Bacterial protein HU dictates the morphology of DNA condensates produced by crowding agents and polyamines

Abstract

Controlling the size and shape of DNA condensates is important in vivo and for the improvement of nonviral gene delivery. Here, we demonstrate that the morphology of DNA condensates, formed under a variety of conditions, is shifted completely from toroids to rods if the bacterial protein HU is present during condensation. HU is a non-sequence-specific DNA binding protein that sharply bends DNA, but alone does not condense DNA into densely packed particles. Less than one HU dimer per 225 bp of DNA is sufficient to completely control condensate morphology when DNA is condensed by spermidine. We propose that rods are favored in the presence of HU because rods contain sharply bent DNA, whereas toroids contain only smoothly bent DNA. The results presented illustrate the utility of naturally derived proteins for controlling the shape of DNA condensates formed in vitro. HU is a highly conserved protein in bacteria that is implicated in the compaction and shaping of nucleoid structure. However, the exact role of HU in chromosome compaction is not well understood. Our demonstration that HU governs DNA condensation in vitro also suggests a mechanism by which HU could act as an architectural protein for bacterial chromosome compaction and organization in vivo.

Figure 1.

PEG-induced DNA condensate morphologies and morphology statistics as a function of HU concentration. (A) TEM image of a representative condensate of linear DNA condensed by PEG 8000 (no HU present). (B) TEM image of a representative condensate produced under identical conditions as in A, except in the presence of 200 nM HU. Scale bar is 100 nm. (C) Relative rod populations versus HU concentration for linear DNA condensed by PEG. Samples were 5 µM DNA bp, 125 mg/ml PEG 8000, 50 mM Tris-HCl, 1 mM EDTA (pH 7.8), 100 mM NaCl, and indicated concentrations of HU dimer. Each rod population measurement reported is the average of counts from three different EM grid preparations.

Figure 2.

Spermidine-induced DNA condensate morphologies and morphology statistics as a function of HU concentration. (A) TEM image of a representative condensate of linear DNA condensed by spermidine (no HU present). (B) TEM image of representative condensates produced under identical conditions as in A, except in the presence of 50 nM HU. (C) TEM image of representative condensates of supercoiled DNA condensed by spermidine (no HU present). (D) TEM image of representative condensates produced under identical conditions as in C, except in the presence of 50 nM HU. Scale bar is 100 nm. (E) Relative rod populations versus HU concentration for linear and supercoiled DNA condensed by spermidine. Samples were 5 µM DNA bp, 700 µM spermidine chloride, 0.33 × TE (pH 7.8), 15 mM NaCl, and indicated concentrations of HU dimer.

Figure 3.

Spermine-induced DNA condensate morphologies and morphology statistics as a function of HU concentration. (A) TEM image of representative condensates of linear DNA condensed by spermine (no HU present). (B) TEM image of representative condensates produced under identical conditions as that shown in A, except in the presence of 100 nM HU. Scale bar is 100 nm. (C) Relative rod populations plotted as a function of HU concentration for linear DNA condensed by spermine. Samples were 5 µM DNA bp, 15 µM spermine chloride, 0.33 × TE (pH 7.8), 15 mM NaCl and indicated concentrations of HU dimer.

Figure 4.

Condensate morphology statistics versus HU concentration for reactions with HU added to DNA before condensation. (A) Relative rod populations versus HU concentration for linear DNA condensed by PEG. Samples were 5 µM DNA bp, 125 mg/ml PEG 8000, 100 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA (pH 7.8), and indicated concentration of HU dimer. (B) Relative rod populations versus HU concentration for linear (circle) and supercoiled DNA (triangle) condensed by spermidine. Samples were 5 µM DNA bp, 700 µM spermidine chloride, 0.33 × TE (pH 7.8), 15 mM NaCl and indicated concentrations of HU dimer. (C) Relative rod populations versus HU concentration for linear DNA condensed by spermine. Samples were 5 µM DNA bp, 15 µM spermine chloride, 0.33 × TE (pH 7.8), 15 mM NaCl and indicated concentrations of HU dimer. Dashed curves are best-fit curves from rod populations measured for corresponding experiments in which HU was added coincident with the condensing agent (see Figures 1–3 for details).

Figure 5.

A model for how HU affects the process of DNA condensation in which rods and toroids are formed. The three stages of DNA condensation in vitro, as described in text, are: rod/toroid nucleation; proto-rod/proto-toroid formation (intramolecular condensation); and condensate growth (intermolecular condensation), which includes strand exchange between condensates (under some conditions). Bold arrows indicate steps that apparently become more favorable in the presence of HU. Black ellipsoids represent HU molecules.