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1.
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.  相似文献   

2.
3.
Gp7 is a minor capsid protein of the Bacillus subtilis bacteriophage SPP1. Homologous proteins are found in numerous phages but their function remained unknown. Deletion of gene 7 from the SPP1 genome yielded a mutant phage (SPP1del7) with reduced burst-size. SPP1del7 infections led to normal assembly of virus particles whose morphology, DNA and protein composition was undistinguishable from wild-type virions. However, only approximately 25% of the viral particles that lack gp7 were infectious. SPP1del7 particles caused a reduced depolarization of the B. subtilis membrane in infection assays suggesting a defect in virus genome traffic to the host cell. A higher number of SPP1del7 DNA ejection events led to abortive release of DNA to the culture medium when compared with wild-type infections. DNA ejection in vitro showed that no detectable gp7 is co-ejected with the SPP1 genome and that its presence in the virion correlated with anchoring of released DNA to the phage particle. The release of DNA from wild-type phages was slower than that from SPP1del7 suggesting that gp7 controls DNA exit from the virion. This feature is proposed to play a central role in supporting correct routing of the phage genome from the virion to the cell cytoplasm.  相似文献   

4.
Tailed bacteriophage particles carry DNA highly pressurized inside the capsid. Challenge with their receptor promotes release of viral DNA. We show that addition of the osmolyte polyethylene glycol (PEG) has two distinct effects in bacteriophage SPP1 DNA ejection. One effect is to inhibit the trigger for DNA ejection. The other effect is to exert an osmotic pressure that controls the extent of DNA released in phages that initiate ejection. We carried out independent measurements of each effect, which is an essential requirement for their quantitative study. The fraction of phages that do not eject increased linearly with the external osmotic pressure. In the remaining phage particles ejection stopped after a defined amount of DNA was reached inside the capsid. Direct measurement of the size of non-ejected DNA by gel electrophoresis at different PEG concentrations in the latter sub-population allowed determination of the external osmotic pressure that balances the force powering DNA exit (47 atm for SPP1 wild-type). DNA exit stops when the ejection force mainly due to repulsion between DNA strands inside the SPP1 capsid equalizes the force resisting DNA insertion into the PEG solution. Considering the turgor pressure in the Bacillus subtilis cytoplasm the energy stored in the tight phage DNA packing is only sufficient to power entry of the first 17% of the SPP1 chromosome into the cell, the remaining 83% requiring application of additional force for internalization.  相似文献   

5.
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 in the course of ejection. In this work, we argue by a combination of experimental techniques (osmotic suppression without DNase I monitored by UV absorbance, pulse-field electrophoresis, and cryo-transmission electron microscopy 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 resist 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.  相似文献   

6.
Recent work has shown that pressures inside dsDNA phage capsids can be as high as many tens of atmospheres; it is this pressure that is responsible for initiation of the delivery of phage genomes to host cells. The forces driving ejection of the genome have been shown to decrease monotonically as ejection proceeds, and hence to be strongly dependent on the genome length. Here we investigate the effects of ambient salts on the pressures inside phage-λ, for the cases of mono-, di-, and tetravalent cations, and measure how the extent of ejection against a fixed osmotic pressure (mimicking the bacterial cytoplasm) varies with cation concentration. We find, for example, that the ejection fraction is halved in 30 mM Mg2+ and is decreased by a factor of 10 upon addition of 1 mM spermine. These effects are calculated from a simple model of genome packaging, using DNA-DNA repulsion energies as determined independently from x-ray diffraction measurements on bulk DNA solutions. By comparing the measured ejection fractions with values implied from the bulk DNA solution data, we predict that the bending energy makes the d-spacings inside the capsid larger than those for bulk DNA at the same osmotic pressure.  相似文献   

