Isolation and attempted introduction of sugar alcohol-utilizing bacteria in the sheep rumen

R.J. WALLACE AND N.D. WALKER. 1993. Bacteria that use sorbitol, xylitol, maltitol and dulcitol (galactitol) were isolated from the sheep rumen following enrichments in which bacteria were grown in rumen fluid medium where the sugar alcohol was the only added energy source. Only isolates obtained with sorbitol and maltitol grew sufficiently rapidly to be considered for enrichment by the sugar alcohol in vivo. Isolate SS2, a strain of Selenomonas ruminantium var. lactilytica which grew on sorbitol at 0.87 h^-1 was selected for further study and a rifampicin-resistant mutant, SS2/R5, was isolated to facilitate tracking in the mixed population. Despite an initial transient increase in numbers, a significant population of S. ruminantium SS2/R5 failed to establish in sheep which were dosed twice daily with 10 g of sorbitol. Continuous infusion of sorbitol increased numbers only slightly compared with twice-daily dosing. In vitro experiments indicated that strain SS2/R5 grew less well in the presence of other rumen organisms, particularly ciliate protozoa, than in pure culture. Furthermore, the concentration of sorbitol in vivo was lower than predicted from in vitro experiments, indicating that sorbitol was absorbed rapidly from the rumen. Similar observations were made with xylitol, dulcitol and maltitol. Proposed enrichment strategies that use sugar alcohols or other materials to support the growth of introduced bacteria will thus have to take account of the combined problems of microbe-microbe interactions and the loss of the compounds by absorption from the rumen.     

Chemoattractant effects of sugars and their related compounds on black abalone Haliotis discus

The chemoattractant properties of sugars and their related compounds were statistically estimated on the basis of an exploratory behavior of black abalone Haliotis discus. Six monosaccharides, three disaccharides, six sugar alcohols, six glycosides and two artificial sweeteners were tested. The active compounds were: glucose and galactose (monosaccharides), maltose (disaccharides), sorbitol, mannitol, maltitol, dulcitol and erythritol (sugar alcohols), all the glycosides, and saccharin (artificial sweeteners). Maltose, dulcitol and particularly phyllodukin showed the highest activity. The chemoattractant properties of maltose and phyllodukin increased as concentrations increased. The activity of phyllodulcin was higher at all concentrations tested than that of maltose.     

Breakdown of different peptides byPrevotella (Bacteroides) ruminicola and mixed microorganisms from the sheep rumen

Several di-, tri-, and oligopeptides were incubated individually in vitro with rumen fluid from two sheep receiving a mixed grass hay/concentrate diet and with washed cells ofPrevotella (formerlyBacteroides)ruminicola M384 andP. ruminicola B₁4. The rates of breakdown of most peptides were similar in the rumen fluid from the two sheep. Acidic and proline-containing peptides tended to be more slowly degraded than neutral or basic peptides. The dipeptide at the N-terminus of higher peptides was observed as an early product of hydrolysis, confirming that a dipeptidyl aminopeptidase type of activity was present. The relative rates of breakdown of dipeptides byP. ruminicola were different from that of rumen fluid, but the hydrolysis of higher peptides followed a similar pattern, and dipeptides from the N-terminus were detected as early products.     

Sequence Analysis of the Plasmid pRRI2 from the Rumen Bacterium Prevotella ruminicola 223/M2/7 and the Use of pRRI2 in Prevotella/Bacteroides Shuttle Vectors

pRRI2 is a small cryptic plasmid from the rumen bacterium Prevotella ruminicola 223/M2/7 which has been used for the construction of shuttle vectors (pRH3 and pRRI207) that replicate in many Bacteroides/Prevotella strains as well as in Escherichia coli. Sequence analysis of pRRI2 reveals that it is a 3240-bp plasmid carrying two clear open reading frames. Rep, encoded by ORF1, shows 48 and 47% amino acid sequence identity with RepA proteins from Bacteroides vulgatus and Bacteroides fragilis, respectively. ORF2, named Pre, shares 34% amino acid sequence identity with a putative plasmid recombination protein from the Flavobacterium spp. plasmid pFL1 and 30% amino acid sequence identity with BmpH from B. fragilis Tn5520. Disruption of ORF1 with HindIII prevents replication and maintenance in Bacteroides spp. hosts, but shuttle vectors carrying pRRI2 interrupted within ORF2, by EcoRI*, are able to replicate. pRRI2 shows no significant similarity with the only other P. ruminicola plasmid to have been studied previously, pRAM4.     

