Simple conjugation and outgrowth procedures for tagging vibrios with GFP, and factors affecting the stable expression of the gfp tag

Aim: Our goal was to develop a simple system for tagging wild-type marine bacteria with gfp. Methods and Results: Escherichia coli strain CC118λpir carrying the conjugative helper plasmid pEVS104 and the gfp-containing plasmid pKV111 was used to transfer gfp to Vibrio recipients. Four different media were tested for their ability to support the growth of recipients, but not the E. coli donor, to allow powerful enrichment of gfp-tagged wild-type vibrios from mating mixes. Forty-three vibrio strains, representing 39 different species, were successfully tagged with gfp using the conjugative transfer from E. coli followed by selective outgrowth at 15°C on ZoBell 2216E agar containing 0･5% sodium alginate. Using this outgrowth medium, colonies of GFP-expressing vibrio clones were detectable within 4 days. The percentage of visibly fluorescent cells in three representative GFP-tagged vibrios was higher at 15°C than at 20 or 25°C (c. 50% vs. 45% or 40%, respectively), and was also higher during the aerobic rather than the anaerobic culturing (c. 50% vs. 35%, respectively). Conclusions: We found a simple selective outgrowth technique that enabled us to isolate a wide variety of GFP-tagged marine vibrios following the conjugative transfer of gfp from E. coli. Significance and Impact of the Study: Tagging cells with GFP and related fluorescent proteins is a powerful approach for investigating the bacteria in situ, particularly during the colonization of hosts. The simple and cost-effective outgrowth condition described in this study could be applied to construct a wide variety gfp-tagged marine bacteria