title: Dual labeling of Pseudomonas putida with fluorescent proteins for in situ monitoring of conjugal transfer of the TOL plasmid.
abstract: We describe here a dual-labeling technique involving the green fluorescent protein (GFP) and the red fluorescent protein (DsRed) for in situ monitoring of horizontal gene transfer via conjugation. A GFPmut3b-tagged derivative of narrow-host-range TOL plasmid (pWWO) was delivered to Pseudomonas putida KT2442, which was chromosomally labeled with dsRed by transposon insertion via biparental mating. Green and red fluorescent proteins were coexpressed in donor P. putida cells. Cells expressing both fluorescent proteins were smaller in size than cells expressing GFP alone. Donors and transconjugants in mixed culture or sludge samples were discriminated on the basis of their fluorescence by using confocal laser scanning microscopy. Conjugal plasmid transfer frequencies on agar surfaces and in sludge microcosms were determined microscopically without cultivation. This method worked well for in situ monitoring of horizontal gene transfer in addition to tracking the fate of microorganisms released into complex environments. To the best of our knowledge, this is the first study that discusses the coexpression of GFP and DsRed for conjugal gene transfer studies.
authors.affiliation: Institute of Water Quality and Waste Management, Technical University of Munich, Garching 85748, Germany.
authors.forename: Y Venkata
authors.initials: YV
authors.lastname: Nancharaiah
authors.forename: Pierre
authors.initials: P
authors.lastname: Wattiau
authors.forename: Stefan
authors.initials: S
authors.lastname: Wuertz
authors.forename: Stephan
authors.initials: S
authors.lastname: Bathe
authors.forename: S Venkata
authors.initials: SV
authors.lastname: Mohan
authors.forename: Peter A
authors.initials: PA
authors.lastname: Wilderer
authors.forename: Martina
authors.initials: M
authors.lastname: Hausner
mesh_terms: D018441:Biofilms; D003227:Conjugation, Genetic; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism*; D010957:Plasmids; D016958:Pseudomonas putida / Q000235:genetics*
chemical_list: D008164:Luminescent Proteins; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins