FIG. 3.
Generation of reporter fusions using GFP. (A) Simplified scheme of regulatory cascades of the SPI1 and SPI2 regulons. Various environmental and nutritional factors affect the expression of S. enterica virulence genes. Global regulatory systems, such as PhoPQ, SirA/BarA, or EnvZ/OmpR, modulate expression. The regulatory systems HilA and SsrAB have central roles in controlling expression of genes within SPI1 and SPI2, respectively, or further loci outside of the SPI. (B) A transcriptional fusion (RBS-start codon fusion) of GFPmut3 to the promoter of sseJ was generated by Red-mediated recombination. The resulting strain was grown in PCN−P or PCN+P medium. Growth in media with low concentrations of Pi results in the expression of genes of the SPI2 regulon. S. enterica serovar Typhimurium strains without a GFP reporter or harboring plasmid pFPV25.1 for constitutive expression of GFP were used as negative and positive controls, respectively. Bacteria were analyzed for the GFP fluorescence by flow cytometry. (C) A set of clones harboring an sseJ::GFP fusion was confirmed by PCR for proper insertion of the reporter cassette and analyzed by flow cytometry as described for panel B.