Mu-seq reveals previously undetected long-range contact patterns in the Escherichia coli nucleoid

The spatial organization of the Escherichia coli chromosome is a key determinant of its functional and regulatory landscape, but widely used chromatin conformation capture methods, such as Hi-C, provide an incomplete view of the underlying three-dimensional genome connectivity. Here, we introduce Mu-seq, a massively multiplexed Mu transposition-based approach that maps genome-wide DNA-DNA proximity in live cells without crosslinking. By distributing engineered, barcoded Mu prophages throughout the chromosome and tracking their replicative transposition events, Mu-seq generates dense, uniform sampling of physical contacts across the genome, enabling a direct, in vivo snapshot of chromosome architecture. Applying this method in E. coli, we constructed a contact dataset spanning over 200,000 unique loci, revealing interaction patterns not accessible to current crosslinking-based techniques. Mu-seq findings can be broken down into three layers. First, it recapitulates polymer-like enrichment of short-range contacts. Second, contacts identified by Mu-seq are strongly shaped by chromatin state: transcriptionally active and accessible regions disproportionately contribute to long-range connectivity, whereas protein-occupied domains, including H-NS-enriched regions, are depleted of insertions and contacts. Third, beyond distance-dependent polymer behavior and chromatin accessibility, Mu-seq reveals specific long-range associations among functionally related loci, such as ribosomal operons. These findings suggest that bacterial chromosome organization can bring functionally related regions into shared three-dimensional neighborhoods even when they are widely separated along the linear genome. Together, these findings support a layered model of nucleoid organization, in which transcriptionally active loops extend outward from a dense, protein-rich axial core. Mu-seq thus provides a powerful, complementary in vivo framework for probing bacterial chromosome architecture, uncovering regulatory and structural features of genome organization that are largely invisible to crosslinking-based approaches.