The use of 16S rRNA-targeted oligonucleotide probes to study competition between ruminal fibrolytic bacteria: development of probes for Ruminococcus species and evidence for bacteriocin production.

A total of six oligonucleotide probes, complementary to the 16S rRNA, were evaluated for quantitative and determinative studies of Ruminococcus albus and Ruminococcus flavefaciens. On the basis of specificity studies, probes for R. albus (probe RAL196) and R. flavefaciens (probe RFL196) were selected to quantitate these species in mixed culture. In combination with a Fibrobacter succinogenes S85 subspecies probe (SUB1) and a domain Bacteria (formerly kingdom Eubacteria) probe (EUB338), they were used to quantitate these species competing in mixed cultures for cellobiose as the carbon source. In dicultures containing R. albus 8 and F. succinogenes S85, competition was not observed. However, R. flavefaciens FD-1 eventually outcompeted F. succinogenes S85 when cellobiose was the substrate. When R. albus 8 and R. flavefaciens FD-1 were grown together on cellobiose medium, R. albus 8 outcompeted R. flavefaciens FD-1, resulting in undetectable R. flavefaciens 16S rRNA only 1 to 3 h after inoculation, suggesting production of an antagonistic compound by R. albus 8 during rapid growth on soluble substrates. Further, when R. albus 8, R. flavefaciens FD-1, and F. succinogenes S85 were grown together in a triculture, R. flavefaciens FD-1 16S rRNA was detectable for only 2 h after inoculation, while R. albus 8 and F. succinogenes S85 showed a similar competition pattern to that of the dicultures. The results show that the Ruminococcus probes were effective in the measurement of relative populations of selected R. albus and R. flavefaciens strains during in vitro competition studies with F. succinogenes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Competition for cellulose among three predominant ruminal cellulolytic bacteria under substrate-excess and substrate-limited conditions.

Three predominant ruminal cellulolytic bacteria (Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, and Ruminococcus albus 7) were grown in different binary combinations to determine the outcome of competition in either cellulose-excess batch culture or in cellulose-limited continuous culture. Relative populations of each species were estimated by using signature membrane-associated fatty acids and/or 16S rRNA-targeted oligonucleotide probes. Both F. succinogenes and R. flavefaciens coexisted in cellulose-excess batch culture with similar population sizes (58 and 42%, respectively; standard error, 12%). By contrast, under cellulose limitation R. flavefaciens predominated (> 96% of total cell mass) in coculture with F. succinogenes, regardless of whether the two strains were inoculated simultaneously or whether R. flavefaciens was inoculated into an established culture of F. succinogenes. The predominance of R. flavefaciens over F. succinogenes under cellulose limitation is in accord with the former's more rapid adherence to cellulose and its higher affinity for cellodextrin products of cellulose hydrolysis. In batch cocultures of F. succinogenes and R. albus, the populations of the two species were similar. However, under cellulose limitation, F. succinogenes was the predominant strain (approximately 80% of cell mass) in cultures simultaneously coinoculated with R. albus. The results from batch cocultures of R. flavefaciens and R. albus were not consistent within or among trials: some experiments yielded monocultures of R. albus (suggesting production of an inhibitory agent by R. albus), while others contained substantial populations of both species. Under cellulose limitation, R. flavefaciens predominated over R. albus (85 and 15%, respectively), as would be expected by the former's greater adherence to cellulose. The retention of R. albus in the cellulose-limited coculture may result from a combination of its ability to utilize glucose (which is not utilizable by R. flavefaciens), its demonstrated ability to adapt under selective pressure in the chemostat to utilization of lower concentrations of cellobiose, a major product of cellulose hydrolysis, and its possible production of an inhibitory agent.     

Utilization of individual cellodextrins by three predominant ruminal cellulolytic bacteria.

Growth of the ruminal bacteria Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, and R. albus 7 followed Monod kinetics with respect to concentrations of individual pure cellodextrins (cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose). Under the conditions tested, R. flavefaciens FD-1 possesses the greatest capacity to compete for low concentrations of these cellodextrins.     

Gas-liquid chromatography for evaluating polysaccharide degradation by Ruminococcus flavefaciens C94 and Bacteroides succinogenes S85.

