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1.
Rosanne Hertzberger Jos Arents Henk L. Dekker R. David Pridmore Christof Gysler Michiel Kleerebezem M. Joost Teixeira de Mattos 《Applied and environmental microbiology》2014,80(7):2229-2239
Hydrogen peroxide production is a well-known trait of many bacterial species associated with the human body. In the presence of oxygen, the probiotic lactic acid bacterium
Lactobacillus johnsonii NCC 533 excretes up to 1 mM H2O2, inducing growth stagnation and cell death. Disruption of genes commonly assumed to be involved in H2O2 production (e.g., pyruvate oxidase, NADH oxidase, and lactate oxidase) did not affect this. Here we describe the purification of a novel NADH-dependent flavin reductase encoded by two highly similar genes (LJ_0548 and LJ_0549) that are conserved in lactobacilli belonging to the Lactobacillus acidophilus group. The genes are predicted to encode two 20-kDa proteins containing flavin mononucleotide (FMN) reductase conserved domains. Reductase activity requires FMN, flavin adenine dinucleotide (FAD), or riboflavin and is specific for NADH and not NADPH. The Km for FMN is 30 ± 8 μM, in accordance with its proposed in vivo role in H2O2 production. Deletion of the encoding genes in L. johnsonii led to a 40-fold reduction of hydrogen peroxide formation. H2O2 production in this mutant could only be restored by in trans complementation of both genes. Our work identifies a novel, conserved NADH-dependent flavin reductase that is prominently involved in H2O2 production in L. johnsonii. 相似文献
2.
Genome analysis of Lactobacillus johnsonii NCC533 has been recently completed. One of its annotated genes, lj0569, encodes the protein having the conserved domain of glycoside hydrolase family 31. Its homolog gene (ljag31) in L. johnsonii NBRC13952 was cloned and expressed using an Escherichia coli expression system, resulting in poor production of recombinant LJAG31 protein due to inclusion body formation. Production of soluble recombinant LJAG31 was improved with high concentration of NaCl in medium, possible endogenous chaperone induction by benzyl alcohol, and over-expression of GroES–GroEL chaperones. Recombinant LJAG31 was an α-glucosidase with broad substrate specificity toward both homogeneous and heterogeneous substrates. This enzyme displayed higher specificity (in terms of kcat/Km) toward nigerose, maltulose, and kojibiose than other natural substrates having an α-glucosidic linkage at the non-reducing end, which suggests that these sugars are candidates for prebiotics contributing to the growth of L. johnsonii. To our knowledge, LJAG31 is the first bacterial α-1,3-glucosidase to be characterized with a high kcat/Km value for nigerose [α-d-Glcp-(1 → 3)-d-Glcp]. Transglucosylation of 4-nitrophenyl α-d-glucopyranoside produced two 4-nitrophenyl disaccharides (4-nitrophenyl α-nigeroside and 4-nitrophenyl α-isomaltoside). These hydrolysis and transglucosylation properties of LJAG31 are different from those of mold (Acremonium implicatum) α-1,3-glucosidase of glycoside hydrolase family 31. 相似文献
3.
Scott Wushke Victor Spicer Xiang Li Zhang Brian Fristensky Oleg V. Krokhin David B. Levin Nazim Cicek Richard Sparling 《Systematic and applied microbiology》2017,40(5):245-253
Caldibacillus debilis GB1 is a facultative anaerobe isolated from a thermophilic aero-tolerant cellulolytic enrichment culture. There is a lack of representative proteomes of facultative anaerobic thermophilic Bacillaceae, exploring aerobic/anaerobic expression. The C. debilis GB1 genome was sequenced and annotated, and the proteome characterized under aerobic and anaerobic conditions while grown on cellobiose. The draft sequence of C. debilis GB1 contains a 3,340,752 bp chromosome and a 5,386 bp plasmid distributed over 49 contigs. Two-dimensional liquid chromatography mass spectrometry/mass spectrometry was used with Isobaric Tags for Relative and Absolute Quantification (iTRAQ) to compare protein expression profiles, focusing on energy production and conversion pathways. Under aerobic conditions, proteins in glycolysis and pyruvate fermentation pathways were down-regulated. Simultaneously, proteins within the tricarboxylic acid cycle, pyruvate dehydrogenase, the electron transport chain, and oxygen scavenging pathways showed increased amounts. Under anaerobic conditions, protein levels in fermentation pathways were consistent with the generated end-products: formate, acetate, ethanol, lactate, and CO2. Under aerobic conditions CO2 and acetate production was consistent with incomplete respiration. Through a direct comparison with gene expression profiles from Escherichia coli, we show that global regulation of core metabolism pathways is similar in thermophilic and mesophilic facultative anaerobes of the Phylum Proteobacteria and Firmicutes. 相似文献
4.
