Curation Information

Publication
Epoxide-mediated CifR repression of cif gene expression utilizes two binding sites in Pseudomonas aeruginosa.;Ballok AE, Bahl CD, Dolben EL, Lindsay AK, St Laurent JD, Hogan DA, Madden DR, O'Toole GA;Journal of bacteriology 2012 Oct; 194(19):5315-24 [22843844]
TF
CifR [Q9HZR6, view regulon]
Reported TF sp.
Pseudomonas aeruginosa UCBPP-PA14
Reported site sp.
Pseudomonas aeruginosa UCBPP-PA14
Created by
Dinara Sagitova
Curation notes
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Experimental Process

EMSA confirmed CifR binding to the cifR-morB intergenic region. This binding was disrupted upon addition of the CifR substrate EBH. Additional EMSA experiment with two smaller, non-overlapping promoter fragments showed that cifR-morB intergenic region contained two CifR binding sites. By performing EMSA with smaller DNA fragments, the CifR binding sites were narrowed down further. morB and cifR transcription start sites were determined by 5'RACE. CifR binding was abolished after a 6 bp long mutation was inserted into the center of both putative sites. Western analysis showed that the same 6 bp long mutation in the morB proximal site caused an increase in Cif production. qRT-PCR performed using wild-type and 6 bp mutant strains confirmed that CifR represses morB and cifR gene expression.

Transcription Factor Binding Sites


TATTTGTATCGATCACTA
TATCTGTATCGGTCGCTA
TATTTGTATCGATCACTA
TATCTGTATCGGTCGCTA

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
TATTTGTATCGATCACTA PA14_26140,
... ... PA14_26140 morB PA14_26110 PA14_26090
Experimental technique details EMSA (ECO:0001807) - Experimental technique details qRT-PCR [RNA] (ECO:0001808) - Experimental technique details Site directed mutagenesis (ECO:0005667) - Experimental technique details Visual sequence inspection (nan) - repressor not specified
TATCTGTATCGGTCGCTA morB,
... ... morB PA14_26110 PA14_26090 PA14_26140
Experimental technique details EMSA (ECO:0001807) - Experimental technique details qRT-PCR [RNA] (ECO:0001808) - Experimental technique details Site directed mutagenesis (ECO:0005667) - Experimental technique details Visual sequence inspection (nan) - Experimental technique details Western blot (quantitative) expression analysis (ECO:0000279) - repressor not specified