ELECTROPORATION OF ALCALIGENES-EUTROPHUS WITH (MEGA) PLASMIDS AND GENOMIC DNA FRAGMENTS

Electroporation was used as a tool to explore the genetics of the heav y-metal-resistant strain Alcaligenes eutrophus CH34. A 12.9-kb A. eutr ophus-Escherichia coli shuttle vector, pMOL850, was constructed to opt imize electroporation conditions. This vector is derived from the E. c oil plasmid pSUP202 and contains the replication region of the A. eutr ophus megaplasmid pMOL28. Electroporation was used to transform A. eut rophus CH34 derivatives with megaplasmids (sizes up to 240 kb), and tr ansformants were selected for resistance to heavy metals. Electroporat ion was also performed with endonuclease-digested genomic DNA. Transfo rmation of markers affecting lysine biosynthesis (lysA194) and biosynt hesis of the siderophore alcaligin E were observed. Transfer of the no nselected markers pheB332 and aro-333, linked to lysA194, confirmed th e intervention of homologous recombination. However, during transforma tion of ale::Tn5-Tc, illegitimate recombination and transposition were also observed as an alternative for the inheritance of the Tn5-Tc mar kers.