1. MGH-PGA Genomic Analysis of Stress and Inflammation: Pseudomonas aeruginosa Strain PA14 Non-Redundant Mutant Library Nicole T. Liberati, Dan G. Lee, Jacinto M. Villanueva and Frederick M. Ausubel Department of Molecular Biology Massachusetts General Hospital

2. How Good is the Throughput for Screening for P. aeruginosa Virulence Factors? 32 Pathogenicity genes identified out of 8,000 mutants screened. Genome size is ~6.5 Mb: ~5500 non-essential genes. Computer simulation: Screen ~30,000 random insertions to reach 95% saturation. Construct non-redundant library by sequencing 30,000 transposon insertion sites. –Limited number of mutants to screen (~5500). –Learn which genes are NOT involved in a particular process.

3. 1st PCR Reaction 2nd PCR Reaction PCR Cleanup and Sequencing 1 2 3 Arbitrary PCR to Amplify Sequence Adjacent to Transposon Insertion Transposon

4. PA14 Non-Redundant Library Protocol Plates with PA14 Transposon Mutants Cultures in 384 well plates Arbitrary PCR 1 96 well plates Arbitrary PCR 2 96 well plates Sequencing QBOT MySQL PA14 Database PA14 & PA01 Genomes Robot Biomek Robot Biomek 4. BLAST analysis 3. PHRED trimming 1. catalog 2. Record correspondences Biomek

5. Current Status of the Non-Redundant Library 45 x 384 (17,280) mutants generated Projected date of completion: 12/31/03 Insertions are reasonably random: 20 40 60 80 100 0 # insertions/10Kb Chromosomal Location 1,000,0002,000,0003,000,0004,000,0005,000,0006,000,000 TnphoA Mariner

6. Gm resistance Mariner Transposase Amp resistance pir-dependent ori Inserted into the PA14 genome: T7 Promoter** Tra genes pMAR 2xT7 lacZ T7 Promoter Mariner: pMFLGM.GB*- 2xT7 (pMAR 2XT7) 29 bp IR** 29 bp IR ** From Eric Rubin’s lab* From John Mekalanos’s lab

7. The PA14 Transposon Insertion Mutant Database (http://pga.mgh.harvard.edu/cgi-bin/pa14/mutants/retrieve.cgi) Authors: Daniel G. Lee, Nicole T. Liberati, Jonathan M. Urbach, Eric VanHelene, Jacinto M Villanueva,Tao Wei Frederick M. Ausubel

8. Database Request Page (http://pga.mgh.harvard.edu/cgi-bin/pa14/mutants/retrieve.cgi)

9. How Good is the Throughput for Screening for P. aeruginosa Virulence Factors? 32 Pathogenicity genes identified out of 8,000 mutants screened. Genome size is ~6.5 Mb: ~5500 non-essential genes. Screen ~30,000 random insertions to reach 95% saturation. Screen in several model hosts to identify all virulence factors. Screening for mutant phenotypes is rate limiting. Construct non-redundant library by sequencing 30,000 transposon insertion sites.

10. An arrayed collection of P. aeruginosa strains containing a known disruption in each non-essential open reading frame (ORF) in the genome Wild typeMutant #1Mutant #2 Non-Redundant Library Advantages : Limited number of mutants to screen (~5500). Learn which genes are NOT involved in a particular process.

11. Transposon: Kan r E. Coli P. aeruginosa Transposase Generation of Transposon Insertion Mutations Select for kanamycin-resistant P. aeruginosa

12. Non-redundant mutants from PA14 library A CB ABC Control Growth Condition (rich media) “red” probe Experimental Growth Condition (murine CF lung) “green” probe #1 #2 #3 Gene A oligos Gene B oligos Gene C oligos Microarray: 2 oligonucleotides Flanking each Insertion in the library “TraSH”* Methodology: Detection of the Absence of Transposon Mutant(s) in a Pool of Mutants *Transposon-Site Hybridization Sassetti, C. M., Boyd, D. H., and Rubin, E. J., (2001). PNAS 9812712-12717Badarinarayana, V., et al. (2001) Nature Biotechnology 19:1060-5

13. ~6.5 Mb ~5,500 non-essential genes ~5,225 non-redundant mutants 27,500 insertions 32,500 insertions Transposon Mutagenesis 5 fold excess Recovery/Sequencing failures Non-Redundant Library Size Choose most 5’ insertion in each ORF