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

Abstract

SummaryIn vitro transcription starting from the promoters of the Listeria monocytogenes genes hly, plcA, actA, mpl, prfA and iap has been studied. Whereas transcription from P hly , P plcA and P actA is strictly PrfA-dependent, that from P iap , P prfA1/2 and, unexpectedly, also from P mpl is independent. Initiation of in vitro transcription at all tested promoters except P prfA requires high concentrations of ATP but not GTP. The nucleotides required in higher concentrations for efficient in vitro transcription are always included in the first three nucleotides of the corresponding transcript. RNA polymerase prepared from L. monocytogenes cultured either in rich culture medium (RNAP BHI ), exposed to heat shock conditions (RNAP 48 ) or conditioned in minimal essential medium (RNAP MEM ) shows significant differences in the transcription efficiencies when transcription is initiated at these promoters. Transcription starting from the PrfAdependent promoters P actA and P hly is enhanced with RNAP 48 and RNAP MEM (in relation to P iap -mediated transcription), while transcription from the other promoters is reduced when compared with RNAP BHI . These data suggest that in vivo transcription of the genes actA and hly may not function optimally with RNA polymerase loaded with the vegetative sigma factor 43, but may require a modified RNA polymerase, possibly loaded with an alternative sigma factor.

σB- and PrfA-Dependent Transcription of Genes Previously Classified as Putative Constituents of theListeria monocytogenesPrfA Regulon

“…After 2 hours at 378C with shaking, 8 mL of RNAprotectÔ was added to each tube to stabilize the RNA for subsequent RNA purification, as detailed below. Quantitative RT-PCR analysis of transcript levels for plcA, which is positively regulated by PrfA (Mengaud et al, 1991;Bohne et al, 1994;Sheehan et al, 1995;Lalic-Multhaler et al, 2001), validated that this charcoal exposure protocol increased PrfA-dependent transcription by >1 log (Fig. 1).…”

Glycerol Metabolism and PrfA Activity inListeria monocytogenes

“…In addition, we purified PrfA using buffers without glycerol and determined the specific activities of both PrfA preparations in the previously established in vitro runoff transcription assay using reaction buffers with and without glycerol (6,29,30). In vitro transcription was initiated at the PrfA-dependent hpt (uhpT) promoter (Phpt) as previously described (48).…”

LipA, a Tyrosine and Lipid Phosphatase Involved in the Virulence of Listeria monocytogenes

“…Six of the PrfA-regulated genes are clustered on Listeria pathogenicity island 1 (13,21,50). Some genes, such as inlA, inlB, inlC, mpl, and hly, can be expressed from both PrfA-dependent and PrfAindependent promoters (20,(32)(33)(34). Other genes expressed independently of PrfA, such as iap and aut, also contribute to Listeria virulence (11,32).…”