Transcript C. elegans as a Model Host for Human Bacterial Pathogens

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
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.
Arbitrary PCR to Amplify Sequence
Adjacent to Transposon Insertion
1
Transposon
1st PCR Reaction
2
2nd PCR Reaction
3
PCR Cleanup and Sequencing
PA14 Non-Redundant Library Protocol
QBOT
Robot
Plates with
PA14 Transposon
Mutants
PA14 & PA01
Genomes
Cultures in
384 well plates
Biomek
Robot
Arbitrary
PCR 1
96 well plates
1. catalog
MySQL
PA14
Database
4. BLAST
analysis
Biomek
Arbitrary
PCR 2
96 well plates
2. Record
correspondences
3. PHRED
trimming
Biomek
Sequencing
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:
# insertions/10Kb
100
80
TnphoA
60
Mariner
40
20
0
1,000,000
2,000,000
3,000,000
4,000,000
Chromosomal Location
5,000,000
6,000,000
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
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
Database Request Page
(http://pga.mgh.harvard.edu/cgi-bin/pa14/mutants/retrieve.cgi)
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.
Simulation of 30,0 00 random transposon insertions in the P. aeruginosa genome
No insertions
1 insertion
2+ insertions
Total
•
# of Genes
301.6 (5.4%)
615.2 (11%)
4,653.2 (83.5%)
5,570
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.
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.
Generation of Transposon Insertion Mutations
Transposase
E. Coli
Transposon: Kanr
P. aeruginosa
Select for kanamycin-resistant P. aeruginosa
“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
A
Microarray:
2 oligonucleotides
Flanking each
Insertion in the
library
B
Gene A oligos
Badarinarayana, V., et al. (2001) Nature Biotechnology 19:1060-5
C
#3
A
Gene B oligos
B
C
Gene C oligos
Sassetti, C. M., Boyd, D. H., and Rubin, E. J., (2001). PNAS 9812712-12717
Non-Redundant Library Size
~5,500 non-essential genes
Transposon Mutagenesis
5 fold excess
27,500 insertions
~6.5 Mb
Recovery/Sequencing
failures
32,500 insertions
Choose most 5’
insertion in each ORF
~5,225 non-redundant mutants