Identification of methanol-regulated promoter sequences from the facultative methylotrophic bacterium...

A promoter-probe vector (pHX200) was constructed using the broad-host-range cosmid pLA2917 and a promoterless xylE gene of Pseudomonas as the reporter gene. Insertion of the cloned promoter fragment of the methanol dehydrogenase large subunit gene moxF (methanol oxidation) in front of the xylE gene in pHX200V-47 resulted in high-level expression of the xylE gene product--catechol 2,3-dioxygenase--in Methylobacterium 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 the 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 promoter clones were identified that originate from three gene clusters (groups V, VI and VII) on the M. organophilum XX chromosome involved in methanol oxidation. Vector pHX200 and its derivatives were successfully mobilized into cells of three phylogenetically diverse methylotrophic bacteria: Methylophilus methylotrophus AS1, Methylobacterium extorquens AM1 and Methylobacterium sp. DM4. The reporter gene (xylE) was functionally expressed in all three bacteria with the aid of a proper promoter. Transcriptional fusions of methanol-regulated promoters with the xylE gene were mobilized into Mox- mutants of M. organophilum XX and M. extorquens AM1 to study the roles of methanol oxidation genes, especially regulatory genes. It appeared that vector pHX200 is an efficient promoter probe with wide host-range and an excellent tool for studies of structure and function of promoters/regulators.