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Journal of Dairy Science Aug 1996Ruminant animals depend on cellulolytic ruminal bacteria to digest cellulose, but these bacteria cannot resist the low ruminal pH that modern feeding practices can... (Review)
Review
Ruminant animals depend on cellulolytic ruminal bacteria to digest cellulose, but these bacteria cannot resist the low ruminal pH that modern feeding practices can create. Because the cellulolytic bacteria cannot grow on cellobiose at low pH, pH sensitivity is a general aspect of growth and not just a limitation of the cellulases per se. Acid-resistant ruminal bacteria have evolved the capacity to let their intracellular pH decrease, maintain a small pH gradient across the cell membrane, and prevent an intracellular accumulation of VFA anions. Cellulolytic bacteria cannot grow with a low intracellular pH, and an increase in pH gradient leads to anion toxicity. Prevotella ruminicola cannot digest native cellulose, but it grows at low pH and degrades the cellulose derivative, carboxymethylcellulose. The Prevotella ruminicola carboxymethylcellulase cannot bind to cellulose, but a recombinant enzyme having the Prevotella ruminicola catalytic domain and a binding domain from Thermomonspora fusca was able to bind and had cellulase activity that was at least 10-fold higher. Based on these results, gene reconstruction offers a means of converting Prevotella ruminicola into a ruminal bacterium that can digest cellulose at low pH.
Topics: Animals; Bacteria; Cellulose; Dextrins; Fermentation; Genetic Engineering; Hydrogen-Ion Concentration; Rumen
PubMed: 8880476
DOI: 10.3168/jds.S0022-0302(96)76510-4 -
Reproduction, Nutrition, Development 1998The molecular weight, amino acid composition and hydrophobicity of the peptide residue produced by hydrolysis of protein by two strains of Prevotella ruminicola (23 and...
In vitro study of molecular weight, hydrophobicity and amino acid composition of peptides during breakdown of a casein hydrolysate by two strains of Prevotella ruminicola.
The molecular weight, amino acid composition and hydrophobicity of the peptide residue produced by hydrolysis of protein by two strains of Prevotella ruminicola (23 and S17/3) were determined. These last two characteristics could play a role in the control of proteolysis. Both strains produced dipeptidyl peptidases (DAP-1) but only P. ruminicola 23 synthesised alanine aminopeptidase. The area of 3-5 kDa peptides decreased, while the peptides directly assimilable by bacteria (0.5-1 kDa and < 0.5 kDa) increased with strain S17/3, but decreased with P. ruminicola 23. The amino acid compositions showed that the proportions of these compounds changed little with time and there was proline enrichment. Similarly, reverse phase HPLC showed no evidence of enrichment of the culture medium by hydrophobic peptides during the growth phase of P. ruminicola. These experiments show that the changes in the various peptide classes resulting from the hydrolysis and uptake of peptides by P. ruminicola differed with time and depended on the strain used. The nature of the enzyme activity and the use of other nitrogen sources may explain the difference between the two strains.
Topics: Amino Acids; Ammonia; CD13 Antigens; Caseins; Chemical Phenomena; Chemistry, Physical; Hydrolysis; Molecular Weight; Organoplatinum Compounds; Peptides; Prevotella
PubMed: 9923009
DOI: 10.1051/rnd:19980509 -
Animal Nutrition (Zhongguo Xu Mu Shou... Mar 2020Rumen fermentation parameters and microbiota were evaluated in 3 in vitro rumen fermentation experiments after addition of chestnut tannins (CWT) or an extract from...
