Curation Information

Publication
Global position analysis of the Pseudomonas aeruginosa quorum-sensing transcription factor LasR.;Gilbert KB, Kim TH, Gupta R, Greenberg EP, Schuster M;Molecular microbiology 2009 Sep; 73(6):1072-85 [19682264]
TF
LasR [P25084, view regulon]
Reported TF sp.
Pseudomonas aeruginosa PAO1
Reported site sp.
Pseudomonas aeruginosa PAO1
Created by
Erill Lab
Curation notes
-

Experimental Process

ChIP-chip analysis with LasR. ChIP on w-t and lasR rhlR mutant. Sites identified by motif discovery (CONSENSUS) and statistical validation (PATSER) on significantly enriched ChIP peaks. Expression data inferred from previous DNA array from same authors (PubMed: 12644476).

ChIP assay conditions
LasR_Paur: We employed ChIP-chip to identify direct targets of LasR in vivo. We used early-stationary phase P. aeruginosa cultures grown in buffered Lennox LB. These conditions are identical to our previous transcriptome analysis, allowing direct comparison of data sets, and they result in high-level induction of QS target genes (Schuster et al., 2003). We chose the wild type strain expressing lasR in its native context to capture protein-DNA interactions under physiologically relevant condition
ChIP notes
Cells were inoculated from mid-logarithmic phase cultures to initial optical densities of 0.01. The experimental strain was P. aeruginosa PAO1, and the nonspecific control strain was an isogenic lasR, rhlR double mutant.Ten ml aliquots were cross- Gilbert et al. Page 8 Mol Microbiol. Author manuscript; available in PMC 2010 September 1. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript linked by the addition of formaldehyde to a final concentration of 1% and incubation at room temperature for 15 min with gentle agitation. The cross-linking reaction was quenched by the addition of fresh glycine to a final concentration of 125 mM and incubation at room temperature for 10 min. Next, the cells were washed twice in 10 ml of cold 1xTBS (20 mM Tris-HCL, pH 7.5, 150 mM NaCl) and the pellets were frozen at −80°C. After thawing, cells were resuspended in 1 ml immunoprecipitation (IP) buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% deoxycholic acid) supplemented with 250x diluted Protease Inhibitor Cocktail (Set III, Novagen) and 1 mg/ml lysozyme. The suspension was incubated at 37°C for 15 minutes. Samples were chilled on ice and sonicated three times for 10 seconds each using a Branson microtip sonicator at an output of 0.4 with no pulse. A 25 μl aliquot from the cleared lysate was set aside as the total DNA control. P. aeruginosa whole-genome microarrays, including all intergenic regions, were custommade by Nimblegen and consisted of 60-mers tiled with a spacing of 30 bp. The ChIP DNA and control input DNA were differentially labeled, hybridized to one array, and scanned. Separate arrays were used for wild type (specific) and lasR rhlR mutant (non-specific) samples. Intensity data (signals) were normalized within each array and also across all arrays, using Loess normalization. This procedure resulted in comparable array signals for the entire data set in terms of mean signal and variance. After this two-step normaliza

Transcription Factor Binding Sites


CCATCTGACATGTAGG
CCTGGAGACCAGGCTG
CAATCAGAAATGTCCC
CTAACAAAAGACATAG
CCGTCAGTATTGTTAG
CTGTGAAATCTGGCAG
CTACCAGATCTGGCAG
CTGTCGGTTCGCATAG
CTGCCCATAATCCCCG
CTATCAGTTCTGTAGT
CTGTGAGATTTGGGAG
CTACCTGTTTTGGTAG
CTATCAGAATGAATAG
CTATCCATTCTGATGG
CCGACAAATCTGTGAC
CTGGCCGATGGGCGCG
CTATGCGTCCGGACAG
CTATCCGTTCTGTCCC
GTACCTGAACTGCCAG
CTATCTGAAATGCGAG
CCATCTGACATGTAGG
CCTGGAGACCAGGCTG
CAATCAGAAATGTCCC
CTAACAAAAGACATAG
CCGTCAGTATTGTTAG
CTGTGAAATCTGGCAG
CTACCAGATCTGGCAG
CTGTCGGTTCGCATAG
CTGCCCATAATCCCCG
CTATCAGTTCTGTAGT
CTGTGAGATTTGGGAG
CTACCTGTTTTGGTAG
CTATCAGAATGAATAG
CTATCCATTCTGATGG
CCGACAAATCTGTGAC
CTGGCCGATGGGCGCG
CTATGCGTCCGGACAG
CTATCCGTTCTGTCCC
GTACCTGAACTGCCAG
CTATCTGAAATGCGAG

