The importance of homologous recombination in the generation of large deletions in hybrid plasmids in Amycolatopsis mediterranei

The cloning vector pRL60 was developed previously as a tool for genetic man ipulations in Amycolatopsis mediterranei, which produces the commercially a nd medicinally important antibiotic rifamycin. Here, a method based on intr aplasmid recombinations is described for the construction of smaller plasmi ds in A. mediterranei, which also helped in delimiting the origin of replic ation (pA-rep) of the parent plasmid. The strategy involved the cloning of a selectable marker, erythromycin resistance gene (ermE), onto plasmids pUL AM2 and pULVK2A (derivatives of pRL1), followed by selection of the hybrid or concatemeric plasmids pRL50 and pRL80 (with large homologous repeats) in Escherichia coli GM2163. These hybrid plasmids were then transferred to A. medirerranei DSM 40773 by electroporation, with selection in the presence of different antibiotics. During the process of transformation and selectio n in A. mediterranei, pRL50 and pRL80 underwent intraplasmid recombinations , yielding derivatives that retained a common region essential for maintena nce and replication, as well as the selected resistance genes. This approac h produced several smaller plasmids designated pRL51, pRL52, pRL53, pRL60, pRL81, and pRL82. These plasmids, isolated from A. mediterranei DSM 40773, could be transferred to different Amycolatopsis strains at transformation e fficiencies ranging from 0.7 x 10(2) to 4 x 10(4) transformants/mu g DNA. T he electroporation parameters under which maximum transformation efficienci es were obtained varied from strain to strain. Since the isolation of plasm id DNA from Amycolatopsis strains were extremely difficult, a convenient an d rapid method of direct transfer of plasmid DNA, i.e., electroduction, was also developed in which the above-described shuttle plasmids were transfer red directly from A. mediterranei to E. coli In addition, the sequence of t he minimal (pA-rep, similar to 1.0 kb) of plasmid pRL51 was determined. The nucleotide base sequence of the pA-rep region did not have any clear simil arity to the DNA or amino acid sequences in various databases, suggesting t hat it is unique. (C) 2000 Academic Press.