Isolation of the Escherichia coli nucleoid.

Numerous protocols for the isolation of bacterial nucleoids have been described based on treatment of cells with sucrose-lysozyme-EDTA and subsequent lysis with detergents in the presence of counterions (e.g., NaCl, spermidine). Depending on the lysis conditions both envelope-free and envelope-bound nucleoids could be obtained, often in the same lysate. To investigate the mechanism(s) involved in compacting bacterial DNA in the living cell, we wished to isolate intact nucleoids in the absence of detergents and high concentrations of counterions. Here, we compare the general lysis method using detergents with a procedure involving osmotic shock of Escherichia coli spheroplasts that resulted in nucleoids free of envelope fragments. After staining the DNA with DAPI (4',6-diamidino-2-phenylindole) and cell lysis by either isolation procedure, free-floating nucleoids could be readily visualized in fluorescence microscope preparations. The detergent-salt and the osmotic-shock nucleoids appeared as relatively compact structures under the applied ionic conditions of 1 M and 10 mM, respectively. RNase treatment caused no dramatic changes in the size of either nucleoid.

Depletion-induced demixing in aqueous protein-polysaccharide solutions.

We explore the separation of aqueous protein-polysaccharide solutions into two liquid phases. In particular, we have studied the combinations beta-lactoglobulin/pullulan, alpha-lactalbumin/pullulan, and other examples from the literature under a variety of conditions such as varying salt content, pH (in most cases at the isoelectric point), and protein radius. We restrict ourselves to relatively small proteins (globular) and long polysaccharide chains. The mechanism behind the phase separation is explained in terms of the depletion interaction (i.e., the cross-interaction) in a suspension of small spheres (proteins) immersed in a semidilute solution of coils (polysaccharide) forming an entangled network. Weak attractions between the spheres have been taken into account by assuming the formation of small clusters. As a general rule, we find that the depletion free energy per protein particle governing the protein partitioning in the phase equilibrium is linear in the polysaccharide concentration over the whole range of experimentally accessible coexistence curves. Furthermore, the proportionality constant is shown to be a very useful quantity to understand the characteristics of the coexistence curves. The linearity thus found is supported by theoretical arguments developed by de Gennes and Odijk.

Polymer-mediated compaction and internal dynamics of isolated Escherichia coli nucleoids.

Nucleoids of Escherichia coli were isolated by osmotic shock under conditions of low salt in the absence of added polyamines or Mg(2+). As determined by fluorescence microscopy, the isolated nucleoids in 0.2 M NaCl are expanded structures with an estimated volume of about 27 microm(3) according to a procedure based on a 50% threshold for the fluorescence intensity. The nucleoid volume is measured as a function of the concentration of added polyethylene glycol. The collapse is a continuous process, so that a coil-globule transition is not witnessed. The Helmholtz free energy of the nucleoids is determined via the depletion interaction between the DNA helix and the polyethylene glycol chains. The resulting compaction relation is discussed in terms of the current theory of branched DNA supercoils and it is concluded that the in vitro nucleoid is crosslinked in a physical sense. Despite the congested and crosslinked state of the nucleoid, the relaxation rate of its superhelical segments, as monitored by dynamic light scattering, turns out to be purely diffusional. At small scales, the nucleoid behaves as a fluid.

Depletion theory of protein transport in semi-dilute polymer solutions.

We consider the effect of polymer depletion on the transport (diffusion and electrophoresis) of small proteins through semi-dilute solutions of a flexible polymer. A self-consistent field theory may be set up in the important case of quasi-ideal interactions when the protein is small enough. Dynamic depletion, the reorganization of the depletion layer as the protein diffuses, is computed within a free-draining approximation. The transport of the dressed particle (protein + depletion layer) is tackled by extending Ogston's analysis of probe diffusion through fibrous networks to the case of a probe diffusing through a semi-dilute polymer inhomogeneous on the scale of the polymer correlation length. The resulting exponential retardation agrees almost quantitatively with that found in recent electrophoresis experiments of small proteins in polymer solutions that have been ascertained to be semi-dilute (S. P. Radko and A. Chrambach, Electrophoresis, 17:1094-1102, 1996; Biopolymers, 4:183-189, 1997).

Electrostatic-undulatory theory of plectonemically supercoiled DNA.

We present an analytical calculation of the electrostatic interaction in a plectonemic supercoil within the Poisson-Boltzmann approximation. Undulations of the supercoil strands arising from thermal motion couple nonlinearly with the electrostatic interaction, giving rise to a strong enhancement of the bare interaction. In the limit of fairly tight winding, the free energy of a plectonemic supercoil may be split into an elastic contribution containing the bending and torsional energies and an electrostatic-undulatory free energy. The total free energy of the supercoil is minimized according to an iterative scheme, which utilizes the special symmetry inherent in the usual elastic free energy of the plectoneme. The superhelical radius, opening angle, and undulation amplitudes in the radius and pitch are obtained as a function of the specific linking difference and the concentration of monovalent salt. Our results compare favorably with the experimental values for these parameters of Boles et al. (1990. J. Mol. Biol. 213:931-951). In particular, we confirm the experimental observation that the writhe is a virtually constant fraction of the excess linking number over a wide range of superhelical densities. Another important prediction is the ionic strength dependence of the plectonemic parameters, which is in reasonable agreement with the results from computer simulations.

Hexagonally packed DNA within bacteriophage T7 stabilized by curvature stress.

A continuum computation is proposed for the bending stress stabilizing DNA that is hexagonally packed within bacteriophage T7. Because the inner radius of the DNA spool is rather small, the stress of the curved DNA genome is strong enough to balance its electrostatic self-repulsion so as to form a stable hexagonal phase. The theory is in accord with the microscopically determined structure of bacteriophage T7 filled with DNA within the experimental margin of error.

Osmotic compaction of supercoiled DNA into a bacterial nucleoid.

A theory is presented of the phase separation of supercoiled DNA into a nucleoid in a bacterial cell. The suspension consists of DNA interacting with globular proteins in excess salt. A cross virial between DNA and a protein is computed as well as the DNA self-energy arising from excluded volume. The cellular parameters of Escherichia coli would appear to be compatible with the thermodynamic equilibrium derived theoretically. The state of superhelical DNA in the nucleoid could be liquid crystalline and rippled.

Polymer- and salt-induced toroids of hexagonal DNA.

A model is proposed for polymer- and salt-induced toroidal condensates of DNA, based on a recent theory of the undulation enhancement of the electrostatic interaction in the bulk hexagonal phase of semiflexible polyions. In a continuum approximation, the thermodynamic potential of a monomolecular toroid may be split up in bulk, surface, and curvature contributions. With the help of an approximate analytical minimization procedure, the optimal torus dimensions are calculated as a function of the concentrations of inert polymer and added salt. The stability of the torus is analyzed in terms of its surface tension and a bulk melting criterion. The theory should be applicable to psi-toroids that are not too thick.

Growth of linear charged micelles.

The electrostatics of micellar growth is reviewed and extended for solutions containing excess salt. In dilute solution the expansion of a linear micelle with increasing salt concentration is explained for a wide range of ionic strength. When the micellar charge density is very high, counterions condense nonuniformly onto the micellar rod. In that case the micelle may contract upon the addition of salt. In semidilute solutions the excluded-volume effect is an additional factor complicating the ionic strength dependence of micellar growth.