chromosome display highly transcribed genes at their boundaries, indicating a role of transcription in the formation of a CID boundary. More direct evidence came from a finding that the placement of a highly transcribed gene at a position where no boundary was present created a new CID boundary in the C. crescentus chromosome. However, not all CID boundaries correlated with highly transcribed genes in the E. coli chromosome suggesting that other unknown factors are also responsible for the formation of CID boundaries and supercoiling diffusion barriers. Macrodomains Plectonemic DNA loops organized as topological domains or CIDs appear to coalesce further to form large spatially distinct domains called macrodomains (MDs). In E. coli, MDs were initially identified as large segments of the genome whose DNA markers localized together (co-localized) in fluorescence in situ hybridization (FISH) studies. A large genomic region (~1-Mb) covering oriC (origin of chromosome replication) locus co-localized and was called Ori macrodomain. Likewise, a large genomic region (~1-Mb) covering the replication terminus region (ter) co-localized and was called Ter macrodomain. MDs were later identified based on how frequently pairs of lambda att sites that were inserted at various distant locations in the chromosome recombined with each other. In this recombination-based method, a MD was defined as a large genomic region whose DNA sites can primarily recombine with each other, but not with those outside of that MD. The recombination-based method confirmed the Ori and Ter MDs that were identified in FISH studies and identified two additional MDs. The two additional MDs were formed by the additional ~1-Mb regions flanking the Ter and were referred to as Left and Right. These four MDs (Ori, Ter, Left, and Right) composed most of the genome, except for two genomic regions flanking the Ori. These two regions (NS-L and NS-R) were more flexible and non-structured compared to a MD as DNA sites in them recombined with DNA sites located in MDs on both sides. The genetic position of oriC appears to dictate the formation of MDs, because repositioning of oriC by genetic manipulation results in the reorganization of MDs. For example, genomic regions closest to the oriC always behave as an NS regardless of DNA sequence and regions further away always behave as MDs. The Hi-C technique further confirmed a hierarchical spatial organization of CIDs in the form of macrodomains. In other words, CIDs of a macrodomain physically interacted with each other more