DNA Ejection Research Articles

Features of membrane receptors in bacterial multiplication process and necessary conditions for phage infection of bacteria.

According to the obtained experimental results, the thermal shock (from 37 to 53 °C) not only stops the multiplication process of Escherichia coli bacteria, but also causes bacterial titer to decrease gradually. After this period lasting up to 1 hour, the bacterial cells continue to grow. A similar type of response was observed when bacteria were subjected to acid shock. Increasing acidity of media leads to decrease of bacterial growth process, and finally, their titer curve sharply falls over time. Also, interesting results were obtained about necessary conditions for infecting the bacteria by phages. Particularly, DNA injection from phages into bacterial cells requires most of corresponding bacterial membrane receptors to be occupied by phages. We suppose that this occurs due to autocrine phenomenon when the signaling molecules block the DNA ejection from phage particles. This effect lasts until a certain number of phage particles are attached to the membrane. After that, DNA injection from phage head into the cytoplasm takes place and the process of bacterial infection begins. The real number of phages in a stock is by several orders higher than the number of plaque-forming units in a given stock, which is determined by a classical double-layer agar method.

Finding of widespread viral and bacterial revolution dsDNA translocation motors distinct from rotation motors by channel chirality and size.

BackgroundDouble-stranded DNA translocation is ubiquitous in living systems. Cell mitosis, bacterial binary fission, DNA replication or repair, homologous recombination, Holliday junction resolution, viral genome packaging and cell entry all involve biomotor-driven dsDNA translocation. Previously, biomotors have been primarily classified into linear and rotational motors. We recently discovered a third class of dsDNA translocation motors in Phi29 utilizing revolution mechanism without rotation. Analogically, the Earth rotates around its own axis every 24 hours, but revolves around the Sun every 365 days.ResultsSingle-channel DNA translocation conductance assay combined with structure inspections of motor channels on bacteriophages P22, SPP1, HK97, T7, T4, Phi29, and other dsDNA translocation motors such as bacterial FtsK and eukaryotic mimiviruses or vaccinia viruses showed that revolution motor is widespread. The force generation mechanism for revolution motors is elucidated. Revolution motors can be differentiated from rotation motors by their channel size and chirality. Crystal structure inspection revealed that revolution motors commonly exhibit channel diameters larger than 3 nm, while rotation motors that rotate around one of the two separated DNA strands feature a diameter smaller than 2 nm. Phi29 revolution motor translocated double- and tetra-stranded DNA that occupied 32% and 64% of the narrowest channel cross-section, respectively, evidencing that revolution motors exhibit channel diameters significantly wider than the dsDNA. Left-handed oriented channels found in revolution motors drive the right-handed dsDNA via anti-chiral interaction, while right-handed channels observed in rotation motors drive the right-handed dsDNA via parallel threads. Tethering both the motor and the dsDNA distal-end of the revolution motor does not block DNA packaging, indicating that no rotation is required for motors of dsDNA phages, while a small-angle left-handed twist of dsDNA that is aligned with the channel could occur due to the conformational change of the phage motor channels from a left-handed configuration for DNA entry to a right-handed configuration for DNA ejection for host cell infection.ConclusionsThe revolution motor is widespread among biological systems, and can be distinguished from rotation motors by channel size and chirality. The revolution mechanism renders dsDNA void of coiling and torque during translocation of the lengthy helical chromosome, thus resulting in more efficient motor energy conversion.

Bacteriophage P22 tailspike: structure of the complete protein and function of the interdomain linker.

Attachment of phages to host cells, followed by phage DNA ejection, represents the first stage of viral infection of bacteria. Salmonella phage P22 has been extensively studied, serving as an experimental model for bacterial infection by phages. P22 engages bacteria by binding to the sugar moiety of lipopolysaccharides using the viral tailspike protein for attachment. While the structures of the N-terminal particle-binding domain and the major receptor-binding domain of the tailspike have been analyzed individually, the three-dimensional organization of the intact protein, including the highly conserved linker region between the two domains, remained unknown. A single amino-acid exchange in the linker sequence made it possible to crystallize the full-length protein. Two crystal structures of the linker region are presented: one attached to the N-terminal domain and the other present within the complete tailspike protein. Both retain their biological function, but the mutated full-length tailspike displays a retarded folding pathway. Fitting of the full-length tailspike into a published cryo-electron microscopy map of the P22 virion requires an elastic distortion of the crystal structure. The conservation of the linker suggests a role in signal transmission from the distal tip of the molecule to the phage head, eventually leading to DNA ejection.