Quantitative data format: ChIP enrichment. Ratio w-t vs. lasR/rhlR mutant. Range: 2.1-12

CCATCTGACATGTAGG 6.9
CCTGGAGACCAGGCTG 2.7
CAATCAGAAATGTCCC 6.6
CTAACAAAAGACATAG 2.4
CCGTCAGTATTGTTAG 6.4
CTGTGAAATCTGGCAG 3.6
CTACCAGATCTGGCAG 2.4
CTGTCGGTTCGCATAG 3.8
CTGCCCATAATCCCCG 5.7
CTATCAGTTCTGTAGT 4.1
CTGTGAGATTTGGGAG 2.4
CTACCTGTTTTGGTAG 2.2
CTATCAGAATGAATAG 12.0
CTATCCATTCTGATGG 4.3
CCGACAAATCTGTGAC 7.5
CTGGCCGATGGGCGCG 6.5
CTATGCGTCCGGACAG 2.7
CTATCCGTTCTGTCCC 4.0
GTACCTGAACTGCCAG 2.6
CTATCTGAAATGCGAG 3.9

Gene Regulation

Regulated genes for each binding site are displayed below. Gene regulation diagrams show binding sites, positively-regulated genes, negatively-regulated genes, both positively and negatively regulated genes, genes with unspecified type of regulation. For each indvidual site, experimental techniques used to determine the site are also given.

