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1.
Viruses ; 15(12)2023 12 12.
Article in English | MEDLINE | ID: mdl-38140651

ABSTRACT

The following hypothesis proposes non-diffusive, environmental bacteriophage (phage) motion. (1) Some phage-hosting, motile bacteria undergo chemotaxis down ATP concentration gradients to escape lysis-inducing conditions, such as phage infection. (2) Some phages respond by non-infective binding to the motile bacteria. (3) When the bacteria reach a lower ATP concentration, which is a condition that signals increased density of phage-susceptible bacteria, the phage converts, Trojan-horse-like, to productive binding and infection. This hypothesis was previously proposed for Bacillus thuringiensis siphophage 0105phi7-2. It is tested here and confirmed with the following observations. (1) B. thuringiensis is found, macroscopically, preferentially located at low ATP concentrations when propagated in-gel after inoculation in the center of an artificially generated ATP concentration gradient. (2) Inoculating phage 0105phi7-2 at the bacteria inoculation site, 2-3 h after inoculation of bacteria, results in cell lysing activity that moves with the bacteria, without a visible trail of lysis. Trojan-horse-like behavior is consistent with only biofilm-inhabiting phages because environmental selection for this behavior requires limited fluid flows. We propose using artificial ATP concentration gradients to instigate Trojan-horse-like phage behavior during phage therapy of bacterial biofilms.


Subject(s)
Bacillus thuringiensis , Bacteriophages , Phage Therapy , Biofilms , Adenosine Triphosphate
2.
Int J Mol Sci ; 24(10)2023 May 18.
Article in English | MEDLINE | ID: mdl-37240285

ABSTRACT

Diversity of phage propagation, physical properties, and assembly promotes the use of phages in ecological studies and biomedicine. However, observed phage diversity is incomplete. Bacillus thuringiensis siphophage, 0105phi-7-2, first described here, significantly expands known phage diversity, as seen via in-plaque propagation, electron microscopy, whole genome sequencing/annotation, protein mass spectrometry, and native gel electrophoresis (AGE). Average plaque diameter vs. plaque-supporting agarose gel concentration plots reveal unusually steep conversion to large plaques as agarose concentration decreases below 0.2%. These large plaques sometimes have small satellites and are made larger by orthovanadate, an ATPase inhibitor. Phage head-host-cell binding is observed by electron microscopy. We hypothesize that this binding causes plaque size-increase via biofilm evolved, ATP stimulated ride-hitching on motile host cells by temporarily inactive phages. Phage 0105phi7-2 does not propagate in liquid culture. Genomic sequencing/annotation reveals history as temperate phage and distant similarity, in a virion-assembly gene cluster, to prototypical siphophage SPP1 of Bacillus subtilis. Phage 0105phi7-2 is distinct in (1) absence of head-assembly scaffolding via either separate protein or classically sized, head protein-embedded peptide, (2) producing partially condensed, head-expelled DNA, and (3) having a surface relatively poor in AGE-detected net negative charges, which is possibly correlated with observed low murine blood persistence.


Subject(s)
Bacillus thuringiensis , Bacteriophages , Animals , Mice , Bacillus thuringiensis/genetics , Sepharose , Bacteriophages/genetics , DNA , Whole Genome Sequencing , Genome, Viral
3.
J Alzheimers Dis ; 88(2): 429-438, 2022.
Article in English | MEDLINE | ID: mdl-35662126

