Dominance of <Emphasis Type="Italic">Prevotella</Emphasis> and low abundance of classical ruminal bacterial species in the bovine rumen revealed by relative quantification real-time PCR

Relative quantification real-time PCR was used to quantify several bacterial species in ruminal samples from two lactating cows, each sampled 3 h after feeding on two successive days. Abundance of each target taxon was calculated as a fraction of the total 16S rRNA gene copies in the samples, using taxon-specific and eubacterial domain-level primers. Bacterial populations showed a clear predominance of members of the genus Prevotella, which comprised 42% to 60% of the bacterial rRNA gene copies in the samples. However, only 2% to 4% of the bacterial rRNA gene copies were represented by the classical ruminal Prevotella species Prevotella bryantii, Prevotella ruminicola and Prevotella brevis. The proportion of rRNA gene copies attributable to Fibrobacter succinogenes, Ruminococcus flavefaciens, Selenomonas ruminantium and Succinivibrio dextrinosolvens were each generally in the 0.5% to 1% range. Proportions for Ruminobacter amylophilus and Eubacterium ruminantium were lower (0.1% to 0.2%), while Butyrivibrio fibrisolvens, Streptococcus bovis, Ruminococcus albus and Megasphaera elsdenii were even less abundant, each comprising <0.03% of the bacterial rRNA gene copies. The data suggest that the aggregate abundance of the most intensively studied ruminal bacterial species is relatively low and that a large fraction of the uncultured population represents a single bacterial genus.     

Activity and properties of fumarate reductase in ruminal bacteria

Fumarate-reducing bacteria were sought from the main ruminal bacteria. Fibrobacter succinogenes, Selenomonas ruminantium subsp. ruminantium, Selenomonas ruminantium subsp. lactilytica, and Veillonella parvula reduced fumarate by using H(2) as an electron donor. Ruminococcus albus, Prevotella ruminicola, and Anaerovibrio lipolytica consumed fumarate, although they did not oxidize H(2). Of these bacteria, V. parvula, two strains of Selenomonas, and F. succinogenes had a high capacity to reduce fumarate. In all the fumarate-reducing bacteria examined, fumarate reductase existed in the membrane fraction. Based on the activity per cell mass and the affinity of fumarate reductase to fumarate, these bacteria were divided into two groups, which corresponded to the capacity to use H(2): A group of bacteria with higher activity and affinity were able to use H(2) as an electron donor for fumarate reduction. The bacteria in this group should gain an advantage over the bacteria in another group in fumarate reduction in the rumen. Cellulose digestion by R. albus was improved by fumarate reduction by S. lactilytica as a result of an increased growth of R. albus, which may have been caused by the fact that S. lactilytica immediately consumed H(2) produced by R. albus. Thus fumarate reduction may play an important role in keeping a low partial pressure of H(2) in the rumen.     

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.     

Diet-dependent shifts in the bacterial population of the rumen revealed with real-time PCR

A set of PCR primers was designed and validated for specific detection and quantification of Prevotella ruminicola, Prevotella albensis, Prevotella bryantii, Fibrobacter succinogenes, Selenomonas ruminantium-Mitsuokella multiacida, Streptococcus bovis, Ruminococcus flavefaciens, Ruminobacter amylophilus, Eubacterium ruminantium, Treponema bryantii, Succinivibrio dextrinosolvens, and Anaerovibrio lipolytica. By using these primers and the real-time PCR technique, the corresponding species in the rumens of cows for which the diet was switched from hay to grain were quantitatively monitored. The dynamics of two fibrolytic bacteria, F. succinogenes and R. flavefaciens, were in agreement with those of earlier, culture-based experiments. The quantity of F. succinogenes DNA, predominant in animals on the hay diet, fell 20-fold on the third day of the switch to a grain diet and further declined on day 28, with a 57-fold reduction in DNA. The R. flavefaciens DNA concentration on day 3 declined to approximately 10% of its initial value in animals on the hay diet and remained at this level on day 28. During the transition period (day 3), the quantities of two ruminal prevotella DNAs increased considerably: that of P. ruminicola increased 7-fold and that of P. bryantii increased 263-fold. On day 28, the quantity of P. ruminicola DNA decreased 3-fold, while P. bryantii DNA was still elevated 10-fold in comparison with the level found in animals on the initial hay diet. The DNA specific for another xylanolytic bacterium, E. ruminantium, dropped 14-fold during the diet switch and was maintained at this level on day 28. The concentration of a rumen spirochete, T. bryantii, decreased less profoundly and stabilized with a sevenfold decline by day 28. The variations in A. lipolytica DNA were not statistically significant. After an initial slight increase in S. dextrinosolvens DNA on day 3, this DNA was not detected at the end of the experiment. S. bovis DNA displayed a 67-fold increase during the transition period on day 3. However, on day 28, it actually declined in comparison with the level in animals on the hay ration. The amount of S. ruminantium-M. multiacida DNA also increased eightfold following the diet switch, but stabilized with only a twofold increase on day 28. The real-time PCR technique also uncovered differential amplification of rumen bacterial templates with the set of universal bacterial primers. This observation may explain why some predominant rumen bacteria have not been detected in PCR-generated 16S ribosomal DNA libraries.     

