## 4 Role of DNA Topology and Homeostatic Regulation of Supercoiling Response
Importantly, Rho is associated with RNAP throughout the transcription cycle allosterically trapping the transcription complex [74; 80; 81] and preventing the formation of deleterious R-loops (a loop formed by single-stranded RNA base-paired with one strand of duplex DNA with the complementary DNA strand being displaced) at supercoiling-dependent stable RNA operons [82], as well as precluding antisense transcription [83] and transcriptional read-through [79], potentially increased under conditions of DNA hypernegative supercoiling. Concomitant activation of sigma _rpoE_ and anti-sigma _rseAB_ genes by hyperenegative DNA supercoiling (Table 1) suggests an induction of envelope stress under hypernegative supercoiling regimen [84] and supports the notion that the \(\sigma\) and anti-\(\sigma\) factors are often co-transcribed to ensure the maintenance of stoichiometric levels [85]. Again in keeping with homeostatic control of DNA supercoiling, DNA relaxation induced by addition of nonfloxacin to LZ41 WT strain (and its derivatives lacking the NAPs), encoding nonfloxacin-resistant TopoIV (i.e., gyrase is inhibited, whereas TopoIV is not), activates the _gyrA_ and _gyrB_ genes encoding the DNA gyrase subunits. DNA relax \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline
**Condition** & \multicolumn{4}{c}{**Hypernegative Supercoiling**} & \multicolumn{4}{c}{**DNA relaxation**} \\ \hline
**Strain,** & & & & & & & & \\
**Background** & **LZ54 WT** & **\(\Delta\)_fis_ & **\(\Delta\)_lms_ & **\(\Delta\)_fis/lms_** & **LZ41 WT** & **\(\Delta\)_fis_ & **\(\Delta\)_lms_ & **\(\Delta\)_fis/lms_** \\ \hline Genes of NAPs & & & & _lrp_ & & & \\ and global TFs & _fis_ & _lrp_ & & _hupB_ & _hupA_ & _firr_ & _firr_ & _firr_ \\  & & & & _sipA_ & & & & \\ \hline Genes of RNAP & _crl_ & _musG_ & _crl_ & _musG_ & _musG_ & & & & _greA_ & _greA_ \\ modulators & _musB_ & _rho_ & _musG_ & _rho_ & & & & \\ \hline Genes of the & & & _rpoE_ & & & & & \\ RNAP sigma and & & & _rseAB_ & _rpoE_ & _rseA_ & _rpoH_ & _rpoD_ & _rpoD_ & _rpoD_ \\ anti-sigma factors & & & & & & & _ficcl_ & _fliA_ \\ \hline Topoisomerase & _yrdD_ & _yrdD_ & _yrdD_ & _gyrA_ & & & & _gyrA_ \\ genes & _parC_ & _parC_ & _parC_ & _parE_ & _gyrB_ & _gyrB_ & _gyrB_ & _gyrB_ \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Relevant genes up-regulated under conditions of nonfloxacin-induced hypernegative DNA supercoiling and DNA relaxation in exponentially growing _E.coli_ LZ54 and LZ41 wild-type (WT) strains and their mutant derivatives lacking the NAP-encoding genes _fis_, _lms_ or both (data from [15] and unpublished results). ation also activates the genes encoding the sigma factors RpoD, RpoH and the secondary channel modulator GreA (Table 1). Activation of this latter is consistent with its role in ppGpp-DksA regulatory subnetwork [40; 86] and might counteract the down-regulation of ribosomal RNA promoters under conditions of DNA relaxation [87; 88]. The _E. coli rpoH_ gene encoding the heat shock \(\sigma^{32}\) (aka \(\sigma^{\text{H}}\)) factor is sensitive to changes of DNA supercoiling, and DNA relaxation induced by novobiocin treatment of _B. subtilis_, was shown to decrease transcription from both the ribosomal RNA promoters and the promoters dependent on alternative sigma factors including \(\sigma^{\text{H}}\)[89; 90]. The RNAP \(\sigma^{70}\) subunit encoded by _rpoD_ gene confers preference for highly negatively supercoiled templates [91] and its overexpression can increase the global DNA negative superhelicity [39], suggesting a compensatory activation of _rpoD_ expression counteracting the norfloxacin-induced DNA relaxation especially in LZ41 cells lacking the NAPs and hence, their buffering effects due to constraint of negative supercoils. Among the genes encoding the NAPs, _fis_, _lrp_, _lupB_, _liftA_ and _stpA_ are activated by hypernegative supercoiling, while _liftB_, _lupA_, and the global TFs _crp_ and _fnr_ are activated by DNA relaxation (Table 1).