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 -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.
SummaryElements essential for PrfA-dependent transcription were analysed on two promoters of Listeria monocytogenes , the PrfA-dependent promoter of the phospholipase gene plcA (P plcA ) and a putative promoter of the aroA gene (P aroA2 ) which contains a similar PrfA-binding site and a similar ----10 box as P plcA but does not function as PrfA-dependent promoter. We constructed a series of hybrid plcA-aroA promoters by exchanging corresponding sequence elements of these two 'promoters'. The results showed that the two critical elements of PrfA-dependent promoters, the PrfA-box and the ----10 box, can be functionally exchanged as long as the distance in between is maintained to 22 or 23 bp. However, the interspace sequence and the sequence downstream of the ----10 box of P aroA2 were strongly inhibitory for PrfAdependent transcription. A detailed analysis of these two sequences revealed that the RNA polymerase binding site being part of the actual in vivo and in vitro used aroA promoter (P aroA1 ) and a sequence immediately downstream of the putative ----10 site, possibly blocking the formation of the open complex, were responsible for the inhibition of PrfA-dependent transcription from P aroA2 . Taking into consideration the lessons learned from this study we were able to construct a functional PrfA-dependent aroA promoter.
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