Characterization of alcohol dehydrogenase genes of derepressible wild-type Alcaligenes eutrophus H16 and constitutive mutants

Abstract

The nucleotide sequence of the gene that encodes the fermentative, derepressible alcohol dehydrogenase (ADH) in Alcaligenes eutrophus H16 and of adjacent regions was recently determined. Two potential -10 regions resembling the Escherichia coli sigma 70 consensus sequence were identified 77 and 93 nucleotides upstream of the structural gene. By determination of the 5' mRNA terminus of the wild-type adh gene, the proximal -10 region was identified as responsible for adh expression under derepressive conditions. Transcription started seven nucleotides downstream of this region, at position 388. Sequence analysis of seven mutants expressing the adh gene under aerobic conditions revealed mutations in one or the other potential -10 region. In all seven strains, the mutations restored the invariant T of the E. coli promoter consensus sequence. Mutants altered in the proximal -10 region transcribed the adh gene under aerobic conditions with the same 5' mRNA terminus as in the wild type; gene expression was impaired very little under aerobic conditions. Mutants altered in the distal -10 region also transcribed the adh gene aerobically but were still partially derepressible. The 5' mRNA terminus was seven nucleotides downstream of the distal -10 region, at position 372. When these mutants were cultivated under conditions of restricted oxygen supply, the adh gene was transcribed from both -10 regions, resulting in the synthesis of two mRNA species with different 5' termini.