Charged residues in the H-NS linker drive DNA binding and gene silencing in single cells

Abstract

Nucleoid-associated proteins (NAPs) facilitate chromosome organization in bacteria, but the precise mechanism remains elusive. H-NS is a NAP that also plays a major role in silencing pathogen genes. We used genetics, single-particle tracking in live cells, superresolution microscopy, atomic force microscopy, and molecular dynamics simulations to examine H-NS/DNA interactions in single cells. We discovered a role for the unstructured linker region connecting the N-terminal oligomerization and C-terminal DNA binding domains. In the present work we demonstrate that linker amino acids promote engagement with DNA. In the absence of linker contacts, H-NS binding is significantly reduced, although no change in chromosome compaction is observed. H-NS is not localized to two distinct foci; rather, it is scattered all around the nucleoid. The linker makes DNA contacts that are required for gene silencing, while chromosome compaction does not appear to be an important H-NS function.

Keywords: H-NS; atomic force microscopy; nucleoid-associated proteins; single-particle tracking; superresolution microscopy.

Fig. 1.

Substitutions in the H-NS linker affect gene regulation. (A) L is the linker (amino acids 80–94) and DBD is the DNA binding domain. Original linker residues are in red, additional positive charged residues in blue, alanines in green, and glutamines in black. (B) Swarming motility assay of the linker substitution strains. The relative diameters of the swarm were normalized to the wild-type H-NS (100%). The mean was calculated from three independent experiments with at least two replicates.

Fig. 2.

DNA binding is impaired in H-NS linker mutants. (A) The csgD promoter in the absence of H-NS protein. (B) Wild-type H-NS binds and polymerizes along the DNA, forming a stiffened filament. Linker mutants lacking the linker (C) or with the polyQ linker (D) form small foci (yellow arrows) due to reduced binding affinity (SI Appendix, Fig. S3) and fail to polymerize. Addition of positive charges improves binding, leading to DNA polymerization in E and F. The K2R2 mutant (G) binds similarly to the wild-type protein. Two panels show different representative images. All proteins were present at 600 nM, and binding was performed in 50 mM KCl, 2 mM MgCl₂, 10 mM Tris⋅HCl (pH 7.4) buffer. (H) Relative height distribution histograms were obtained from DNA contours of each experiment. (Scale bar: 200 nm.)

Fig. 3.

Two-color sequential SMLM of H-NS-PAmCherry and DNA-EdU-Alexa Fluor 647 (A647). Wild-type H-NS (A) and ΔL (B). Top left in A and B are images of H-NS localization, and right panels in A and B are nucleoid localization. Merged images of H-NS and the nucleoid are in the bottom left. Bottom right panels in A and B include the outline of the nucleoids in cyan. Boxes outlined with dotted white lines indicate a 0.9-µm-thick line drawn along the long axis of the cell integrating pixel values across the width of the line creating the intensity profiles in C and D. The indices i to vi correspond to intensity profile plots in C and D. C and D are intensity profiles of H-NS (orange) and DNA (cyan) localizations for wild-type H-NS (C) and ΔL (D). (Scale bars: 0.5 µm.)

Fig. 4.

SptPALM of wild-type H-NS and ΔL. (A) Representative cells and tracks of wild-type and ΔL strains. (B) Apparent diffusion coefficient D* histogram obtained from the slope of the first three points of the MSD plot calculated from individual tracks. (C) Diffusion map constructed from the D* obtained from B by converting the x–y vector coordinates of all of the spots in the tracks into pixel coordinates (50-nm size). The color map ranges from D* = 0 (red) to 1.5 µm²/s (dark blue). Values >1.5 µm²/s were also dark blue pixels. (D) CPD plot of both wild-type and ΔL strains. Blue circles represent the experimental data, and red lines represent the fit of the data to SI Appendix, Eq. 3. (Scale bars: 0.5 µm.)

Fig. 5.

Two of nine snapshots from independent trajectories of MD simulations with different initial conformations. The C terminus of H-NS (amino acids 95–137) is indicated in green and the linker (amino acids 80–94) indicated in magenta. All trajectories feature a stable attraction between DNA and the H-NS linker. Key residues forming H-bonds are shown in stick representation and H-bonds are shown in black dashed lines. See SI Appendix, Fig. S14 and Movie S1 for remaining snapshots.