REGULATION OF EXPRESSION OF THE ETHANOL DEHYDROGENASE GENE (ADHE) IN ESCHERICHIA-COLI BY CATABOLITE REPRESSOR ACTIVATOR PROTEIN CRA

The adhE gene encodes ethanol dehydrogenase and is located at min 27.9 of the Escherichia coli chromosome. Expression of adhE is about 10-fo ld higher in cells grown anaerobically than in cells grown aerobically and is dependent on both transcriptional and posttranscriptional fact ors. In this study, a trans-regulatory element repressing adhE express ion was characterized by genetic and biochemical approaches. A mutatio n downregulating adhE expression was mapped at min 2 of the chromosome . DNA sequence analysis revealed a missense mutation in the cra gene, formerly known as fruR. The cra gene encodes a catabolite repressor-ac tivator protein (Cra) involved in the modulation of carbon flow in E. coli. The mutant protein (Cra) sustained an Arg148-->His substitution causing 1.5- and 3-fold stronger repression of adhE transcription und er anaerobic and aerobic conditions, respectively. By contrast, era nu ll mutants displayed 1.5- and 4-fold increased adhE transcription unde r those conditions. Disruption of the era gene did not abolish the ana erobic activation of the adhE gene but diminished it twofold. Cra and Cra were purified as fusion proteins tagged with an N-terminal 6xHis element. In vitro, both fusion proteins showed binding to the adhE pro moter region and to the control fruB promoter region, which is a known Cra target. However, only 6xHis-tagged Cra, and not 6xHis-Cra, was d isplaced from the DNA target by the effector, fructose-1-phosphate (F1 P), suggesting that the mutant protein is locked in a promoter-binding conformation and is no longer responsive to F1P. We suggest that Cra helps to tighten the control of adhE transcription under aerobic condi tions by its repression.