ABSTRACT

Protein amyloid-ß (Aß) oligomers with ß-sheet-like backbone (ß-structured) form extracellular amyloid plaques associated with Alzheimer's disease (AD). However, the relationship to AD is not known. Some investigations suggest that the toxic Aß component has α-sheet-like backbone (α-structured) subsequently detoxified by intracellular α-to-ß conversion before plaque formation. Our objective is to compare this latter hypothesis with observations made by electron microscopy of thin sections of AD-cerebral cortex. We observe irregular, 200-2,000 nm, intracellular, lipofuscin-like inclusions. Some are light-staining and smooth. Others are dark-staining and made granular by fibers that are usually overlapping and are sometimes individually seen. Aspects unusual for lipofuscin include 1) dark and light inclusions interlocking as though previously one inclusion, 2) dark inclusion-contained 2.6 nm thick sub-fibers that are bent as though α-structured, and 3) presence of inclusions in lysosomes and apparent transfer of dark inclusion material to damaged, nearby lysosomal membranes. These data suggest the following additions to α-structure-based hypotheses: 1) Lipofuscin-associated, α-structured protein toxicity to lysosomal membranes is in the chain of AD causation; 2) α-to-ß detoxification of α-structured protein occurs in lipofuscin and causes dark-to-light transition that, when incomplete, is the origin of cell-to-cell transmission essential for development of AD.


Subject(s)
Alzheimer Disease , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Humans , Lipofuscin , Lysosomes/metabolism , Plaque, Amyloid/metabolism
4.
Viruses ; 13(10)2021 10 18.
Article in English | MEDLINE | ID: mdl-34696524

ABSTRACT

Phage G is recognized as having a remarkably large genome and capsid size among isolated, propagated phages. Negative stain electron microscopy of the host-phage G interaction reveals tail sheaths that are contracted towards the distal tip and decoupled from the head-neck region. This is different from the typical myophage tail contraction, where the sheath contracts upward, while being linked to the head-neck region. Our cryo-EM structures of the non-contracted and contracted tail sheath show that: (1) The protein fold of the sheath protein is very similar to its counterpart in smaller, contractile phages such as T4 and phi812; (2) Phage G's sheath structure in the non-contracted and contracted states are similar to phage T4's sheath structure. Similarity to other myophages is confirmed by a comparison-based study of the tail sheath's helical symmetry, the sheath protein's evolutionary timetree, and the organization of genes involved in tail morphogenesis. Atypical phase G tail contraction could be due to a missing anchor point at the upper end of the tail sheath that allows the decoupling of the sheath from the head-neck region. Explaining the atypical tail contraction requires further investigation of the phage G sheath anchor points.


Subject(s)
Myoviridae/ultrastructure , Viral Tail Proteins/ultrastructure , Bacteriophages/metabolism , Bacteriophages/ultrastructure , Capsid/metabolism , Capsid Proteins/metabolism , Cryoelectron Microscopy/methods , Myoviridae/genetics , Viral Tail Proteins/genetics , Viral Tail Proteins/metabolism , Virion/metabolism , Virion/ultrastructure
5.
Antibiotics (Basel) ; 10(6)2021 Jun 16.
Article in English | MEDLINE | ID: mdl-34208477

ABSTRACT

Blood-borne therapeutic phages and phage capsids increasingly reach therapeutic targets as they acquire more persistence, i.e., become more resistant to non-targeted removal from blood. Pathogenic bacteria are targets during classical phage therapy. Metastatic tumors are potential future targets, during use of drug delivery vehicles (DDVs) that are phage derived. Phage therapy has, to date, only sometimes been successful. One cause of failure is low phage persistence. A three-step strategy for increasing persistence is to increase (1) the speed of lytic phage isolation, (2) the diversity of phages isolated, and (3) the effectiveness and speed of screening phages for high persistence. The importance of high persistence-screening is illustrated by our finding here of persistence dramatically higher for coliphage T3 than for its relative, coliphage T7, in murine blood. Coliphage T4 is more persistent, long-term than T3. Pseudomonas chlororaphis phage 201phi2-1 has relatively low persistence. These data are obtained with phages co-inoculated and separately assayed. In addition, highly persistent phage T3 undergoes dispersal to several murine organs and displays tumor tropism in epithelial tissue (xenografted human oral squamous cell carcinoma). Dispersal is an asset for phage therapy, but a liability for phage-based DDVs. We propose increased focus on phage persistence-and dispersal-screening.