The anoxygenic green sulfur bacteria (GSBs) assimilate CO2 autotrophically through the reductive (reverse) tricarboxylic acid (RTCA) cycle. Some organic carbon sources, such as acetate and pyruvate, can be assimilated during the phototrophic growth of the GSBs, in the presence of CO2 or HCO3−. It has not been established why the inorganic carbonis required for incorporating organic carbon for growth and how the organic carbons are assimilated. In this report, we probed carbon flux during autotrophic and mixotrophic growth of the GSB Chlorobaculum tepidum. Our data indicate the following: (a) the RTCA cycle is active during autotrophic and mixotrophic growth; (b) the flux from pyruvate to acetyl-CoA is very low and acetyl-CoA is synthesized through the RTCA cycle and acetate assimilation; (c) pyruvate is largely assimilated through the RTCA cycle; and (d) acetate can be assimilated via both of the RTCA as well as the oxidative (forward) TCA (OTCA) cycle. The OTCA cycle revealed herein may explain better cell growth during mixotrophic growth with acetate, as energy is generated through the OTCA cycle. Furthermore, the genes specific for the OTCA cycle are either absent or down-regulated during phototrophic growth, implying that the OTCA cycle is not complete, and CO2 is required for the RTCA cycle to produce metabolites in the TCA cycle. Moreover, CO2 is essential for assimilating acetate and pyruvate through the CO2-anaplerotic pathway and pyruvate synthesis from acetyl-CoA. 相似文献
5.
Carbon dioxide effects on ethanol production, pyruvate decarboxylase, and alcohol dehydrogenase activities in anaerobic sweet potato roots 总被引:1,自引:1,他引:0
The effect of varied anaerobic atmospheres on the metabolism of sweet potato (Ipomoea batatas [L.] Lam.) roots was studied. The internal gas atmospheres of storage roots changed rapidly when the roots were submerged under water. O2 and N2 gases disappeared quickly and were replaced by CO2. There were no appreciable differences in gas composition among the four cultivars that were studied. Under different anaerobic conditions, ethanol concentration in the roots was highest in a CO2 environment, followed by submergence and a N2 environment in all the cultivars except one. A positive relationship was found between ethanol production and pyruvate decarboxylase activity from both 100% CO2-treated and 100% N2-treated roots. CO2 atmospheres also resulted in higher pyruvate decarboxylase activity than did N2 atmospheres. Concentrations of CO2 were higher within anaerobic roots than those in the ambient anaerobic atmosphere. The level of pyruvate decarboxylase and ethanol in anaerobic roots was proportional to the ambient CO2 concentration. The measurable activity of pyruvate decarboxylase that was present in the roots was about 100 times less than that of alcohol dehydrogenase. Considering these observations, it is suggested that the rate-limiting enzyme for ethanol biosynthesis in sweet potato storage roots under anoxia is likely to be pyruvate decarboxylase rather than alcohol dehydrogenase. 相似文献
6.