Site sequence Regulated genes Gene diagram Experimental techniques TF function TF type
CCATCTGACATGTAGG plcB, PA0027, PA0028,
... ... plcB PA0027 PA0028 aroE hemF PA0029 PA0030 betC PA0032
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details DNA-array expression analysis (ECO:0005525) - Experimental technique details Motif-discovery (ECO:0005558) - activator dimer
CCTGGAGACCAGGCTG bphP,
... ... bphP bphO PA4115 PA4118 aph PA4120
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details DNA-array expression analysis (ECO:0005525) - Experimental technique details Motif-discovery (ECO:0005558) - activator dimer
CAATCAGAAATGTCCC PA5184,
... ... PA5184 PA5183 PA5182 PA5181.1 PA5181 PA5180 PA5179 PA5185 PA5186 PA5187 PA5188 PA5189 PA5190
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details DNA-array expression analysis (ECO:0005525) - Experimental technique details Motif-discovery (ECO:0005558) - activator dimer
CTAACAAAAGACATAG mvfR,
... ... mvfR phnB phnA pqsE pqsD pqsC pqsB pqsA nadA PA1005
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details DNA-array expression analysis (ECO:0005525) - Experimental technique details Motif-discovery (ECO:0005558) - activator dimer
CCGTCAGTATTGTTAG xcpP, xcpQ,
... ... xcpP xcpQ xcpR xcpS xcpT xcpU xcpV xcpW xcpX xcpY xcpZ PA3094.3 PA3094.2 PA3094.1 PA3106 metZ purF
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details DNA-array expression analysis (ECO:0005525) - Experimental technique details Motif-discovery (ECO:0005558) - activator dimer
CTGTGAAATCTGGCAG rhlA, rhlB,
... ... rhlA rhlB rhlR PA3480 PA3481 metG PA3483 PA3484 PA3485
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details DNA-array expression analysis (ECO:0005525) - Experimental technique details Motif-discovery (ECO:0005558) - activator dimer
CTACCAGATCTGGCAG PA0122,
... ... PA0122 PA0121 PA0120 PA0119 PA0123
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details DNA-array expression analysis (ECO:0005525) - Experimental technique details Motif-discovery (ECO:0005558) - activator dimer
CTGTCGGTTCGCATAG nuh,
... ... nuh PA0142 PA0144 PA0145
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details DNA-array expression analysis (ECO:0005525) - Experimental technique details Motif-discovery (ECO:0005558) - activator dimer
CTGCCCATAATCCCCG PA0144,
... ... PA0144 nuh PA0142 PA0145
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details DNA-array expression analysis (ECO:0005525) - Experimental technique details Motif-discovery (ECO:0005558) - activator dimer
CTATCAGTTCTGTAGT PA0855,
... ... PA0855 fumC2 PA0853 PA0856 bolA
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details DNA-array expression analysis (ECO:0005525) - Experimental technique details Motif-discovery (ECO:0005558) - activator dimer
CTGTGAGATTTGGGAG pqsA, pqsB, pqsC, pqsD, pqsE,
... ... pqsA pqsB pqsC pqsD pqsE ogt cupC3 cupC2 cupC1 phnA phnB mvfR
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details DNA-array expression analysis (ECO:0005525) - Experimental technique details Motif-discovery (ECO:0005558) - activator dimer
CTACCTGTTTTGGTAG PA1656, PA1657, PA1658, PA1659,
... ... PA1656 PA1657 PA1658 PA1659 PA1655 PA1654 PA1653 PA1652
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details DNA-array expression analysis (ECO:0005525) - Experimental technique details Motif-discovery (ECO:0005558) - activator dimer
CTATCAGAATGAATAG kynB, kynU,
... ... kynB kynU PA2082
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details DNA-array expression analysis (ECO:0005525) - Experimental technique details Motif-discovery (ECO:0005558) - activator dimer
CTATCCATTCTGATGG pvdS,
... ... pvdS pvdG pvdL PA2427 PA2428
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details DNA-array expression analysis (ECO:0005525) - Experimental technique details Motif-discovery (ECO:0005558) - activator dimer
CCGACAAATCTGTGAC PA2939,
... ... PA2939 PA2938 PA2937 PA2936 PA2935 cif PA2933 morB cifR PA2930 PA2929 PA2940 PA2941 PA2942 PA2942.1
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details DNA-array expression analysis (ECO:0005525) - Experimental technique details Motif-discovery (ECO:0005558) - activator dimer
CTGGCCGATGGGCGCG PA3535,
... ... PA3535 PA3534 PA3533 PA3532 bfrB
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details DNA-array expression analysis (ECO:0005525) - Experimental technique details Motif-discovery (ECO:0005558) - activator dimer
CTATGCGTCCGGACAG flp,
... ... flp rcpC rcpA tadZ tadA tadB tadC tadD PA4298 tadG pprB fppA PA4294 pprA PA4292 PA4291 PA4290 PA4289 PA4288 PA4287 PA4286 recC recB recD PA4282 sbcD
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details DNA-array expression analysis (ECO:0005525) - Experimental technique details Motif-discovery (ECO:0005558) - activator dimer
CTATCCGTTCTGTCCC PA5232,
... ... PA5232 PA5231 PA5230 PA5229 PA5233
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details DNA-array expression analysis (ECO:0005525) - Experimental technique details Motif-discovery (ECO:0005558) - activator dimer
GTACCTGAACTGCCAG PA0805, PA0806
... ... PA0805 PA0806
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details DNA-array expression analysis (ECO:0005525) - Experimental technique details Motif-discovery (ECO:0005558) - activator dimer
CTATCTGAAATGCGAG phnC, amrZ,
... ... phnC amrZ PA3383 phnE PA3381 PA3380 PA3379 PA3378 PA3377 PA3376 PA3375 PA3374 PA3373 PA3372 PA3371 PA3370 PA3369
Experimental technique details ChIP-chip (ECO:0006007) - Experimental technique details DNA-array expression analysis (ECO:0005525) - Experimental technique details Motif-discovery (ECO:0005558) - activator dimer