Identification of the DNA binding sites of PerA, the transcriptional activator of the bfp and per operons in enteropathogenic Escherichia coli.
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 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.
Ibarra
J Antonio
JA
Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62251, México.
Villalba
Miryam I
MI
Puente
José Luis
JL
D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D036781:Fimbriae Proteins / Q000235:genetics* / Q000378:metabolism; D010861:Fimbriae, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009876:Operon / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D014158:Transcription, Genetic
C104901:BfpA protein, E coli; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; C120886:bfpT protein, E coli; D036781:Fimbriae Proteins