A chromosomal loop anchor mediates bacterial genome organization

Nucleoprotein complexes play an integral role in genome organization of both eukaryotes and prokaryotes. Apart from their role in locally structuring and compacting DNA, several complexes are known to influence global organization by mediating long-range anchored chromosomal loop formation leading to spatial segregation of large sections of DNA. Such megabase (Mb) range interactions are ubiquitous in eukaryotes, but have not been demonstrated in prokaryotes. Here, using a novel genome-wide sedimentation based approach, we found that a transcription factor, Rok, forms large nucleoprotein complexes in the bacterium Bacillus subtilis. Using chromosome conformation capture (Hi-C) and live-imaging of DNA loci, we show that these complexes robustly interact with each other over large distances. Importantly, these Rok dependent long-range interactions lead to anchored chromosomal loop formation, thereby spatially isolating large sections of DNA, as previously observed for insulator proteins in eukaryotes. RNA-seq, sedimentation assay, and Hi-C performed on wild type B. subtilis and rok mutant strains. We used sedimentation based DNA coverage to identify large chromosomal complexes. Briefly, in this assay cell lysates are mildly treated with DNase and then separated using untracentrifugation on sucrose steps. The top and dense fraction is then analysed for loss or gain of DNA coverage to identify large DNA-protein complexes.