Figure 3
Primer extension analysis of sagA/pel from wild type, sagA/pel mutant and kidney-recovered strains. Bacteria were grown overnight at 37°C with 10% CO2 in THY broth. RNA was extracted and sagA/pel was detected using a primer extension assay and sagA/pel-specific primers. Equal amounts of RNA were loaded in each lane. Lane 1, RNA isolated from a wild type CS101 strain. Lane 2, RNA from an isogenic sagA/pel::Tn917 mutant. Lanes 3 and 4, RNA isolated from 2 different KR variants of the sagA/pel mutant. The larger sagA/pel transcript is indicated as t1, the smaller as t2. The DNA sequence in the lower part of the figure is from the region around the sagA/pel promoter. These represent bases 598514–598593 in the S. pyogenes genome [43]. The bases in italics are the putative -10 region of the sagA/pel promoter. The overlined regions are 16, 6 and 6 base inverted repeats. The bold letters are the 5'-ends of the t1 and t2 transcripts as determined by primer extension. The position of the Tn917 insertion is indicated by the ^ symbol. Note that the site of insertion is slightly different from what was previously reported [4]. The t2 transcript was not detected in the KR variants even at longer exposures.