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Peer-reviewedCC BYVirulence

ISKpn28 transposition and genomic alterations in λ-Red recombinants of hypervirulent Klebsiella pneumoniae: Whole-genome sequencing and phenotypic analysis

  • Seong Hun Jeong1
  • Ju Yeong Lee2
  • Keon Young Lee1
  • Miri Hyun1
  • Ji Yeon Lee1
  • Michael McClelland3
  • Jin Kyung Kim2
  • Hyun ah Kim1
  1. 1Department of Infectious Diseases, Keimyung University Dongsan Hospital, Keimyung University School of Medicine, Keimyung University, Daegu, Republic of Korea
  2. 2Department of Microbiology, Keimyung University School of Medicine, Daegu, Republic of Korea
  3. 3Department of Microbiology and Molecular Genetics, University of California, Irvine, CA, USA
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Abstract

The λ-Red recombination system is commonly used to investigate gene function in Klebsiella pneumoniae; however, unintended secondary genomic alterations in recombinant strains may remain undetected by locus-specific validation methods such as polymerase chain reaction and Sanger sequencing. Following rmpA mutant construction in the clinical hypervirulent K. pneumoniae (hvKp) strain K255, two recombinant derivatives showed identical results on locus-specific validation, confirming the intended rmpA replacement. However, the strains differed in uronic acid content and interaction with murine macrophages. We therefore performed whole-genome sequencing (WGS) incorporating both short- and long-read data to investigate potential secondary genomic alterations. Both recombinant derivatives harbored an additional chromosomal copy of ISKpn28 within the nlpD gene. The K255ΔrmpA-1 strain additionally exhibited a chromosomal deletion of 55,652 base pairs, whereas K255ΔrmpA-2 harbored an ISKpn74-associated plasmid inversion. These findings provide genomic evidence of ISKpn28 transposition in recombinant derivatives of the clinical hvKp strain K255, and highlight the potential value of WGS incorporating long-read sequencing for assessing genome-wide integrity in λ-Red-derived recombinant hvKp strains harboring ISKpn28.

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Cite

BibTeXRIS
BibTeX
@article{jeong2026iskpn28,
  title = {{ISKpn28 transposition and genomic alterations in λ-Red recombinants of hypervirulent Klebsiella pneumoniae: Whole-genome sequencing and phenotypic analysis}},
  author = {Seong Hun Jeong and Ju Yeong Lee and Keon Young Lee and Miri Hyun and Ji Yeon Lee and Michael McClelland and Jin Kyung Kim and Hyun ah Kim},
  journal = {Virulence},
  year = {2026},
  month = nov,
  doi = {10.1080/21505594.2026.2732382},
  url = {https://doi.org/10.1080/21505594.2026.2732382},
  note = {CC BY},
}
RIS
TY  - JOUR
TI  - ISKpn28 transposition and genomic alterations in λ-Red recombinants of hypervirulent Klebsiella pneumoniae: Whole-genome sequencing and phenotypic analysis
AU  - Seong Hun Jeong
AU  - Ju Yeong Lee
AU  - Keon Young Lee
AU  - Miri Hyun
AU  - Ji Yeon Lee
AU  - Michael McClelland
AU  - Jin Kyung Kim
AU  - Hyun ah Kim
PY  - 2026
DA  - 2026/11/02
JO  - Virulence
DO  - 10.1080/21505594.2026.2732382
UR  - https://doi.org/10.1080/21505594.2026.2732382
AB  - The λ-Red recombination system is commonly used to investigate gene function in Klebsiella pneumoniae; however, unintended secondary genomic alterations in recombinant strains may remain undetected by locus-specific validation methods such as polymerase chain reaction and Sanger sequencing. Following rmpA mutant construction in the clinical hypervirulent K. pneumoniae (hvKp) strain K255, two recombinant derivatives showed identical results on locus-specific validation, confirming the intended rmpA replacement. However, the strains differed in uronic acid content and interaction with murine macrophages. We therefore performed whole-genome sequencing (WGS) incorporating both short- and long-read data to investigate potential secondary genomic alterations. Both recombinant derivatives harbored an additional chromosomal copy of ISKpn28 within the nlpD gene. The K255ΔrmpA-1 strain additionally exhibited a chromosomal deletion of 55,652 base pairs, whereas K255ΔrmpA-2 harbored an ISKpn74-associated plasmid inversion. These findings provide genomic evidence of ISKpn28 transposition in recombinant derivatives of the clinical hvKp strain K255, and highlight the potential value of WGS incorporating long-read sequencing for assessing genome-wide integrity in λ-Red-derived recombinant hvKp strains harboring ISKpn28.
SN  - 2150-5608
SN  - 2150-5594
ER  -