Incompatibility groups ofPseudomonasplasmids revisited: comprehensive analysis of R-factors and their replicons

Abstract

ABSTRACTBacterial plasmid incompatibility (Inc) groups, IncP-1 to IncP-14, have long been recognised as R-factors. These factors harbour antimicrobial resistance genes (ARGs) inPseudomonasspecies (PIncs). Despite their importance, some PIncs plasmid remain underexplored at the sequence level. This study attempts to address this gap by determining the complete nucleotide sequences of several key plasmids. Using BLAST search and AlphaFold3-based protein structure prediction, we identified replication initiation proteins (RIPs) and origins of vegetative replication (oriV) for these plasmids. Notably, mini-plasmids containing these RIP andoriVsequences successfully replicated inPseudomonas aeruginosa. Through pangenome analyses using the PLSDB database with the experimentally identified RIP gene reference, 2,351/59,895 plasmids were classified, and their core and accessory genes, including ARGs, were detected. Furthermore, phylogenetic analyses of the RIPs with publicly available protein sequences enabled the grouping of existing PIncs into distinct evolutionary lineages, providing a robust framework for identifying RIPs across a broader range of previously uncharacterised plasmids. The RIPs of 8,860 plasmids in the PLSDB were newly assigned. These findings offer crucial insights into the complex landscape of plasmid-mediated ARG dissemination, marking a significant advancement in the understanding ofPseudomonasplasmids across clinical and environmental contexts.