An obligately anaerobic thermophilic sporeforming sulfate-reducing bacterium, named strain CAMZ, was isolated from a benzoate enrichment from a 58°C thermophilic anaerobic bioreactor. The cells of strain CAMZ were 0.7 m by 2–5 m rods with pointed ends, forming single cells or pairs. Spores were central, spherical, and caused swelling of the cells. The Gram stain was negative. Electron donors used included lactate, pyruvate, acetate and other short chain fatty acids, short chain alcohols, alanine, and H2/CO2. Lactate and pyruvate were oxidized completely to CO2 with sulfate as electron acceptor. Sulfate was required for growth on H2/CO2, and both acetate and sulfide were produced from H2/CO2-sulfate. Sulfate, thiosulfate, or elemental sulfur served as electron acceptors with lactate as the donor while sulfite, nitrate, nitrite, betaine, or a hydrogenotrophic methanogen did not. The optimum temperature for growth of strain CAMZ was 55–60°C and the optimum pH value was 6.5. The specific activities of carbon monoxide dehydrogenase of cells of strain CAMZ grown on lactate, H2/CO2, or acetate with sulfate were 7.2, 18.1, and 30.8 mol methyl viologen reduced min–1 [mg protein]–1, respectively, indicating the presence of the CO/Acetyl-CoA pathway in this organism. The mol%-G+C of strain CAMZ's DNA was 49.7. The new species name Desulfotomaculum thermoacetoxidans is proposed for strain CAMZ. 相似文献
7.
Following incubation of mesophilic methanogenic floccular sludge from a lab-scale upflow anaerobic sludge bed reactor used to treat cattle manure wastewater, a stable 5-aminosalicylate-degrading enrichment culture was obtained. Subsequently, a Citrobacter freundii strain, WA1, was isolated from the 5-aminosalicylate-degrading methanogenic consortium. The methanogenic enrichment culture degraded 5-aminosalicylate completely to CH4, CO2 and NH4
+, while C. freundii strain WA1 reduced 5-aminosalicylate with simultaneous deamination to 2-hydroxybenzyl alcohol during anaerobic growth with electron donors such as pyruvate, glucose or serine. When grown on pyruvate, C. freundii WA1 converted 3-aminobenzoate to benzyl alcohol and also reduced benzaldehyde to benzyl alcohol. Pyruvate was fermented to acetate, CO2, H2 and small amounts of lactate, succinate and formate. Less lactate (30%) was produced from pyruvate when C. freundii WA1 grew with 5-aminosalicylate as co-substrate. 相似文献
8.
Metabolism of One-Carbon Compounds by the Ruminal Acetogen Syntrophococcus sucromutans 总被引:2,自引:1,他引:1
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Syntrophococcus sucromutans is the predominant species capable of O demethylation of methoxylated lignin monoaromatic derivatives in the rumen. The enzymatic characterization of this acetogen indicated that it uses the acetyl coenzyme A (Wood) pathway. Cell extracts possess all the enzymes of the tetrahydrofolate pathway, as well as carbon monoxide dehydrogenase, at levels similar to those of other acetogens using this pathway. However, formate dehydrogenase could not be detected in cell extracts, whether formate or a methoxyaromatic was used as electron acceptor for growth of the cells on cellobiose. Labeled bicarbonate, formate, [1-14C] pyruvate, and chemically synthesized O-[methyl-14C]vanillate were used to further investigate the catabolism of one-carbon (C1) compounds by using washed-cell preparations. The results were consistent with little or no contribution of formate dehydrogenase and pointed out some unique features. Conversion of formate to CO2 was detected, but labeled formate predominantly labeled the methyl group of acetate. Labeled CO2 readily exchanged with the carboxyl group of pyruvate but not with formate, and both labeled CO2 and pyruvate predominantly labeled the carboxyl group of acetate. No CO2 was formed from O demethylation of vanillate, and the acetate produced was position labeled in the methyl group. The fermentation pattern and specific activities of products indicated a complete synthesis of acetate from pyruvate and the methoxyl group of vanillate. 相似文献
9.
