Proton correlation nuclear magnetic resonance study of anaerobic metabolism of Escherichia coli

Ogino, Takashi; Arata, Y.; Fujiwara, S.; Shoun, Hirofumi; Beppu, Teruhiko

doi:10.1021/bi00615a022

Cited by 23 publications

(19 citation statements)

References 10 publications

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“…We monitored the synthesis of fermentation products by performing in vivo nuclear magnetic resonance (NMR) scans of whole cultures (1,22,23). B. subtilis cells were grown aerobically in E medium (per liter; 0.2 g of MgSO 4 ⅐ 7H 2 O, 2 g of citric acid, 13.1 g of K 2 HPO 4 , 3.5 g of NaNH 4 PO 4 ⅐ 4H 2 O, pH 7.0) containing 1% glucose, 1% sodium pyruvate, 0.2% glutamate, trace element solutions, and the auxotrophic requirement.…”

Section: Methodsmentioning

confidence: 99%

Characterization of anaerobic fermentative growth of Bacillus subtilis: identification of fermentation end products and genes required for growth

et al. 1997

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Bacillus subtilis can grow anaerobically by respiration with nitrate as a terminal electron acceptor. In the absence of external electron acceptors, it grows by fermentation. Identification of fermentation products by using in vivo nuclear magnetic resonance scans of whole cultures indicated that B. subtilis grows by mixed acid-butanediol fermentation but that no formate is produced. An ace mutant that lacks pyruvate dehydrogenase (PDH) activity was unable to grow anaerobically and produced hardly any fermentation product. These results suggest that PDH is involved in most or all acetyl coenzyme A production in B. subtilis under anaerobic conditions, unlike Escherichia coli, which uses pyruvate formate lyase. Nitrate respiration was previously shown to require the ResDE two-component signal transduction system and an anaerobic gene regulator, FNR. Also required are respiratory nitrate reductase, encoded by the narGHJI operon, and moaA, involved in biosynthesis of a molybdopterin cofactor of nitrate reductase. The resD and resDE mutations were shown to moderately affect fermentation, but nitrate reductase activity and fnr are dispensable for fermentative growth. A search for genes involved in fermentation indicated that ftsH is required, and is also needed to a lesser extent for nitrate respiration. These results show that nitrate respiration and fermentation of B. subtilis are governed by divergent regulatory pathways.Recent studies have shown that Bacillus subtilis, which had been widely believed to be a strict aerobe, can grow anaerobically in the presence of nitrate (3,8,13,15,20,25,27,29,34). Respiratory nitrate reductase encoded by the narGHJI operon (3) was shown to be responsible for nitrate respiration (13, 15). Mutations in moaA (formerly narA), the product of which shows homology to the Escherichia coli moaA gene product (26), impair nitrate respiration, probably by conferring a defect in the biosynthesis of the nitrate reductase cofactor (8). Transcription of narGHJI and narK (required for nitrite extrusion [3]) is induced by oxygen limitation, and the induction is completely abolished by mutations in fnr, the second gene of the narK-fnr operon (3,15). FNR is known to be a global anaerobic gene regulator in E. coli and has amino acid sequence similarity to the catabolite activator protein (30). In E. coli, the activity of FNR, but not the expression of fnr, was shown to be stimulated by anaerobiosis. This is believed to be due to the cluster of cysteine residues in the amino terminus of the protein that may play a role in modulating FNR activity by a mechanism involving bound iron (9, 10, 37). Unlike E. coli, in which fnr expression is weakly repressed by anaerobiosis, fnr expression in B. subtilis is strongly induced by oxygen limitation (3,20). Anaerobic induction of fnr transcription is controlled at two levels. First, fnr transcription at an intergenic fnr-specific promoter is activated by oxygen limitation and requires phosphorylated ResD, the production of which depends on a cognate histidine se...

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Section: Methodsmentioning

confidence: 99%

Characterization of anaerobic fermentative growth of Bacillus subtilis: identification of fermentation end products and genes required for growth

et al. 1997

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“…These experiments were modeled on the work of Ogino et al (15,16), who monitored the synthesis of fermentation products by performing in vivo NMR scans of whole cultures. E. coli cells were grown in M9 medium (pH 7.2) in the presence of 100 mM glucose as the sole carbon source.…”

Section: Methodsmentioning

confidence: 99%

“…As far as we know, no systematic survey has been done to relate the redox level of the fermentable substrate to the composition of the fermentation product. Here we report such a study based on the use of in vivo nuclear magnetic resonance (NMR) of anaerobic cultures (15,16). The use of in vivo NMR avoids compromising anaerobic conditions by withdrawing samples, and it simultaneously analyzes all fermentation products.…”

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confidence: 99%

Anaerobic fermentation balance of Escherichia coli as observed by in vivo nuclear magnetic resonance spectroscopy

