Rapid gene inactivation in Pseudomonas aeruginosa

A rapid and efficient method to inactivate genes in Pseudomonas aeruginosa has been developed. It is based on pKnockout vectors which carry either a g entamicin or a streptomycin/spectinomycin resistance cassette allowing for selection in P. aeruginosa where these vectors do not replicate. A PCR frag ment of the gene of interest carrying 5'- and 3'-truncations is cloned into a pKnockout vector. mobilized into P. aeruginosa, and subsequently integra ted into the chromosomal copy of the target gene. The orientation of the Fr agment determines whether (i) the target gene is disrupted without blocking the transcription of downstream genes or (ii) the insertion exerts a polar effect thereby leading to inactivation of a whole operon. (C) 2000 Federat ion of European Microbiological Societies. published by Elsevier Science B. V. All rights reserved.