Spatial organization of DNA sequences directs the assembly of bacterial chromatin by a nucleoid-associated protein

Abstract

Structural differentiation of bacterial chromatin depends on cooperative binding of abundant nucleoid-associated proteins at numerous genomic DNA sites and stabilization of distinct long-range nucleoprotein structures. Histone-like nucleoid-structuring protein (H-NS) is an abundant DNA-bridging, nucleoid-associated protein that binds to an AT-rich conserved DNA sequence motif and regulates both the shape and the genetic expression of the bacterial chromosome. Although there is ample evidence that the mode of H-NS binding depends on environmental conditions, the role of the spatial organization of H-NS-binding sequences in the assembly of long-range nucleoprotein structures remains unknown. In this study, by using high-resolution atomic force microscopy combined with biochemical assays, we explored the formation of H-NS nucleoprotein complexes on circular DNA molecules having different arrangements of identical sequences containing high-affinity H-NS-binding sites. We provide the first experimental evidence that variable sequence arrangements result in various three-dimensional nucleoprotein structures that differ in their shape and the capacity to constrain supercoils and compact the DNA. We believe that the DNA sequence-directed versatile assembly of periodic higher-order structures reveals a general organizational principle that can be exploited for knowledge-based design of long-range nucleoprotein complexes and purposeful manipulation of the bacterial chromatin architecture.

H‐NS, IHF, and DnaA lead to changes in nucleoid organizations, replication initiation, and cell division

“…In agreement with previous reports , we found that both the absence of H‐NS or the presence of extra H‐NS led to a dramatic increase in the number of cells with uncompacted nucleoids in a medium‐independent manner (Figure ). An increase in the number of cells with uncompacted nucleoids was also observed in the hns 206 mutant (Figure ), indicating that the availability of H‐NS and fully functional H‐NS is required for correct nucleoid compaction.…”

“…At least 12 distinct types of NAPs have been described in E. coli while IHF, HU, Fis, and H‐NS are well characterized . The 15.5 kDa H‐NS protein is believed to play an important role in nucleoid compaction and organization by bridging distant DNA segments . Also IHF, as a member of nucleoid‐associated proteins, promotes nucleoid compaction .…”

Coherent Domains of Transcription Coordinate Gene Expression During Bacterial Growth and Adaptation

“…Osmotic shock, besides redirecting RNAP binding in the genome [85], could also affect the binding of NAPs, which depends on both sequence composition and DNA topology [86][87][88][89][90]. For example, the binding of the global repressor H-NS [91] is modulated by ionic composition, sequence organization, and DNA topology [88,[92][93][94][95][96][97]. H-NS preferentially binds A/T-rich sequences and represses transcription by bridging DNA duplexes and forming inter-wound filaments [98,99].…”

Bacterial H-NS contacts DNA at the same irregularly spaced sites in both bridged and hemi-sequestered linear filaments

“…As a first step toward in vitro reconstitution of bacterial chromatin, we compared H-NS filaments formed on purified, deproteinized genomic DNA (gDNA) to in vivo H-NS binding patterns determined by ChIP-seq. We predicted that H-NS filaments would nucleate at AT-rich, high-affinity sites Japaridze et al, 2017;Lang et al, 2007) but that strong contributions of other DBPs to H-NS filament locations would make the in vitro and in vivo patterns different. To define locations of H-NS filament formation on sheared gDNA, we captured the reconstituted filaments by binding to nitrocellulose filters, and then determined 6/11/2020 filament locations by HT-sequencing of DNA recovered from the filters.…”