6.
Viruses ; 12(4)2020 04 07.
Article in English | MEDLINE | ID: mdl-32272774

ABSTRACT

We review some aspects of the rapid isolation of, screening for and characterization of jumbo phages, i.e., phages that have dsDNA genomes longer than 200 Kb. The first aspect is that, as plaque-supporting gels become more concentrated, jumbo phage plaques become smaller. Dilute agarose gels are better than conventional agar gels for supporting plaques of both jumbo phages and, prospectively, the even larger (>520 Kb genome), not-yet-isolated mega-phages. Second, dilute agarose gels stimulate propagation of at least some jumbo phages. Third, in-plaque techniques exist for screening for both phage aggregation and high-in-magnitude, negative average electrical surface charge density. The latter is possibly correlated with high phage persistence in blood. Fourth, electron microscopy of a thin section of a phage plaque reveals phage type, size and some phage life cycle information. Fifth, in-gel propagation is an effective preparative technique for at least some jumbo phages. Sixth, centrifugation through sucrose density gradients is a relatively non-destructive jumbo phage purification technique. These basics have ramifications in the development of procedures for (1) use of jumbo phages for phage therapy of infectious disease, (2) exploration of genomic diversity and evolution and (3) obtaining accurate metagenomic analyses.


Subject(s)
Bacteriophages/classification , Bacteriophages/isolation & purification , Electrophoresis, Agar Gel/methods , Genome, Viral , Bacteriophages/ultrastructure , Centrifugation, Density Gradient , DNA , Genomics , Metagenomics , Microscopy, Electron
7.
Pharmaceuticals (Basel) ; 13(1)2020 Jan 18.
Article in English | MEDLINE | ID: mdl-31963711

ABSTRACT

Increased knowledge of virus assembly-generated particles is needed for understanding both virus assembly and host responses to virus infection. Here, we use a phage T3 model and perform electron microscopy (EM) of thin sections (EM-TS) of gel-supported T3 plaques formed at 30 °C. After uranyl acetate/lead staining, we observe intracellular black particles, some with a difficult-to-see capsid. Some black particles (called LBPs) are larger than phage particles. The LBP frequency is increased by including proflavine, a DNA packaging inhibitor, in the growth medium and increasing plaque-forming temperature to 37 °C. Acidic phosphotungstate-precipitate (A-PTA) staining causes LBP substitution by black rings (BRs) that have the size and shape expected of hyper-expanded capsid containers for LBP DNA. BRs are less frequent in liquid cultures, suggesting that hyper-expanded capsids evolved primarily for in-gel (e.g., in-biofilm) propagation. BR-specific A-PTA staining and other observations are explained by α-sheet intense structure of the major subunit of hyper-expanded capsids. We hypothesize that herpes virus triggering of neurodegenerative disease occurs via in-gel propagation-promoted (1) generation of α-sheet intense viral capsids and, in response, (2) host production of α-sheet intense, capsid-interactive, innate immunity amyloid protein that becomes toxic. We propose developing viruses that are therapeutic via detoxifying interaction with this innate immunity protein.

8.
BMC Res Notes ; 12(1): 560, 2019 Sep 05.
Article in English | MEDLINE | ID: mdl-31488211

ABSTRACT

OBJECTIVE: Our immediate objective is to determine whether infectivity of lytic podophage T3 has a relatively high persistence in the blood of a mouse, as suggested by previous data. Secondarily, we determine whether the T3 surface has changed during this mouse passage. The surface is characterized by native agarose gel electrophoresis (AGE). Beyond our current data, the long-term objective is optimization of phages chosen for therapy of all bacteremias and associated sepsis. RESULTS: We find that the persistence of T3 in mouse blood is higher by over an order of magnitude than the previously reported persistence of (1) lysogenic phages lambda and P22, and (2) lytic phage T7, a T3 relative. We explain these differences via the lysogenic character of lambda and P22, and the physical properties of T7. For the future, we propose testing a new, AGE-based strategy for rapidly screening for high-persistence, lytic, environmental podophages that have phage therapy-promoting physical properties.


