The Afipia toolbox and its use to isolate flagellar mutants.

Afipia felis, a Gram-negative alphaproteobacterium, has been implicated as one of the causative agents of cat scratch disease. To identify and begin to examine the virulence traits of this organism, we developed and tested a highly efficient transposon delivery system and a stable plasmid vector expressing green fluorescent protein. The transposome system is based on a Tn5-derived transposon and a phage restriction endonuclease type I inhibitor. Electroporation of this construct produced a library of >2600 mutants, which were screened for flagella biosynthesis mutants using a monoclonal antibody to Afipia flagellin. Insertion loci for two selected mutants were located in the genes for flagellin and flagellin biosynthesis FlhA, confirming the validity of the approach.