ABSTRACT
Objective To investigate the relationship between PrfA-dependent promoters and PrfA regulation. Methods LacZ reporter gene fusions used to investigate the inhibitory elements for PrfA-dependent transcription were carried on two promoters of Listeria monocytogenes: a PrfA-dependent promoter of the phospholipase gene pica (PplcA) and a putative promoter of the aroA gene (ParoA2) which contains a similar PrfA-binding site (PrfA-box) and a similar-10 box as PplcA but does not function as PrfA-dependent promoter. A series of hybrid plcA-aroA promoters by exchanging corresponding sequence elements of these two "promoters" were constructed and incorporated into upstream of a promoterless lacZ gene. The variant promoter-lacZ transcriptional fusions were then electroporated into L. monocytogenes wild-type strain P14, prfA mutant P14a and prfA deletion mutant A42, respectively. The expression level of PrfA is the highest in the P14a and the lowest in A42. The corresponding transcription activities of hybrid promoters were measured by the β-galactosidase assay. Results 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 22 or 23 bp. However, the interspace sequence and the sequence downstream of the -10 box of ParoA2 were strongly inhibitory for PrfA-dependent transcription. Conclusion Downstream sequence together -10 box of ParoA2 might fold into a hairpin structure when present in a single stranded DNA and possibly block the formation of the transcriptional initiation open complex, hence, inhibit the PrfA-dependent transcription from ParoA2.