Rumen fermentation parameters and microbiota were evaluated in 3 in vitro rumen fermentation experiments after addition of chestnut tannins (CWT) or an extract from Bertoni (SB) to substrates. A control (CTR) substrate was fermented alone or added with 1.5% of CWT or SB extracts in a batch culture system (Exp. 1, fermentation in 500 mL for 24 h) and in a subsequent continuous culture system (Exp. 2, fermentation in 2 L bottles for 9 d). Experiment 3 used the fermentation system of Exp. 1 and tested 7 doses of each extract added to CTR (additions of 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.2% and 1.4% for 48 h). The addition of CWT lowered ( < 0.01) the in vitro rumen ammonia concentration in all experiments and reduced the protozoa counts in Exp. 1 ( < 0.05). In contrast, the SB extract did not modify the ammonia concentrations, but significantly lowered the protozoa counts in all 3 experiments (reduction of 47% and 20% in Exp. 1 and 2, < 0.05; and a quadratic reduction in Exp. 3, = 0.63, < 0.01). Neither extract affected the fermentation in terms of gas production (Exp. 1 and 3) nor volatile fatty acids (VFA) yield (Exp. 1 and 2), if we exclude a reduction at the highest CWT concentration in Exp. 3. Changes in VFA profile were induced by CWT and were limited to reductions in the iso-valerate ( < 0.01, in Exp. 2) and iso-butyrate levels ( < 0.01, Exp. 2). The CWT increased the abundance of and and decreased that of ( < 0.01, < 0.05 and < 0.05, respectively). The SB extract increased the relative abundance of ( < 0.05). Both of the studied substances had an impact on rumen metabolism, with SB reducing protozoa counts and CWT lowering the rumen ammonia concentration. The effects of both extracts on the rumen were appreciable at low dietary doses, and the negative impacts on fermentation were limited to the reduction in protein degradation with the addition of CWT.
PubMed: 32211529
DOI: 10.1016/j.aninu.2019.11.009 -
Journal of Bacteriology Oct 1962White, D. C. (Rockefeller Institute, New York, N.Y.), M. P. Bryant, and D. R. Caldwell. Cytochrome-linked fermentation in Bacteroides ruminicola. J. Bacteriol....
White, D. C. (Rockefeller Institute, New York, N.Y.), M. P. Bryant, and D. R. Caldwell. Cytochrome-linked fermentation in Bacteroides ruminicola. J. Bacteriol. 84:822-828. 1962-Previous studies showed that Bacteroides ruminicola, an anaerobic, saccharolytic, ruminal bacterium, ferments glucose with the production of succinic, acetic, and formic acids, requires a large amount of CO(2), and most strains require heme for growth. Difference spectra of cell suspensions of both heme-requiring strain 23, B. ruminicola subsp. ruminicola, and heme-independent strain GA33, B. ruminicola subsp. brevis, showed the presence of a cytochrome (absorption maxima at 560 mmu, near 530 mmu, and 428 mmu) similar to cytochrome b. This cytochrome and flavoprotein (trough at 450 mmu) in the cells, reduced by endogenous metabolism, were oxidized on addition of air, CO(2), oxalacetate, malate, or fumarate but no oxidation occurred in the presence of succinate, malonate, lactate, pyruvate, aspartate, citrate, NO(3) (-), SO(4) (=), 2-n-heptyl or hydroxyquinoline-N-oxide (HOQNO), amytal or azide. The oxidation of these cellular pigments by fumarate was not inhibited by CN(-), CO, malonate, succinate, amytal, or HOQNO. Glucose and reduced diphosphopyridine nucleotide (DPNH), but not succinate, reduced the pigments in frozen-thawed cells previously exposed to air for 4 hr at room temperature. The results suggest that this cytochrome and flavoprotein form an electron transport system for fumarate reduction to succinate by DPNH generated by glycolysis, and that succinate is produced via CO(2) condensation with pyruvate or phosphoenolpyruvate and with oxalacetate, malate, and fumarate as intermediates. A pigment similar to cytochrome o (absorption maxima at 570, 555, and 416 mmu) was observed when reduced cells were treated with CO and compared to reduced cells, but there was no detectable cytochrome oxidase activity. The function of this pigment is obscure. No peroxidase or catalase activity was detected in either strain. Pyridine hemochromogens of both strains indicate one major heme, a protoheme-like pigment, with absorption in the alpha region maximum at 556 mmu. As B. ruminicola is one of the most numerous of rumen bacteria and ferments a wide variety of carbohydrates of importance in ruminant rations, cytochrome must be of importance in electron transport in rumen contents, a highly anaerobic environment.
