Muralytic Activities of Ruminococcus albus 8

Ruminococcus albus 8 was cultured with isolated alfalfa cell walls as the carbon source. The culture broth was assayed for muralytic enzyme activities. The effect, with respect to the production of such muralytic enzymes, of growing the microorganism on different carbon sources was also investigated. Also, the rates of solubilization and utilization by R. albus of individual alfalfa cell wall sugars during a 96-h growth period were examined.     

Albusin B, a bacteriocin from the ruminal bacterium Ruminococcus albus 7 that inhibits growth of Ruminococcus flavefaciens

An approximately 32-kDa protein (albusin B) that inhibited growth of Ruminococcus flavefaciens FD-1 was isolated from culture supernatants of Ruminococcus albus 7. Traditional cloning and gene-walking PCR techniques revealed an open reading frame (albB) encoding a protein with a predicted molecular mass of 32,168 Da. A BLAST search revealed two homologs of AlbB from the unfinished genome of R. albus 8 and moderate similarity to LlpA, a recently described 30-kDa bacteriocin from Pseudomonas sp. strain BW11M1.     

Cellulase from Ruminococcus albus and Mixed Rumen Microorganisms

Cellulase in the cultural filtrates of Ruminococcus albus and cellulase extracted from mixed rumen microorganisms were investigated with acid-swollen cellulose and carboxymethylcellulose as substrates. Maximal activity occurred at approximately pH 5.8 and 47 C. Apparent Michaelis constants (K_m) varied between 0.53 and 0.02% carboxymethylcellulose, depending on the level of activity and the method of assay. R. albus cellulase has a lower K_m value than the enzyme extracted from mixed rumen microorganisms. Antisera from rabbits immunized with a cellulase preparation from R. albus inhibited the cellulolytic activity of both systems. Based on the relative degree of inhibition, approximately 20% of the cellulase of the mixed rumen microorganisms was immunologically similar to R. albus cellulase. Ratios of activity in different assay techniques showed the two sources of activity to be similar in the mechanisms of degradation. However, glucose is the main product of cellulose degradation by mixed rumen microorganisms, and cellobiose is the product of degradation by R. albus.     

Identification of Ruminococcus flavefaciens as the Predominant Cellulolytic Bacterial Species of the Equine Cecum

Detection and quantification of cellulolytic bacteria with oligonucleotide probes showed that Ruminococcus flavefaciens was the predominant species in the pony and donkey cecum. Fibrobacter succinogenes and Ruminococcus albus were present at low levels. Four isolates, morphologically resembling R. flavefaciens, differed from ruminal strains by their carbohydrate utilization and their end products of cellobiose fermentation.     

Phenylacetic and Phenylpropionic Acids Do Not Affect Xylan Degradation by Ruminococcus albus

Since the addition of either ruminal fluid or a combination of phenylacetic and phenylpropionic acids (PAA/PPA) has previously been shown to dramatically improve cellulose degradation and growth of Ruminococcus albus, it was of interest to determine the effects of these additives on xylan-grown cultures. Although cell-bound xylanase activity increased when either PAA/PPA or ruminal fluid was added to the growth medium, total xylanase did not change, and neither of these supplements affected the growth or xylan-degrading capacity of R. albus 8. Similarly, neither PAA/PPA nor ruminal fluid affected xylan degradation by multiple strains of R. albus when xylan prepared from oat spelts was used as a carbohydrate source. These results show that the xylanolytic potential of R. albus is not conditional on the availability of PAA/PPA or other components of ruminal fluid.     

Phenylpropanoic Acid: Growth Factor for Ruminococcus albus

Phenylpropanoic acid accounted for part of the stimulatory effect of rumen fluid on the rate of growth and of cellulose digestion by cultures of Ruminococcus albus strain 8 grown on a chemically defined medium. As little as 3 μM concentration gave maximum response.     

