ICE in ruminant Mycoplasma

Integrative and conjugative elements (ICEs) are major drivers of horizontal gene transfer (HGT) in bacteria, yet the contribution of the recipient cells in ICE transfer remains poorly understood. Using the ruminant pathogens Mycoplasma agalactiae and Mycoplasma bovis as minimal models, we combined genome-wide transposon mutagenesis with high-throughput mating assays to study recipient-encoded factors involved in ICE acquisition. The surface-exposed lipoprotein P48 was identified as the primary determinant of ICE uptake in both species. Structural and functional assays revealed that P48 acts as a substrate-binding component of an ABC transporter with nucleoside-binding capacity. A point mutation disrupting nucleoside binding was sufficient to severely impair ICE acquisition, establishing that nucleoside recognition by P48 is required for conjugative transfer. However, ICE uptake did not require nucleoside transport, as inactivation of the permease component of the associated ABC transporter conferred resistance to nucleoside analog toxicity but not to ICE invasion. Loss of P48 also triggered transcriptional activation of vestigial ICE genes, suggesting that surface recognition may influence the intracellular state of the recipient. Remarkably, ICE transmission from recipient-derived donors was unaffected by loss of P48, underscoring its role as an acquisition-specific factor. Together, our study uncovers a novel, surface-exposed recipient factor required for efficient ICE transfer in mycoplasmas and establishes nucleotide binding as a central function in conjugation. By demonstrating that recipient-expressed functions can directly determine the success of ICE dissemination, this work challenges the donor-centric paradigm of bacterial conjugation and suggests new strategies to limit horizontal gene flow in pathogenic and synthetic mycoplasmas.