Fermentation of xylans by Butyrivibrio fibrisolvens and other ruminal bacteria.

The ability of Butyrivibrio fibrisolvens and other ruminal bacteria (6 species, 18 strains) to ferment a crude xylan from wheat straw or to ferment xylans from larchwood or oat spelts was studied. Liquid cultures were monitored for carbohydrate utilization, cell growth (protein), and fermentation acid production. B. fibrisolvens 49, H17c, AcTF2, and D1 grew almost as well on one or more of the xylans as they did on cellobiose-maltose. B. fibrisolvens 12, R28, A38, X10C34, ARD22a, and X6C61 exhibited moderate growth on xylans. Partial fermentation of xylans was observed with Bacteroides ruminicola B14, Bacteroides succinogenes S85, Ruminococcus albus 7, Ruminococcus flavefaciens C94 and FD1, and Succinivibrio dextrinosolvens 22B. All xylans tested appeared to have a small fraction of carbohydrate that supported low levels of growth of nonxylanolytic strains such as Selenomonas ruminantium HD4. Compared to growth on hexoses, the same array of fermentation acids was produced upon growth on xylans for most strains; however, reduced lactate levels were observed for B. fibrisolvens 49 and Selenomonas ruminantium HD4. Measurements of enzyme activities of B. fibrisolvens AcTF2, 49, H17c, and D1 indicated that the xylobiase activities were cell associated and that the xylanase activities were predominantly associated with the culture fluid. The pattern of expression of these enzymes varied both between strains and between the carbon sources on which the strains were grown.     

Fermentation of xylans by Butyrivibrio fibrisolvens and other ruminal bacteria

The ability of Butyrivibrio fibrisolvens and other ruminal bacteria (6 species, 18 strains) to ferment a crude xylan from wheat straw or to ferment xylans from larchwood or oat spelts was studied. Liquid cultures were monitored for carbohydrate utilization, cell growth (protein), and fermentation acid production. B. fibrisolvens 49, H17c, AcTF2, and D1 grew almost as well on one or more of the xylans as they did on cellobiose-maltose. B. fibrisolvens 12, R28, A38, X10C34, ARD22a, and X6C61 exhibited moderate growth on xylans. Partial fermentation of xylans was observed with Bacteroides ruminicola B14, Bacteroides succinogenes S85, Ruminococcus albus 7, Ruminococcus flavefaciens C94 and FD1, and Succinivibrio dextrinosolvens 22B. All xylans tested appeared to have a small fraction of carbohydrate that supported low levels of growth of nonxylanolytic strains such as Selenomonas ruminantium HD4. Compared to growth on hexoses, the same array of fermentation acids was produced upon growth on xylans for most strains; however, reduced lactate levels were observed for B. fibrisolvens 49 and Selenomonas ruminantium HD4. Measurements of enzyme activities of B. fibrisolvens AcTF2, 49, H17c, and D1 indicated that the xylobiase activities were cell associated and that the xylanase activities were predominantly associated with the culture fluid. The pattern of expression of these enzymes varied both between strains and between the carbon sources on which the strains were grown.     

Utilization of nucleic acids by Selenomonas ruminantium and other ruminal bacteria.

Species of ruminal bacteria were screened for the ability to grow in media containing RNA or DNA as the energy source. Bacteroides ruminicola D31d and Selenomonas ruminantium HD4, GA192, and D effectively used RNA for growth, but not DNA. B. ruminicola D31d was able grow on nucleosides but not on bases or ribose. The S. ruminantium strains were able to grow when provided with either nucleosides or ribose but not bases. Strains of S. ruminantium, but not B. ruminicola D31d, were also able to use nucleosides as nitrogen sources. These data suggest that RNA fermentation may be a general characteristic of S. ruminantium.     

