Transformation of the phytopathogenic bacterium Clavibacter michiganense subsp. michiganense by electroporation and development of a cloning vector

We constructed a cloning vector for use in the plant pathogenic bacterium Clavibacter michiganense subsp. michiganense. The vector pDM100 consists of a 3.2-kb restriction fragment of the Clavibacter plasmid pCMl joined to a pBR325 derivative carrying the neomycin phosphotransferase of transposon TnS and the gentamicin acetyltransferase of Tnl696. Both antibiotic resistance genes are efficiently expressed in C. michiganense subsp. michiganense. Although polyethylene glycol-mediated transfection of spheroplasts with the DNA of the C. michiganense subsp. michiganense-specific bacteriophage CMP1 yielded about 3 x 103 transfectants per,ug of DNA, in transformations with plasmid DNA only a very few transformants were obtained. However, the transformation efficiency could be improved by electroporation of intact cells, giving about 2 x 103 transformants per,ug of plasmid DNA. Since a transformation procedure and a cloning vector are now available, pathogenicity in C. michiganense subsp. michiganense can now be analyzed genetically. Clavibacter michiganense is a gram-positive coryneform bacterium which is a pathogen for a variety of plants of agricultural importance such as tomato, potato, and maize (7, 8). Although strains isolated from different host plants are closely related, the host range is a strain-specific feature