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1.
Ramezani M  Resmer KL  White RL 《The FEBS journal》2011,278(14):2540-2551
The pathways of glutamate catabolism in the anaerobic bacterium Fusobacterium varium, grown on complex, undefined medium and chemically defined, minimal medium, were investigated using specifically labelled (13)C-glutamate. The metabolic end-products acetate and butyrate were isolated from culture fluids and derivatized for analysis by nuclear magnetic resonance and mass spectrometry. On complex medium, labels from L-[1-(13)C]glutamate and L-[4-(13)C]glutamate were incorporated into C1 of acetate and equally into C1/C3 of butyrate, while label derived from L-[5-(13)C]glutamate was not incorporated. The isotopic incorporation results and the detection of glutamate mutase and 3-methylaspartate ammonia lyase in cell extracts are most consistent with the methylaspartate pathway, the best known route of glutamate catabolism in Clostridium species. When F. varium was grown on defined medium, label from L-[4-(13)C]glutamate was incorporated mainly into C4 of butyrate, demonstrating a major role for the hydroxyglutarate pathway. Upon addition of coenzyme B(12) or cobalt ion to the defined medium in replicate experiments, isotope was located equally at C1/C3 of butyrate in accord with the methylaspartate pathway. Racemization of D-glutamate and subsequent degradation of L-glutamate via the methylaspartate pathway are supported by incorporation of label into C2 of acetate and equally into C2/C4 of butyrate from D-[3-(13)C]glutamate and the detection of a cofactor-independent glutamate racemase in cell extracts. Together the results demonstrate a major role for the methylaspartate pathway of glutamate catabolism in F. varium and substantial participation of the hydroxyglutarate pathway when coenzyme B(12) is not available.  相似文献   

2.
Glutamate is a major source of energy for Fusobacterium species but its mode of catabolism has not hitherto been elucidated. Cell suspensions of F. nucleatum and F. varium, as representative species from the oral cavity and gastrointestinal tract, respectively, both decarboxylated position-labelled glutamate but by different pathways. 14CO2 was released only from C-5 by F. nucleatum whereas F. varium decarboxylated glutamate at either C-1 or C-5. In both species, 2 mols of glutamate fermented yielded 2 mols of acetate and 1 mol of butyrate, suggesting the possibility of three metabolic pathways: the 2-oxoglutarate, mesaconate and 4-aminobutyrate pathways. Enzymes representative of the three pathways were assayed for in cell-free extracts of fusobacteria. All species tested possessed high levels of both glutamate dehydrogenase and 2-oxoglutarate reductase, indicating the presence of the 2-oxoglutarate pathway. Enzymes representative of the mesaconate pathway were detected in F. sulci, F. ulcerans, F. mortiferum and F. varium, while the latter two species also possessed the 4-aminobutyrate pathway. The pathways of glutamate catabolism therefore bore no relationship to the site of isolation of the fusobacteria tested but instead correlated with their chemotaxonomic properties. Thus, F. varium, F. mortiferum, F. ulcerans and F. sulci, which possess a peptidoglycan structure based on diaminopimelic acid, have either two or three pathways for glutamate catabolism whereas F. nucleatum and other species that have a lanthionine-based murein metabolized glutamate solely by the 2-oxoglutarate pathway.  相似文献   

3.
A selective medium which allows detection of relatively small numbers of Fusobacterium varium in fecal specimens is described. Blood-agar containing 50 mug of rifampin per ml inhibits the growth of many species of Bacteriodes and of F. fusi-forme/nucleatum but allows good growth of F. varium and most strains of F. mortiferum. Quantitative cultures of 11 fecal specimens were done on rifampin and other selective and nonselective media. F. varium was recovered in counts of 10(6) and 10(7) per gram from two specimens on rifampin only. A third specimen yielded 10(10)F. varium on several media, including rifampin. Some Eubacterium and Clostridium species also grew on rifampin, and these ordinarily were distinguished from the Fusobacterium by colony morphology. This medium is of value in fecal flora studies and should be useful with other kinds of specimens where mixtures of organisms are common.  相似文献   

