Summary: A promoter-probe vector (pHX200) was constructed using the broad-host-range cosmid pLA2917 and a promoterless gene of as the reporter gene. Insertion of the cloned promoter fragment of the methanol dehydrogenase large subunit gene (ethanol idation) in front of the gene in pHX200V-47 resulted in high-level expression of the gene product – catechol 2,3-dioxygenase – in XX. The specific activity of the enzyme was four times higher in methanol-grown XX culture than in succinate-grown culture. Interestingly, the insertion of the same fragment in the opposite orientation in front of the 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 XX chromosome involved in methanol oxidation. Vector pHX200 and its derivatives were successfully mobilized into cells of three phylogenetically diverse methylotrophic bacteria: AS1, AM1 and sp. DM4. The reporter gene () was functionally expressed in all three bacteria with the aid of a proper promoter. Transcriptional fusions of methanol-regulated promoters with the gene were mobilized into Mox mutants of XX and 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.


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