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Generation and maintenance of the circularized multimeric IS26-associated translocatable unit encoding multidrug
Masamune Aihara1,2, Yasuhiro Gotoh3, Saki Shirahama4
1Department of Clinical Chemistry and Laboratory Medicine, Kyushu University Hospital, Fukuoka, Japan. aihara.masamune.402@m.kyushu-u.ac.jp.
Abstract:
In gram-negative bacteria, IS26 often exists in multidrug resistance (MDR) regions, forming a pseudocompound transposon (PCTn) that can be tandemly amplified. It also generates a circular intermediate called the "translocatable unit (TU)", but the TU has been detected only by PCR. Here, we demonstrate that in a Klebsiella pneumoniae MDR clone, mono- and multimeric forms of the TU were generated from the PCTn in a preexisting MDR plasmid where the inserted form of the TU was also tandemly amplified. The two modes of amplification were reproduced by culturing the original clone under antimicrobial selection pressure, and the amplified state was maintained in the absence of antibiotics. Mono- and multimeric forms of the circularized TU were generated in a RecA-dependent manner from the tandemly amplified TU, which can be generated in RecA-dependent and independent manners. These findings provide novel insights into the dynamic processes of genome amplification in bacteria.
Insights
This study reveals how the IS26 element in gram-negative bacteria amplifies its multidrug resistance (MDR) regions and forms circular translocatable units (TUs). These amplified states persist even without antibiotic pressure, offering insights into bacterial genome dynamics.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- IS26 elements are frequently found in multidrug resistance (MDR) regions of gram-negative bacteria.
- IS26 forms pseudocompound transposons (PCTn) capable of tandem amplification.
- A circular intermediate, the translocatable unit (TU), is generated but previously only detected via PCR.
Purpose of the Study:
- To investigate the generation and amplification of TUs from PCTn in a Klebsiella pneumoniae MDR clone.
- To understand the dynamics of TU formation and amplification under antimicrobial selection.
- To elucidate the role of RecA in the amplification processes.
Main Methods:
- Utilized a Klebsiella pneumoniae MDR clone containing an MDR plasmid with IS26 elements.
- Applied antimicrobial selection pressure to induce and study amplification.
- Analyzed mono- and multimeric forms of the TU and their amplification states.
- Investigated the RecA dependency of TU generation and amplification.
Main Results:
- Mono- and multimeric forms of the TU were generated from the PCTn in the MDR plasmid.
- The inserted TU form also underwent tandem amplification within the plasmid.
- Amplification was induced by antimicrobial selection and maintained without antibiotics.
- Circularized TUs were generated in a RecA-dependent manner from tandemly amplified TUs.
- Tandemly amplified TUs could be generated via RecA-dependent and independent pathways.
Conclusions:
- Demonstrated direct generation of mono- and multimeric TUs from PCTn in a K. pneumoniae MDR clone.
- Showcased that IS26-mediated amplification can be stable, persisting post-selection.
- Highlighted the dual role of RecA in TU formation and amplification, providing novel insights into bacterial genome plasticity.
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