4.
The phylogenetic interrelationships of 14 members of the genus Fusobacterium were investigated by performing a comparative analysis of the 16S rRNA sequences of these organisms. The sequence data revealed considerable intrageneric heterogeneity. The four species Fusobacterium nucleatum (including F. nucleatum subsp. nucleatum, F. nucleatum subsp. polymorphum, "F. nucleatum subsp. fusiforme," and "F. nucleatum subsp. animalis"), Fusobacterium alocis, Fusobacterium periodonticum, and Fusobacterium simiae, which colonize oral cavities, exhibited high levels of sequence homology with each other and formed a distinct group within the genus. Fusobacterium mortiferum, Fusobacterium varium, and Fusobacterium ulcerans also formed a phylogenetically coherent group, as did the two species Fusobacterium gonidiaformans and Fusobacterium necrophorum. Fusobacterium russii and Fusobacterium necrogenes displayed no specific relationship with any of the other fusobacteria. The sequence data are discussed in the context of previous physiological and chemical findings.  相似文献   

5.
Metabolic footprinting of the anaerobic bacterium Fusobacterium varium demonstrated the accumulation of six carboxylic acids as metabolic end-products and revealed specific growth requirements and utilization capabilities towards amino acids. Guided by (1)H NMR determinations of residual amino acids in spent medium, a modified chemically defined minimal medium (CDMM*) was developed by minimizing the amino acid composition while satisfying nutritional requirements to support abundant growth of F. varium. Quantitative determinations of carboxylate salts and residual substrates were readily performed by (1)H NMR analysis of lyophilized residues from CDMM* cultures without interference from initial medium components. Only small concentrations of alanine, arginine, glycine, isoleucine, leucine, methionine, proline and valine were required to support growth of F. varium, whereas larger quantities of aspartate, asparagine, cysteine, glutamine, glutamate, histidine, lysine, serine and threonine were utilized, most likely as energy sources. Both bacterial growth and the distribution of carboxylate end-products depended on the composition of the chemically defined medium. In cultures provided with glucose as the primary energy source, the accumulation of butyrate and lactate correlated with growth, consistent with the regeneration of reduced coenzyme formed by the oxidative steps of glucose catabolism.  相似文献   

6.
An immunolatex reagent was developed from antiserum to Fusobacterium varium, an obligate anaerobe isolated from the colon of the cockroach, Periplaneta americana L. A new technique that enabled the preparation of a highly efficient immunolatex conjugate was used to localize the bacterium with scanning electron microscopy, in situ, in the mixed gut contents.  相似文献   

7.
The etiology of ulcerative colitis (UC) is unknown, while an exacerbating factor of this disease is associated with infectious agents. Recently, Fusobacterium varium has been found in the mucosa of a significant number of patients with UC. The aim of this study was to estimate the prevalence of F. varium infection based on serology, evaluate the relationship between F. varium seropositivity and UC, and determine the clinical characteristics of infected UC individuals. Seropositive patients were determined by immunoblotting with F. varium ATCC 8501 antigen. We also identified cross-reactive protein spots by peptide mass mapping analysis. These protein spots showed putative caseinolytic protease protein, putative translation elongation factor G, and putative enolase. Immunoblotting with F. varium antigen revealed signals with sera from 45 (40.2%) of the 112 UC patients and 20 (15.6%) of the 128 healthy controls, respectively ( P <0.01). In terms of disease activity, seropositive UC patients were more likely to have clinically severe disease than seronegative UC patients. Disease location in seropositive patients was more extensive than the seronegative patients. In conclusion, F. varium is a predominant infection in the UC population and is a potential pathogen of UC.  相似文献   

8.
W Chen  K Ohmiya    S Shimizu 《Applied microbiology》1986,52(4):612-616
Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.  相似文献   

9.
Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.  相似文献   

10.
A new selective medium (JVN) for the isolation of Fusobacterium spp. from clinical material is described. The medium incorporates josamycin, vancomycin and norfloxacin (at 3, 4 and 1 microgram/ml, respectively) as the selective agents, plus 5% defibrinated horse blood in Fastidious Anaerobe Agar Base (Lab M). This formula allowed luxuriant growth of all 82 strains (eight recognized species) of fusobacteria tested, while significantly inhibiting 51/51 (100%) strains of facultative anaerobes and 45/51 (88%) strains of other obligate anaerobes. JVN medium allowed the successful isolation of strains of Fusobacterium naviforme, F. nucleatum and F. necrophorum from the gingivae of 9/16 healthy volunteers, and strains of F. varium and F. mortiferum from faecal suspensions seeded with these organisms.  相似文献   