Correction: A Novel Method to Couple Electrophysiological Measurements and Fluorescence Imaging of Suspended Lipid Membranes: The Example of T5 Bacteriophage DNA Ejection

Correction: A Novel Method to Couple Electrophysiological Measurements and Fluorescence Imaging of Suspended Lipid Membranes: The Example of T5 Bacteriophage DNA Ejection

Molecular Competition between Large and Small Polyethylene-Glycols (PEGs) Partitioning into OMPC Porin Channels

Molecular crowding by polyethylene glycols (PEGs) has been employed in a wide range of inquiries into molecular structure and dynamics. Investigating the partitioning of crowded polymers into biological nanopores reveals properties also seen in, e.g., DNA ejection and compaction into viral capsids, as well as in facilitated molecule transport. Capitalizing on our previous work on the partitioning of binary PEG mixtures into the benchmark Alpha-Hemolysin nanopore, we have now studied partitioning of mono- and polydisperse polymer solutions into comparably sized, yet differently structured, diffusion channels. We find that in the presence of large non-partitioning PEG 3400, the small, otherwise equipartitioning PEG 200 partitions disproportionately. Specifically, with 15% PEG 3400, an added 15% PEG 200 reduces channel conductance by 65% (compared with channel conductance in polymer-free solutions), while 15% PEG 200 alone reduces channel conductance by only 45%. We employed open-channel noise analysis to investigate channel diffusion properties of individual polymers, and to compare them with their properties in bulk water solution. In this way we demonstrate and quantify crowding-assisted transport through nanopores in mixed-polymer systems.

A novel method to couple electrophysiological measurements and fluorescence imaging of suspended lipid membranes: the example of T5 bacteriophage DNA ejection.

We present an innovative method to couple electrophysiological measurements with fluorescence imaging of functionalized suspended bilayers. Our method combines several advantages: it is well suited to study transmembrane proteins that are difficult to incorporate in suspended bilayers, it allows single molecule resolution both in terms of electrophysiological measurements and fluorescence imaging, and it enables mechanical stimulations of the membrane. The approach comprises of two steps: first the reconstitution of membrane proteins in giant unilamellar vesicles; then the formation of a suspended bilayer spanning a 5 to 15 micron-wide aperture that can be visualized by high NA microscope objectives. We exemplified how the technique can be used to detect in real time the translocation of T5 DNA across the bilayer during its ejection from the bacteriophage capsid.

Topological friction strongly affects viral DNA ejection

Bacteriophages initiate infection by releasing their double-stranded DNA into the cytosol of their bacterial host. However, what controls and sets the timescales of DNA ejection? Here we provide evidence from stochastic simulations which shows that the topology and organization of DNA packed inside the capsid plays a key role in determining these properties. Even with similar osmotic pressure pushing out the DNA, we find that spatially ordered DNA spools have a much lower effective friction than disordered entangled states. Such spools are only found when the tendency of nearby DNA strands to align locally is accounted for. This topological or conformational friction also depends on DNA knot type in the packing geometry and slows down or arrests the ejection of twist knots and very complex knots. We also find that the family of (2, 2k+1) torus knots unravel gradually by simplifying their topology in a stepwise fashion. Finally, an analysis of DNA trajectories inside the capsid shows that the knots formed throughout the ejection process mirror those found in gel electrophoresis experiments for viral DNA molecules extracted from the capsids.

Assessing the Conformational Changes of pb5, the Receptor-binding Protein of Phage T5, upon Binding to Its Escherichia coli Receptor FhuA

Within tailed bacteriophages, interaction of the receptor-binding protein (RBP) with the target cell triggers viral DNA ejection into the host cytoplasm. In the case of phage T5, the RBP pb5 and the receptor FhuA, an outer membrane protein of Escherichia coli, have been identified. Here, we use small angle neutron scattering and electron microscopy to investigate the FhuA-pb5 complex. Specific deuteration of one of the partners allows the complete masking in small angle neutron scattering of the surfactant and unlabeled proteins when the complex is solubilized in the fluorinated surfactant F6-DigluM. Thus, individual structures within a membrane protein complex can be described. The solution structure of FhuA agrees with its crystal structure; that of pb5 shows an elongated shape. Neither displays significant conformational changes upon interaction. The mechanism of signal transduction within phage T5 thus appears different from that of phages binding cell wall saccharides, for which structural information is available.

