Abstract
This study aimed to reconstruct the classification system of Pseudomonas-derived plasmids and establish a comprehensive database. We applied replicon typing based on nucleotide sequences of replication initiation proteins (RIPs) and replication origins (oriV). RIPs and oriVs were experimentally identified from representative plasmids, confirming their replication and transfer abilities. A curated reference library (repP library) was constructed and used to reclassify 846 Pseudomonas plasmids in PLSDB, with 279 (~30%) newly assigned to PInc groups. RIP analysis revealed that ~60% of these plasmids have winged-helix (WH) domain-containing RIPs, and ~20% (180 plasmids) carry AEP-type RIPs. Similar patterns were found across all PLSDB plasmids. We also developed a method to determine host ranges using GFP-based conjugation and droplet digital PCR (ddPCR) with water-in-oil emulsions. This study provides a refined framework for plasmid classification and host annotation, supporting advances in microbial genomics and antimicrobial resistance research.
