Disentangling polymer confinement from specific-folding interactions reveals the drivers of E. coli chromosome organization.

The three-dimensional organization of the bacterial chromosome is critical for gene regulation. Chromosome conformation capture (Hi-C) has enabled genome-wide mapping of chromosomal folding, yet ensemble-averaged contact maps entangle biologically specific-folding interactions (SFIs) with nonspecific polymer compaction and mask single-cell heterogeneity. Here, we developed a polymer-based simulation framework in E. coli to address these limitations. We found that a null model of 300,000 random polymer configurations recapitulated the global chromosomal organization observed in Hi-C data, establishing that most Hi-C signals reflect generic polymer behavior in a confined volume. Contrasting null-model predictions with experimental Hi-C data isolated a small subset of SFIs (< 7%) and generated a specific-fold ensemble of 20,000 single-cell conformations that reproduced chromosomal interaction domains and single-cell heterogeneity. SFIs were enriched in the ter region, reduced nucleoid accessibility, colocalized with cryptic prophages, and depleted in positively supercoiled regions. H-NS and MatP emerged as major chromosome-wide and local determinants of SFIs, respectively. Furthermore, high-SFI regions correlated with stress-adaptive genes, whereas low-SFI regions harbored housekeeping genes. Together, our results established that a small number of biologically encoded SFIs superimposed on a polymer background shape the E. coli chromosome and gene expression, providing a quantitative framework for dissecting chromosome architecture and function.