PrfAK 220 T did not form a PrfA-DNA complex in electrophoretic mobility shift assays, but low concentrations of CI complexes (PrfAK 220 T-RNA polymerase-DNA complex) were formed by adding RNA polymerase, suggesting that PrfA interacted with RNA polymerase in solution in the absence of DNA. However, the data showed that the PrfAK 220 T protein is dimerized just as well as its wild-type counterpart, but does not bind to PrfA-boxes. The substitution of the lysine at position 220 occurred in the helix H.
This substitution inactivated PrfA, since expression of the PrfAK 220 T mutant gene in an EGDprfA strain did not restore the haemolytic and phosphatidylcholine phospholipase C activities, in contrast to the wild-type prfA gene. These strains exhibited no expression of PrfA-regulated proteins and thus no virulence.
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: PrfAK 220 T. A Naturally Occurring Mutation K 220 T in the Pleiotropic Activator PrfA of Listeria Monocytogenes Results in a Loss of Virulence Due to Decreasing DNA-Binding AffinityĮnergy Technology Data Exchange (ETDEWEB)
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