RESUMO
The rumen ecosystem harbours a galaxy of microbes working in syntrophy to carry out a metabolic cascade of hydrolytic and fermentative reactions. This fermentation process allows ruminants to harvest nutrients from a wide range of feedstuff otherwise inaccessible to the host. The interconnection between the ruminant and its rumen microbiota shapes key animal phenotypes such as feed efficiency and methane emissions and suggests the potential of reducing methane emissions and enhancing feed conversion into animal products by manipulating the rumen microbiota. Whilst significant technological progress in omics techniques has increased our knowledge of the rumen microbiota and its genome (microbiome), translating omics knowledge into effective microbial manipulation strategies remains a great challenge. This challenge can be addressed by modelling approaches integrating causality principles and thus going beyond current correlation-based approaches applied to analyse rumen microbial genomic data. However, existing rumen models are not yet adapted to capitalise on microbial genomic information. This gap between the rumen microbiota available omics data and the way microbial metabolism is represented in the existing rumen models needs to be filled to enhance rumen understanding and produce better predictive models with capabilities for guiding nutritional strategies. To fill this gap, the integration of computational biology tools and mathematical modelling frameworks is needed to translate the information of the metabolic potential of the rumen microbes (inferred from their genomes) into a mathematical object. In this paper, we aim to discuss the potential use of two modelling approaches for the integration of microbial genomic information into dynamic models. The first modelling approach explores the theory of state observers to integrate microbial time series data into rumen fermentation models. The second approach is based on the genome-scale network reconstructions of rumen microbes. For a given microorganism, the network reconstruction produces a stoichiometry matrix of the metabolism. This matrix is the core of the so-called genome-scale metabolic models which can be exploited by a plethora of methods comprised within the constraint-based reconstruction and analysis approaches. We will discuss how these methods can be used to produce the next-generation models of the rumen microbiome.
Assuntos
Microbiota , Rúmen , Animais , Rúmen/metabolismo , Ruminantes/metabolismo , Metagenoma , Fermentação , Metano/metabolismoRESUMO
BACKGROUND: The large intestine is a colonization site of beneficial microbes complementing the nutrition of cattle but also of zoonotic and animal pathogens. Here, we present the first global gene catalog of cattle fecal microbiomes, a proxy of the large intestine microbiomes, from 436 metagenomes from six countries. RESULTS: Phylogenomics suggested that the reconstructed genomes and their close relatives form distinct branches and produced clustering patterns that were reminiscent of the metagenomics sample origin. Bacterial taxa had distinct metabolic profiles, and complete metabolic pathways were mainly linked to carbohydrates and amino acids metabolism. Dietary changes affected the community composition, diversity, and potential virulence. However, predicted enzymes, which were part of complete metabolic pathways, remained present, albeit encoded by different microbes. CONCLUSIONS: Our findings provide a global insight into the phylogenetic relationships and the metabolic potential of a rich yet understudied bacterial community and suggest that it provides valuable services to the host. However, we tentatively infer that members of that community are not irreplaceable, because similar to previous findings, symbionts of complex bacterial communities of mammals are expendable if there are substitutes that can perform the same task. Video Abstract.
Assuntos
Bactérias , Metagenômica , Aminoácidos , Animais , Bactérias/genética , Carboidratos , Bovinos , Intestino Grosso , Mamíferos/microbiologia , FilogeniaRESUMO
AIMS: To monitor the effect of a live yeast additive on feedstuff colonization by targeted fibrolytic micro-organisms and fibre degradation in the cow rumen. METHODS AND RESULTS: Abundance of adhering fibrolytic bacteria and fungi on feedstuffs incubated in sacco in the cow rumen was quantified by qPCR and neutral detergent fibre (NDF) degradation was measured. Saccharomyces cerevisiae I-1077 (SC) increased the abundance of fibre-associated Fibrobacter succinogenes on wheat bran (WB) and that of Ruminococcus flavefaciens on alfalfa hay (AH) and wheat silage (WS). The greatest effect was observed on the abundance of Butyrivibrio fibrisolvens on AH and soya hulls (SH) (P < 0·001). Fungal biomass increased on AH, SH, WS and WB in the presence of SC. NDF degradation of AH and SH was improved (P < 0·05) with SC supplementation. CONCLUSIONS: Live yeasts enhanced microbial colonization of fibrous materials, the degree of enhancement depended on their nature and composition. As an effect on rumen pH was not likely to be solely involved, the underlying mechanisms could involve nutrient supply or oxygen scavenging by the live yeast cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Distribution of this microbial additive could be an interesting tool to increase fibre digestion in the rumen and thereby improve cow feed efficiency.