D. V. Fedosov D. A. Podkopaeva M. L. Miroshnichenko E. A. Bonch-Osmolovskaya A. V. Lebedinsky M. Yu. Grabovich 《Microbiology》2006,75(2):119-124
This work is concerned with the metabolism of Caldithrix abyssi—an anaerobic, moderately thermophilic bacterium isolated from deep-sea hydrothermal vents of the Mid-Atlantic Ridge and representing a new, deeply deviated branch within the domain Bacteria. Cells of C. abyssi grown on acetate and nitrate, which was reduced to ammonium, possessed nitrate reductase activity and contained cytochromes of the b and c types. Utilization of acetate occurred as a result of the operation of the TCA and glyoxylate cycles. During growth of C. abyssi on yeast extract, fermentation with the formation of acetate, propionate, hydrogen, and CO2 occurred. In extracts of cells grown on yeast extract, acetate was produced from pyruvate with the involvement of the following enzymes: pyruvate: ferredoxin oxidoreductase (2.6 μmol/(min mg protein)), phosphate acetyltransferase (0.46 μmol/(min mg protein)), and acetate kinase (0.3 μmol/(min mg protein)). The activity of fumarate reductase (0.14 μmol/(min mg protein)), malate dehydrogenase (0.17 μmol/(min mg protein)), and fumarate hydratase (1.2 μmol/(min mg protein)), as well as the presence of cytochrome b, points to the formation of propionate via the methyl-malonyl-CoA pathway. The activity of antioxidant enzymes (catalase and superoxide dismutase) was detected. Thus, enzymatic mechanisms have been elucidated that allow C. abyssi to switch from fermentation to anaerobic respiration and to exist in the gradient of redox conditions characteristic of deep-sea hydrothermal vents. 相似文献
10.
Andrea Michel Abigail Koch-Koerfges Karin Krumbach Melanie Brocker Michael Bott 《Applied and environmental microbiology》2015,81(21):7496-7508
Corynebacterium glutamicum, a model organism in microbial biotechnology, is known to metabolize glucose under oxygen-deprived conditions to l-lactate, succinate, and acetate without significant growth. This property is exploited for efficient production of lactate and succinate. Our detailed analysis revealed that marginal growth takes place under anaerobic conditions with glucose, fructose, sucrose, or ribose as a carbon and energy source but not with gluconate, pyruvate, lactate, propionate, or acetate. Supplementation of glucose minimal medium with tryptone strongly enhanced growth up to a final optical density at 600 nm (OD600) of 12, whereas tryptone alone did not allow growth. Amino acids with a high ATP demand for biosynthesis and amino acids of the glutamate family were particularly important for growth stimulation, indicating ATP limitation and a restricted carbon flux into the oxidative tricarboxylic acid cycle toward 2-oxoglutarate. Anaerobic cultivation in a bioreactor with constant nitrogen flushing disclosed that CO2 is required to achieve maximal growth and that the pH tolerance is reduced compared to that under aerobic conditions, reflecting a decreased capability for pH homeostasis. Continued growth under anaerobic conditions indicated the absence of an oxygen-requiring reaction that is essential for biomass formation. The results provide an improved understanding of the physiology of C. glutamicum under anaerobic conditions. 相似文献
11.
Syntrophic Association by Cocultures of the Methanol- and CO2-H2-Utilizing Species Eubacterium limosum and Pectin-Fermenting Lachnospira multiparus During Growth in a Pectin Medium 总被引:3,自引:2,他引:1
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Lachnospira multiparus grew very well in an anaerobic 0.2% pectin medium, whereas Eubacterium limosum, which utilizes methanol, H2-CO2, and lactate, did not. Cocultures of the two species grew at a somewhat more rapid growth rate than did L. multiparus alone and almost doubled the amount of growth as measured by optical density. In model experiments with cultures transferred once a day with a 2-day retention time, L. multiparus produced mainly acetate, methanol, ethanol, formate, lactate, CO2, and H2 from pectin. The coculture produced one-third more acetate, and butyrate and CO2 were the only other significant end products. The results are discussed in relationship to microbial metabolic interactions and interspecies hydrogen transfer. 相似文献
12.
