Positive Selection of Mutations Leading to Loss or Reduction of Transcriptional Activity of PrfA, the Central Regulator ofListeria monocytogenesVirulence

Abstract

Transcription factor PrfA controls the expression of virulence genes essential forListeria monocytogenespathogenesis. 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 listerialiapgene, encoding the p60 protein, is lethal if overexpressed inBacillus subtilis. A plasmid in which theiapgene is placed under the control of the PrfA-dependenthlypromoter was constructed and introduced intoB. subtilis. This strain was rapidly killed when expression ofiapwas induced by introduction of a second plasmid carryingprfA. Two classes ofB. subtilissurvivor mutants were identified: one carried mutations iniap, and the second carried mutations inprfA. Sequence analysis of the defectiveprfAgenes identified mutations in three regions of the PrfA protein: region A, between amino acids 58 and 67 in the β-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.