Utilization of nucleic acids by Selenomonas ruminantium and other ruminal bacteria.

Species of ruminal bacteria were screened for the ability to grow in media containing RNA or DNA as the energy source. Bacteroides ruminicola D31d and Selenomonas ruminantium HD4, GA192, and D effectively used RNA for growth, but not DNA. B. ruminicola D31d was able grow on nucleosides but not on bases or ribose. The S. ruminantium strains were able to grow when provided with either nucleosides or ribose but not bases. Strains of S. ruminantium, but not B. ruminicola D31d, were also able to use nucleosides as nitrogen sources. These data suggest that RNA fermentation may be a general characteristic of S. ruminantium.     

Phosphoenolpyruvate-dependent phosphorylation of hexoses by ruminal bacteria: evidence for the phosphotransferase transport system.

Six species of ruminal bacteria were surveyed for the phosphoenolpyruvate (PEP)-dependent phosphorylation of glucose. Selenomonas ruminantium HD4, Streptococcus bovis JB1, and Megasphaera elsdenii B159 all showed significant activity, but Butyrivibrio fibrisolvens 49, Bacteroides succinogenes S85, and Bacteroides ruminicola B1(4) showed low rates of PEP-dependent phosphorylation and much higher rates in the presence of ATP. S. ruminantium HD4, S. bovis JB1, and M. elsdenii B159 also used PEP to phosphorylate the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DG). Rates of 2-DG phosphorylation with ATP were negligible for S. bovis JB1 and M. elsdenii B159, but toluene-treated cells of S. ruminantium HD4 phosphorylated 2-DG in the presence of ATP as well as PEP. Cell-free extracts of S. ruminantium HD4 used ATP but not PEP to phosphorylate glucose and 2-DG. Since PEP could serve as a phosphoryl donor in toluene-treated cells but not in cell-free extracts, there was evidence for membrane and hence phosphotransferase system involvement in the PEP-dependent activity. The ATP-dependent phosphorylating enzymes from S. ruminantium HD4 and S. bovis JB1 had molecular weights of approximately 48,000 and were not inhibited by glucose 6-phosphate. Based on these criteria, they were glucokinases rather than hexokinases. The S. ruminantium HD4 glucokinase was competitively inhibited by 2-DG and mannose, sugars that differ from glucose in the C-2 position. Since 2-DG was a competitive inhibitor of glucose, the same enzyme probably phosphorylates both sugars. The S. bovis JB1 glucokinase was not inhibited by either 2-DG or mannose and had a higher Km and Vmax for glucose.     

Phosphoenolpyruvate-dependent phosphorylation of hexoses by ruminal bacteria: evidence for the phosphotransferase transport system

Six species of ruminal bacteria were surveyed for the phosphoenolpyruvate (PEP)-dependent phosphorylation of glucose. Selenomonas ruminantium HD4, Streptococcus bovis JB1, and Megasphaera elsdenii B159 all showed significant activity, but Butyrivibrio fibrisolvens 49, Bacteroides succinogenes S85, and Bacteroides ruminicola B1(4) showed low rates of PEP-dependent phosphorylation and much higher rates in the presence of ATP. S. ruminantium HD4, S. bovis JB1, and M. elsdenii B159 also used PEP to phosphorylate the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DG). Rates of 2-DG phosphorylation with ATP were negligible for S. bovis JB1 and M. elsdenii B159, but toluene-treated cells of S. ruminantium HD4 phosphorylated 2-DG in the presence of ATP as well as PEP. Cell-free extracts of S. ruminantium HD4 used ATP but not PEP to phosphorylate glucose and 2-DG. Since PEP could serve as a phosphoryl donor in toluene-treated cells but not in cell-free extracts, there was evidence for membrane and hence phosphotransferase system involvement in the PEP-dependent activity. The ATP-dependent phosphorylating enzymes from S. ruminantium HD4 and S. bovis JB1 had molecular weights of approximately 48,000 and were not inhibited by glucose 6-phosphate. Based on these criteria, they were glucokinases rather than hexokinases. The S. ruminantium HD4 glucokinase was competitively inhibited by 2-DG and mannose, sugars that differ from glucose in the C-2 position. Since 2-DG was a competitive inhibitor of glucose, the same enzyme probably phosphorylates both sugars. The S. bovis JB1 glucokinase was not inhibited by either 2-DG or mannose and had a higher Km and Vmax for glucose.     

