For the understanding of biological processes, the analysis of gene regulation is of central importance. Despite the availability of high-throughput approaches to the investigation of global gene regulation, the analysis of individual gene expression is of continuing interest for validating and extending data from microarray analyses or other parallel approaches.
A variety of reporter genes is available to study gene expression, with luciferase and β-galactosidase being the most frequently used enzymes (19
) and green fluorescent protein (GFP) being used for multiple applications in living cells (25
). Most reporters allow the construction of transcriptional fusions to a regulatory element of interest and the construction of translational fusions, resulting in the synthesis of hybrid proteins. In addition to the study of the regulation of the synthesis of a given protein, translational fusions are also of interest for monitoring the fate of a protein, for example, degradation, subcellular localization, secretion into the extracellular space, or translocation into other cells.
Generation of reporter gene fusions conventionally involves either the cloning and fusion of regulatory elements to a reporter gene or random mutagenesis with transposons harboring the reporter gene followed by identification fusions. The latter approach is undirected but results in the generation of single-copy reporter fusions in the native chromosomal context. A common problem is the introduction of foreign regulatory elements, such as promoters and terminators present in the transposon. In contrast, cloning is directed but usually results in the presence of the regulatory elements in several copies on an episomal element. The directed generation of single-copy chromosomal reporter fusions is also possible. However, such approaches previously involved extensive genetic manipulation, such as the generation of recombinant suicide vectors or bacteriophages, and/or resulted in the integration of the reporter fusion into a different chromosomal context (examples in references 5
, and 22
The development of methods using the Red recombinase for genetic engineering opened a path to rational and rapid deletion of genes, operons, or larger genomic elements (4
) in Escherichia coli
, Salmonella enterica
, and a range of other bacteria. The Red recombinase approach has also been used to epitope tag chromosomal genes (26
) and to recombine fusions of bacterial promoter elements with GFP (9
) into a defined position on the bacterial chromosome. Another modification allowed Flp recombinase (FLP)-mediated integration of lacZ
reporter plasmids into FLP target (FRT) sites that remained on the bacterial chromosome for Red-mediated gene deletions (7
). We have also utilized Red-mediated recombination to integrate expression cassettes for heterologous proteins into the chromosome of S. enterica
We previously demonstrated that the Red recombination approach can be used to integrate recombinant expression cassettes into the chromosome of S. enterica
serovar Typhimurium (10
). We also envisaged the use of this recombination technique for the generation of precise gene fusions with reporter genes. Previous studies showed that Red recombination allowed the precise construction of translational fusions of chromosomal genes to epitope tags (26
). Here, we describe a Red recombinase-based approach that allows the rapid and precise construction of transcriptional and translational reporter gene fusions in the bacterial chromosome. The availability of a set of template plasmids will allow the rapid construction of single-copy reporter gene fusions that are precisely targeted to chromosomal locations.