A naturally occurring mutation K220T in the pleiotropic activator PrfA of Listeria monocytogenes results in a loss of virulence due to decreasing DNA-binding affinity.

The sequencing of prfA, encoding the transcriptional regulator of virulence genes, in 26 low-virulence field Listeria monocytogenes strains showed that eight strains exhibited the same single amino-acid substitution: PrfAK220T. These strains exhibited no expression of PrfA-regulated proteins and thus no virulence. This substitution inactivated PrfA, since expression of the PrfAK220T mutant gene in an EGDDeltaprfA strain did not restore the haemolytic and phosphatidylcholine phospholipase C activities, in contrast to the wild-type prfA gene. The substitution of the lysine at position 220 occurred in the helix alphaH. However, the data showed that the PrfAK220T protein is dimerized just as well as its wild-type counterpart, but does not bind to PrfA-boxes. PrfAK220T did not form a PrfA-DNA complex in electrophoretic mobility shift assays, but low concentrations of CI complexes (PrfAK220T-RNA polymerase-DNA complex) were formed by adding RNA polymerase, suggesting that PrfA interacted with RNA polymerase in solution in the absence of DNA. Formation of some transcriptionally active complexes was confirmed by in vitro runoff transcription assays and quantitative RT-PCR. Crystallographic analyses described the structure of native PrfA and highlighted the key role of allosteric changes in the activity of PrfA and especially the role of the Lys220 in the conformation of the helix-turn-helix (HTH) motif.

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

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.