Molecular cloning and analysis ofadhR, a protein regulating alcohol fermentation in Escherichia coli

Previous work suggested that induction of the fermentative alcohol dehydrogenase gene of Escherichia coli is NADH-dependent and that the regulatory proteins FNR or CRP might be involved in the regulatory pathway. We have shown that the presence of mutations in fnr or crp or both together had no effect on expression of the adhE gene. Studies using deletion strains revealed that the first 5' 380 nucleotides of adhE regulatory region could be deleted without any effect on ADH expression. Primer extension analysis revealed two transcription start sites at position --272 and --647 relative to the start codon.; The adhR gene, encoding a regulatory protein for the fermentative alcohol dehydrogenase, was cloned using l 3C5 of the Kohara collection as the source of DNA. The gene was subsequently subcloned into a variety of plasmids in order to test its physiological importance. We constructed a disrupted derivative of the adhR gene by inserting a kanamycin cassette within the coding region. When integrated into the chromosome, the adhR::Kan had three distinct physiological consequences: first, it prevented growth on acetate minimal medium. Second, it prevented anaerobic growth on highly reduced carbon sources. Third, it caused an 8-fold decrease in the level of ADH expression anaerobically. We sequenced the adhR gene which codes for 368 amino acids and constructed a protein fusion with AdhR fused to the maltose binding protein. The purified protein was shown to bind a 386 bp fragment within the upstream regulatory region of adhE. Binding of AdhR was NAD(H)-dependent.; We have isolated mutants that no longer show normal regulation of adhE. These mutants are able to overproduce ADH when glucuronate, which does not generate any reducing equivalents when metabolized, is used as carbon source. The mutations responsible for this phenotype appear to be located at the adhR locus.