Fig. 4: Impact of the strong gyrase site (SGS) on transcription-induced counter-silencing.

a Representative flow cytometry profiles from cultures of strains MA14692, MA14752 and MA14793 (left, middle and right panel, respectively) grown to early stationary phase in the presence or absence of AHTc. MA14692 contains the unmodified P^tettetA-T^hisL construct. In strain MA14752 a 189 bp fragment encompassing phage Mu SGS is inserted mid-way between tetA and hilD. Strain MA14793 carries a mutant SGS (mut2, denoted by an asterisk) with sequence changes at the position of the gyrase cleavage site (see text and Supplementary Fig. 5 for details). b Percentage of cells expressing hilA-GFP^SF in n = 4 independent repeats of the analysis in (a). c Quantification of hilD mRNA levels from the cultures used in (b). RNA from n = 4 or n = 5 independent experiments was quantified by RT-qPCR as described in the legend to Fig. 1. d Representative flow cytometry profiles from cultures of strains MA14696, MA14755 and MA14756 (left, middle and right panel, respectively) grown as in (a). Strain MA14696 carries a P^tetcat-T^hisL cassette at the exact same position as the constructs in (a). Strains MA14755 and MA14756 are derivatives of MA14696 carrying wt SGS and a mutant SGS (mut1, denoted by an asterisk), respectively (see text and Supplementary Fig. 5 for details). e Percentage of cells expressing hilA-GFP^SF in n = 4 independent repeats of the analysis in (d). f Quantification of hilD mRNA levels from the cultures used in (e). RNA from n = 3 or n = 4 independent experiments was quantified as described in (c). The error bars in (b, c, e, f) represent mean values ± SD. Statistical significance was determined by one-way ANOVA with either Šidák’s (b, e) or Tukey’s (c, f) multiple comparisons test. Adjusted P values were 0.029 and 0.052 in (b) (-SGS/ + SGS and +SGS/ + SGS* comparisons, respectively), 0.0005, 0,0931 and 0.0224 in (c) (-SGS/ + SGS, -SGS/ + SGS* and +SGS/ + SGS* comparisons, respectively), 0.014 and 0.034 in (e) (-SGS/ + SGS and +SGS/ + SGS* comparisons, respectively) and <0.0001, 0.0016 and <0.0001 in (f) (-SGS/ + SGS, -SGS/ + SGS* and +SGS/ + SGS* comparisons, respectively); (ns, P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). Source data are provided as a Source Data file.