Positive selection of mutations leading to loss or reduction of transcriptional activity of PrfA, the central regulator Listeria monocytogenes virulence

Transcription factor PrfA controls the expression of virulence genes essent ial for Listeria monocytogenes pathogenesis. To gain insight into the struc ture-function relationship of PrfA, we devised a positive-selection system to isolate mutations reducing or abolishing transcriptional activity. The s ystem 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 p romoter was constructed and introduced into B. subtilis. This strain was ra pidly killed when expression of iap was induced by introduction of a second plasmid carrying prfA. Two classes of B. subtilis survivor mutants were id entified: one carried mutations in iap, and the second carried mutations in prfA. Sequence analysis of the defective pr A genes identified mutations i n three regions of the PrfA protein: region A, between amino acids 58 and 6 7 in the beta -roll domain of PrfA; region B, between amino acids 169 and 1 93, 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.