Fig. 4
From: Methylation of rRNA as a host defense against rampant group II intron retrotransposition
Characterization of top-12 mutants relative to control. a. Retrotransposition analysis. RTP assays were performed in low-throughput for each mutant to quantify retrotransposition frequencies. Representative plates are shown for the rlmH::ISS1 mutant and the isogenic wild-type control strain. Retrotransposition frequencies were calculated by dividing the CFU on selective media by that on nonselective media. b. Retrotransposition frequencies. Each mutant was evaluated relative to wild-type IL1403 containing pLNRK-RIG, omitting extreme outliers. Individual data points are shown, with a horizontal bar at the mean with standard deviation. For all data points, see Additional File 1: Fig. S10. c. DNA dot blots. Dot blots were performed to measure the amount of donor plasmid (pLNRK-RIG). For data, see Additional File 1: Fig. S11. d. Northern blots. Intron RNA level (precursor and spliced intron) was normalized against 16S rRNA. For gels, see Additional File 1: Fig. S12. e. Primer extension analysis. Splicing efficiency was calculated as the amount of spliced intron divided by the sum of spliced intron plus precursor. For gels, see Additional File 1: Fig. S13. f. Western blots. The amount of intron-encoded protein, LtrA, was normalized to total protein. For blots, see Additional File 1: Fig. S14. g. Induction from the PnisA promoter. The plasmid pLNRK-GFP reported GFP fluorescence. Values are normalized GFP of each mutant relative to the wild type after 3 h of induction (green, induced; black, uninduced). For all data, see Additional File 1: Fig. S15. h. Retrohoming analysis. Assays were performed with wild type and rlmH::ISS1 containing pLNRK-RIG (intron donor) and pMN1343 (homing site). No significant difference was observed between rlmH::ISS1 and control (p = 0.51). i. Complementation assays. Assays were performed with (blue) or without (red) complementation plasmids (CP) and reported relative to the respective wild-type control. Complementation significantly reduced the RTP frequency of rlmH::ISS1 (p < 0.05)