Insights from public database sequences related to the replication initiation protein TrfA of the IncP-1 plasmid RK2

Replicon typing identifies sequences similar to known DNA replication initiators and is widely used to detect specific plasmid groups (e.g., IncP-1) in genome and metagenome sequencing data. However, the characteristics of these homologous sequences in public databases have not been systematically assessed, making it difficult to determine whether detecting a specific replicon type reliably indicates the presence of a particular plasmid group. Here, we conducted amino acid sequence alignments to identify sequences similar to the replication initiation protein TrfA of the IncP-1 plasmid RK2 in the NCBI non-redundant (nr) database. In the nr nucleotide database, TrfA-matched nucleotide sequences were found across diverse taxonomic groups and replicons, including complete and partial plasmids and chromosomes. In total, 76 protein sequences from the reference plasmid RK2 were screened against the nucleotide sequences of the trfA-harboring plasmids to identify candidate IncP-1 plasmids. TrfA-related proteins, originating from bacterial chromosomes, plasmids, and phages, were selected from the nr amino acid database and used to infer phylogenetic trees. Our phylogenetic analyses reveal that TrfA homologs have diverged through vertical inheritance within IncP-1 and horizontal gene transfer across replicons and taxa. These findings caution against overreliance on single-gene replicon typing to infer plasmid group identity from sequence data.