Physical structure, genetic content and expression of the alkBAC operon

We cloned sequences of the alk (alkane utilization) operon of Pseudomonas and characterized them physically and genetically. These sequences were used to construct a DNA restriction map of the alkBAC region. We physically mapped alk::Tn7 insertions and delta alkBA deletions, and we were able to show complementation or marker rescue of alk point mutations by cloned DNA sequences. Our results confirmed the existence of an operon containing structural loci encoding activities for membrane alkane hydroxylase component (alkB), soluble alkane hydroxylase component (alkA) and membrane alcohol dehydrogenase (alkC). Physical mapping of alkC::Tn7 insertions and complementation of alkC point mutations by cloned sequences from the alkBA region showed that we were previously mistaken in inferring the existence of a separate unlinked alkC cluster. Studies with an alkB-lacZ transcription fusion construct established that the operon is transcribed in the order alkBAC and is under positive regulation by alkR regulatory functions.