The rise of antibiotic resistance poses a growing global health challenge, complicating the treatment of bacterial infections. This issue is intensified by horizontal gene transfer, specifically through conjugative plasmids, carrying not only antibiotic resistance genes but also the necessary machinery for their transfer. A suitable model for studying this mechanism is the conjugative plasmid pIP501, encoding a type IV secretion system (T4SS) which consists of 15 transfer genes (traA - traO) and is arranged in a single operon. pIP501 serves as a valuable model for investigating Gram-positive bacterial conjugative transfer.
Our study employed markerless gene knockouts in pIP501, utilizing in vivo biparental mating assays with Enterococcus faecalis and other Firmicutes strains to understand the functions of transfer proteins. Results highlighted the essential nature of 13 out of 15 transfer proteins. Interestingly, TraO, a putative surface adhesin, was found non-essential, demonstrating only a transfer rate reduction. Nevertheless, TraO is assumed to play a crucial role in facilitating contact between donor and recipient cells prior to conjugative transfer.
To broaden our understanding of TraO across bacterial species, we generated new pIP501 and traO deletion strains. Diverse donor-recipient combinations enabled a comparative analysis of transfer rates among strains, encompassing such as Enterococcus and Streptococcus. Future investigations should focus on the role of TraO domains, necessitating complementation experiments with truncated TraO variants in inter- and intraspecies biparental mating assays. Furthermore, understanding TraO's structure can provide valuable insights into its localization, fold and domain functions.