Structural characterization of T7 tail machinery reveals a conserved tubular structure among otherPodoviridaefamily members and suggests a common mechanism for DNA delivery

Bacteriophage tail complexes play an essential role in host recognition and DNA delivery during virus infection. These molecular machines are composed of a tubular structure surrounded by fibers, with a central channel that acts as a conduit for DNA ejection. The T7 tail complex is formed by four proteins: connector (gp8), gatekeeper (gp11), nozzle (gp12), and fibers (gp17). Previous biochemical and structural studies allowed definition of the stoichiometry and order of assembly of these proteins. Here we compared the tail complex from other Podoviridae phages that infect bacteria with Gram− type envelopes (K1E, P-SSP7, and e15), and found strong similarities with the T7 nozzle; this was supported by sequence alignment and secondary structure prediction studies. These similarities were not observed in the new reconstruction of protein p9 presented here, which builds the hexameric nozzle of ϕ29, a virus that infects Gram+ bacteria. The results suggest that the Podoviridae nozzle has evolved to adapt to mem...

Structure, Adsorption to Host, and Infection Mechanism of Virulent Lactococcal Phage p2

Lactococcal siphophages from the 936 and P335 groups infect the Gram-positive bacterium Lactococcus lactis using receptor binding proteins (RBPs) attached to their baseplate, a large multiprotein complex at the distal part of the tail. We have previously reported the crystal and electron microscopy (EM) structures of the baseplates of phages p2 (936 group) and TP901-1 (P335 group) as well as the full EM structure of the TP901-1 virion. Here, we report the complete EM structure of siphophage p2, including its capsid, connector complex, tail, and baseplate. Furthermore, we show that the p2 tail is characterized by the presence of protruding decorations, which are related to adhesins and are likely contributed by the major tail protein C-terminal domains. This feature is reminiscent of the tail of Escherichia coli phage λ and Bacillus subtilis phage SPP1 and might point to a common mechanism for establishing initial interactions with their bacterial hosts. Comparative analyses showed that the architecture of the phage p2 baseplate differs largely from that of lactococcal phage TP901-1. We quantified the interaction of its RBP with the saccharidic receptor and determined that specificity is due to lower k(off) values of the RBP/saccharidic dissociation. Taken together, these results suggest that the infection of L. lactis strains by phage p2 is a multistep process that involves reversible attachment, followed by baseplate activation, specific attachment of the RBPs to the saccharidic receptor, and DNA ejection.

The Tip of the Tail Needle Affects the Rate of DNA Delivery by Bacteriophage P22

The P22-like bacteriophages have short tails. Their virions bind to their polysaccharide receptors through six trimeric tailspike proteins that surround the tail tip. These short tails also have a trimeric needle protein that extends beyond the tailspikes from the center of the tail tip, in a position that suggests that it should make first contact with the host’s outer membrane during the infection process. The base of the needle serves as a plug that keeps the DNA in the virion, but role of the needle during adsorption and DNA injection is not well understood. Among the P22-like phages are needle types with two completely different C-terminal distal tip domains. In the phage Sf6-type needle, unlike the other P22-type needle, the distal tip folds into a “knob” with a TNF-like fold, similar to the fiber knobs of bacteriophage PRD1 and Adenovirus. The phage HS1 knob is very similar to that of Sf6, and we report here its crystal structure which, like the Sf6 knob, contains three bound L-glutamate molecules. A chimeric P22 phage with a tail needle that contains the HS1 terminal knob efficiently infects the P22 host, Salmonella enterica, suggesting the knob does not confer host specificity. Likewise, mutations that should abrogate the binding of L-glutamate to the needle do not appear to affect virion function, but several different other genetic changes to the tip of the needle slow down potassium release from the host during infection. These findings suggest that the needle plays a role in phage P22 DNA delivery by controlling the kinetics of DNA ejection into the host.

Herpes Virus Genome, The Pressure Is On

Herpes simplex virus type 1 (HSV-1) packages its micrometers-long double-stranded DNA genome into a nanometer-scale protein shell, termed the capsid. Upon confinement within the capsid, neighboring DNA strands experience repulsive electrostatic and hydration forces as well as bending stress associated with the tight curvature required of packaged DNA. By osmotically suppressing DNA release from HSV-1 capsids, we provide the first experimental evidence of a high internal pressure of tens of atmospheres within a eukaryotic human virus, resulting from the confined genome. Furthermore, the ejection is progressively suppressed by increasing external osmotic pressures, which reveals that internal pressure is capable of powering ejection of the entire genome from the viral capsid. Despite billions of years of evolution separating eukaryotic viruses and bacteriophages, pressure-driven DNA ejection has been conserved. This suggests it is a key mechanism for viral infection and thus presents a new target for antiviral therapies.

