## 5 Interdependence of the Network Elements  
Since the genes encoding DNA topoisomerases, NAPs and transcription machinery components are interconnected in a homeostatic network [1; 3], the mutations in RNAP genes can affect the expression of genes encoding the NAPs and vice versa. In _E. coli_ strains with mutated NAP genes, the \(\sigma^{70}\) and \(\sigma^{5}\) factor levels vary distinctly with the background--as evident both in crude cell extracts and in heparin column purified preparations of RNAP (Figure 2). This suggests that the NAPs modulate not only the expression levels of sigma factors, but also the sigma factor composition of the holoenzyme. In turn, in extracts of cells carrying mutations in the beta subunit of RNAP (the so-called \"stringent\" RNAP mutants mimicking the effect of the stringent response regulator ppGpp) [97], the NAP levels are correspondingly altered during bacterial growth (Figure 3). Furthermore, deletion of _rpoZ_ gene encoding the RNAP \(\omega\) subunit increases both the RNAP \(\sigma^{5}/\sigma^{70}\) ratio in the cells and the RpoS impact in the transcript profile, concomitantly modulating the impacts of the NAPs and global TFs and favoring transcription of Rel genes responding to DNA relaxation [39]. Conversely, a decrease of the RNAP \(\sigma^{5}/\sigma^{70}\) ratio in _rpoZ_ cells induced by overexpression of RpoD, increases the impact of the latter on the transcript profile and again, modulates the impacts of the NAPs and global TFs (Table 2) favoring transcription of Hyp genes responding to high levels of negative superhelicity [39]. These \(\sigma\) factor-dependent changes in regulatory impacts of the NAPs and global TFs are reflected in up-regulation of corresponding regulons and overlapping subsets thereof--the couplons (Table 2; the organization of couplons will be enlarged upon below). Figure 2: Variation of the \(\sigma^{70}\) and \(\sigma^{5}\) factor levels in _E. coli_ strains with mutated NAP genes. Graphical representation of quantitative Western blots of RNA polymerase subunits detected in crude and heparin column-purified protein extracts of _E. coli_ CSH50 wild type (wt), _AlhfAB_, _Sfis_, _Alnns_, _Alnps_, _Alnps_, _Alnps_, _Alnps_ and _Alrp_ mutant cells during exponential growth phase (OD\({}_{600}\) = 0.5) using mouse monoclonal antibodies raised against RpoD (\(\sigma^{70}\)) and RpoS (\(\sigma^{5}\)) and normalized against RpoC (\(\lx@sectionsign\)’). (**A**). Comparison of the relative (crude cell extract) to effective (heparin protein extract) amount of RpoD (\(\sigma^{70}\)) (**B**). Comparison of the relative (crude cell extract) to effective (heparin protein extract) amount of RpoS (\(\sigma^{5}\)). Note that in crude extracts the normalized levels of RpoS constitute about one third of those of RpoD. Bars indicate the standard errors of two biological replicates. _E. coli_ CSH50 wild type and isogenic NAP mutant strains were grown in 3l of dYT medium (1.6% tryptone 1% yeast extract, 0.05% NaCl) in BIOSTAT Bplus fermentor Sartorius AB, Göttingen, Germany). Conditions were set to 37 \({}^{\circ}\)C, pH 7.5, 500 rpm, and pO2 of 100% at the start of the growth, and carefully controlled throughout the growth. For more details see “Experimental procedure to Figure 2” in the Supplementary data file. Figure 3: Variation of the NAP levels in _E. coli_ CSH50 cells carrying mutations in the beta subunit of RNAP (“stringent” RNAP mutants; described in [97]), during the various growth stages. Western blots of FIS, H-NS, IHF and Dps proteins in crude protein extracts of: (**A**) rpoB3370 (T563P), (**B**) rpoB3449 (D532A) and (**C**) rpoB114 (S531F) strains analyzed during early (exponential), middle (transition to stationary) and late (stationary) growth phases. Overnight cultures (after 12 h of growth in a shaker at 37 \({}^{\circ}\)C) were inoculated in rich dYT medium at O.D\({}_{600}\) of 0.1.