Curation Information

Publication: Choline catabolism, σ⁵⁴ factor and NtrC are required for the full expression of the Pseudomonas aeruginosa phosphorylcholine phosphatase gene.;Massimelli MJ, Sánchez DG, Buchieri MV, Olvera L, Beassoni PR, Schweizer HP, Morett E, Lisa AT;Microbiological research 2011 Jul 20; 166(5):380-90 [20869215]
TF: RpoN [P49988, view regulon]
Reported TF sp.: Pseudomonas aeruginosa PAO1
Reported site sp.: Pseudomonas aeruginosa PAO1
Created by: Dinara Sagitova
Curation notes: -

Experimental Process

LacZ reporter assays in wild-type and rpoN mutant strains showed that β-galactosidase activity was highly reduced (≅75%) in the mutant. Site-directed mutagenesis in the putative −12 element reduced pchP expression by 95%. The pchP transcriptional start site was determined by 5' RACE experiment. The presence of TSS 14 nucleotides downstream of the putative σ54 −12 element, provided further evidence that this promoter was transcribed by this sigma factor. Additionally, a sequence resembling an IHF binding site was identified in the pchP promoter region.

Transcription Factor Binding Sites

Sites reported in the paper
Sites matched in the genome

GGCGCAGGGTTTGC

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
GGCGCAGGGTTTGC	pchP		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 Site directed mutagenesis (ECO:0005667) Site directed mutagenesis - ECO:0005667 Target-specific mutation, as opposed to non-specific mutation. In the context of TF-binding sites, site-directed mutagenesis is typically used to establish/confirm the specific sequence and location of a site, often in tandem with EMSA. Different positions of a putative binding site are mutated to non-consensus (or random) bases and binding to the mutated site is evaluated through EMSA or other means. Often implemented only in conserved motif positions or serially through all positions of a site. - Experimental technique details Visual sequence inspection (nan) Visual sequence inspection - nan Visual inspection of promoter region to identify putative binding sites based on previous knowledge -	activator	not specified