Antibiotic resistance patterns and plasmids of ruminal strains of Bacteroides ruminicola and Bacteroides multiacidus

Summary Plasmids were recovered and are described from three ruminal Bacteroides strains — 23, 223/M2/7 and 46/5(2). Although all three were originally isolated as Bacteroides ruminicola, 46/5(2) is shown here to be a strain of Bacteroides multiacidus, a species not previously described from the rumen. An 11.7 kbp plasmid present in strain 46/5(2) gave the same digestion pattern with Sal I and Sma I as a plasmid in B. multiacidus subgroup b type strain P208-58. Strains 46/5(2) and P208-58 both showed resistance to tetracycline, as did B. ruminicola strain 223/M2/7. B. multiacidus P208-58, and, to a lesser degree, B. ruminicola 23, showed resistance to ampicillin. Four other strains of B. ruminicola and one of B. multiacidus in which plasmids were not detected were sensitive to both antibiotics. It has still to be established whether these resistance traits are plasmid or chromosomally coded.     

Sensitivity of antennal gustatory receptor neurones to aphid honeydew sugars in the carabid Anchomenus dorsalis

Using electrophysiology, the stimulating effect of 13 sugars and three sugar alcohols (each at a concentration of 100 mm ) to antennal gustatory receptor neurones (GRNs) is tested in the carabid beetle Anchomenus dorsalis (Pontoppidan, 1763) (Coleoptera, Carabidae). Maltose, sucrose, glucose and raffinose are the most stimulating sugars for the sugar‐sensitive neurone (SuN), evoking 6.7–18.6 spikes s^?1 in fed insects, whereas the others had little or no effect. The firing rate of the antennal GRNs is not affected by any of the tested sugar alcohols, dulcitol, inositol and sorbitol. Additionally, concentration/response curves for sucrose and maltose are obtained in the range 0.01–100 mm . The responses of beetles starved for 96 h to this range of sucrose are two‐ to three‐fold higher compared with those of fed beetles. The presence of a terminal α‐glucose unit is an important feature of the molecular structure determining the stimulating properties of the two disaccharides, maltose and sucrose, as well as glucose. The other monosaccharide unit of the molecule is also of great importance in determining the stimulating properties of various disaccharides. The sensitivity of the SuN to the four most prevalent aphid honeydew sugars suggests that A. dorsalis uses these chemicals as sensory cues when searching for aphids as prey.     

A newEscherichia coli: Bacteroides shuttle vector,pRRI207, based on theBacteroides ruminicola plasmid replicon pRRI2

A 3.4kb cryptic plasmid, pRRI2, was isolated fromBacteroides ruminicola 223/M2/7 and used as the basis for a newBacteroides/Escherichia shuttle vector. Constructs were made incorporating pRRI2, aBacteroides erythromycin/clindamycin resistance marker and theE. coli pUC8-derived vector pHG165. One of these, pRRI207 (11 kb), was capable of introduction into strains belonging to four different species ofBacteroides (B. uniformis, B. distasonis, B. thetaiotaomicron, orB. ruminicola) either by conjugal transfer fromE. coli or by electrotransformation. pRRI207 carries several unique restriction sites derived from the pUC8 multiple cloning site. Only one of sixB. ruminicola strains tested was used successfully as a recipient for pRRI207, indicating that further modifications to transfer procedures or marker genes may be needed for wider application in this species.     