A facultatively anaerobic spirochete isolated from a high-salinity pond grew optimally when 0.75 M NaCl, 0.2 M MgSO4, and 0.01 M CaCl2 were present in media containing yeast extract, peptone, and a carbohydrate. The organism failed to grow when any one of these three salts was omitted from the medium. Aerobically-grown colonies of the spirochete were red, whereas anaerobically-grown colonies showed no pigmentation. Non-pigmented mutants of the spirochete were isolated.The spirochete used carbohydrates, but not amino acids, as energy sources. Glucose was fermented to CO2, H2, ethanol, acetate, and a small amount of lactate. Determinations of radioactivity in products formed from glucose-1-14C and enzymatic assays indicated that glucose was dissimilated to pyruvate mainly via the Embden-Meyerhof pathway. Pyruvate was metabolized through a clostridial-type clastic reaction.Cells growing acrobically performed an incomplete oxidation of glucose mainly to CO2 and acetate. Comparison of aerobic and anaerobic growth yields indicated that oxidative phosphorylation occurred in cells growing aerobically. The guanine + cytosine content of the DNA of the spirochete was 62 moles%. It is proposed that the spirochete described herein be considered a new species and that it be namedSpirochaeta halophila. 相似文献
13.
Hydrogen peroxide production by Lactobacillus johnsonii NCC 533 and its role in anti-Salmonella activity 总被引:1,自引:0,他引:1
The human intestinal isolate Lactobacillus johnsonii NCC 533 (La1) is a probiotic strain with well-documented antimicrobial properties. Previous research has identified the production of lactic acid and bacteriocins as important factors, but that other unidentified factors are also involved. We used the recently published genome sequence of L. johnsonii NCC 533 to search for novel antipathogen factors and identified three potential gene products that may catalyze the synthesis of the known antimicrobial factor hydrogen peroxide, H(2)O(2). In this work, we confirmed the ability of NCC 533 as well as eight different L. johnsonii strains and Lactobacillus gasseri to produce H(2)O(2) when resting cells were incubated in the presence of oxygen, and that culture supernatant containing NCC 533-produced H(2)O(2) was effective in killing the model pathogen Salmonella enterica serovar Typhimurium SL1344 in vitro. 相似文献
14.
PA-1, a Versatile Anaerobe Obtained in Pure Culture, Catabolizes Benzenoids and Other Compounds in Syntrophy with Hydrogenotrophs, and P-2 plus Wolinella sp. Degrades Benzenoids 总被引:5,自引:4,他引:1
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Methanogenic enrichments catabolizing 13 mM phenylacetate or 4 mM phenol were established at 37°C, using a 10% inoculum from a municipal anaerobic digester. By using agar roll tubes of the basal medium plus 0.1% yeast extract-25 mM fumarate, a hydrogenotrophic lawn of Wolinella succinogenes and phenol or phenylacetate, strains P-2 and PA-1, respectively, were isolated in coculture with W. succinogenes. With the lawn deleted, PA-1 was isolated in pure culture. Strain P-2 is apparently a new species of anaerobic, motile, gram-negative, spindle-shaped, small rod that as yet has been grown only in coculture with W. succinogenes. It used phenol, hydrocinnamate, benzoate, and phenylacetate as energy sources. Product recovery by the coculture, per mole of phenol and 4.4 mol of fumarate used, included 2.03, 0.12, 0.08, and 3.23 mol, respectively, of acetate, propionate, butyrate, and succinate. Carbon recovery was 75% and H recovery was 80%, although CO2 and a few other possible products were not determined. That P-2 is an obligate proton-reducing acetogen and possible pathways for its degradation of phenol are discussed. Strain PA-1 is apparently a new species of anaerobic, motile, relatively small, gram-negative rod. It utilized compounds such as phenylacetate, hydrocinnamate, benzoate, phenol, resorcinol, gallate, 4-aminophenol, 2-aminobenzoate, pyruvate, Casamino Acids, and aspartate as energy sources in coculture with W. succinogenes. Per mole of phenylacetate and 1.44 mol of fumarate used, 1.04, 0.53, and 0.78 mol of acetate, propionate, and succinate, respectively, were recovered from the coculture. Only about 50% of the carbon and H were recovered. In coculture with Methanospirillum hungatei, 0.96 mol of acetate and 0.25 mol of methane were recovered per mol of pyruvate used; 0.90 mol of acetate and 0.33 mol of methane, per mol of fumarate used; 0.93 mol of acetate and 0.54 mol of methane, per mol of aspartate used; and 1.71 mol of acetate and 0.57 mol of methane, per mol of glucose used. Carbon and H recoveries, assuming CO2 and ammonia were produced in stoichiometric amounts, were 97 and 98% for pyruvate, 72.5 and 82% for fumarate, 96.5 and 98% for aspartate, and 61.8 and 76% for glucose. No explanation such as contamination could be found for the fact that the coculture PA-1 plus Wolinella sp. did not use glucose; after growth with M. hungatei on pyruvate, however, the latter coculture used glucose. The PA-1 pure culture produced 0.86 mol of propionate per mol of succinate used during growth. PA-1 produced a small amount of H2. Strain PA-1 is the most versatile anaerobic bacterium yet known that catabolizes monobenzenoids in the absence of electron acceptors such as sulfate or nitrate. 相似文献
15.
