ROLE OF NAD IN REGULATING THE ADHE GENE OF ESCHERICHIA-COLI

The fermentative alcohol dehydrogenase of Escherichia coli is encoded by the adhE gene, which is induced under anaerobic conditions but repr essed in air. Previous work suggested that induction of adhE might dep end 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 amoun ts of NADH. Expression of adhE was monitored both by assay of alcohol dehydrogenase activity and by expression of phi(adhE'-lacZ) gene fusio ns. The expression of the adhE gene correlated with the ratio of NADH to NAD(+) The role of NADH in eliciting adhE induction was supported b y a variety of treatments known to change the ratio of NADH to NAD(+) or alter the total NAD(+)-plus-NADH pool. Blocking the electron transp ort chain, either by mutation or by chemical inhibitors, resulted in t he artificial induction of the adhE gene under aerobic conditions. Con versely, 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 su pplementation with exogenous NAD or nicotinic acid. In merodiploid str ains 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 ad hE 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 inc reased induction of the phi(adhE'-lacZ) fusion. Moreover, mutations gi ving elevated levels of active AdhE protein decreased the induction of the phi(adhE'-lacZ) fusion. This finding suggests that the enzymatic activity of the AdhE protein modulates the level of NADH under anaerob ic conditions, thus indirectly regulating its own expression.