Subject(s)
Bacteremia/therapy , Bacteriophage T3/physiology , Phage Therapy/methods , Sepsis/therapy , Animals , Bacteremia/blood , Bacteriolysis , Bacteriophage T7/physiology , Female , Mice, Inbred C57BL , Sepsis/blood
9.
BMC Res Notes ; 11(1): 699, 2018 Oct 04.
Article in English | MEDLINE | ID: mdl-30286794

ABSTRACT

OBJECTIVE: Our immediate objective is to test the data-suggested possibility that in-agarose gel bacterial propagation causes gel fiber dislocation and alteration of cell distribution. We also test the further effect of lowering water activity. We perform these tests with both Gram-negative and Gram-positive bacteria. Data are obtained via electron microscopy of thin sections, which provides the first images of both bacteria and gel fibers in gel-supported bacterial lawns. The long-term objective is analysis of the effects of in-gel propagation on the DNA packaging of phages. RESULTS: We find that agarose gel-supported cells in lawns of Escherichia coli and Lysinibacillus (1) are primarily in clusters that increase in size with time and are surrounded by gel fibers, and (2) sometimes undergo gel-induced, post-duplication rotation and translation. Bacterial growth-induced dislocation of gel fibers is observed. One reason for clustering is that clustering promotes growth by increasing the growth-derived force applied to the gel fibers. Reactive force exerted by gel on cells explains cell movement. Finally, addition to growth medium of 0.94 M sucrose causes cluster-associated E. coli cells to become more densely packed and polymorphic. Shape is determined, in part, by neighboring cells, a novel observation to our knowledge.


Subject(s)
Agar , Bacillaceae/physiology , Bacterial Physiological Phenomena , Escherichia coli/physiology , Gels , Microscopy, Electron
10.
Viruses ; 10(6)2018 06 06.
Article in English | MEDLINE | ID: mdl-29882754

ABSTRACT

Studies of phage capsids have at least three potential interfaces with nanomedicine. First, investigation of phage capsid states potentially will provide therapies targeted to similar states of pathogenic viruses. Recently detected, altered radius-states of phage T3 capsids include those probably related to intermediate states of DNA injection and DNA packaging (dynamic states). We discuss and test the idea that some T3 dynamic states include extensive α-sheet in subunits of the capsid’s shell. Second, dynamic states of pathogenic viral capsids are possible targets of innate immune systems. Specifically, α-sheet-rich innate immune proteins would interfere with dynamic viral states via inter-α-sheet co-assembly. A possible cause of neurodegenerative diseases is excessive activity of these innate immune proteins. Third, some phage capsids appear to have characteristics useful for improved drug delivery vehicles (DDVs). These characteristics include stability, uniformity and a gate-like sub-structure. Gating by DDVs is needed for (1) drug-loading only with gate opened; (2) closed gate-DDV migration through circulatory systems (no drug leakage-generated toxicity); and (3) drug release only at targets. A gate-like sub-structure is the connector ring of double-stranded DNA phage capsids. Targeting to tumors of phage capsid-DDVs can possibly be achieved via the enhanced permeability and retention effect.


Subject(s)
Antineoplastic Agents/metabolism , Capsid/chemistry , Capsid/metabolism , Drug Carriers/metabolism , Nanomedicine/methods , Bacteriophage T3/chemistry , Bacteriophage T3/physiology , Humans , Protein Binding , Protein Conformation
11.
Biophys Rev ; 10(2): 583-596, 2018 Apr.
Article in English | MEDLINE | ID: mdl-29243090

ABSTRACT

Mature double-stranded DNA bacteriophages have capsids with symmetrical shells that typically resist disruption, as they must to survive in the wild. However, flexibility and associated dynamism assist function. We describe biochemistry-oriented procedures used to find previously obscure flexibility for capsids of the related phages, T3 and T7. The primary procedures are hydration-based buoyant density ultracentrifugation and purified particle-based cryo-electron microscopy (cryo-EM). We review the buoyant density centrifugation in detail. The mature, stable T3/T7 capsid is a shell flexibility-derived conversion product of an initially assembled procapsid (capsid I). During DNA packaging, capsid I expands and loses a scaffolding protein to form capsid II. The following are observations made with capsid II. (1) The in vivo DNA packaging of wild type T3 generates capsid II that has a slight (1.4%), cryo-EM-detected hyper-expansion relative to the mature phage capsid. (2) DNA packaging in some altered conditions generates more extensive hyper-expansion of capsid II, initially detected by hydration-based preparative buoyant density centrifugation in Nycodenz density gradients. (3) Capsid contraction sometimes occurs, e.g., during quantized leakage of DNA from mature T3 capsids without a tail.

