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

Ion-dependent dynamics of DNA ejections for bacteriophage lambda

We study the control parameters that govern the dynamics of in vitro DNA ejection in bacteriophage lambda. Past work has demonstrated that bacteriophage DNA is highly pressurized; this pressure has been hypothesized to help drive DNA ejection. Ions influence this process by screening charges on DNA; however, a systematic variation of salt concentrations to explore these effects has not been undertaken. To study the nature of the forces driving DNA ejection, we performed in vitro measurements of DNA ejection in bulk and at the single-phage level. We present measurements on the dynamics of ejection and on the self-repulsion force driving ejection. We examine the role of ion concentration and identity in both measurements, and show that the charge of counter-ions is an important control parameter. These measurements show that the frictional force acting on the ejecting DNA is subtly dependent on ionic concentrations for a given amount of DNA in the capsid. We also present evidence that phage DNA forms loops during ejection; we confirm that this effect occurs using optical tweezers. We speculate this facilitates circularization of the genome in the cytoplasm.Comment: David Wu and David Van Valen contributed equally to this project. 28 pages (including supplemental information), 4 figure

Physics of Viral Infectivity: Energetics of Genome Ejection

All viruses that infect bacteria, plant, or animal cells involve a genome (RNA or DNA) that is encapsidated by a rigid protein shell. After delivery of the viral genome into the host cell, new capsid proteins, which are encoded by viral DNA or RNA, are expressed and self-assembled into new viral capsids. The main objective of my research was to study the key physical factors of genome ejection that control the viral life cycle. One of the main steps in the viral life cycle is genome ejection, which is considered a physical process. It has recently been shown that the ejection of the viral genome from the phage capsid is driven by internal pressure reaching tens of atmospheres. This pressure is partially responsible for the delivery of the viral genome into the host cell, thus making it central in the infection process. In this doctoral thesis, direct measurements of the energy associated with genome ejection are presented. The viral capsid is “opened” by the LamB receptor protein in vitro, and ejection is measured using microcalorimetry. The energy of genome ejection from the viral capsid is obtained as a function of the relative packaging density of the viral genome. A DNA phase transition was observed by measuring the ejection enthalpy as a function of temperature. Recent in vitro experiments have shown that DNA ejection from the phage can be restricted by the surrounding osmotic pressure when ejected DNA is digested by DNase I during the course of ejection. The most important finding of this work was that the ejection of an intact genome (i.e. undigested) in a crowded environment is enhanced or even completed by the 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 resist it, while in vitro ejection is extremely dependent on the pressure within the virus capsid. The effect of internal pressure on the infection of a bacterial cell is unknown. A microfluidic technique was employed to monitor individual cells and determine the distribution of lysis due to infection in relation to the capsid pressure. The probability of lysis was found to decrease markedly with decreasing capsid pressure

Influence of Internal Capsid Pressure on Viral Infection by Phage λ

Ejection of the genome from the virus, phage λ, is the initial step in the infection of its host bacterium. In vitro, the ejection depends sensitively on internal pressure within the virus capsid; however, the in vivo effect of internal pressure on infection of bacteria is unknown. Here, we use microfluidics to monitor individual cells and determine the temporal distribution of lysis due to infection as the capsid pressure is varied. The lysis probability decreases markedly with decreased capsid pressure. Of interest, the average lysis times remain the same but the distribution is broadened as the pressure is lowered

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 to 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 work, 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

Temperature Induced Phase Transition of Encapsidated Phage DNA

Double-stranded DNA bacteriophages are highly stressed under the high internal pressure that is described as stored energy. Most of them eject their genome into the host bacteria via receptor binding. In the previous work, isothermal titration calorimtery (ITC) was used, for first time, to directly measure the heat released during DNA ejection from the phage lambda, triggered in solution by solubilized LamB receptor. In the current study, using ITC, we investigate the ejection energy from the capsid of phage lambda packaged with 94-110% genome, at a wider temperature range between 18 and 40°C. We experimentally show that DNA undergoes phase transition when exposed to different temperatures. The event of phase transition was also confirmed by differential scanning calorimetry (DSC)