Topics: Animals; Bacteroides; Cytochromes; Electron Transport; Energy Metabolism; Fermentation; Fumarates; Heme; Lactates; Malates; Oxidation-Reduction; Oxidoreductases; Prevotella ruminicola; Pyruvates; Succinates; Succinic Acid
PubMed: 14000291
DOI: 10.1128/jb.84.4.822-828.1962 -
Journal of Dairy Science Aug 1996Studies using 15N have indicated that as much as 50% of the microbial mass turns over before N passes to the lower gut, and this N recycling significantly decreases the... (Review)
Review
Studies using 15N have indicated that as much as 50% of the microbial mass turns over before N passes to the lower gut, and this N recycling significantly decreases the availability of microbial protein. Protozoa digest bacteria and smaller protozoa, but bacterial protein can turn over even if protozoa are not present. Fibrobacter succinogenes cultures lyse even when they are growing, and the lysis rate is independent of growth rate. When extracellular sugar is depleted, F. succinogenes secretes an extracellular proteinase that inactivates the autolysins. This method of autolytic regulation decreases the turnover of stationary cells. Bacteriophage and anaeroplasma can cause lysogeny, but, as yet, there is little proof that these processes are important determinants of bacterial turnover in vivo. Dietary manipulations (e.g., salt feeding and particle size reduction) that increase liquid and solid dilution rates can increase bacterial flow by decreasing bacterial residence time and turnover. Some dead ruminal bacteria are able to maintain their cellular integrity, and the ratio of dead to live cells in ruminal fluid may be as great as 10:1. Bacterial survival appears to be at least partially explained by the method of sugar transport. When bacteria rely solely on mechanisms of ion-coupled sugar symport, an energized membrane is necessary for the reinitiation of growth. If group translocation (phosphotransferase system) is the mechanisms of transport, uptake can be driven by phosphoenolpyruvate, and an energized membrane and the storage of intracellular reserve materials are not an absolute criteria for survival. In some cases, N deprivation accelerates death. When Prevotella ruminicola was limited for N under conditions of excess energy, methylglyoxal production caused a rapid decrease in viability. The impact of bacterial death in the rumen is not clear-cut. If the rate of fermentation is zero-order with respect to cell concentration (substrate-limited), cell death would have little impact on digestion.
Topics: Animals; Bacterial Physiological Phenomena; Bacteriolysis; Colony Count, Microbial; Eukaryota; Nitrogen Isotopes; Rumen
PubMed: 8880474
DOI: 10.3168/jds.S0022-0302(96)76508-6 -
Applied and Environmental Microbiology Aug 1995Two genes concerned with xylan degradation were found to be closely linked in the ruminal anaerobe Prevotella ruminicola B(1)4, being separated by an intergenic region...
A xylan hydrolase gene cluster in Prevotella ruminicola B(1)4: sequence relationships, synergistic interactions, and oxygen sensitivity of a novel enzyme with exoxylanase and beta-(1,4)-xylosidase activities.
Two genes concerned with xylan degradation were found to be closely linked in the ruminal anaerobe Prevotella ruminicola B(1)4, being separated by an intergenic region of 75 nucleotides. xynA is shown to encode a family F endoxylanase of 369 amino acids, including a putative amino-terminal signal peptide. xynB encodes an enzyme of 319 amino acids, with no obvious signal peptide, that shows 68% amino acid identity with the xsa product of Bacteroides ovatus and 31% amino acid identity with a beta-xylosidase from Clostridium stercorarium; together, these three enzymes define a new family of beta-(1,4)-glycosidases. The activity of the cloned P. ruminicola xynB gene product, but not that of the xynA gene product, shows considerable sensitivity to oxygen. Studied under anaerobic conditions, the XynB enzyme was found to act as an exoxylanase, releasing xylose from substrates including xylobiose, xylopentaose, and birch wood xylan, but was relatively inactive against oat spelt xylan. A high degree of synergy (up to 10-fold stimulation) was found with respect to the release of reducing sugars from oat spelt xylan when XynB was combined with the XynA endoxylanase from P. ruminicola B(1)4 or with endoxylanases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17. Pretreatment with a fungal arabinofuranosidase also stimulated reducing-sugar release from xylans by XynB. In P. ruminicola the XynA and XynB enzymes may act sequentially in the breakdown of xylan.
