BACTERIAL PLASMID CONJUGATION ON SEMISOLID SURFACES MONITORED WITH GREEN FLUORESCENT PROTEIN (GFP) FROM AEQUOREA-VICTORIA AS A MARKER

Horizontal transfer of the TOL plasmid was examined in Pseudomonas put ida (Pp) KT2442 micro-colonies on semisolid agar surfaces. Horizontal gene transfer is usually studied in large populations where all inform ation is based on average estimates of the transfer events in the enti re population. We have used the green fluorescent protein (GFP) from t he jellyfish Aequorea victoria as a plasmid marker, in combination wit h single-cell observations. This provided hitherto unknown details on the distribution of cells active in conjugation, In the present study, donor cells containing the gfp gene expressed from the bacteriophage T7 Phi 10 promoter on the TOL plasmid, and recipient cells expressing the corresponding phage RNA polymerase allowed us to monitor the occur rence of ex-conjugants as green fluorescent cells upon illumination wi th blue light (470-490 nm). Further, the recipients were labeled with the luxAB genes to distinguish micro-colonies of donor cells from reci pient cells. We conclude that conjugal plasmid transfer in Pp KT2442 c ells on semi-solid surfaces occurs mainly during a short period of tim e after the initial contact of donors and recipients, indicating that spread of the TOL plasmid is limited in static, but viable cultures.