## 4 DNA Supercoiling Gradient and Genomic Sequence Organization  
DNA supercoiling itself is a continuous variable directly responding to environmental changes [48; 49; 50; 51]. What is the physical basis of this capacity? According to the classic physics definition, continuity finds its expression in the laws of nearby action, connecting only the values of physical quantities at space-time points in the immediate vicinity of one another. In the chromosomal DNA polymer, this \"continuity\" is afforded by the physical connectivity of the nucleotide sequence, where the \"laws of nearby action\" are embodied in stacking interactions between the DNA base pairs \"in the immediate vicinity of one another\". Importantly, these local interactions can be modulated by supercoiling [52]. Figure 1: Regulation of the gene expression of ppGpp synthetase _relA_ and the quorum-sensing regulator _qseB_ in growing wild-type _E. coli_ cells and mutant derivatives lacking the chromatin-shaping proteins FIS and H-NS. Abscissa—time (min) after inoculation of stationary cells in fresh medium. The time intervals 0–100’, 100–300’, and \(>\)300’ contain approximately the early (lag), middle (exponential), and stationary phases, respectively [12]. Ordinate—relative expression in arbitrary units. The color of the curves indicates the genetic background. The _Escherichia coli_ CSH50 overnight cultures were inoculated at an initial OD600 of 0.1 in rich double-yeast-tryptone (dYT) medium and grown in a fermenter under constant pH 7.4 and high aeration (5 L air per min) at 37 \({}^{\circ}\)C for 7 h (420 min). Samples for RNA-seq were taken at intervals after inoculation as indicated and immediately dissolved in ice-cold ethanol–phenol (5% phenol) solution to prevent mRNA degradation. RNA was extracted using the RNeasy Mini kit (Qiagen, Hilden, Germany) and treated with Turbo DNase (Life Technologies, Carlsbad, CA, USA). Subsequent rRNA depletion was carried out using the MicrobExpress kit (Life Technologies), and 0.5 \(\upmu\)g of enriched mRNA of each sample was subjected to RNA-seq (Illumina HiSeq 2000, Illumina, San Diego, CA, USA). In this figure and Figures 2 and 3 time-resolved RNA-seq data of _E. coli_ wild-type, _fis_, and _lns_ mutant strains can be accessed via GSE65244 (NCBI Geo database). How is the spatial gene order translated into the temporal gene expression pattern? The coupling of temporal gene expression to spatial gene order is facilitated by the physical continuity of the chromosomal DNA polymer on the one hand and its topological closure on the other. Topological closure imparts the ability to directionally transfer the information over distance [53], which can be facilitated by DNA-bending NAPs [54; 55; 56; 57]. The sequential character of gene expression implies directionality, which most readily can be associated with the process of replication, proceeding bi-directionally along both replichores from OriC toward Ter. Indeed, it was observed that gene transcription is temporally coupled to the event of gene replication during the bacterial cell cycle [58; 59]. The link between gene replication and gene expression depends on the coupling of the transient supercoiling imbalance and supercoil diffusion induced by translocating replisomes [60; 61] to gene transcription [62; 63]. In addition to replication, the transcription of multiple exceptionally strong ribosomal RNA operons oriented from OriC toward Ter induces negative and positive supercoils, respectively, in the wake and ahead of the translocating RNAPs [64]. This directionally opposite diffusion of negative and positive supercoils potentially generates a global topological asymmetry along the chromosomal OriC-Ter axis. In _E. coli_, this imbalance is enhanced by a significantly (5-10 times) higher frequency of gyrase binding sites around the chromosomal OriC pole compared to Ter [6; 14; 65; 66].