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Sample records for bacteriophage phi29 dna

  1. Fingerprinting of Peptides with a Large Channel of Bacteriophage Phi29 DNA Packaging Motor.

    PubMed

    Ji, Zhouxiang; Wang, Shaoying; Zhao, Zhengyi; Zhou, Zhi; Haque, Farzin; Guo, Peixuan

    2016-09-01

    Nanopore technology has become a highly sensitive and powerful tool for single molecule sensing of chemicals and biopolymers. Protein pores have the advantages of size amenability, channel homogeneity, and fabrication reproducibility. But most well-studied protein pores for sensing are too small for passage of peptide analytes that are typically a few nanometers in dimension. The funnel-shaped channel of bacteriophage phi29 DNA packaging motor has previously been inserted into a lipid membrane to serve as a larger pore with a narrowest N-terminal constriction of 3.6 nm and a wider C-terminal end of 6 nm. Here, the utility of phi29 motor channel for fingerprinting of various peptides using single molecule electrophysiological assays is reported. The translocation of peptides is proved unequivocally by single molecule fluorescence imaging. Current blockage percentage and distinctive current signatures are used to distinguish peptides with high confidence. Each peptide generated one or two distinct current blockage peaks, serving as typical fingerprint for each peptide. The oligomeric states of peptides can also be studied in real time at single molecule level. The results demonstrate the potential for further development of phi29 motor channel for detection of disease-associated peptide biomarkers. PMID:27435806

  2. Supercoiled DNA wraps around the bacteriophage phi 29 head-tail connector.

    PubMed Central

    Turnquist, S; Simon, M; Egelman, E; Anderson, D

    1992-01-01

    Supercoiled pBR322 DNA wraps around the outside of the isolated Bacillus subtilis bacteriophage phi 29 head-tail connector, the crux of the DNA packaging machine of the viral precursor capsid or prohead. The contour length of the supercoiled DNA, determined by EM, decreased by approximately 180 base pairs for each connector bound. Mass and radial density determinations by scanning transmission EM showed that the increased mass of the connector-DNA complex relative to the connector alone was equivalent to approximately 170 base pairs of DNA and was located around the outside of the connector. Topoisomerase I treatment of the complexes followed by deproteinization suggested that supercoils were restrained by the connectors. Connectors bound linear and open-circular plasmid DNAs inefficiently but were not wrapped by these DNAs. The wrapping of supercoiled DNA around the isolated phi 29 connector is hypothesized to reflect the initiation phase of the normal process of DNA packaging. Packaging substrates would be supercoiled, wrapped by the connector, linearized, and translocated by rotation of the connector relative to the viral capsid with the aid of ATP hydrolysis. Images PMID:1438237

  3. Structural changes of bacteriophage [phi]29 upon DNA packaging and release

    SciTech Connect

    Xiang, Y.; Morais, M.C.; Battisti, A.J.; Grimes, S.; Jardine, P.J.; Anderson, D.L.; Rossmann, M.G.

    2008-04-24

    Cryo-electron microscopy three-dimensional reconstructions have been made of mature and of emptied bacteriophage {phi}29 particles without making symmetry assumptions. Comparisons of these structures with each other and with the {phi}29 prohead indicate how conformational changes might initiate successive steps of assembly and infection. The 12 adsorption capable 'appendages' were found to have a structure homologous to the bacteriophage P22 tailspikes. Two of the appendages are extended radially outwards, away from the long axis of the virus, whereas the others are around and parallel to the phage axis. The appendage orientations are correlated with the symmetry-mismatched positions of the five-fold related head fibers, suggesting a mechanism for partial cell wall digestion upon rotation of the head about the tail when initiating infection. The narrow end of the head-tail connector is expanded in the mature virus. Gene product 3, bound to the 5-foot ends of the genome, appears to be positioned within the expanded connector, which may potentiate the release of DNA-packaging machine components, creating a binding site for attachment of the tail.

  4. Experimental test of connector rotation during DNA packaging into bacteriophage phi29 capsids.

    PubMed

    Hugel, Thorsten; Michaelis, Jens; Hetherington, Craig L; Jardine, Paul J; Grimes, Shelley; Walter, Jessica M; Falk, Wayne; Anderson, Dwight L; Bustamante, Carlos

    2007-03-01

    The bacteriophage phi29 generates large forces to compact its double-stranded DNA genome into a protein capsid by means of a portal motor complex. Several mechanical models for the generation of these high forces by the motor complex predict coupling of DNA translocation to rotation of the head-tail connector dodecamer. Putative connector rotation is investigated here by combining the methods of single-molecule force spectroscopy with polarization-sensitive single-molecule fluorescence. In our experiment, we observe motor function in several packaging complexes in parallel using video microscopy of bead position in a magnetic trap. At the same time, we follow the orientation of single fluorophores attached to the portal motor connector. From our data, we can exclude connector rotation with greater than 99% probability and therefore answer a long-standing mechanistic question. PMID:17311473

  5. The bacteriophage straight phi29 portal motor can package DNA against a large internal force.

    PubMed

    Smith, D E; Tans, S J; Smith, S B; Grimes, S; Anderson, D L; Bustamante, C

    2001-10-18

    As part of the viral infection cycle, viruses must package their newly replicated genomes for delivery to other host cells. Bacteriophage straight phi29 packages its 6.6-microm long, double-stranded DNA into a 42 x 54 nm capsid by means of a portal complex that hydrolyses ATP. This process is remarkable because entropic, electrostatic and bending energies of the DNA must be overcome to package the DNA to near-crystalline density. Here we use optical tweezers to pull on single DNA molecules as they are packaged, thus demonstrating that the portal complex is a force-generating motor. This motor can work against loads of up to 57 pN on average, making it one of the strongest molecular motors reported to date. Movements of over 5 microm are observed, indicating high processivity. Pauses and slips also occur, particularly at higher forces. We establish the force-velocity relationship of the motor and find that the rate-limiting step of the motor's cycle is force dependent even at low loads. Notably, the packaging rate decreases as the prohead is filled, indicating that an internal force builds up to approximately 50 pN owing to DNA confinement. Our data suggest that this force may be available for initiating the ejection of the DNA from the capsid during infection. PMID:11607035

  6. Construction of Bacteriophage Phi29 DNA Packaging Motor and its Applications in Nanotechnology and Therapy

    PubMed Central

    Lee, Tae Jin; Schwartz, Chad; Guo, Peixuan

    2010-01-01

    Nanobiotechnology involves the creation, characterization, and modification of organized nanomaterials to serve as building blocks for constructing nanoscale devices in technology and medicine. Living systems contain a wide variety of nanomachines and highly ordered structures of macromolecules. The novelty and ingenious design of the bacterial virus phi29 DNA packaging motor and its parts inspired the synthesis of this motor and its components as biomimetics. This 30-nm nanomotor uses six copies of an ATP-binding pRNA to gear the motor. The structural versatility of pRNA has been utilized to construct dimers, trimers, hexamers, and patterned superstructures via the interaction of two interlocking loops. The approach, based on bottom-up assembly, has also been applied to nanomachine fabrication, pathogen detection and the delivery of drugs, siRNA, ribozymes, and genes to specific cells in vitro and in vivo. Another essential component of the motor is the connector, which contains 12 copies of a protein gp10 to form a 3.6-nm central channel as a path for DNA. This article will review current studies of the structure and function of the phi29 DNA packaging motor, as well as the mechanism of motion, the principle of in vitro construction, and its potential nanotechnological and medical applications. PMID:19495981