12.
Viruses ; 9(5)2017 05 19.
Article in English | MEDLINE | ID: mdl-28534826

ABSTRACT

Adenosine triphosphate (ATP) cleavage powers packaging of a double-stranded DNA (dsDNA) molecule in a pre-assembled capsid of phages that include T3. Several observations constitute a challenge to the conventional view that the shell of the capsid is energetically inert during packaging. Here, we test this challenge by analyzing the in vitro effects of ATP on the shells of capsids generated by DNA packaging in vivo. These capsids retain incompletely packaged DNA (ipDNA) and are called ipDNA-capsids; the ipDNA-capsids are assumed to be products of premature genome maturation-cleavage. They were isolated via preparative Nycodenz buoyant density centrifugation. For some ipDNA-capsids, Nycodenz impermeability increases hydration and generates density so low that shell hyper-expansion must exist to accommodate associated water. Electron microscopy (EM) confirmed hyper-expansion and low permeability and revealed that 3.0 mM magnesium ATP (physiological concentration) causes contraction of hyper-expanded, lowpermeability ipDNA-capsids to less than mature size; 5.0 mM magnesium ATP (border of supraphysiological concentration) or more disrupts them. Additionally, excess sodium ADP reverses 3.0 mM magnesium ATP-induced contraction and re-generates hyper-expansion. The Nycodenz impermeability implies assembly perfection that suggests selection for function in DNA packaging. These findings support the above challenge and can be explained via the assumption that T3 DNA packaging includes a back-up cycle of ATP-driven capsid contraction and hyper-expansion.


Subject(s)
Adenosine Triphosphate/pharmacology , Bacteriophage T3/genetics , Capsid/drug effects , DNA Packaging , DNA, Viral/genetics , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Bacteriophage T3/metabolism , Bacteriophage T3/ultrastructure , Capsid/metabolism , Capsid/ultrastructure , Capsid Proteins/genetics , DNA, Viral/metabolism , Electrophoresis, Polyacrylamide Gel , Microscopy, Electron , Permeability/drug effects , Virus Assembly/drug effects
13.
Bacteriophage ; 6(4): e1268664, 2016.
Article in English | MEDLINE | ID: mdl-28090387

ABSTRACT

We argue that a paradigm shift is needed in the analysis of phage DNA packaging. We then test a prediction of the following paradigm shift-engendering hypothesis. The motor of phage DNA packaging has two cycles: (1) the well-known packaging ATPase-driven (type 1) cycle and (2) a proposed back-up, shell expansion/contraction-driven (type 2) cycle that reverses type 1 cycle stalls by expelling accidentally packaged non-DNA molecules. We test the prediction that increasing the cellular concentration of all macromolecules will cause packaging-active capsids to divert to states of hyper-expansion and contraction. We use a directed evolution-derived, 3-site phage T3 mutant, adapted to propagation in concentrated bacterial cytoplasm. We find this prediction correct while discovering novel T3 capsids previously obscure.