11.
W Chen  K Ohmiya    S Shimizu 《Applied microbiology》1987,53(3):542-548
Intergeneric protoplast fusion between Fusobacterium varium (Pcs Glu+) and Enterococcus faecium (Pcr Glu-) was performed under strictly anaerobic conditions to improve dehydrodivanillin (DDV) degradation. The fusion frequency obtained from the selective medium (Pc+ Glu-) was about 0.9 X 10(-5) to 1.3 X 10(-5). The seven fusants isolated were all gram-negative anaerobes with rod shapes like that of F. varium and with main phenotypical properties of cocci like those of E. faecium such as esculin and starch hydrolysis, milk clotting, and lactate production. Five fusants showed enhanced DDV degradation activities that were 2 to 4 times higher than those of parental strains. Genetic relatedness between a fusant (FE7) and the parents was estimated by DNA-DNA Southern blot hybridization with 32P-labeled chromosomal DNA fragments of F. varium and E. faecium as respective probes. The fusant FE7 presented a very high cross-hybridization with both probes, indicating a high DNA homology between the fusant and both parental strains. Almost all the fusants obtained here have stably kept the properties described above for about 2 years. These results suggest that intergeneric gene transfer takes place through protoplast fusion and that the fusants that were obtained are stable recombinants.  相似文献   

12.
Intergeneric protoplast fusion between Fusobacterium varium (Pcs Glu+) and Enterococcus faecium (Pcr Glu-) was performed under strictly anaerobic conditions to improve dehydrodivanillin (DDV) degradation. The fusion frequency obtained from the selective medium (Pc+ Glu-) was about 0.9 X 10(-5) to 1.3 X 10(-5). The seven fusants isolated were all gram-negative anaerobes with rod shapes like that of F. varium and with main phenotypical properties of cocci like those of E. faecium such as esculin and starch hydrolysis, milk clotting, and lactate production. Five fusants showed enhanced DDV degradation activities that were 2 to 4 times higher than those of parental strains. Genetic relatedness between a fusant (FE7) and the parents was estimated by DNA-DNA Southern blot hybridization with 32P-labeled chromosomal DNA fragments of F. varium and E. faecium as respective probes. The fusant FE7 presented a very high cross-hybridization with both probes, indicating a high DNA homology between the fusant and both parental strains. Almost all the fusants obtained here have stably kept the properties described above for about 2 years. These results suggest that intergeneric gene transfer takes place through protoplast fusion and that the fusants that were obtained are stable recombinants.  相似文献   

13.
A selective medium for Fusobacterium spp.   总被引:1,自引:1,他引:0  
J.S. BRAZIER, D.M. CITRON AND E.J.C. GOLDSTEIN, 1991. A new selective medium (JVN) for the isolation of Fusobacterium spp. from clinical material is described. The medium incorporates josamycin, vancomycin and norfloxacin (at 3, 4 and 1 μg/ml, respectively) as the selective agents, plus 5% defibrinated horse blood in Fastidious Anaerobe Agar Base (Lab M). This formula allowed luxuriant growth of all 82 strains (eight recognized species) of fusobacteria tested, while significantly inhibiting 51/51 (100%) strains of facultative anaerobes and 45/51 (88%) strains of other obligate anaerobes. JVN medium allowed the successful isolation of strains of Fusobacterium naviforme, F. nucleatum and F. necrophorum from the gingivae of 9/16 healthy volunteers, and strains of F. varium and F. mortiferum from faecal suspensions seeded with these organisms.  相似文献   