  7. Signals at the bacteriophage phi 29 DNA replication origins required for protein p6 binding and activity.

    PubMed Central

    Serrano, M; Gutiérrez, J; Prieto, I; Hermoso, J M; Salas, M

    1989-01-01

    Protein p6 of Bacillus subtilis phage phi 29 binds specifically to the ends of the viral DNA that contain the replication origins, giving rise to a nucleoprotein structure. DNA regions recognized by protein p6 have been mapped by deletion analysis and DNase I footprinting. Main protein p6-recognition signals have been located between nucleotides 62 and 125 at the right phi 29 DNA end and between nucleotides 46 and 68 at the left end. In addition, recognition signals are also present at other sites within 200-300 bp at each phi 29 DNA end. Protein p6 does not seem to recognize a specific sequence in the DNA, but rather a structural feature, which could be bendability. The formation of the protein p6-DNA nucleoprotein complex is likely to be the structural basis for the protein p6 activity in the initiation of replication. Images PMID:2767056

  8. Circularly permuted viral pRNA active and specific in the packaging of bacteriophage phi 29 DNA.

    PubMed

    Zhang, C; Trottier, M; Guo, P

    1995-03-10

    A viral-encoded 120-base pRNA has been shown to have an essential role in the packaging of bacteriophage phi 29 DNA. The finding that both the 5'- and 3'-termini of the pRNA are proximate and crucial for biological function (C. Zhang, C. Lee, and P. Guo, 1994, Virology, 201, 77-85) prompted investigation of the activity of circularly permuted pRNAs (cpRNA) and of the expandability and essentiality of bases extending from the termini. A 117-base pRNA with a deletion of three bases downstream of the proximal terminus was active in DNA packaging. Concatemeric DNAs containing two tandem pRNA genes separated by a short or a long loop sequence were constructed. The cpRNAs from these DNA templates were transcribed in vitro and shown to be active in phi 29 DNA packaging, with activity comparable to the parental (noncircularly permuted) pRNA, indicating that neither of the loops tested affected the activity and folding of the cpRNA. As few as four bases were sufficient to serve as a loop for the terminal 180 degree turn, and a loop as long as 27 bases did not affect the cpRNA structure and function. Eight cpRNAs were constructed to assess the effect of openings within the wild-type pRNA structure. Opening of the bulge at residue 38 did not affect cpRNA activity, but opening the bulge at residue 55 greatly reduced it. Although the sequence of the 5',3'-terminal loop was not important for the folding and activity of the cpRNA, the activities of cpRNAs with openings at individual bulges or hairpins were different, indicating that each region plays a different role in pRNA folding and function. Our results indicate that it is possible to generate active circularly permuted pRNA by assigning internal sites of the pRNA as new 3'- and 5'-termini. The creation of new variable ends makes the labeling of internal bases of the pRNA molecule possible and will facilitate the analysis of pRNA secondary and tertiary structure. PMID:7533964

  9. Single pore translocation of folded, double-stranded, and tetra-stranded DNA through channel of bacteriophage phi29 DNA packaging motor.

    PubMed

    Haque, Farzin; Wang, Shaoying; Stites, Chris; Chen, Li; Wang, Chi; Guo, Peixuan

    2015-01-01

    The elegant architecture of the channel of bacteriophage phi29 DNA packaging motor has inspired the development of biomimetics for biophysical and nanobiomedical applications. The reengineered channel inserted into a lipid membrane exhibits robust electrophysiological properties ideal for precise sensing and fingerprinting of dsDNA at the single-molecule level. Herein, we used single channel conduction assays to quantitatively evaluate the translocation dynamics of dsDNA as a function of the length and conformation of dsDNA. We extracted the speed of dsDNA translocation from the dwell time distribution and estimated the various forces involved in the translocation process. A ∼35-fold slower speed of translocation per base-pair was observed for long dsDNA, a significant contrast to the speed of dsDNA crossing synthetic pores. It was found that the channel could translocate both dsDNA with ∼32% of channel current blockage and with ∼64% for tetra-stranded DNA (two parallel dsDNA). The calculation of both cross-sectional areas of the dsDNA and tetra-stranded DNA suggested that the blockage was purely proportional to the physical space of the channel lumen and the size of the DNA substrate. Folded dsDNA configuration was clearly reflected in their characteristic current signatures. The finding of translocation of tetra-stranded DNA with 64% blockage is in consent with the recently elucidated mechanism of viral DNA packaging via a revolution mode that requires a channel larger than the dsDNA diameter of 2 nm to provide room for viral DNA revolving without rotation. The understanding of the dynamics of dsDNA translocation in the phi29 system will enable us to design more sophisticated single pore DNA translocation devices for future applications in nanotechnology and personal medicine. PMID:25890769

  10. Single Pore Translocation of Folded, Double-Stranded, and Tetra-stranded DNA through Channel of Bacteriophage Phi29 DNA Packaging Motor

    PubMed Central

    Haque, Farzin; Wang, Shaoying; Stites, Chris; Chen, Li; Wang, Chi; Guo, Peixuan

    2015-01-01

    The elegant architecture of the channel of bacteriophage phi29 DNA packaging motor has inspired the development of biomimetics for biophysical and nanobiomedical applications. The reengineered channel inserted into a lipid membrane exhibits robust electrophysiological properties ideal for precise sensing and fingerprinting of dsDNA at the single-molecule level. Herein, we used single channel conduction assays to quantitatively evaluate the translocation dynamics of dsDNA as a function of the length and conformation of dsDNA. We extracted the speed of dsDNA translocation from the dwell time distribution and estimated the various forces involved in the translocation process. A ~35-fold slower speed of translocation per base pair was observed for long dsDNA, a significant contrast to the speed of dsDNA crossing synthetic pores. It was found that the channel could translocate both dsDNA with ~32% of channel current blockage and ~64% for tetra-stranded DNA (two parallel dsDNA). The calculation of both cross-sectional areas of the dsDNA and tetra-stranded DNA suggested that the blockage was purely proportional to the physical space of the channel lumen and the size of the DNA substrate. Folded dsDNA configuration was clearly reflected in their characteristic current signatures. The finding of translocation of tetra-stranded DNA with 64% blockage is in consent with the recently elucidated mechanism of viral DNA packaging via a revolution mode that requires a channel larger than the dsDNA diameter of 2 nm to provide room for viral DNA revolving without rotation. The understanding of the dynamics of dsDNA translocation in the phi29 system will enable us to design more sophisticated single pore DNA translocation devices for future applications in nanotechnology and personal medicine. PMID:25890769

  11. Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: prohead restoration for DNA-gp3 packaging and assembly.

