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FEMS Microbiology Ecology Jul 2000Molecular biology approaches were employed to examine the genetic diversity of bacteria from the Cytophaga/Flexibacter/Bacteroides (CFB) phylum in the rumen of cattle....
Molecular biology approaches were employed to examine the genetic diversity of bacteria from the Cytophaga/Flexibacter/Bacteroides (CFB) phylum in the rumen of cattle. By this means we were able to identify cultured strains that represent some of the larger CFB clusters previously identified only by PCR amplification and sequencing. Complete 16S rDNA sequences were obtained for 16 previously isolated rumen strains, including the type strains of Prevotella ruminicola, P. bryantii, P. brevis and P. albensis to represent a wide range of diversity. Phylogenetic analysis of cultured strains revealed the existence of three clusters of ruminal CFB: (i) a cluster of Prevotella strains, which have been found only in the rumen, including the two type strains, P. brevis GA33(T) and P. ruminicola 23(T); (ii) Prevotella spp. that cluster with prevotellas from other ecological niches such as the oral cavity and which include the type strains, P. bryantii B(1)4(T) and P. albensis M384(T); (iii) two Bacteroides spp. strains clustering with B. forsythus of oral origin. In order to establish whether the cultivated isolates cover the whole range of ruminal CFB genetic diversity, 16S rRNA gene sequences were amplified and cloned from DNA extracted from the same rumen samples (one cow in Slovenia, one in Scotland and three in Japan). Sequencing and phylogenetic analysis of 16S rRNA genes confirmed the existence of two superclusters of ruminal Prevotella, one exclusively ruminal and the other including non-ruminal species. In the case of ruminal Bacteroides spp., however, phylogenetic analysis revealed the existence of three new superclusters, one of which has as yet no cultivable counterpart. Interestingly, these Bacteroides clusters were represented almost exclusively by clone libraries from the Japanese cattle and only three sequences were from the European cattle. This study agrees with previous analyses in showing that rumen Prevotella/Bacteroides strains exhibit a remarkable degree of genetic diversity and suggests that different strain groupings may differ greatly in their recovery by cultural methods. The most important conclusion, however, is that cultured strains can be identified that represent some of the larger clusters previously identified only by PCR amplification and sequencing.
PubMed: 10922505
DOI: 10.1111/j.1574-6941.2000.tb00728.x -
Current Microbiology Oct 1999The current research was aimed at comparing proteolytic activities among ruminal Prevotella spp. Growth rates of Prevotella sp. 2202, Prevotella ruminicola D31d, P....
The current research was aimed at comparing proteolytic activities among ruminal Prevotella spp. Growth rates of Prevotella sp. 2202, Prevotella ruminicola D31d, P. brevis GA33, P. albensis M384, and P. bryantii B(1)4 varied with N source, and no one N source produced the fastest growth in all species. Proteolytic activity was greatest with casein compared with peptides, AA, and NH(4)Cl in all species. Proteolytic activity of Prevotella sp. 2202, P. brevis GA33, and P. bryantii B(1)4 was modulated by N source. With gelatin co-polymerized SDS-PAGE, the extracellular activities of the Prevotella spp. showed wide variation in number, size, and type of proteases. Prevotella sp. 2202 and P. albensis M384 produced metalloproteases of low molecular weight (40 kDa). P. ruminicola D31d produced one cysteine protease (100-200 kDa) and two metalloproteases (90-100 kDa). P. brevis GA33 generated a diffuse clearing zone (95-160 kDa) containing serine, cysteine, and metalloproteases. P. bryantii B(1)4 produced a metalloprotease greater than 200 kDa in size. The molecular sizes provided are estimations and served only to differentiate among the bacterial species in this study. Large variations in proteolytic activities among species and the known genetic diversity of the Prevotella taxon suggested that targeting this bacterial assemblage for genetic manipulation in order to alter the bacterial impact on ruminal protein degradation would be difficult.
Topics: Animals; Culture Media; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Nitrogen; Prevotella; Protease Inhibitors; Rumen
PubMed: 10486053
DOI: 10.1007/s002849900443 -
Applied and Environmental Microbiology Aug 1997The glutamate dehydrogenase (GDH) activities for the type strains of Prevotella ruminicola (strain 23), Prevotella brevis (strain GA33), and Prevotella bryantii (strain...
