Genetic footprinting with mariner-based transposition in Pseudomonas aeruginosa

The complete DNA sequence of Pseudomonas aeruginosa provides an opportunity to apply functional genomics to a major human pathogen, A comparative geno mics approach combined with genetic footprinting was used as a strategy to identify genes required for viability in P. aeruginosa, Use of a highly eff icient in vivo mariner transposition system in P. aeruginosa facilitated th e analysis of candidate genes of this class. We have developed a rapid and efficient allelic exchange system by using the I-Scel homing endonuclease i n conjunction with in vitro mariner mutagenesis to generate mutants within targeted regions of the P. aeruginosa chromosome for genetic footprinting a nalyses. This technique for generating transposon insertion mutants should be widely applicable to other organisms that are not naturally transformabl e or may lack well developed in vivo transposition systems. We tested this system with three genes in P, aeruginosa that have putative essential homol ogs in Haemophilus influenzae, We show that one of three H. influenzae esse ntial gene homologs is needed for growth in P, aeroginosa, validating the p racticality of this comparative genomics strategy to identify essential gen es in P. aeruginosa.