Cleavage of di- and tripeptides by Prevotella ruminicola

The final step in the conversion of protein to amino acids by the common Gram-negative rumen bacterium, Prevotella (formerly Bacteroides) ruminicola , is the cleavage of di- and tripeptides. Dipeptidase and tripeptidase activities were predominantly cytoplasmic, and toluene treatment increased the rate of Ala₂ and Ala₃ hydrolysis by whole cells, suggesting that transport limited the rate of hydrolysis of extracellular di- and tripeptides. The hydrolysis of Ala₂ and Ala₃ by whole cells was not affected by protonophores, ionophores or dicyclohexylcarbodiimide, but Ala₂ hydrolysis by EDTA-treated cells was inhibited by the Ca²⁺/H⁺ ionophore, tetronasin. Ala₃ hydrolysis was not affected by protonophores or ionophores in EDTA-treated cells. The dipeptidase of strain M384 was inhibited > 99% by 1,10-phenanthroline and 39% by EDTA but not other protease inhibitors, consistent with the enzyme being a metalloprotease. Tripeptidase was insensitive to protease inhibitors, except for a 33% inhibition by EDTA. Cleavage of tripeptides occurred at the bond adjacent to the N-terminal amino acid. Distinct di-, tri- and oligopeptidase peaks were obtained by anion-exchange liquid chromatography of disrupted cells. Banding patterns on native PAGE using activity staining also indicated that P. ruminicola M384 had separate single dipeptidase and tripeptidase enzymes which hydrolysed a range of peptides. The dipeptidase of strain M384 was different from other strains of P. ruminicola: strains GA33 and B₁4 had activities which ran at the same R_f; strain GA33 had another band of lower activity; strain 23 had two bands different from those of the other strains. The tripeptidases ran at the same R_f for the different strains. Dipeptidase activity of all strains was inhibited by 1,10-phenanthroline on gels. Gel permeation chromatography indicated that the M_r of the dipeptidases from strains M384 and B₁4 were 115 000 and 114 500 respectively, and 112 500 and 121 500 for the corresponding tripeptidases. Thus the metabolism of small peptides by P. ruminicola involves separate permeases and intracellular peptidases for di- and tripeptides.     

Identification and Mutational Analysis of <Emphasis Type="Italic">Escherichia coli</Emphasis> Sorbitol-Enhanced Glucose-Repressed <Emphasis Type="Italic">srlA</Emphasis> Promoter Expressed in LB Medium by Using Homologous Recombination and One-Round PCR Products

Escherichia coli has been used for recombinant protein production for many years. However, no native E. coli promoters have been found for constitutive expression in LB medium. To obtain high-expression E. coli promoters active in LB medium, we inserted various promoter regions upstream of eEmRFP that encodes a red fluorescent protein. Among the selected promoters, only colonies of srlA promoter transformants turned red on LB plate. srlA is a gene that regulates sorbitol utilization. The addition of sorbitol enhanced eEmRFP expression but glucose and other sugars repressed, indicating that srlAp is a sorbitol-enhanced glucose-repressed promoter. To analyze the srlAp sequence, a novel site-directed mutagenesis method was developed. Since we demonstrated that homologous recombination in E. coli could occur between 12-bp sequences, 12-bp overlapping sequences were attached to the set of primers that were designed to produce a full-length plasmid, denoted “one-round PCR product.” Using this method, we identified that the srlA promoter region was 100 bp. Further, the sequence adjacent to the start codon was found to be essential for high expression, suggesting that the traditionally used restriction enzyme sites for cloning in the promoter region have hindered expression. The srlA-driven expression system and DNA manipulation with one-round PCR products are useful tools in E. coli genetic engineering.     

Toxicity of volatile fatty acids at rumen pH prevents enrichment ofEscherichia coli by sorbitol in rumen contents

Attempts were made to establishEscherichia coli ML308 in the sheep rumen by inoculating it in combination with the slowly metabolized sugar alcohol, sorbitol. Numbers were determined by plating dilutions on nutrient agar containing the chromogenic -galactoside, 5-bromo-4-chloro-3-indolyl--d-galactoside. This strain, alac-constitutive mutant, produced distinctive blue colonies.E. coli ML308 failed to grow in rumen fluid, despite being able to grow rapidly on sorbitol and in rumen fluid at pH 7.0. Its growth rate was depressed by relatively small drops in pH in the presence of volatile fatty acids (VFA), such that normal pH's of 6.2–6.6 in rumen contents were inhibitory. The indigenous remen bacterium,Streptococcus bovis, was much more resistant to the combination of high VFA concentrations and low pH. The success of this and similar strategies for the introduction of new organisms with beneficial new properties will, therefore, depend on the organism's having a tolerance to VFA over a range of rumen pH that enables them to survive in the same way as native species.     