16.
The hyperthermophilic anaerobic eubacterium Thermotoga maritima was grown on glucose as carbon and energy source. During growth 1 mol glucose was fermented to 2 mol acetate, 2 mol CO2 and 4 mol H2. The molar growth yicld on glucose (Yglucose) was about 45 g cell dry mass/mol glucose. In the presence of elemental sulfur growing cultures of T. maritima converted 1 mol glucose to 2 mol acetate, 2 mol CO2 about 0.5 mol H2 and about 3.5 mol H2S. Yglucose was about 45 g/mol. Cell extracts contained all enzymes of the Embden-Meyerhof pathway: hexokinase (0.29 U/mg, 50°C), glucose-6-phosphate isomerase (0.56 U/mg, 50°C), phosphofructokinase (0.19 U/mg, 50° C), fructose-1,6-bisphosphate aldolase (0.033 U/mg, 50°C), triosephosphate isomerase (6.3 U/mg, 50°C), glyceraldehyde-3-phosphate dehydrogenase (NAD+ reducing: 0.63 U/mg, 50°C), phosphoglycerate kinase (3.7 U/mg, 50°C), phosphoglycerate mutase (0.4 U/mg, 50°C); enolase (4 U/mg, 80°C), pyruvate kinase (0.05 U/mg, 50°C). Furthermore, cell extracts contained pyruvate: ferredoxin oxidoreductasee (0.43 U/mg, 60°C); NADH: ferredoxin oxidoreductase (benzylviologen reduction: 0.46 U/mg, 80°C); hydrogenase (benzylviologen reduction: 15 U/mg, 80°C), phosphate acetyltransferase (0.13 U/mg, 80°C), acetate kinase (1.2 U/mg, 55°C), lactate dehydrogenase (0.16 U/mg, 80°C) and pyruvate carboxylase (0.02 U/mg, 50°C). The findings indicate that the hyperthermophilic eubacterium T. maritima ferments sugars (glucose) to acetate, CO2 and H2 involving the Embden-Meyerhof pathway, phosphate acetyltransferase and acetate kinase. Thus, the organism differs from the hyperthermophilic archaeon Pyrococcus furiosus which ferments sugars to acetate, CO2 and H2 involving a modified non-phosphorylated Entner-Doudoroff pathway and acetyl-CoA synthetase (ADP forming). 相似文献
17.
