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 pCM1 joined to a pBR325 derivative carrying the neomycin phosphotransferase of transposon Tn5 and the gentamicin acetyltransferase of Tn1696. 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 10(3) transfectants per microgram 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 10(3) transformants per microgram 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.
Meletzus
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Gentechnologie/Mikrobiologie, Fakultät für Biologie, Universität Bielefeld, Federal Republic of Germany.
Eichenlaub
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R
D000192:Actinomycetales / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004558:Electric Stimulation; D004926:Escherichia coli / Q000235:genetics; D005821:Genetic Techniques; D005822:Genetic Vectors; D010641:Phenotype; D010957:Plasmids; D015183:Restriction Mapping; D014162:Transfection; D014169:Transformation, Bacterial
D004269:DNA, Bacterial