Assuntos
Ração Animal/microbiologia , Bactérias/metabolismo , Fibras na Dieta/metabolismo , Fungos/metabolismo , Rúmen/microbiologia , Leveduras/metabolismo , Ração Animal/análise , Animais , Bactérias/crescimento & desenvolvimento , Bovinos , Suplementos Nutricionais/análise , Digestão , Fungos/crescimento & desenvolvimento , Reação em Cadeia da Polimerase em Tempo Real , Rúmen/metabolismo , Silagem/análise , Silagem/microbiologia , Glycine max/metabolismo , Glycine max/microbiologia , Triticum/metabolismo , Triticum/microbiologia , Leveduras/crescimento & desenvolvimentoRESUMO
The potential of dietary supplements of 2 live yeast strains (Saccharomyces cerevisiae) or camelina oil to lower ruminal methane (CH4) and carbon dioxide (CO2) production and the associated effects on animal performance, rumen fermentation, rumen microbial populations, nutrient metabolism, and milk fatty acid (FA) composition of cows fed grass silage-based diets were examined. Four Finnish Ayrshire cows (53±7 d in milk) fitted with rumen cannula were used in a 4×4 Latin square with four 42-d periods. Cows received a basal total mixed ration (control treatment) with a 50:50 forage-to-concentrate ratio [on a dry matter (DM) basis] containing grass silage, the same basal total mixed ration supplemented with 1 of 2 live yeasts, A or B, administered directly in the rumen at 10(10) cfu/d (treatments A and B), or supplements of 60g of camelina oil/kg of diet DM that replaced concentrate ingredients in the basal total mixed ration (treatment CO). Relative to the control, treatments A and B had no effects on DM intake, rumen fermentation, ruminal gas production, or apparent total-tract nutrient digestibility. In contrast, treatment CO lowered DM intake and ruminal CH4 and CO2 production, responses associated with numerical nonsignificant decreases in total-tract organic matter digestibility, but no alterations in rumen fermentation characteristics or changes in the total numbers of rumen bacteria, methanogens, protozoa, and fungi. Compared with the control, treatment CO decreased the yields of milk, milk fat, lactose, and protein. Relative to treatment B, treatment CO improved nitrogen utilization due to a lower crude protein intake. Treatment A had no influence on milk FA composition, whereas treatment B increased cis-9 10:1 and decreased 11-cyclohexyl 11:0 and 24:0 concentrations. Treatment CO decreased milk fat 8:0 to 16:0 and total saturated FA, and increased 18:0, 18:1, 18:2, conjugated linoleic acid, 18:3n-3, and trans FA concentrations. Decreases in ruminal CH4 production to treatment CO were related, at least in part to lowered DM intake, whereas treatments had no effect on ruminal CH4 emission intensity (g/kg of digestible organic matter intake or milk yield). Results indicated that live yeasts A and B had no influence on animal performance, ruminal gas production, rumen fermentation, or nutrient utilization in cows fed grass silage-based diets. Dietary supplements of camelina oil decreased ruminal CH4 and CO2 production, but also lowered the yields of milk and milk constituents due to an adverse effect on intake.
