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Journal of Bacteriology, October 2001, p. 5562-5570, Vol. 183, No. 19
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.19.5562-5570.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Positive Selection of Mutations Leading to Loss or Reduction of Transcriptional Activity of PrfA, the Central Regulator of Listeria monocytogenes Virulence

M. Herler,1 A. Bubert,1,dagger M. Goetz,1 Y. Vega,2 J. A. Vazquez-Boland,2 and W. Goebel1,*

Biocenter of the University of Würzburg (Microbiology), Würzburg, Germany,1 and Molecular Bacterial Pathogenesis Group, Veterinary Faculty, Complutense University, 28040 Madrid, Spain2

Received 24 April 2001/Accepted 9 July 2001

Transcription factor PrfA controls the expression of virulence genes essential for Listeria monocytogenes pathogenesis. To gain insight into the structure-function relationship of PrfA, we devised a positive-selection system to isolate mutations reducing or abolishing transcriptional activity. The system is based on the observation that the listerial iap gene, encoding the p60 protein, is lethal if overexpressed in Bacillus subtilis. A plasmid in which the iap gene is placed under the control of the PrfA-dependent hly promoter was constructed and introduced into B. subtilis. This strain was rapidly killed when expression of iap was induced by introduction of a second plasmid carrying prfA. Two classes of B. subtilis survivor mutants were identified: one carried mutations in iap, and the second carried mutations in prfA. Sequence analysis of the defective prfA genes identified mutations in three regions of the PrfA protein: region A, between amino acids 58 and 67 in the beta -roll domain of PrfA; region B, between amino acids 169 and 193, which corresponds to the DNA-binding helix-turn-helix motif; and region C, comprising the 38 C-terminal amino acids of PrfA, which form a leucine zipper-like structure. PrfA proteins with mutations in regions B and C were unable to bind to the PrfA-binding site in the target DNA, while mutations in region A resulted in a protein still binding the target DNA but unable to form a stable complex with RNA polymerase and initiate transcription in vitro.

* Corresponding author. Mailing address: Biocenter of the University of Würzburg (Microbiology), Würzburg, Germany. Phone: 49 931 8884401. Fax: 49 931 8884402. E-mail: goebel{at}biozentrum.uni-wuerzburg.de.

dagger Present address: Scientific Laboratories Products, Merck KGaA, Darmstadt, Germany.

Journal of Bacteriology, October 2001, p. 5562-5570, Vol. 183, No. 19
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.19.5562-5570.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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