14.
Potrykus J  White RL  Bearne SL 《Proteomics》2008,8(13):2691-2703
The butyrate-producing anaerobe Fusobacterium varium is an integral constituent of human gut microflora. Unlike many gut microorganisms, F. varium is capable of fermenting both amino acids and glucose. Although F. varium has been implicated in beneficial as well as pathological bacterium-host interactions, its genome has not been sequenced. To obtain a better understanding of the metabolic processes associated with amino acid fermentation by F. varium, we used a gel-based proteomic approach to examine the changes in the soluble proteome accompanying the utilization of eight different growth substrates: glucose, L- and D-glutamate, L-histidine, L- and D-lysine, and L- and D-serine. Using LC-MS/MS to analyze approximately 25% of the detected protein spots, we were able to identify 47 distinct proteins. While the intracellular concentrations of enzymes characteristic of a catabolic pathway for a specific amino acid were selectively increased in response to the presence of an excess of that amino acid in the growth medium, the concentrations of the core acetate-butyrate pathway enzymes remained relatively constant. Our analysis revealed (i) high intracellular concentrations of glutamate mutase and beta-methylaspartate ammonia-lyase under all growth conditions, underscoring the importance of the methylaspartate pathway of glutamate catabolism in F. varium (ii) the presence of two enzymes of the hydroxyglutarate pathway of glutamate degradation in the proteome of F. varium ((R)-2-hydroxyglutaryl-CoA dehydratase and NAD-specific glutamate dehydrogenase) specifically when L-glutamate was the main energy source (iii) the presence of genes in the genome of F. varium encoding each of the enzymes of the hydroxyglutarate pathway (iv) the presence of both L- and D-serine ammonia-lyases (dehydratases) which permit F. varium to thrive on either L- or D-serine, respectively, and (v) the presence of aspartate-semialdehyde dehydrogenase and dihydrodipicolinate synthase, consistent with the ability of F. varium to synthesize meso-2,6-diaminopimelic acid as a component of its peptidoglycan. Proteins involved in other cellular processes, including oxidation-reduction reactions, protein synthesis and turnover, and transport were also identified.  相似文献   

15.
Potrykus J  Mahaney B  White RL  Bearne SL 《Proteomics》2007,7(11):1839-1853
A proteome survey and MS analysis were conducted to investigate glucose metabolism in Fusobacterium varium, a butyrate-producing constituent of the indigenous human gut microflora. The bacterium was capable of catabolizing glucose as the main energy source via the Embden-Meyerhof-Parnas pathway. 2-DE analyses revealed that the apparent concentrations of the six identified glycolytic enzymes (pyruvate kinase, enolase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase, and glyceraldehyde-3-phosphate dehydrogenase) were specifically increased in response to the presence of glucose in the chemically defined minimal growth medium, and did not diminish when the medium was additionally supplemented with L-glutamate, an amino acid readily fermented by members of the Fusobacterium genus. A substrate pool depletion study revealed that the sugar, and not the amino acid, is the more efficient growth substrate. Both proteomics and substrate pool depletion studies revealed that F. varium can simultaneously utilize both glucose and L-glutamate as energy sources. Enzymes involved in L-glutamate metabolism were also identified, including an NAD-dependent glutamate dehydrogenase and two enzymes of the methylaspartate pathway of L-glutamate catabolism (glutamate mutase and methylaspartate ammonia-lyase). Their apparent intracellular concentrations were elevated when the bacterium was cultured in media supplemented with excess L-glutamate. Our observation that the apparent concentrations of specific proteins were elevated in response to a particular growth substrate supplied as an energy source provides the first evidence for the presence of a nutrient-responsive mechanism governing intracellular protein concentration in F. varium.  相似文献   

16.
A scheme for differentiating Fusobacterium spp. and Leptotrichia spp. from Bac-teroides spp, was devised after examining 114 strains of fusobacteria and asac-charolytic bacteroides (17 reference strains and 97 clinical isolates). Sensitivity to a 300 μg/ml plate of phosphomycin and an acid reaction on a lysine plate were found to be reliable for differentiating Fusobacterium spp. and L. buccalis from Bacteroides Using a short set of simple cultural and biochemical tests, isolates could be identified as F. necrophorum, F. necrogenes, F. nucleatum, F. varium or L. buccalis . These tests were: indole, lecithinase, phosphatase, DNase and gas production, aesculin and casein hydrolysis, greening of casein/methylene blue agar, nitrite reduction, bile tolerance and haemolysis on horse blood agar.  相似文献   

