Some chemical and physical properties of extracellular polysaccharides produced by Butyrivibrio fibrisolvens strains.

Most strains of Butyrivibrio fibrisolvens are known to produce extracellular polysaccharides (EPs). However, the rheological and functional properties of these EPs have not been determined. Initially, 26 strains of Butyrivibrio were screened for EP yield and apparent viscosities of cell-free supernatants. Yields ranged from less than 1.0 to 16.3 mg per 100 mg of glucose added to the culture. Viscosities ranged from 0.71 to 5.44 mPa.s. Five strains (CF2d, CF3, CF3a, CE51, and H10b) were chosen for further screening. The apparent viscosity of the EP from each of these strains decreased by only 50 to 60% when the shear rate was increased from 20 to 1,000 s-1. Strain CE51 produced the EP having the highest solution viscosity. A detailed comparison of shear dependency of the EP from strain CF3 with xanthan gum showed that this EP was less shear sensitive than xanthan gum and, at a shear rate of 1,000 s-1, more viscous. EPs from strains CF3 and H10b were soluble over a wide range of pH (1 to 13) in 80% (vol/vol) ethanol-water or in 1% (wt/vol) salt solutions. The pH of 1% EP solutions was between 4.5 and 5.5. Addition of acid increased solution viscosities, whereas addition of base decreased viscosity. EPs from strains CF3, CE51, and H10b displayed qualitatively similar infrared spectra. Calcium and sodium were the most abundant minerals in the three EPs. The amounts of magnesium, calcium, and iron varied considerably among the EPs, but the potassium contents remained relatively constant.     

Neutral sugar composition of extracellular polysaccharides produced by strains of Butyrivibrio fibrisolvens

The extracellular polysaccharides (EPSs) produced by 37 isolates presently classified as Butyrivibrio species (or more specifically as Butyrivibrio fibrisolvens) were purified from glucose-grown cultures. The neutral sugar compositions of these EPSs were determined by both thin-layer and gas-liquid chromatographic techniques. Results showed that while the neutral sugar composition of the EPS was constant for a given strain, it varied considerably between strains. In addition, several acidic components in the EPS, of both known and unknown structure, were detected artifactually as acetylated lactones, the acetylated alditols derived from these lactone(s), or both. Two novel components, L-altrose and the acidic sugar 4-O-[1-carboxyethyl]-D-galactose, were common constituents of the EPS from some strains of B. fibrisolvens. These and other EPS compositional features were used to sort isolates of B. fibrisolvens into groups which may have taxonomic significance. A scheme for sorting isolates into these groups, and the relative relationships between groups, is proposed.     

Structural studies of the extracellular polysaccharide produced by Butyrivibrio fibrisolvens strain H10b

The extracellular polysaccharide produced by Butyrivibrio fibrisolvens strain H10b, when grown under strictly anaerobic conditions with glucose as carbohydrate source, has been studied by chemical and spectroscopic techniques. The results demonstrate that the polysaccharide consists of hexasaccharide repeating units with the following structure: [structure: see text] The isolated polysaccharide was found to be approximately 65% acetylated at O-2 of the 3-O-[(S)-1-carboxyethyl]-beta-D-Glcp residue. The absolute configuration of the 1-carboxyethyl groups was determined by circular dichroism.     

Esterase activities in Butyrivibrio fibrisolvens strains

Thirty strains of Butyrivibrio fibrisolvens isolated in diverse geographical locations were examined for esterase activity by using naphthyl esters of acetate, butyrate, caprylate, laurate, and palmitate. All strains possessed some esterase activity, and high levels of activity were observed with strains 49, H17c, S2, AcTF2, and LM8/1B. Esterase activity also was detected in other ruminal bacteria (Bacteroides ruminicola, Selenomonas ruminantium, Ruminobacter amylophilus, and Streptococcus bovis). For all B. fibrisolvens strains tested, naphthyl fatty acid esterase activity paralleled culture growth and was predominantly cell associated. With strains 49, CF4c, and S2, the activity was retained by protoplasts made from whole cells. Esterase activity was detected with all strains when grown on glucose, and some strains showed higher activity levels when grown on other substrates (larchwood xylan or citrus pectin). When nitrophenyl esters of fatty acids were used to measure esterase activity, generally four- to sevenfold-higher activity levels were detected, and with a number of strains substantial levels were found in the culture fluid. Cultures of these strains (H17c, NOR37, D1, and D30g) contained xylanase and acetyl xylan esterase activities, neither of which was associated to any great extent with the cells. Acetyl xylan esterase has not been previously detected in ruminal bacteria and may be important to overall digestion of forage by these organisms.     

