Effective Mutagenesis of Vibrio fischeri by Using Hyperactive Mini-Tn5 Derivatives

We have developed a transposon mutagenesis system for Vibrio fischeri ES114 that utilizes a hyperactive mutant Tn5 transposase (E54K and M56A) and optimized transposon ends. Using a conjugation-based procedure, we obtained independent single-insertion mini-Tn5 mutants at a rate of similar to 10(-6). This simple and inexpensive technique represents a significant improvement over previous methods for transposon mutagenesis of V. fischeri and should be applicable to many other bacteria.