Under anaerobic conditions and in the absence of alternative electron acceptors Escherichia coli converts sugars to a mixture of products by fermentation. The major soluble products are acetate, ethanol, acetate and formate with smaller amounts of succinate. In addition the gaseous products hydrogen and carbon dioxide are produced in substantial amounts. The pathway generating fermentation products is branched and the flow down each branch is varied in response both to the pH of the culture medium and the nature of the fermentation substrate. In particular, the ratio of the various fermentation products is manipulated in order to balance the number of reducing equivalents generated during glycolytic breakdown of the substrate. The enzymes and corresponding genes involved in these fermentation pathways are described. The regulatory responses of these genes and enzymes are known but the details of the underlying regulatory mechanisms are still obscure.

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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...

Escherichia coli NZN111 is blocked in the ability to grow fermentatively on glucose but gave rise spontaneously to a mutant that had this ability. The mutant carries out a balanced fermentation of glucose to give approximately 1 mol of succinate, 0.5 mol of acetate, and 0.5 mol of ethanol per mol of glucose. The causative mutation was mapped to the ptsG gene, which encodes the membrane-bound, glucose-specific permease of the phosphotransferase system, protein EIICB glc . Replacement of the chromosomal ptsG gene with an insertionally inactivated form also restored growth on glucose and resulted in the same distribution of fermentation products. The physiological characteristics of the spontaneous and null mutants were consistent with loss of function of the ptsG gene product; the mutants possessed greatly reduced glucose phosphotransferase activity and lacked normal glucose repression. Introduction of the null mutant into strains not blocked in the ability to ferment glucose also increased succinate production in those strains. This phenomenon was widespread, occurring in different lineages of E. coli, including E. coli B.

Under anaerobic conditions, especially a t low pH, Escherichia coli converts pyruvate to D-lactate by means of an NADH-linked lactate dehydrogenase (LDH). This LDH is present in substantial basal levels under all conditions but increases approximately 10-fold at low pH. The ldhA gene, encoding the fermentative lactate dehydrogenase of E. coli, was cloned using iZ10E6 of the Kohara collection as the source of DNA. The ldhA gene was subcloned on a 2.8 kb Mlul-Mlul fragment into a multicopy vector and the region encompassing the gene was sequenced. The ldhA gene of E. coli was highly homologous to genes for other D-lactate-specif ic dehydrogenases but unrelated to those for the L-lactate-specific enzymes. We constructed a disrupted derivative of the ldhA gene by inserting a kanamycin resistance cassette into the unique Kpnl site within the coding region. When transferred to the chromosome, the ldhA: :Kan construct abolished the synthesis of the D-LDH completely. When present in high copy number, the ldhA gene was greatly overexpressed, suggesting escape from negative regulation. Cells expressing high levels of the D-LDH grew very poorly, especially in minimal medium. This poor growth was largely counteracted by supplementation with high alanine or pyruvate concentrations, suggesting that excess LDH converts the pyruvate pool to lactate, thus creating a shortage of 3-carbon metabolic intermediates. Using an IdhA-cat gene fusion construct w e isolated mutants which no longer showed pH-dependent regulation of the ldhA gene. Some of these appeared to be in the pta gene, which encodes phosphotransacetylase, suggesting the possible involvement of acetyl phosphate in ldhA regulation.

The genes encoding essential enzymes of the fermentative pathway for ethanol production in Zymomonas mobilis, an obligately ethanologenic bacterium, were inserted into Escherichia coli under the control of a common promoter. Alcohol dehydrogenase II and pyruvate decarboxylase from Z. mobilis were expressed at high levels in E. coli, resulting in increased cell growth and the production of ethanol as the principal fermentation product from glucose. These results demonstrate that it is possible to change the fermentation products of an organism, such as E. coli, by the addition of genes encoding appropriate enzymes which form an alternative system for the regeneration of NAD+.

