High-efficiency transposon mutagenesis by electroporation of a Pseudomonas fluorescens strain

Abstract

A method is described for mutagenesis of Pseudomonas fluorescens strains by electroporation with the transposon delivery vector pUT/mini-Tn5 Km. The transposition process was shown to be optimal at 12.5 kV cm-1 for a pulse time (Bowen and Koslak, 1992) of about 4 ms. The Pseudomonas fluorescens L6.5 target strain exhibited maximal electrocompetence when harvested at the middle of the exponential growth phase. As many as 7.7 10(5) mutants per picomole of delivery vector (7.5 kb) could be obtained, and these kanamycin-resistant mutants were shown to have lost the pUT plasmid. By external calibration with plasmids of increasing size (from 11.5 to 60.1 kb), the efficiency of the transformation process was evaluated to be approximately 1.31 x 10(8) transformants per picomole of delivery vector. Efficiency of the transposition process was 0.58%. This rapid method was used to tag for the cloning three independent chromosomal loci responsible for the Alk+ phenotype of Pseudomonas fluorescens L6.5 strain.