Fig. 4: The H-NS multimerization region blocks H-NS mediated DNA bridging in vitro and in vivo.

a H-NS-39 interferes with H-NS mediated DNA bridging in vitro. The bar chart illustrates the amount of radiolabelled DNA recovered by H-NS mediated bridging interactions with biotin labelled ISAba13. The radiolabelled sequence corresponds to the H-NS bound regulatory DNA upstream of genes encoding the type 6 secretion system of A. baumannii (Fig. S3a). Where present, H-NS was used at a final concentration of 6 μM. The bridging interaction is disrupted by H-NS-39, added at final concentrations between 0.5 and 10 μM. Results are the average of three independent experiments and error bars indicate standard deviation from the mean. The schematic above the graph illustrates the procedure. DNA fragments are shown as solid lines and H-NS is in green. Individual H-NS molecules possess surfaces for DNA binding (circle), dimerisation (dark green semi-circle) and multimerization (pale green semi-circle). H-NS-39 (red) consists of the multimerization surface only. Different letters above bars indicate significantly different groups according to an unpaired one-way ANOVA with Tukey’s HSD test (P = 6.63e⁻¹¹). Source data are provided as a Source Data file. b H-NS-39 does not bind DNA or interfere with binding of H-NS to DNA in vitro. Results of an electrophoretic mobility shift assay to measure binding of H-NS to the regulatory region of genes encoding the type 6 secretion system of A. baumannii, and the impact of H-NS-39. Where present, H-NS and H-NS-39 were used at final concentrations of 6 μM and 1–10 μM respectively. The schematic is as described for (a). The experiment was done twice. c Global DNA binding by H-NS in vivo is the same in the presence and absence of H-NS-39. The panel shows two heatmaps, each representing the A. baumannii chromosome divided into 1 kb sections. Sections are coloured according to the H-NS ChIP-seq binding signal in the absence (top) or presence (bottom) of ectopic H-NS-39 expression. The Pearson correlation coefficient (r) between datasets is shown. The raw H-NS ChIP-seq read depths are provided in Supplementary Data 1. d Global 10 kb resolution 3C-seq contact maps are the same in the presence and absence of H-NS-39. The heatmaps illustrate interaction frequencies between 10 kb sections of the A. baumannii chromosome, measured by 3C-seq, in the presence and absence of H-NS-39. Axes indicate the genomic location of each bin in the pair. Individual sections are coloured according to the number of interactions between the two corresponding chromosomal locations. The contact matrix values, and explorable versions of each matrix, are in Supplementary Data files 2–4. e H-NS-39 alters short range interactions in 1 kb resolution 3C-seq contact maps. The heatmaps illustrate interaction frequencies between 1 kb sections of the A. baumannii chromosome, measured by 3C-seq, in the presence and absence of H-NS-39. An interaction pattern indicative of a loop is marked by a blue triangle. Signal in this region is lost in the presence of H-NS-39. The locations of genes (red arrows) are also shown alongside H-NS binding patterns determined by ChIP-seq with or without expression of H-NS-39. The contact matrix values, and explorable versions of each matrix, are in Supplementary Data files 2–4.