FIG 1.
Multimerization of plasmids carrying blaIMP-6 in E. coli. (A) Ladder bands on a Southern blot using a blaIMP-6 probe following S1-PFGE. Southern blotting with a blaIMP-6 probe following PFGE of S1-digested genomic DNA from E. coli isolates E042, E044, E058, E059, E114, and E244 revealed ladder patterns, whereas that of K. pneumoniae isolate E188 carrying a plasmid with blaIMP-6 (pE188_IMP6) revealed a single band. Lane M, DNA size marker (lambda ladder; Bio-Rad). (B) Ladder band pattern of the wild type and transconjugant. The ladder band on a Southern blot with a blaIMP-6 probe following S1-PFGE in isolate E044 was detected in a pE044_IMP6 conjugant E. coli isolate as well. (C) Comparison of plasmids pE044_IMP6 and pE188_IMP6. pE044_IMP6 consisted of pE188_IMP6 juxtaposed with three sets of transposase genes of the IS91 family. Block arrows indicate confirmed or putative open reading frames and their orientations. Arrow size is proportional to the predicted open reading frame length. The color code is as follows: red, carbapenem resistance gene; yellow, other antimicrobial resistance gene; light blue, conjugative transfer gene; blue, mobile element. Putative, hypothetical, or unknown genes are represented by gray arrows. (D) DNA fragments contained in each ladder band included only the sequence of pE044_IMP6. DNA fragments extracted from bands at 50, 100, 150, and 200 kbp by S1-PFGE were sequenced by an Illumina MiSeq system. Mean and maximum depths of sequence reads mapped against pE044_IMP6 and the full-length chromosome indicated that each band consisted of multimerized plasmid pE044_IMP6. (E) Size distribution of plasmid reads obtained from ultra-long-read sequencing. The top and bottom of the plots are drawn in different y-axis value ranges. The frequencies of the integer multiplication of relative length were higher than surrounding ones. (F) Box plot in each bin of size distribution. The matched ratio was calculated as the ratio of the number of matched nucleotides to the read length. (G) Comparison of the genomic structures of the longest sequence read and plasmid pE044_IMP6. The longest read obtained from ultra-long-read sequencing was comprised of almost four multimerized monomer plasmids. (H) pE044_IMP6 multimerized by recA. Southern blotting with a blaIMP-6 probe following S1-PFGE was conducted for E. coli isolates E188 and E044, pE188_IMP6 transformants ME188 and JW188 (recA-deficient ME188), and pE044_IMP6 transformants ME044 and JW044 (recA-deficient ME044).