Degradation and utilization by Butyrivibrio fibrisolvens H17c of xylans with different chemical and physical properties.

Hemicelluloses, mainly xylans, can be a major component of diets consumed by ruminants and undergo various degrees of microbial digestion in the rumen. The ability of Butyrivibrio fibrisolvens, a major xylanolytic ruminal species, to degrade and utilize nine chemically and physically different xylans for growth was examined. The arabinoxylans used included two isolated from corncobs (CCX-A and CCX-B), a native xylan excreted by corn cell tissue cultures (CX), an oxalic acid-treated, arabinose-depleted CX, and oat spelt xylan. Except for CCX-A, these xylans were extensively converted within 3 h of growth to acid-alcohol-soluble forms that remained at high levels for the duration of culture growth. These xylans contain mainly xylose and arabinose with small amounts of uronic acids. For a given xylan, all three components were used at about the same rate and extent. During the early stages of growth B. fibrisolvens also rapidly solubilized glucuronoxylans from birchwood, larchwood, 4-O-methylglucuronoxylan, and the xylose homopolymer xylan isolated from beechwood (BEWX). In contrast to the findings for the arabinoxylans, little acid-alcohol-soluble carbohydrate remained in these cultures after 9 h of growth, except for BEWX. Initially, with birchwood, larchwood, and 4-O-methylglucuronoxylan the uronic acid components were preferentially used over the xylose. Final xylan utilization measured at 72 h for all xylans varied from 57% for CCX-A to 92% for BEWX and was correlated with the initial 12-h utilization rate for a given xylan. Since CCX-A and BEWX are both highly water insoluble, this aspect did not appear to influence overall utilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Esterase activities in Butyrivibrio fibrisolvens strains.

Thirty strains of Butyrivibrio fibrisolvens isolated in diverse geographical locations were examined for esterase activity by using naphthyl esters of acetate, butyrate, caprylate, laurate, and palmitate. All strains possessed some esterase activity, and high levels of activity were observed with strains 49, H17c, S2, AcTF2, and LM8/1B. Esterase activity also was detected in other ruminal bacteria (Bacteroides ruminicola, Selenomonas ruminantium, Ruminobacter amylophilus, and Streptococcus bovis). For all B. fibrisolvens strains tested, naphthyl fatty acid esterase activity paralleled culture growth and was predominantly cell associated. With strains 49, CF4c, and S2, the activity was retained by protoplasts made from whole cells. Esterase activity was detected with all strains when grown on glucose, and some strains showed higher activity levels when grown on other substrates (larchwood xylan or citrus pectin). When nitrophenyl esters of fatty acids were used to measure esterase activity, generally four- to sevenfold-higher activity levels were detected, and with a number of strains substantial levels were found in the culture fluid. Cultures of these strains (H17c, NOR37, D1, and D30g) contained xylanase and acetyl xylan esterase activities, neither of which was associated to any great extent with the cells. Acetyl xylan esterase has not been previously detected in ruminal bacteria and may be important to overall digestion of forage by these organisms.     

Fermentation of glucose and xylose in ruminal strains of Butyrivibrio fibrisolvens

Metabolism of glucose and xylose and parameters of growth were investigated in strains of Butyrivibrio fibrisolvens ATCC 19171 and CE 51. In the strain ATCC 19171, the composition of fermentation end-products was the same in cultures supplied with glucose and xylose. The strain CE 51 produced more volatile fatty acids and less lactate from xylose than from glucose. Cells of this strain grown on xylose possessed phosphoketolase activity (EC 4.1.2.9). In both strains the production of cell dry matter and growth rate were higher in cultures supplied with glucose. In xylose-grown cultures butyrivibrios tended to convert more substrate carbon into metabolites and less into cellular material than in cultures grown on glucose.     

Detection and characterization of extracellular proteases in Butyrivibrio fibrisolvens H17c

Summary The detection and approximate molecular weights of extracellular serine protease isoenzymes produced by Butyrivibrio fibrisolvens H17c were determined by gelatin-PAGE. Nine bands of protease activity with apparent molecular weights of approximately 101000, 95000, 87000, 80000, 76000, 68000, 63000, 54000 and 42000 were detected after gelatin-PAGE of supernatants from exponential phase cultures. A tenth serine protease band with an apparent molecular weight of approximately 32000 was detected in stationary phase cells. The activities of all ten protease bands were inhibited by a serine protease inhibitor but their activities were not affected by inhibitors of trypsin-like enzymes or metallo-, sulphydryl-and carboxylproteases. The activity of all ten exoprotease bands was optimal between pH 6.0 and 7.5. The ten exoprotease bands were only detected in media containing trypticase or casamino acids as nitrogen sources. Production of the ten protease bands was not affected by the carbohydrate source.     

