Abstract
The β-isopropylmalate dehydrogenase (β-IPMDH) gene of Acetobacter aceti No. 1023, which complemented the leuB mutation of Escherichia coli, was cloned and expressed in E. coll. Plasmids pCAL1 and pCAL4, carrying a 5.44 kilobase pairs (kb) HindIII-fragment in the opposite orientation, conferred the same β-IPMDH activity as that of the wild type strain of E. coli. Restriction mapping and deletion analysis indicated that the β-IPMDH gene was located on a 1.65kb AatII-HindIII fragment. Plasmids pMVL1 and pMVL2, composing the cloned β-IPMDH gene and plasmid pMV102, a plasmid indigenous to Acetobacter, were constructed as plasmid cloning vectors which allow selection of leu+ transformants in an A. aceti leu- host. The A. aceti leu- host was obtained through the insertional inactivation occurring as a result of homologous recombination between the chromosome of A. aceti and the cloned β-IPMDH gene, which was disrupted by insertion of the kanamycin resistance gene from pACYC177 into the BamHI site in the AatII-HindIII fragment. This system constitutes a relatively simple technique for gene disruption or replacement in Acetobacter that requires a single transformation.
