Elementary 3D organization of active and silenced E. coli genome.

Unravelling how genomes are spatially organized and how their three-dimensional (3D) architecture drives cellular functions remains a major challenge in biology(1,2). In bacteria, genomic DNA is compacted into a highly ordered, condensed state called nucleoid(3-5). Despite progress in characterizing bacterial 3D genome architecture over recent decades(6-8), the fine structure and functional organization of the nucleoid remain elusive due to low-resolution contact maps from methods such as Hi-C(9-11). Here we developed an enhanced Micro-C chromosome conformation capture, achieving 10-base pair (bp) resolution. This ultra-high-resolution analysis reveals elemental spatial structures in the Escherichia coli nucleoid, including chromosomal hairpins (CHINs) and chromosomal hairpin domains (CHIDs). These structures, organized by histone-like proteins H-NS and StpA, have key roles in repressing horizontally transferred genes. Disruption of H-NS causes drastic reorganization of the 3D genome, decreasing CHINs and CHIDs, whereas removing both H-NS and StpA results in their complete disassembly, increased transcription of horizontally transferred genes and delayed growth. Similar effects are observed with netropsin, which competes with H-NS and StpA for AT-rich DNA binding. Interactions between CHINs further organize the genome into isolated loops, potentially insulating active operons. Our Micro-C analysis reveals that all actively transcribed genes form distinct operon-sized chromosomal interaction domains (OPCIDs) in a transcription-dependent manner. These structures appear as square patterns on Micro-C maps, reflecting continuous contacts throughout transcribed regions. This work unveils the fundamental structural elements of the E. coli nucleoid, highlighting their connection to nucleoid-associated proteins and transcription machinery.