A simple system for converting lacZ to gfp reporter fusions in diverse bacteria

We describe new plasmids that facilitate the rapid conversion of lacZ fusions to gfp transcriptional fusions in bacteria. The exchange is based on a double recombination between lacZ sequences in a suicide vector and the recipient chromosome. The suicide vector is a mobilizable, conditionally replicative plasmid that contains the gene for gfp with flanking lacZ homology and is derived from a broad host range plasmid that has been successfully used in a wide range of bacterial species. The technique was used to convert lacZ reporter fusions to gfp fusions in Escherichia coli, Bordetella bronchiseptica and Agrobacterium tumefaciens. Green fluorescent protein expression in the new recombinants reflected the beta galactosidase expression in the parent strains. GFP is particularly useful for rapid quantification of gene expression in real time and in single cells. As a demonstration of an application of this system, we studied the induction of virE transcription by the VirA/VirG two-component system in A. tumefaciens in response to various levels of phenolic inducer. Analysis of GFP fluorescence in single cells revealed that at intermediate levels of inducer the population of cells was remarkably heterogeneous. The tools described here will be useful for general studies of transcriptional regulation as well as for applications that require spatial and temporal identification of gene expression, such as in the study of biofilms, and interactions between bacteria and their environment.