Introduction Analysing gene expression of pathogenic bacteria during infection at single cell level is a challenge. Most studies have to cope with averaged data, levelling subpopulations and loss of spatial information. This is especially problematic when considering the adaptations to diverse intracellular niches throughout various infection stages. The use of fluorescent reporters enables the temporal and spatial tracking of individual cell gene expression and allows isolation of observed subpopulations for subsequent downstream analyses.

Goals We aimed to develop a fluorescent reporter vector for Staphylococcus aureus to examine up to three different physiological states during in vivo infection. The vector should enable the easy adaptation of the system to the respective question, but must be transmitted stably and provide an internal reference.

Methods The reporter vector pTricolor contains the three codon-optimized fluorescent reporter genes dtomato, gfp and mcerulean. One reporter is constitutively expressed from an optimized pgi-SigA promoter and allows copy-number correction and normalization between different experiments. The remaining two reporter genes can be placed under control of target promoters of interest. Reporter gene expression is monitored by fluorescence microscopy.

Results We demonstrate that pTricolor exhibits stable transmission for at least eight days in vitro and 24 hours during infection under non-selective conditions. The fluorescent reporters can be expressed independently of each other and genetic modifications can be easily implemented. In an initial study, the reporter genes were placed under the control of a SigB dependent (clpL) and a CodY dependent promoter (saHPF), respectively. The detected reporter signals revealed subpopulations of intracellular S. aureus in an infection experiment using S9 host cells.

Summary pTricolor enables the analysis of differential gene expression for up to two S. aureus target genes during internalization and long-term infection in a standardised and spatiotemporal manner. Thus, it is a suitable reporter vector to investigate subpopulations and face central questions of virulence regulation.