Figure 7

Gene expression and Tn5 transposition assays in strains harboring disruptions of global transcriptional regulators. (A) β-galactosidase activity for isogenic strains (wt, hfq⁻, crp⁻ and lrp⁻) harboring the TCF in single-copy in the chromosome (DBH303 and derivatives). Cells were grown to mid-log phase in Luria broth (LB). Mean and standard error values of duplicate experiments, each of which included at least three replicates, are shown. (B) IS50-lacZ transcript levels. Total RNA was extracted from cells described in panel (A), and subjected to RT-PCR. (C) Western blot analysis of Crp levels in cellular extracts from wt and hfq⁻ cells grown in LB. As a negative control, crp⁻ cells were also analyzed. A representative image is shown in the inset. Crp levels were normalized to GroES, which is known to be insensitive to hfq status [19]. (D) Tn5 transposition from the chromosome of DBH179 (wt) and DBH345 (crp⁻) was measured by the conjugal ‘mating out’ assay as described in Methods. The data is from a single experiment wherein five independent clones of each strain were tested. Mean and standard error values are shown. The average transposition frequency was 1.70 × 10⁻⁴ events per mL of mating mix for the wt strain and for purposes of comparison this value was set at 1 and the ‘crp’ value was normalized to this. In two other independent experiments the fold increase in Tn5 transposition for crp⁻ versus wt did not differ by more than 20% compared to the experiment shown (data not shown). For experiments in (A-C), mean and standard error values from at least three independent isolates are shown.