Two predominant rumen cellulolytic bacteria, Ruminococcus flavefaciens C94 and Bacteroides succinogenes S85, were incubated with ground filter paper (Whatman no. 1), cattle manure fiber, wheat straw, Kentucky bluegrass, alfalfa, and corn silage as substrates. Analyses of the initial substrate and the recovered residue after 48 h of static incubation showed that R. flavefaciens C94 was quantitatively more effective than B. succinogenes S85 in degrading total dry matter (32.3% versus 16.1%). However, B. succinogenes S85 demonstrated a qualitative advantage in degrading the hemicellulose and hemicellulosic sugars of particular substrates. R. flavefaciens degraded a mean 29.7% of the cellulose and 35.6% of the hemicellulose in the various substrates, whereas B. succinogenes degraded a mean 17.9 and 31.6% of these fractions, respectively. Gas-liquid chromatography was an important aid in characterizing the polysaccharide-degrading capabilities of these rumen species.     

Differential protein phosphorylation-dephosphorylation in response to carbon source in Ruminococcus flavefaciens FD-1

AIMS: The aims of this study were to study the effect of cellobiose or cellulose as a carbon source on the differential protein phosphorylation-dephosphorylation of cytoplasmic and membrane-associated proteins from Ruminococcus flavefaciens FD-1. METHODS AND RESULTS: SDS-PAGE analysis was used to compare in vitro labelled proteins (32P-ATP) isolated from R. flavefaciens FD-1 grown on either cellobiose or cellulose as the carbon source. Distinctly different protein phosphorylation patterns were detected depending on carbon source and cell fraction. Analysis of the nature of the phosphorylated proteins indicates that phosphorylated proteins from cellobiose grown cultures are phosphorylated on serine residues, whereas phosphorylated proteins from cellulose grown cultures are phosphorylated on threonine residues. CONCLUSIONS: The results of this comparative analysis show a shift from serine phosphorylation of proteins to a threonine phosphorylation when R. flavefaciens FD-1 cells are grown on cellulose as opposed to cellobiose. There appears to be a role for these phosphorylation events in sensing the carbon source for growth and regulating co-ordinated metabolism in R. flavefaciens FD-1. SIGNIFICANCE AND IMPACT OF THE STUDY: We have demonstrated that there is a protein phosphorylation system in R. flavefaciens FD-1 that may be the primary sensing system for carbon source by R. flavefaciens FD-1 and the further regulation of gene expression related to cellulose degradation.     

Adhesion of cellulolytic ruminal bacteria to barley straw

Adhesion of the cellulolytic ruminal bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes to barley straw was measured by incubating bacterial suspensions with hammer-milled straw for 30 min, filtering the mixtures through sintered glass filters, and measuring the optical densities of the filtrates. Maximum adhesion of both species occurred at pH 6.0 and during mid- to late-exponential phase. Adhesion was saturable at 33 and 23 mg (dry weight) g of straw for R. flavefaciens and F. succinogenes, respectively. Methyl cellulose and carboxymethyl cellulose inhibited adhesion by 24 to 33%. Competition between species was determined by measuring characteristic cell-associated enzyme activities in filtrates of mixtures incubated with straw; p-nitrophenyl-beta-d-lactopyranoside hydrolysis was used as a marker for F. succinogenes, while either beta-xylosidase or carboxymethyl cellulase was used for R. flavefaciens, depending on the other species present. R. flavefaciens had no influence on F. succinogenes adhesion, and F. succinogenes had only a minor (<20%) effect on R. flavefaciens adhesion. The noncellulolytic ruminal bacteria Bacteroides ruminicola and Selenomonas ruminantium had no influence on adhesion of either cellulolytic species, although these organisms also adhered to the straw. We concluded that R. flavefaciens and F. succinogenes have separate, specific adhesion sites on barley straw that are not obscured by competition with non-cellulolytic species.     

NMR study of cellulose and wheat straw degradation by Ruminococcus albus 20

Cellulose and wheat straw degradation by Ruminococcus albus was monitored using NMR spectroscopy. In situ solid-state (13)C-cross-polarization magic angle spinning NMR was used to monitor the modification of the composition and structure of cellulose and (13)C-enriched wheat straw during the growth of the bacterium on these substrates. In cellulose, amorphous regions were not preferentially degraded relative to crystalline areas by R. albus. Cellulose and hemicelluloses were also degraded at the same rate in wheat straw. Liquid state two-dimensional NMR experiments were used to analyse in detail the sugars released in the culture medium, and the integration of NMR signals enabled their quantification at various times of culture. The results showed glucose and cellodextrin accumulation in the medium of cellulose cultures; the cellodextrins were mainly cellotriose and accumulated to up to 2 mm after 4 days. In the wheat straw cultures, xylose was the main soluble sugar detected (1.4 mm); arabinose and glucose were also found, together with some oligosaccharides liberated from hemicellulose hydrolysis, but to a much lesser extent. No cellodextrins were detected. The results indicate that this strain of R. albus is unable to use glucose, xylose and arabinose for growth, but utilizes efficiently xylooligosaccharides. R. albus 20 appears to be less efficient than Fibrobacter succinogenes S85 for the degradation of wheat straw.     

