A High-Throughput, Genome-Wide, Phenotypic Screen for
Genes Regulating Quorum Sensing in Pseudomonas aeruginosa
CDC/Janice Carr
© 2006 Lee et al
Tim Gobble
Dr. Martin Schuster
Department of Microbiology
Pseudomonas aeruginosa
CDC/Janice Carr
© 2006 Lee et al
1st most common cause of hospital-acquired pneumonia (17%)
3rd most common cause of urinary-tract infections (11%)
5th most common isolate from all sites (11%)
Pseudomonas aeruginosa
CDC/Janice Carr
© 2006 Lee et al
50% mortality rates for infections involving:
Cancer
Cystic fibrosis
Severe burns
Quorum Sensing in Vibrio fischeri
Autoinducer
LuxI
Quorum Sensing in Vibrio fischeri
LuxR
© 2003 Walt Disney Pictures
Transcription
of lux operon
Quorum Sensing in P. aeruginosa
LasI
LasR
RhlR
RhlI
Quorum Sensing in P. aeruginosa
las Regulators
las & rhl Regulators
las System
rhl System
las-regulated
rhl-regulated
•Elastase
•Rhamnolipids
•Pyocyanin
las
& rhlofregulated
2-5%
chromosome
•LasA
Non-Redundant Mutant Library

5,459 P. aeruginosa PA14 mutants

Subset of parental PA14 transposon insertion
library (Liberati et al., 2006)

Each mutant has a unique gene disabled by
transposon insertion
5’
3’
Transposon
3’
5’
5’
3’
3’
5’
Non-Redundant Mutant Library

5,459 P. aeruginosa PA14 mutants

Subset of parental PA14 transposon insertion
library (Liberati et al., 2006)

Each mutant has a unique gene disabled by
transposon insertion
5’
3’
Transposon
3’
5’
Quorum Sensing in P. aeruginosa
las Regulators
las & rhl Regulators
las System
rhl System
las-regulated
rhl-regulated
•Elastase
•Rhamnolipids
•Pyocyanin
las & rhl regulated
•LasA
Milk Plate Assay Extracellular proteolysis (Elastase)
Wild Type
lasR-
Quorum Sensing in P. aeruginosa
las Regulators
las & rhl Regulators
las System
rhl System
las-regulated
rhl-regulated
•Elastase
•Rhamnolipids
•Pyocyanin
las & rhl regulated
•LasA
Staphylolytic Activity Assay
FIGURE2.
2. Lysis
Lysisof
ofboiled
boiledStaphylococcus
Staphylococcusaureus
aureuscells
cellsby
byLasA
LasA
FIGURE
proteaseininliquid
liquidculture
culturesupernatant
supernatant
protease
0.25
0.2
OD600
wt
lasI
0.15
lasR
rhlR
0.1
rhlI
vfr
0.05
0
0
10
20
30
Time (min)
40
50
60
Pyocyanin Production Assay
Relative pyocyanin production – OD310 of liquid culture
supernatant
Rhamnolipid Production Assay
rhlR-
Disabled
Gene
Milk-plate
Assay
Staphylolytic
Act. Assay
Pyocyanin
Production
Rhamnolipid
Production
Wildtype
++
++
++
++
lasR
-
-
-
+
lasI
-
-
-
+/++
rhlR
++
+/-
-
-
rhlI
++
+/-
-
+/++
suhB
-
-
+/-
+/-
gidA
+
-
-
-
lepA
-
-
+
++
Possible Modes of Regulation
 suhB
 May carry a riboswitch in non-coding region of mRNA
 Riboswitch – structural RNA element within non-coding
regions of some mRNAs that directly sense metabolites and
directly regulate gene expression
 gidA
 May play a role in moderating translational fidelity
 lepA
 Newly discribed ribosomal elongation factor that
back-translocates the ribosome
Additional Studies
• Confirm transposon location
• Complementation
– Add plasmid with suhB, gidA or lepA genes
– Add plasmid with lasR gene
Acknowledgements

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
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Howard Hughes Medical Institute
Undergraduate Research Innovation
Scholarship & Creativity (URISC)
Dr. Martin Schuster
Daniel Lee
Frederick Ausubel & Nicole Liberati
Department of Microbiology
OSU Center for Genome Research and
Biocomputing
Dr. Kevin Ahern