Some effects of glucose and ammonia on protein synthesis by rumen bacteria

Summary Four species of ruminal anaerobic bacteria:Bacteroides ruminicola Succinivibrio dexrinosolvesn, Selenomonas ruminantium, andStreptococcus bovis, were used in growth experiments to study the effects of ammonia-nitrogen concentration, at several levels of fermentable energy, on nutrient utilization and protein synthesis. The optimal available ammonia concentration for use without wastage by most of the organisms was 5.4 mmol/l, butBacteroides ruminicola used as much as 27 mmol/l ammonia. All the organisms used 27 mmol/l glucose completely. Differences were found among the organisms in the optimal available glucose and ammonia concentrations associated with maximal total protein formation and maximal protein formation per unit of glucose or ammonia used during growth. The glucose concentration associated with most efficient conversion of glucose to protein (5.4 mmol/l) byBacteroides ruminicola andSuccinivibrio dextrinosolvens was five-fold less than that associated with most efficient protein formation per unit of glucose used byStreptococcus bovis andSelenomonas ruminantium. Under nutritional conditions associated with ruminant productivity, it is likely that, for both microbe and animal, substantial nutrient waste occurs.

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Resumen Los efectos de la concenración de N-amónico, considerando distintos niveles de energía fermentable, en la utilización de nutrientes y en la síntesis de proteínas se estudiaron mediante experiencias de crecimiento de cuatro bacterias del rumen:Bacteroides ruminicola, Succinovibrio dexrinosolvens, Selenomonas ruminantum yStreptococcus bovis. La concentración óptima de amoniaco, es decir aquella utilizada sin despilfarro, fue de 5.4 mmol/l para la maoría de los microorganismos, exceptuando aBacteroides ruminicola que podía llegar a utilizar hasta 27 mmol/l. Todos los microorganismos utilizaron 27 mmol/l de glucosa. Las concentraciones óptimas de amoniaco y glucosa asociadas bien sea a la máxima formación de proteínas totales a a la máxima formación de proteínas por unidad de glucosa o amoniaco utilizadas durante el crecimiento fueron distintas entre los diferentes organismos estudiados. La conversión en proteína a partir de glucosa se realizó más eficazmente a concentraciones de 5.4 mmol/l de esta paraBacteroides ruminicola ySuccinovibrio dextrinosolvens, concentración cinco veces menor que la utilizada porStreptococcuus bovis ySelenomonas ruminantium. Considerando las condiciones de nutrición ligadas a la producción de rumiantes es probable que tanto en relación al microorganismo como en relación al animal se produzca un despilfarro de nutrientes importante.

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Résumé Quatre espèces de bactéries anaérobies du rumen (Bacteroides ruminicola, Succinivibrio dextrinosolvens, Selenomonas ruminantium etStreptococcus bovis) ont été utilisées dans des expériences de croissance ayant pour but d'étudier, en fonction de plusieurs niveaux d'énergie fermentescible, l'effet de la concentration en azote ammoniacal sur l'utilisation des substances nutritives et la synthèse protéique. Pour la plupart des organismes, la concentation maximum en ammoniaque permettant d'éviter une perte est de 5,4 mmol/l. Toutefois,Bacteroides ruminicola peut utiliser jusqu'à 27 mmol/l d'ammoniaque. Tous les organismes utilisent complétement une quantité de glucose de 27 mmol/l. Des différences entre les organismes ont été constatées en ce qui concerne les concentrations optimales en glucose et en ammoniaque qui assurent une croissance totale et une production de protéines maximale par unité de glucose et d'ammoniaque utilisée. ChezBacterioides ruminicola etSuccinivibrio dextrinosolvens, la concentration en glucose assurant la conversion la plus efficace du glucose en protéines (5,4 mmol/l) est 5 fois moindre que dans le cas deStreptococcus bovis etSelenomonas ruminantium. Il est probable que, dans les conditions nutritionnelles utilisées en élevage des ruminants, des quantités importantes d'aliments soient perdues.

     

Heterotrophic Growth of Two Strains of the Acido-Thermophilic Red Alga Galdieria sulphuraria

The acido-and thermophilic red alga Galdieria sulphuraria (Galdieri)Merola grows under mixo- and heterotrophic conditions on 27different sugars and sugar alcohols as sole carbon source. Weseparated two strains from an isolate originally collected atMt. Lawu (Indonesia). These strains are indistinguishable ingrowth and pigmentation under autotrophic conditions. However,under heterotrophic conditions, strain 074 W lost most of itspigments whereas strain 074 G stayed green on all substratestested. Strain 074 G had the highest pigment content when grownon sugar alcohols. Usually the alga exhibited a short lag-phasefollowed by logarithmic growth. However, when transferred fromauto- to heterotrophic conditions a lag-period of about 45 dayswas observed with the sugar alcohol dulcitol. Similarly, longlag-periods were also noticed for strain 074 G grown on D-mannitoland for strain 074 W grown on D-ribose. The length of the lag-phaseis a function of the length of the previous culture under autotrophicconditions. This enormous versatility in the heterotrophic growthof Galdieria sulphuraria presents an ideal system to study themetabolism of rare sugars and sugar alcohols. (Received November 21, 1994; Accepted March 9, 1995)     

