Osmotic pressure: resisting or promoting DNA ejection from phage

Recent in vitro experiments have shown that DNA ejection from bacteriophage can be partially stopped by surrounding osmotic pressure when ejected DNA is digested by DNase I on the course of ejection. We argue in this work by combination of experimental techniques (osmotic suppression without DNaseI monitored by UV absorbance, pulse-field electrophoresis, and cryo-EM visualization) and simple scaling modeling that intact genome (i.e. undigested) ejection in a crowded environment is, on the contrary, enhanced or eventually complete with the help of a pulling force resulting from DNA condensation induced by the osmotic stress itself. This demonstrates that in vivo, the osmotically stressed cell cytoplasm will promote phage DNA ejection rather than resisting it. The further addition of DNA-binding proteins under crowding conditions is shown to enhance the extent of ejection. We also found some optimal crowding conditions for which DNA content remaining in the capsid upon ejection is maximum, which correlates well with the optimal conditions of maximum DNA packaging efficiency into viral capsids observed almost 20 years ago. Biological consequences of this finding are discussed

DNA heats up : Energetics of genome ejection from phage revealed by isothermal titration calorimetry

Most bacteriophages are known to inject their double-stranded DNA into bacteria upon receptor binding in an essentially spontaneous way. This downhill thermodynamic process from the intact virion toward the empty viral capsid plus released DNA is made possible by the energy stored during active packaging of the genome into the capsid. Only indirect measurements of this energy have been available until now using either single-molecule or osmotic suppression techniques. In this paper, we describe for the first time the use of isothermal titration calorimetry to directly measure the heat released (or equivalently the enthalpy) during DNA ejection from phage lambda, triggered in solution by a solubilized receptor. Quantitative analyses of the results lead to the identification of thermodynamic determinants associated with DNA ejection. The values obtained were found to be consistent with those previously predicted by analytical models and numerical simulations. Moreover, the results confirm the role of DNA hydration in the energetics of genome confinement in viral capsids.Comment: 24 pages including figures and tabl

The effect of genome length on ejection forces in bacteriophage lambda

A variety of viruses tightly pack their genetic material into protein capsids that are barely large enough to enclose the genome. In particular, in bacteriophages, forces as high as 60 pN are encountered during packaging and ejection, produced by DNA bending elasticity and self-interactions. The high forces are believed to be important for the ejection process, though the extent of their involvement is not yet clear. As a result, there is a need for quantitative models and experiments that reveal the nature of the forces relevant to DNA ejection. Here we report measurements of the ejection forces for two different mutants of bacteriophage lambda, lambda b221cI26 and lambda cI60, which differ in genome length by ~30%. As expected for a force-driven ejection mechanism, the osmotic pressure at which DNA release is completely inhibited varies with the genome length: we find inhibition pressures of 15 atm and 25 atm, respectively, values that are in agreement with our theoretical calculations

Challenging packaging limits and infectivity of phage λ

The terminase motors of bacteriophages have been shown to be among the strongest active machines in the biomolecular world, being able to package several tens of kilobase pairs of viral genome into a capsid within minutes. Yet these motors are hindered at the end of the packaging process by the progressive build-up of a force resisting packaging associated with already packaged DNA. In this experimental work, we raise the issue of what sets the upper limit on the length of the genome that can be packaged by the terminase motor of phage λ and still yield infectious virions, and the conditions under which this can be efficiently performed. Using a packaging strategy developed in our laboratory of building phage λ from scratch, together with plaque assay monitoring, we have been able to show that the terminase motor of phage λ is able to produce infectious particles with up to 110% of the wild-type (WT) λ-DNA length. However, the phage production rate, and thus the infectivity, decreased exponentially with increasing DNA length, and was a factor of 103 lower for the 110% λ-DNA phage. Interestingly, our in vitro strategy was still efficient in fully packaging phages with DNA lengths as high as 114% of the WT length, but these viruses were unable to infect bacterial cells efficiently. Further, we demonstrated that the phage production rate is modulated by the presence of multivalent ionic species. The biological consequences of these finding are discussed

