Role of NAD in regulating the adhE gene of Escherichia coli

Abstract

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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A Mutant in theADH1Gene ofChlamydomonas reinhardtiiElicits Metabolic Restructuring during Anaerobiosis

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