Assuntos
Brassicaceae/química , Bovinos/metabolismo , Metano/biossíntese , Óleos de Plantas/administração & dosagem , Rúmen/metabolismo , Saccharomyces cerevisiae/fisiologia , Animais , Dióxido de Carbono/metabolismo , Dieta/veterinária , Suplementos Nutricionais , Digestão/efeitos dos fármacos , Ácidos Graxos/análise , Feminino , Fermentação , Lactação/efeitos dos fármacos , Lactose/metabolismo , Leite/química , Óleos de Plantas/farmacologia , Poaceae , Rúmen/efeitos dos fármacos , Rúmen/microbiologia , SilagemRESUMO
Protozoa are commensal eukaryotes in the rumen of herbivores. Protozoa are large producers of hydrogen, which is utilized by methanogenic archaea to produce methane, a greenhouse gas. The removal of protozoa from the rumen (defaunation) decreases methanogenesis, but also negatively affects fiber digestion, which is the main function of the rumen. The aim of this study was to examine the effect of long-term defaunation on the structure of the microbiota and particularly methanogenic archaea and fibrolytic bacteria to better understand the microbial mechanisms responsible for the decrease in methanogenesis and fibrolysis. The trial was conducted in 5 adult sheep subjected successively to long-term defaunation (2 yr), refaunation (12 wk), and short-term defaunation (10 wk). Methanogens were enumerated by quantitative PCR targeting the rrs (16S ribosomal RNA subunit) and mcrA (methyl coenzyme-M reductase) genes. The rrs gene was used to quantify the 3 major culturable rumen cellulolytic bacterial species (i.e., Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens) and total bacteria. Bacterial and methanogen diversity was also examined by PCR-DGGE (PCR-denaturing gradient gel electrophoresis) analysis targeting the rrs and mcrA genes, respectively. Total rumen bacterial density estimated as rrs copies per gram of DM of rumen content increased in response to long- and short-term defaunation (+1 log, P < 0.001), but without noticeable shifts in diversity. Defaunation increased the rrs copies per gram of DM of rumen content of R. albus and R. flavefaciens (+2 log, P < 0 0.001), but did not affect that of F. succinogenes. Despite a 20% reduction in methane emission in the 2 defaunated periods, the mcrA and rrs copies of methanogens per gram of DM of rumen content increased (+1 log, P < 0.001) in the absence of protozoa, whereas the diversity of the dominant methanogenic community was not modified. This study shows no major difference between long- and short-term defaunation in abundance and diversity of bacteria and archaea. It also provides evidence that monitoring the abundance and diversity of methanogens is not sufficient to comprehend the microbial mechanisms leading to a reduction in methane emissions by ruminants. This study also reports for the first time in sheep a selective effect of defaunation on the abundance of cellulolytic bacterial species.
Assuntos
Celulose/metabolismo , Fibrobacter/fisiologia , Methanobacteriaceae/fisiologia , Rúmen/microbiologia , Ruminococcus/fisiologia , Ovinos/fisiologia , Animais , Masculino , Methanobacteriaceae/efeitos dos fármacos , Methanobacteriaceae/genética , Oxirredutases/genética , Oxirredutases/metabolismo , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Ruminococcus/efeitos dos fármacos , Ruminococcus/genéticaRESUMO
AIMS: To characterize fibre degradation, colonization and fermentation, and xylanase activity of two xylanolytic bacteria Bacteroides xylanisolvens XB1A(T) and Roseburia intestinalis XB6B4 from the human colon. METHODS AND RESULTS: The bacteria grew well on all the substrates chosen to represent dietary fibres: wheat and corn bran, pea, cabbage and leek fibres, and also on purified xylans. Roseburia intestinalis colonized the substrates more efficiently than Bact. xylanisolvens. For the two bacteria, 80-99% of the total xylanase activity was associated with the cells whatever the substrate and time of growth. Optimal specific activities of cells were obtained on oat spelt xylan; they were higher than those previously measured for xylanolytic bacteria from the human gut. Roseburia intestinalis produced high molecular mass xylanases (100-70 kDa), while Bact. xylanisolvens produced lower molecular mass enzymes, including a cell-associated xylanase of 37 kDa. CONCLUSIONS: The two bacteria display very high xylanolytic activity on the different substrates. Differences were observed on substrate attachment and enzyme systems, suggesting that the two species occupy different niches within the gut microbiota. SIGNIFICANCE AND IMPACT OF THE STUDY: This study characterizes xylan degradation by two major species of the human intestine.
