Curation Information

Publication: Integration of cyclic di-GMP and quorum sensing in the control of vpsT and aphA in Vibrio cholerae.;Srivastava D, Harris RC, Waters CM;Journal of bacteriology 2011 Nov; 193(22):6331-41 [21926235]
TF: VpsR [Q9KU59, view regulon]
Reported TF sp.: Vibrio cholerae O1 biovar El Tor str. N16961
Reported site sp.: Vibrio cholerae O1 biovar El Tor str. N16961
Created by: Erill Lab
Curation notes: -

Experimental Process

The vpsT and aphA promoters were shown to be inducible with c-di-GMP using lux assays. Sites for Lrp, HapR and VpsR were predicted by comparison to known consensus. The transcriptional start site of vpsT was determined with RACE PCR. EMSAs with multiple size fragments confirmed that the VpsR sites were located upstream of -119 from TSS. Expression assays with vpsR and hapR mutants using multiple size fragments also showed that it they are required for induction of both promoters and that the predicted sites are likely responsible for such induction.

Transcription Factor Binding Sites

Sites reported in the paper
Sites matched in the genome

TTTTCATTATTGAGAATA
TTCTTAATATTGAGAAGG

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
TTTTCATTATTGAGAATA	VC2647,		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 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 Luciferase reporter assay (ECO:0005648) Luciferase reporter assay - ECO:0005648 A type of reporter assay . The idea is identical to a betagalactosidase assay, but here fluorescence of luciferase gene product is used as the reporter. -	activator	monomer
TTCTTAATATTGAGAAGG	VCA0952		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 Luciferase reporter assay (ECO:0005648) Luciferase reporter assay - ECO:0005648 A type of reporter assay . The idea is identical to a betagalactosidase assay, but here fluorescence of luciferase gene product is used as the reporter. -	activator	monomer