The glutamate dehydrogenase (GDH) activities for the type strains of Prevotella ruminicola (strain 23), Prevotella brevis (strain GA33), and Prevotella bryantii (strain B(1)4) were assessed by a combination of enzyme assays and analysis of migration patterns of GDH proteins following nondenaturing polyacrylamide gel electrophoresis. Unlike results with most other prokaryotes, but similar to results with other members of the family Bacteroidaceae, NADPH-utilizing specific activity was greatest in all species following ammonia-limited growth. Similar also to previous findings with P. bryantii, the NAD(P)H-utilizing GDH activity of P. ruminicola can be attributed to a single protein. However, P. brevis produces an additional GDH protein(s) in response to growth with peptides. These results conclusively demonstrate that all type strains of the ruminal Prevotella sp. grouping possess GDH activity.
Topics: Ammonia; Electrophoresis, Polyacrylamide Gel; Glutamate Dehydrogenase; Nitrogen; Peptides; Prevotella
PubMed: 9251223
DOI: 10.1128/aem.63.8.3314-3317.1997 -
FEMS Microbiology Letters May 1997Growth of Prevotella ruminicola strains B(1)4 (subsp. brevis) and D31d (subsp. ruminicola), was inhibited by protamine, a polycationic, low molecular mass protein....
Growth of Prevotella ruminicola strains B(1)4 (subsp. brevis) and D31d (subsp. ruminicola), was inhibited by protamine, a polycationic, low molecular mass protein. Results showed that protamine has a bacteriocidal effect when present in concentrations exceeding 30 micrograms ml-1. Protamine exerted its toxic effects by disrupting the outer membrane, which was demonstrated by: (i) an increased sensitivity to hydrophobic antibiotics (novobiocin and monensin) and (ii) release of the periplasmic enzyme alkaline phosphatase following short-term exposure to protamine. Although the concentrations of protamine inhibitory to P. ruminicola are relatively low, the effects of such a compound are probably too broad to permit its successful use in terms of manipulating ruminal proteolysis.
Topics: Alkaline Phosphatase; Anti-Bacterial Agents; Cell Membrane; Microbial Sensitivity Tests; Monensin; Novobiocin; Prevotella; Protamines
PubMed: 9163910
DOI: 10.1111/j.1574-6968.1997.tb10353.x -
International Journal of Systematic... Apr 1997Selected phenotypic characteristics of isolates of Prevotella ruminicola (formerly Bacteroides ruminicola) were studied in order to establish whether the characteristics... (Comparative Study)
Comparative Study
Phenotypic diversity among ruminal isolates of Prevotella ruminicola: proposal of Prevotella brevis sp. nov., Prevotella bryantii sp. nov., and Prevotella albensis sp. nov. and redefinition of Prevotella ruminicola.
Selected phenotypic characteristics of isolates of Prevotella ruminicola (formerly Bacteroides ruminicola) were studied in order to establish whether the characteristics of genotypic strain groups established previously on the basis of 16S ribosomal DNA sequences differed systematically. Among strains formerly considered P. ruminicola subsp. brevis, strains related to strain GA33T (T = type strain) typically failed to produce carboxymethyl cellulase (CMCase) activity detectable by plate assays and failed to ferment xylose, while strains related to strain B14T produced abundant CMCase and fermented xylose. We propose that strains related to GA33T, which have DNA G + C contents between 45 and 51 mol%, should be assigned to a new species, Prevotella brevis, and that strains related to B14T, which have DNA G + C contents between 39 and 43 mol%, should be assigned to another new species, Prevotella bryantii. Most of the isolates formerly classified as P. ruminicola subsp. ruminicola strains produced CMCase and had DNA G + C contents between 45 and 51 mol%, and we propose that these organisms should be placed in the redefined species P. ruminicola. A small group of isolates that have lower G + C contents are assigned to another new species, Prevotella albensis. Most P. brevis and P. bryantii strains produced abundatn extracellular DNase activity. Proteinase activities (as determined by [14C]casein hydrolysis) varied widely between strains, and P. brevis strains exhibited the highest mean activity. All strains produced dipeptidyl peptidase activity, but the relative activities against different peptide substrates exhibited by P. bryantii, P. albensis, and P. brevis differed systematically. The phenotypic differences among the newly defined species suggest that they may occupy distinct niches within the rumen ecosystem.
