Fig. 4

Confirming DNA phosphorothioation and DNA restriction activities encoded by dndBCDE and dptFGH operons. a Equal amounts of genomic DNA isolated from NADC 6564 and other strains listed on the right side of the gel picture was either treated or not treated with iodine. The treated DNA was loaded in to lanes marked with + and untreated DNA loaded in to lanes marked with – signs. Smearing effect in the lane loaded with iodine-treated DNA is indicative of DNA degradation compared to the presence of intact DNA (approximately 25 to 50 kb in size based on the size of molecular weight markers loaded in lanes marked M) in lanes loaded with untreated DNA. b and c Confirmation of S-DNA modification-dependent restriction activity encoded by the dpt operon. The pUC18 plasmid DNA lacking S-DNA modification was electroporated into strains NADC 6564 (dpt operon-positive) and EDL933 (dpt operon-negative) and recovery of this plasmid was determined by comparing number of colonies produced on LB agar-carbenicillin (100 μg/mL) by the electroporated cultures of these strains (b). Absolute colony counts shown represent mean plus and minus standard deviation of three independent experiments (c). d Table showing comparisons of nucleotide sequence homology of peaX gene encoded on the IE to other four pea genes located elsewhere on the chromosome of NADC 6564 and to mcr genes encoding colistin resistance in E. coli. Numbers in colored checker boxes represent homology between compared genes numbered 1–7 and labeled with corresponding gene name on the outer margins of the Table. e Graph showing the plot of minimum inhibitory concentration (MIC) of colistin in strain NADC 6564 compared to strains EDL933 and Sakai lacking the peaX gene and the 39 kb insertion element. E. coli TOP10 (sensitive to colistin) and E. coli BEAR119605 (resistant to colistin) were used as negative and positive controls in the MIC assay. Results are mean plus the standard deviation of three independent assays. **** = p < 0.0001