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Updated: Jul 7, 2026

Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing
Published on: July 7, 2020
Transposable modules generated by a single copy of insertion sequence ISPme1 and their influence on structure and
Dariusz Bartosik1, Mateusz Putyrski, Lukasz Dziewit
1Department of Bacterial Genetics, Institute of Microbiology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland. bartosik@biol.uw.edu.pl
Abstract:
We demonstrated that a single copy of insertion sequence ISPme1 can mobilize adjacent segments of genomic DNA of Paracoccus methylutens DM12, which leads to the generation of diverse transposable elements of various size and DNA contents. All elements (named transposable modules [TMos]) contain ISPme1 (placed at the 5' ends of the elements) and have variable 3'-end regions of between 0.5 and 5 kb. ISPme1 was shown to encode an outwardly oriented promoter, which may activate the transcription of genes transposed within TMos in evolutionarily distinct hosts. TMos may therefore be considered to be natural systems enabling gene capture, expression, and spread. However, unless these elements have been inserted into a highly conserved genetic context to enable a precise definition of their termini, it is extremely difficult or even impossible to identify them in bacterial genomes by in silico sequence analysis. We showed that TMos are present in the chromosome and plasmids of strain DM12. Sequence analysis of plasmid pMTH1 (32 kb) revealed that four TMos, previously identified with a trap vector, pMEC1, comprise 87% of its genome. Repeated TMos within pMTH1 may stimulate other structural rearrangements resulting from homologous recombination between long repeat sequences. This illustrates that TMos may play a significant role in shaping the structure of natural plasmids, which consequently may have a great impact on the evolution of plasmid genomes.
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