Poster Presentation BacPath 13: Molecular Analysis of Bacterial Pathogens Conference 2015

IS26-mediated excision of Translocatable Units (#152)

Christopher J. Harmer 1 , Ruth M. Hall 1
  1. University Of Sydney, Darlington, NSW, Australia

IS26 plays a key role in disseminating antibiotic resistance genes in Gram-negative bacteria. IS26-flanked structures resemble class I transposons, but we have recently shown that some of the features of IS26 movement do not resemble those of the IS and class I transposons studied to date. The novel IS26 movement mechanism involves a new family of mobile genetic elements called Translocatable Units (TU), made up of the central mobilized region with a single copy of IS26. This study aimed to examine IS26-mediated TU formation.

Most IS26-bounded transposons are stable. However, we fortuitously identified a kanamycin resistance “transposon”, Tn4352B (IS26-aphA1a-IS26), from which the TU was readily lost when recA- cells were grown without kanamycin selection. This ability was traced to the presence of two G nucleotides adjacent to the end of one of the copies of IS26. The sequence flanking Tn4352B influences the frequency of TU loss, but the two G’s and Tnp26 were essential. In the kanamycin-susceptible cells, only a single copy of IS26 remained. Hence, a single IS26 and the complete central region, the predicted TU, had been excised. The TU junction was detected by PCR. The circular TU carrying the kanamycin resistance gene aphA1a was generated in vitro and transformed into kanamycin-sensitive cells carrying an IS26-containing plasmid.Inkanamycin-resistant transformants the TU had been incorporated adjacent to the existing IS26, re-forming Tn4352B. These experiments have established that Tnp26 catalyzes precise excision of the TU. The role of the additional G’s is unclear but they do not increase tnp26 transcription. 

An additional unidentified factor in Escherichia coli may be influencing TU excision. TU loss from Tn4352B in both high-copy pUC plasmids and low-copy plasmids in E. coli was observed to occur. In contrast, Tn4352B was stable in Klebsiella pneumonia. This phenomenon needs to be explored further.