A highly efficient transposon mutagenesis system for the tomato pathogen Clavibacter michiganensis subsp michiganensis

A transposon mutagenesis system for Clavibacter michiganensis subsp. michig anensis was developed based on antibiotic resistance transposons that were derived from the insertion element IS1409 from Arthrobacter sp. strain TM1 NCIB12013. As a prerequisite, the electroporation efficiency was optimized by using unmethylated DNA and treatment of the cells with glycine such that about 5 x 10(6) transformants per jag of DNA were generally obtained. Elec troporation of C. michiganensis subsp. michiganensis with a suicide vector carrying transposon Tn1409C resulted in approximately 1 x 10(3) transposon mutants per mug of DNA and thus is suitable for saturation mutagenesis. Ana lysis of Tn1409C insertion sites suggests a random mode of transposition. T ransposition of Tn1409C was also demonstrated for other subspecies of C. mi chiganensis.