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PubMed1997

Peptidases of the rumen bacterium, Prevotella ruminicola.

Prevotella (formerly Bacteroides) ruminicola is a numerous rumen bacterium which plays a significant role in the metabolism of proteins and peptides in the rumen. Measurement of the hydrolysis of synthetic aminopeptidase substrates by sonicated extracts and whole cells of different species of rumen bacteria indicated that P. ruminicola had the greatest range and specific activity of dipeptidyl peptidases among the species tested. Streptococcus bovis hydrolysed some dipeptidyl peptidase substrates to a lesser extent, and several species broke down Ala2-p-nitroanilide, including Ruminobacter amylophilus, Ruminococcus spp. and Veillonella parvula. Dipeptidyl peptidases, which cleave dipeptides from the amino-terminus of longer peptides, were much more active than aminopeptidases removing single amino acids in P. ruminicola. Ion-exchange chromatography of sonicated extracts of P. ruminicola M384 revealed at least four distinct activities: one hydrolysed Ala2-p-nitroanilide, ValAla-p-nitroanilide, Ala4and Ala5; another was an O2-sensitive activity hydrolysing GlyArg-4-methoxynapthylamide, ArgArg-4-methoxynaphthylamide, Gly5 and ValGlySerGlu, similar to dipeptidyl peptidase type I DPP-1); a third hydrolysed GlyPro-p-nitroanilide and GlyPro-4-methoxynapthylamide and was similar to dipeptidyl peptidase type IV XDPP-4); a fourth broke down LysAla-4-methoxynaphthylamide. All of the enzymes, and particularly those active against Ala2-p-nitroanilide and GlyPro-p-nitroanilide, were inhibited by serine protease inhibitors, and all except DPP-4 were inhibited by EDTA. Both DPP-1 and the enzyme hydrolysing LysAla-4-methoxynaphthylamide were inhibited strongly by iodoacetate. DPP-4 was inhibited completely by diprotin A. Competitive inhibition experiments suggested that DPP-1 was less important than the other enzymes in the breakdown of peptide mixtures.

Journal Article↗

PubMedDate not supplied

What is currently known about the molecular mechanisms of colonisation resistance.

Anaerobes are the main bacteria which inhabit the digestive tract. They have adapted their development to many environmental factors such as host secretions, corresponding to control mechanisms which allow a relative stability of microflora in healthy humans. These control mechanisms include the control of endogenous and exogenous bacterial populations which corresponds to bacterial antagonism occurring in the digestive tract of mammals and represents one of its main functions. The main antagonism to study is colonisation resistance since this function of the gut microflora is responsible for the prevention of many intestinal diseases. However, only a few things are currently known about this function and the main questions which need attention are: (1) which bacteria are responsible for colonisation resistance? and (2) what are the molecular mechanisms involved in such a function? In order to answer these questions, we need to study simplified models based on gnotobiotic animals. Two models are currently being studied in France. One corresponds to the colonisation resistance of C. perfringens which seems to be linked to the in vivo secretions of an antimicrobial substance by a Ruminococcus species. The other corresponds to the colonisation resistance of C. difficile whose mechanisms are different and seem to be linked to an interaction between mucin and microflora.

Journal Article↗

PubMed1997

H2/CO2 metabolism in acetogenic bacteria isolated from the human colon.

The present work reports on autotrophic metabolism in four H2/CO2-utilizing acetogenic bacteria isolated from the human colon (two Clostridium species, one Streptococcus species, and Ruminococcus hydrogenotrophicus). H2/CO2-utilization by these human acetogenic strains occurred during both exponential and stationary phases of growth. Acetate was the major metabolite produced by all isolates following the stoichiometric equation of reductive acetogenesis. Furthermore, the ability of these acetogenic bacteria to incorporate 13CO2 into acetate in the presence of H2 in the gas phase demonstrated the utilization of the reductive pathway of acetate formation from a one-carbon compound. Energy conservation during the autotrophic metabolism in colonic acetogens might involve sodium- or proton-chemiosmotic mechanisms. A sodium-dependent ATP generation was only demonstrated in one Clostridium species, whereas sodium could be replaced by potassium in other strains. The minimal thresholds of hydrogen uptake were determined and varied from 1100 to 3680 ppm depending on the acetogenic strain. These values appeared higher than those measured for the colonic methanogen,Methanobrevibacter smithii.

