiotic resistance genes are plasmid borne. Evidence is presented to suggest that the presence of oxygen may affect the quality of the DNA obtained from Ruminococcus.

Methylcellulose inhibition of exo-beta-1,4-glucanase A from Ruminococcus flavefaciens FD-1.

A homogeneous preparation of exo-beta-1,4-glucanase A from Ruminococcus flavefaciens FD-1 was competitively inhibited by low concentrations (less than 3 mM) of methylcellulose. The enzyme was also sensitive to the surfactant properties of methylcellulose at high methylcellulose concentrations.

Purification and characterization of an exo-beta-1,4-glucanase from Ruminococcus flavefaciens FD-1.

An exo-beta-1,4-glucanase (Exo A) from Ruminococcus flavefaciens FD-1 was purified to homogeneity and characterized. Enzyme activity was monitored during purification by using the substrate p-nitrophenyl-beta-D-cellobioside (NPC). Over 85% of the NPC activity was found to be extracellular once the filter paper was degraded (7 days). Culture supernatant was harvested, and the protein was concentrated by ultrafiltration. The retentate (greater than or equal to 300,000 Mr), containing most of the activity against NPC, was then fractionated with a TSK DEAE-5PW column. This yielded a sharp major peak of NPC enzyme activity, followed by a broader, less active area that appeared to contain at least six minor peaks of lower enzymatic activity. Further purification was achieved by chromatography with a hydroxylapatite column. Finally, gel filtration chromatography yielded a homogeneous enzyme (Exo A) as determined by silver stains of both sodium dodecyl sulfate- and nondenaturing electrophoresis gels. Substrate specificity experiments and the products of cellulose digestion indicate that the enzyme was an exo-beta-1,4-glucanase. Exo A required Ca2+ for maximal activity and had an apparent Km of 3.08 mM for NPC, with a Vmax of 0.298 mumol/min per mg of protein. The enzyme had an Mr of 230,000, as determined by gel filtration chromatography, and was a dimer of 118,000-Mr subunits. The N-terminal amino acid sequence of the enzyme is presented.

Cloning and expression in Escherichia coli of a cellulase gene from Ruminococcus flavefaciens.

An endoglucanase gene of Ruminococcus flavefaciens FD1 was cloned on the vector pEcoR251 to form the recombinant plasmid pMEB200. The cloned endoglucanase gene showed carboxymethylcellulase enzyme activity but no degradation of Avicel (FMC Corp., Philadelphia, Pa.) or filter paper. Carboxymethylcellulase activity was found during the late-exponential-growth phase and accumulated in the periplasmic fraction. Enzyme production was not subject to catabolite repression by glucose.

Effect of phenolic monomers on ruminal bacteria.

Ruminal bacteria were subjected to a series of phenolic compounds in various concentrations to acquire fundamental information on the influence on growth and the potential limits to forage utilization by phenolic monomers. Ruminococcus albus 7, Ruminococcus flavefaciens FD-1, Butyrivibrio fibrisolvens 49, and Lachnospira multiparus D-32 were tested against 1, 5, and 10 mM concentrations of sinapic acid, syringaldehyde, syringic acid, ferulic acid, vanillin, vanillic acid, p-coumaric acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, and hydrocinnamic acid. Responses were variable and dependent on the phenolic compound and microbial species. Compounds especially toxic (i.e., resulting in poor growth, effect on several species, dose-related response) were p-coumaric acid and p-hydroxybenzaldehyde, and adaptation to the toxins did not occur after three 24-h periods. Syringic, p-hydroxybenzoic, and hydrocinnamic acids stimulated growth of all four species and also stimulated filter paper degradation by R. flavefaciens. None of the stimulatory compounds supported microbial growth in the absence of carbohydrates. In vitro dry matter digestibility of cellulose (Solka-Floc) was not stimulated by any of the phenolic compounds (10 mM), but the cinnamic acids and benzoic aldehydes (10 mM) reduced (P less than 0.05) digestion by the mixed population in ruminal fluid. Growth of R. flavefaciens in the presence of p-hydroxybenzoic acid (10 mM) or p-coumaric acid (5 mM) resulted in recognizable alterations in cell ultrastructure. Both phenolics caused a reduction in cell size (P less than 0.05), and p-coumaric acid caused a reduction in capsular size (P less than 0.05) and produced occasional pleomorphic cells.