SPECIFIC BINDING OF THE LISTERIA-MONOCYTOGENES TRANSCRIPTIONAL REGULATOR PRFA TO TARGET SEQUENCES REQUIRES ADDITIONAL FACTOR(S) AND IS INFLUENCED BY IRON

The PrfA protein, which is a member of the Crp/Fnr family of prokaryot ic transcription activators, regulates the virulence genes of Listeria monocytogenes. In this work, specific binding of PrfA to its target D NA was determined by electrophoretic mobility-shift assays (EMSAs) usi ng cell-free extracts from the two L. monocytogenes strains EGD and NC TC 7973. PrfA-specific binding differs between the two strains, even w hen the concentration of PrfA was adjusted to similar levels. Both str ains exhibited increased PrfA-specific binding after a shift into mini mal essential medium (MEM) without showing a significant change in the amount of PrfA protein, relative to extracts from bacteria grown in b rain-heart infusion (BHI). The purified PrfA protein from strain EGD p roduced in Escherichia coli did not exhibit specific binding to the ta rget DNA but did so upon addition of PrfA-free extracts from various L isteria species and Bacillus subtilis. The observed activation of PrfA seems to be caused by a PrfA-activating factor (Paf), which is probab ly a protein since elevated temperature, but not RNase treatment, dest royed the activation potential of such PrfA-free extracts. Moreover, f ractionation of these extracts by sucrose gradient centrifugation yiel ded the Paf activity in a fraction sedimenting at 3.2 S. Specific bind ing of PrfA-containing extracts from strain EGD to the hly and actA pr omoter sequences was strongly inhibited by iron, whereas that of extra cts from strain NCTC 7973 was only slightly reduced. The iron effect s eems to be mediated by Paf rather than by PrfA itself.