Interactions between anaerobic cellulolytic bacteria and fungi in the presence of Methanobrevibacter smithii

A mixed inoculum of cellulolytic rumen bacteria depressed straw degradation by a mixed culture of cellulolytic fungi grown in the presence of Methanobrevibacter smithii. The inhibitory effect appeared to be caused by Ruminococcus albus strain JI and R. flavefaciens strain 007. Ruminococcus albus strain J1 also depressed straw degradation by the fungi, but R. albus strain SY3 and three strains of Bacteroides (Fibrobacter) succinogenes tested showed little or no inhibitory activity. It seems that some ruminococci show competitive or antagonistic activity towards certain rumen fungi.     

β-Glucanase expression by Ruminococcus flavefaciens FD-1

Abstract Ruminococcus flavefaciens has been hypothesized to produce cellulase constitutively. We have studied the effect of carbon source, either cellobiose or cellulose, on the production of cellulase in batch cultures of R. flavefaciens FD-1. Total CMCase and ¹⁴C-cellulase activity was approximately 2-fold higher in cellobiose grown cells than in cellulose grown cells, whereas p-nitrophenyl-β- d -cellobiosidase (PNPCase) activity was not affected by culture conditions. The addition of cellulose to cells growing on cellobiose did not alter the amount or rate of PNPCase and ¹⁴C-cellulase production. Northern blot analysis of mRNAs produced by R. flavefaciens FD-1 grown using either cellobiose or cellulose as the substrate indicated that two of the four β-glucanase genes cloned from R. flavefaciens FD-1 were only expressed in cells grown with cellulose as the substrate. Although the adherence of cells and cellulase enzyme to native cellulose can complicate interpretations of these data, the results indicate that cellulase synthesis by R. flavefaciens is differentially regulated by carbon source.     

Cellulolytic bacteria in buffalo rumen

The influence of three different feeds, wheat straw, sorghum and berseem, on total and cellulolytic bacterial counts in the buffalo rumen at different time intervals from 0 to 8 h after feeding was studied. Berseem feeding supported maximum growth of rumen bacteria in general and cellulolytic bacteria in particular. Wheat straw supported the poorest growth.
The types of cellulolytic bacteria recovered from the rumen of adult buffaloes were Ruminococcus albus, R. flavefaciens, Bacteroides succinogenes, Butyrivibrio fibrisolvens, Clostridium lochheadii, Cl. longisporum and other Clostridium spp. Cellulolytic cocci were present in smaller numbers than rod forms in the rumen of wheat-straw-fed buffaloes, whereas the cocci outnumbered rod forms in sorghum-and berseem-fed buffaloes.     

Effect of soluble carbohydrates on digestion of cellulose by pure cultures of rumen bacteria.

The rate of cellulose digestion in the presence of either glucose or cellobiose was studied for the three predominant species of cellulolytic rumen bacteria: Ruminococcus albus, Ruminococcus flavefaciens, and Bacteroides succinogenes. When a soluble carbohydrate was added to cellulose broth, the lag phase of cellulose digestion was shortened. Presumably, this was due to greater numbers of bacteria, because increasing the size of the inoculum had a similar effect. Cellulose digestion occurred simultaneously with utilization of the soluble carbohydrate. The rate of cellulose digestion slowed markedly for B. succinogenes and R. flavefaciens and slowed less for R. albus after the cellobiose or glucose had been utilized, and was accompanied by a decrease in pH. Both the rate and the extent of cellulose digestion were partially inhibited when the initial pH of the medium was 6.3 or below. R. albus appeared to be less affected by a low-pH medium than were B. succinogenes and R. flavefaciens. When a soluble carbohydrate was added to the fermentation during the maximum-rate phase of cellulose digestion, the rate of cellulose digestion was not affected until after the soluble carbohydrate had been depleted and the pH had decreased markedly. Prolonged exposure of the bacteria to a low pH had little if any effect on their subsequent ability to digest cellulose. Cellulase activity of intact bacterial cells appeared to be constitutive in nature for these three species of rumen bacteria.     

