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

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Experimental Process

Regulation by LasR confirmed by beta-gal assays.

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

• Sites reported in the paper
• Sites matched in the genome
• Site-specific quantitative values
• High-throughput data

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.