A DNA-binding protein senses DNA superhelicity to switch between bridging and nucleoprotein filament formation

The torsional state of DNA encodes regulatory information beyond sequence, yet how chromosome-associated proteins read this mechanical signal to switch between distinct functional outputs remains poorly understood. Using real-time single-molecule fluorescence imaging on topologically constrained DNA, we demonstrate that DNA superhelical polarity is the primary determinant of H-NS binding mode with direct consequences for chromosomal domain organization and gene silencing. On positively supercoiled DNA, H-NS assembles into nucleoprotein filaments at AT-rich loci, acting as a topological barrier that confines plectoneme diffusion. On negatively supercoiled DNA, H-NS switches to bridging mode, immobilizing plectonemes at AT-rich sites. Upon changes in DNA topology, H-NS dynamically switches between binding modes within seconds. An oligomerization-deficient mutant retains bridging but cannot form filaments, confirming the mechanistic distinction. On multi-AT-locus DNA, helicity-driven mode selection produces self-organized “chromosomal” architecture: some sites stochastically capture the plectoneme and bridge, while remaining sites assemble insulating filaments. These findings establish DNA superhelical polarity as the master switch governing H-NS binding mode and “chromosomal” domain organization, with broad implications for transcriptional regulation in bacteria.