Effect of soluble carbohydrates on digestion of cellulose by pure cultures of rumen bacteria

The rate of cellulose digestion in the presence of either glucose or cellobiose was studied for the three predominant species of cellulolytic rumen bacteria: Ruminococcus albus, Ruminococcus flavefaciens, and Bacteroides succinogenes. When a soluble carbohydrate was added to cellulose broth, the lag phase of cellulose digestion was shortened. Presumably, this was due to greater numbers of bacteria, because increasing the size of the inoculum had a similar effect. Cellulose digestion occurred simultaneously with utilization of the soluble carbohydrate. The rate of cellulose digestion slowed markedly for B. succinogenes and R. flavefaciens and slowed less for R. albus after the cellobiose or glucose had been utilized, and was accompanied by a decrease in pH. Both the rate and the extent of cellulose digestion were partially inhibited when the initial pH of the medium was 6.3 or below. R. albus appeared to be less affected by a low-pH medium than were B. succinogenes and R. flavefaciens. When a soluble carbohydrate was added to the fermentation during the maximum-rate phase of cellulose digestion, the rate of cellulose digestion was not affected until after the soluble carbohydrate had been depleted and the pH had decreased markedly. Prolonged exposure of the bacteria to a low pH had little if any effect on their subsequent ability to digest cellulose. Cellulase activity of intact bacterial cells appeared to be constitutive in nature for these three species of rumen bacteria.     

Development and use of competitive PCR assays for the rumen cellulolytic bacteria: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens

Competitive PCR assays were developed for the enumeration of the rumen cellulolytic bacterial species: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens. The assays, targeting species-specific regions of 16S rDNA, were evaluated using DNA from pure culture and rumen digesta spiked with the relevant cellulolytic species. Minimum detection levels for F. succinogenes, R. albus and R. flavefaciens were 1-10 cells in pure culture and 10(3-4) cells per ml in mixed culture. The assays were reproducible and 11-13% inter- and intra-assay variations were observed. Enumeration of the cellulolytic species in the rumen and alimentary tract of sheep found F. succinogenes dominant (10(7) per ml of rumen digesta) compared to the Ruminococcus spp. (10(4-6) per ml). The population size of the three species did not change after the proportion of dietary alfalfa hay was increased. All three species were detected in the rumen, omasum, caecum, colon and rectum. Numbers of the cellulolytic species at these sites varied within and between animals.     

Interactions between rumen bacterial strains during the degradation and utilization of the monosaccharides of barley straw cell-walls

Pure cultures and pair-combinations of strains representative of the rumen cellulolytic species Ruminococcus flavefaciens, Fibrobacter succinogenes and Butyrivibrio fibrisovens were grown on cell-wall materials from barley straw. Of the pure cultures, R. flavefaciens solubilized straw most rapidly. The presence of B. fibrisolvens , which was unable to degrade straw extensively in pure culture, increased the solubilization of dry matter by R. flavefaciens and the solubilization of cell-wall carbohydrates by both R. flavefaciens and F. succinogenes. During fermentation, both R. flavefaciens and F. succinogenes released bound glucose and free and bound arabinose and xylose into solution. The accumulation of these sugars, especially arabinose and xylose, was greatly reduced in co-cultures containing B. fibrisolvens , suggesting that significant interspecies cross feeding of the products of hemicellulose hydrolysis (particularly soluble bound xylose released by F. succinogenes ) occurs during straw degradation by mixed cultures containing this species.     

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.     

Molecular beacons: trial of a fluorescence-based solution hybridization technique for ecological studies with ruminal bacteria.

Molecular beacons are fluorescent probes developed for solution rather than membrane hybridization. We have investigated the utility of these probes to study rumen microbial ecology. Two cellulolytic species, Ruminococcus albus and Fibrobacter succinogenes, were tested. Membrane and solution hybridizations gave similar results in competition experiments with cocultures of R. albus 8 and F. succinogenes S85.     

The effect of ammonia treatment on the solubilization of straw and the growth of cellulolytic rumen bacteria

Pre-treatment of straw with anhydrous ammonia increased its susceptibility to solubilization by the predominant cellulolytic bacteria from the rumen, Bacteroides succinogenes, Ruminococcus albus and R. flavefaciens. Ammonia treatment also increased the production of microbial protein and fermentation products by all three species. Scanning electron microscope observations of straw during digestion suggested that the attack of straw by these bacteria was accompanied by the formation of substantial numbers of adherent microcolonies.     

