A single substitution in the putative helix-turn-helix motif of the pleiotropic activator PrfA attenuates Listeria monocytogenes virulence

Mol Microbiol. 1996 May;20(4):785-97. doi: 10.1111/j.1365-2958.1996.tb02517.x.

Abstract

PrfA, the regulator of virulence-gene expression in the pathogenic bacterium Listeria monocytogenes, displays sequence similarity to members of the CAP-FNR family of transcriptional regulators. To test the functional significance of this similarity, we constructed and analysed substitutions of two amino acids of PrfA predicted to contact DNA, i.e. Ser-184 and Ser-183. Substitution of Ser-184 by Ala reduced DNA binding and virulence-gene activation, and attenuated the virulence in a mouse model of infection. In contrast, substitution of Ser-183 by Ala had the opposite effect in these functional assays. A 17bp DNA sequence, which includes a putative PrfA site, was shown to be sufficient for target-site recognition by PrfA and PrfA-S183A. Our results strongly support the hypothesis that PrfA is a structural and functional homologue of CAP. In addition, they establish a clear correlation between DNA binding by PrfA, virulence-gene activation, and virulence.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Base Sequence
  • Cyclic AMP Receptor Protein / chemistry
  • DNA / metabolism
  • Helix-Turn-Helix Motifs*
  • Hemolysin Factors / metabolism
  • Kinetics
  • Lac Operon
  • Listeria monocytogenes / genetics
  • Listeria monocytogenes / pathogenicity*
  • Mice
  • Molecular Sequence Data
  • Peptide Termination Factors
  • Sequence Alignment
  • Structure-Activity Relationship
  • Trans-Activators / chemistry*
  • Trans-Activators / genetics

Substances

  • Bacterial Proteins
  • Cyclic AMP Receptor Protein
  • Peptide Termination Factors
  • Trans-Activators
  • DNA