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Abbreviations:DBD -DNA binding domain ; DBP -DNA binding protein ; FeBABE -Fe(III) (S)-1-(p-Bromoacetamidobenzyl)ethylenediaminetetraacetate ; Fe·EDTA-H-NS -H-NS derivatized with a the Fe 2+• EDTA nuclease ; MD -molecular dynamic ; ·OH -hydroxyl radical ; RNAP -RNA polymerase 6/11/2020 ABSTRACT Gene silencing in bacteria is mediated by chromatin proteins, of which Escherichia coli H-NS is a paradigmatic example. H-NS forms multimeric nucleoprotein filaments with either one or two DNA duplexes, but the structures of these linear or bridged filaments remains unknown. Neither the determinants of H-NS genomic distribution nor the arrangements of H-NS DNA binding domains (DBDs) in linear or bridged filaments are currently defined. To address these questions, which are central to understanding gene silencing in bacteria, we developed a hybrid approach that combines a new method for in vitro chromatin reconstitution and mapping (Nitro-seq), ChIP-seq, nuclease-tagged DBDs to map DNA contacts, ·OH footprinting, molecular dynamics,