TraN variants mediate conjugation species specificity of IncA/C, IncH and Acinetobacter baumannii plasmids

IncA/C and IncH plasmids commonly carry antimicrobial resistance genes, notably bla _NDM-1 . Although these plasmids disseminate among Gram-negative pathogens via conjugation, the mechanisms underlying mating pair stabilisation (MPS) and conjugation species specificity remain poorly understood. In IncF plasmids, MPS is mediated by interactions between outer membrane proteins (OMP) encoded by the plasmids in the donor (TraN) and by the chromosome in the recipient. Using the Plascad database, we extracted 1,436 TraN sequences: 62.5% (898/1,436), mainly in IncF plasmids, are 550–660aa (we renamed TraN short, TraN _S ); 15% (216/1,436), in IncA/C plasmids, are 880–950aa (TraN medium, TraN _M ); and 11% (160/1,436), in IncH plasmids, are 1,050–1,070aa (TraN long, TraN _L ). A group of six TraN from Acinetobacter baumannii plasmids (891aa) were designated TraN V-shaped (TraN _V ). Like TraN _S , TraN _M and TraN _L contain base and distal tip domains essential for conjugation, whereas TraN _V has a base and two distinct tip domains forming a V-shaped structure. TraN _M , TraN _L and TraN _V determine conjugation species specificity, with TraN _L cooperating with OmpA. Tip swapping reverses conjugation specificity, revealing how TraN _M and TraN _L diversity influence plasmid host range and AMR dissemination. Our new data reveal the molecular basis of plasmid host specificity and broaden our understanding of how conjugation drives the dissemination of antimicrobial resistance genes among clinically relevant bacteria.