Single-copy green fluorescent protein gene fusions allow accurate measurement of Salmonella gene expression in vitro and during infection of mammalian cells.

The increasing incidence of infectious diseases is driving renewed efforts to understand the interactions between bacterial pathogens and their hosts at a new level. This has prompted research to study the transcriptional response of bacteria during infection. The past decade has seen the development of a number of techniques for identifying in vivo-induced bacterial genes and for monitoring gene induction in complex environments. Genetic tools for visualization of gene expression in situ have also been devised. Fluorescent reporters have been developed for studying bacterial infection from both the host point of view and the bacterial point of view (44, 57, 60). The green fluorescent protein (GFP) from Aequoria victoria is an excellent tool for monitoring gene expression, because it is naturally fluorescent without any exogenous cofactor or substrate (9). Variants of GFP that have different spectral characteristics, folding properties, levels of chromophore brightness, or half-lives have broadened the spectrum of possible applications for GFP (4). Measurement of fluorescence by flow cytometry has revolutionized the use of fluorescent reporter genes in bacteria (27) and now allows observation of gene expression at the level of individual bacterial cells. Plasmid-borne gfp fusions have already been used to monitor gene expression in Salmonella strains (6, 61), as well as in other gram-negative or gram-positive bacteria, including Escherichia coli (42), Serratia liquefaciens (2), Erwinia herbicola (37), Staphylococcus aureus (45), Listeria monocytogenes (64), and Streptococcus gordonii (24). Such fusions have proven to be useful for identifying bacterial genes induced during infection of eukaryotic cells or animal hosts and have allowed some assessment of bacterial gene expression levels in such complex environments. However, the variation in plasmid copy number that can occur in individual bacterial cells makes plasmid-borne gene fusions unsuitable for measuring differences in gene expression between single bacteria (34, 35). Furthermore, plasmid-borne GFP fusions are useful only for reporting the induction of genes; any reduction in fluorescence may simply reflect plasmid loss, because it is not possible to guarantee that plasmids are maintained in every bacterial cell in plant or animal hosts. Indeed, high expression levels lead to accumulation of toxic levels of GFP in bacterial cells, which results in dramatic plasmid loss (63; I. Hautefort, J. M. Sidebotham, and J. C. D. Hinton, unpublished data). Hopkins et al. showed that a pBR322-derived plasmid-borne gfp fusion was unstable in wild-type Salmonella enterica serovar Typhimurium strain 14028 during infection of BALB/c mice, while it remained stable in a PhoPc mutant (29). More recently, it was reported that a plasmid-borne tac-gfp fusion was not stable in a wild-type Salmonella strain during murine infection (6). This study showed that the level of plasmid loss was related to promoter strength, meaning that the levels of expression of different promoters cannot be compared by using plasmid-borne gene fusions. Our interest in determining the diversity of gene expression within bacterial populations in the host prompted us to develop an improved approach for construction of gfp transcriptional fusions that could be used reliably during infection. We reasoned that integration of single-copy gene fusions onto the bacterial chromosome was the best way to ensure genetic stability. Single-copy gfp gene fusions have been used to monitor the activity of the marRAB promoter in large populations of Salmonella serovar Typhimurium (46). This approach involves inactivation of the gene of interest, making it unsuitable for studying virulence genes during infection. Recently, the activity of the isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible spac hybrid promoter (65) was successfully monitored by using a single copy of gfpmut2 as a reporter in Bacillus subtilis (41). We performed preliminary experiments involving expression of single-copy gfpmut1 fusions in Salmonella; and we discovered that the GFPmut1 variant, which is even brighter than GFPmut2 (11), was only fluorescent enough to report particularly high levels of promoter activity in Salmonella during infection (Hautefort et al., unpublished). We overcame this problem by using the GFP+ variant (51), which carries the GFPuv mutations F99S, M153T, and V163A (12) along with the EGFP mutations F64L and S65T (11), resulting in better folding of the protein coupled with enhanced brightness. In this study we describe the use of GFP+-based single-copy gene fusions to measure Salmonella serovar Typhimurium gene induction in vitro and during infection of mammalian cells.