Topics: Amino Acid Sequence; Animals; Base Sequence; DNA, Bacterial; Endo-1,4-beta Xylanases; Genes, Bacterial; Genetic Linkage; Molecular Sequence Data; Multigene Family; Prevotella; Rumen; Sequence Homology, Amino Acid; Xylans; Xylosidases; beta-Glucosidase
PubMed: 7487028
DOI: 10.1128/aem.61.8.2958-2964.1995 -
FEMS Microbiology Letters Apr 201016S rRNA gene-based analysis of rumen Prevotella was carried out to estimate the diversity and diet specificity of bacteria belonging to this genus. Total DNA was...
16S rRNA gene-based analysis of rumen Prevotella was carried out to estimate the diversity and diet specificity of bacteria belonging to this genus. Total DNA was extracted from the rumen digesta of three sheep fed two diets with different hay-to-concentrate ratios (10 : 1 and 1 : 2). Real-time PCR quantification of Prevotella revealed that the relative abundance of this genus in the total rumen bacteria was up to 19.7%, while the representative species Prevotella bryantii and Prevotella ruminicola accounted for only 0.6% and 3.8%, respectively. Denaturing gradient gel electrophoresis analysis for Prevotella revealed shifts in the community composition with the diet. Analysis of 16S rRNA gene clone libraries showed significant differences (P=0.001) between clones detected from the sheep on the diets with different hay-to-concentrate ratios. The majority (87.8%) of Prevotella clones had <97% sequence similarity with known rumen Prevotella. These data suggest that uncultured Prevotella is more abundant than known Prevotella and that members of this genus appear to have specific metabolic niches.
Topics: Animals; Cluster Analysis; DNA, Bacterial; DNA, Ribosomal; Diet; Electrophoresis, Polyacrylamide Gel; Genetic Variation; Metagenome; Molecular Sequence Data; Nucleic Acid Denaturation; Phylogeny; Prevotella; RNA, Ribosomal, 16S; Rumen; Sequence Analysis, DNA; Sheep
PubMed: 20158525
DOI: 10.1111/j.1574-6968.2010.01911.x -
International Journal of Microbiology 2017A xylanase gene with a 1,116-bp open reading frame, encoding an endo--1,4-xylanase, was cloned from a chicken cecum metagenome. The translated XynA protein consisted of...
A xylanase gene with a 1,116-bp open reading frame, encoding an endo--1,4-xylanase, was cloned from a chicken cecum metagenome. The translated XynA protein consisted of 372 amino acids including a putative signal peptide of 23 amino acids. The calculated molecular mass of the mature XynA was 40,013 Da, with a theoretical pI value of 5.76. The amino acid sequence of XynA showed 59% identity to endo--1,4-xylanase from and and 58% identity to that from . XynA has two conserved motifs, DVVNE and TEXD, containing two active site glutamates and an invariant asparagine, characteristic of GH10 family xylanase. The gene without signal peptide sequence was cloned and fused with thioredoxin protein (Trx.Tag) in pET-32a plasmid and overexpressed in Tuner™(DE3)pLysS. The purified mature XynA was highly salt-tolerant and stable and displayed higher than 96% of its catalytic activity in the reaction containing 1 to 4 M NaCl. It was only slightly affected by common organic solvents added in aqueous solution to up to 5 M. This chicken cecum metagenome-derived xylanase has potential applications in animal feed additives and industrial enzymatic processes requiring exposure to high concentrations of salt and organic solvents.
PubMed: 28751915
DOI: 10.1155/2017/4018398 -
Frontiers in Cellular and Infection... 2020Gut dysbiosis has been associated with several disease outcomes including diabetes in human populations. Currently, there are no studies of the gut microbiome...