Biotransformation of 2,4,6-Trinitrotoluene by Pure Culture Ruminal Bacteria

Twenty-one ruminal bacteria species were tested for their ability to degrade 2,4,6-trinitrotoluene (TNT) within 24 h. Butyrivibrio fibrisolvens, Fibrobacter succinogenes, Lactobacillus vitulinus, Selenomonas ruminantium, Streptococcus caprinus, and Succinivibrio dextrinosolvens were able to completely degrade 100 mg/L TNT, with <5% of the original TNT recovered as diaminonitrotoluene metabolites. Eubacterium ruminantium, Lactobacillus ruminis, Ruminobacter amylophilus, Streptococcus bovis, and Wolinella succinogenes were able to completely degrade 100 mg/L TNT, with 23–60% of the TNT recovered as aminodinitrotoluene and/or diaminonitrotoluene metabolites. Clostridium polysaccharolyticum, Megasphaera elsdenii, Prevotella bryantii, Prevotella ruminicola, Ruminococcus albus, and Ruminococcus flavefaciens were able to degrade 80–90% of 100 mg/L TNT. Desulfovibrio desulfuricans subsp. desulfuricans, Prevotella albensis, and Treponema bryantii degraded 50–80% of the TNT. Anaerovibrio lipolytica was completely inhibited by 100 mg/L TNT. These results indicate that a variety of rumen bacteria is capable of transforming TNT.     

The cellular location of Prevotella ruminicola beta-1,4-D-endoglucanase and its occurrence in other strains of ruminal bacteria.

Prevotella ruminicola B(1)4, TC1-1, TF1-3, and TS1-5 all produced immunologically cross-reacting 88- and 82-kDa carboxymethyl cellulases (CMCases). P. ruminicola 23, 118B, 20-63, and 20-78 had much lower CMCase activities, and Western blots (immunoblots) showed no cross-reaction with the B(1)4 CMCase antiserum. Fibrobacter succinogenes S85 and Selenomonas ruminantium HD4 and D produced CMCase, but these enzymes were smaller and did not cross-react with the B(1)4 CMCase antiserum. The B(1)4 CMCase antiserum inhibited the B(1)4, TC1-1, TF1-3, and TS1-5 CMCase activities and agglutinated these cells, but it had no effect on the other strains or species. On the basis of these results, the B(1)4 CMCase is a strain-specific enzyme that is located on the outside surface of the cells. P. ruminicola B(1)4 cultures, grown on sucrose, did not have significant CMCase activity, but these cells could bind purified 88- and 82-kDa CMCase but not 40.5-kDa CMCase. Because the 40.5-kDa CMCase is a fully active, truncated form of the CMCase, it appears that the N-terminal domain of the 88-kDa B(1)4 CMCase anchors the CMCase to the cells. Cells grown on cellobiose produced at least 10-fold more CMCase than the sucrose-grown cells, and the cellobiose-grown cells could only bind 15% as much CMCase as sucrose-grown cells. Virtually all of the CMCase activity of exponentially growing cultures was cell associated, but CMCase activity was eventually detected in the culture supernatant. On the basis of the observation that the 88-kDa CMCase was gradually converted to the 82-kDa CMCase when cultures reached the stationary phase without a change in specific activity, it appears that the 82-kDa protein is probably a proteolytic degradation product of the 88-kDa CMCase.     

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.     

Interaction of ruminal bacteria in the production and utilization of maltooligosaccharides from starch.

The degradation and utilization of starch by three amylolytic and one nonamylolytic species of ruminal bacteria were studied. Pure cultures of Streptococcus bovis JB1, Butyrivibrio fibrisolvens 49, and Bacteroides ruminicola D31d rapidly hydrolyzed starch and maltooligosaccharides accumulated. The major starch hydrolytic products detected in S. bovis cultures were glucose, maltose, maltotriose, and maltotetraose. In addition to these oligosaccharides, B. fibrisolvens cultures produced maltopentaose. The products of starch hydrolysis by B. ruminicola were even more complex, yielding glucose through maltotetraose, maltohexaose, and maltoheptaose but little maltopentaose. Selenomonas ruminantium HD4 grew poorly on starch, digested only a small portion of the available substrate, and generated no detectable oligosaccharides as a result of cultivation in starch containing medium. S. ruminantium was able to grow on a mixture of maltooligosaccharides and utilize those of lower degree (less than 10) of polymerization. A coculture system containing S. ruminantium as a dextrin-utilizing species and each of the three amylolytic bacteria was developed to test whether the products of starch hydrolysis were available for crossfeeding to another ruminal bacterium. Cocultures of S. ruminantium and S. bovis contained large numbers of S. bovis but relatively few S. ruminantium and exhibited little change in the pattern of maltooligosaccharides observed for pure cultures of S. bovis. In contrast, S. ruminantium was able to compete with B. fibrisolvens and B. ruminicola for these growth substrates. When grown with B. fibrisolvens, S. ruminantium grew to high numbers and maltooligosaccharides accumulated to a much lesser degree than in cultures of B. fibrisolvens alone. S. ruminantium-B. ruminicola cultures contained large numbers of both species, and maltooligosaccharides never accumulated in these cocultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Utilization of xylooligosaccharides by selected ruminal bacteria.