Incorporation of [15N]Ammonia by the Cellulolytic Ruminal Bacteria Fibrobacter succinogenes BL2, Ruminococcus albus SY3, and Ruminococcus flavefaciens 17

The origin of cell nitrogen and amino acid nitrogen during growth of ruminal cellulolytic bacteria in different growth media was investigated by using ¹⁵NH₃. At high concentrations of peptides (Trypticase, 10 g/liter) and amino acids (15.5 g/liter), significant amounts of cell nitrogen of Fibrobacter succinogenes BL2 (51%), Ruminococcus flavefaciens 17 (43%), and Ruminococcus albus SY3 (46%) were derived from non-NH₃-N. With peptides at 1 g/liter, a mean of 80% of cell nitrogen was from NH₃. More cell nitrogen was formed from NH₃ during growth on cellobiose compared with growth on cellulose in all media. Phenylalanine was essential for F. succinogenes, and its ¹⁵N enrichment declined more than that of other amino acids in all species when amino acids were added to the medium.     

Naturally Occurring DNA Transfer System Associated with Membrane Vesicles in Cellulolytic Ruminococcus spp. of Ruminal Origin

A genetic transformation system with similarities to those reported for gram-negative bacteria was found to be associated with membrane vesicles of the ruminal cellulolytic genus Ruminococcus. Double-stranded DNA was recovered from the subcellular particulate fraction of all the cellulolytic ruminococci examined. Electron microscopy revealed that the only particles present resembled membrane vesicles. The likelihood that the DNA was associated with membrane vesicles (also known to contain cellulosomes) was further supported by the adherence of the particles associated with the subcellular DNA to cellulose powder added to culture filtrates. The particle-associated DNA comprised a population of linear molecules ranging in size from <20 kb to 49 kb (Ruminococcus sp. strain YE73) and from 23 kb to 90 kb (Ruminococcus albus AR67). Particle-associated DNA from R. albus AR67 represented DNA derived from genomic DNA of the host bacterium having an almost identical HindIII digestion pattern and an identical 16S rRNA gene. Paradoxically, particle-associated DNA was refractory to digestion with EcoRI, while the genomic DNA was susceptible to extensive digestion, suggesting that there is differential restriction modification of genomic DNA and DNA exported from the cell. Transformation using the vesicle-containing fraction of culture supernatant of Ruminococcus sp. strain YE71 was able to restore the ability to degrade crystalline cellulose to two mutants that were otherwise unable to do so. The ability was heritable and transferred to subsequent generations. It appears that membrane-associated transformation plays a role in lateral gene transfer in complex microbial ecosystems, such as the rumen.     

Adherence of the Gram-Positive Bacterium Ruminococcus albus to Cellulose and Identification of a Novel Form of Cellulose-Binding Protein Which Belongs to the Pil Family of Proteins

The adherence of Ruminococcus albus 8 to crystalline cellulose was studied, and an affinity-based assay was also used to identify candidate cellulose-binding protein(s). Bacterial adherence in cellulose-binding assays was significantly increased by the inclusion of either ruminal fluid or micromolar concentrations of both phenylacetic and phenylpropionic acids in the growth medium, and the addition of carboxymethylcellulose (CMC) to assays decreased the adherence of the bacterium to cellulose. A cellulose-binding protein with an estimated molecular mass following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of ~21 kDa, designated CbpC, was present in both cellobiose- and cellulose-grown cultures, and the relative abundance of this protein increased in response to growth on cellulose. Addition of 0.1% (wt/vol) CMC to the binding assays had an inhibitory effect on CbpC binding to cellulose, consistent with the notion that CbpC plays a role in bacterial attachment to cellulose. The nucleotide sequence of the cbpC gene was determined by a combination of reverse genetics and genomic walking procedures. The cbpC gene encodes a protein of 169 amino acids with a calculated molecular mass of 17,655 Da. The amino-terminal third of the CbpC protein possesses the motif characteristic of the Pil family of proteins, which are most commonly involved with the formation of type 4 fimbriae and other surface-associated protein complexes in gram-negative, pathogenic bacteria. The remainder of the predicted CbpC sequence was found to have significant identity with 72- and 75-amino-acid motifs tandemly repeated in the 190-kDa surface antigen protein of Rickettsia spp., as well as one of the major capsid glycoproteins of the Chlorella virus PBCV-1. Northern blot analysis showed that phenylpropionic acid and ruminal fluid increase cbpC mRNA abundance in cellobiose-grown cells. These results suggest that CbpC is a novel cellulose-binding protein that may be involved in adherence of R. albus to substrate and extends understanding of the distribution of the Pil family of proteins in gram-positive bacteria.     