    PubMed Central

    Bjornsti, M A; Reilly, B E; Anderson, D L

    1985-01-01

    The DNA-protein complex DNA-gp3 of phi 29 is efficiently packaged into purified proheads with the aid of plasmid-derived gp16. The filled heads can be assembled to phage by addition of an extract providing the products for neck-tail assembly (Bjornsti et al., J. Virol. 50:766-772, 1984). However, purified proheads lost their competence to package DNA-gp3 upon storage for 2 months at 4 degrees C. Competence was restored by complementation with extracts of certain mutant-infected cells, and these experiments demonstrated that late proteins were not involved; restoration obtained with 4-8-14--infected cells was indistinguishable from that obtained with 7-8-14--infected cells. 2-8-14- and 3-8-14- extracts restored about one-third of the capacity to package exogenous DNA-gp3. A 1-8-14- extracts restored activity to package 20.6% of the DNA-gp3 added, but phage were not produced. PMID:3919187

  12. My life with bacteriophage phi29.

    PubMed

    Salas, Margarita

    2012-12-28

    This article is a survey of my scientific work over 52 years. During my postdoctoral stay in Severo Ochoa's laboratory, I determined the direction of reading of the genetic message, and I discovered two proteins that I showed to be involved in the initiation of protein synthesis. The work I have done in Spain with bacteriophage ϕ29 for 45 years has been very rewarding. I can say that I was lucky because I did not expect that ϕ29 would give so many interesting results, but I worked hard, with a lot of dedication and enthusiasm, and I was there when the luck arrived. I would like to emphasize our work on the control of ϕ29 DNA transcription and, in particular, the finding for the first time of a protein covalently linked to the 5'-ends of ϕ29 DNA that we later showed to be the primer for the initiation of phage DNA replication. Very relevant was the discovery of the ϕ29 DNA polymerase, with its properties of extremely high processivity and strand displacement capacity, together with its high fidelity. The ϕ29 DNA polymerase has become an ideal enzyme for DNA amplification, both rolling-circle and whole-genome linear amplification. I am also very proud of the many brilliant students and collaborators with whom I have worked over the years and who have become excellent scientists. This Reflections article is not intended to be the end of my scientific career. I expect to work for many years to come. PMID:23124207

  13. Structure and Function Study of Phi29 DNA packaging motor

    NASA Astrophysics Data System (ADS)

    Fang, Huaming

    A powerful nanomotor is employed by the tailed dsDNA virus to package the genome into a preformed protein shell during the process of replication. The bacteriophage phi29 is an excellent model for investigating the viral DNA packaging mechanism. The phi29 DNA packaging motor is composed of three ring structures: the dodecameric connector ring, the hexameric pRNA ring and the hexameric ATPase gp16 ring. The connector is the central hub for the DNA to enter and to exit. There are four positively charged lysine rings scattered inside the highly negatively charged connector channel. It is speculated that these positive charged lysine rings may play active roles during DNA packaging in many models. To test this prevalent view, the basic lysine residues were mutated to neutral alanines and the pH environment was altered. Amazingly, the results were beyond expectation. Neither the DNA translocation nor the one-way traffic property of the channel were measurably influenced by the alteration of the charge of lysine residues when the basic lysine residues mutated to neutral alanines or the pH environment changed to acid or basic. The ATPase or the terminase is the central part of the viral DNA packaging motor. The phi29 ATPase is highly hydrophobic and tends to aggregate in solution. A green fluorescent protein tag (eGFP) fused to the N-terminus of gp16 enhanced its solubility and stability. The eGFP-gp16 showed similar activity to wild type gp16 and was easily detected by fluorescent instruments. The interaction between eGFP-gp16 and DNA in the various conditions were investigated by electrophoretic mobility shift assay, FRET and sucrose gradient. gamma-S-ATP dramatically increased gp16 binding affinity to DNA and ATP, ADP, phosphate could release gp16 from gp16-DNA-gamma-S-ATP complex. The sliding of gp16 out of the gp16-DNA-gamma-S-ATP complex could be blocked by addition of Steptavidin to ends of dsDNA which is conjugated with biotins. Also, we found that six eGFP-gp16

  14. Construction of RNA nanocages by re-engineering the packaging RNA of Phi29 bacteriophage

    NASA Astrophysics Data System (ADS)

    Hao, Chenhui; Li, Xiang; Tian, Cheng; Jiang, Wen; Wang, Guansong; Mao, Chengde

    2014-05-01

    RNA nanotechnology promises rational design of RNA nanostructures with wide array of structural diversities and functionalities. Such nanostructures could be used in applications such as small interfering RNA delivery and organization of in vivo chemical reactions. Though having impressive development in recent years, RNA nanotechnology is still quite limited and its programmability and complexity could not rival the degree of its closely related cousin: DNA nanotechnology. Novel strategies are needed for programmed RNA self-assembly. Here, we have assembled RNA nanocages by re-engineering a natural, biological RNA motif: the packaging RNA of phi29 bacteriophage. The resulting RNA nanostructures have been thoroughly characterized by gel electrophoresis, cryogenic electron microscopy imaging and dynamic light scattering.

  15. Robust properties of membrane-embedded connector channel of bacterial virus phi29 DNA packaging motor.

    PubMed

    Jing, Peng; Haque, Farzin; Vonderheide, Anne P; Montemagno, Carlo; Guo, Peixuan

    2010-10-01

    Biological systems contain highly-ordered macromolecular structures with diverse functions, inspiring their utilization in nanotechnology. A motor allows linear dsDNA viruses to package their genome into a preformed procapsid. The central component of the motor is the portal connector that acts as a pathway for the translocation of dsDNA. The elegant design of the connector and its channel motivates its application as an artificial nanopore (Nature Nanotechnology, 4, 765-772). Herein, we demonstrate the robust characteristics of the connector of the bacteriophage phi29 DNA packaging motor by single pore electrophysiological assays. The conductance of each pore is almost identical and is perfectly linear with respect to the applied voltage. Numerous transient current blockade events induced by dsDNA are consistent with the dimensions of the channel and dsDNA. Furthermore, the connector channel is stable under a wide range of experimental conditions including high salt and pH 2-12. The robust properties of the connector nanopore made it possible to develop a simple reproducible approach for connector quantification. The precise number of connectors in each sheet of the membrane was simply derived from the slopes of the plot of voltage against current. Such quantifications led to a reliable real time counting of DNA passing through the channel. The fingerprint of DNA translocation in this system has provided a new tool for future biophysical and physicochemical characterizations of DNA transportation, motion, and packaging. PMID:20523933

  16. The effect of N- or C-terminal alterations of the connector of bacteriophage phi29 DNA packaging motor on procapsid assembly, pRNA binding, and DNA packaging

