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R B Hespell

Publications and source records attributed to R B Hespell.

At least 19 recordsLinked to original sources
PubMed1995

Differentiation of ruminal and human Streptococcus bovis strains by DNA homology and 16s rRNA probes.

Streptococcus bovis is commonly present in the rumen, but strains of S. bovis have also occasionally been isolated from human blood or fecal samples. Studies were undertaken with 16s rRNA gene sequences and DNA hybridizations to define the genetic relationships between these two groups of strains. Ruminal strains were found to yield genomic DNA restriction endonuclease digest patterns different from human strains when either the 16s rRNA gene amplified from ruminal S. bovis strain JB1 or a conserved universal 23s rRNA fragment was used as probes. A DNA probe based on the V1 region of the 16s rRNA of S. bovis JB1 was found to hybridize to DNAs of other ruminal S. bovis strains (K27FF4, 21-09-6C, five new ruminal isolates, and weak hybridization was found with DNAs from S. bovis 33317 (type strain), S. equinus 9812, and six other ruminal isolates. No hybridization occurred with strains representing different major human biotypes/homology groups (43143, 43144, 27960, V1387). All ruminal S. bovis strains had a guanosine plus cytosine DNA content of 37.4-38.8 mol% and, based on DNA-DNA genomic hybridizations, could be separated into two homology groups, one of which included S. equinus 9812 and S. bovis 33317. Both ruminal groups had less than 38% DNA homology to the human strains, indicating ruminal strains are clearly two separate species distinct from the human strains.

Animals↗
PubMed1995

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)

Animals↗
PubMed1991

Introduction of the Bacteroides ruminicola xylanase gene into the Bacteroides thetaiotaomicron chromosome for production of xylanase activity.

The xylanase gene from the ruminal bacterium Bacteroides ruminicola 23 is highly expressed in colonic Bacteroides species when carried on plasmid pVAL-RX. In order to stabilize xylanase expression in the absence of antibiotic selection, the xylanase gene was introduced into the chromosome of Bacteroides thetaiotaomicron 5482 by using suicide vector pVAL-7. Xylanase activity in the resulting strain, B. thetaiotaomicron BTX, was about 30% of that observed in B. thetaiotaomicron 5482 containing the xylanase gene on pVAL-RX. The data obtained from continuous culture experiments using antibiotic-free medium showed that expression of xylanase activity in strain BTX was extremely stable, with no demonstrated loss of the inserted xylanase gene over 60 generations, with dilution rates from 0.42 to 0.03 h-1. In contrast, the plasmid-borne xylanase gene was almost completely lost by 60 generations in the absence of antibiotic selection. Incubation of strain BTX with oatspelt xylan resulted in the degradation of more than 40% of the xylan to soluble xylooligomers. The stability of xylanase expression in B. thetaiotaomicron BTX suggests that this microorganism might be suitable for introduction into the rumen and increased xylan degradation.

Animals↗
PubMed1991

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.

Bacteroidaceae↗
PubMed1991

Conjugal transfer of Tn916, Tn916 delta E, and pAM beta 1 from Enterococcus faecalis to Butyrivibrio fibrisolvens strains.

Anaerobic filter matings of Butyrivibrio fibrisolvens H17c, CF3, D1, or GS113, representing different DNA relatedness groups, were done with Enterococcus faecalis CG110, which contains chromosomally inserted Tn916. Tetracycline-resistant transconjugants were obtained with each mating pair at average frequencies of 4.4 x 10(-6) (per recipient) and 5.2 x 10(-6) (per donor). The transfer frequencies of Tn916 into B. fibrisolvens varied 5- to 10-fold with mating time, strain, and growth stage. By using Southern hybridization with pAM120 as the probe, Tn916 was shown to insert at one or more separate chromosomal sites for each strain of B. fibrisolvens. Retransfer of Tn916 from B. fibrisolvens H17c or CF3 to E. faecalis OG1-X or JH 2-2 or to B. fibrisolvens D1 or GS113 could not be shown. Matings of E. faecalis RH110, which contains chromosomally inserted Tn916 delta E, with B. fibrisolvens 49, H17c, D1, CF3, GS113, or VV-1 resulted in erythromycin-resistant transconjugants at average frequencies of 5.3 x 10(-7) (per recipient) and 2.5 x 10(-7) (per donor). Tn916 delta E was shown by Southern hybridization with pAM120 to insert at one or more sites in the chromosome of each strain. B. fibrisolvens H17c was anaerobically filter mated with E. faecalis JH 2-SS, which contains pAM beta 1. Erythromycin-resistant transconjugants were obtained at frequencies of 2 x 10(-5) (per recipient) and 6 x 10(-5) (per donor). The presence of pAM beta 1 in these transconjugants could not be shown by agarose gel electrophoresis of plasmid minilysates but could be shown by Southern hybridization analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteroidaceae↗
PubMed1991

