Our goal was to develop a robust tagging method that can be used to track bacterial strains
in vivo. To address this challenge, we adapted two existing systems: a modular plasmid-based reporter system (pCS26) that has been used for high-throughput gene expression studies in Salmonellaand Escherichia coliand Tn 7transposition. We generated kanamycin- and chloramphenicol-resistant versions of pCS26 with bacterial luciferase, green fluorescent protein (GFP), and mCherry reporters under the control of σ 70 -dependent promoters to provide three different levels of constitutive expression. We improved upon the existing Tn 7system by modifying the delivery vector to accept pCS26 constructs and moving the transposase genes from a nonreplicating helper plasmid into a temperature-sensitive plasmid that can be conditionally maintained. This resulted in a 10- to 30-fold boost in transposase gene expression and transposition efficiencies of 10 −8 to 10 −10 in Salmonella entericaserovar Typhimurium and E. coliAPEC O1, whereas the existing Tn 7system yielded no successful transposition events. The new reporter strains displayed reproducible signaling in microwell plate assays, confocal microscopy, and in vivoanimal infections. We have combined two flexible and complementary tools that can be used for a multitude of molecular biology applications within the Enterobacteriaceae. This system can accommodate new promoter-reporter combinations as they become available and can help to bridge the gap between modern, high-throughput technologies and classical molecular genetics. IMPORTANCEThis article describes a flexible and efficient system for tagging bacterial strains. Using our modular plasmid system, a researcher can easily change the reporter type or the promoter driving expression and test the parameters of these new constructs in vitro. Selected constructs can then be stably integrated into the chromosomes of desired strains in two simple steps. We demonstrate the use of this system in Salmonellaand E. coli, and we predict that it will be widely applicable to other bacterial strains within the Enterobacteriaceae. This technology will allow for improved in vivoanalysis of bacterial pathogens.