Ultrasound‐mediated gene delivery systems by AG73‐modified bubble liposomes

Targeted gene delivery to neovascular vessels in tumors is considered a promising strategy for cancer therapy. We previously reported that "Bubble liposomes" (BLs), which are ultrasound (US) imaging gas-encapsulating liposomes, were suitable for US imaging and gene delivery. When BLs are exposed to US, the bubble is destroyed, creating a jet stream by cavitation, and resulting in the instantaneous ejection of extracellular plasmid DNA (pDNA) or other nucleic acids into the cytosol. We developed AG73 peptide-modified Bubble liposomes (AG73-BL) as a targeted US contrast agent, which was designed to attach to neovascular tumor vessels and to allow specific US detection of angiogenesis (Negishi et al., Biomaterials 2013, 34, 501-507). In this study, to evaluate the effectiveness of AG73-BL as a gene delivery tool for neovascular vessels, we examined the gene transfection efficiency of AG73-BL with US exposure in primary human endothelial cells (HUVEC). The transfection efficiency was significantly enhanced if the AG73-BL attached to the HUVEC was exposed to US compared to the BL-modified with no peptide or scrambled peptide. In addition, the cell viability was greater than 80% after transfection with AG73-BL. These results suggested that after the destruction of the AG73-BL with US exposure, a cavitation could be effectively induced by the US exposure against AG73-BL binding to the cell surface of the HUVEC, and the subsequent gene delivery into cells could be enhanced. Thus, AG73-BL may be useful for gene delivery as well as for US imaging of neovascular vessels.

Physical Evolution of Pressure-Driven Viral Infection

Physical Evolution of Pressure-Driven Viral Infection

Sophisticated Modeling Uncovers Atomic DNA Structure in Bacteriophage Φ29 Cavity

Sophisticated Modeling Uncovers Atomic DNA Structure in Bacteriophage Φ29 Cavity

Langevin dynamics simulation of DNA ejection from a phage

We have performed Langevin dynamics simulations of a coarse-grained model of ejection of dsDNA from Φ29 phage. Our simulation results show significant variations in the local ejection speed, consistent with experimental observations reported in the literature for both in vivo and in vitro systems. In efforts to understand the origin of such variations in the local speed of ejection, we have investigated the correlations between the local ejection kinetics and the packaged structures created at various motor forces and chain flexibility. At lower motor forces, the packaged DNA length is shorter with better organization. On the other hand, at higher motor forces typical of realistic situations, the DNA organization inside the capsid suffers from significant orientational disorder, but yet with long orientational correlation times. This in turn leads to lack of registry between the direction of the DNA segments just to be ejected and the direction of exit. As a result, a significant amount of momentum transfer is required locally for successful exit. Consequently, the DNA ejection temporarily slows down exhibiting pauses. This slowing down occurs at random times during the ejection process, completely determined by the particular starting conformation created by prescribed motor forces. In order to augment our inference, we have additionally investigated the ejection of chains with deliberately changed persistence length. For less inflexible chains, the demand on the occurrence of large momentum transfer for successful ejection is weaker, resulting in more uniform ejection kinetics. While being consistent with experimental observations, our results show the nonergodic nature of the ejection kinetics and call for better theoretical models to portray the kinetics of genome ejection from phages.