Topics: Animals; Deoxyribonucleases; Endopeptidases; Fermentation; Genetic Variation; Genotype; Phenotype; Polysaccharide-Lyases; Prevotella; RNA, Bacterial; RNA, Ribosomal, 16S; Rumen; Species Specificity; Terminology as Topic
PubMed: 9103611
DOI: 10.1099/00207713-47-2-284 -
International Journal of Systematic... Apr 1994A high degree of genetic diversity among 29 strains of Prevotella (Bacteroides) ruminicola from the rumen was revealed by comparing restriction fragment length... (Comparative Study)
Comparative Study
A high degree of genetic diversity among 29 strains of Prevotella (Bacteroides) ruminicola from the rumen was revealed by comparing restriction fragment length polymorphisms in 16S rRNA genes, sodium dodecyl sulfate-polyacrylamide gel profiles of total-cell proteins, and G + C contents of chromosomal DNAs. In order to obtain information on phylogenetic relationships, the sequences of a 389-bp region of the 16S rRNA gene, including variable regions 4 and 5, were compared for 10 strains. These 10 strains formed a single group when their sequences were compared with 16S ribosomal DNA sequences from other species, including Bacteroides spp. from the human colon. On the other hand, the great genetic distances between many P. ruminicola strains, including P. ruminicola subsp. brevis B(1)4 and GA33 and P. ruminicola 23T (T = type strain), support the hypothesis that these organisms should be reclassified into new species. We identified signature oligonucleotides based on 16S ribosomal DNA sequences that distinguished strains related to strains 23T, B(1)4, GA33, and M384, as well as an oligonucleotide that specifically recognized all but one of the Bacteroides and Prevotella strains tested. On the basis of the priming activities of these signature oligonucleotides in PCR reactions and on other criteria, we concluded that 12 of the original 29 strains were related to strain 23T, 4 were related to strain B(1)4, and 4 were related to strain GA33. While there are clear grounds for subdividing the species P. ruminicola on the basis of genotypic differences, it is appropriate to delay formal reclassification until further work on the phenotypic differentiation of the new groups is completed.
Topics: Animals; Bacterial Proteins; Bacteroides; Base Sequence; Cloning, Molecular; DNA, Ribosomal; Molecular Sequence Data; Oligonucleotides; Phylogeny; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; RNA, Ribosomal, 16S; Rumen; Sequence Analysis, DNA; Sequence Homology, Nucleic Acid
PubMed: 7910475
DOI: 10.1099/00207713-44-2-246 -
Applied and Environmental Microbiology May 1992New strains with enhanced resistance to monensin were developed from Prevotella (Bacteroides) ruminicola subsp. ruminicola 23 and P. ruminicola subsp. brevis GA33 by...
New strains with enhanced resistance to monensin were developed from Prevotella (Bacteroides) ruminicola subsp. ruminicola 23 and P. ruminicola subsp. brevis GA33 by stepwise exposure to increasing concentrations of monensin. The resulting resistant strains (23MR2 and GA33MR) could initiate growth in concentrations of monensin which were 4 to 40 times greater than those which inhibited the parental strains. Resistant strains also showed enhanced resistance to nigericin and combinations of monensin and nigericin but retained sensitivity to lasalocid. Glucose utilization in cultures of the monensin-sensitive strains (23 and GA33) and one monensin-resistant strain (23MR2) was retarded but not completely inhibited when logarithmic cultures were challenged with monensin (10 mg/liter). Monensin challenge of cultures of the two monensin-sensitive strains (23 and GA33) was characterized by 78 and 51% decreases in protein yield (milligrams of protein per mole of glucose utilized), respectively. Protein yields in cultures of resistant strain 23MR2 were decreased by only 21% following monensin challenge. Cell yields and rates of glucose utilization by resistant strains GA33MR were not decreased by challenge with 10 mg of monensin per liter. Resistant strains produced greater relative proportions of propionate and less acetate than the corresponding sensitive strains. The relative amounts of succinate produced were greater in cultures of strains 23, GA33, and 23MR2 following monensin challenge. However, only minor changes in end product formation were associate with monensin challenge of resistant strain GA33MR. These results suggest that monensin has significant effects on both the growth characteristics and metabolic activities of these predominant, gram-negative ruminal bacteria.
