A Novel Conjugation System in AMR-associated pELF-type Linear Plasmids of Enterococcus faecium.

The pELF-type linear plasmid is a unique transconjugative plasmid that disseminates various antimicrobial resistance (AMR) genes (e.g., vancomycin resistance in vancomycin-resistant Enterococcus faecium, [VRE]), leading to hospital outbreaks worldwide. Despite evidence of its role in AMR gene expansion, the molecular mechanisms underlying conjugative transfer of pELF-type plasmids have been poorly understood due to their unique linear structure and the lack of convenient genetic engineering tools adapted to E. faecium strain. Herein, we focused on a putative transconjugation-associated gene cluster, named the trapELF cluster, encoded on pELF2, a vanA-type vancomycin resistance gene-harboring plasmid. RNA-seq analysis of the pELF2 plasmid identified a putative operon containing an ftsK-like ATPase-encoding gene and an additional 10 genes as candidates for conjugative transfer of pELF-type linear plasmids. We constructed isogenic deletion mutants for these 10 genes and revealed that two genes, designated traC and traD, are essential for the conjugative transfer of pELF2 to a recipient strain. Inducible overexpression of the tra genes in trans from an ectopic shuttle vector confirmed the essentiality of traC and traD for conjugative transfer. Promoter assay using a luciferase reporter plasmid revealed that the upstream region of the putative tra operon possesses functional promoter activity characterized by constitutive strong transcriptional expression. Furthermore, we observed membrane localization for a fluorescent protein fusion of TraC, but not for that of TraD. Based on these findings, we propose a working model for the unique molecular machinery for the transconjugation of pELF-type linear plasmids. Author SummaryEnterococci are among the opportunistic pathogens of greatest global concern due to their multidrug resistance, and it is well established that various plasmids mediate horizontal transfer of resistance genes in this species, converting susceptible strains into resistant ones. While most enterococcal plasmids have a circular structure, a linear pELF-type plasmid was recently discovered. Since then, pELF-type plasmids have been reported worldwide as important vehicles for antimicrobial resistance genes, but their mechanism of transfer has remained unknown. In this study, we have, for the first time, successfully identified the genes required for the conjugative transfer of pELF-type plasmids. We confirmed that these genes are absent from known conjugative plasmids, yet are highly conserved among pELF-type plasmid sequences deposited in public databases. Taken together, these findings suggest that pELF-type plasmids possess a unique transfer mechanism that reflects their distinctive molecular architecture.