Sequence of a cellulase gene from the rumen anaerobe Ruminococcus flavefaciens 17

Summary A cellulase gene (endA) was isolated from a library of Ruminococcus flavefaciens strain 17 DNA fragments inserted in pUC13. The endA product showed activity against acid-swollen cellulose, carboxymethyl-cellulose, lichenan, cellopentaose and cellotetraose, but showed no activity against cellotriose or binding to avicel. Nucleotide sequencing indicated an encoded product of 455 amino acids which showed significant sequence similarity (ranging from 56% to 61%) with three endoglucanases from Ruminococcus albus, and with Clostridium thermocellum endoglucanase E. Little relatedness was found with a cellodextrinase previously isolated from R. flavefaciens FD1.     

Cell surface enzyme attachment is mediated by family 37 carbohydrate-binding modules, unique to Ruminococcus albus

The rumen bacterium Ruminococcus albus binds to and degrades crystalline cellulosic substrates via a unique cellulose degradation system. A unique family of carbohydrate-binding modules (CBM37), located at the C terminus of different glycoside hydrolases, appears to be responsible both for anchoring these enzymes to the bacterial cell surface and for substrate binding.     

Nutritional Interdependence Among Rumen Bacteria, Bacteroides amylophilus, Megasphaera elsdenii, and Ruminococcus albus

Nutritional interdependence among three representatives of rumen bacteria, Bacteroides amylophilus, Megasphaera elsdenii, and Ruminococcus albus, was studied with a basal medium consisting of minerals, vitamins, cysteine hydrochloride, and NH₄⁺. B. amylophilus grew well in the basal medium supplemented with starch and produced branched-chain amino acids after growth ceased. When cocultured with B. amylophilus in the basal medium supplemented with starch and glucose, amino acid-dependent M. elsdenii produced an appreciable amount of branched-chain fatty acids, which are essential growth factors for cellulolytic R. albus. A small addition of starch (0.1 to 0.3%) to the basal medium containing glucose and cellobiose brought about successive growth of the three species in the order of B. amylophilus, M. elsdenii, and R. albus, and successive growth was substantiated by the formation of branched-chain amino acids and fatty acids in the culture. Supplementation with 0.5% starch, however, failed to support the growth of R. albus. On the basis of these results, the effects of supplementary starch or branched-chain fatty acids on cellulose digestion in the rumen was discussed.     

Fermentation of Insoluble Cellulose by Continuous Cultures of Ruminococcus albus

The hydrolysis and fermentation of insoluble cellulose (Avicel) by continuous cultures of Ruminococcus albus 7 was studied. An anaerobic continuous culture apparatus was designed which permitted gas collection, continuous feeding, and wasting at different retention times. The operation of the apparatus was controlled by a personal computer. Cellulose destruction ranged from ca. 30 to 70% for hydraulic retention times of 0.5 to 2.0 days. Carbon recovery in products was 92 to 97%, and the oxidation-reduction ratios ranged from 0.91 to 1.15. The total product yield (biomass not included) per gram of cellulose (expressed as glucose) was 0.83 g g⁻¹, and the ethanol yield was 0.41 g g⁻¹. The product yield was constant, indicating that product formation was growth linked.     

Kinetics of Insoluble Cellulose Fermentation by Continuous Cultures of Ruminococcus albus

Data from analyses of continuous culture fermentation of insoluble cellulose by Ruminococcus albus 7 were used to derive constants for the rate of cellulose hydrolysis and fermentation, growth yield, and maintenance. Cellulose concentration was 1% in the nutrient reservoir, and hydraulic retention times of 0.5, 1.0, 1.5, 1.75, and 2.0 days were used. Concentrations of reducing sugars in the cultures were negligible (less than 1%) compared with the amount of hydrolyzed cellulose, indicating that cellulose hydrolysis was the rate-limiting step of the fermentation. The rate of utilization of cellulose depended on the steady-state concentration of cellulose and was first order with a rate constant (k) of 1.18 day⁻¹. The true microbial growth yield (Y) was 0.11 g g⁻¹, the maintenance coefficient (m) was 0.10 g g⁻¹ h⁻¹, and the maximum Y_ATP was 7.7 g of biomass (dry weight) mol of ATP⁻¹.     

