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Dual Labeling of Pseudomonas putida with Fluorescent Proteins for In Situ Monitoring of Conjugal Transfer of the TOL Plasmid

Y. Venkata Nancharaiah, Pierre Wattiau, Stefan Wuertz, Stephan Bathe, S. Venkata Mohan,^|| Peter A. Wilderer, and Martina Hausner^*

Institute of Water Quality and Waste Management, Technical University of Munich, Garching 85748, Germany

Received 22 November 2002/ Accepted 7 May 2003

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.

Present address: Water and Steam Chemistry Laboratory, BARC Facilities, Kalpakkam 603012, India.

Present address: Biological Engineering Unit, Catholic University of Louvain, B-1348 Louvain-la-Neuve, Belgium.

Present address: Department of Civil and Environmental Engineering, University of California, Davis, CA 95616.

^|| Present address: Biochemical and Environmental Engineering Centre, Indian Institute of Chemical Technology, Hyderabad 500 007, India.

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