The ability of ruminal bacteria to utilize xylooligosaccharides was examined. Xylooligosaccharides were prepared by partially hydrolyzing oat spelt xylan in phosphoric acid. This substrate solution was added (0.2%, wt/vol) to a complex medium containing yeast extract and Trypticase that was inoculated with individual species of ruminal bacteria, and growth and utilization were monitored over time. All of the xylanolytic bacteria examined were able to utilize this oligosaccharide mixture as a growth substrate. Butyrivibrio fibrisolvens, Eubacterium ruminantium, and Ruminococcus albus used xylooligosaccharides and whole, unhydrolyzed xylan to similar extents, while Prevotella ruminicola used twice as much xylooligosaccharides as xylan (76 versus 34%). Strains of Selenomonas ruminantium were the only nonxylanolytic species that were able to grow on xylooligosaccharides. The ability of individual S. ruminantium strains to utilize xylooligosaccharides was correlated with the presence of xylosidase and arabinosidases activities.     

Interaction of ruminal bacteria in the production and utilization of maltooligosaccharides from starch.

The degradation and utilization of starch by three amylolytic and one nonamylolytic species of ruminal bacteria were studied. Pure cultures of Streptococcus bovis JB1, Butyrivibrio fibrisolvens 49, and Bacteroides ruminicola D31d rapidly hydrolyzed starch and maltooligosaccharides accumulated. The major starch hydrolytic products detected in S. bovis cultures were glucose, maltose, maltotriose, and maltotetraose. In addition to these oligosaccharides, B. fibrisolvens cultures produced maltopentaose. The products of starch hydrolysis by B. ruminicola were even more complex, yielding glucose through maltotetraose, maltohexaose, and maltoheptaose but little maltopentaose. Selenomonas ruminantium HD4 grew poorly on starch, digested only a small portion of the available substrate, and generated no detectable oligosaccharides as a result of cultivation in starch containing medium. S. ruminantium was able to grow on a mixture of maltooligosaccharides and utilize those of lower degree (less than 10) of polymerization. A coculture system containing S. ruminantium as a dextrin-utilizing species and each of the three amylolytic bacteria was developed to test whether the products of starch hydrolysis were available for crossfeeding to another ruminal bacterium. Cocultures of S. ruminantium and S. bovis contained large numbers of S. bovis but relatively few S. ruminantium and exhibited little change in the pattern of maltooligosaccharides observed for pure cultures of S. bovis. In contrast, S. ruminantium was able to compete with B. fibrisolvens and B. ruminicola for these growth substrates. When grown with B. fibrisolvens, S. ruminantium grew to high numbers and maltooligosaccharides accumulated to a much lesser degree than in cultures of B. fibrisolvens alone. S. ruminantium-B. ruminicola cultures contained large numbers of both species, and maltooligosaccharides never accumulated in these cocultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of tetronasin and monensin on fermentation, microbial numbers and the development of ionophore-resistant bacteria in the rumen

The Gram-negative rumen bacteria Fibrobacter succinogenes S85, Prevotella ruminicola M384 and Veillonella parvula L59 were grown in media containing successively increasing concentrations of the ionophores, monensin and tetronasin. All three species became more resistant to the ionophore with which they were grown. Increased resistance to one ionophore caused increased resistance to the other, and cross-resistance to another ionophore—lasalocid—and an antibiotic—avoparcin. Recovery of tetronasin-resistant bacteria from the rumen of monensin-fed sheep increased and vice versa, indicating that similar cross-resistance occurred in vivo.     

