A plasmid-encoded Klebsiella michiganensis HipBA type II toxin–antitoxin system makes a significant contribution to plasmid maintenance

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Shutt-McCabe, J, Hoyles, L ORCID: https://orcid.org/0000-0002-6418-342X and McVicker, G ORCID: https://orcid.org/0000-0002-6967-5968, 2024. A plasmid-encoded Klebsiella michiganensis HipBA type II toxin–antitoxin system makes a significant contribution to plasmid maintenance. In: Klebsiella Epidemiology and Biology Symposium 2024, Paris, France, 20-22 November 2024.

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Abstract

Introduction: HipBA is considered a chromosome-encoded type II toxin–antitoxin (TA) system, implicated in persister formation. HipA toxin acts as a serine/threonine kinase that inhibits cell growth, while the HipB antitoxin neutralizes the toxin. Through bioinformatics analysis, we found HipBA on a plasmid of Klebsiella michiganensis PS_Koxy4. In this study, we characterized this plasmid-encoded HipBA system.

Methods: SLING was used to identify hipBA in the genome of PS_Koxy4, and across the K. oxytoca species complex (KoSC) (n=860 genomes). hipA from pPSKoxy_4_2 was cloned into the arabinose-inducible expression vector pBAD33, and its expression induced in Escherichia coli NEB 5-alpha. hipB was cloned into the pBAD33-compatible vector pGM101 under its native promoter. Viability assays were conducted to determine whether HipB nullified the effects of HipA in Escherichia coli and Klebsiella oxytoca NCTC 13727. A non-toxic mutant of hipA was generated by passage of the expression vector through K. oxytoca. A pSTAB vector combining the IncA/C replication origin from pPSKoxy_4_2 with the sacB-neoR cassette from pIB279 was constructed, with or without the hipBA locus from pPSKoxy_4_2. These plasmids were transformed independently into either E. coli or K. oxytoca to observe plasmid stability in both E. coli and Klebsiella lacking native plasmids and any conflicting antibiotic resistance.

Results: The plasmid-encoded HipBA type II TA system of K. michiganensis PS_Koxy4 is a functional and highly effective means of effecting plasmid stabilization. HipA is highly toxic to K. oxytoca, to the extent that the bacterium mutates the toxin to abrogate its effects. Examining the diversity of plasmid- and chromosome-encoded HipBA across the KoSC, we found at least four additional variants of plasmid-encoded HipBA that are distinct from KoSC chromosome-encoded HipBA. These variants remain to be characterized.

Discussion and conclusions: Bacterial TA systems contribute to the stability of mobile genetic elements through a phenomenon called addiction. Prior to this study, only four type II TA systems (vapBC, mazEF, vagCD, kacAT) had been experimentally validated for Klebsiella (pneumoniae). Here we characterize the first plasmid-encoded variant of HipBA. This work expands our knowledge both of plasmid stabilization in clinically relevant Klebsiella spp. and mobile genetic elements more broadly.

Item Type:	Conference contribution
Description:	Poster
Creators:	Shutt-McCabe, J., Hoyles, L. and McVicker, G.
Date:	November 2024
Identifiers:	Number Type 2459311 Other
Divisions:	Schools > School of Science and Technology
Record created by:	Laura Borcherds
Date Added:	27 Jun 2025 10:12
Last Modified:	27 Jun 2025 10:31
Related URLs:	Organisation
URI:	https://irep.ntu.ac.uk/id/eprint/53838