Bacterial Population Adherent to the Epithelium on the Roo of the Dorsal Rumen of Sheep

By anaerobic procedures, the total number of adherent bacteria was determined on tissue samples obtained from the roof of the dorsal rumen of three sheep. After four washings, 1.91 × 10⁷, 0.34 × 10⁷, and 1.23 × 10⁷ bacteria per cm² were still attached to the rumen epithelium in sheep 1, 2, and 3, respectively. A total of 95 strains of bacteria were isolated from these three samples. Based on morphology, Gram stain, anaerobiosis, motility, and fermentation end products, they were presumptively identified as follows: Butyrivibrio fibrisolvens, 30 strains; atypical Butyrivibrio, 5 strains; Bacteroides ruminicola, 22 strains; Lactobacillus, 1 strain; and unknown Bacteroides species, 37 strains. For sheep 3, washing the rumen epithelium a total of 10 times reduced the adherent bacterial population by 93% (8.4 × 10⁵ bacteria per cm²). Of 30 strains isolated from this sample, 22 were presumptively identified as Butyrivibrio and Bacteroides types. These results suggest that the epithelium on the roof of the dorsal rumen is primarily colonized by two genera of bacteria, Butyrivibrio and Bacteroides. Most Butyrivibrio and Bacteroides ruminicola strains appeared to be similar to previously isolated rumen strains. However, the unknown Bacteroides species differed considerably from the three species of this genus which are commonly isolated from rumen contents.     

Incidence of apple fruit and leaf surface metabolites on Cydia pomonella oviposition

Water soluble metabolites identified from surfaces of apple tree fruit and leaf stimulate oviposition in Cydia pomonella L. The effects of two artificial blends of primary metabolites representing fruit and leaf surfaces, respectively, and of components within the blends were examined on egg-laying after two time periods: 3 min and 25 min of darkness. An artificial mixture of six metabolites, viz., three sugar alcohols (sorbitol, quebrachitol, and myo-inositol) and three sugars (glucose, fructose, and sucrose) did stimulate egg-laying. Fructose, sorbitol, and myo-inositol are important components of the stimulatory blend. Contact durations may induce variations in egg-laying responses. After 3 min of darkness, there were no differences in numbers of females laying eggs nor in the numbers of eggs laid on cloths treated with the complete blends and the controls. There were, however, clear effects of groups of compounds and of individual compounds. Reduced blends without sugars and sugar alcohols were in many cases significantly less stimulatory than the complete blends and the controls. After 25 min of darkness, the proportions of females laying eggs as well as the numbers of eggs were higher after treatment with the complete blends than on the controls. The proportions of females laying eggs on cloths treated with the reduced blends were rather similar to the controls, whereas there were still significant effects on the numbers of eggs laid after treatment with reduced blends derived from fruits but not from leaves.     

Establishment and growth characteristics of a cell suspension culture of Marchantia polymorpha L. with high chlorophyll content

A green-pigmented cell suspension culture of Marchantia polymorpha was established using the medium of Murashige and Skoog with addition of organic acids of the tricarboxylic-acid cycle, vitamins and sugars plus sugar alcohols, exclusion of kinetin, and replacement of sucrose with glucose. In continuous light, the cells grew exponentially for ca. 10 days; in the dark, they grew only to a slight extent. The light-grown cells contained well-developed chloroplasts, and chlorophyll content reached almost twice that of the intact gametophyte.     

13C-NMR spectroscopy as a tool to study organic osmolytes in the mangrove red algal genera Bostrychia and Stictosiphonia (Ceramiales)

Field and culture samples of the red algal genera Bostrychia and Stictosiphonia from all around the world were analyzed for the polyols D-sorbitol and dulcitol, that function as osmolytes, as well as for the heteroside digeneaside by using ¹³C-NMR spectroscopy and HPLC. While all plants exhibited D-sorbitol, the occurrence of dulcitol and digeneaside was highly variable. Therefore, different types of low molecular weight carbohydrate distribution patterns were found in Bostrychia and Stictosiphonia. The presence of D-sorbitol seems to be a reliable chemosystematic character for both genera, because no other red alga is known to contain this compound. The lack of dulcitol may be correlated with the geographical origin of the Bostrychia and Stictosiphonia samples: while all tropical isolates exhibited both sugar alcohols, in cold-temperate plants only D-sorbitol was determined. In warm-temperate species, however, both polyol distribution types may occur. These data are discussed in terms of possible temperature sensitivity of the dulcitol pathway. However, the biological function of digeneaside (the main photo-assimilated compound in members of the order Ceramiales) is still obscure.     

