Fig. 1
From: Methylation of rRNA as a host defense against rampant group II intron retrotransposition
Mutant library generation and characterization. a. Intron-donor plasmid pLNRK-RIG. The retrotransposition indicator gene (RIG) is located within the Ll.LtrB group II intron (red), which is under the control of the nisin promotor (PnisA). RIG contains a kanamycin resistance gene (black boxed arrow) with its own promoter (Pkan opposite to the direction of transcription), interrupted by a group I intron (gpI, white box). The Ll.LtrB intron is flanked by partial native exons and contains its own intron-encoded protein, LtrA. The pLNRK backbone contains a chloramphenicol resistance gene (camR) [21]. b. Retrotransposition assays identify retrotransposition events through the acquisition of kanamycin resistance (KanR) after the group I intron is spliced out. c. ISS1-mediated mutagenesis schematic. Cells containing the intron donor plasmid, pLNRK-RIG, were transformed with pGh4:ISS1 and selected for erythromycin resistance. Cultures containing both plasmids were grown to allow for transposition of the ISS1 transposon, followed by a temperature shift to remove the temperature sensitive pGh4:ISS1, screened for the loss of erythromycin resistance, and storage in 96-well plates alongside various controls. For details, see Additional File 1: Fig. S1 d. Southern blot analysis of the library for ISS1 copy number. A random subset of 50 ISS1 mutants were probed for ISS1 insertions using Southern blotting. Dark blue dot indicates a strain had multiple insertions. For all data, see Additional File 1: Fig. S2. e. Distribution of the ISS1 mutant library insertions (blue) around the L. lactis IL1403 chromosome (black). Sequencing and analysis of the library resulted in the identification of 746 unique ISS1 insertions. f. Comparison of functional coverage of the ISS1 mutant library (blue) against the L. lactis genome (black), based on relative abundance of COG categories. Asterisks represent a significant difference (p < 0.05) as determined by a hypergeometric test. A key of all COG categories is in Additional File 1: Table S3