Journal Article↗

PubMed1997

Degradation and utilization of xylans by the rumen anaerobe Prevotella bryantii (formerly P. ruminicola subsp. brevis) B(1)4.

Freshly harvested whole cells from cultures of P. bryantii B(1)4 grown with oat spelt xylan (OSX) as an energy source showed less than 25% of the enzyme activity against OSX, and less than 15% of the activity against birchwood xylan (BWX) and carboxymethylcellulose, that was detectable in sonicated cell preparations. This indicates that much of this hydrolytic activity is either periplasmic, membrane-associated or intracellular and may be concerned with the processing of transported oligosaccharides.P. bryantii B(1)4 cultures were able to utilise up to 45% and 51% of the total pentose present in OSX and BWX, respectively, after 24 h, but could utilize 84% of a water-soluble fraction of BWX. Analysis of the xylan left undegraded after incubation with P. bryantii showed that while xylose and arabinose were removed to a similar extent, uronic acids were utilized to a greater extent than xylose. Predigestion of xylans with two cloned xylanases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens gave little increase in overall pentose utilization suggesting that external P. bryantii xylanases are as effective as the cloned R. flavefaciens enzymes in releasing products that can be utilised by P. bryantii cells. The xylanase system of P. bryantiiis able to efficiently utilise not only xylo-oligosaccharides but also larger water-soluble xylan fragments.

Journal Article↗

PubMed1997

Effect of Methanobrevibacter sp MF1 inoculation on glycoside hydrolase and polysaccharide depolymerase activities, wheat straw degradation and volatile fatty acid concentrations in the rumen of gnotobiotically-reared lambs.

Four naturally born lambs were placed in sterile isolators 24 h after birth before the natural establishment of cellulolytic microorganisms and archaea methanogens. At the age of 6 weeks they were inoculated with pure cultures of the strains FD1 and 007 of Ruminococcus flavefaciens and at the age of 4 months with a pure culture of Methanobrevibacter sp. MF1. Following the establishment of MF1, the population of R. flavefaciens slightly increased in the rumen of the four lambs, there was also an increase in straw degradation, in the activity of some glycoside and polysaccharide hydrolases of the adherent microbial populations and in the concentration of acetate in ruminal contents.

Journal Article↗

PubMed2002

Development of a membrane-array method for the detection of human intestinal bacteria in fecal samples.

A membrane-array method was developed for the detection of human intestinal bacteria in fecal samples without using the expensive microarray-arrayer and laser-scanner. The 16S rDNA sequences of 20 predominant human intestinal bacterial species were used to design oligonucleotide probes. Three 40-mer oligonucleotides specific for each bacterial species (total 60 probes) were synthesized and applied to nitrocellulose membranes. Digoxigenin (DIG)-labeled 16S rDNAs were amplified by polymerase chain reaction (PCR) from human fecal samples or pure cultured bacteria using two universal primers, and were hybridized to the membrane-array. Hybridization signals were read by NBT/BCIP color development. The 20 intestinal bacterial species tested were Bacteroides thetaiotaomicron, B. vulgatus, B. fragilis, B. distasonis, Clostridium clostridiiforme, C. leptum, Fusobacterium prausnitzii, Peptostreptococcus productus, Ruminococcus obeum, R. bromii, R. callidus, R. albus, Bifidobacterium longum, B. adolescentis, B. infantis, Eubacterium biforme, E. aerofaciens, Lactobacillus acidophilus,Escherichia coli, and Enterococcus faecium. The two universal primers were able to amplify full size 16S rDNA from all of the 20 bacterial species tested. The hybridization results indicated that the membrane-array method is a reliable technique for the detection of predominant human intestinal bacteria in the fecal samples. The result was also confirmed by using specific PCR methods for these bacteria.

