In vivo de-amplification of a multi-resistance pseudo-compound transposon in Escherichia coli

Pulmones, R.; Moyo, S. J.; Tesfay, B.; Gidabayda, J.; Justine, M.; Hoyland Lohr, I.; Blomberg, B.; Wagstaff, S. P.; Langeland, N.; Roberts, A. P.

doi:10.64898/2026.03.06.710075

bioRxiv · 10.64898/2026.03.06.710075

In vivo de-amplification of a multi-resistance pseudo-compound transposon in Escherichia coli

Pulmones, R.·Moyo, S. J.·Tesfay, B.·Gidabayda, J.·Justine, M.·Hoyland Lohr, I.·Blomberg, B.·Wagstaff, S. P.·Langeland, N.·Roberts, A. P.

View original source ↗Export metadata ↓

Abstract

The rapid expansion of antimicrobial resistance (AMR) among Gram-negative pathogens presents a major clinical challenge, particularly in vulnerable populations such as infants. The dissemination of resistance is often mediated by mobile genetic elements (MGEs) that can mobilise antimicrobial resistance genes (ARGs) both within and between genomes. The insertion sequence (IS) IS26, is a MGE with the ability to replicate itself and associated ARGs, create translocatable units (TU), and produce tandem arrays of ARGs. Here we present the 18-week in vivo evolution of a community-acquired multi-drug resistant (MDR) Escherichia coli colonising an infant gut, characterised by a de-amplification of an IS26-mediated tandem array of ARGs. The hybrid-assembled ancestral and descendant strain genomes show an evolutionary rate of 10.22 SNPs per genome per year. Independent analysis of the hybrid genome assembly, and of Oxford Nanopore Technologies (ONT) and Illumina read-mapping support the existence of at least five copies of a TU (Tn3-like(tnpA)-tetR-tetA-yedA-{Delta}Tn1721(tnpA) -IS26-aac(6)-Ib-cr-blaOXA-1-{Delta}catB3-IS26) in the ancestor and only one in the descendant. Despite de-amplification, no change in fitness (p = 0.275) and piperacillin-tazobactam susceptibility (TZP) was observed. In contrast, gentamicin susceptibility increased, in the absence of known associated mutations. This study provides insight into IS26-mobility dynamics in vivo and their implications for AMR, within the rapidly changing environment of the neonatal gut.

Explore related subjects

microbiology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Pulmones, R., Moyo, S. J., Tesfay, B., Gidabayda, J., Justine, M., Hoyland Lohr, I., Blomberg, B., Wagstaff, S. P., Langeland, N., Roberts, A. P.. 2026-03-06. In vivo de-amplification of a multi-resistance pseudo-compound transposon in Escherichia coli. https://doi.org/10.64898/2026.03.06.710075

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Cryo-EM Structure of Mycobacteriophage D29 Reveals a Specialized Portal-Capsid Junction and a Unique 13-Subunit Baseplate Collar

Mycobacteriophages are promising agents against drug-resistant mycobacterial infections, yet their structural organization and host-recognition mechanisms remain poorly understood. Here, we determined cryo-EM structures of the capsid, connector, tail tube, and baseplate of the lytic, broad-host-range mycobacteriophage D29, revealing a near-complete virion architecture. At the capsid-portal interface, five copies of the previously uncharacterized protein gp19.1 bridge adjacent capsid subunits around the dodecameric portal, potentially reinforcing the symmetry-mismatched vertex. At the baseplate, 13 copies of gp6 assemble into a collar surrounding the sixfold-symmetric gp23-gp27 core. Remarkably, this 6:13 symmetry mismatch is accommodated through six distinct gp6- gp27 interfaces involving 12 gp6 subunits, leaving one subunit largely disengaged from the core. This asymmetric organization produces a tilted, outward-flaring collar that presents an extensive surface with the potential to engage the host cell. Whole-cell binding and plaque-competition assays further implicate the gp6 collar and the flexible trimeric central fiber gp31 in host recognition. Together, these findings reveal distinct structural adaptations at symmetry-mismatched interfaces and provide a structural framework for understanding mycobacteriophage assembly and host engagement.

microbiology↗

bioRxiv2026

A Luciferase Reporter of Ester Prodrug Delivery to the Cytosol of Intracellular Mycobacteria

The bacterial cell envelope restricts access to cytoplasmic targets and is a principal reason that otherwise potent antibacterial compounds fail in cells. Masking an anionic carboxylic acid as an ester is a common way to cross that barrier, but the strategy succeeds only if the target organism also hydrolyzes the ester once the compound arrives. These two requirements, permeation and intracellular unmasking, are rarely measured together in live bacterial cells, and existing reporters cannot specify where unmasking occurred. Here we describe a bioluminescent platform that resolves both steps in real time in live mycobacteria using carboxylate esters of D-luciferin. Luciferase requires that an ester both reach the bacterial cytoplasm and be hydrolyzed there, which makes light a compartment-specific readout of productive delivery. Across a series of alkyl esters in Mycobacterium smegmatis, chain length governed permeation while branching adjacent to the carbonyl governed esterase processing, indicating that the two parameters can be tuned independently. The n-butyl ester gave the strongest signal in broth, in infected macrophages, and in the pathogen Mycobacterium avium, whereas the tert-butyl ester was inert in Mycobacterium avium and unmasking of the whole series was attenuated at phagosomal pH. Ester activation is therefore both species- and compartment-dependent, and masking groups are best selected in the organism and environment in which they must act.

microbiology↗

bioRxiv2026

Flexible phosphorus allocation to the phosphoproteome underpins diatom regulatory adaptations to fluctuating phosphate supply

Diatoms are major drivers of marine phosphorus (P) cycling and are highly adapted to cope with variable P supply. P-starved diatoms can sense increases in P availability within seconds, accumulating this limiting resource through luxury polyphosphate (polyP) storage. Yet, how diatoms coordinate such responses is unknown. Protein phosphorylation is a core mechanism for environmental signal transduction in eukaryotes, but how cells balance investment in protein phosphorylation against competing physiological demands during P limitation is poorly understood. Here, we characterise system-wide remodelling of the Phaeodactylum tricornutum phosphoproteome in response to P deficiency and subsequent resupply. We identify widespread proteome dephosphorylation across 44% of phosphosites, revealing a previously unrecognised P-sparing strategy in marine phytoplankton. Following P resupply, diatoms orchestrate extensive phosphoproteome reprogramming within minutes. We characterise previously unrecognized components for early recovery from P limitation, including a novel environmentally prevalent intracellular H+/phosphate transporter strongly conserved in diverse diatoms. We also identify a vacuolar Ca2+-dependent protein kinase (CDPK) associated with polyP-storage granules. Mutant and inhibitor analyses demonstrate a role for Ca2+- and CDPK-signalling in the regulation of luxury polyP accumulation. Our findings reveal that recovery from P starvation is mediated by rapid phosphoregulatory control of intracellular P trafficking, organellar ATP generation coupled with luxury P storage, enabling diatoms to exploit newly available P and thrive in fluctuating P regimes. Collectively, our study uncovers the phosphoproteome as a flexible intracellular P reservoir and establishes mechanistic links between intracellular P budgeting, signalling and the marine P cycle.

microbiology↗