The effect of genome length on ejection forces in bacteriophage lambda

Department of Physics, California Institute of Technology, Pasadena, 91125, USA.
Virology (Impact Factor: 3.28). 06/2006; 348(2):430-6. DOI: 10.1016/j.virol.2006.01.003
Source: PubMed

ABSTRACT 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, lambdab221cI26 and lambdacI60, which differ in genome length by approximately 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, for the short and long genomes, respectively, values that are in agreement with our theoretical calculations.

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    • "The force driving ejection of the protein from P22 arises from the confined DNA and is expected to be similar to that associated with the ejection of DNA from λ phage , which like P22 has a T¼ 7 capsid and is about the same size . Grayson et al . ( 2006 ) carried out osmotic suppression measurements on λ for the 48 . 5 kb wild - type genome and a 37 . 7 kb mutant and found that the ejection was completely inhibited at pressures of 20 – 25 and 10 – 15 atm , respectively . The pressure required to inhibit the E proteins that we have observed for P22 originates from 43 . 5 kb - length of "
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    ABSTRACT: Double-stranded DNA bacteriophages are highly pressurized, providing a force driving ejection of a significant fraction of the genome from its capsid. In P22-like Podoviridae, internal proteins ("E proteins") are packaged into the capsid along with the genome, and without them the virus is not infectious. However, little is known about how and when these proteins come out of the virus. We employed an in vitro osmotic suppression system with high-molecular-weight polyethylene glycol to study P22 E protein release. While slow ejection of the DNA can be triggered by lipopolysaccharide (LPS), the rate is significantly enhanced by the membrane protein OmpA from Salmonella. In contrast, E proteins are not ejected unless both OmpA and LPS are present and their ejection when OmpA is present is largely complete before any genome is ejected, suggesting that E proteins play a key role in the early stage of transferring P22 DNA into the host. Copyright © 2015 Elsevier Inc. All rights reserved.
    Virology 07/2015; 485:128-134. DOI:10.1016/j.virol.2015.07.006 · 3.28 Impact Factor
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    • "In the in vitro λ experiments [36] [37] [38] [39], phage virions are immersed in a solution containing PEG and/or DNA condensing agents, and DNA is ejected when triggered by the LamB receptor protein. "
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    ABSTRACT: Bacteriophages, phages for short, are viruses of bacteria. The majority of phages contain a double-stranded DNA genome packaged in a capsid at a density of ∼500 mg ml(-1). This high density requires substantial compression of the normal B-form helix, leading to the conjecture that DNA in mature phage virions is under significant pressure, and that pressure is used to eject the DNA during infection. A large number of theoretical, computer simulation and in vitro experimental studies surrounding this conjecture have revealed many--though often isolated and/or contradictory--aspects of packaged DNA. This prompts us to present a unified view of the statistical physics and thermodynamics of DNA packaged in phage capsids. We argue that the DNA in a mature phage is in a (meta)stable state, wherein electrostatic self-repulsion is balanced by curvature stress due to confinement in the capsid. We show that in addition to the osmotic pressure associated with the packaged DNA and its counterions, there are four different pressures within the capsid: pressure on the DNA, hydrostatic pressure, the pressure experienced by the capsid and the pressure associated with the chemical potential of DNA ejection. Significantly, we analyze the mechanism of force transmission in the packaged DNA and demonstrate that the pressure on DNA is not important for ejection. We derive equations showing a strong hydrostatic pressure difference across the capsid shell. We propose that when a phage is triggered to eject by interaction with its receptor in vitro, the (thermodynamic) incentive of water molecules to enter the phage capsid flushes the DNA out of the capsid. In vivo, the difference between the osmotic pressures in the bacterial cell cytoplasm and the culture medium similarly results in a water flow that drags the DNA out of the capsid and into the bacterial cell.
    Physical Biology 12/2010; 7(4):045006. DOI:10.1088/1478-3975/7/4/045006 · 3.14 Impact Factor
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    • "The first group of studies uses a continuum representation of DNA and the apparatus of statistical mechanics. These methods have been able to reflect the basic physics of DNA confinement and to estimate thermodynamic properties such as forces and free energy (Grayson et al., 2006; Kindt et al., 2001; Purohit et al., 2003; Purohit et al., 2005; Tzlil et al., 2003). The continuum approach has also been successful in explaining the role of the osmotic pressure during genome ejection (Evilevitch et al., 2008). "
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    ABSTRACT: Electrostatic interactions play an important role in both packaging of DNA inside bacteriophages and its release into bacterial cells. While at physiological conditions DNA strands repel each other, the presence of polyvalent cations such as spermine and spermidine in solutions leads to the formation of DNA condensates. In this study, we discuss packaging of DNA into bacteriophages P4 and Lambda under repulsive and attractive conditions using a coarse-grained model of DNA and capsids. Packaging under repulsive conditions leads to the appearance of the coaxial spooling conformations; DNA occupies all available space inside the capsid. Under the attractive potential both packed systems reveal toroidal conformations, leaving the central part of the capsids empty. We also present a detailed thermodynamic analysis of packaging and show that the forces required to pack the genomes in the presence of polyamines are significantly lower than those observed under repulsive conditions. The analysis reveals that in both the repulsive and attractive regimes the entropic penalty of DNA confinement has a significant non-negligible contribution into the total energy of packaging. Additionally we report the results of simulations of DNA condensation inside partially packed Lambda. We found that at low densities DNA behaves as free unconfined polymer and condenses into the toroidal structures; at higher densities rearrangement of the genome into toroids becomes hindered, and condensation results in the formation of non-equilibrium structures. In all cases packaging in a specific conformation occurs as a result of interplay between bending stresses experienced by the confined polymer and interactions between the strands.
    Journal of Structural Biology 11/2010; 174(1):137-46. DOI:10.1016/j.jsb.2010.11.007 · 3.23 Impact Factor
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