    PubMed Central

    Cai, Ying; Xiao, Feng; Guo, Peixuan

    2010-01-01

    Double-stranded DNA viruses package their genomes into procapsids via an ATP-driven nanomotor. This ingenious motor configuration has inspired the development of biomimetics in nanotechnology. Bacteriophage ϕ29 DNA-packaging motor has been a popular tool in nanomedicine. To provide information for further motor modification, conjugation, labeling, and manufacturing, the connector protein gp10 of the ϕ29 DNA packaging motor was truncated, mutated, and extended. A 25-residue deletion or a 14-residue extension at the C terminus of gp10 did not affect procapsid assembly. A 42–amino acid extension at the N terminus did not interfere with the procapsid assembly but significantly decreased the DNA-packaging efficiency. DNA-packaging activity was restored upon protease cleavage of the extended region. Replacing the N-terminal peptide containing arginine and lysine with a histidine-rich peptide did not affect procapsid assembly but completely inhibited the packaging RNA (pRNA) binding to the connector and hindered subsequent DNA packaging. These results indicate that (1) the N-terminal arginine-lysine residues play a critical role in pRNA binding but are not essential for procapsid assembly; (2) the connector core, but not the flexible N- or C-terminal domains, is responsible for signaling the procapsid assembly; (3) pRNA binds to the connector as a result of electrostatic interactions between the polyanionic nature of nucleic acids and the cationic side groups of the amino acids, similar to RNA binding to Tat or polyArg. PMID:18201942

  17. Rapid Detection and Identification of a Pathogen's DNA Using Phi29 DNA Polymerase

    SciTech Connect

    Xu, Y.; Dunn, J.; Gao, S.; Bruno, J. F.; Luft, B. J.

    2008-10-31

    Zoonotic pathogens including those transmitted by insect vectors are some of the most deadly of all infectious diseases known to mankind. A number of these agents have been further weaponized and are widely recognized as being potentially significant biothreat agents. We describe a novel method based on multiply-primed rolling circle in vitro amplification for profiling genomic DNAs to permit rapid, cultivation-free differential detection and identification of circular plasmids in infectious agents. Using Phi29 DNA polymerase and a two-step priming reaction we could reproducibly detect and characterize by DNA sequencing circular DNA from Borrelia burgdorferi B31 in DNA samples containing as little as 25 pg of Borrelia DNA amongst a vast excess of human DNA. This simple technology can ultimately be adapted as a sensitive method to detect specific DNA from both known and unknown pathogens in a wide variety of complex environments.

  18. Structure of bacteriophage [phi]29 head fibers has a supercoiled triple repeating helix-turn-helix motif

    SciTech Connect

    Xiang, Ye; Rossmann, Michael G.

    2011-12-22

    The tailed bacteriophage {phi}29 capsid is decorated with 55 fibers attached to quasi-3-fold symmetry positions. Each fiber is a homotrimer of gene product 8.5 (gp8.5) and consists of two major structural parts, a pseudohexagonal base and a protruding fibrous portion that is about 110 {angstrom} in length. The crystal structure of the C-terminal fibrous portion (residues 112-280) has been determined to a resolution of 1.6 {angstrom}. The structure is about 150 {angstrom} long and shows three distinct structural domains designated as head, neck, and stem. The stem region is a unique three-stranded helix-turn-helix supercoil that has not previously been described. When fitted into a cryoelectron microscope reconstruction of the virus, the head structure corresponded to a disconnected density at the distal end of the fiber and the neck structure was located in weak density connecting it to the fiber. Thin section studies of Bacillus subtilis cells infected with fibered or fiberless {phi}29 suggest that the fibers might enhance the attachment of the virions onto the host cell wall.

  19. Duality of polynucleotide substrates for Phi29 DNA polymerase: 3′→5′ RNase activity of the enzyme

    PubMed Central

    Lagunavicius, Arunas; Kiveryte, Zivile; Zimbaite-Ruskuliene, Vilma; Radzvilavicius, Tomas; Janulaitis, Arvydas

    2008-01-01

    Phi29 DNA polymerase is a small DNA-dependent DNA polymerase that belongs to eukaryotic B-type DNA polymerases. Despite the small size, the polymerase is a multifunctional proofreading-proficient enzyme. It catalyzes two synthetic reactions (polymerization and deoxynucleotidylation of Phi29 terminal protein) and possesses two degradative activities (pyrophosphorolytic and 3′→5′ DNA exonucleolytic activities). Here we report that Phi29 DNA polymerase exonucleolyticaly degrades ssRNA. The RNase activity acts in a 3′ to 5′ polarity. Alanine replacements in conserved exonucleolytic site (D12A/D66A) inactivated RNase activity of the enzyme, suggesting that a single active site is responsible for cleavage of both substrates: DNA and RNA. However, the efficiency of RNA hydrolysis is ∼10-fold lower than for DNA. Phi29 DNA polymerase is widely used in rolling circle amplification (RCA) experiments. We demonstrate that exoribonuclease activity of the enzyme can be used for the target RNA conversion into a primer for RCA, thus expanding application potential of this multifunctional enzyme and opening new opportunities for RNA detection. PMID:18230765

  20. The bacteriophage phi29 head-tail connector imaged at high resolution with the atomic force microscope in buffer solution.

    PubMed

    Müller, D J; Engel, A; Carrascosa, J L; Vélez, M

    1997-05-15

    The surfaces of two- and three-dimensional phi29 connector crystals were imaged in buffer solution by atomic force microscopy (AFM). Both topographies show a rectangular unit cell with dimensions of 16.5 nm x 16.5 nm. High resolution images of connectors from the two-dimensional crystal surface show two connectors per unit cell confirming the p42(1)2 symmetry. The height of the connector was estimated to be at least 7.6 nm, a value close to that found in previous studies using different techniques. The 12 subunits of the wide connector domain were clearly resolved and showed a right-handed vorticity. The channel running along the connector had a diameter of 3.7 nm in the wide domain, while it was 1.7 nm in the narrow domain end, thus suggesting a tronco-conical channel shape. Moreover, the narrow connector end appears to be rather flexible. When the force applied to the stylus was between 50 and 100 pN, the connector end was fully extended. At forces of approximately 150 pN, these ends were pushed towards the crystal surface. The complementation of the AFM data with the three-dimensional reconstruction obtained from electron microscopy not only confirmed the model proposed, but also offers new insights that may help to explain the role of the connector in DNA packing. PMID:9184202

  1. Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase.

    PubMed

    Manrao, Elizabeth A; Derrington, Ian M; Laszlo, Andrew H; Langford, Kyle W; Hopper, Matthew K; Gillgren, Nathaniel; Pavlenok, Mikhail; Niederweis, Michael; Gundlach, Jens H

    2012-04-01

    Nanopore technologies are being developed for fast and direct sequencing of single DNA molecules through detection of ionic current modulations as DNA passes through a pore's constriction. Here we demonstrate the ability to resolve changes in current that correspond to a known DNA sequence by combining the high sensitivity of a mutated form of the protein pore Mycobacterium smegmatis porin A (MspA) with phi29 DNA polymerase (DNAP), which controls the rate of DNA translocation through the pore. As phi29 DNAP synthesizes DNA and functions like a motor to pull a single-stranded template through MspA, we observe well-resolved and reproducible ionic current levels with median durations of ∼28 ms and ionic current differences of up to 40 pA. Using six different DNA sequences with readable regions 42-53 nucleotides long, we record current traces that map to the known DNA sequences. With single-nucleotide resolution and DNA translocation control, this system integrates solutions to two long-standing hurdles to nanopore sequencing. PMID:22446694