Selenium uptake by Butyrivibrio fibrisolvens and Bacteroides ruminicola

Abstract The uptake and incorporation of ⁷⁵[Se]selenite by Butyrivibrio fibrisolvens and Bacteroides ruminicola were by constitutive systems. Rates of uptake were higher in chemostat culture than in batch culture and there may be some inducible component. Uptake of [⁷⁵Se]selenite was distinct from sulphate or selenate transport, since sulphate and selenate did not inhibit selenite uptake, nor could sulphate or selenate uptake be demonstrated in these organisms. Selenite uptake in B. fibrisolvens had and apparent K _m of 1.74 mM and a V _max of 109 ng Se · min⁻¹· (mg protein)⁻¹. An apparent K _m of 1.76 mM and V _max of 1.5 μg Se · min⁻¹· (mg protein)⁻¹ was obtained for B. ruminicola . [⁷⁵Se]Selenite uptake by both organisms was partially sensitive to inhibition by 2,4-DNP. Uptake by B. fibrisolvens was also partially inhibited by azide and arsenate and in B. ruminicola it was partially inhibited by fluoride. CCCP, CPZ, DCCD or quinine did not inhibit uptake in either B. fibrisolvens or B. ruminicola . Selenite transport by both organisms was sensitive to IAA and NEM and was strongly inhibited by sulphite and nitrite. [⁷⁵Se]Selenite was converted to selenocystine, selenohomocystine and selenomethionine by B. fibrisolvens. B. ruminicola did not incorporate [⁷⁵Se]selenite into organic compounds, but did reduce it to red elemental selenium.     

Digestion of structural polysaccharides of Panicum and vetch hays by the rumen bacterial strains Fibrobacter succinogenes BL2 and Butyrivibrio fibrisolvens D1

The rumen bacterial strains Fibrobacter succinogenes BL2 and Butyrivibrio fibrisolvens D1, were grown in monocultures and pair combination on cell walls (CW) of two tropical hays: Panicum (grass) and vetch (legume), and their ability to solubilize and utilize CW structural carbohydrate was determined. With respect to both substrates, F. succinogenes BL2 was a better solubilizer of CW carbohydrate than B. fibrisolvens D1. However, the solubilization of Panicum constituents by any bacterial monoculture and co-culture was higher than that of vetch. Complementary interaction between B. fibrisolvens D1 and F. succinogenes BL2 was identified only with respect to carbohydrate utilization, but not with the extent of CW solubilization, which was determined mainly by the F. succinogenes strain. In both substrates, utilization of solubilized cellulose by BL2 monocultures was high (86.4–97.5%), whereas that of solubilized xylan and hemicellulose was much lower (35.2–41.6%). Under scanning electron microscopy visualization, the BL2 bacterial cell mass attached to and colonized on CW particles was characterized by the appearance of protuberant structures known as polycellulosome complexes on their surface topology. Correspondence to: J. Miron     

Identification of l-iduronic acid as a constituent of the major extracellular polysaccharide produced by Butyrivibrio fibrisolvens strain X6C61

Abstract Butyrivibrio fibrisolvens strain X6C61 produces two extracellular polysaccharides (EPS-I and EPS- II) separable by anion-exchange chromatography. The neutral sugar constituents of EPS-I were identified by gas-liquid chromatography (GLC) as the alditol acetates of rhamnose, mannose, galactose, glucose, and an unidentified component. These results were confirmed using thin-layer chromatography (TLC). Neutral sugar analysis of EPS-II, which eluted from DEAE-Sephadex at 0.4 M NaCl, yielded the alditol acetates of rhamnose, galactose, glucose, and idose. However, idose was not found when hydrolysates of EPS-II were analysed by TLC. Further investigations showed that the iditol hexaacetate detected via GLC was an artifact of the commonly-used procedures for neutral sugar analysis. This compound was instead generated from l -iduronic acid, as shown by GLC-MS studies.     

Enzymology of butyrate formation by Butyrivibrio fibrisolvens.

