FIG. 4.

Generation of reporter fusions using luc. Clones harboring a transcriptional fusion of luc to the sifA promoter or the sopE2 promoter were generated by Red-mediated integration. For each fusion, clones were confirmed by PCR for proper insertion of the reporter cassette and a set of confirmed clones were subjected to luciferase activity assays. (A) The expression levels of various clones of the transcriptional sifA::luc fusion (RBS-start codon fusion, strain 635) after FLP-mediated deletion of the aph cassette were compared to those of the parental strain (strain 607.3-9). (B) The expression of the sifA::luc fusion of strain 635 was analyzed during culture in PCN+P (open bars) and PCN−P (filled bars) media. The OD₆₀₀ of the bacterial culture was determined (circles). (C) A transcriptional fusion (operon fusion) of luc to gyrA was generated, and the resulting strain was analyzed as for panel B. (D) A transcriptional fusion (RBS-start codon fusion) of luc to sopE2, encoding an effector protein of the SPI1 T3SS, was generated. The expression kinetics was analyzed at various time points of culture in LB broth. Expression was analyzed in the wild-type background and in strains deficient in hilA or sirA, encoding regulators of the SPI1 regulon. Luciferase activities are means and standard deviations from three assays. Circles, OD₆₀₀.