Use and Evaluation of a pES213-Derived Plasmid for the Constitutive Expression of gfp Protein in Pathogenic Vibrios: a Tagging Tool for In Vitro Studies

Abstract

Insertion of green fluorescent protein (GFP) into bacterial cells for constitutive expression is a powerful tool for the localization of species of interest within complex mixtures. Here, we demonstrate and evaluate the efficacy of the pES213-derived donor plasmid pVSV102 (gfp Kn^r) as a conjugative tool for the tagging of Vibrio and related species (termed vibrios). Using a triparental mating assay assisted by the helper plasmid pEVS104 (tra trb Kn^r), we successfully tagged 12 species within the Vibrionaceae family representing 8 of the proposed clades. All transconjugant strains demonstrated bright fluorescence and were readily differentiable within complex mixtures of nontagged cells. Plasmid retention was assessed using persistence and subculture experimentation. Persistence experiments evaluated plasmid loss over time for nonsubcultured samples inoculated into antibiotic-free media and sterile artificial seawater, whereas subculture trials evaluated plasmid loss following one to four subculture passages. Strong plasmid retention (≥80%) was observed in persistence experiments for all transconjugant strains for up to 48 h in both antibiotic-free media and artificial seawater with the exception of Vibrio cholerae, which showed a substantial decline in media after 24 h. Subculturing experiments also demonstrated strong plasmid stability, with all transconjugant strains showing ≥80% retention after four subculture passages. The results of this research suggest that pVSV102 is a stable GFP plasmid for the tagging of a broad range of vibrios. IMPORTANCE Prior research has suggested that the use of Aliivibrio fischeri-derived donor plasmids with the pES213 origin of replication may provide increased plasmid stability for the tagging of vibrios compared to Escherichia coli-derived p15A plasmids. Here, we present a structured protocol for conjugation-based tagging of vibrios using the pES213-derived plasmid pVSV102 and evaluate the plasmid stability of tagged strains. These methods and the resulting transconjugant strains provide important standardized tools to facilitate experimentation requiring the use of traceable vibrio strains. Furthermore, the determination of the species-specific plasmid stability provides an estimation of the anticipated level of plasmid loss under the given set of culture conditions. This estimation can be used to reduce the occurrence of experimental biases introduced by plasmid drift.

Keywords: GFP tag; Vibrio; green fluorescent protein.