Structure of the Pseudomonas putida alkBAC operon. Identification of transcription and translation products

The structural genes of the Pseudomonas oleovorans alk (alkane utilization) system, which are localized on the alkBAC operon, were cloned as a 16.9-kilobase pair EcoRI fragment. We have measured the length and determined the position of the alkBAC operon on this fragment by electron microscopy of R-loops. Furthermore, the 7.3-kilobase pair long alkBAC operon was analyzed for translation products in Escherichia coli minicells. Using a spectrum of overlapping subclones, six different proteins were identified. Starting from the alkBAC promotor, these polypeptides had molecular masses of 41, 15, 49, 58, 59, and 20 kDa, respectively. The 41-kDa protein was identified as alkane hydroxylase by reaction with a specific antibody. The 15- and 49-kDa peptides are soluble components of the alkane hydroxylase complex. The 58-kDa protein is most likely involved in alkanol dehydrogenase activity.