  2. Preparation of Phi29 DNA Polymerase Free of Amplifiable DNA Using Ethidium Monoazide, an Ultraviolet-Free Light-Emitting Diode Lamp and Trehalose

    PubMed Central

    Takahashi, Hirokazu; Yamazaki, Hiroyuki; Akanuma, Satoshi; Kanahara, Hiroko; Saito, Toshiyuki; Chimuro, Tomoyuki; Kobayashi, Takayoshi; Ohtani, Toshio; Yamamoto, Kimiko; Sugiyama, Shigeru; Kobori, Toshiro

    2014-01-01

    We previously reported that multiply-primed rolling circle amplification (MRPCA) using modified random RNA primers can amplify tiny amounts of circular DNA without producing any byproducts. However, contaminating DNA in recombinant Phi29 DNA polymerase adversely affects the outcome of MPRCA, especially for negative controls such as non-template controls. The amplified DNA in negative control casts doubt on the result of DNA amplification. Since Phi29 DNA polymerase has high affinity for both single-strand and double-stranded DNA, some amount of host DNA will always remain in the recombinant polymerase. Here we describe a procedure for preparing Phi29 DNA polymerase which is essentially free of amplifiable DNA. This procedure is realized by a combination of host DNA removal using appropriate salt concentrations, inactivation of amplifiable DNA using ethidium monoazide, and irradiation with visible light from a light-emitting diode lamp. Any remaining DNA, which likely exists as oligonucleotides captured by the Phi29 DNA polymerase, is degraded by the 3′-5′ exonuclease activity of the polymerase itself in the presence of trehalose, used as an anti-aggregation reagent. Phi29 DNA polymerase purified by this procedure has little amplifiable DNA, resulting in reproducible amplification of at least ten copies of plasmid DNA without any byproducts and reducing reaction volume. This procedure could aid the amplification of tiny amounts DNA, thereby providing clear evidence of contamination from laboratory environments, tools and reagents. PMID:24505243

  3. Novel application of Phi29 DNA polymerase: RNA detection and analysis in vitro and in situ by target RNA-primed RCA

    PubMed Central

    Lagunavicius, Arunas; Merkiene, Egle; Kiveryte, Zivile; Savaneviciute, Agne; Zimbaite-Ruskuliene, Vilma; Radzvilavicius, Tomas; Janulaitis, Arvydas

    2009-01-01

    We present a novel Phi29 DNA polymerase application in RCA-based target RNA detection and analysis. The 3′→5′ RNase activity of Phi29 DNA polymerase converts target RNA into a primer and the polymerase uses this newly generated primer for RCA initiation. Therefore, using target RNA-primed RCA, padlock probes may be targeted to inner RNA sequences and their peculiarities can be analyzed directly. We demonstrate that the exoribonucleolytic activity of Phi29 DNA polymerase can be successfully applied in vitro and in situ. These findings expand the potential for detection and analysis of RNA sequences distanced from 3′-end. PMID:19244362

  4. The highly conserved amino acid sequence motif Tyr-Gly-Asp-Thr-Asp-Ser in alpha-like DNA polymerases is required by phage phi 29 DNA polymerase for protein-primed initiation and polymerization.

    PubMed Central

    Bernad, A; Lázaro, J M; Salas, M; Blanco, L

    1990-01-01

    The alpha-like DNA polymerases from bacteriophage phi 29 and other viruses, prokaryotes and eukaryotes contain an amino acid consensus sequence that has been proposed to form part of the dNTP binding site. We have used site-directed mutants to study five of the six highly conserved consecutive amino acids corresponding to the most conserved C-terminal segment (Tyr-Gly-Asp-Thr-Asp-Ser). Our results indicate that in phi 29 DNA polymerase this consensus sequence, although irrelevant for the 3'----5' exonuclease activity, is essential for initiation and elongation. Based on these results and on its homology with known or putative metal-binding amino acid sequences, we propose that in phi 29 DNA polymerase the Tyr-Gly-Asp-Thr-Asp-Ser consensus motif is part of the dNTP binding site, involved in the synthetic activities of the polymerase (i.e., initiation and polymerization), and that it is involved particularly in the metal binding associated with the dNTP site. Images PMID:2191296

  5. Genetic analysis of bacteriophage phi 29 of Bacillus subtilis: integration and mapping of reference mutants of two collections.

    PubMed Central

    Mellado, R P; Moreno, F; Viñuela, E; Salas, M; Reilly, B E; Anderson, D L

    1976-01-01

    Reference mutants of Bacillus subtilis phage phi 29 of the Madrid and Minneapolis collections were employed to construct a genetic map. Suppressor-sensitive and temperature-sensitive mutants were assigned to 17 cistrons by quantitative complementation. Three-factor crosses were used to assign an unambiguous order for the 17 cistrons. Recombination frequencies determined by two-factor crosses were used to construct a linear genetic map of 24.4 recombination units. The genes were numbered sequentially from left to right (1 to 17) according to their relative map position. PMID:822174

  6. In vitro transcription of the Bacillus subtilis phage phi 29 DNA by Bacillus subtilis and Escherichia coli RNA polymerases.

    PubMed Central

    Sogo, J M; Lozano, M; Salas, M

    1984-01-01

    The Escherichia coli RNA polymerase bound to phage phi 29 DNA has been visualized by electron microscopy. Thirteen specific binding sites have been observed at 1.7,2.6,5.5,10.4,13.7,25.2,25.7,26.3,33.5,59.5,69.2,91.7 and 99.6 DNA length units and they have been named A1,A1I,A1II,A1III,A1IV,A2,A2I, A3, A4,B1,B1I,C1 and C2, respectively. The binding sites A1,A2,A3,B1,C1 and C2 coincide with those found with Bacillus subtilis RNA polymerase. The transcription of phage phi 29 DNA with B. subtilis or E. coli RNA polymerases has been studied. With the B. subtilis RNA polymerase eight transcripts were found, starting at positions corresponding to the binding sites A1, A1III, A2,A3,B1I,B2,C1 and C2, respectively. With the E. coli RNA polymerase the same transcripts were found and a new one starting at position corresponding to the A4 binding site. The RNAs starting at binding sites A1,A1III,A2,B1I, B2,C1 and C2 are transcribed from right to left, as expected for early RNA. The RNAs which initiate at positions A3 and A4 are transcribed from left to right and probably correspond to late RNAs. Images PMID:6322128

  7. Phi29 Connector-DNA Interactions Govern DNA Crunching and Rotation, Supporting the Check-Valve Model.

    PubMed

    Kumar, Rajendra; Grubmüller, Helmut

    2016-01-19

    During replication of the ϕ29 bacteriophage inside a bacterial host cell, a DNA packaging motor transports the viral DNA into the procapsid against a pressure difference of up to 40 ± 20 atm. Several models have been proposed for the underlying molecular mechanism. Here we have used molecular dynamics simulations to examine the role of the connector part of the motor, and specifically the one-way revolution and the push-roll model. We have focused at the structure and intermolecular interactions between the DNA and the connector, for which a near-complete structure is available. The connector is found to induce considerable DNA deformations with respect to its canonical B-form. We further assessed by force-probe simulations to which extent the connector is able to prevent DNA leakage and found that the connector can act as a partial one-way valve by a check-valve mechanism via its mobile loops. Analysis of the geometry, flexibility, and energetics of channel lysine residues suggested that this arrangement of residues is incompatible with the observed DNA packaging step-size of ∼2.5 bp, such that the step-size is probably determined by the other components of the motor. Previously proposed DNA revolution and rolling motions inside the connector channel are both found implausible due to structural entanglement between the DNA and connector loops that have not been resolved in the crystal structure. Rather, in the simulations, the connector facilitates minor DNA rotation during the packaging process compatible with recent optical-tweezers experiments. Combined with the available experimental data, our simulation results suggest that the connector acts as a check-valve that prevents DNA leakage and induces DNA compression and rotation during DNA packaging. PMID:26789768