The effect of ammonia treatment on the solubilization of straw and the growth of cellulolytic rumen bacteria

Pre-treatment of straw with anhydrous ammonia increased its susceptibility to solubilization by the predominant cellulolytic bacteria from the rumen, Bacteroides succinogenes, Ruminococcus albus and R. flavefaciens. Ammonia treatment also increased the production of microbial protein and fermentation products by all three species. Scanning electron microscope observations of straw during digestion suggested that the attack of straw by these bacteria was accompanied by the formation of substantial numbers of adherent microcolonies.     

The hydrolysis of lucerne cell-wall monosaccharide components by monocultures or pair combinations of defined ruminal bacteria

The defined ruminal bacterial strains Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD1, Ruminococcus albus 7, Butyrivibrio fibrisolvens D1, and Bacteroides ruminicola GA33 were grown, in monocultures or as combinations of pair strains, on isolated lucerne cell-walls (CW) as the sole carbohydrate substrate. Fibrobacter succinogenes S85 was the dominant strain determining extent of CW hydrolysis in all combinations with S85. The hydrolysis of cellulose, xylan, hemicellulose side-sugars, and total CW monosaccharides by pure S85 were: 58·8, 47·3, 66·9 and 57·0%, respectively. The strains combination S85 plus D1 comprised the highest complementary effect, increasing significantly the hydrolysis of cellulose and total CW monosaccharides by 16% and 13%, respectively, above the values obtained by pure S85. This complementation was expressed also in growth pattern of bacteria.
The monocultures of FD1, D1 and GA33 had very little hydrolytic effect on lucerne cellulose, but higher effects on xylan and hemicellulose side-sugars. The combinations D1 plus GA33 and 7 plus GA33 were complementary in the hydrolysis of all CW polysaccharides. The combinations FD1 plus D1, FD1 plus GA33, and 7 plus D1 were complementary only with respect to hemicellulose hydrolysis. On the other hand, the cellulolytic combinations S85 plus FD1, S85 plus 7 and FD1 plus 7 demonstrated negative interactions in lucerne CW polysaccharides hydrolysis.
Under scanning electron microscopy (SEM), S85 comprised the most dense layer of bacterial cell mass attached to and colonized on CW particles. The cell surface topology of the cellulolytic strains S85, FD1 and 7 attached to CW particles was specified by a coat of characteristic protuberant structures.     

The hydrolysis of lucerne cell-wall monosaccharide components by monocultures or pair combinations of defined ruminal bacteria

The defined ruminal bacterial strains Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD1, Ruminococcus albus 7, Butyrivibrio fibrisolvens D1, and Bacteroides ruminicola GA33 were grown, in monocultures or as combinations of pair strains, on isolated lucerne cell-walls (CW) as the sole carbohydrate substrate. Fibrobacter succinogenes S85 was the dominant strain determining extent of CW hydrolysis in all combinations with S85. The hydrolysis of cellulose, xylan, hemicellulose side-sugars, and total CW monosaccharides by pure S85 were: 58.8, 47.3, 66.9 and 57.0%, respectively. The strains combination S85 plus D1 comprised the highest complementary effect, increasing significantly the hydrolysis of cellulose and total CW monosaccharides by 16% and 13%, respectively, above the values obtained by pure S85. This complementation was expressed also in growth pattern of bacteria. The monocultures of FD1, D1 and GA33 had very little hydrolytic effect on lucerne cellulose, but higher effects on xylan and hemicellulose side-sugars. The combinations D1 plus GA33 and 7 plus GA33 were complementary in the hydrolysis of all CW polysaccharides. The combinations FD1 plus D1, FD1 plus GA33, and 7 plus D1 were complementary only with respect to hemicellulose hydrolysis. On the other hand, the cellulolytic combinations S85 plus FD1, S85 plus 7 and FD1 plus 7 demonstrated negative interactions in lucerne CW polysaccharides hydrolysis. Under scanning electron microscopy (SEM), S85 comprised the most dense layer of bacterial cell mass attached to and colonized on CW particles. The cell surface topology of the cellulolytic strains S85, FD1 and 7 attached to CW particles was specified by a coat of characteristic protuberant structures.