Adult↗

PubMed1992

Effects of glycerol on the growth, adhesion, and cellulolytic activity of rumen cellulolytic bacteria and anaerobic fungi.

The effect of glycerol on the growth, adhesion, and cellulolytic activity of two rumen cellulolytic bacterial species, Ruminococcus flavefaciens and Fibrobacter succinogenes subsp. succinogenes, and of an anaerobic fungal species, Neocallimastix frontalis, was studied. At low concentrations (0.1-1%), glycerol had no effect on the growth, adhesion, and cellulolytic activity of the two bacterial species. However, at a concentration of 5%, it greatly inhibited their growth and cellulolytic activity. Glycerol did not affect the adhesion of bacteria to cellulose. The growth and cellulolytic activity of N. frontalis were inhibited by glycerol, increasingly so at higher concentrations. At a concentration of 5%, glycerol totally inhibited the cellulolytic activity of the fungus. Thus, glycerol can be added to animal feed at low concentrations.

Animals↗

PubMed1993

Analyses of the gene and amino acid sequence of the Prevotella (Bacteroides) ruminicola 23 xylanase reveals unexpected homology with endoglucanases from other genera of bacteria.

The DNA sequence for the xylanase gene from Prevotella (Bacteroides) ruminicola 23 was determined. The xylanase gene encoded for a protein with a molecular weight of 65,740. An apparent leader sequence of 22 amino acids was observed. The promoter region for expression of the xylanase gene in Bacteroides species was identified with a promoterless chloramphenicol acetyltransferase gene. A region of high amino acid homology was found with the proposed catalytic domain of endoglucanases from several organisms, including Butyrivibrio fibrisolvens, Ruminococcus flavefaciens, and Clostridium thermocellum. The cloned xylanase was found to exhibit endoglucanase activity against carboxymethyl cellulose. Analysis of the codon usage for the xylanase gene found a bias towards G and C in the third position in 16 of 18 amino acids with degenerate codons.

Amino Acid Sequence↗

PubMedDate not supplied

Comparative in vitro activity of cefpodoxime against anaerobes other than Bacteroides fragilis.

To assess the in vitro activity of cefpodoxime against anaerobic respiratory tract and oropharyngeal pathogens 77 strains belonging to 18 gram-negative and 7 gram-positive species were studied by means of agar dilution tests. For comparison cefuroxime, amoxicillin, amoxicillin + clavulanic acid and clindamycin were also tested. Cefpodoxime was found to be active at concentrations of less than or equal to 0.125 mg/l against Prevotella oralis, Prevotella buccalis, Prevotella bivia, Porphyromonas asaccharolytica, Bacteroides corporis, Bacteroides gracilis, Fusobacterium necrophorum, Fusobacterium naviforme and Propionibacterium acnes. Prevotella oris, Prevotella buccae, Fusobacterium nucleatum, Peptostreptococcus asaccharolyticus, and Ruminococcus bromii were inhibited at concentrations of less than or equal to 1 mg/l and Prevotella denticola, Prevotella melaninogenica, Prevotella intermedia, Porphyromonas gingivalis, Bacteroides pneumosintes, and Peptostreptococcus micros at concentrations of less than or equal to 4 mg/l. Strains of Veillonella parvula were inhibited by cefpodoxime at 0.25-8 mg/l, and single strains of Peptostreptococcus anaerobius and Peptostreptococcus magnus showed MICs of 32 and 64 mg/l, respectively. The results obtained warrant the use of cefpodoxime in therapy of anaerobic and mixed aerobic-anaerobic infections of the upper and lower respiratory tract and similar infections not involving Bacteroides fragilis.

Anti-Bacterial Agents↗

PubMed1988

Synergism of rumen microbial hydrolases during degradation of plant polymers.

In isolated mixture of exocellular enzymes of rumen bacteria Ruminococcus flavefaciens, Butyrivibrio fibrisolvens and rumen fungus Neocallimastix frontalis, specific activities of cellulases, hemicellulases and glycosidases were determined. The highest specific activities were shown mostly for proteins of N. frontalis.

