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A Highly Efficient Transposon Mutagenesis System for the Tomato Pathogen Clavibacter michiganensis subsp. michiganensis

November 2001 , Volume 14 , Number  11
Pages  1,312 - 1,318

Oliver Kirchner , Karl-Heinz Gartemann , Eva-Maria Zellermann , Rudolf Eichenlaub , and Annette Burger

Fakultät für Biologie, Lehrstuhl für Mikrobiologie/Gentechnologie, Universität Bielefeld, Universitätsstr. 25, D-33615 Bielefeld, Germany

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Accepted 23 July 2001.

A transposon mutagenesis system for Clavibacter michiganensis subsp. michiganensis 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 × 106 transformants per μg of DNA were generally obtained. Electroporation of C. michiganensis subsp. michiganensis with a suicide vector carrying transposon Tn1409C resulted in approximately 1 × 103 transposon mutants per μg of DNA and thus is suitable for saturation mutagenesis. Analysis of Tn1409C insertion sites suggests a random mode of transposition. Transposition of Tn1409C was also demonstrated for other subspecies of C. michiganensis.

© 2001 The American Phytopathological Society