Osmotic pressure inhibition of DNA ejection from phage

Abstract

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 , 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.V iral capsids are often highly pressurized. In the case of double-stranded DNA bacteriophages, for example, there are large forces acting to push the genome out through the tail of the viral particle. Recent experiments (1) and theory (2, 3) have established that the forces inside these capsids reach very high values (of order 50 pN) when the genomes are fully packaged. These forces are estimated to correspond to pressures of the order of 50 atm (1 atm ϭ 101.3 kPa; refs. 1-4). Such high values of stress result from the DNA being strongly bent and confined. Specifically, the inner diameter of the capsid is comparable to the DNA persistence length (50 nm), and the typical interaxial spacings are small enough (2.5 nm; ref. 5) that neighboring chains experience strong repulsions. It is precisely these forces that drive the ejection of the genome into the host cell after the tail of the capsid has been bound to its receptor protein in the outer cell membrane; equivalently, it is these forces that must be exerted by the motor protein responsible for packaging of the viral genome (1).Previous in vitro experiments have demonstrated that DNA ejection is fast (occurring on a time scale of seconds) and complete when phage capsids are opened by their receptors in aqueous solution (6). The capsids are permeable to water and salt ions, so there is no difference in hydrostatic pressure between the inside and outside of the capsid, and osmotic equilibrium is also maintained (7). The pressure difference between the capsid and the solution is therefore associated only with the confinement of the DNA. When the capsid is opened, ejection proceeds until this pressure difference falls to zero. Clearly, at this point the force driving the ejection has also dropped to zero. For the in vivo situation, however, ejection does not occur into a simple salt solution. The DNA is ejected into a highly concentrated colloidal suspension, the cell cytoplasm, which is characterized by high concentrations of proteins and other macromolecular structures. In general, then, one expects this colloidal solution to give rise to a force that resists the entry of DNA (8). More explicitly, because the ejection force associated with stress inside the capsid drops monotonically as the ejection proceeds, there will be a...

Measurements of Single DNA Molecule Packaging Dynamics in Bacteriophage λ Reveal High Forces, High Motor Processivity, and Capsid Transformations

“…This force is notably two to three-fold lower than that measured for ϕ29 in a similar ionic condition 6 . However, it is in good agreement with the λ phage DNA ejection force measured by osmotic pressure experiments 35,36 and predicted by theoretical calculations 23,36 . Specifically, an osmotic pressure of ~15 atm was found to be necessary to suppress DNA ejection from a λ mutant having a 41.5 kbp truncated genome 36 , corresponding to an ejection force of ~15 pN 20 .…”

Role of osmotic and hydrostatic pressures in bacteriophage genome ejection

“…(7) indicates that both osmotic and hydrostatic pressure gradients between the environment and the capsid become zero. These situations correspond to the osmotic suppression experiments [6][7][8][9]12,13]; complete suppression of ejection yields an estimate of the osmotic pressures in a mature phage particle: ∼25 atm for λ, ∼47 atm for SPP1, and ∼16 atm for the deletion mutant T5st(0).…”

“…Keeping in mind that the model applies only to equilibrium states and not to the dynamics or molecular mechanism of the process, it successfully explains in vitro data for phage λ DNA ejection. Such experiments include the addition of capsid-permeable DNA-condensing agents or capsid-impermeable osmolytes to arrest ejection when the DNA remaining in the capsid reaches equilibrium with the buffer [6][7][8][9] and time-resolved light scattering [10].…”

Modeling and simulation of the mechanical response from nanoindentation test of DNA-filled viral capsids

“…The solid material of the virus is described by four parameters, two for the capsid and two for the core. The osmotic pressure inside the capsid, caused by the strong bending of the DNA and the repulsive forces between the neighboring negatively charged chains is of order 2.0 × 10 −3 − 4.0 × 10 −3 GPa, see [12]. The osmotic pressure is added in to the model as an extra pressure at each node in the FE-model, both on the capsid and on the core.…”

“…In case of phage λ, it has been shown that the capsid strength approximately matches the force exerted by the DNA on the capsid walls [2,12]. This suggests that the capsid strength determines the maximum length of DNA that can be packaged, which in turn is also correlated with the strength of the packaging motor complex [2,13].…”