Direct Detection and Quantification of Horizontal Gene Transfer by Using Flow Cytometry and gfp as a Reporter Gene Søren J. Sørensen, 1 Anders H. Sørensen, 1 Lars H. Hansen, 1 Gunnar Oregaard, 1 Duncan Veal 2 1 Department of General Microbiology, Institute of Molecular Biology, University of Copenhagen, Sølvgade 83 H, DK 1307, Copenhagen, Denmark 2 Centre for Fluorimetric Applications in Biotechnology, Department of Biological Sciences, Macquarie University, Sydney, New South Wales 2109, Australia Received: 23 September 2002 / Accepted: 2 October 2002 Abstract. A new cultivation-independent method for studying conjugal gene transfer between bacteria was evaluated. The method was based on direct detection and enumeration of donor and transconjugant bacterial cells by flow cytometry. Specific detection of transconjugants was obtained by using a conjugative plasmid tagged with a reporter gene (gfp) encoding green fluorescent protein. A chromo- somal encoded repressor (lacI q1 ) repressed expression of GFP in the donor bacteria. Enumeration of the donor cells was performed after induction of GFP expression by the addition of inducer isopropyl-thio- -D-galactoside (IPTG). The method presented here provided simple and precise quantification of horizontal gene transfer between both Escherichia coli and Pseudomonas putida strains. The spread of plasmids carrying antimicrobial resistance or other undesirable functions has come under increased scrutiny in recent years. Despite several years of re- search, there is still a considerable lack of information on the importance of horizontal gene transfer in bacterial communities. Most studies on horizontal gene transfer in natural environments have been based on cultivation techniques [8, 13, 15]. Since only a fraction of the total microbial population can be studied with these methods, the significance of these studies can be questioned [16]. Here we describe a cultivation-independent method for the detection and quantification of horizontal gene transfer among bacteria in natural environments. For this purpose, we have used a conjugative plasmid (pKJK10) with a fluorescent marker gene (gfp). In brief, the gfp gene marker on the plasmid is fused to a synthetic lac promoter. The lacI q1 gene was introduced into the chro- mosome of the donor bacteria in order to facilitate re- pression of gfp expression in the donor cells. Green fluorescence can be induced in the donor cells by the addition of the gratuitous inducer isopropyl-thio--D- galactoside (IPTG). After transfer of the plasmid to re- cipient bacteria lacking the chromosomally encoded re- pressor, the gfp gene will be expressed, and the transconjugant cells can be detected by their green fluo- rescence. Bacterial populations can be analyzed and sorted by flow cytometry according to cell size, surface structure, and fluorescence properties. By utilizing the fluores- cently labeled reporter plasmids described above, hori- zontal gene transfer was detected and quantified by flow cytometry, and transconjugant cells were isolated by fluorescence-activated cell sorting. Materials and Methods Bacterial strains and media. All bacterial strains and plasmids used in this study are listed in Table 1. Bacterial strains were routinely grown overnight at 30°C in ABTGC broth (AB basal salts + 0.1% wt/vol thiamine + 0.2% wt/vol glucose + 0.5% wt/vol casaminoacids) [2] supplemented with appropriate antibiotics and washed three times in PBS [12] before use. PBS was also used for serial dilutions for plating and direct counting in epi-fluorescence microscopy (EFM). A modified Mab buffer [17] containing 50 mM tetra sodium pyrophosphate and 0.05% Tween 80 was used as diluent for flow cytometric analysis. LB [12] modified to low salt conditions (4 g NaCl/L), LB(4), was used as general growth medium throughout the study. Construction of the pUTI q1 mini-Tn5. The lacI q1 gene was excised from p2172 [11] as an XmnI-FspI fragment and ligated into a HincII- digested pUC18-NotI plasmid [7]. Thereafter, the gene was excised as a NotI fragment and ligated into the unique NotI site in pUT Kn-res [9]. Correspondence to: S. J. Sørensen; email: SJS@MERMAID.MOLBIO. KU.DK CURRENT MICROBIOLOGY Vol. 47 (2003), pp. 129 –133 DOI: 10.1007/s00284-002-3978-0 Current Microbiology An International Journal © Springer-Verlag New York Inc. 2003