Butyrivibrio fibrisolvens is a major butyrate-forming species in the bovine and ovine rumen. The enzymology of butyrate formation from pyruvate was investigated in cell-free extracts of B. fibrisolvens D1. Pyruvate owas oxidized to acetylcoenzyme A (CoA) in the presence of CoA.SH and benzyl viologen or flavin nucleotides. The bacterium uses thiolase, beta-hydroxybutyryl-CoA dehydrogenase, crotonase, and crotonyl-CoA reductase to form butyryl-CoA from acetyl-CoA. Reduction of acetoacetyl-CoA to beta-hydroxybutyryl-CoA was faster with NADH than with NADPH. Crotonyl-CoA was reduced to butyryl-CoA by NADH, but not by NADPH, only in the presence of flavin nucleotides. Reduction of flavin nucleotides by NADH was much slower than the flavin-dependent reduction of crotonyl-CoA. This indicates that flavoproteins rather than free flavin participated in the reduction of crotonyl-CoA. Butyryl-CoA was converted to butyrate by phosphate butyryl transferase and butyrate kinase.     

Pentose transport by the ruminal bacterium Butyrivibrio fibrisolvens

Abstract Butyrivibrio fibrisolvens is a fibrolytic ruminal bacterium that degrades hemicellulose and ferments the resulting pentose sugars. Washed cells of strain D1 accumulated radiolabelled xylose ( K _m= 1.5 μ M) and arabinose ( K _m= 0.2 μ M) when the organism was grown on xylose, arabinose, or glucose, but cultures grown on sucrose or cellobiose had little capacity to transport pentose. Glucose and xylose inhibited transport of each other non-competitively. Both sugars were utilized preferentially over arabinose, but since they did not inhibit transport of arabinose, it appeared that the preference was related to an internal metabolic step. Although the protonmotive force was completely abolished by ionophores, cells retained some ability to transport pentose. In contrast, the metabolic inhibitors iodoacetate, arsenate, and fluoride had little effect on protonmotive force but caused a large decrease in intracellular ATP and xylose and arabinose uptake. These results suggested that high-affinity, ATP-dependent mechanisms were responsible for pentose transport and hexose sugars affected the utilization of xylose and arabinose.     

An analysis of the extracellular xylanases and cellulases of Butyrivibrio fibrisolvens H17c

The extracellular xylanase and cellulase components of Butyrivibrio fibrisolvens H17c were investigated. Two major peaks of enzyme activity were eluted by hydroxylapatite chromatography and designated complex A (CA), having cellulase activity, and complex B (CB) having predominantly xylanase activity but with some activity on carboxymethyl cellulose (CMC). CB was further purified on a DE-52 column and subjected to gel filtration. The xylanase and CMCase activities eluted in a single peak with an apparent molecular mass greater than thyroglobulin (Mr 669,000). CMC xymograms of polyacrylamide gels electrophoresed under non-denaturing conditions indicated the presence of five bands with CMCase activity from CA and eight from CB. Xylan xymograms under the same conditions indicated the presence of four bands of activity in CB. Under mild denaturing conditions the xylanase activity in CB was found in 11 bands with molecular mass ranging from 45 to 180 and the CMCase activity in three bands with molecular mass ranging from 45 kDa to 60 kDa. This indicates that CB exists as a multi-subunit protein aggregate of xylanases, some of which also have cellulase activity.     

Identification of L-altrose in the extracellular polysaccharide from Butyrivibrio fibrisolvens strain CF3

Abstract Butyrivibrio fibrisolvens strain CF3, a stricyly anaerobic bacterial isolate from the ovine cecum, produces extracellular polysaccharides (EPS) when grown on a defined medium containing glucose as the carbon source. EPS were purified from culture supernatants and their monosaccharide composition was determined by two different procedures. Analysis of EPS hydrolysates by thin-layer chromatography (TLC) yielded spots coincident with standard glucose, altrose, and 1,6-anhydroaltrose. These results were corroborated by both gas-liquid chromatography (GLC) and GLC-mass spectroscopy (GLC-MS) of alditol acetates prepared from EPS hydrolysates. Purification of the altrose from EPS hydrolysates was accomplished by preparative paper and column chromatography. Polarimetry demonstrated the isolated altrose to have the L -configuration. The occurrence of this hexose in nature has not yet been reported.     

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.     