Animals↗

PubMed1995

Production of caproic acid by cocultures of ruminal cellulolytic bacteria and Clostridium kluyveri grown on cellulose and ethanol.

Ruminal cellulolytic bacteria (Fibrobacter succinogenes S85 or Ruminococcus flavefaciens FD-1) were combined with the non-ruminal bacterium Clostridium kluyveri and grown together on cellulose and ethanol. Succinate and acetate produced by the cellulolytic organisms were converted to butyrate and caproate only when the culture medium was supplemented with ethanol. Ethanol (244 mM) and butyrate (30 mM at pH 6.8) did not inhibit cellulose digestion or product formation by S85 or FD-1; however caproate (30 mM at pH 6.8) was moderately inhibitory to FD-1. Succinate consumption and caproate production were sensitive to culture pH, with more caproic acid being produced when the culture was controlled at a pH near neutrality. In a representative experiment under conditions of controlled pH (at 6.8) 6.0 g cellulose l-1 and 4.4 g ethanol l-1 were converted to 2.6 g butyrate l-1 and 4.6 g caproate l-1. The results suggest that bacteria that efficiently produce low levels of ethanol and acetate or succinate from cellulose should be useful in cocultures for the production of caproic acid, a potentially useful industrial chemical and bio-fuel precursor.

Animals↗

PubMed2001

Regulation of H(+)-ATPase synthesis in response to reduced pH in ruminal bacteria.

The capacity of ruminal bacteria to regulate H(+)-ATPase synthesis in response to reduced pH was investigated to explain acid tolerance. The activity of H(+)-ATPase in Streptococcus bovis, an acid-tolerant bacterium, was 2.2-fold higher at pH 4.5 than at pH 5.5. The increase in the amount of H(+)-ATPase protein was similar, suggesting that the increase in H(+)-ATPase activity is owing to the increase in H(+)-ATPase synthesis. The level of atp-mRNA at pH 4.5 was 2.5-fold higher than at pH 5.5, indicating that H(+)-ATPase synthesis is regulated at the transcriptional level, responding to low pH. In Ruminococcus albus, an acid-sensitive bacterium, H(+)-ATPase activity, the amount of H(+)-ATPase protein, and the level of atp-mRNA at pH 7.0 were similar to the values at pH 6.0, the lowest pH permitting growth. This result suggests that R. albus is incapable of enhancing H(+)-ATPase synthesis at low pH. Thus, acid tolerance appeared to be related to the capacity to augment the synthesis of H(+)-ATPase responding to low pH.

Animals↗

PubMed2000

The effect of a methanogen, Methanobrevibacter smithii, on the growth rate, organic acid production, and specific ATP activity of three predominant ruminal cellulolytic bacteria.

Three predominant ruminal cellulolytic organisms, Fibrobacter succinogenes S85, Ruminococcus albus 8, and R. flavefaciens FD-1, were cultured with a methanogen, Methanobrevibacter smithii. Growth rates, bacterial protein, organic acids, and methane production were measured. When grown in diculture with the methanogen, a fermentative advantage was observed with F. succinogenes S85 as seen by an increase in specific rate of ATP production and organic acid concentration. The introduction of the methanogen did not improve the growth rate, organic acid yield, or specific rate of ATP production for R. albus 8. The growth rate and amount of organic acid end products increased when R. flavefaciens FD-1 was cultured with the methanogen; however, the specific activity of ATP production did not increase.

Adenosine Triphosphate↗

PubMed1990

Detection of glycoproteins separated by nondenaturing polyacrylamide gel electrophoresis using the periodic acid-Schiff stain.