The fermentative alcohol dehydrogenase of Escherichia coli is encoded by the adhE gene, which is induced under anaerobic conditions but repressed in air. Previous work suggested that induction of adhE might depend on NADH levels. We therefore directly measured the NAD ؉ and NADH levels for cultures growing aerobically and anaerobically on a series of carbon sources whose metabolism generates different relative amounts of NADH. Expression of adhE was monitored both by assay of alcohol dehydrogenase activity and by expression of (adhE-lacZ) gene fusions. The expression of the adhE gene correlated with the ratio of NADH to NAD ؉ . The role of NADH in eliciting adhE induction was supported by a variety of treatments known to change the ratio of NADH to NAD ؉ or alter the total NAD ؉ -plus-NADH pool. Blocking the electron transport chain, either by mutation or by chemical inhibitors, resulted in the artificial induction of the adhE gene under aerobic conditions. Conversely, limiting NAD synthesis, by introducing mutational blocks into the biosynthetic pathway for nicotinic acid, decreased the expression of adhE under anaerobic conditions. This, in turn, was reversed by supplementation with exogenous NAD or nicotinic acid. In merodiploid strains carrying deletion or insertion mutations abolishing the synthesis of AdhE protein, an adhE-lacZ fusion was expressed at nearly 10-fold the level observed in an adhE ؉ background. Introduction of mutant adhE alleles producing high levels of inactive AdhE protein gave results equivalent to those seen in absence of the AdhE protein. This finding implies that it is the buildup of NADH due to lack of enzyme activity, rather than the absence of the AdhE protein per se, which causes increased induction of the (adhE-lacZ) fusion. Moreover, mutations giving elevated levels of active AdhE protein decreased the induction of the (adhE-lacZ) fusion. This finding suggests that the enzymatic activity of the AdhE protein modulates the level of NADH under anaerobic conditions, thus indirectly regulating its own expression.The cofactor NAD plays a key role in many biological oxidation-reduction reactions. The maintenance of bacterial metabolism depends on these redox reactions both for biosynthetic intermediates and for the reducing equivalents generated. Thus, any investigation of the regulation of the metabolism of facultative anaerobes must consider the potential role of this cofactor. In Escherichia coli, the glycolytic pathway and tricarboxylic acid cycle are the major source of metabolic intermediates and the reduced cofactor, NADH (30). Without NADH reoxidation, the NAD ϩ pool would be quickly depleted, halting cellular metabolism and growth. When oxygen is present, reducing equivalents from NADH are transferred to the electron transport chain (ETC), generating H 2 O and a membrane potential which is used to synthesize ATP (31). This regenerates NAD ϩ for use in subsequent reactions. When enterobacteria grow anaerobically, an electron transport system is available only if alternate elect...

Mutants of Escherichia coli deficient in the fermentative NAD-linked lactate dehydrogenase (ldh) have been isolated. These mutants showed no growth defects under anaerobic conditions unless present together with a defect in pyruvate formate lyase (pfl (12,14,22). The conversion of pyruvate to lactic acid under anaerobic conditions is catalyzed by a third enzyme (28). This is a soluble, NAD-linked enzyme that is specific for the production of D-lactic acid (27,28). The fermentative LDH has been purified and is allosterically activated by its substrate, pyruvate (28,29). The fermentative LDH is found in both aerobically and anaerobically grown cultures (27,28 fermentative LDH. Such ldh mutants were characterized to assess the physiological importance of lactate production. MATERIALS AND METHODSBacterial strains, media, and genetic methods. All bacteria were strains of E. coli K-12 except for strain Cl, which is the E. coli C type strain (Table 1). Rich broth contained (per liter) tryptone (10 g), NaCI (5 g), and yeast extract (1 g). Minimal medium M9 (15) was supplemented with carbon sources at 0.4% (wt/vol) and, where appropriate, with amino acids (50 mg/liter). Solid media contained 1.5%a (wt/vol) Difco Bacto-Agar. Anaerobic growth was performed in Oxoid anaerobic jars under an H2-CO2 atmosphere generated by Oxoid gas-generating kits. All anaerobic growth media were supplemented with trace elements Fe (50 ,uM), Se (5 ,M), Mo (5 ,uM), and Mn (5 p.M) as previously described (8, 34). Colonies were stained for pyruvate formate lyase by the benzyl viologen procedure (31). Methods for mutagenesis with ethyl methane sulfona,te, for transduction with phage P1 vir, and for mapping with the Hfr::TnlO set (32) have been detailed in recent publications from this laboratory (1, 34).Enzyme assays. Soluble extracts were made by breaking cells in the French press followed by ultracentrifugation to remove membrane fragments. This procedure was previously used to prepare extracts for the assay of alcohol dehydrogenase (5, 6). The NAD-linked LDH was assayed by a slight modification of the method of Tarmy and Kaplan (28). One unit of LDH activity is 1 A340 unit per min.NMR. The nuclear magnetic resonance (NMR) experiments were modeled on the work of Ogino et al. (16,17), who monitored the synthesis of fermentation products by in vivo NMR scans of whole cultures. Cells were grown in M9 medium (pH 7.2) with 0.1 M glucose as the sole carbon source. When the cell density reached approximately 5 x 108/ml the cells were collected by centrifugation at 7,000 rpm (5,900 x g) for 2 min at 5'C. The cell pellets were suspended in M9 buffer and washed twice with the same buffer. The cells were finally suspended in M9 buffer (pH 7.2) containing 0.1 M glucose and anaerobic trace metals at a cell density of 5 x 108/ml. A 0.9-ml sample of cell suspension was placed in