In vitro transcription of PrfA-dependent and -independent genes of Listeria monocytogenes

In vitro transcription starting from the promoters of the Listeria monocyto genes genes hly, plcA, actA, mpl, prfA and iap has been studied. Whereas tr anscription from P-hly, P-plcA and P-actA is strictly PrfA-dependent, that from P-iap, P-prfA1/2 and, unexpectedly, also from P-mpi is independent. In itiation of in vitro transcription at all tested promoters except P-prfA re quires high concentrations of ATP but not GTP. The nucleotides required in higher concentrations for efficient in vitro transcription are always inclu ded in the first three nucleotides; of the corresponding transcript. RNA po lymerase prepared from L. monocytogenes cultured either in rich culture med ium (RNAP(BHI)), exposed to heat shock conditions (RNAP(48)) or conditioned in minimal essential medium (RNAP(MEM)) shows significant differences in t he transcription efficiencies when transcription is initiated at these prom oters. Transcription starting from the PrfA-dependent promoters P-actA and P-hly is enhanced with RNAP48 and RNAPMEM (in relation to Pip-mediated tran scription), while transcription from the other promoters is reduced when co mpared with RNAP(BHI)-These data suggest that in vivo transcription of the genes actA and hly may not function optimally with RNA polymerase loaded wi th the vegetative sigma factor 43, but may require a modified RNA polymeras e, possibly loaded with an alternative sigma factor.