Growth Phase-Dependent Chromosome Condensation and Heat-Stable Nucleoid-Structuring Protein Redistribution in Escherichia coli under Osmotic Stress

Abstract

The heat-stable nucleoid-structuring (H-NS) protein is a global transcriptional regulator implicated in coordinating the expression of over 200 genes in Escherichia coli, including many involved in adaptation to osmotic stress. We have applied superresolved microscopy to quantify the intracellular and spatial reorganization of H-NS in response to a rapid osmotic shift. We found that H-NS showed growth phase-dependent relocalization in response to hyperosmotic shock. In stationary phase, H-NS detached from a tightly compacted bacterial chromosome and was excluded from the nucleoid volume over an extended period of time. This behavior was absent during rapid growth but was induced by exposing the osmotically stressed culture to a DNA gyrase inhibitor, coumermycin. This chromosomal compaction/H-NS exclusion phenomenon occurred in the presence of either potassium or sodium ions and was independent of the presence of stress-responsive sigma factor σ^S and of the H-NS paralog StpA.IMPORTANCE The heat-stable nucleoid-structuring (H-NS) protein coordinates the expression of over 200 genes in E. coli, with a large number involved in both bacterial virulence and drug resistance. We report on the novel observation of a dynamic compaction of the bacterial chromosome in response to exposure to high levels of salt. This stress response results in the detachment of H-NS proteins and their subsequent expulsion to the periphery of the cells. We found that this behavior is related to mechanical properties of the bacterial chromosome, in particular, to how tightly twisted and coiled is the chromosomal DNA. This behavior might act as a biomechanical response to stress that coordinates the expression of genes involved in adapting bacteria to a salty environment.

Keywords: Escherichia coli; H-NS; chromosome organization; nucleoid-associated proteins; osmotic stress; stress response.

FIG 1

Dynamics of chromosome compaction following osmotic shock. (A and B) Absolute, projected chromosome area (A) and the area fraction (i.e., normalized to the projected area of the cell) (B), for both exponential-phase and stationary-phase cells, up to 60 min after osmotic shock. Error bars represent standard deviations of the means (see Table S1 in the supplemental material).

FIG 2

SRRF images of exponential-growth-phase dynamic response to osmotic stress (300 mM KCl). From left to right, t = 0, 5, 10, and 45 min postinduction. (A to D) H-NS (H-NS-mEos3.2_FLAG). (E to H) Chromosome. (I to L) Merged images. The merged images were magnified to show details (magnified regions are indicated by boxes in the images above the merged images). All scale bars are 2 μm.

FIG 3

SRRF images of stationary-phase dynamic response to osmotic stress (300 mM KCl). From left to right, t = 0, 5, 10, and 45 min postinduction. (A to D) H-NS-mEos3.2_FLAG. (E to H) Chromosome. (I to L) Merged images. The merged images were magnified to show details (magnified regions are indicated by boxes in the images above the merged images). All scale bars are 2 μm. (M and N) Major-axis (M) and minor-axis (N) normalized intensity cross sections of H-NS distribution at 30 min postinduction. Intensity traces from individual bacteria (40 cells) are shown, with the average given by the dashed line (mean pixel values are indicated by circles).

FIG 4

Analysis of cells treated with coumermycin (Table S2). (A) SRRF images of exponential-growth-phase cells subjected to osmotic stress treatment (300 mM KCl) for 30 min without (left column) or with (right column) coumermycin (Coum; 5 μg/ml). From top to bottom, H-NS-mEos3.2_FLAG, DAPI-stained chromosome, and merged images. Scale bars are 2 μm. (B and C) Comparison of the projected chromosome area (B) and area fraction (C) between exponential growth phase and stationary phase cells. Shown are results for control cells, cells subjected to osmotic shock (KCl), and cells exposed to KCl plus coumermycin.

FIG 5

Chromatin immunoprecipitation of H-NS_HA at four H-NS-bound loci (proV [upper left], xapR [upper right], yjcF [lower left], and bglF [lower right]) during osmotic shock (KCl) or gyrase inhibition with coumermycin (Coum) or both. Cells, diluted in M9 medium from overnight cultures, were grown to the indicated growth phase. At 30 min after addition of KCl or coumermycin or both, cells were fixed with formaldehyde and DNA-protein complexes were immunoprecipitated as described in Materials and Methods. Enrichment of bound DNA was quantified by real-time PCR and normalized to the amount of DNA in the sample prior to immunoprecipitation. Values above or adjacent to bars indicate the mean levels of ChIP efficiency.

FIG 6

Transcript levels of H-NS-regulated genes in the stationary phase during osmotic shock. RNA was extracted from stationary-phase cultures 30 min after treatment with 300 mM KCl. RNA was reverse transcribed, and transcript levels were measured by quantitative real-time PCR. Fold change is reported compared to identical cultures to which KCl had not been added. All transcripts levels were normalized to those of gyrB as a control. Values above bars represent mean values corresponding to fold changes in expression.