  8. Programmable folding of fusion RNA in vivo and in vitro driven by pRNA 3WJ motif of phi29 DNA packaging motor

    PubMed Central

    Shu, Dan; Khisamutdinov, Emil F.; Zhang, Le; Guo, Peixuan

    2014-01-01

    Misfolding and associated loss of function are common problems in constructing fusion RNA complexes due to changes in energy landscape and the nearest-neighbor principle. Here we report the incorporation and application of the pRNA-3WJ motif of the phi29 DNA packaging motor into fusion RNA with controllable and predictable folding. The motif included three discontinuous ∼18 nucleotide (nt) fragments, displayed a distinct low folding energy (Shu D et al., Nature Nanotechnology, 2011, 6:658–667), and folded spontaneously into a leading core that enabled the correct folding of other functionalities fused to the RNA complex. Three individual fragments dispersed at any location within the sequence allowed the other RNA functional modules to fold into their original structures with authentic functions, as tested by Hepatitis B virus ribozyme, siRNA, and aptamers for malachite green (MG), spinach, and streptavidin (STV). Only nine complementary nucleotides were present for any two of the three ∼18-nt fragments, but the three 9 bp branches were so powerful that they disrupted other double strands with more than 15 bp within the fusion RNA. This system enabled the production of fusion complexes harboring multiple RNA functionalities with correct folding for potential applications in biotechnology, nanomedicine and nanotechnology. We also applied this system to investigate the principles governing the folding of RNA in vivo and in vitro. Temporal production of RNA sequences during in vivo transcription caused RNA to fold into different conformations that could not be predicted with routine principles derived from in vitro studies. PMID:24084081

  9. Construction and 3-D computer modeling of connector arrays with tetragonal to decagonal transition induced by pRNA of phi29 DNA-packaging motor.

    PubMed

    Guo, Yin Yin; Blocker, Forrest; Xiao, Feng; Guo, Peixuan

    2005-06-01

    The bottom-up assembly of patterned arrays is an exciting and important area in current nanotechnology. Arrays can be engineered to serve as components in chips for a virtually inexhaustible list of applications ranging from disease diagnosis to ultrahigh-density data storage. In attempting to achieve this goal, a number of methods to facilitate array design and production have been developed. Cloning and expression of the gene coding for the connector of the bacterial virus phi29 DNA-packaging motor, overproduction of the gene products, and the in vitro construction of large-scale carpet-like arrays composed of connector are described in this report. The stability of the arrays under various conditions, including varied pH, temperature and ionic strength, was tested. The addition of packaging RNA (pRNA) into the array caused a dramatic shift in array structure, and resulted in the conversion of tetragonal arrays into larger decagonal structures comprised of both protein and RNA. RNase digestion confirmed that the conformational shift was caused by pRNA, and that RNA was present in the decagons. As has been demonstrated in biomotors, conformational shift of motor components can generate force for motor motion. The conformational shift reported here can be utilized as a potential force-generating mechanism for the construction of nanomachines. Three-dimensional computer models of the constructed arrays were also produced using a variety of connector building blocks with or without the N- or C-terminal sequence, which is absent from the current published crystal structures. Both the connector array and the decagon are ideal candidates to be used as templates to build patterned suprastructures in nanotechnology. PMID:16060143

  10. Oriented single directional insertion of nanochannel of bacteriophage SPP1 DNA packaging motor into lipid bilayer via polar hydrophobicity.

    PubMed

    Zhou, Zhi; Ji, Zhouxiang; Wang, Shaoying; Haque, Farzin; Guo, Peixuan

    2016-10-01

    Insertion of biological nanopore into artificial membrane is of fundamental importance in nanotechnology. Many applications require control and knowledge of channel orientation. In this work, the insertion orientation of the bacteriophage SPP1 and phi29 DNA packaging motors into lipid membranes was investigated. Single molecule electrophysiological assays and Ni-NTA-nanogold binding assays revealed that both SPP1 and phi29 motor channels exhibited a one-way traffic property for TAT peptide translocation from N- to C-termini of the protein channels. SPP1 motor channels preferentially inserts into liposomes with their C-terminal wider region facing inward. Changing the hydrophobicity of the N- or C-termini of phi29 connector alters the insertion orientation, suggesting that the hydrophobicity and hydrophilicity of the termini of the protein channel governs the orientation of the insertion into lipid membrane. It is proposed that the specificity in motor channel orientation is a result of the hydrophilic/hydrophobic interaction at the air/water interface when the protein channels are incorporating into liposome membranes. PMID:27529454

  11. Crystal structure of 3WJ core revealing divalent ion-promoted thermostability and assembly of the Phi29 hexameric motor pRNA

    PubMed Central

    Zhang, Hui; Endrizzi, James A.; Shu, Yi; Haque, Farzin; Sauter, Claude; Shlyakhtenko, Lyudmila S.; Lyubchenko, Yuri; Guo, Peixuan; Chi, Young-In

    2013-01-01

    The bacteriophage phi29 DNA packaging motor, one of the strongest biological motors characterized to date, is geared by a packaging RNA (pRNA) ring. When assembled from three RNA fragments, its three-way junction (3WJ) motif is highly thermostable, is resistant to 8 M urea, and remains associated at extremely low concentrations in vitro and in vivo. To elucidate the structural basis for its unusual stability, we solved the crystal structure of this pRNA 3WJ motif at 3.05 Å. The structure revealed two divalent metal ions that coordinate 4 nt of the RNA fragments. Single-molecule fluorescence resonance energy transfer (smFRET) analysis confirmed a structural change of 3WJ upon addition of Mg2+. The reported pRNA 3WJ conformation is different from a previously published construct that lacks the metal coordination sites. The phi29 DNA packaging motor contains a dodecameric connector at the vertex of the procapsid, with a central pore for DNA translocation. This portal connector serves as the foothold for pRNA binding to procapsid. Subsequent modeling of a connector/pRNA complex suggests that the pRNA of the phi29 DNA packaging motor exists as a hexameric complex serving as a sheath over the connector. The model of hexameric pRNA on the connector agrees with AFM images of the phi29 pRNA hexamer acquired in air and matches all distance parameters obtained from cross-linking, complementary modification, and chemical modification interference. PMID:23884902