Special issue on physical virology

Compared to organisms such as bacteria and eukaryotic cells, viruses have such a low level of complexity that they were at first regarded as being proto-organisms, at the borderline between living and inanimate matter. This view was gradually superseded by the recognition that viruses are highly efficient organisms which have evolved in order to hijack the “active” biological machinery of the infected cells. Recent studies revealed how physical mechanisms are used by the viruses for guiding all salient steps of their “life cycle” This fact has also strongly motivated the recent upsurge of theoretical and experimental studies addressing the molecular basis of the key problems in virology. Specifically, the topics that are presently investigated most intensively include the following: the self-assembly and maturation of viral capsids, the functioning of the molecular motors that loads the viral genome inside preformed capsids, the conformational arrangement of the DNA or RNA inside capsids, the physical forces at play during the ejection of the viral DNA into the host cell, and the electrostatic interactions between the nucleic acids and coat proteins and virus-based nano-composites. The abovementioned efforts in characterizing the key molecular aspects of single viral particles are complemented by the very active research in the modeling of viral epidemics where, again, physics-based statistical mechanical approaches are widely used. A “physical virology” topical conference held at the ICTP in 2012 provided a timely state-of-the art perspective on the subject that is rapidly progressing because of the ongoing advancements in experimental techniques (cryo-EM imaging and single-molecule manipulation) and theoretical ones (large-scale simulations). It came at a very timely junction as the first Gordon conference on physical virology was held in 2009 in Galveston, Texas, and the latest one in Ventura, California, in 2013, which could be taken as the defining events in the establishment of the whole field of physical virology. This special issue of the Journal of Biological Physics is entirely dedicated to physical virology and aims to present a broad overview of the current state of the art in various subtopics of physical virology and stimulate additional activity in this rapidly developing field.

DNA bending-induced phase transition of encapsidated genome in phage

The DNA structure in phage capsids is determined by DNA–DNA interactions and bending energy. The effects of repulsive interactions on DNA interaxial distance were previously investigated, but not the effect of DNA bending on its structure in viral capsids. By varying packaged DNA length and through addition of spermine ions, we transform the interaction energy from net repulsive to net attractive. This allowed us to isolate the effect of bending on the resulting DNA structure. We used single particle cryo-electron microscopy reconstruction analysis to determine the interstrand spacing of double-stranded DNA encapsidated in phage λ capsids. The data reveal that stress and packing defects, both resulting from DNA bending in the capsid, are able to induce a long-range phase transition in the encapsidated DNA genome from a hexagonal to a cholesteric packing structure. This structural observation suggests significant changes in genome fluidity as a result of a phase transition affecting the rates of viral DNA ejection and packaging.

Effects of pulling forces, osmotic pressure, condensing agents and viscosity on the thermodynamics and kinetics of DNA ejection from bacteriophages to bacterial cells: a computational study

In this work, we report on simulations of double-stranded DNA (dsDNA) ejection from bacteriophage ϕ29 into a bacterial cell. The ejection was studied with a coarse-grained model, in which viral dsDNA was represented by beads on a torsion-less string. The bacteriophage’s capsid and the bacterial cell were defined by sets of spherical constraints. To account for the effects of the viscous medium inside the bacterial cell, the simulations were carried out using a Langevin dynamics protocol. Our simplest simulations (involving constant viscosity and no external biasing forces) produced results compatible with the push–pull model of DNA ejection, with an ejection rate significantly higher in the first part of ejection than in the latter parts. Additionally, we performed more complicated simulations, in which we included additional factors such as external forces, osmotic pressure, condensing agents and ejection-dependent viscosity. The effects of these factors (independently and in combination) on the thermodynamics and kinetics of DNA ejection were studied. We found that, in general, the dependence of ejection forces and ejection rates on the amount of DNA ejected becomes more complex if the ejection is modeled with a broader, more realistic set of parameters and influences (such as variation in the solvent’s viscosity and the application of an external force). However, certain combinations of factors and numerical parameters led to the opposition of some ejection-driving and ejection-inhibiting influences, ultimately causing an apparent simplification of the ejection profiles.

Effect of temperature and capsid tail on the packing and ejection of viral DNA.

We use a simulation technique based on molecular dynamics and stochastic rotation model to present the effect of temperature and capsid tail on the packaging and ejection processes of semiflexible polymers. We consider two types of solvents, a good solvent, where the polymer is neutral and repulsion interactions among its various sections are favored, and one where the polymer is charged, giving rise to extra electrostatic reaction. For tailless capsids, we find that packing a neutral polymer is slightly slower at higher temperatures whereas its ejection is slightly slower at lower temperatures. We find the same trend for a charged polymer but the effect is noticeably larger. At a high enough temperature, we notice that packing a charged polymer can be stopped. On the other hand, at fixed temperature and regardless whether the polymer is charged, packing is much easier for a capsid with a tail whereas ejection is much slower. The effect of including the tail on the dynamics of a charged polymer, in particular, is rather significant: more packing fraction is facilitated at higher temperatures due to more ordered polymer configuration inside the capsid. In contrast, during ejection the tail traps the last remaining beads for quite some time before allowing full ejection. We interpret these results in terms of entropic and electrostatic forces.