Curation Information

Publication: Transcriptional organization and regulation of the Vibrio anguillarum heme uptake gene cluster.;Mouriño S, Osorio CR, Lemos ML, Crosa JH;Gene 2006 Jun 7; 374():68-76 [16515846]
TF: Fur [P37736, view regulon]
Reported TF sp.: Vibrio anguillarum 775
Reported site sp.: Vibrio anguillarum 775
Created by: Dinara Sagitova
Curation notes: -

Experimental Process

Transcription start sites were determined by primer extension experiments with RNA isolated from V.anguillarum H775-3, a strain deficient in anguibactin biosynthesis. The authors identified a putative Fur box based on E.coli Fur box consensus. Primer extension analysis with RNA obtained from V.anguillarum H775-3 grown in iron rich conditions suggested Fur-mediated repression of huvA since no signal was observed. Similarly, the huvXZ and tonB1exbB1D1-huvBCD genes were shown to be regulated by Fur. Fur-mediated repression of these genes was confirmed by measuring the β-galactosidase activities of uvA∷lacZ, huvX∷lacZ and tonB1∷lacZ. The results of this assay showed that expression was high in low-iron medium and low after the addition of iron to the medium. Additionally, lacZ reporter assay in the fur mutant strain showed that β-galactosidase activity was high under both high- and low-iron conditions.

Transcription Factor Binding Sites

Sites reported in the paper
Sites matched in the genome

AATGATAGTAATTACCATT
AATAAGATCAATTATCAAT

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
AATGATAGTAATTACCATT	VAA_01392		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 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. - 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 -	repressor	not specified
AATAAGATCAATTATCAAT	VAA_01389,		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 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. - 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 -	repressor	not specified