Assuntos
Bacteroides/enzimologia , Colo/microbiologia , Fibras na Dieta/metabolismo , Fermentação , Bactérias Gram-Positivas/enzimologia , Xilosidases/metabolismo , Bacteroides/crescimento & desenvolvimento , Bacteroides/isolamento & purificação , Metabolismo dos Carboidratos , Bactérias Gram-Positivas/crescimento & desenvolvimento , Bactérias Gram-Positivas/isolamento & purificação , Humanos , Xilanos/metabolismoRESUMO
Ruminant production is under increased public scrutiny in terms of the importance of cattle and other ruminants as major producers of the greenhouse gas methane. Methanogenesis is performed by methanogenic archaea, a specialised group of microbes present in several anaerobic environments including the rumen. In the rumen, methanogens utilise predominantly H2 and CO2 as substrates to produce methane, filling an important functional niche in the ecosystem. However, in addition to methanogens, other microbes also have an influence on methane production either because they are involved in hydrogen (H2) metabolism or because they affect the numbers of methanogens or other members of the microbiota. This study explores the relationship between some of these microbes and methanogenesis and highlights some functional groups that could play a role in decreasing methane emissions. Dihydrogen ('H2' from this point on) is the key element that drives methane production in the rumen. Among H2 producers, protozoa have a prominent position, which is strengthened by their close physical association with methanogens, which favours H2 transfer from one to the other. A strong positive interaction was found between protozoal numbers and methane emissions, and because this group is possibly not essential for rumen function, protozoa might be a target for methane mitigation. An important function that is associated with production of H2 is the degradation of fibrous plant material. However, not all members of the rumen fibrolytic community produce H2. Increasing the proportion of non-H2 producing fibrolytic microorganisms might decrease methane production without affecting forage degradability. Alternative pathways that use electron acceptors other than CO2 to oxidise H2 also exist in the rumen. Bacteria with this type of metabolism normally occupy a distinct ecological niche and are not dominant members of the microbiota; however, their numbers can increase if the right potential electron acceptor is present in the diet. Nitrate is an alternative electron sinks that can promote the growth of particular bacteria able to compete with methanogens. Because of the toxicity of the intermediate product, nitrite, the use of nitrate has not been fully explored, but in adapted animals, nitrite does not accumulate and nitrate supplementation may be an alternative under some dietary conditions that deserves to be further studied. In conclusion, methanogens in the rumen co-exist with other microbes, which have contrasting activities. A better understanding of these populations and the pathways that compete with methanogenesis may provide novel targets for emissions abatement in ruminant production.
RESUMO
AIM: To examine the effect of concentrate and yeast additive on the number of cellulolytic bacteria in the rumen of sheep. METHODS AND RESULTS: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens were quantified using real-time PCR (targeting 16S rDNA) in parallel to cellulolytic flora enumeration with cultural techniques. Whatever the conditions tested, R. flavefaciens was slightly more abundant than F. succinogenes, with both species outnumbering R. albus. Before feeding, the shift from hay to hay plus concentrate diet had no effect on rumen pH and on the number of the three specie; while after feeding, the concentrate-supplemented diet induced a decrease (-1 log) of the number of the three species concomitant with the rumen acidification. Overall, the presence of the live yeast resulted in a significant increase (two- to fourfold) of the Ruminococci. CONCLUSION: The use of real-time PCR allowed us to show changes in the number of cellulolytic bacterial species in vivo in response to diet shift and additives that could not be as easily evidenced by classical microbial methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the understanding of the negative impact of readily fermentable carbohydrates on rumen cellulolysis and the beneficial effect of yeast on rumen fermentation.
Assuntos
Ração Animal , Celulose/metabolismo , Bactérias Gram-Positivas/isolamento & purificação , Rúmen/microbiologia , Carneiro Doméstico/microbiologia , Leveduras , Animais , Metabolismo dos Carboidratos , Contagem de Colônia Microbiana , DNA Bacteriano/análise , Suplementos Nutricionais , Fermentação , Fibrobacter/genética , Fibrobacter/isolamento & purificação , Fibrobacter/metabolismo , Bactérias Gram-Positivas/metabolismo , Masculino , Sondas de Oligonucleotídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Rúmen/metabolismo , Ruminococcus/genética , Ruminococcus/isolamento & purificação , Ruminococcus/metabolismo , Carneiro Doméstico/metabolismoRESUMO
Wheat straw degradation by Fibrobacter succinogenes was monitored by nuclear magnetic resonance (NMR) spectroscopy and chemolytic methods to investigate the activity of an entire fibrolytic system on an intact complex substrate. In situ solid-state NMR with 13C cross-polarization magic angle spinning was used to monitor the modification of the composition and structure of lignocellulosic fibers (of 13C-enriched wheat straw) during the growth of bacteria on this substrate. There was no preferential degradation either of amorphous regions of cellulose versus crystalline regions or of cellulose versus hemicelluloses in wheat straw. This suggests either a simultaneous degradation of the amorphous and crystalline parts of cellulose and of cellulose and hemicelluloses by the enzymes or degradation at the surface at a molecular scale that cannot be detected by NMR. Liquid-state two-dimensional NMR experiments and chemolytic methods were used to analyze in detail the various sugars released into the culture medium. An integration of NMR signals enabled the quantification of oligosaccharides produced from wheat straw at various times of culture and showed the sequential activities of some of the fibrolytic enzymes of F. succinogenes S85 on wheat straw. In particular, acetylxylan esterase appeared to be more active than arabinofuranosidase, which was more active than alpha-glucuronidase. Finally, cellodextrins did not accumulate to a great extent in the culture medium.
