TY - JOUR
T1 - Identification of the DNA binding sites of PerA, the transcriptional activator of the bfp and per operons in enteropathogenic Escherichia coli
AU - Ibarra, J. Antonio
AU - Villalba, Miryam I.
AU - Puente, José Luis
PY - 2003/5
Y1 - 2003/5
N2 - The bundle-forming pilus (BFP) is an important virulence factor for enteropathogenic Escherichia coli (EPEC). Genes involved in its biogenesis and regulation are tightly regulated by PerA (BfpT), a member of the AraC/XylS family of transcriptional regulators. The aim of this work was to purify PerA and determine its association with bfpA and perA (bfpT) regulatory regions by electrophoretic mobility shift and DNase I footprinting assays. PerA was purified as a maltose-binding protein (MBP) fusion, which was capable of complementing bfpA expression and which was able to restore the localized adherence phenotype of an EPEC perA mutant strain. Upstream of bfpA and perA, MBP-PerA recognized with similar affinity asymmetric nucleotide sequences in which a 29-bp-long AT-rich consensus motif was identified. These DNA motifs share 66% identity and were previously shown, by deletion analysis, to be involved in the PerA-dependent expression of both genes. Interestingly, in perA, this motif spans the sequence between positions -75 and -47, approximately one helix turn upstream of the -35 promoter sequence, while in bfpA, it spans the sequence between positions -83 and -55, approximately two helix turns upstream from the promoter. An additional PerA binding site was identified at the 5′ end end of the bfpA structural gene, which was not required for its activation. Experiments with LexA-PerA fusions suggested that PerA acts as a monomer to activate the transcription of both perA and bfpA, in contrast to what has been documented for other members of this family of transcriptional regulators.
AB - The bundle-forming pilus (BFP) is an important virulence factor for enteropathogenic Escherichia coli (EPEC). Genes involved in its biogenesis and regulation are tightly regulated by PerA (BfpT), a member of the AraC/XylS family of transcriptional regulators. The aim of this work was to purify PerA and determine its association with bfpA and perA (bfpT) regulatory regions by electrophoretic mobility shift and DNase I footprinting assays. PerA was purified as a maltose-binding protein (MBP) fusion, which was capable of complementing bfpA expression and which was able to restore the localized adherence phenotype of an EPEC perA mutant strain. Upstream of bfpA and perA, MBP-PerA recognized with similar affinity asymmetric nucleotide sequences in which a 29-bp-long AT-rich consensus motif was identified. These DNA motifs share 66% identity and were previously shown, by deletion analysis, to be involved in the PerA-dependent expression of both genes. Interestingly, in perA, this motif spans the sequence between positions -75 and -47, approximately one helix turn upstream of the -35 promoter sequence, while in bfpA, it spans the sequence between positions -83 and -55, approximately two helix turns upstream from the promoter. An additional PerA binding site was identified at the 5′ end end of the bfpA structural gene, which was not required for its activation. Experiments with LexA-PerA fusions suggested that PerA acts as a monomer to activate the transcription of both perA and bfpA, in contrast to what has been documented for other members of this family of transcriptional regulators.
UR - http://www.scopus.com/inward/record.url?scp=0037406928&partnerID=8YFLogxK
U2 - 10.1128/JB.185.9.2835-2847.2003
DO - 10.1128/JB.185.9.2835-2847.2003
M3 - Artículo
C2 - 12700263
AN - SCOPUS:0037406928
SN - 0021-9193
VL - 185
SP - 2835
EP - 2847
JO - Journal of Bacteriology
JF - Journal of Bacteriology
IS - 9
ER -