## 8 Spatial Organization of Regulons and Couplons  
Given the revealed correspondence between the chromosomal order and temporal expression of genes during the passage of cells from early to exponential to stationary phase, how are the spatial organisation and the temporal expression of genes comprised in regulons and couplons related? The genomic targets of both the NAPs and the RNAP sigma factors demonstrate a peculiar spatial organization [103]. For example, the regulons of RpoD (major \(\sigma^{70}\) factor, predominating during active growth and transcribing the bulk of genes) and RpoS (stationary phase or stress \(\sigma^{\text{S}}\) factor predominating on cessation of growth) demonstrate a conspicuous opposite bias of spatial organization, being enriched respectively at the Ori and Ter ends of the chromosome (Supplementary Figure S1). Also the RpoH (heat shock sigma factor) and RpoE (extracytoplasmic sigma factor) regulons show a bias towards the OriC end, whereas RpoN (the nitrogen limitation sigma factor) shows an opposite bias on the left and right replichores. Similarly, the regulons of the NAPs and global TFs, CRP and Fnr, show distinct spatial patterns and all, perhaps except those of Fnr and IHF, show a relative enrichment in the OriC half of the chromosome (Supplementary Figure S2). Interestingly, spatial organization of couplons reveals an apparent \(\sigma\) factor-dependent peculiarity. For example, genomic distributions of all the couplons of NAPs involving the major \(\sigma^{70}\) factor (RpoD), positively correlate with the spatial pattern of the regulons of NAPs rather than with that of RpoD (Figure 5A,C; Supplementary Figures S3 and S4). In contrast, the spatial patterns of couplons of the NAPs involving alternative sigma factors (ASFs, meaning all the sigma factors except the 'house-keeping' sigma RpoD) are positively correlated to the patterns of ASF regulons (Figure 5B,D; Supplementary Figure S5). The difference between the spatial organization of the major RpoD and ASF couplons is perhaps unsurprising, given that the vegetative RNAP\(\sigma^{70}\) holoenzyme binds the vast majority of genomic promoters, whereas the holoenzymes assembled with ASFs which govern more focused functions, bind only subsets thereof [113]. Since the observed biases in the impacts of regulons on spatial organization of couplons are conspicuous, the pertinent question is how these impacts are related to the temporal expression of couplons. Figure 5: Correlation between the genomic organization of regulons and couplons. (**A**). Genomic organisation of the FIS and RpoD regulons and FIS/RpoS couplons. The circular chromosome is divided into the right (top) and left (bottom) replichores. The origin of replication is situated on the left, the terminus on the right. The red and blue colors respectively indicate a significant enrichment (Z score > 2) and depletion (Z score < \(-\)2) in relative frequency of the regulon or FIS/RpoD couplon genes. Note that the pattern of FIS/RpoD couplons is largely coinciding with that of the FIS regulon (dashed box). (**B**). Genomic organisation of the FIS and RpoS regulons and FIS/RpoS couplons. Note that the pattern of FIS/RpoS couplons largely coincides with that of the RpoS regulon (dashed box). Enrichment of regulon genes was determined by comparison of the number of regulon genes within the 100 kb window with the number of regulon genes within random samples with the same number of genes. Z-scores were derived from 10.000 random samples. (**C**). Pearson correlations (ordinate) between the genomic organisation of NAP/RpoD couplons and cognate NAP regulons (indicated on abscissa, the first four columns in the graph.