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. 2010 Jun 11;76(15):5124–5139. doi: 10.1128/AEM.03107-09

FIG. 2.

FIG. 2.

Mapping of the L. seeligeri and L. monocytogenes prfA transcriptional start sites through 5′ RACE PCR on RNA isolated from bacteria grown without aeration at 37°C. (A) Agarose gel electrophoresis of 5′ RACE PCR products generated using RNA from stationary-phase L. monocytogenes (lanes 2 to 4) and L. seeligeri (lanes 5 to 8) and prfA-specific primers. Lanes 1 and 9, DNA marker; lanes 2 and 5, PCR on untailed L. monocytogenes and L. seeligeri cDNA, respectively (included to identify unspecific PCR products which would show up in this reaction); lane 3, PCR on tailed L. monocytogenes cDNA; lanes 6 and 7, PCR on tailed L. seeligeri cDNA (run in duplicate); lanes 4 and 8, negative PCR controls (no template). Arrows mark PCR product that was excised for cloning and sequencing to determine transcriptional start sites; the weak larger product found in L. seeligeri RACE PCR did not map to any apparent promoter site. (B) DNA sequence of the prfA promoter region in L. monocytogenes (strain 10403S; GenBank accession no. NZ_AARZ00000000) and L. seeligeri (strain FSL S4-039; determined in this study). The first (5′) nt shown here is 154 and 162 nt upstream of the start codon for L. monocytogenes and L. seeligeri, respectively; the fragments used for complementation all start upstream of the first nt shown here. The L. monocytogenes P1- and P2prfA promoters, including the σA- and σB-dependent promoters, are indicated as previously reported (13, 14, 45); −10 and −35 sequences are marked in bold and underlined; the PrfA binding box (55) is marked by a broken line (—) beneath the sequence; homologous sequences in L. seeligeri are also indicated in the same way. Translational start sites (ATG for L. monocytogenes and GTG for L. seeligeri) are indicated in bold and underlined. Transcriptional start sites mapped by RACE PCR in L. seeligeri and L. monocytogenes grown without aeration are indicated by an asterisk (*) (for L. monocytogenes grown without aeration, the P2prfA start site was identified in all 8 RACE PCR clones sequenced; for L. seeligeri grown without aeration, the P2prfA start site was identified in 13 of 14 RACE PCR clones sequenced).