We observed that published methods for staining glycoproteins in sodium dodecyl sulfate-polyacrylamide gels did not stain glycoproteins separated by nondenaturing polyacrylamide gel electrophoresis. Therefore, the periodic acid-Schiff stain for glycoprotein was adapted for use with proteins analyzed by nondenaturing polyacrylamide gel electrophoresis. Following nondenaturing polyacrylamide gel electrophoresis proteins were denatured in situ by incubation with aqueous 2% sodium dodecyl sulfate, 5% 2-mercaptoethanol, 40% ethanol, and 5% acetic acid at 90 degrees C for 15 min followed by periodic acid-Schiff staining. This modified procedure will detect at least 0.2 micrograms protein-associated carbohydrate. Omission of the periodic acid treatment from the protocol was used as a control to detect nonspecific staining of some proteins. This modified procedure was validated using both purified glycoproteins and extracellular culture fluid containing carbohydrate-associated proteins of Ruminococcus flavefaciens.

Bacteriological Techniques↗

PubMed1991

Deantigenation of human erythrocytes by bacterial glycosidases--evidence for the noninvolvement of medium-sized glycosphingolipids in the Dolichos biflorus lectin hemagglutination.

Fresh human A1 erythrocytes, washed and pretreated in phosphate buffer with or without papain, were incubated at 37 degrees C with blood group-degrading enzymes from the human fecal Ruminococcus torques strain IX-70. The effects were assayed as changes in hemagglutination patterns, and blood group activities of alkali stable glycolipid extracts from the enzyme-treated cells using Dolichos biflorus anti-A1 lectin, Ulex europaeus type 1 anti-H lectin, and various monoclonal anti-A antibodies. Hemolysis was negligible (less than or equal to 1% after 6 h), and the osmotic fragility increased slightly only after papain treatment. The papain-untreated A1 erythrocytes lost D. biflorus agglutinability within minutes at room temperature with the unfractionated bacterial enzyme mixture IX-70 (42 mU 1,3-alpha-N-acetylgalactosaminidase (alpha-GalNAc'ase)/ml), but remained A active by strong agglutination with BioClone anti-A antibody even after 6 h of incubation. Thin layer chromatographic (TLC) immunostaining of extracted lipids showed hydrolysis of D. biflorus binding glycosphingolipids with more than six monosaccharides after 1 h, i.e., at a slower rate than the loss of D. biflorus agglutinability. Disappearance of these glycosphingolipids after 1 h paralleled the appearance of U. europaeus agglutinability and the strong binding of this lectin to glycolipid extracts in TLC immunoassays. A partly purified 1,3-alpha-GalNAc'ase (XI-117) (100 mU/ml) and a 1,2-alpha-fucosidase fraction (XI-50) containing alpha-GalNAc'ase (10 mU/ml) did not degrade blood group A active glycosphingolipids but completely abolished the D. biflorus agglutinability within 6 h. Papain pretreatment exposed U. europaeus receptors on the cell surface without changing the A1 hemagglutination pattern. It also facilitated a complete degradation of D. biflorus and U. europaeus reactive glycolipids with the IX-70 enzyme mixture within 6 h. The D. biflorus lectin was a good discriminator of A1/A2 subjects using erythrocyte lipid extracts but had a low affinity for the blood group A type 3 and type 4 glycosphingolipids in the TLC-overlay technique. In conclusion this study shows that (i) loss of D. biflorus A1 hemagglutination does not correlate with a loss of D. biflorus binding glycosphingolipids and (ii) loss of D. biflorus binding glycosphingolipids does not correlate with a loss of D. biflorus agglutinability. The results indicate that the serological D. biflorus agglutinability of A1 erythrocytes is not dependent on medium-sized glycosphingolipids (hexa- to dodecaglycosylceramides).

ABO Blood-Group System↗

PubMed1990

Metabolism of glycyrrhetic acid by rat liver microsomes: glycyrrhetinate dehydrogenase.