Introduction of Tn916 and pAM beta 1 into Streptococcus bovis JB1 by conjugation.

The transposon Tn916 and self-mobilizing plasmid pAM beta 1 were conjugated from Enterococcus faecalis to the ruminal bacterium Streptococcus bovis JB1. Transconjugants were identified by resistance to tetracycline (Tn916) or erythromycin (pAM beta 1) and by Southern hybridization analyses. Transfer frequencies were 7.0 x 10(-6) and 1.0 x 10(-6) per recipient cell for Tn916 and pAM beta 1, respectively. The transconjugants JB1/Tn916 and JB1/pAM beta 1 were used as donors for matings with E. faecalis, Bacillus subtilis, and the ruminal bacterium Butyrivibrio fibrisolvens. While pAM beta 1 was successfully transferred to all three organisms, Tn916 was transferred only into B. subtilis and B. fibrisolvens at very low frequencies. This is the first report of conjugal DNA transfers between two ruminal organisms.

Conjugation, Genetic↗
PubMed1991

Phylogenetic analysis of the spirochetes.

The 16S rRNA sequences were determined for species of Spirochaeta, Treponema, Borrelia, Leptospira, Leptonema, and Serpula, using a modified Sanger method of direct RNA sequencing. Analysis of aligned 16S rRNA sequences indicated that the spirochetes form a coherent taxon composed of six major clusters or groups. The first group, termed the treponemes, was divided into two subgroups. The first treponeme subgroup consisted of Treponema pallidum, Treponema phagedenis, Treponema denticola, a thermophilic spirochete strain, and two species of Spirochaeta, Spirochaeta zuelzerae and Spirochaeta stenostrepta, with an average interspecies similarity of 89.9%. The second treponeme subgroup contained Treponema bryantii, Treponema pectinovorum, Treponema saccharophilum, Treponema succinifaciens, and rumen strain CA, with an average interspecies similarity of 86.2%. The average interspecies similarity between the two treponeme subgroups was 84.2%. The division of the treponemes into two subgroups was verified by single-base signature analysis. The second spirochete group contained Spirochaeta aurantia, Spirochaeta halophila, Spirochaeta bajacaliforniensis, Spirochaeta litoralis, and Spirochaeta isovalerica, with an average similarity of 87.4%. The Spirochaeta group was related to the treponeme group, with an average similarity of 81.9%. The third spirochete group contained borrelias, including Borrelia burgdorferi, Borrelia anserina, Borrelia hermsii, and a rabbit tick strain. The borrelias formed a tight phylogenetic cluster, with average similarity of 97%. THe borrelia group shared a common branch with the Spirochaeta group and was closer to this group than to the treponemes. A single spirochete strain isolated fromt the shew constituted the fourth group. The fifth group was composed of strains of Serpula (Treponema) hyodysenteriae and Serpula (Treponema) innocens. The two species of this group were closely related, with a similarity of greater than 99%. Leptonema illini, Leptospira biflexa, and Leptospira interrogans formed the sixth and most deeply branching group. The average similarity within this group was 83.2%. This study represents the first demonstration that pathogenic and saprophytic Leptospira species are phylogenetically related. The division of the spirochetes into six major phylogenetic clusters was defined also by sequence signature elements. These signature analyses supported the conclusion that the spirochetes represent a monophylectic bacterial phylum.