Assuntos
Fibras na Dieta/metabolismo , Fibrobacter/metabolismo , Triticum/metabolismo , Animais , Celulose/metabolismo , Meios de Cultura , Fibrobacter/crescimento & desenvolvimento , Técnicas In Vitro , Cinética , Lignina/metabolismo , Espectroscopia de Ressonância Magnética , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismo , Rúmen/microbiologiaRESUMO
Sodium gradients (DeltapNa) were measured in resting cells of Fibrobacter succinogenes by in vivo 23Na nuclear magnetic resonance using Tm(DOTP)5- [thulium(III) 1,4,7,10-tetraazacyclododecane-N',N",N"'-tetramethylenephosphonate] as the shift reagent. This bacterium was able to maintain a DeltapNa of -55 to -40 mV for extracellular sodium concentrations ranging from 30 to 200 mM. Depletion of Na+ ions during the washing steps led to irreversible damage (modification of glucose metabolism and inability to maintain a sodium gradient).
Assuntos
Bactérias Anaeróbias/fisiologia , Rúmen/microbiologia , Cloreto de Sódio/metabolismo , Animais , Espectroscopia de Ressonância Magnética/métodos , Compostos Organometálicos/metabolismo , Compostos Organofosforados/metabolismo , Isótopos de Sódio/metabolismoRESUMO
1D and 2D NMR experiments were used to analyse the synthesis of various metabolites by resting cells of Fibrobacter succinogenes S85 when incubated with [1-(13)C]glucose, in both extracellular and cellular media. Besides the expected glycogen, succinate, acetate, glucose-1-P and glucose-6-P, maltodextrins and cellodextrins were detected. Maltodextrins were excreted into the external medium. They were found to have linear structures with a maximum degree of polymerization (DP) of about 6 or 7 units. Cellodextrins were located in the cells (cytoplasm and/or periplasm), and their DP was < or = 4. Both labelled (1-(13)C and 6-(13)C) and unlabelled maltodextrins and cellodextrins were detected, showing the contribution of carbohydrate cycling in F. succinogenes, including the reversal of glycolysis and the futile cycle of glycogen. The mechanisms of these oligosaccharide syntheses are discussed.
Assuntos
Bactérias/metabolismo , Celulose/análogos & derivados , Celulose/biossíntese , Dextrinas/biossíntese , Glucose/metabolismo , Polissacarídeos/biossíntese , Animais , Meios de Cultura , Maltose , Ressonância Magnética Nuclear Biomolecular/métodos , Rúmen/microbiologiaRESUMO
The endoglucanase activity of cells and extracellular culture fluid of Fibrobacter succinogenes S85 grown on glucose, cellobiose, soluble polysaccharides (beta-glucan, lichenan) and intact plant polysaccharides, was compared. The specific activity of cells grown on cellulose or forages was 6- to 20-fold higher than that of cells grown on soluble substrates, suggesting an induction of endoglucanases by the insoluble substrates. The ratios of cells to extracellular culture fluid endoglucanase activities measured in cultures grown on sugars or insoluble polysaccharides suggested that the endoglucanases induced by the insoluble polysaccharides remained attached to the cells. The mRNA of all the F. succinogenes glycoside hydrolase genes sequenced so far were then quantified in cells grown on glucose, cellobiose or cellulose. The results show that all these genes were transcribed in growing cells, and that they are all overexpressed in cultures grown on cellulose. Endoglucanase-encoding endB and endA(FS) genes, and xylanase-encoding xynC gene appeared the most expressed genes in growing cells. EGB and ENDA are thus likely to play a major role in cellulose degradation in F. succinogenes.
