Curation Information

Publication: Role of the Pseudomonas aeruginosa las and rhl quorum-sensing systems in rhlI regulation.;de Kievit TR, Kakai Y, Register JK, Pesci EC, Iglewski BH;FEMS microbiology letters 2002 Jun 18; 212(1):101-6 [12076794]
TF: LasR [P25084, view regulon]
Reported TF sp.: Pseudomonas aeruginosa PAO1
Reported site sp.: Pseudomonas aeruginosa PAO1
Created by: Matthew Coveyou
Curation notes: -

Experimental Process

Investigation of the quorum-sensing regulation of P. aeruginoas PAO1 gene rhlI began with identification of the transcriptional start site via primer extension. The same assay showed reduced transcription in a lasR-mutant strain. A putative las-box was identified from comparison with lux-box sequences. Beta-gal assay was applied to five mutant strains for comparison with wild-type expression: lasR-, lasI-, rhlR-, rhlI-, and lasI-/rhlI-. Mutant strains lasR-, lasI-, and lasI-/rhlI- showed dramatic loss of expression, while levels in rhlR- and rhlI- strains did not fall below 75% of the wild-type, indicating primary regulation by the lasR/lasI system. Addition of respective autoinducers restored expression in lasI- and rhlI- mutants. Assays were repeated with partial las-box deletions in all strains, and expression fell dramatically in all five and the wild-type.

Transcription Factor Binding Sites

Sites reported in the paper
Sites matched in the genome

CCCTACCAGATCTGGCAGG

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
CCCTACCAGATCTGGCAGG	rhlI		Experimental technique details Beta-gal reporter assay Beta-gal reporter assay Reporter assay using the beta-galactosidase (lacZ) gene. The lacZ gene is typically fused to the promoter of interest. Differential regulation of the promoter mediated by the TF is assessed by induction of the system and evaluation of lacZ expression. Bacteria expressing lacZ appear blue when grown on a X-gal medium. The assay is often performed using a plasmid borne construction on a lacZ(def) strain. - Experimental technique details Consensus search (ECO:0005624) Consensus search - ECO:0005624 This is a weak form of in-silico search, in which the consensus sequence for the motif is compared to genomic positions and the number of mismatches (between candidate site and consensus) is used as a measure of site-quality. - Experimental technique details Multiple sequence alignment (MSA) (ECO:0005556) Multiple sequence alignment (MSA) - ECO:0005556 MSA can be used to align promoter regions and highlight regions of conservation that may be indicative of transcription factor binding sites. - Experimental technique details Primer Extension assay (ECO:0005657) Primer Extension assay - ECO:0005657 Primer Extension Assay is a simple method of assessing expression levels. The total RNA produced by the culture is isolated. A short synthetic oligonucleotide primer (complementary to a short sequence on the target RNA) is radiolabelled (usually with 32P) and the primer anneals to the RNA. Reverse transcriptase then extends the RNA producing cDNA. The cDNA is analyzed on a polyacrylamide gel. The amount of cDNA in a band on the gel is proportional to the amount of initial RNA, thus providing expression levels of RNA from the culture. -	activator	not specified