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

Microbiology. 2006 Dec;152(Pt 12):3485-3496. doi: 10.1099/mic.0.29008-0.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • ADP Ribose Transferases / genetics
  • Amino Acid Substitution
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism*
  • Bacterial Toxins / genetics
  • Binding Sites / genetics
  • Consensus Sequence
  • Cyclic AMP Receptor Protein / genetics*
  • Cyclic AMP Receptor Protein / metabolism*
  • DNA Footprinting
  • DNA, Bacterial / analysis
  • DNA, Bacterial / genetics
  • DNA, Intergenic
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Escherichia coli / genetics
  • Escherichia coli Proteins / metabolism
  • Exotoxins / genetics
  • Genes, Regulator
  • Genetic Complementation Test
  • Lac Operon
  • Mutagenesis, Site-Directed
  • Mutation
  • Promoter Regions, Genetic
  • Protein Binding / genetics
  • Pseudomonas aeruginosa / genetics*
  • Pseudomonas aeruginosa / metabolism*
  • Receptors, Cyclic AMP / metabolism
  • Trans-Activators / genetics
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism*
  • Transcription, Genetic*
  • Virulence Factors / genetics
  • beta-Galactosidase / biosynthesis

Substances

  • Bacterial Proteins
  • Bacterial Toxins
  • Cyclic AMP Receptor Protein
  • DNA, Bacterial
  • DNA, Intergenic
  • DNA-Binding Proteins
  • Escherichia coli Proteins
  • Exotoxins
  • LasR protein, Pseudomonas aeruginosa
  • Receptors, Cyclic AMP
  • RegA protein, Bacteria
  • Trans-Activators
  • Transcription Factors
  • Vfr protein, Pseudomonas aeruginosa
  • Virulence Factors
  • cyclic AMP receptor protein, E coli
  • ADP Ribose Transferases
  • toxA protein, Pseudomonas aeruginosa
  • beta-Galactosidase