Pseudomonas aeruginosa

For Pseudomonas aeruginosa, the leading cause of death in patients with cystic fibrosis, two large-scale libraries of mutants were generated in two different projects using multipurpose Tn5 derivatives, which were introduced into the genome by conjugation. The mini-transposons harbored promoterless phoA and lacZ [27] or Photorhabdus luminescens luxCDABE reporter genes (http://pseudomutant. pseudomonas.com/) capable of generating translational fusions if appropriately inserted in a target gene. In both cases, the identification of the transposon insertion sites, which clearly represents the limiting step in this approach where large number of mutants are to be analyzed, relied on less laborious methods than in the previous effort. Briefly, efficient PCR techniques, either inverse PCR (http:// pseudomutant.pseudomonas.com/) or a two-stage semidegenerate PCR [27], were used to amplify the transposon insertion sites and the PCR products were directly sequenced. In the first project [27], where 42 240 mutants were arrayed, 36154 of the transposon insertions sites (80%) were successfully mapped, of which 30100 were unique. This yielded insertions in 4892 different ORFs, representing 87.8% of the annotated coding sequences in the P. aeruginosa genome. Since it was estimated that 300-400 of the 678 ORFs in which transposon insertions were not recovered are likely to be essential, the mutational coverage was probably around 94%, which is a major achievement. In the second project (http://pseudomutant. pseudomonas.com/), although the coverage was more modest, 2519 insertion sites were mapped, yielding insertion in 1284 different ORFs. Interestingly, in

1253 mutants, the mini-transposon generated active translational fusions with the P. luminescens lux genes. Importantly, the potential of the larger collection of mutants in producing essentially complete lists of candidate genes implicated in various biological processes has been validated by the results of two simple screens aimed at phenotypes with well-understood genetic bases [27], these being twitching motility and prototrophic growth. In both cases, all the genes expected from earlier studies and several previously undescribed candidates were identified.

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