Regulated phase transitions of bacterial chromatin: a non-enzymatic pathway for generic DNA protection

Abstract

The enhanced stress resistance exhibited by starved bacteria represents a central facet of virulence, since nutrient depletion is regularly encountered by pathogens in their natural in vivo and ex vivo environments. Here we explore the notion that the regular stress responses, which are mediated by enzymatically catalyzed chemical transactions and promote endurance during the logarithmic growth phase, can no longer be effectively induced during starvation. We show that survival of bacteria in nutrient-depleted habitats is promoted by a novel strategy: finely tuned and fully reversible intracellular phase transitions. These non-enzymatic transactions, detected and studied in bacteria as well as in defined in vitro systems, result in DNA sequestration and generic protection within tightly packed and highly ordered assemblies. Since this physical mode of defense is uniquely independent of enzymatic activity or de novo protein synthesis, and consequently does not require energy consumption, it promotes virulence by enabling long-term bacterial endurance and enhancing antibiotic resistance in adverse habitats.

Fig. 1. Electron microscopy and X-ray scattering patterns of E.coli cells. (A) Wild-type E.coli at mid-logarithmic phase. The dark particles are ribosomes. The ribosome-free spaces contain chromatin. (B) Escherichia coli at mid-logarithmic phase, stained solely with the DNA-specific reagent osmium-ammine-SO₂. The irregular spreading of chromatin over the cytoplasm is indicated. (C) Dps-overproducing cell induced at mid-logarithmic phase and further incubated for 48 h. (D) Wild-type E.coli incubated for 48 h following the onset of the stationary phase. (E) Same as (D), but stained with the DNA-specific reagent osmium-ammine-SO₂. Samples were prepared by the cryo-fixation method. Scale bars are 400 nm (A and B) and 150 nm (C–E). (F) X-ray scattering patterns from intact wild-type E.coli cells, presented as a difference profile, which is obtained by subtracting the scattering curve of mid-logarithmic phase bacteria, in which no bands are discerned, from the scattering profile of E.coli cells incubated for 48 h following the onset of stationary phase.

Fig. 2. Effects of Mg²⁺ on DNA–Dps co-crystallization. Dps and closed- circular DNA (at a protein:DNA weight ratio of 1:5) were incubated for 10 min in 10 mM Tris pH 7.0, 20 mM NaCl, 0.4 mM EDTA, with the following Mg²⁺ concentrations: (A) no Mg²⁺ added, large DNA–Dps co-crystals (as the assembly shown here, which spans the whole field of the micrograph) are regularly detected; (B) 1.0 mM, only small ordered complexes are obtained; (C) 3.0 mM, no co-crystalline assemblies can be detected under such conditions. The magnification in (A), (B) and (C) is identical; scale bar is 150 nm.

Fig. 3. Effects of Mg²⁺ on Dps-mediated DNA protection. (A) Dps-mediated DNA protection. Lane 1, linear DNA (pBluescript, 2958 bp, linearized with EcoRI); lane 2, linear DNA treated with DNase I (1 U, 5 min at room temperature); lanes 3–7, DNase I treatment of Dps–DNA complexes at the following Dps:DNA weight ratios: 0.5, 1.0, 2.0, 3.0 and 5.0, respectively. (B) Mg²⁺ effects. Dps–DNA complexes (5:1 w/w protein:DNA ratio) with or without added Mg²⁺ were treated with DNase I. The Mg²⁺ row designates the concentration of Mg²⁺ in mM; the Dps and DNase I rows designate the presence or absence of these substances in the reaction mixtures. Note that nuclease activity is preserved at the whole range of [Mg²⁺] used, as indicated by control experiments consisting of DNA, DNase I and Mg²⁺ at various concentrations, but no Dps (left lane of each [Mg²⁺]).

Fig. 4. Electron microscopy of Dps^– cells and of an in vitro DNA cholesteric phase. (A and B) Dps^– cells from cultures incubated for 6 days following the onset of a stationary phase, exhibiting a cholesteric DNA organization. Dps^– bacteria harvested following shorter incubation periods revealed morphological patterns identical to those exhibited by wild-type E.coli at mid-logarithmic phase. (C) Magnification of the central region of the cell depicted in (A). Scale bars in (A), (B) and (C) are 150, 300 and 100 nm, respectively. (D) Electron micrograph (provided by Dr A.Leforestier) of an in vitro cholesteric phase of pure DNA molecules (146 bp; 200 mg/ml). The sample has been freeze-fractured following cryo-fixation at an oblique angle to the cholesteric stratification (Leforestier and Livolant, 1993). The nested arcs that characterize the cholesteric phase are detected in both the in vivo and in vitro samples.