Flow cytometric and microscopic analysis of GFP-tagged Pseudomonas fluorescens bacteria

Abstract

The gene encoding the green fluorescent protein (GFP) from the jellyfish Aequorea victoria was assembled into an expression cassette for bacteria by fusing it to the T7 gene10 ribosome binding site and the strong, constitutive promoter PpsbA from Amaranthus hybridus. By using Tn5-based transposon delivery systems, Pseudomanas fluorescens bacteria were chromosomally tagged with gfp. We demonstrate that expression of a single copy gfp gene is sufficient to permit the visualization of bacteria by epifluorescence and laser confocal microscopy and detection by flow cytometry. The green fluorescent phenotype was detectable in all growth phases even under nutrient-limited conditions.