Screening the TB Transposon Library for Mutants Attenuated in Virulence 
A signature tagged mutagenesis (STM) library was previously generated in the virulent P. aeruginosa CF isolate, TB, using the plasposon vector pTnModOGm [20],[35].
Auxotrophic mutants were removed from the library by pre-screening post-conjugation TB isolates onto minimal media and selecting only clones, which grew normally.
This pre-screening eliminated clones that might have exhibited attenuated virulence based upon reduced growth or viability, rather than loss of a specific virulence function.
After this initial segregation, 2,200 clones were picked into a 96-well plate and tested with the C. elegans high throughput-killing assay.
Each mutant was tested at least four times using this assay.
The mutants selected were segregated into three groups based upon their level of virulence attenuation as compared to the TB parental strain, which killed 85% of worms under the test conditions.
In group 1, we included 187 mutants (9% of total library) which allowed more than 50% worm survival, group 2 between 30% and 50% (536 mutants, 25%), and group 3 below 30% (1450 mutants, 65%), approaching the same level of killing as the parental isolate.
Within group 1, the 12 most highly attenuated isolates, classified as group 1A (0.4% of the total library), were further studied.
When cultured in the presence of these mutants more than 70% of worms survived, representing a near 5-fold reduction in virulence compared to TB.
