Some essential elements on the inlC promoter for prfA-dependent regulation in Listeria monocytogenes

To study some essential elements of a PrfA-dependent promoter of Listeria monocytogenes, a series of promoter mutants incorporated into upstream of a promoterless lacZ gene were constructed from a known listerial PrfA-dependent promoter, inlC promoter, by PCR-mediated site-directed mutagenesis and recombinant PCR technique and then electroporated into L. monocytogenes wild-type strain P14, prfA * mutant Pl4a and prfA deletion mutant A42. The corresponding transcription activities of altered promoters were measured by the beta-galactosidase assay. The results showed that a PrfA-box-like sequence ("pseudo-PrfA-box"), TTAACAGCGTTTGTTAA, 22bp downstream of the transcriptional start site of PinlC had no ability to enhance or inhibit the PrfA-dependent transcription of inlC promoter, even it was modified to the "ideal PrfA-box" TTAACATTTGTTAA. However, there was almost no PrfA-dependent transcriptional activity from the mutants deletion of the inlC original PrfA-box. Moreover, altered spacing between 3'-end of the PrfA-box and 5'-end of the -10 box in the inlC promoter region affected transcription efficiency dramatically, which also happened in another promoter-dependent promoter, plcA promoter. Those above suggested that besides the "PrfA-box", additional unknown PrfA-dependent promoter structure(s) or sequence(s) might be required for the PrfA binding to the promoter and initiation of transcription. Furthermore, the distance between the PrfA-box and the -10 box should be fixed to 22 or 23bp for the PrfA-dependent transcription.