Alam

Clark

1989

J Bacteriol

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Fermenting anaerobic cultures of Escherichia coli were observed by the nonintrusive technique of in vivo, whole-culture nuclear magnetic resonance. Fermentation balances were calculated for hexoses, pentoses, sugar alcohols, and sugar acids. Substrates more reduced than glucose yielded more of the highly reduced fermentation product ethanol, whereas more-oxidized substrates produced more of the less-reduced fermentation product acetate. These relationships were made more obvious by the introduction of ldhA mutations, which abolished lactate production, and Afrd mutations, which eliminated succinate. When grown anaerobically on sugar alcohols such as sorbitol, E. coli produced ethanol in excess of the amount calculated by the standard fermentation pathways. Reducing equlivalents must be recycled from formate to account for this excess of ethanol. In mutants deficient in hydrogenase (hydB), ethanol production from sorbitol was greatly decreased, implying that hydrogen gas released from formate by the formate-hydrogen lyase system may be partially recycled, in the wild type, to increase the yield of the highly reduced fermentation product ethanol.The well-known laboratory organism Escherichia coli is a facultative anaerobe. In the absence of oxygen and alternative electron acceptors such as nitrate (11), E. coli ferments sugars and their derivatives to a mixture of acids and ethanol (D. P. Clark, FEMS Microbiol. Rev., in press). This has been referred to as the mixed-acid fermentation because a mixture of acetic, lactic, formic, and succinic acids is produced (8,19). The ratio of these products has been measured by classical chemical methods for glucose fermentation under various culture conditions (7,17,20). Generally speaking, the overall proportion of lactic acid increases under acidic conditions, while the ratio of ethanol to acetate remains approximately 1:1 (1, 8). We have shown that the fermentative lactate dehydrogenase, which is responsible for lactic acid synthesis, is conjointly induced by acidic and anaerobic conditions (13). On the other hand, earlier reports that the nature of the buffer affects the fermentation product ratio (8,20) were not confirmed (13). We believe that the difference in buffering strength and pK between various buffers may give the illusory appearance of such an effect. However, when these factors are allowed for, only the overall pH maintained is significant (13).A factor which greatly affects the fermentation product ratio is the redox level of the substrate relative to glucose. Thus, sugar alcohols, which are more reduced than the corresponding hexoses, must yield a higher proportion of more-reduced fermentation products in order to achieve hydrogen balance (Clark, in press). As far as we know, no systematic survey has been done to relate the redox level of the fermentable substrate to the composition of the fermentation product. Here we report such a study based on the use of in vivo nuclear magnetic resonance (NMR) of anaerobic cultures (15, 16). The use of in vivo NMR avoids...

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“…Observation of fermentation products by NMR. These experiments were modeled on the work of Ogino and coworkers (23,24), who monitored the synthesis of fermentation products by performing in vivo nuclear magnetic resonance (NMR) scans of whole cultures. E. coli were grown in M9 medium (pH 7.2) in the presence of 0.1 M glucose as the sole carbon source.…”

Section: Methodsmentioning

confidence: 99%

Escherichia coli derivatives lacking both alcohol dehydrogenase and phosphotransacetylase grow anaerobically by lactate fermentation

Gupta

Clark

1989

J Bacteriol

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Escherichia coli mutants lacking alcohol dehydrogenase (adh mutants) cannot synthesize the fermentation product ethanol and are unable to grow anaerobically on glucose and other hexoses. Similarly, phosphotransacetylase-negative mutants (pta mutants) neither excrete acetate nor grow anaerobically. However, when a strain carrying an adh deletion was selected for anaerobic growth on glucose, spontaneous pta mutants were isolated. Strains carrying both adh and pta mutations were observed by in vivo nuclear magnetic resonance and shown to produce lactic acid as the major fermentation product. Various combinations of ad/h pta double mutants regained the ability to grow anaerobically on hexoses, by what amounts to a homolactic fermentation. Unlike wild-type strains, such adh pta double mutants were unable to grow anaerobically on sorbitol or on glucuronic acid. The growth properties of strains carrying various mutations affecting the enzymes of fermentation are discussed in terms of redox balance.

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Proton correlation nuclear magnetic resonance study of anaerobic metabolism of Escherichia coli

Cited by 23 publications

References 10 publications

Characterization of anaerobic fermentative growth of Bacillus subtilis: identification of fermentation end products and genes required for growth

Characterization of anaerobic fermentative growth of Bacillus subtilis: identification of fermentation end products and genes required for growth

Anaerobic fermentation balance of Escherichia coli as observed by in vivo nuclear magnetic resonance spectroscopy

Escherichia coli derivatives lacking both alcohol dehydrogenase and phosphotransacetylase grow anaerobically by lactate fermentation

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