Curation Information

Publication: The pH-dependent expression of the urease operon in Streptococcus salivarius is mediated by CodY.;Huang SC, Burne RA, Chen YY;Applied and environmental microbiology 2014 Jun 20; (): [24951785]
TF: CodY [A0A074IWI7, view regulon]
Reported TF sp.: Streptococcus salivarius 57.I
Reported site sp.: Streptococcus salivarius 57.I
Created by: Matthew Coveyou
Curation notes: -

Experimental Process

A putative CodY binding site on the ureI promoter was identified by comparison with the CodY consensus binding site. CAT reporter assay and urease assays were both used to demonstrate repression of the ureI promoter by CodY. ChIP-PCR of ureC mRNA demonstrated lower transcription rates in the wild type than in the CodY mutant strain, confirming negative regulation. Data also demonstrated CodY to be modulated by growth pH, and suggested ureI is regulated by additional transcription factors. EMSA confirmed specific binding of CodY to the ureI promoter.

Transcription Factor Binding Sites

Sites reported in the paper
Sites matched in the genome

AAATTTCTGAAAATT

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
AAATTTCTGAAAATT	ureI, ureA, ureB, ureC, ureE, ureF, ureG, ureD, ureM, ureQ, ureO,		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 CAT reporter gene assays (ECO:0005617) CAT reporter gene assays - ECO:0005617 A reporter assay based on the fusion of a promoter of interest with the cat gene, which produces chloramphenicol acetyltransferase, capable of being acetylated by acetyl CoA. The amount of acetylated chloramphenicol is directly proportional to the amount of CAT enzyme present. - Experimental technique details ChIP-PCR (ECO:0005620) ChIP-PCR - ECO:0005620 ChIP-chip (and to a lesser degree ChIP-Seq) results are often validated with ChIP-PCR, in which a PCR with specific primers is performed on the pulled-down DNA. As in the case of RNASeq, there are many variations of these main techniques. - 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 -	repressor	not specified