MGH-PGA Genomic Analysis of Stress and Inflammation:

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

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

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: 100 TnphoA 80 Mariner 60 # insertions/10Kb 40 20 0 1,000,000 2,000,000 3,000,000 4,000,000 5,000,000 6,000,000 Chromosomal Location

6. Mariner: pMFLGM.GB*- 2xT7 (pMAR 2XT7) lacZ Mariner Transposase 29 bp IR** pir-dependent ori T7 Promoter** pMAR 2xT7 Gm resistance T7 Promoter Tra genes 29 bp IR Amp resistance Inserted into the PA14 genome: * From John Mekalanos’s lab ** From Eric Rubin’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. Non-Redundant Library An arrayed collection of P. aeruginosa strains containing a known disruption in each non-essential open reading frame (ORF) in the genome Wild type Mutant #1 Mutant #2 Advantages: Limited number of mutants to screen (~5500). Learn which genes are NOT involved in a particular process.

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

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

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