A TRANSPOSON FOR GREEN FLUORESCENT PROTEIN TRANSCRIPTIONAL FUSIONS - APPLICATION FOR BACTERIAL TRANSPORT EXPERIMENTS

The movement of bacteria through groundwater is a poorly understood pr ocess. Factors such as soil porosity and mineralogy, heterogeneity of soil particle size, and response of the bacteria to their environment contribute to the pattern of bacterial flow, The identification of tra nsported bacteria is often a limiting factor in both laboratory and he ld transport experiments, Two bacterial strains were modified for use in bacterial transport experiments: a strain of Escherichia coli harbo ring the pGFP plasmid and a strain of Pseudomonas putida modified with a Tn5 derivative, Tn5GFP1. The Tn5GFP1 transposon incorporates the ge ne (gfp) encoding green fluorescent protein (GFP) and can be used to m utagenize Gram(-) bacteria. Fluorescent colonies were suspended in pho sphate-buffered saline (PBS) at a concentration of approx. 10(9) bacte ria/ml. A 10-cm glass column packed with quartz sand (diameter range 1 77-250 mu m) was equilibrated with PBS prior to the forced flow introd uction of the bacteria, Collected fractions were analyzed and the bact eria quantitated using a fluorescence spectrometer. Results demonstrat e that the bacteria can be accurately tracked using their fluorescence , and that the intensity of the signal can be used to determine a C/Co ratio for the transported bacteria. The data show a rapid breakthroug h of the bacteria followed by a characteristic curve pattern. A lower limit of detection of 10(5) cells was estimated based on these experim ents, The Tn5GFP1 transposon should become a valuable tool for labelin g bacteria.