Eric Raspaud

Osmotic pressure inhibition of DNA ejection from phage

Bacterial viral capsids in aqueous solution can be opened in vitro by addition of their specific receptor proteins, with consequent full ejection of their genomes. We demonstrate that it is possible to control the extent of this ejection by varying the external osmotic pressure. In the particular case of bacteriophage λ, the ejection is 50% inhibited by osmotic pressures (of polyethylene glycol) comparable to those operative in the cytoplasm of host bacteria; it is completely suppressed by a pressure of 20 atmospheres. Furthermore, our experiments monitor directly a dramatic decrease of the stress inside the unopened phage capsid upon addition of polyvalent cations to the host solution, in agreement with many recent theories of DNA interactions.

Are liquid crystalline properties of nucleosomes involved in chromosome structure and dynamics

Nucleosome core particles correspond to the structural units of eukaryotic chromatin. They are charged colloids, 101 Å in diameter and 55 Å in length, formed by the coiling of a 146/147 bp DNA fragment (50 nm) around the histone protein octamer. Solutions of these particles can be concentrated, under osmotic pressure, up to the concentrations found in the nuclei of living cells. In the presence of monovalent cations (Na+), nucleosomes self-assemble into crystalline or liquid crystalline phases. A lamello-columnar phase is observed at ‘low salt’ concentrations, while a two-dimensional hexagonal phase and a three-dimensional quasi-hexagonal phase form at ‘high salt’ concentrations. We followed the formation of these phases from the dilute isotropic solutions to the ordered phases by combining cryoelectron microscopy and X-ray diffraction analyses. The phase diagram is presented as a function of the monovalent salt concentration and applied osmotic pressure. An alternative method to condense nucleosomes is to induce their aggregation upon addition of divalent or multivalent cations (Mg2+, spermidine3+ and spermine4+). Ordered phases are also found in the aggregates. We also discuss whether these condensed phases of nucleosomes may be relevant from a biological point of view.

Bacteriophage T5 DNA Ejection under Pressure

The transfer of the bacteriophage genome from the capsid into the host cell is a key step of the infectious process. In bacteriophage T5, DNA ejection can be triggered in vitro by simple binding of the phage to its purified Escherichia coli receptor FhuA. Using electrophoresis and cryo-electron microscopy, we measure the extent of DNA ejection as a function of the external osmotic pressure. In the high pressure range (7-16 atm), the amount of DNA ejected decreases with increasing pressure, as theoretically predicted and observed for lambda and SPP1 bacteriophages. In the low and moderate pressure range (2-7 atm), T5 exhibits an unexpected behavior. Instead of a unique ejected length, multiple populations coexist. Some phages eject their complete genome, whereas others stop at some nonrandom states that do not depend on the applied pressure. We show that contrarily to what is observed for the phages SPP1 and lambda, T5 ejection cannot be explained as resulting from a simple pressure equilibrium between the inside and outside of the capsid. Kinetics parameters and/or structural characteristics of the ejection machinery could play a determinant role in T5 DNA ejection.

DNA condensed by protamine: a "short" or "long" polycation behavior.

Using centrifugation assay and light scattering measurements, we study the condensation of DNA by the salmon protamine, a highly basic protein carrying 21 positive charges out of 30 amino acids, in the presence of a high amount of monovalent salt. The DNA condensation is followed by a macroscopic phase separation. It occurs while a large amount of polycations remains freely diffusing in the bulk. A similar behavior was described before for small multivalent ions in diluted DNA solution in a lower salt range. Sensitivity to the salt is however amplified when increasing the charge of polycations. Indeed, a high power-law dependence is observed here with an exponent 11. This variation agrees with the power-law dependence that characterizes the binding of small polycations to DNA. In other words, we show that protamines behave like small polycations in the diluted DNA-high salt regime, while they behave like other large polycations in the diluted DNA-low salt regime as shown in a previous study. In addition, instead of the classical view where binding of polycations to DNA is supposed to trigger DNA condensation in low and moderate salt conditions, we propose that, under high salt conditions, the potential presence of a DNA dense phase triggers the binding of protamines to DNA.