7.
8.
P16 is a virion protein and, as such, is incorporated into the phage head as a step in morphogenesis. The role of P16 in assembly is not essential since particles are formed without this protein which appear normal by electron microscopy. P16 is essential when the particle infects a cell in the following cycle of infection. In the absence of functional P16, the infection does not appear to proceed beyond release of phage DNA from the capsid. No known genes are expressed, no DNA is transcribed, and the host cell survives the infection, continuing to grow and divide normally. The P16 function is required only during infection for the expression of phage functions. Induction in the absence of P16 proceeds with the expression of early and late genes and results in particle formation. P16 must be incorporated during morphogenesis into progeny particles after both infection and induction for the progeny to be infectious. The P16 function is necessary for transduction as well as for infection. Its activity is independent of new protein synthesis and it is not under immunity control. P16 can act in trans, but appears to act preferentially on the phage or phage DNA with which it is packaged. The data from complementation studies are compatible with P16 release from the capsid with the phage DNA. In the absence of P16 the infection is blocked, but the phage genome is not degraded. The various roles which have been ruled out for P16 are: (i) an early regulatory function, (ii) an enzymatic activity necessary for phage production, (iii) protection of phage DNA from host degradation enzymes, (iv) any generalized alteration of the host cell, (v) binding parental DNA to the replication complex, and (vi) any direct involvement in the replication of P22 DNA. P16 can be responsible for: (i) complete release of the DNA and disengagement from the capsid, (ii) bringing the released DNA to some necessary cell site or compartment such as the cytoplasm, (iii) removal of other virion proteins from the injected DNA, and (iv) alterations of the structure of the injected DNA.  相似文献   

9.
The irreversible binding of bacteriophages to their receptor(s) in the host cell surface triggers release of the naked genome from the virion followed by transit of viral DNA to the host cell cytoplasm. We have purified, for the first time, a receptor from a Gram-positive bacterium that is active to trigger viral DNA ejection in vitro. This extracellular region ("ectodomain") of the Bacillus subtilis protein YueB (YueB780) was a 7 S elongated dimer forming a 36.5-nm-long fiber. YueB780 bound to the tail tip of bacteriophage SPP1. Although a stable receptor-phage interaction occurred between 0 and 37 degrees C, complete blocking of phage DNA release or partial ejection events were observed at temperatures below 15 degrees C. We also showed that the receptor was exposed to the B. subtilis surface. YueB differed structurally from phage receptors from Gram-negative bacteria. Its properties revealed a fiber spanning the full length of the 30-nm-thick peptidoglycan layer. The fiber is predicted to be anchored in the cell membrane through transmembrane segments. These features, highly suitable for a virus receptor in Gram-positive bacteria, are very likely shared by a large number of phage receptors.  相似文献   

10.
The transfer of the bacteriophage genome from the capsid into the host cell is a key step of the infectious process. In bacteriophage T5, DNA ejection can be triggered in vitro by simple binding of the phage to its purified Escherichia coli receptor FhuA. Using electrophoresis and cryo-electron microscopy, we measure the extent of DNA ejection as a function of the external osmotic pressure. In the high pressure range (7-16 atm), the amount of DNA ejected decreases with increasing pressure, as theoretically predicted and observed for λ and SPP1 bacteriophages. In the low and moderate pressure range (2-7 atm), T5 exhibits an unexpected behavior. Instead of a unique ejected length, multiple populations coexist. Some phages eject their complete genome, whereas others stop at some nonrandom states that do not depend on the applied pressure. We show that contrarily to what is observed for the phages SPP1 and λ, T5 ejection cannot be explained as resulting from a simple pressure equilibrium between the inside and outside of the capsid. Kinetics parameters and/or structural characteristics of the ejection machinery could play a determinant role in T5 DNA ejection.  相似文献   

11.
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 λ, 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.  相似文献   

12.
In this study we present a bacteriophage isolated from the Great Salt Plains National Wildlife Refuge (GSP) that is shown to have a genome size of 340 kb, unusually large for a bacterial virus. Transmission electron microscopy analysis of the virion showed this to be a Myoviridae, the first reported to infect the genus Halomonas. This temperate phage, PhigspC, exhibits a broad host range, displaying the ability to infect two different Halomonas spp. also isolated from the GSP. The phage infection process demonstrates a high level of tolerance towards temperature, pH and salinity; however, free virions are rapidly inactivated in water unless supplemented with salt. We show that susceptibility to osmotic shock is correlated with the density of the packaged DNA (rho(pack)). Lysogens of Halomonas salina GSP21 were detrimental to host fitness at 10% salinity, but the lysogen was able to grow faster than the wild type at 20% salinity. From these results we propose that the extensive genome of PhigspC may encode environmentally relevant genes (ERGs); genes that are perhaps not essential for the phage life cycle but increase host and phage fitness in some environmental conditions.  相似文献   