Assuntos
Proteínas de Bactérias/biossíntese , Celulase/metabolismo , Glicosídeo Hidrolases/biossíntese , Celobiose , Meios de Cultura , Primers do DNA , Regulação Enzimológica da Expressão Gênica , Glucose , Glicosídeo Hidrolases/genética , RNA Mensageiro/análise , TrissacarídeosRESUMO
The enzymatic properties of two endoglucanases from Fibrobacter succinogenes, EGB and EGC, were analysed. EGB and EGC were purified from recombinant Escherichia coli cultures expressing their gene. The failure of purification of EGB by classical techniques led us to produce antipeptide antibodies that allowed immunopurification of the protein from E. coli as well as its detection in F. succinogenes cultures. Synthetic peptides were selected from the predicted primary structure of EGB, linked to bovine serum albumin and used as immunogens to obtain specific antibodies. One of the polyclonal antipeptide antisera was used to purify EGB. EGC was purified by affinity chromatography with Ni-NTA resin. The endo mode of action of the two enzymes on carboxymethyl-cellulose was different. The values of K(m) and V(max) were respectively 13.6 mg/ml and 46 micromol/min mg protein for EGB, and 7 mg/ml and 110 micromol/min mg protein for EGC. The reactivity of the antipeptide and the anti-EGC sera with F. succinogenes proteins of molecular mass different from that of EGB and EGC produced in E. coli suggested post-translational modification of the two enzymes in F. succinogenes cultures. Expression of endB and endC genes in F. succinogenes was confirmed by RT-PCR.
Assuntos
Proteínas de Bactérias/análise , Celulase/análise , Bactérias Anaeróbias Gram-Negativas/enzimologia , Animais , Proteínas de Bactérias/metabolismo , Bovinos , Celulase/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Fibrobacter succinogenes S85, a cellulolytic rumen bacterium, is very efficient in degrading lignocellulosic substrates and could be used to develop a biotechnological process for the treatment of wastes. In this work, the metabolism of cellulose by F. succinogenes S85 was investigated using in vivo 13C NMR and 13C-filtered spin-echo difference 1H NMR spectroscopy. The degradation of unlabelled cellulose synthesised by Acetobacter xylinum was studied indirectly, in the presence of [1-13C]glucose, by estimating the isotopic dilution of the final bacterial fermentation products (glycogen, succinate, acetate). During the pre-incubation period of F. succinogenes cells with cellulose fibres, some cells ('non-adherent') did not attach to the solid material. Results for 'adherent' cells showed that about one fourth of the glucose units entering F. succinogenes metabolism originated from cellulose degradation. A huge reversal of succinate metabolism pathway and production of large amounts of unlabelled acetate which was observed during incubation with glucose only, was found to be much decreased in the presence of solid substrate. The synthesis of glucose 6-phophate was slightly increased in the presence of cellulose. Results clearly showed that 'non-adherent' cells were able to metabolise glucose very efficiently; consequently the metabolic state of these cells was not responsible for their 'non-adherence' to cellulose fibre.
Assuntos
Biofilmes , Celulose/metabolismo , Bactérias Anaeróbias Gram-Negativas/metabolismo , Espectroscopia de Ressonância Magnética , Acetatos/análise , Acetatos/metabolismo , Animais , Aderência Bacteriana/fisiologia , Isótopos de Carbono , Bovinos , Fermentação/efeitos dos fármacos , Fermentação/fisiologia , Glucose/metabolismo , Glucose/farmacologia , Glicogênio/análise , Glicogênio/metabolismo , Prótons , Rúmen/microbiologia , Ácido Succínico/análise , Ácido Succínico/metabolismoRESUMO
We report for the first time the cloning and characterisation of a protozoal enzyme involved in plant cell wall polysaccharide degradation. A cDNA library was constructed from the ruminal protozoan Polyplastron multivesiculatum and a stable clone expressing xylanase activity was isolated. The encoded enzyme belongs to the glycoside hydrolase family 11, and phylogenetic analysis indicates a closer relationship with catalytic domains from Gram-positive bacteria than the other fibrolytic eukaryotes from the rumen, the anaerobic fungi.