Anaerobic Production of Extracellular Polysaccharide by Butyrivibrio fibrisolvens nyx

Anaerobic production of extracellular polysaccharide (EP) was examined, using a previously uncharacterized, obligately anaerobic rumen isolate, Butyrivibrio fibrisolvens nyx, which produced an EP that was rheologically similar to xanthan gum. The main objectives were to determine the nutritional requirements and conditions which promoted EP production by strain nyx. Strain nyx was grown anaerobically in defined and semidefined media. In addition to carbohydrate and nitrogen sources, strain nyx required acetic acid, folic acid, biotin, and pyridoxamine. Strain nyx produced similar amounts of EP at 35 to 40°C. Conditions that improved growth usually improved EP production. Of the carbohydrates tested, glucose supported the fastest growth and most EP production, followed by sucrose, xylose, and lactose. Strain nyx utilized ammonium sulfate, urea, or vitamin-free casein hydrolysate as nitrogen sources for growth and EP production. At 2 and 20 g/liter, respectively, ammonium sulfate and vitamin-free casein hydrolysate supported about the same rates of growth and EP production. EP was not produced in the lag or stationary phases, and EP production was exponential during exponential cell growth. Based on the results of this work, anaerobic EP production with B. fibrisolvens nyx could reduce energy costs for industrial EP production compared with the cost of aerated systems. Finally, this work demonstrated that, under appropriate growth conditions, a gastrointestinal tract (ruminal) microorganism produced high levels of EP.     

Some chemical and physical properties of nisin, a small-protein antibiotic produced by Lactococcus lactis

Nisin is a small gene-encoded antimicrobial protein produced by Lactococcus lactis that contains unusual dehydroalanine and dehydrobutyrine residues. The reactivity of these residues toward nucleophiles was explored by reacting nisin with a variety of mercaptans. The kinetics of reaction with 2-mercaptoethane-sulfonate and thioglycolate indicated that the reaction pathway includes a binding step. Reaction of nisin at high pH resulted in the formation of multimeric products, apparently as a result of intramolecular and intermolecular reactions between nucleophilic groups and the dehydro residues. One of the nucleophiles had a pKa of about 9.8. The unique vinyl protons of the dehydro residues that give readily identifiable proton nuclear magnetic resonances were used to observe the addition of nucleophiles to the dehydro moiety. After reaction with nucleophiles, nisin lost its antibiotic activity and no longer showed the dehydro resonances, indicating that the dehydro groups had been modified. The effect of pH on the solubility of nisin was determined; the solubility was quite high at low pH (57 mg/ml at pH 2) and was much lower at high pH (0.25 mg/ml at pH 8 to 12), as measured before significant pH-induced chemical modification had occurred. High-performance liquid chromatography on a C18 column was an effective technique for separating unmodified nisin from its reaction products. The cyanogen bromide cleavage products of nisin were about 90% less active toward inhibition of bacterial spore outgrowth than was native nisin. These results are consistent with earlier observations, which suggested that the dehydro residues of nisin have a role in the mechanism of antibiotic action, in which they act as electrophilic Michael acceptors toward nucleophiles in the cellular target.     

4-O-(1-carboxyethyl)-D-galactose. A new acidic sugar from the extracellular polysaccharide produced by Butyrivibrio fibrisolvens strain 49.

The structure of a new acidic sugar from the extracellular polysaccharide of Butyrivibrio fibrisolvens strain 49 was determined as 4-O-(1-carboxyethyl)-D-galactose on the basis of 13C-n.m.r. and 1H-n.m.r. spectroscopy, m.s. and chemical degradation studies.     

Xylose and arabinose utilization by the rumen bacterium Butyrivibrio fibrisolvens

Abstract The rumen bacterium Butyrivibrio fibrisolvens strain D1 co-utilized xylose and glucose in batch culture, but there was a marked preference for glucose over arabinose. When both pentoses were provided, xylose was preferred over arabinose. Strain D1 co-utilized a combination of either pentose and cellobiose, but preferred pentoses over maltose. Pentose sugars were depleted less rapidly in the presence of sucrose than controls containing only pentose. In contrast, B. fibrisolvens strain A38 exhibited a strong preference for disaccharides, including maltose, over either xylose or arabinose. Theoretical maximum growth yields for strain D1v in single-substrate continuous culture were highest for sucrose and cellobiose and the maintenance energy coefficient for arabinose was at least 3.8-fold greater than for other substrates. We suggest that B. fibrisolvens may have evolved a mechanism to utilize certain sugars before arabinose in order to avoid this high maintenance energy expenditure.