Locus of enterocyte effacement-encoded regulator (Ler) of pathogenic Escherichia coli competes off histone-like nucleoid-structuring protein (H-NS) through noncooperative DNA binding

Abstract

The locus of enterocyte effacement-encoded regulator (Ler) of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) functions to activate transcription of virulence genes silenced by the histone-like nucleoid-structuring protein (H-NS). Despite its important role in the bacterial gene regulation, the binding mode of Ler to DNA and its mechanism in alleviating genes repressed by H-NS are largely unknown. In this study, we use magnetic tweezers to demonstrate that Ler binds extended DNA through a largely noncooperative process, which results in DNA stiffening and DNA folding depending on protein concentration. We also show that Ler can replace prebound H-NS on DNA over a range of potassium and magnesium concentrations. Our findings reveal the DNA binding properties of Ler and shed light to further understand the anti-silencing activity of Ler.

Keywords: Atomic Force Microscopy; DNA-binding Protein; Gene Regulation; H-NS; Ler; Magnetic Tweezers; Protein-DNA Interaction; Single Molecule Biophysics.

FIGURE 1.

Single molecule stretching and imaging experiments on Ler·DNA complexes. A, force-extension curves generated from single DNA stretching experiments on Ler·DNA complexes at various protein concentrations using the force-scanning procedure detailed under “Experimental Procedures.” Reduction in DNA extension accompanied with hysteresis between the force-decrease (FD) and force-increase (FI) curve was observed at low protein concentration. At higher protein concentrations, Ler stiffened the DNA, indicated by higher extension compared with naked DNA. B, force-extension curves obtained by the force-jumping procedure detailed under “Experimental Procedures,” which prevents DNA folding, show increased DNA extension that commensurate with Ler concentration. The error bars represent the standard deviation obtained from three successive force-jumping data from the same DNA molecule. C–F, representative AFM images of Ler·DNA complexes at various protein concentrations in 50 mm KCl: naked DNA (C), DNA complexed with 30 nm Ler (D), 300 nm Ler (E), and 3 μm Ler (F). Ler binding on DNA results in more compact nucleoprotein structures. At 3 μm Ler, co-existing extended nucleoprotein complexes were also observed. G, line profiles of naked DNA (magenta), DNA complexed with 30 nm Ler (green), DNA complexed with 300 nm Ler (blue), and DNA complexed with 3 μm Ler (orange) as indicated by the lines in C–F. All the AFM images are 0.7 × 0.7 μm in size.

FIGURE 2.

Single molecule characterization of Ler and H-NS binding to DNA. A, Ler·DNA complex's bending persistence length and contour length were quantified using the data generated from force-jumping procedure by fitting with the Marko-Siggia formula in panel. B, average occupancy from multiple independent experiments at each concentration was fitted with the Hill equation. The values of the Hill coefficient and dissociation constant of Ler binding to DNA were obtained from the average values of each individual experiment. C, H-NS·DNA complex's bending persistence length and contour length in stiffening binding mode were quantified using the same method as A. D, values of the Hill coefficient and dissociation constant of H-NS binding to DNA were quantified using the same method as in B. The error bars for each panel represent the mean ± S.E., and the error bars in B and D were calculated based on the error propagation from the standard error of persistence length measurements in A and C, respectively, at each protein's concentration obtained from multiple independent experiments.

FIGURE 3.

Ler·DNA complex response to environmental factors. A, Ler·DNA complex response to KCl concentration. Ler binding to DNA was reduced as KCl concentration was increased, as apparent in the reduction of nucleoprotein stiffness at 400 mm KCl. The data points at 50 mm KCl (magenta) overlap the data points at 100 mm KCl (green) and 200 mm KCl (blue) and are not visible in the panel. B, Ler·DNA complexes were relatively insensitive to variations in magnesium concentration. The force-decrease curves were largely unaffected as the magnesium concentration is varied from 2 to 10 mm MgCl₂, whereas an increased hysteresis was observed between the force-decrease and force-increase curves. C, Ler·DNA complexes were relatively insensitive to pH. The force-decrease curves were largely unaffected as pH is varied from 8.8 to 6.8, although an increased hysteresis was observed between the force-decrease and force-increase curves. D, Ler·DNA complex binding was reduced when the temperature was increased from 23 to 37 °C, indicated by the decrease in DNA extension and increased level of hysteresis at higher temperatures.

FIGURE 4.

Ler replaces H-NS in stiffening mode to form a stable Ler·DNA complex. A, at 50 mm KCl, H-NS forms rigid nucleoprotein filaments, which can be replaced by Ler when the concentration of Ler was progressively increased in the presence of 600 nm H-NS. This is indicated by the decrease in nucleoprotein stiffness to the level of Ler nucleoprotein complex (light gray region) when Ler concentration in solution exceeds 100 nm. B, DNA complexed with 1.2 μm H-NS resulted in rigid nucleoprotein filaments. C–E, DNA was first incubated in 1.2 μm H-NS for 30 min before various concentrations of Ler were introduced in the solution for another 20 min (0.6 μm Ler for C, 1.2 μm Ler for D, and 2.4 μm Ler for E). Some parts of the H-NS filaments were compacted by Ler proteins, indicating that Ler can replace H-NS filaments. F, line profiles of DNA complexed with 1.2 μm H-NS (magenta), H-NS pre-coated DNA with 0.6 μm Ler (green), H-NS pre-coated DNA with 1.2 μm Ler (blue), and H-NS pre-coated DNA with 2.4 μm Ler (orange) as indicated by the lines in B–E. All the AFM images are 0.7 × 0.7 μm in size.

FIGURE 5.

Effects of variations in KCl and MgCl₂ concentrations on Ler and H-NS competition for DNA binding. A, at 200 mm KCl and 600 nm H-NS, the measured extension was close to that of naked DNA. Increasing Ler concentrations while maintaining 600 nm H-NS gave progressive increases in DNA extension. The resulting force-extension curve approaches the predicted curve for saturated Ler-bound DNA when Ler concentration exceeds 300 nm. B, at 50 mm KCl, 10 mm MgCl₂, where H-NS predominantly induces DNA bridging with negligible DNA stiffening, Ler replaced H-NS when the concentration of Ler was progressively increased while maintaining 600 nm H-NS. This is indicated by reduced DNA folding and an increase in the extension of protein·DNA complexes. At 600 nm Ler, the curves are close to the predicted curve for Ler nucleoprotein complex (light gray region).

FIGURE 6.

Models of Ler binding to DNA and its role in gene regulation. At nonsaturated condition or low tension, Ler can bind DNA in the wrapped mode. At a higher concentration (Conc.) of Ler or high tension, Ler binds DNA in an unwrapped mode in a largely noncooperative process to form Ler·DNA nucleoprotein complex array. H-NS, however, forms rigid nucleoprotein filament at low MgCl₂ concentrations (<5 mm) through a cooperative binding process. The formation of nucleoprotein filament may silence genes by blocking RNA polymerases from accessing the promoters or blocking translocation of RNA polymerases. Ler can replace H-NS nucleoprotein filaments in some environmental conditions (such as low KCl concentration). This replacement of H-NS by Ler, together with Ler's lack of cooperativity, may serve as the basis to understand Ler's anti-silencing activity.