14.
Proc Natl Acad Sci U S A ; 111(43): E4606-14, 2014 Oct 28.
Article in English | MEDLINE | ID: mdl-25313071

ABSTRACT

Many dsDNA viruses first assemble a DNA-free procapsid, using a scaffolding protein-dependent process. The procapsid, then, undergoes dramatic conformational maturation while packaging DNA. For bacteriophage T7 we report the following four single-particle cryo-EM 3D reconstructions and the derived atomic models: procapsid (4.6-Å resolution), an early-stage DNA packaging intermediate (3.5 Å), a later-stage packaging intermediate (6.6 Å), and the final infectious phage (3.6 Å). In the procapsid, the N terminus of the major capsid protein, gp10, has a six-turn helix at the inner surface of the shell, where each skewed hexamer of gp10 interacts with two scaffolding proteins. With the exit of scaffolding proteins during maturation the gp10 N-terminal helix unfolds and swings through the capsid shell to the outer surface. The refolded N-terminal region has a hairpin that forms a novel noncovalent, joint-like, intercapsomeric interaction with a pocket formed during shell expansion. These large conformational changes also result in a new noncovalent, intracapsomeric topological linking. Both interactions further stabilize the capsids by interlocking all pentameric and hexameric capsomeres in both DNA packaging intermediate and phage. Although the final phage shell has nearly identical structure to the shell of the DNA-free intermediate, surprisingly we found that the icosahedral faces of the phage are slightly (∼4 Å) contracted relative to the faces of the intermediate, despite the internal pressure from the densely packaged DNA genome. These structures provide a basis for understanding the capsid maturation process during DNA packaging that is essential for large numbers of dsDNA viruses.


Subject(s)
Bacteriophage T7/chemistry , Capsid/chemistry , Cryoelectron Microscopy , Image Processing, Computer-Assisted , Models, Molecular , Bacteriophage T7/ultrastructure , Capsid/ultrastructure , Capsid Proteins/chemistry , DNA Packaging , Protein Binding , Protein Structure, Secondary , Virus Assembly
15.
Virology ; 456-457: 157-70, 2014 May.
Article in English | MEDLINE | ID: mdl-24889235

ABSTRACT

DNA packaging of phages phi29, T3 and T7 sometimes produces incompletely packaged DNA with quantized lengths, based on gel electrophoretic band formation. We discover here a packaging ATPase-free, in vitro model for packaged DNA length quantization. We use directed evolution to isolate a five-site T3 point mutant that hyper-produces tail-free capsids with mature DNA (heads). Three tail gene mutations, but no head gene mutations, are present. A variable-length DNA segment leaks from some mutant heads, based on DNase I-protection assay and electron microscopy. The protected DNA segment has quantized lengths, based on restriction endonuclease analysis: six sharp bands of DNA missing 3.7-12.3% of the last end packaged. Native gel electrophoresis confirms quantized DNA expulsion and, after removal of external DNA, provides evidence that capsid radius is the quantization-ruler. Capsid-based DNA length quantization possibly evolved via selection for stalling that provides time for feedback control during DNA packaging and injection.


Subject(s)
Bacteriophage T3/physiology , Bacteriophage T3/ultrastructure , DNA, Viral/metabolism , Mutation , Virion/physiology , Virion/ultrastructure , Virus Assembly , Bacteriophage T3/genetics , DNA, Viral/ultrastructure , Deoxyribonuclease I/metabolism , Electrophoresis , Microscopy, Electron, Transmission , Virion/genetics
16.
Bacteriophage ; 4(4): e961869, 2014.
Article in English | MEDLINE | ID: mdl-26713220

ABSTRACT

Drug development has typically been a primary foundation of strategy for systematic, long-range management of pathogenic cells. However, drug development is limited in speed and flexibility when response is needed to changes in pathogenic cells, especially changes that produce drug-resistance. The high replication speed and high diversity of phages are potentially useful for increasing both response speed and response flexibility when changes occur in either drug resistance or other aspects of pathogenic cells. We present strategy, with some empirical details, for (1) using modern molecular biology and biophysics to access these advantages during the phage therapy of bacterial infections, and (2) initiating use of phage capsid-based drug delivery vehicles (DDVs) with procedures that potentially overcome both drug resistance and other present limitations in the use of DDVs for the therapy of neoplasms. The discussion of phage therapy includes (a) historical considerations, (b) changes that appear to be needed in clinical tests if use of phage therapy is to be expanded, (c) recent work on novel phages and its potential use for expanding the capabilities of phage therapy and (d) an outline for a strategy that encompasses both theory and practice for expanding the applications of phage therapy. The discussion of DDVs starts by reviewing current work on DDVs, including work on both liposomal and viral DDVs. The discussion concludes with some details of the potential use of permeability constrained phage capsids as DDVs.