Molecular Exchange in Colloidal Dispersions

This thesis is a study of molecular exchange between the aggregates in a colloidal dispersion. The problem of oil molecular transport at resolubilization of big oil drops by smaller microemulsion droplets is considered as an experimental model system. The resolubilization kinetics was measured through a temperature jump into a droplet microemulsion phase from a two-phase region of microemulsion droplets and separating oil. The relaxation process was monitored by following the turbidity of the system. A quantitative model for the solubilization kinetics was formulated on the basis of experimental observations. The effects of concentrations and sizes of droplets are treated within a framework of a cell model. New computer simulation approach for molecular exchange studies is also introduced. The simulation model describes discreet spherical aggregates moving in a Brownian motion at the same time as small molecules, such as oil or surfactant, are allowed to exchange between the aggregates. The model allows a detailed study of the local exchange between neighbor micelles as well as the collective long-range exchange. The theoretical analysis from both models showed that as a result of high droplet concentration, the molecular exchange between the droplets occurs via diffusive monomer transport only in the vicinity of the droplets’ surface, and not across the entire system as predicted by the infinite dilution limit approximation. A quantitative agreement between the simulation, the cell model, and experiment was obtained in description of the resolubilization process. In addition, structure and transport properties of weakly charged oil-in-water microemulsion droplets, were also studied. Static and dynamic properties such as osmotic pressure, osmotic compressibility, self-diffusion, collective diffusion, and zero shear viscosity were analyzed by experimental and theoretical techniques

Effects of condensing agent and nuclease on the extent of ejection from phage lambda

We have recently demonstrated, that DNA ejection from bacteriophage I can be partially or completely suppressed in vitro by external osmotic pressure. This suggests that DNA ejection from phage is driven by an internal mechanical force consisting of DNA bending and DNA-DNA electrostatic repulsion energies. In the present work we investigate the extent to which DNA ejection is incomplete at zero osmotic external pressure when phage is opened with its receptor in vitro. The DNA fragment remaining in the capsid and the tail that is no longer bent or compressed sand hence for which there is no internal driving force for ejections is shown not to be ejected. We also demonstrate that DNA can be "pulled" out from the capsid by DNase I acting as a DNA binding protein or spermine acting as a DNA condensing agent. In particular, cryo electron microscopy and gel electrophoresis experiments show the following: (i) DNA ejection from bacteriophage I incubated in vitro with its receptor is incomplete at zero external osmotic force, with several persistence lengths of DNA remaining inside the phage capsid, if no nuclease ( DNase I) or DNA condensing agent ( spermine) is present in the host solution; (ii) in the presence of both DNase I and spermine in the host solution, 60% (approximate to 29 kbp) of wild-type lambda DNA (48.5 kbp) remains unejected inside the phage capsid, in the form of an unconstrained toroidal condensate; (iii) with DNase I added, but no spermine, the ejection is complete; (iv) with spermine, but without DNase I added, all the DNA is again ejected, and organized as a toroidal condensate outside

Physics of viral infectivity : Matching genome length with capsid size

In this work, we review recent advances in the field of physical virology, presenting both experimental and theoretical studies on the physical properties of viruses. We focus on the double-stranded DNA (dsDNA) bacteriophages as model systems for all of the dsDNA viruses both prokaryotic and eukaryotic. Recent studies demonstrate that the DNA packaged into many dsDNA viral capsids is highly pressurized, which provides a force for the first step of passive injection of viral DNA into either bacterial or eukaryotic cells. Moreover, specific studies on capsid strength show a strong correlation between genome length and capsid size and robustness. The implications of these newly appreciated physical properties of a viral particle with respect to the infection process are discussed

DNA ejection from bacteriophage: Towards a general behavior for osmotic-suppression experiments

We present in this work in vitro measurements of the force ejecting DNA from two distinct bacteriophages (T5 and lambda using the osmotic-suppression technique. Our data are analyzed by revisiting the current theories of DNA packaging in spherical capsids. In particular we show that a simplified analytical model based on bending considerations only is able to account quantitatively for the experimental findings. Physical and biological consequences are discussed

Influence of Internal DNA Pressure on Stability and Infectivity of Phage λ.

Viruses must remain infectious while in harsh extracellular environments. An important aspect of viral particle stability for double-stranded DNA viruses is the energetically unfavorable state of the tightly confined DNA chain within the virus capsid creating pressures of tens of atmospheres. Here, we study the influence of internal genome pressure on the thermal stability of viral particles. Using differential scanning calorimetry to monitor genome loss upon heating, we find that internal pressure destabilizes the virion, resulting in a smaller activation energy barrier to trigger DNA release. These experiments are complemented by plaque assay and electron microscopy measurements to determine the influence of intra-capsid DNA pressure on the rates of viral infectivity loss. At higher temperatures (65-75°C), failure to retain the packaged genome is the dominant mechanism of viral inactivation. Conversely, at lower temperatures (40-55°C), a separate inactivation mechanism dominates, which results in non-infectious particles that still retain their packaged DNA. Most significantly, both mechanisms of infectivity loss are directly influenced by internal DNA pressure, with higher pressure resulting in a more rapid rate of inactivation at all temperatures