17.
Identification of fusobacteria in a routine diagnostic laboratory   总被引:3,自引:0,他引:3  
A scheme for differentiating Fusobacterium spp. and Leptotrichia spp. from Bacteroides spp. was devised after examining 114 strains of fusobacteria and asaccharolytic bacteroides (17 reference strains and 97 clinical isolates). Sensitivity to a 300 micrograms/ml plate of phosphomycin and an acid reaction on a lysine plate were found to be reliable for differentiating Fusobacterium spp. and L. buccalis from Bacteroides. Using a short set of simple cultural and biochemical tests, isolates could be identified as F. necrophorum, F. necrogenes, F. nucleatum, F. varium or L. buccalis. These tests were: indole, lecithinase, phosphatase, DNase and gas production, aesculin and casein hydrolysis, greening of casein/methylene blue agar, nitrite reduction, bile tolerance and haemolysis on horse blood agar.  相似文献   

18.
Two Pathways of Glutamate Fermentation by Anaerobic Bacteria   总被引:12,自引:6,他引:6  
Two pathways are involved in the fermentation of glutamate to acetate, butyrate, carbon dioxide, and ammonia-the methylaspartate and the hydroxyglutarate pathways which are used by Clostridium tetanomorphum and Peptococcus aerogenes, respectively. Although these pathways give rise to the same products, they are easily distinguished by different labeling patterns of the butyrate when [4-(14)C]glutamate is used as substrate. Schmidt degradation of the radioactive butyrate from C. tetanomorphum yielded equally labeled propionate and carbon dioxide, whereas nearly all the radioactivity of the butyrate from P. aerogenes was recovered in the corresponding propionate. This procedure was used as a test for the pathway of glutamate fermentation by 15 strains (9 species) of anaerobic bacteria. The labeling patterns of the butyrate indicate that glutamate is fermented via the methylaspartate pathway by C. tetani, C. cochlearium, and C. saccarobutyricum, and via the hydroxyglutarate pathway by Acidaminococcus fermentans, C. microsporum, Fusobacterium nucleatum, and F. fusiformis. Enzymes specific for each pathway were assayed in crude extracts of the above organisms. 3-Methylaspartase was found only in clostridia which use the methylaspartate pathway, including Clostridium SB4 and C. sticklandii, which probably degrade glutamate to acetate and carbon dioxide by using a second amino acid as hydrogen acceptor. High levels of 2-hydroxyglutarate dehydrogenase were found exclusively in organisms that use the hydroxyglutarate pathway. The data indicate that only two pathways are involved in the fermentation of glutamate by the bacteria analyzed. The methylaspartate pathway appears to be used only by species of Clostridium, whereas the hydroxyglutarate pathway is used by representatives of several genera.  相似文献   

19.
Pathway of lysine degradation in Fusobacterium nucleatum.   总被引:5,自引:3,他引:2       下载免费PDF全文
Lysine was fermented by Fusobacterium nucleatum ATCC 25586 with the formation of about 1 mol each of acetate and butyrate. By the use of [1-14C]lysine or [6-14C]lysine, acetate and butyrate were shown to be derived from both ends of lysine, with acetate being formed preferentially from carbon atoms 1 and 2 and butyrate being formed preferentially from carbon atoms 3 to 6. This indicates that the lysine carbon chain is cleaved between both carbon atoms 2 and 3 and carbon atoms 4 and 5, with the former predominating [1-14C]acetate was also extensively incorporated into butyrate, preferentially into carbon atoms 3 and 4. Cell-free extracts of F. nucleatum were shown to catalyze the reactions of the 3-keto,5-aminohexanoate pathway of lysine degradation, previously described in lysine-fermenting clostridia. The 3-keto,5-aminohexanoate cleavage enzyme was partially purified and shown to have properties much like those of the clostridial enzyme. We conclude that both the pathway and the enzymes of lysine degradation are similar in F. nucleatum and lysine-fermenting clostridia.  相似文献   

20.
Urease activities of anaerobic bacteria that constituted predominant gut flora were examined. It was demonstrated that some strains of Eubacterium aerofaciens, E. lentum, and Peptostreptococcus products produced urease. They were the most numerous species in human feces. All strains of Bifidobacterium infantis and some strains of Bacteroides multiacidus, B. bifidum, Clostridium symbiosum, Fusobacterium necrophorum, F. varium, Lactobacillus fermentum, Peptococcus asaccharolyticus, and P. prevotii produced urease. The optimum pH of the Lactobacillus urease was found to be 4.0, whereas the pH value of B. multiacidus urease was 8.0.  相似文献   

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