Assuntos
Cilióforos/enzimologia , Rúmen/parasitologia , Xilosidases/genética , Xilosidases/metabolismo , Sequência de Aminoácidos , Anaerobiose , Animais , Sequência de Bases , Cilióforos/genética , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Glucanos/metabolismo , Bactérias Gram-Positivas/enzimologia , Bactérias Gram-Positivas/genética , Immunoblotting , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Xilano Endo-1,3-beta-Xilosidase , Xilanos/metabolismo , Xilosidases/químicaRESUMO
The effect of the presence of ammonia on [1-13C]glucose metabolism in the rumen fibrolytic bacterium Fibrobacter succinogenes S85 was studied by 13C and 1H nuclear magnetic resonance (NMR). Ammonia halved the level of glycogen storage and increased the rate of glucose conversion into acetate and succinate 2.2-fold and 1.4-fold, respectively, reducing the succinate-to-acetate ratio. The 13C enrichment of succinate and acetate was precisely quantified by 13C-filtered spin-echo difference 1H-NMR spectroscopy. The presence of ammonia did not modify the 13C enrichment of succinate C-2 (without ammonia, 20.8%, and with ammonia, 21.6%), indicating that the isotopic dilution of metabolites due to utilization of endogenous glycogen was not affected. In contrast, the presence of ammonia markedly decreased the 13C enrichment of acetate C-2 (from 40 to 31%), reflecting enhanced reversal of the succinate synthesis pathway. The reversal of glycolysis was unaffected by the presence of ammonia as shown by 13C-NMR analysis. Study of cell extracts showed that the main pathways of ammonia assimilation in F. succinogenes were glutamate dehydrogenase and alanine dehydrogenase. Glutamine synthetase activity was not detected. Glutamate dehydrogenase was active with both NAD and NADP as cofactors and was not repressed under ammonia limitation in the culture. Glutamate-pyruvate and glutamate-oxaloacetate transaminase activities were evidenced by spectrophotometry and 1H NMR. When cells were incubated in vivo with [1-13C]glucose, only 13C-labeled aspartate, glutamate, alanine, and valine were detected. Their labelings were consistent with the proposed amino acid synthesis pathway and with the reversal of the succinate synthesis pathway.
Assuntos
Bactérias Anaeróbias Gram-Negativas/metabolismo , Ácido Acético/metabolismo , Aminoácidos/biossíntese , Amônia/metabolismo , Amônia/farmacologia , Animais , Carbono/metabolismo , Isótopos de Carbono , Glucose/metabolismo , Bactérias Anaeróbias Gram-Negativas/efeitos dos fármacos , Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética , Nitrogênio/metabolismo , Rúmen/microbiologia , Ácido Succínico/metabolismoRESUMO
The metabolism of glucose and cellobiose, products of cellulose hydrolysis, was investigated in four cellulolytic strains of the genus Fibrobacter: Fibrobacter succinogenes S85, 095, HM2 and Fibrobacter intestinalis NR9. In vivo 13C nuclear magnetic resonance was used to quantify the relative contribution of glucose and cellobiose to metabolite production, glycogen storage and cellodextrins synthesis in these four strains. The same features were found in all four strains of the genus Fibrobacter metabolizing simultaneously glucose and cellobiose: i) differential metabolism of glucose and cellobiose; glucose seems preferentially used for glycogen storage and energy production, while part of cellobiose seems to be diverted from glycolysis, ii) synthesis of cellodextrins, mainly from cellobiose not entering into glycolysis, iii) accumulation of glucose 6-phosphate, iv) simultaneous presence of cellobiose phosphorylase and cellobiase activities. Although genetically diverse, the Fibrobacter genus appears to possess a marked homogeneity in its carbon metabolism.
