IDENTIFICATION OF METHANOL-REGULATED PROMOTER SEQUENCES FROM THE FACULTATIVE METHYLOTROPHIC BACTERIUM METHYLOBACTERIUM-ORGANOPHILUM XX

A promoter-probe vector (pHX200) was constructed using the broad-host- range cosmid pLA2917 and a promoterless xylE gene of Pseudomonas as th e reporter gene. insertion of the cloned promoter fragment of the meth anol dehydrogenase large subunit gene moxF (methanol oxidation) in fro nt of the xylE gene in pHX200V-47 resulted in high-level expression of the xylE gene product - catechol 2,3-dioxygenase - in Methylobacteriu m organophilum XX. The specific activity of the enzyme was four times higher in methanol-grown M. organophilum XX culture than in succinate- grown culture. Interestingly, the insertion of the same fragment in th e opposite orientation in front of the xylE gene (pHX200V-74) also led to elevated catechol 2,3-dioxygenase activity. This promoter activity was also methanol regulated. A total of 21 methanol-regulated promote r clones were identified that originate from three gene clusters (grou ps V, VI and VII) on the M. organophilum XX chromosome involved in met hanol oxidation. Vector pHX200 and its derivatives were successfully m obilized into cells of three phylogenetically diverse methylotrophic b acteria: Methylophilus methylotrophus AS1, Methylobacterium extorquens AM1 and Methylobacterium sp. DM4. The reporter gene (xylE) was functi onally expressed in all three bacteria with the aid of a proper promot er. Transcriptional fusions of methanol-regulated promoters with the x ylE gene were mobilized into Mox- mutants of M. organophilum XX and M. extorquens AMI to study the roles of ethanol oxidation genes, especia lly regulatory genes. It appeared that vector pHX200 is an efficient p romoter probe with wide host-range and an excellent tool for studies o f structure and function of promoters/regulators.