Animals↗
PubMed1990

Heterologous expression of the Bacteroides ruminicola xylanase gene in Bacteroides fragilis and Bacteroides uniformis.

A cloned xylanase gene from the ruminal bacterium Bacteroides ruminicola 23 was transferred by conjugation into the colonic species Bacteroides fragilis and Bacteroides uniformis by using the Escherichia coli-Bacteroides shuttle vector pVAL-1. The cloned gene was expressed in both species, and xylanase specific activity in crude extracts was found to be at least 1400-fold greater than that found in the B. ruminicola strain. Analysis of crude extract proteins from the recombinant B. fragilis by SDS-PAGE demonstrated a new 60,000 molecular weight protein. The xylanase activity expressed in both E. coli and B. fragilis was capable of degrading xylan to xylooligosaccharides in vitro. This is the first demonstration that colonic Bacteroides species can express a gene from a ruminal Bacteroides species.

Bacteroides↗
PubMed1990

The genes for three xylan-degrading activities from Bacteroides ovatus are clustered in a 3.8-kilobase region.

Genes coding for three xylan-degrading activities, xylanase, xylosidase, and arabinosidase, were simultaneously cloned from the colonic anaerobic organism Bacteriodes ovatus. The genes for the three enzymes were located on a 3.8-kilobase EcoRI genomic insert and were cloned by using plasmid pUC18. All three activities were expressed in Escherichia coli JM83, and all were cell associated. Expression of the xylanase gene was independent from expression of the xylosidase and arabinosidase genes, whereas expression of the latter two genes appeared to be coordinated. Restriction endonuclease analysis of the arabinosidase and xylosidase genes and partial purification of these enzyme activities from E. coli suggested that these activities were catalyzed by a bifunctional protein or two proteins of very similar molecular weight. All three enzyme activities were regulated in B. ovatus in response to the carbon source used for growth. This is the first report of the cloning and expression of B. ovatus genes.

Bacteroides↗
PubMed1990

Physiology and genetics of xylan degradation by gastrointestinal tract bacteria.

Hemicelluloses or xylans are major components (35%) of plant materials. For ruminant animals, about 50% of the dietary xylans are degraded, but only small amounts of xylans are degraded in the lower gut of nonruminant animals and humans. In the rumen, the major xylanolytic species are Butyrivibrio fibrisolvens and Bacteroides ruminicola. In the human colon, Bacteroides ovatus and Bacteroides fragilis subspecies "a" are major xylanolytic bacteria. Xylans are chemically complex, and their degradation requires multiple enzymes. Expression of these enzymes by gut bacteria varies greatly among species. Butyrivibrio fibrisolvens makes extracellular xylanases but Bacteroides species have cell-bound xylanase activity. Biochemical characterization of xylanolytic enzymes from gut bacteria has not been done. A xylosidase gene has been cloned from B. fibrosolvens 113. The data from DNA hybridizations using a xylanase gene cloned from B. fibrisolvens 49 indicate this gene may be present in other B. fibrisolvens strains. A cloned xylanase from Bact. ruminicola was transferred to and highly expressed in Bact. fragilis and Bact. uniformis. Arabinosidase and xylosidase genes from Bact. ovatus have been cloned and both activities appear to be catalyzed by a single, bifunctional, novel enzyme. Continued research in genetic and biochemical areas will provide knowledge and insights for manipulation of digestion at the gut level and improved understanding of colonic fiber digestion.

Animals↗
PubMed1989

Identification of the Butyrivibrio fibrisolvens xylosidase gene (xylB) coding region and its expression in Escherichia coli.

The gene encoding the principal Butyrivibrio fibrisolvens xylosidase (xylB) has been cloned and expressed in Escherichia coli under the control of the lac promoter. The coding region for this gene was localized within a 3.2-kilobase B. fibrisolvens DNA fragment in pUC18. A new protein band was observed in recombinant E. coli containing xylB. This protein (approximately 60,000 molecular weight) was presumed to be the xylosidase monomer. The optimal pH (5.5) and substrate range for the recombinant and native xylosidases appeared identical. Both enzymes hydrolyzed xylo-oligosaccharides with chain lengths of 2 to 5 and both were inactive on xylan.

