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...

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...

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