17.
Proc Natl Acad Sci U S A ; 110(17): 6811-6, 2013 Apr 23.
Article in English | MEDLINE | ID: mdl-23580619

ABSTRACT

Motor-driven packaging of a dsDNA genome into a preformed protein capsid through a unique portal vertex is essential in the life cycle of a large number of dsDNA viruses. We have used single-particle electron cryomicroscopy to study the multilayer structure of the portal vertex of the bacteriophage T7 procapsid, the recipient of T7 DNA in packaging. A focused asymmetric reconstruction method was developed and applied to selectively resolve neighboring pairs of symmetry-mismatched layers of the portal vertex. However, structural features in all layers of the multilayer portal vertex could not be resolved simultaneously. Our results imply that layers with mismatched symmetries can join together in several different relative orientations, and that orientations at different interfaces assort independently to produce structural isomers, a process that we call combinatorial assembly isomerism. This isomerism explains rotational smearing in previously reported asymmetric reconstructions of the portal vertex of T7 and other bacteriophages. Combinatorial assembly isomerism may represent a new regime of structural biology in which globally varying structures assemble from a common set of components. Our reconstructions collectively validate previously proposed symmetries, compositions, and sequential order of T7 portal vertex layers, resolving in tandem the 5-fold gene product 10 (gp10) shell, 12-fold gp8 portal ring, and an internal core stack consisting of 12-fold gp14 adaptor ring, 8-fold bowl-shaped gp15, and 4-fold gp16 tip. We also found a small tilt of the core stack relative to the icosahedral fivefold axis and propose that this tilt assists DNA spooling without tangling during packaging.


Subject(s)
Bacteriophage T7/genetics , Capsid Proteins/genetics , Capsid/ultrastructure , Genome, Viral/genetics , Models, Molecular , Virus Assembly/genetics , Cryoelectron Microscopy , Escherichia coli , Imaging, Three-Dimensional
18.
Electrophoresis ; 33(2): 352-65, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22222979

ABSTRACT

We find a new aspect of DNA packaging-associated structural fluidity for phage T3 capsids. The procedure is (i) glutaraldehyde cross-linking of in vivo DNA packaging intermediates for the stabilization of structure and then (ii) determining effective radius by two-dimensional agarose gel electrophoresis (2D-AGE). The intermediates are capsids with incompletely packaged DNA (ipDNA) and without an external DNA segment; these intermediates are called ipDNA-capsids. We initially increase the production of ipDNA-capsids by raising NaCl concentration during in vivo DNA packaging. By 2D-AGE, we find a new state of contracted shell for some particles of one previously identified ipDNA-capsid. The contracted shell-state is found when the ipDNA length/mature DNA length (F) is above 0.17, but not at lower F. Some contracted-shell ipDNA-capsids have the phage tail; others do not. The contracted-shell ipDNA-capsids are explained by premature DNA maturation cleavage that makes accessible a contracted-shell intermediate of a cycle of the T3 DNA packaging motor. The analysis of ipDNA-capsids, rather than intermediates with uncleaved DNA, provides a simplifying strategy for a complete biochemical analysis of in vivo DNA packaging.


Subject(s)
Bacteriophage T3/chemistry , Capsid/chemistry , DNA Packaging/physiology , Electrophoresis, Agar Gel/methods , Electrophoresis, Gel, Two-Dimensional/methods , Bacteriophage T3/genetics , Cross-Linking Reagents/pharmacology , DNA Packaging/drug effects , DNA, Viral/chemistry , Glutaral/pharmacology , Particle Size , Sodium Chloride/pharmacology
19.
J Mol Biol ; 397(2): 361-74, 2010 Mar 26.
Article in English | MEDLINE | ID: mdl-20122936

