Curation Information

Publication: The transcriptional regulator AlgR controls cyanide production in Pseudomonas aeruginosa.;Carterson AJ, Morici LA, Jackson DW, Frisk A, Lizewski SE, Jupiter R, Simpson K, Kunz DA, Davis SH, Schurr JR, Hassett DJ, Schurr MJ;Journal of bacteriology 2004 Oct; 186(20):6837-44 [15466037]
TF: AlgR [P26275, view regulon]
Reported TF sp.: Pseudomonas aeruginosa PAO1
Reported site sp.: Pseudomonas aeruginosa PAO1
Created by: Grace Chandler
Curation notes: -

Experimental Process

S1 nuclease protection analysis using an algR knockout mutant identified lower cyanide levels in the mutant, demonstrating the role of AlgR as an activator of the hcnAB operon. EMSA confirmed AlgR binding to the hcnA promoter region which contains a previously identified AlgR binding site. Quantitative phenotypic analysis of HCN production showed AlgR activates cyanide production in the mucoid isolates and represses HCN production in non-mucoid isolates.

Transcription Factor Binding Sites

Sites reported in the paper
Sites matched in the genome

GAACGACGG

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
GAACGACGG	hcnA, hcnB,		Experimental technique details Ad-hoc quantitative phenotype observation (ECO:0005676) Ad-hoc quantitative phenotype observation - ECO:0005676 A quantifiable phenotypic trait (e.g. glucose intake) is assessed in a quantitative manner after induction of the regulatory mechanism and used as a natural reporter.. The observable phenotypic trait should be described in the experimental notes. - Experimental technique details EMSA (ECO:0001807) EMSA - ECO:0001807 Electro-mobility shift-assays (or gel retardation assays) are a standard way of assessing TF-binding. A fragment of DNA of interest is amplified and labeled with a fluorophore. The fragment is left to incubate in a solution containing abundant TF and non-specific DNA (e.g. randomly cleaved DNA from salmon sperm, of all things) and then a gel is run with the incubated sample and a control (sample that has not been in contact with the TF). If the TF has bound the sample, the complex will migrate more slowly than unbound DNA through the gel, and this retarded band can be used as evidence of binding. The unspecific DNA ensures that the binding is specific to the fragment of interest and that any non-specific DNA-binding proteins left-over in the TF purification will bind there, instead of on the fragment of interest. EMSAs are typically carried out in a bunch of fragments, shown as multiple double (control+experiment) lanes in a wide picture. Certain additional controls are run in at least one of the fragments to ascertain specificity. In the most basic of these, specific competitor (the fragment of interest or a known positive control, unlabelled) is added to the reaction. This should sequester the TF and hence make the retardation band disappear, proving that the binding is indeed specific - Experimental technique details S1 nuclease protection (ECO:0005666) S1 nuclease protection - ECO:0005666 This is a technique used to detect typically mRNA with greater precision than Northern blot or RT-PCR. Therefore, it is commonly used to assess gene expression (specifically transcription), even though it is a protection experiment. In S1 nuclease protection, extracted RNA is hybridized with complementary DNA probes and expose to S1 nuclease to degrade all RNA that is not bound to probes. The remaining (non-degraded) RNA is typically run on a gel to detect the size (and label) of the probe and determine which RNA it is. -	dual	other