3-Phenylpropanoic Acid Improves the Affinity of Ruminococcus albus for Cellulose in Continuous Culture

A continuous-culture device, adapted for use with solid substrates, was used to evaluate the effects of 3-phenylpropanoic acid (PPA) upon the ability of the South African strain Ruminococcus albus Ce63 to ferment cellulose. Steady states of fermentation were established with a dilution rate of 0.17 h⁻¹, and the extent and volumetric rates of cellulose fermentation were determined over four consecutive days. When the growth medium contained no additions (control), 25 μM phenylacetate alone, 25 μM PPA alone, or 25 μM each of phenylacetate and PPA, the extent of cellulose hydrolysis was determined to be 41.1, 35.7, 90.2, and 86.9%, respectively, and the volumetric rate of cellulose hydrolysis was 103.0, 97.9, 215.5, and 230.4 mg liter⁻¹ h⁻¹, respectively. To evaluate the effect of PPA availability on affinity for cellulose, the values for dilution rate and extent of cellulose hydrolysis were used in combination with values for maximum specific growth rate determined from previous studies of growth rates and kinetics of cellulose hydrolysis. The findings support the contention that PPA maintains a competitive advantage for R. albus when grown in a dynamic, fiber-rich environment.     

Effect of 3-Phenylpropanoic Acid on Capsule and Cellulases of Ruminococcus albus 8

The morphology and cellulases of Ruminococcus albus 8 were markedly affected by the inclusion of 3-phenylpropanoic acid (PPA) in a defined growth medium. PPA-grown bacteria produced substantial quantities of cell-bound cellulase, as well as a very high-molecular-weight extracellular enzyme and lesser amounts of two low-molecular-weight enzymes. PPA-deprived bacteria produced greater total amounts of cellulase, but all of it exists in soluble, low-molecular-weight forms. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the availability of PPA did not affect the kinds of proteins produced, but the distribution of two major proteins between cells and supernatant was PPA dependent. These two proteins (85 and 102 kilodaltons) were primarily associated with the cells of PPA-grown bacteria but were found chiefly in the supernatants of PPA-deprived cultures. Examination of thin sections of PPA-grown R. albus 8 by transmission electron microscopy showed a lobed ruthenium red-staining capsule surrounding the cell wall, as well as small vesicular structures (diameter, 0.05 to 0.06 μm) which appeared to aggregate into larger spherical units (diameter, 0.2 to 0.3 μm). In contrast, thin sections of PPA-deprived cells were devoid of vesicles and showed little or no capsule surrounding the cells.     

Purification and properties of the Endo-1,4-β-glucanase from Ruminococcus albus and its gene product in Escherichia coli

Endoglucanases, EGI and EgI, were produced from the same Ruminococcus albus gene in R. albus and recombinant Escherichia coli, respectively. EGI was purified from R. albus culture supernatant and EgI was extracted from the transformant E. coli (JM101/pURA1) and purified. The purified enzymes EGI and EgI revealed maximum endoglucanase activity at a same pH of 6.8 and a temperature of 37°C. Both enzymes were stable at temperatures below 30°C. In addition, about 10% of their original activities were conserved even after boiling for 10 min. Amino acid sequences of both enzymes at the N-terminal (Ala-Ala-Asp-Glu-Ser-Glu-Thr-Glu-Asn-Val-Pro-Val-Ser-Gln-Thr-His--) were consistent with each other. The antiserum against EgI reacted with both EgI and EGI, indicating that both their protein moieties were the same immunologically. However, the molecular size of EGI (43,000) was larger than that of EgI (39,000) due to the presence of sugar moiety. The specific activity (54 units/mg) of EGI was almost double that (27 units/mg) of EgI. EGI was immunologically different from the endoglucanase purified in the previous paper [Ohmiya et al.: Carbohydrate Res., 166, 145–155 (1987)].