Diversity of Extracellular Proteolytic Activities Among Prevotella Species from the Rumen

The current research was aimed at comparing proteolytic activities among ruminal Prevotella spp. Growth rates of Prevotella sp. 2202, Prevotella ruminicola D31d, P. brevis GA33, P. albensis M384, and P. bryantii B₁4 varied with N source, and no one N source produced the fastest growth in all species. Proteolytic activity was greatest with casein compared with peptides, AA, and NH₄Cl in all species. Proteolytic activity of Prevotella sp. 2202, P. brevis GA33, and P. bryantii B₁4 was modulated by N source. With gelatin co-polymerized SDS-PAGE, the extracellular activities of the Prevotella spp. showed wide variation in number, size, and type of proteases. Prevotella sp. 2202 and P. albensis M384 produced metalloproteases of low molecular weight (40 kDa). P. ruminicola D31d produced one cysteine protease (100–200 kDa) and two metalloproteases (90–100 kDa). P. brevis GA33 generated a diffuse clearing zone (95–160 kDa) containing serine, cysteine, and metalloproteases. P. bryantii B₁4 produced a metalloprotease greater than 200 kDa in size. The molecular sizes provided are estimations and served only to differentiate among the bacterial species in this study. Large variations in proteolytic activities among species and the known genetic diversity of the Prevotella taxon suggested that targeting this bacterial assemblage for genetic manipulation in order to alter the bacterial impact on ruminal protein degradation would be difficult. Received: 12 January 1999 / Accepted: 19 May 1999     

Tetrapyrrole utilization by protoheme-synthesizing anaerobes

Protoheme-synthesizing anaerobic bacteria were grown in the presence of metal-free deuteroporphyrin IX or selected metal chelates of deuteroporphyrin IX to determine whether anaerobes that synthesize protoheme-containing cytochrome b de novo would use preformed tetrapyrroles. Characteristic reduced versus oxidized difference spectra of whole cells and pyridine hemochromogen spectra revealed thatSelenomonas ruminantium subsp.lactilytica andBacteroides succinogenes synthesized protoheme-containing b-type cytochromes de novo during growth in the presence of the manganese or magnesium chelates of deuteroporphyrin IX, but thatBacteroides ruminicola subsp.brevis synthesized deuteroheme-containing cytochrome b under the same conditions. During growth of the latter organism in the presence of the vanadium, molybdenum, cobalt, or nickel chelates of deuteroporphyrin, protoheme cytochrome b was formed.Bacteroides ruminicola subsp.brevis had a unique and metal-specific ability to use preformed tetrapyrroles for cytochrome synthesis; that ability was absent in other cytochrome-synthesizing rumen bacteria.     

Phenotypic Characterization of Polysaccharidases Produced by Four Prevotella Type Strains

Four ruminal Prevotella type strains, P. ruminicola JCM8958^T, P. bryantii B₁4^T, P. albensis M384^T, and P. brevis ATCC19188^T, were characterized for polysaccharide-degrading activities with the reducing sugar release assay and zymogram analyses. Carboxymethylcellulase, xylanase, and polygalacturonate (PG)-degrading enzyme activities were determined in cultures grown on oat spelt xylan, xylose, arabinose, cellobiose, and glucose as sole growth substrates. P. ruminicola and P. albensis showed carboxymethylcellulase induction patterns. When xylan was supplied as a sole growth substrate, xylanase activities produced by P. bryantii and P. albensis were at least 18- and 11-fold higher, respectively, than during growth on other carbohydrates, suggesting that the regulation of the xylanases was highly specific to xylan. All strains constitutively produced PG-degrading enzymes. The corresponding activity of P. bryantii was more than 40-fold higher than in other strains. Zymogram analyses routinely detected the presence of high-molecular-weight (100–170 kDa) polysaccharide-degrading enzymes in ruminal Prevotella. Characteristics of the polysaccharide-degrading activities showed diversity of ruminal Prevotella species. Received: 29 November 1999 / Accepted: 1 February 2000