13.
14.
The generalized transducing double-stranded DNA bacteriophage ES18 has an icosahedral head and a long noncontractile tail, and it infects both rough and smooth Salmonella enterica strains. We report here the complete 46,900-bp genome nucleotide sequence and provide an analysis of the sequence. Its 79 genes and their organization clearly show that ES18 is a member of the lambda-like (lambdoid) phage group; however, it contains a novel set of genes that program assembly of the virion head. Most of its integration-excision, immunity, Nin region, and lysis genes are nearly identical to those of the short-tailed Salmonella phage P22, while other early genes are nearly identical to Escherichia coli phages lambda and HK97, S. enterica phage ST64T, or a Shigella flexneri prophage. Some of the ES18 late genes are novel, while others are most closely related to phages HK97, lambda, or N15. Thus, the ES18 genome is mosaically related to other lambdoid phages, as is typical for all group members. Analysis of virion DNA showed that it is circularly permuted and about 10% terminally redundant and that initiation of DNA packaging series occurs across an approximately 1-kbp region rather than at a precise location on the genome. This supports a model in which ES18 terminase can move substantial distances along the DNA between recognition and cleavage of DNA destined to be packaged. Bioinformatic analysis of large terminase subunits shows that the different functional classes of phage-encoded terminases can usually be predicted from their amino acid sequence.  相似文献   

15.
Replication and packaging of choleraphage phi 149 DNA.   总被引:1,自引:1,他引:0       下载免费PDF全文
R Chowdhury  A Ray  P Ray    J Das 《Journal of virology》1987,61(12):3999-4006
The intercellular replication of the circularly permuted DNA of choleraphage phi 149 involves a concatemeric DNA structure with a size equivalent to six genome lengths. The synthesis of both monomeric and concatemeric DNAs during replication of phi 149 occurred in the cytoplasm. The concatemers served as the substrate for the synthesis of mature phage DNA, which was eventually packaged by a headful mechanism starting from a unique pac site in the concatemeric DNA. Packaging of DNA into phage heads involved binding of concatemeric DNA to the cell membrane. A scheme involving sequential packaging of five headfuls proceeding in the counterclockwise direction from the pac site is proposed. After infection under high-phosphate conditions, the concatemeric DNA intermediates were not formed, although synthesis of monomeric molecules was unaffected.  相似文献   

16.
Tailed bacteriophages and herpesviruses consist of a structurally well conserved dodecameric portal at a special 5-fold vertex of the capsid. The portal plays critical roles in head assembly, genome packaging, neck/tail attachment, and genome ejection. Although the structures of portals from phages φ29, SPP1, and P22 have been determined, their mechanistic roles have not been well understood. Structural analysis of phage T4 portal (gp20) has been hampered because of its unusual interaction with the Escherichia coli inner membrane. Here, we predict atomic models for the T4 portal monomer and dodecamer, and we fit the dodecamer into the cryo-electron microscopy density of the phage portal vertex. The core structure, like that from other phages, is cone shaped with the wider end containing the “wing” and “crown” domains inside the phage head. A long “stem” encloses a central channel, and a narrow “stalk” protrudes outside the capsid. A biochemical approach was developed to analyze portal function by incorporating plasmid-expressed portal protein into phage heads and determining the effect of mutations on head assembly, DNA translocation, and virion production. We found that the protruding loops of the stalk domain are involved in assembling the DNA packaging motor. A loop that connects the stalk to the channel might be required for communication between the motor and the portal. The “tunnel” loops that project into the channel are essential for sealing the packaged head. These studies established that the portal is required throughout the DNA packaging process, with different domains participating at different stages of genome packaging.  相似文献   

17.
The translocation of genetic material from the viral capsid to the cell is an essential part of the viral infection process. Whether the energetics of this process is driven by the energy stored within the confined nucleic acid or cellular processes pull the genome into the cell has been the subject of discussion. However, in vitro studies of genome ejection have been limited to a few head-tailed bacteriophages with a double-stranded DNA genome. Here we describe a DNA release system that operates in an archaeal virus. This virus infects an archaeon Haloarcula hispanica that was isolated from a hypersaline environment. The DNA-ejection velocity of His1, determined by single-molecule experiments, is comparable to that of bacterial viruses. We found that the ejection process is modulated by the external osmotic pressure (polyethylene glycol (PEG)) and by increased ion (Mg2+ and Na+) concentration. The observed ejection was unidirectional, randomly paused, and incomplete, which suggests that cellular processes are required to complete the DNA transfer.  相似文献   