Dissolution of xylose metabolism in Lactococcus lactis

Xylose metabolism, a variable phenotype in strains of Lactococcus lactis, was studied and evidence was obtained for the accumulation of mutations that inactivate the xyl operon. The xylose metabolism operon (xylRAB) was sequenced from three strains of lactococci. Fragments of 4.2, 4.2, and 5.4 kb that included the xyl locus were sequenced from L. lactis subsp. lactis B-4449 (formerly Lactobacillus xylosus), L. lactis subsp. lactis IO-1, and L. lactis subsp. lactis 210, respectively. The two environmental isolates, L. lactis B-4449 and L. lactis IO-1, produce active xylose isomerases and xylulokinases and can metabolize xylose. L. lactis 210, a dairy starter culture strain, has neither xylose isomerase nor xylulokinase activity and is Xyl(-). Xylose isomerase and xylulokinase activities are induced by xylose and repressed by glucose in the two Xyl(+) strains. Sequence comparisons revealed a number of point mutations in the xylA, xylB, and xylR genes in L. lactis 210, IO-1, and B-4449. None of these mutations, with the exception of a premature stop codon in xylB, are obviously lethal, since they lie outside of regions recognized as critical for activity. Nevertheless, either cumulatively or because of indirect affects on the structures of catalytic sites, these mutations render some strains of L. lactis unable to metabolize xylose.     

Fiber-degrading systems of different strains of the genus Fibrobacter

The S85 type strain of Fibrobacter succinogenes, a major ruminal fibrolytic species, was isolated 49 years ago from a bovine rumen and has been used since then as a model for extensive studies. To assess the validity of this model, we compared the cellulase- and xylanase-degrading activities of several other F. succinogenes strains originating from different ruminants, including recently isolated strains, and looked for the presence of 10 glycoside hydrolase genes previously identified in S85. The NR9 F. intestinalis type strain, representative of the second species of the genus, was also included in this study. DNA-DNA hybridization and 16S rRNA gene sequencing first classified the strains and provided the phylogenetic positions of isolates of both species. Cellulase and xylanase activity analyses revealed similar activity profiles for all F. succinogenes strains. However, the F(E) strain, phylogenetically close to S85, presented a poor xylanolytic system and weak specific activities. Furthermore, the HM2 strain, genetically distant from the other F. succinogenes isolates, displayed a larger cellulolytic profile on zymograms and higher cellulolytic specific activity. F. intestinalis NR9 presented a higher cellulolytic specific activity and a stronger extracellular xylanolytic activity. Almost all glycoside hydrolase genes studied were found in the F. succinogenes isolates by PCR, except in the HM2 strain, and few of them were detected in F. intestinalis NR9. As expected, the fibrolytic genes of strains of the genus Fibrobacter as well as the cellulase and xylanase activities are better conserved in closely related phylogenetic isolates.     

Novel molecular features of the fibrolytic intestinal bacterium Fibrobacter intestinalis not shared with Fibrobacter succinogenes as determined by suppressive subtractive hybridization

Suppressive subtractive hybridization was conducted to identify unique genes coding for plant cell wall hydrolytic enzymes and other properties of the gastrointestinal bacterium Fibrobacter intestinalis DR7 not shared by Fibrobacter succinogenes S85. Subtractive clones from F. intestinalis were sequenced and assembled to form 712 nonredundant contigs with an average length of 525 bp. Of these, 55 sequences were unique to F. intestinalis. The remaining contigs contained 764 genes with BLASTX similarities to other proteins; of these, 80% had the highest similarities to proteins in F. succinogenes, including 30 that coded for carbohydrate active enzymes. The expression of 17 of these genes was verified by Northern dot blot analysis. Of genes not exhibiting BLASTX similarity to F. succinogenes, 30 encoded putative transposases, 6 encoded restriction modification genes, and 45% had highest similarities to proteins in other species of gastrointestinal bacteria, a finding suggestive of either horizontal gene transfer to F. intestinalis or gene loss from F. succinogenes. Analysis of contigs containing segments of two or more adjacent genes revealed that only 35% exhibited BLASTX similarity and were in the same orientation as those of F. succinogenes, indicating extensive chromosomal rearrangement. The expression of eight transposases, and three restriction-modification genes was confirmed by Northern dot blot analysis. These data clearly document the maintenance of carbohydrate active enzymes in F. intestinalis necessitated by the preponderance of polysaccharide substrates available in the ruminal environment. It also documents substantive changes in the genome from that of F. succinogenes, which may be related to the introduction of the array of transposase and restriction-modification genes.