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Rob Moran @prob91.bsky.social · 18/12/2025

[14/14] pRUM-like plasmids continue to diversify through the actions of IS1216 and other small MGEs These adaptable MDR platforms contribute to the success of CC17 E. faecium, and have the potential to enter other clones, species or genera through cointegrate formation + HGT Thanks for reading!

Schematic overview of ongoing pRUM-like plasmid evolution. At the centre of the figure is a basic pRUM-like structure, which is surrounded by other genetic elements that can contribute to its evolution. Other elements are linked to the pRUM-like structure by arrows that represent their acquisition or loss, with labels describing the mechanisms involved. These include: small MGEs inserting into the pRUM-like structure, IS1216 from the pRUM-like accessory region mediating adjacent backbone deletions, small plasmids being integrated into pRUM-like structures through IS1216 copy-in cointegrate formation, larger IS1216-containing plasmids or free IS1216 translocatable units being integrated through IS1216 targeted conservative cointegrate formation, and IS1216 translocatable unit loss or cointegrate plasmid resolution via homologous recombination.

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Rob Moran @prob91.bsky.social · 18/12/2025

[13/14] We propose a model for the IS1216-mediated evolution of the pRUM-like lineage from pCANE via an intermediate we call pMOLASSES This seems to have involved an evolutionary trade-off, swapping transfer ability for the plasticity of the IS1216 accessory region 🤔 more on this in the paper!

Schematic outlining our hypothesis for the evolution of pRUM-like plasmids from a pCANE-like ancestor. In part A, a pCANE like structure acquires an IS1216 translocatable unit to generate an intermediate structure called pMOLASSES. In part B, a pMOLASSES structure undergoes IS1216-mediated deletion events that remove parts of its backbone to generate a pRUM-like structure.

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Rob Moran @prob91.bsky.social · 18/12/2025

[12/14] The 1990s plasmid matched the pRUM backbone almost perfectly, but it was larger and didn’t contain IS1216… instead of an accessory region, it had 44 kb that looked like backbone and contained putative conjugation determinants 🚀 This looked like the ancestor of pRUM, so we called it pCANE

Alignment of pRUM and pCANE, showing that pCANE includes a near-perfect match to the entire pRUM-like backbone, plus additional backbone instead of the pRUM accessory region. The additional backbone segment includes putative conjugative transfer determinants (for pilus formation, a coupling protein, ATPase, a relaxase) and establishments determinants (anti-resistriction and single-stranded DNA-binding)

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Rob Moran @prob91.bsky.social · 18/12/2025

[9/14] Importantly, we saw lots of cointegrate formation, generating structures comprised of parts from usually distinct plasmids IS26 family elements are great at this! Several small plasmids have been rolled into pRUM-like structures, including pCOLA and pDRY, named after popular rum mixers 🍹

Overview of pRUM-like plasmid structures showing notable variants generated by IS1216 activity, including the acquisition of entire small plasmids, or parts of larger plasmids (most notably examples usually found in species or genera other than E. faecium)

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Rob Moran @prob91.bsky.social · 18/12/2025

[8/14] Examining plasmid structures revealed enormous variation! Most was in the accessory region, which ranged in size from 809 bp (a lone IS1216) to 285 kb, and included all sorts of acquired genes Multiple IS1216-mediated deletions have also removed adjacent parts of the backbone

Map of a representative pRUM-like plasmid backbone with variation marked across it, including insertions in the backbone, antibiotic resistane genes and plasmid replicons found in the accessory region, and backbone deletions extending out from the IS1216 at the boundaries of the accessory region

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Rob Moran @prob91.bsky.social · 18/12/2025

[7/14] We screened GenBank and found 150 complete pRUM-like plasmid sequences Their mostly E. faecium hosts were isolated from various sources around the world, and represented an array of STs… though all but one were E. faecium CC17

Bar chart showing the numbers of pRUM like plasmids found in Enterococcus faecium of various sequence types - the bars are coloured to show the sources of isolation for E. faecium hosts, which include various human clinical samples, human or animal faeces, pet food, or marine sediment

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Rob Moran @prob91.bsky.social · 18/12/2025

[3/14] Two plasmids caught my eye: pHHEf1 and pHHEf2 Their pRUM-like backbones were identical, with ARG-bearing accessory regions in exactly the same position, but the content of those regions was totally different! Strikingly, both were bounded by copies of the insertion sequence IS1216…

Two circular plasmid maps - their backbone is the same, but they have accessory regions that contain different things - vancomycin resistance genes in pHHEf1, and ertythromycin/aminglycoside resistance genes + the integrated small plasmid pCOLA in pHHEf2.... below the circular maps is a linear alignment of the plasmid sequences that confirms their backbones are the same, but their accessory regions, although located in the same backbone position, are completely different

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