Assuntos
Bacteroides/metabolismo , Celobiose/metabolismo , Glucose/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Bacteroides/classificação , Radioisótopos de Carbono , Celulose/análogos & derivados , Celulose/biossíntese , Dextrinas/biossíntese , Enzimas/metabolismo , Glicogênio/metabolismo , Especificidade da EspécieRESUMO
We investigated the carbon metabolism of three strains of Fibrobacter succinogenes and one strain of Fibrobacter intestinalis. The four strains produced the same amounts of the metabolites succinate, acetate, and formate in approximately the same ratio (3.7/1/0.3). The four strains similarly stored glycogen during all growth phases, and the glycogen-to-protein ratio was close to 0.6 during the exponential growth phase. 13C nuclear magnetic resonance (NMR) analysis of [1-13C]glucose utilization by resting cells of the four strains revealed a reversal of glycolysis at the triose phosphate level and the same metabolic pathways. Glycogen futile cycling was demonstrated by 13C NMR by following the simultaneous metabolism of labeled [13C]glycogen and exogenous unlabeled glucose. The isotopic dilutions of the CH2 of succinate and the CH3 of acetate when the resting cells were metabolizing [1-13C]glucose and unlabeled glycogen were precisely quantified by using 13C-filtered spin-echo difference 1H NMR spectroscopy. The measured isotopic dilutions were not the same for succinate and acetate; in the case of succinate, the dilutions reflected only the contribution of glycogen futile cycling, while in the case of acetate, another mechanism was also involved. Results obtained in complementary experiments are consistent with reversal of the succinate synthesis pathway. Our results indicated that for all of the strains, from 12 to 16% of the glucose entering the metabolic pathway originated from prestored glycogen. Although genetically diverse, the four Fibrobacter strains studied had very similar carbon metabolism characteristics.
Assuntos
Bactérias/metabolismo , Glicogênio/metabolismo , Acetatos/metabolismo , Animais , Bactérias/classificação , Isótopos de Carbono , Bovinos , Formiatos/metabolismo , Glucose/metabolismo , Ressonância Magnética Nuclear Biomolecular , Rúmen/microbiologia , Ácido Succínico/metabolismoRESUMO
The glucose metabolism of Fibrobacter succinogenes S85 was studied in detail; key intermediates and alternative pathways were evidenced by NMR and/or enzymatic assays. A high phosphoketolase activity was detected in four strains of Fibrobacter under strictly anaerobic conditions, with ribose-5-phosphate as substrate, no activity was evidenced with fructose-6-phosphate. This is the first report of a pentose phosphates phosphoketolase in bacteria unable to use pentoses. In contrast, the Entner-Doudoroff pathway and the oxidative branch of the pentose phosphate pathway could not be evidenced. Incubation of living cells of F. succinogenes with Na2(13)CO3 confirmed the incorporation of 13CO2 in the carboxylic group of succinate. The presence of fumarase was evidenced by in vivo 4C-NMR using 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO): the enzyme showed a high reversibility under physiological conditions. The production of formate from glucose catabolism was evidenced by enzymatic assay and by NMR and a pyruvate formate lyase activity was detected using strictly anaerobic conditions.
Assuntos
Acetiltransferases/metabolismo , Aldeído Liases/metabolismo , Glucose/metabolismo , Bactérias Anaeróbias Gram-Negativas/metabolismo , Anaerobiose , Dióxido de Carbono/metabolismo , Formiatos/metabolismo , Fumarato Hidratase/metabolismo , Espectroscopia de Ressonância Magnética , Via de Pentose Fosfato , Pentosefosfatos/metabolismo , Fosfoenolpiruvato/metabolismo , Ribosemonofosfatos/metabolismo , Succinatos/metabolismo , Ácido SuccínicoRESUMO
Kinetics of [1-13C]glucose utilization were monitored by in vivo NMR spectroscopy on resting cells of Fibrobacter succinogenes, in the presence of 32 mM [1-13C]glucose, 32 mM [1-13C]glucose and 64 mM unlabelled glucose, or 32 mM [1-13C]glucose and 32 mM unlabelled cellobiose. A similar production of acetate and succinate and a similar storage of glycogen were observed whatever the exogenous substrate. The presence of cellobiose or that of an equivalent amount of glucose did not reduce [1-13C]glucose incorporation to the same extent. Glucose seemed preferentially used for glycogen storage and energy production, while part of the cellobiose appeared to be used for cellodextrin synthesis. Both cellobiase and cellobiose phosphorylase activities were assayed in cell-free extracts. Finally, the intracellular concentration of glucose 6-phosphate was increased by over threefold when cells metabolized cellobiose (alone or in parallel to glucose) as compared with the metabolism of glucose alone.