Topics: Bacterial Proteins; Bacteroides; Drug Resistance, Microbial; Glucose; Ionophores; Kinetics; Microbial Sensitivity Tests; Monensin; Species Specificity
PubMed: 1622231
DOI: 10.1128/aem.58.5.1617-1623.1992 -
The Journal of Infection Nov 1983The pathogenicity of 27 clinical isolates of the Bacteroides melaninogenicus (BM) group and four clinical isolates of B. oralis and B. ruminicola subsp. brevis were...
The pathogenicity of 27 clinical isolates of the Bacteroides melaninogenicus (BM) group and four clinical isolates of B. oralis and B. ruminicola subsp. brevis were investigated by inoculating them into mice and subsequently determining their ability to cause subcutaneous (SC) or intraperitoneal abscesses. Only 11 isolates of BM group and one B. ruminicola induced abscesses in mice, and all were found to be heavily encapsulated on recovery from the abscesses (more than 50 per cent of the organisms were encapsulated). When the other 23 isolates, however, were injected SC in combination with either Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus or Streptococcus pyogenes, abscesses were formed in 16 of the 23 combinations. The Bacteroides spp. recovered from the mixed infection were heavily encapsulated. Capsules also formed in Bacteroides if the organisms were injected together with capsular material or formalin killed cells of K. pneumoniae or encapsulated Bacteroides sp. Once non-encapsulated or only slightly encapsulated strains acquired a capsule, they could induce abscesses on reinoculation into mice.
Topics: Abscess; Animals; Bacteroides; Haemophilus influenzae; Klebsiella pneumoniae; Male; Mice; Prevotella melaninogenica
PubMed: 6141205
DOI: 10.1016/s0163-4453(83)97061-5 -
Acta Paediatrica Scandinavica Nov 1981Aspiration of peritonsillar abscess (quinsy) was aseptically performed in 16 children. Patients' median age was 10 years (range 6 to 17 years), and 12 were males....
Aspiration of peritonsillar abscess (quinsy) was aseptically performed in 16 children. Patients' median age was 10 years (range 6 to 17 years), and 12 were males. Unilateral abscess was present in all but one child. All aspirates were cultured for aerobes and anaerobes and yielded bacterial growth in all patients. Anaerobes were isolated in all patients; in 3 patients (19%), they were the only organism isolated, and in 13 (81%), they were mixed with aerobes. There were 91 anaerobic isolates (5.7 per specimen): 42 Bacteroides sp. (including 23 B. melaninogenicus, 5 B. oralis and 4 B. ruminicola ss. brevis); 18 anaerobic Gram-positive cocci (including 10 Peptostreptococcus sp., 4 Peptococcus sp. and 4 microaerophilic streptococci); 15 Fusobacterium sp.; and 3 Clostridium sp. There were 32 aerobic isolates (2.0 per specimen): 11 gamma-hemolytic streptococci, 8 alpha-hemolytic streptococci, 4 Group A beta-hemolytic streptococci, 4 Haemophilus sp. and 3 S. aureus. Beta-lactamase production was noted in 13 isolates recovered from 11 patients (68%). These were all isolates of S. aureus (3), 8 of 23 B. melaninogenicus (35%), and 2 of 5 B. oralis (40%). Our findings indicate the major role of anaerobic organisms in the polymicrobial etiology of peritonsillar abscesses in children, and demonstrate the presence of many beta-lactamase-producing organisms in two thirds of the patients.
Topics: Adolescent; Bacteria; Bacterial Infections; Child; Female; Humans; Male; Palatine Tonsil; Peritonsillar Abscess; Prevotella melaninogenica; Streptococcus
PubMed: 6119870
DOI: 10.1111/j.1651-2227.1981.tb06235.x -
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