Identification of essential genes for conjugative transfer in antimicrobial resistance-associated pELF-type linear plasmids of opportunistic pathogen Enterococcus faecium.

The pELF-type linear plasmid is a critical mobile genetic element responsible for the dissemination of various antimicrobial resistance (AMR) genes, most notably vancomycin resistance in Enterococcus faecium, which is a leading cause of hospital outbreaks worldwide. Despite their crucial role in the expansion of AMR, the molecular mechanisms underlying the conjugative transfer of these linear plasmids remain poorly understood. In this study, the transfer (tra) region of pELF2, a representative vanA-harboring linear plasmid was characterized. Transcriptomic data suggested that the FtsK/VirD4-type adenosine triphosphatase is encoded within a multi-gene operon. By developing a genetic manipulation framework for E. faecium, an extensive mutational analysis of the tra region was performed and the following three essential genes were identified: traCB4 (a putative VirB4 analog), traDD4 (a VirD4-like coupling protein), and traGB6 (a putative VirB6 analog). These genes are indispensable for conjugative transfer. Reporter assays experimentally confirmed the presence of a functional promoter upstream of the identified tra genes. We confirmed that these genes are highly conserved among pELF-type plasmid sequences deposited in public database. The study findings revealed that pELF-type plasmids utilize highly minimized conjugation machinery, which is similar to unusual systems previously identified in other gram-positive bacteria, such as Streptomyces. This study provides the first molecular insights into the transmission of these clinically important linear plasmids in enterococci and lays a foundation for understanding the dissemination of resistance determinants mediated by atypical mobile genetic elements.