ABSTRACT

Evidence that in vivo bacteriophage T3 DNA packaging includes capsid hyper-expansion that is triggered by lengthening of incompletely packaged DNA (ipDNA) is presented here. This evidence includes observation that some of the longer ipDNAs in T3-infected cells are packaged in ipDNA-containing capsids with hyper-expanded outer shells (HE ipDNA-capsids). In addition, artificially induced hyper-expansion is observed for the outer shell of a DNA-free capsid. Detection and characterization of HE ipDNA-capsids are based on two-dimensional, non-denaturing agarose gel electrophoresis, followed by structure determination with electron microscopy and protein identification with SDS-PAGE/mass spectrometry. After expulsion from HE ipDNA-capsids, ipDNA forms sharp bands during gel electrophoresis. The following hypotheses are presented: (1) T3 has evolved feedback-initiated, ATP-driven capsid contraction/hyper-expansion cycles that accelerate DNA packaging when packaging is slowed by increase in the packaging-resisting force of the ipDNA and (2) each gel electrophoretic ipDNA band reflects a contraction/hyper-expansion cycle.


Subject(s)
Bacteriophage T3/physiology , Capsid/metabolism , DNA Packaging , DNA, Viral/metabolism , Capsid/ultrastructure , Electrophoresis, Agar Gel , Electrophoresis, Polyacrylamide Gel , Mass Spectrometry , Microscopy, Electron , Models, Biological , Viral Proteins/analysis
20.
J Mol Biol ; 384(5): 1384-99, 2008 Dec 31.
Article in English | MEDLINE | ID: mdl-18952096

ABSTRACT

The tightly packaged double-stranded DNA (dsDNA) genome in the mature particles of many tailed bacteriophages has been shown to form multiple concentric rings when reconstructed from cryo-electron micrographs. However, recent single-particle DNA packaging force measurements have suggested that incompletely packaged DNA (ipDNA) is less ordered when it is shorter than approximately 25% of the full genome length. The study presented here initially achieves both the isolation and the ipDNA length-based fractionation of ipDNA-containing T3 phage capsids (ipDNA-capsids) produced by DNA packaging in vivo; some ipDNA has quantized lengths, as judged by high-resolution gel electrophoresis of expelled DNA. This is the first isolation of such particles among the tailed dsDNA bacteriophages. The ipDNA-capsids are a minor component (containing approximately 10(-4) of packaged DNA in all particles) and are initially detected by nondenaturing gel electrophoresis after partial purification by buoyant density centrifugation. The primary contaminants are aggregates of phage particles and empty capsids. This study then investigates ipDNA conformations by the first cryo-electron microscopy of ipDNA-capsids produced in vivo. The 3-D structures of DNA-free capsids, ipDNA-capsids with various lengths of ipDNA, and mature bacteriophage are reconstructed, which reveals the typical T=7l icosahedral shell of many tailed dsDNA bacteriophages. Though the icosahedral shell structures of these capsids are indistinguishable at the current resolution for the protein shell (approximately 15 A), the conformations of the DNA inside the shell are drastically different. T3 ipDNA-capsids with 10.6 kb or shorter dsDNA (<28% of total genome) have an ipDNA conformation indistinguishable from random. However, T3 ipDNA-capsids with 22 kb DNA (58% of total genome) form a single DNA ring next to the inner surface of the capsid shell. In contrast, dsDNA fully packaged (38.2 kb) in mature T3 phage particles forms multiple concentric rings such as those seen in other tailed dsDNA bacteriophages. The distance between the icosahedral shell and the outermost DNA ring decreases in the mature, fully packaged phage structure. These results suggest that, in the early stage of DNA packaging, the dsDNA genome is randomly distributed inside the capsid, not preferentially packaged against the inner surface of the capsid shell, and that the multiple concentric dsDNA rings seen later are the results of pressure-driven close-packing.


Subject(s)
Bacteriophage T3/chemistry , Bacteriophage T3/ultrastructure , Capsid/ultrastructure , DNA Packaging , DNA, Viral/ultrastructure , Bacteriophage T3/genetics , Capsid Proteins/chemistry , Capsid Proteins/ultrastructure , Cryoelectron Microscopy , DNA, Viral/analysis , Electrophoresis, Agar Gel , Electrophoresis, Polyacrylamide Gel , Mass Spectrometry , Nucleic Acid Denaturation , Protein Structure, Quaternary
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