A pathway for conversion of the metabolic intermediate phosphoenolpyruvate (PEP) and the formation of acetate, succinate, formate, and H2 in the anaerobic cellulolytic bacterium Ruminococcus flavefaciens FD-1 was constructed on the basis of enzyme activities detected in extracts of cells grown in cellulose- or cellobiose-limited continuous culture. PEP was converted to acetate and CO2 (via pyruvate kinase, pyruvate dehydrogenase, and acetate kinase) or carboxylated to form succinate (via PEP carboxykinase, malate dehydrogenase, fumarase, and fumarate reductase). Lactate was not formed even during rapid growth (batch culture, µ = 0.35/h). H2 was formed by a hydrogenase rather than by cleavage of formate, and 13C-NMR and14 C-exchange reaction data indicated that formate was produced by CO2 reduction, not by a cleavage of pyruvate. The distribution of PEP into the acetate and succinate pathways was not affected by changing extracellular pH and growth rates within the normal growth range. However, increasing growth rate from 0.017/h to 0.244/h resulted in a shift toward formate production, presumably at the presence of H2. This shift suggested that reducing equivalents could be balanced through formate or H2 production without affecting the yields of the major carbon-containing fermentation endproducts. 相似文献
18.
Jan R. Andreesen Ebtisam El Ghazzawi Gerhard Gottschalk 《Archives of microbiology》1974,96(1):103-118
In a medium containing a trace element solution and 10-4 M ferrous ions the growth yield ofClostridium formicoaceticum on fructose was 5.5 g of weight per l; in the absence of metal ion solution it was 1 g per l. The specific activity of methyl viologen dependent formate dehydrogenase under both conditions was 0.28 and 0.03 units per mg of protein, respectively. It could be increased to 9.75 units when the growth medium contained 10-4 M tungstate and 10-5 M selenite in addition. Molybdate was only about 40% as effective as tungstate. Tungstate or molybdate could not be replaced by vanadate, selenite not by sulfide. The formate dehydrogenase catalyzed also the reduction of CO2 to formate. The highest rate of formate synthesis was observed when pyruvate served as the reductant. No pyruvate: formate exchange but rapid pyruvate: CO2 exchange could be observed with cell-free extracts ofC. formicoaceticum. Pyruvate is fermented byC. formicoaceticum to yield up to 1.16 mole acetate per mole of pyruvate. Resting cells accumulated some formate in addition to acetate. 相似文献
19.
Abhishek Murarka James M. Clomburg Sean Moran Jacqueline V. Shanks Ramon Gonzalez 《The Journal of biological chemistry》2010,285(41):31548-31558
Pyruvate is located at a metabolic junction of assimilatory and dissimilatory pathways and represents a switch point between respiratory and fermentative metabolism. In Escherichia coli, the pyruvate dehydrogenase complex (PDHC) and pyruvate formate-lyase are considered the primary routes of pyruvate conversion to acetyl-CoA for aerobic respiration and anaerobic fermentation, respectively. During glucose fermentation, the in vivo activity of PDHC has been reported as either very low or undetectable, and the role of this enzyme remains unknown. In this study, a comprehensive characterization of wild-type E. coli MG1655 and a PDHC-deficient derivative (Pdh) led to the identification of the role of PDHC in the anaerobic fermentation of glucose. The metabolism of these strains was investigated by using a mixture of 13C-labeled and -unlabeled glucose followed by the analysis of the labeling pattern in protein-bound amino acids via two-dimensional 13C,1H NMR spectroscopy. Metabolite balancing, biosynthetic 13C labeling of proteinogenic amino acids, and isotopomer balancing all indicated a large increase in the flux of the oxidative branch of the pentose phosphate pathway (ox-PPP) in response to the PDHC deficiency. Because both ox-PPP and PDHC generate CO2 and the calculated CO2 evolution rate was significantly reduced in Pdh, it was hypothesized that the role of PDHC is to provide CO2 for cell growth. The similarly negative impact of either PDHC or ox-PPP deficiencies, and an even more pronounced impairment of cell growth in a strain lacking both ox-PPP and PDHC, provided further support for this hypothesis. The three strains exhibited similar phenotypes in the presence of an external source of CO2, thus confirming the role of PDHC. Activation of formate hydrogen-lyase (which converts formate to CO2 and H2) rendered the PDHC deficiency silent, but its negative impact reappeared in a strain lacking both PDHC and formate hydrogen-lyase. A stoichiometric analysis of CO2 generation via PDHC and ox-PPP revealed that the PDHC route is more carbon- and energy-efficient, in agreement with its beneficial role in cell growth. 相似文献