Green fluorescent protein-based reporter systems for genetic analysis of bacteria including monocopy applications

The green fluorescent protein (GFP) gene, gfp, was used to develop versatile reporter systems for genetic analysis in, and monitoring of bacteria. This reporter system is available on a plasmid and on a mini-transposon located in a suicide delivery plasmid for generation of chromosomal fusions. To achieve sensitivity levels necessary for use in monocopy applications and for detection of single cells, the 3'-end of gfp was replaced by that of a modified gfp gene characterized by a 45-fold stronger fluorescence signal than that exhibited by the natural GFP. This modified gfp gene was also equipped with the strong translation signals of the atpE gene. Transfer of the mini-transposon into two different Pseudomonas spp. and Alcaligenes eutrophus produced random chromosomal fusions, some 5% of which exhibited fluorescence detectable by eye. Individual GFP+ cells were readily observed by fluorescence microscopy.