  12. The bacteriophage DNA packaging machine.

    PubMed

    Feiss, Michael; Rao, Venigalla B

    2012-01-01

    Large dsDNA bacteriophages and herpesviruses encode a powerful ATP-driven DNA-translocating machine that encapsidates a viral genome into a preformed capsid shell or prohead. The key components of the packaging machine are the packaging enzyme (terminase, motor) and the portal protein that forms the unique DNA entrance vertex of prohead. The terminase complex, comprised of a recognition subunit (small terminase) and an endonuclease/translocase subunit (large terminase), cuts viral genome concatemers. The terminase-viral DNA complex docks on the portal vertex, assembling a motor complex containing five large terminase subunits. The pentameric motor processively translocates DNA until the head shell is full with one viral genome. The motor cuts the DNA again and dissociates from the full head, allowing head-finishing proteins to assemble on the portal, sealing the portal, and constructing a platform for tail attachment. A body of evidence from molecular genetics and biochemical, structural, and biophysical approaches suggests that ATP hydrolysis-driven conformational changes in the packaging motor (large terminase) power DNA motion. Various parts of the motor subunit, such as the ATPase, arginine finger, transmission domain, hinge, and DNA groove, work in concert to translocate about 2 bp of DNA per ATP hydrolyzed. Powerful single-molecule approaches are providing precise delineation of steps during each translocation event in a motor that has a speed as high as a millisecond/step. The phage packaging machine has emerged as an excellent model for understanding the molecular machines, given the mechanistic parallels between terminases, helicases, and numerous motor proteins. PMID:22297528

  13. Current advances in Phi29 pRNA biology and its application in drug delivery.

    PubMed

    Ye, Xin; Hemida, Maged; Zhang, Huifang M; Hanson, Paul; Ye, Qiu; Yang, Decheng

    2012-01-01

    Bacteriophage 29 (Phi29) packaging RNA (pRNA) is one of the key components in the viral DNA-packaging motor. It contains two functional domains facilitating the translocation of DNA into the viral capsid by interacting with other elements in the motor and promoting adenosine triphosphates hydrolysis. Through the connection between interlocking loops in adjacent pRNA monomers, pRNA functions in the form of multimer ring in the motor. Previous studies have addressed the unique structure and conformation of pRNA. However, there are different DNA-packaging models proposed for the viral genome transportation mechanism. The DNA-packaging ability and the unique features of pRNA have been attracting efforts to study its potential applications in nanotechnology. The pRNA has been proved to be a promising tool for delivering nucleic acid-based therapeutic molecules by covalent linkage with ribozymes, small interfering RNAs, aptamers, and artificial microRNAs. The flexibility in constructing dimers, trimers, and hexamers enables the assembly of polyvalent nanoparticles to carry drug molecules for therapeutic purposes, cell ligands for target delivery, image detector for drug entry monitoring, and endosome disrupter for drug release. Besides these fascinating pharmacological advantages, pRNA-based drug delivery has also been demonstrated to prolong the drug half life with minimal induction of immune response and toxicity. PMID:22362726

  14. In vitro incorporation of the phage Phi29 connector complex

    SciTech Connect

    Fu Chiyu; Prevelige, Peter E.

    2009-11-10

    The incorporation of the DNA packaging connector complex during lambdoid phage assembly in vivo is strictly controlled-one and only one of the twelve identical icosahedral vertices is differentiated by the inclusion of a portal or connector dodecamer. Proposed control mechanisms include obligate nucleation from a connector containing complex, addition of the connector as the final step during assembly, and a connector-mediated increase in the growth rate. The inability to recapitulate connector incorporation in vitro has made it difficult to obtain direct biochemical evidence in support of one model over another. Here we report the development an in vitro assembly system for the well characterized dsDNA phage Phi29 which results in the co-assembly of connector with capsid and scaffolding proteins to form procapsid-like particles (PLPs). Immuno-electron microscopy demonstrates the specific incorporation of connector vertex in PLPs. The connector protein increases both the yield and the rate of capsid assembly suggesting that the incorporation of the connector in Phi29 likely promotes nucleation of assembly.

  15. Nucleotide sequence of bacteriophage fd DNA.

    PubMed Central

    Beck, E; Sommer, R; Auerswald, E A; Kurz, C; Zink, B; Osterburg, G; Schaller, H; Sugimoto, K; Sugisaki, H; Okamoto, T; Takanami, M

    1978-01-01

    The sequence of the 6,408 nucleotides of bacteriophage fd DNA has been determined. This allows to deduce the exact organisation of the filamentous phage genome and provides easy access to DNA segments of known structure and function. PMID:745987

  16. Bacteriophage replication modules.

    PubMed

    Weigel, Christoph; Seitz, Harald

    2006-05-01

    Bacteriophages (prokaryotic viruses) are favourite model systems to study DNA replication in prokaryotes, and provide examples for every theoretically possible replication mechanism. In addition, the elucidation of the intricate interplay of phage-encoded replication factors with 'host' factors has always advanced the understanding of DNA replication in general. Here we review bacteriophage replication based on the long-standing observation that in most known phage genomes the replication genes are arranged as modules. This allows us to discuss established model systems--f1/fd, phiX174, P2, P4, lambda, SPP1, N15, phi29, T7 and T4--along with those numerous phages that have been sequenced but not studied experimentally. The review of bacteriophage replication mechanisms and modules is accompanied by a compendium of replication origins and replication/recombination proteins (available as supplementary material online). PMID:16594962

  17. Crystal and cryoEM structural studies of a cell wall degrading enzyme in the bacteriophage [psi]29 tail

    SciTech Connect

    Xiang, Ye; Morais, Marc C.; Cohen, Daniel N.; Bowman, Valorie D.; Anderson, Dwight L.; Rossmann, Michael G.

    2009-08-28

    The small bacteriophage {phi}29 must penetrate the {approx}250-{angstrom} thick external peptidoglycan cell wall and cell membrane of the Gram-positive Bacillus subtilis, before ejecting its dsDNA genome through its tail into the bacterial cytoplasm. The tail of bacteriophage {phi}29 is noncontractile and {approx}380 {angstrom} long. A 1.8-{angstrom} resolution crystal structure of gene product 13 (gp13) shows that this tail protein has spatially well separated N- and C-terminal domains, whose structures resemble lysozyme-like enzymes and metallo-endopeptidases, respectively. CryoEM reconstructions of the WT bacteriophage and mutant bacteriophages missing some or most of gp13 shows that this enzyme is located at the distal end of the {phi}29 tail knob. This finding suggests that gp13 functions as a tail-associated, peptidoglycan-degrading enzyme able to cleave both the polysaccharide backbone and peptide cross-links of the peptidoglycan cell wall. Comparisons of the gp13{sup -} mutants with the {phi}29 mature and emptied phage structures suggest the sequence of events that occur during the penetration of the tail through the peptidoglycan layer.