Gut dysbiosis has been associated with several disease outcomes including diabetes in human populations. Currently, there are no studies of the gut microbiome composition in relation to type 2 diabetes (T2D) in Africans. Here, we describe the profile of the gut microbiome in non-diabetic adults (controls) and investigate the association between gut microbiota and T2D in urban West Africans. Gut microbiota composition was determined in 291 Nigerians (98 cases, 193 controls) using fecal 16S V4 rRNA gene sequencing done on the Illumina MiSeq platform. Data analysis of operational taxonomic units (OTU) was conducted to describe microbiome composition and identify differences between T2D and controls. The most abundant phyla were , and . , and were significantly lower in cases than controls ( < 0.001). Feature selection analysis identified a panel of 18 OTUs enriched in cases that included . A panel of 17 OTUs that was enriched in the controls included , and . OTUs with strain-level annotation showing the largest fold-change included (logFC = -3.1; = 4.2 × 10), (logFC = -2.5; = 0.005), (logFC = -1.76; = 0.01), all lower in cases. These findings are notable because supplementation with and has been shown to improve hyperglycemia and reduce insulin resistance in murine models. This first investigation of gut microbiome and diabetes in urban Africans shows that T2D is associated with compositional changes in gut microbiota highlighting the possibility of developing strategies to improve glucose control by modifying bacterial composition in the gut.
Topics: Actinobacteria; Bacteroidetes; Black People; Case-Control Studies; Diabetes Mellitus, Type 2; Dysbiosis; Female; Firmicutes; Gastrointestinal Microbiome; Humans; Hypoglycemic Agents; Male; Metformin; Middle Aged; Nigeria; Urban Health
PubMed: 32158702
DOI: 10.3389/fcimb.2020.00063 -
Applied and Environmental Microbiology Oct 1996Prevotella ruminicola B(1)4 possesses both NADPH- and NADH-linked glutamate dehydrogenase (GDH) activities, with the greatest specific activity being measured from...
The NAD(P)H-dependent glutamate dehydrogenase activities of Prevotella ruminicola B(1)4 can be attributed to one enzyme (GdhA), and gdhA expression is regulated in response to the nitrogen source available for growth.
Prevotella ruminicola B(1)4 possesses both NADPH- and NADH-linked glutamate dehydrogenase (GDH) activities, with the greatest specific activity being measured from ammonia-limited cultures. Relative to cells grown in the presence of 1 mM ammonium chloride, the NADPH-dependent activity was decreased approximately 10-fold when peptides were provided as a nitrogen source. Nondenaturing polyacrylamide gel electrophoresis (PAGE) was used to visualize the GDH protein(s) in cell extracts of P. ruminicola. For all growth conditions tested, only one GDH protein was detectable, and its relative abundance, as well as its reactivity with either NAD(P)+ or NAD(P)H, correlated well with the specific activities measured from whole-cell assays. Consistent with the findings from enzyme assays and PAGE activity gels, Northern (RNA) blot analysis revealed that expression of a gene encoding NAD(P)H-GDH activity was greatest in ammonia-grown cultures and that GDH activity is regulated in response to nitrogen source (ammonia versus peptides), probably at the level of transcription. A gene encoding the NAD(P)H-utilizing GDH activity (gdhA) was cloned, and its nucleotide sequence was determined and shown to contain an open reading frame of 1,332 bp which would encode a polypeptide of 48.8 kDa. The deduced amino acid sequence possesses three highly conserved motifs typical of family I GDHs, but several unique amino acid substitutions within these motifs were evident. These results are discussed within the context of ruminal nitrogen metabolism and the growth efficiency of succinate- and propionate-producing anaerobic bacteria.
Topics: Amino Acid Sequence; Ammonium Chloride; Base Sequence; Cloning, Molecular; DNA, Bacterial; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Genetic Complementation Test; Glutamate Dehydrogenase; Glutamate Dehydrogenase (NADP+); Molecular Sequence Data; Molecular Weight; Nitrogen; Potassium Chloride; Prevotella; RNA, Bacterial; RNA, Messenger; Recombinant Fusion Proteins; Restriction Mapping; Sequence Analysis, DNA; Sequence Homology, Nucleic Acid
PubMed: 8837439
DOI: 10.1128/aem.62.10.3826-3833.1996