Characterization of DNA-binding specificity and analysis of binding sites of the Pseudomonas aeruginosa global regulator, Vfr, a homologue of the Escherichia coli cAMP receptor protein.
Vfr, a global regulator of Pseudomonas aeruginosa virulence factors, is a homologue of the Escherichia coli cAMP receptor protein, CRP. Vfr is 91% similar to CRP and maintains many residues important for CRP to bind cAMP, bind DNA, and interact with RNA polymerase at target promoters. While vfr can complement an E. coli crp mutant in beta-galactosidase production, tryptophanase production and catabolite repression, crp can only complement a subset of Vfr-dependent phenotypes in P. aeruginosa. Using specific CRP binding site mutations, it is shown that Vfr requires the same nucleotides as CRP for optimal transcriptional activity from the E. coli lac promoter. In contrast, CRP did not bind Vfr target sequences in the promoters of the toxA and regA genes. Footprinting analysis revealed Vfr protected sequences upstream of toxA, regA, and the quorum sensing regulator lasR, that are similar to but significantly divergent from the CRP consensus binding sequence, and Vfr causes similar DNA bending to CRP in bound target sequences. Using a preliminary Vfr consensus binding sequence deduced from the Vfr-protected sites, Vfr target sequences were identified upstream of the virulence-associated genes plcN, plcHR, pbpG, prpL and algD, and in the vfr/orfX, argH/fimS, pilM/ponA intergenic regions. From these sequences the Vfr consensus binding sequence, 5'-ANWWTGNGAWNY : AGWTCACAT-3', was formulated. This study suggests that Vfr shares many of the same functions as CRP, but has specialized functions, at least in terms of DNA target sequence binding, required for regulation of a subset of genes in its regulon.
Kanack
Kristen J
KJ
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive, Madison, WI 53706, USA.
Runyen-Janecky
Laura J
LJ
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive, Madison, WI 53706, USA.
Ferrell
Evan P
EP
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive, Madison, WI 53706, USA.
Suh
Sang-Jin
SJ
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive, Madison, WI 53706, USA.
West
Susan E H
SEH
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive, Madison, WI 53706, USA.
D036002:ADP Ribose Transferases / Q000235:genetics; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001427:Bacterial Toxins / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D016384:Consensus Sequence; D002373:Cyclic AMP Receptor Protein / Q000235:genetics* / Q000378:metabolism*; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D021901:DNA, Intergenic; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000378:metabolism; D005098:Exotoxins / Q000235:genetics; D005809:Genes, Regulator; D005816:Genetic Complementation Test; D007763:Lac Operon; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism*; D011953:Receptors, Cyclic AMP / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D037521:Virulence Factors / Q000235:genetics; D001616:beta-Galactosidase / Q000096:biosynthesis; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial; D021901:DNA, Intergenic; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005098:Exotoxins; D011953:Receptors, Cyclic AMP; D015534:Trans-Activators; D014157:Transcription Factors; D037521:Virulence Factors; D001616:beta-Galactosidase; D000097668:Pseudomonas aeruginosa Exotoxin A; C080365:LasR protein, Pseudomonas aeruginosa; C072722:RegA protein, Bacteria; C091090:Vfr protein, Pseudomonas aeruginosa; C047947:cyclic AMP receptor protein, E coli