A single substitution in the putative helix-turn-helix motif of the pleiotropic activator PrfA attenuates Listeria monocytogenes virulence.
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.
Sheehan
B
B
Unité des Interactions Bactéries-Cellules, CNRS URA 1300, Institut Pasteur, Paris, France.
Klarsfeld
A
A
Ebright
R
R
Cossart
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D019077:Helix-Turn-Helix Motifs; D006459:Hemolysin Factors / Q000378:metabolism; D007700:Kinetics; D007763:Lac Operon; D008089:Listeria monocytogenes / Q000235:genetics / Q000472:pathogenicity*; D051379:Mice; D008969:Molecular Sequence Data; D010454:Peptide Termination Factors; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D002373:Cyclic AMP Receptor Protein; D010454:Peptide Termination Factors; D015534:Trans-Activators; D004247:DNA