Figure 4

a: Dependence of electrophoretic mobility of the reporter plasmid pPF1-appY on nalidixin concentration. The plasmids were isolated from the cells grown in LB for 8 h with indicated concentration of the drug. Fractionation was carried out in 0.8% agarose gel in TAE with 2 μg/ml of chloroquine at 1 V/cm during 20 h. Electrophoretic mobilities are presented as densitograms of corresponding gel lanes. b: Dependence of relative emission intensities of EGFP and mCherry on nalidixin concentration. E.coli K12 MG1655 cells, transformed by two different reporter plasmids were cultivated during 8 h at given nalidixin concentration. Cells were harvested, suspended in 1xPBS pH 7.4, sonicated and fluorescence was measured in extracts cleared by centrifugation. Wave lengths for excitation/emission were 484/507 nm and 587/610 nm for EGFP and mCherry, respectively. The data are given as a mean values measured in 5 independent biological replicates ± s.e.m. Filled symbols correspond to the promoters of appY-associated “island”, open – to the fepA/fes regulatory region. Tubes with bacteria cultivated with different concentration of nalidixic acid for 24 h exemplify observed changes in relative promoter activity c: The effect of nalidixin (1.5 μg/ml ) on the level of RNAs transcribed from five indicated promoters, estimated by qRT-PCR. Expression efficiency of each gene (E) was calculated by the formula E = 2^−ΔC(t) and expressed as a percentage of rpoB-mRNA abundance. Deviation bars show standard error (s.e.m.), number of independent biological replicates for fepA, fes, appY(dir) were 5; for fis n = 6; for appY(an) n = 8.