18.
Rescue of abortive T7 gene 2 mutant phage infection by rifampin.   总被引:2,自引:1,他引:1       下载免费PDF全文
Infection of Escherichia coli with T7 gene 2 mutant phage was abortive; concatemeric phage DNA was synthesized but was not packaged into the phage head, resulting in an accumulation of DNA species shorter in size than the phage genome, concomitant with an accumulation of phage head-related structures. Appearance of concatemeric T7 DNA in gene 2 mutant phage infection during onset of T7 DNA replication indicates that the product of gene 2 was required for proper processing or packaging of concatemer DNA rather than for the synthesis of T7 progeny DNA or concatemer formation. This abortive infection by gene 2 mutant phage could be rescued by rifampin. If rifampin was added at the onset of T7 DNA replication, concatemeric DNA molecules were properly packaged into phage heads, as evidenced by the production of infectious progeny phage. Since the gene 2 product acts as a specific inhibitor of E. coli RNA polymerase by preventing the enzyme from binding T7 DNA, uninhibited E. coli RNA polymerase in gene 2 mutant phage-infected cells interacts with concatemeric T7 DNA and perturbs proper DNA processing unless another inhibitor of the enzyme (rifampin) was added. Therefore, the involvement of gene 2 protein in T7 DNA processing may be due to its single function as the specific inhibitor of the host E. coli RNA polymerase.  相似文献   

19.
The assembly intermediates of the Salmonella bacteriophage P22 are well defined but the molecular interactions between the subunits that participate in its assembly are not. The first stable intermediate in the assembly of the P22 virion is the procapsid, a preformed protein shell into which the viral genome is packaged. The procapsid consists of an icosahedrally symmetric shell of 415 molecules of coat protein, a dodecameric ring of portal protein at one of the icosahedral vertices through which the DNA enters, and approximately 250 molecules of scaffolding protein in the interior. Scaffolding protein is required for assembly of the procapsid but is not present in the mature virion. In order to define regions of scaffolding protein that contribute to the different aspects of its function, truncation mutants of the scaffolding protein were expressed during infection with scaffolding deficient phage P22, and the products of assembly were analyzed. Scaffolding protein amino acids 1-20 are not essential, since a mutant missing them is able to fully complement scaffolding deficient phage. Mutants lacking 57 N-terminal amino acids support the assembly of DNA containing virion-like particles; however, these particles have at least three differences from wild-type virions: (i) a less than normal complement of the gene 16 protein, which is required for DNA injection from the virion, (ii) a fraction of the truncated scaffolding protein was retained within the virions, and (iii) the encapsidated DNA molecule is shorter than the wild-type genome. Procapsids assembled in the presence of a scaffolding protein mutant consisting of only the C-terminal 75 amino acids contained the portal protein, but procapsids assembled with the C-terminal 66 did not, suggesting portal recruitment function for the region about 75 amino acids from the C terminus. Finally, scaffolding protein amino acids 280 through 294 constitute its minimal coat protein binding site.  相似文献   

20.
The goal of this work was to investigate how internal and external forces acting on DNA affect the rate of genome ejection from bacteriophage lambda after the ejection is triggered in vitro by a lambda receptor. The rate of ejection was measured with time-resolved static and dynamic light scattering, while varying such parameters as temperature and packaged DNA length, as well as adding DNA-binding proteins to the host solution. We found that temperature has a strong effect on the ejection rate, with an exponential increase of the initial ejection rate as a function of temperature. This can possibly be explained by the temperature-induced conformational changes in the tail pore-forming proteins where the "open" conformation dominates over "closed", at elevated temperatures. The DNA length also had an effect on initial ejection rate, with a nearly linear dependence comparing the three different genomes (37.7, 45.7 and 48.5 kb DNA), with faster ejection rate for longer genomes. Since the initial rate of ejection increases in an almost direct relationship with the length of the genome, the total time needed to eject DNA completely appeared to be nearly constant for all three DNA length phage mutants. The increased initial rate of ejection with increasing DNA length is due to the increased DNA bending and inter-strand repulsion forces for the longer DNA chains. Finally, we also show that addition of non-specific DNA-binding proteins (HU and DNase I) increases the rate of ejection by exerting additional "pulling" forces on the DNA that is being ejected.  相似文献   

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