256 research outputs found
Binding effects in multivalent Gibbs-Donnan equilibrium
The classical Gibbs-Donnan equilibrium describes excess osmotic pressure
associated with confined colloidal charges embedded in an electrolyte solution.
In this work, we extend this approach to describe the influence of multivalent
ion binding on the equilibrium force acting on a charged rod translocating
between two compartments, thereby mimicking ionic effects on force balance
during in vitro DNA ejection from bacteriophage. The subtle interplay between
Gibbs-Donnan equilibrium and adsorption equilibrium leads to a non-monotonic
variation of the ejection force as multivalent salt concentration is increased,
in qualitative agreement with experimental observations
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
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
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
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
Microvascular response in guinea pig skin to histamine challenge with and without application of skin window.
We measured the microvascular response to histamine in guinea pig skin. Histamine (40 mg ml-1) was given either as a skin prick test or applied topically onto a skin window. The skin window was prepared by applying suction and gentle warming to the skin so that a blister was formed, and by removing the top of the blister. The microvascular response was measured as the accumulation of radiolabelled transferrin in the skin in vivo, reflecting a combination plasma exudation and vasodilatation. In the control (saline) challenge, the response was slightly greater in the skin window than after skin prick challenge and the scatter was larger. Histamine challenge resulted in a significant microvascular response with respect to the control situation when measured immediately after provocation for both challenge techniques. Ten minutes after challenge, a smaller response was measured, which was still significantly greater than control for the skin prick challenge, but not for topical provocation using the skin window technique. We conclude that the microvascular response to histamine after provocation with the skin prick technique is similar to that after topical provocation using the skin window technique. The skin window technique may have a lower sensitivity than the skin prick technique owing to a higher scatter in the control situation. This difference should be considered when performing and interpreting studies of the microvascular reaction in the skin
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
Forces During Bacteriophage DNA Packaging and Ejection
The conjunction of insights from structural biology, solution biochemistry,
genetics and single molecule biophysics has provided a renewed impetus for the
construction of quantitative models of biological processes. One area that has
been a beneficiary of these experimental techniques is the study of viruses. In
this paper we describe how the insights obtained from such experiments can be
utilized to construct physical models of processes in the viral life cycle. We
focus on dsDNA bacteriophages and show that the bending elasticity of DNA and
its electrostatics in solution can be combined to determine the forces
experienced during packaging and ejection of the viral genome. Furthermore, we
quantitatively analyze the effect of fluid viscosity and capsid expansion on
the forces experienced during packaging. Finally, we present a model for DNA
ejection from bacteriophages based on the hypothesis that the energy stored in
the tightly packed genome within the capsid leads to its forceful ejection. The
predictions of our model can be tested through experiments in vitro where DNA
ejection is inhibited by the application of external osmotic pressure
Dynamics of DNA Ejection From Bacteriophage
The ejection of DNA from a bacterial virus (``phage'') into its host cell is
a biologically important example of the translocation of a macromolecular chain
along its length through a membrane. The simplest mechanism for this motion is
diffusion, but in the case of phage ejection a significant driving force
derives from the high degree of stress to which the DNA is subjected in the
viral capsid. The translocation is further sped up by the ratcheting and
entropic forces associated with proteins that bind to the viral DNA in the host
cell cytoplasm. We formulate a generalized diffusion equation that includes
these various pushing and pulling effects and make estimates of the
corresponding speed-ups in the overall translocation process. Stress in the
capsid is the dominant factor throughout early ejection, with the pull due to
binding particles taking over at later stages. Confinement effects are also
investigated, in the case where the phage injects its DNA into a volume
comparable to the capsid size. Our results suggest a series of in vitro
experiments involving the ejection of DNA into vesicles filled with varying
amounts of binding proteins from phage whose state of stress is controlled by
ambient salt conditions or by tuning genome length.Comment: 17 pages, 5 figure
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