Glycyrrhetic acid, derived from a main component of liquorice, was converted to 3-ketoglycyrrhetic acid reversibly by rat liver homogenates in the presence of NADPH or NADP+. Glycyrrhetic acid-oxidizing and 3-ketoglycyrrhetic acid-reducing activities were localized in microsomes among the subcellular fractions of rat liver. Glycyrrhetic acid-oxidizing activity and 3-ketoglycyrrhetic acid-reducing activities showed pH optima at 6.3 and 8.5, respectively, and required NADP+ or NAD+ and NADPH or NADH, respectively, indicating that these activities were due to glycyrrhetinate dehydrogenase. The dehydrogenase was not solubilized from the membranes by the treatment with 1 M NaCl or sonication, indicating that the enzyme is a membrane component. The dehydrogenase was solubilized with detergents such as Emalgen 913, Triton X-100 and sodium cholate, and then separated from 3 beta-hydroxysteroid dehydrogenase (5 beta-androstan-3 beta-ol-17-one-oxidizing activity) by butyl-Toyopearl 650 M column chromatography. Partially purified enzyme catalyzed the reversible reaction between glycyrrhetic acid and 3-ketoglycyrrhetic acid, but was inactive toward 3-epiglycyrrhetic acid and other steroids having the 3 beta-hydroxyl group. The enzyme required NADP+ and NADPH for the highest activities of oxidation and reduction, respectively, and NAD+ and NADH for considerable activities, similar to the results with microsomes. From these results the enzyme is defined as glycyrrhetinate dehydrogenase, being quite different from 3 beta-hydroxysteroid dehydrogenase of Ruminococcus sp. from human intestine, which is active for both glycyrrhetic acid and steroids having the 3 beta-hydroxyl group.

3-Hydroxysteroid Dehydrogenases↗

PubMed1990

Hydrophobic cluster analysis: procedures to derive structural and functional information from 2-D-representation of protein sequences.

Hydrophobic cluster analysis (HCA) [15] is a very efficient method to analyse and compare protein sequences. Despite its effectiveness, this method is not widely used because it relies in part on the experience and training of the user. In this article, detailed guidelines as to the use of HCA are presented and include discussions on: the definition of the hydrophobic clusters and their relationships with secondary and tertiary structures; the length of the clusters; the amino acid classification used for HCA; the HCA plot programs; and the working strategies. Various procedures for the analysis of a single sequence are presented: structural segmentation, structural domains and secondary structure evaluation. Like most sequence analysis methods, HCA is more efficient when several homologous sequences are compared. Procedures for the detection and alignment of distantly related proteins by HCA are described through several published examples along with 2 previously unreported cases: the beta-glucosidase from Ruminococcus albus is clearly related to the beta-glucosidases from Clostridum thermocellum and Hansenula anomala although they display a reverse organization of their constitutive domains; the alignment of the sequence of human GTPase activating protein with that of the Crk oncogene is presented. Finally, the pertinence of HCA in the identification of important residues for structure/function as well as in the preparation of homology modelling is discussed.

Amino Acid Sequence↗

PubMed2003

Development of bacterial and bifidobacterial communities in feces of newborn babies.

Microbial 16S rDNA from babies' fecal samples were amplified by PCR, and analysed by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing. PCR-DGGE profiles were used to follow in time the colonization of the intestine by bacteria. Four healthy babies, one baby who received antibiotics and their parents participated to the present study to determine the extent to which administration of antibiotics can modify the bacterial colonization of neonatal human gut and verify the influence of parental factors on the formation of the fecal bacterial community. In the healthy babies, Escherichia coli or bacteria belonging to Clostridium spp. were the initial colonizers rapidly followed by Bifidobacterium, Bacteroides, Clostridium, Streptococcus, Enterococcus and Actinomyces. Bifidobacterium species appeared already after five days in the breast-fed babies while there was a delay in the baby who received a formula based diet during only one day after birth. In each baby two or three bifidobacterial species including B. infantis were found. The observed variations in species were not associated with the feeding changes. The comparison of DGGE profiles of the babies and their parents patterns showed bands with equal migration suggesting a vertical transmission determined by genetic and environmental factors. The brief appearance of pioneer bacteria determined as being E. coli and Enterococcus spp. in the profile from the baby under antibiotic therapy, was succeeded by a small stable community consisting of Ruminococcus species. No Bifidobacterium sequences were detectable in this antibiotic-treated baby in spite of a partly breast-milk diet.

Journal Article↗