Bacteria, Anaerobic↗
PubMed1989

Cloning and expression in Escherichia coli of a xylanase gene from Bacteroides ruminicola 23.

A gene coding for xylanase activity in the ruminal bacterial strain 23, the type strain of Bacteroides ruminicola, was cloned into Escherichia coli JM83 by using plasmid pUC18. AB. ruminicola 23 genomic library was prepared in E. coli by using BamHI-digested DNA, and transformants were screened for xylanase activity on the basis of clearing areas around colonies grown on Remazol brilliant blue R-xylan plates. Six clones were identified as being xylanase positive, and all six contained the same 5.7-kilobase genomic insert. The gene was reduced to a 2.7-kilobase DNA fragment. Xylanase activity produced by the E. coli clone was found to be greater than that produced by the original B. ruminicola strain. Southern hybridization analysis of genomic DNA from the related B. ruminicola strains, D31d and H15a, by using the strain 23 xylanase gene demonstrated one hybridizing band in each DNA.

Animals↗
PubMed1989

Improved assay for quantitating adherence of ruminal bacteria to cellulose.

A quantitative technique suitable for the determination of adherence of ruminal bacteria to cellulose was developed. This technique employs adherence of cells to cellulose disks and alleviates the problem of nonspecific cell entrapment within cellulose particles. By using this technique, it was demonstrated that the adherence of Ruminococcus flavefaciens FD1 to cellulose was inhibited by formaldehyde, methylcellulose, and carboxymethyl cellulose. Adherence was unaffected by acid hydrolysates of methylcellulose, glucose, and cellobiose.

Animals↗
PubMed1988

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.

Animals↗
PubMed1988

Microbial digestion of hemicelluloses in the rumen.

The ruminant animal is dependent upon anaerobic microorganisms for the digestion of plant materials in the rumen. The resultant microbial cells and fermentation acids are primary nutrients for the animal. Polysaccharides, such as hemicelluloses, are only partially degraded because of the chemical complexities of plant materials and biochemical processes involved in degradation.

Animals↗
PubMed1987

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.

Animals↗
PubMed1986

Proteolytic activity of the ruminal bacterium Butyrivibrio fibrisolvens.

The proteolytic activity of Butyrivibrio fibrisolvens, a ubiquitously distributed bacterial species in the gastrointestinal tracts of ruminants and other mammals, was characterized. The relative proteolytic activity (micrograms of azocasein degraded per hour per milligram of protein) varied greatly with the strain: 0 to 1 for strains D1, D16f, E21C, and X6C61; 7 to 15 for strains IL631, NOR37, S2, LM8/1B, and X10C34; and 90 to 590 for strains 12, 49 H17C, CF4c, CF3, CF1B, and R28. The activity levels of the last group of strains were equal to or greater than those found with Bacteroides amylophilus or Bacteroides ruminicola. With the exception of strain R28 activity, 90% or more of the proteolytic activity was associated with the culture fluid and not the cells. Strain 49 produced proteolytic activity constitutively, but the level of activity (units per milligram of protein) was modulated by growth parameters. With various carbohydrates added to the growth medium, the proteolytic activities of strain 49 were positively correlated with the growth rate. However, when the growth rate varied with the use of different nitrogen sources, a similar correlation was not found. The highest activity level was observed with Casamino Acids (1 g/liter), but this level was reduced by ca. 70% with Trypticase (BBL Microbiology Systems, Cockeysville, Md.) or casein (1 g/liter) and by 85% with ammonium chloride (10 mM) as the sole nitrogen source. The addition of ammonium chloride (1 to 10 mM) to media with low levels of Casamino Acids or Trypticase resulted in lower proteolytic activities but not as low as seen when the complex nitrogen sources were increased to high levels (20 g/liter).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