Bacillus subtilis Bacteria Generate an Internal Mechanical Force within a Biofilm

A key issue in understanding why biofilms are the most prevalent mode of bacterial life is the origin of the degree of resistance and protection that bacteria gain from self-organizing into biofilm communities. Our experiments suggest that their mechanical properties are a key factor. Experiments on pellicles, or floating biofilms, of Bacillus subtilis showed that while they are multiplying and secreting extracellular substances, bacteria create an internal force (associated with a -80±25 Pa stress) within the biofilms, similar to the forces that self-equilibrate and strengthen plants, organs, and some engineered buildings. Here, we found that this force, or stress, is associated with growth-induced pressure. Our observations indicate that due to such forces, biofilms spread after any cut or ablation by up to 15-20% of their initial size. The force relaxes over very short timescales (tens of milliseconds). We conclude that this force helps bacteria to shape the biofilm, improve its mechanical resistance, and facilitate its invasion and self-repair.

Mechanical Behavior of a Bacillus subtilis Pellicle

Bacterial biofilms consist of a complex network of biopolymers embedded with microorganisms, and together these components form a physically robust structure that enables bacteria to grow in a protected environment. This structure can help unwanted biofilms persist in situations ranging from chronic infection to the biofouling of industrial equipment, but under certain circumstances it can allow the biofilm to disperse and colonize new niches. Mechanical properties are therefore a key aspect of biofilm life. In light of the recently discovered growth-induced compressive stress present within a biofilm, we studied the mechanical behavior of Bacillus subtilis pellicles, or biofilms at the air-liquid interface, and tracked simultaneously the force response and macroscopic structural changes during elongational deformations. We observed that pellicles behaved viscoelastically in response to small deformations, such that the growth-induced compressive stress was still present, and viscoplastically at large deformations, when the pellicles were under tension. In addition, by using particle imaging velocimetry we found that the pellicle deformations were nonaffine, indicating heterogeneous mechanical properties with the pellicle being more pliable near attachment surfaces. Overall, our results indicate that we must consider not only the viscoelastic but also the viscoplastic and mechanically heterogeneous nature of these structures to understand biofilm dispersal and removal.

Phase diagrams of DNA and poly(styrene-sulfonate) condensed by a poly-cationic protein, the salmon protamine

Protamines are important biomacromolecules in many respects: they compact DNA most efficiently in the sperm head; they are commonly used in the formulation of non-toxic and efficient gene carriers and they have a very interesting structural charge in-between the small multivalent ions and the large liposomes, colloids or long polycations. In this experimental study, we examine in detail how these small basic proteins induce DNA condensation. A study of the phase separation of a synthetic polyelectrolyte, Na-poly(styrene-sulfonate) (PSS), in aqueous solution with protamines also explores the salt-free range domain and completes this work in order to get a general and broad view of the most representative phase diagram. From the two sets of data, phase diagrams and unique representations are proposed. The solubility of DNA or PSS seems to depend on the ionic conditions through two parameters: C+/C− and L/rs. L/rs denotes the length ratio between the polyanion size and the Debye screening length and C+/C− denotes the charge concentration ratio of protamineversusDNA or PSS. Protamines bind to polyanions and condense them concomitantly. The presence of soluble condensed and uncondensed polyanions delimits the different domains of the diagram defined by the axes C+/C− and L/rs. Another representation emerges only when the molar amount of salt prevents the protein binding up to a certain threshold of protein concentration.

A route to self-assemble suspended DNA nano-complexes.

Highly charged polyelectrolytes can self-assemble in presence of condensing agents such as multivalent cations, amphiphilic molecules or proteins of opposite charge. Aside precipitation, the formation of soluble micro- and nano-particles has been reported in multiple systems. However a precise control of experimental conditions needed to achieve the desired structures has been so far hampered by the extreme sensitivity of the samples to formulation pathways. Herein we combine experiments and molecular modelling to investigate the detailed microscopic dynamics and the structure of self-assembled hexagonal bundles made of short dsDNA fragments complexed with small basic proteins. We suggest that inhomogeneous mixing conditions are required to form and stabilize charged self-assembled nano-aggregates in large excess of DNA. Our results should help re-interpreting puzzling behaviors reported for a large class of strongly charged polyelectrolyte systems.