Characterization of the conjugative modules of the pRAS4 plasmid of Aeromonas salmonicida reveals an environmental lineage

Abstract

Conjugative plasmids of Aeromonas salmonicida, the causative agent of furunculosis in salmonids, have long been implicated in the dissemination of antimicrobial resistance in aquaculture. Among these, pRAS4 has remained the least characterized, with no complete published annotation and no molecular description of its conjugation machinery. In the present study, we report the complete sequence of pRAS4 from two A. salmonicida subsp. salmonicida strains, F878/91 and F830/91, isolated from Atlantic salmon (Salmo salar L.) showing characteristic signs of furunculosis on the Western coast of Norway in 1991. Mating experiments produced antibiotic-resistant transconjugants in Escherichia coli DH5α, and antimicrobial susceptibility testing of single-colony-purified transconjugants confirmed selective transfer of the tetracycline-resistance phenotype encoded by pRAS4. Domain based re-annotation using InterProScan identified two previously unassigned coding sequences as canonical components of the conjugation machinery: an 823-amino-acid coding sequence originally annotated as traN was reassigned as a TraG/VirD4-family type IV coupling protein (Pfam PF02534, E = 5.1 × 10−99), and a 366-amino-acid coding sequence originally labelled “hypothetical protein” was identified as a MobA/VirD2 family relaxase (Pfam PF03432, E = 4.7 × 10−14). Together with the canonical virB1–virB11 mating pair formation cluster and the mobC accessory relaxosome gene, these findings establish that pRAS4 encodes a complete and functional MOB_P/MPF_T conjugation system. PlasmidFinder analysis returned no replicon match for pRAS4, and BLASTp of the RepA protein returned no close match for the canonical IncU plasmid RepAs (pRAS1, pAr-32, pRA3). The closest sequence relatives of the pRAS4 RepA, type IV coupling protein and relaxase are environmental plasmids of Acidithiobacillus, Acidovorax, Microvirgula and related β- and γ-proteobacteria, and the pRAS4 backbone shares an extensive synteny with the plasmids pYKCT010 and pYK0414 from uncultured bacteria of Japanese river sediments. These findings indicate that pRAS4 is a self-transmissible R-plasmid carrying a previously uncharacterized environmental plasmid backbone, and the tetracycline Tet A efflux determinant flanked by Tn3 transposases represents a subsequent adaption under aquaculture associated antibiotic selection.