  18. Forces during Bacteriophage DNA Packaging and Ejection

    PubMed Central

    Purohit, Prashant K.; Inamdar, Mandar M.; Grayson, Paul D.; Squires, Todd M.; Kondev, Jané; Phillips, Rob

    2005-01-01

    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 article 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. PMID:15556983

  19. The Conformation of DNA Packaged in Bacteriophage G

    PubMed Central

    Sun, Mao; Serwer, Philip

    1997-01-01

    When packaged in a bacteriophage capsid, double-stranded DNA occupies a cavity whose volume is roughly twice the volume of the DNA double helix. The data thus far have not revealed whether the compactness of packaged bacteriophage DNA is achieved by folding of the DNA, unidirectional winding of the DNA, or a combination of both folding and winding. To assist in discriminating among these possibilities, the present study uses electron microscopy, together with ultraviolet light-induced DNA-DNA cross-linking, to obtain the following information about the conformation of DNA packaged in the comparatively large bacteriophage, G: 1) At the periphery of some negatively stained particles of bacteriophage G, electron microscopy reveals strands of DNA that are both parallel to each other and parallel to the polyhedral bacteriophage G capsid. However, these strands are not visible toward the center of the zone of packaged DNA. 2) Within some positively stained particles, electron microscopy reveals DNA-associated stain in relatively high concentration at comers of the polyhedral bacteriophage G capsid. 3) When cross-linked DNA is expelled from its capsid during preparation for electron microscopy, some DNA molecules consist primarily of a compacted central region, surrounded by DNA strands that appear to be unraveling at multiple positions uniformly distributed around the compacted DNA region. The above results are explained by a previously presented model in which DNA is compacted by folding to form 12 icosahedrally arranged pear-shaped rings. ImagesFIGURE 1FIGURE 2FIGURE 4 PMID:9017221

  20. Global Structure of a Three-Way Junction in a Phi29 Packaging RNA Dimer Determined Using Site-Directed Spin Labeling

    SciTech Connect

    Zhang, Xiaojun; Tung, Chang-Shung; Sowa, Glenna; Hatmal, Ma'mon M.; Haworth, Ian S.; Qin, Peter Z.

    2012-02-08

    The condensation of bacteriophage phi29 genomic DNA into its preformed procapsid requires the DNA packaging motor, which is the strongest known biological motor. The packaging motor is an intricate ring-shaped protein/RNA complex, and its function requires an RNA component called packaging RNA (pRNA). Current structural information on pRNA is limited, which hinders studies of motor function. Here, we used site-directed spin labeling to map the conformation of a pRNA three-way junction that bridges binding sites for the motor ATPase and the procapsid. The studies were carried out on a pRNA dimer, which is the simplest ring-shaped pRNA complex and serves as a functional intermediate during motor assembly. Using a nucleotide-independent labeling scheme, stable nitroxide radicals were attached to eight specific pRNA sites without perturbing RNA folding and dimer formation, and a total of 17 internitroxide distances spanning the three-way junction were measured using Double Electron-Electron Resonance spectroscopy. The measured distances, together with steric chemical constraints, were used to select 3662 viable three-way junction models from a pool of 65 billion. The results reveal a similar conformation among the viable models, with two of the helices (HT and HL) adopting an acute bend. This is in contrast to a recently reported pRNA tetramer crystal structure, in which HT and HL stack onto each other linearly. The studies establish a new method for mapping global structures of complex RNA molecules, and provide information on pRNA conformation that aids investigations of phi29 packaging motor and developments of pRNA-based nanomedicine and nanomaterial.

  1. Determinants of Bacteriophage 933W Repressor DNA Binding Specificity

    PubMed Central

    Bullwinkle, Tammy J.; Samorodnitsky, Daniel; Rosati, Rayna C.; Koudelka, Gerald B.

    2012-01-01

    We reported previously that 933W repressor apparently does not cooperatively bind to adjacent sites on DNA and that the relative affinities of 933W repressor for its operators differ significantly from that of any other lambdoid bacteriophage. These findings indicate that the operational details of the lysis-lysogeny switch of bacteriophage 933W are unique among lambdoid bacteriophages. Since the functioning of the lysis-lysogeny switch in 933W bacteriophage uniquely and solely depends on the order of preference of 933W repressor for its operators, we examined the details of how 933W repressor recognizes its DNA sites. To identify the specificity determinants, we first created a molecular model of the 933W repressor-DNA complex and tested the predicted protein-DNA interactions. These results of these studies provide a picture of how 933W repressor recognizes its DNA sites. We also show that, opposite of what is normally observed for lambdoid phages, 933W operator sequences have evolved in such a way that the presence of the most commonly found base sequences at particular operator positions serves to decrease, rather than increase, the affinity of the protein for the site. This finding cautions against assuming that a consensus sequence derived from sequence analysis defines the optimal, highest affinity DNA binding site for a protein. PMID:22509323

  2. Infection by choleraphage phi 138: bacteriophage DNA and replicative intermediates.

    PubMed Central

    Chowdhury, R; Das, J

    1986-01-01

    Choleraphage phi 138 contains a linear, double-stranded, circularly permuted DNA molecule of 30 X 10(6) daltons or 45 kilobase pairs. Upon infection, the host DNA is degraded, and synthesis of phage-specific DNA is detectable 20 min after infection. The phage utilizes primarily the host DNA degradation products for its own DNA synthesis. A physical map of phi 138 DNA was constructed with the restriction endonucleases Bg/II, HindIII, and PstI. A concatemeric replicative DNA intermediate equivalent to eight mature genome lengths was identified. The concatemer was shown to be the precursor for the synthesis of mature bacteriophage DNA which is subsequently packaged by a headful mechanism. Images PMID:3951021

  3. Capstan Friction Model for DNA Ejection from Bacteriophages

    NASA Astrophysics Data System (ADS)

    Ghosal, Sandip

    2012-12-01

    Bacteriophages infect cells by attaching to the outer membrane and injecting their DNA into the cell. The phage DNA is then transcribed by the cell’s transcription machinery. A number of physical mechanisms by which DNA can be translocated from the phage capsid into the cell have been identified. A fast ejection driven by the elastic and electrostatic potential energy of the compacted DNA within the viral capsid appears to be used by most phages, at least to initiate infection. In recent in vitro experiments, the speed of DNA translocation from a λ phage capsid has been measured as a function of ejected length over the entire duration of the event. Here, a mechanical model is proposed that is able to explain the observed dependence of exit velocity on ejected length, and that is also consistent with the accepted picture of the geometric arrangement of DNA within the viral capsid.

  4. DNA Poised for Release in Bacteriophage ø29

    PubMed Central

    Tang, Jinghua; Olson, Norman; Jardine, Paul J.; Grimes, Shelley; Anderson, Dwight L.; Baker, Timothy S.

    2008-01-01

    SUMMARY We present here the first asymmetric, three-dimensional reconstruction of a tailed dsDNA virus, the mature bacteriophage ϕ29, at sub-nanometer resolution. This structure reveals the rich detail of the asymmetric interactions and conformational dynamics of the ϕ29 protein and DNA components, and provides novel insight into the mechanics of virus assembly. For example, the dodecameric head-tail connector protein undergoes significant rearrangement upon assembly into the virion. Specific interactions occur between the tightly packed dsDNA and the proteins of the head and tail. Of particular interest and novelty, a ~60Å diameter toroid of dsDNA was observed in the connector-lower collar cavity. The extreme deformation that occurs over a small stretch of DNA is likely a consequence of the high pressure of the packaged genome. This toroid structure may help retain the DNA inside the capsid prior to its injection into the bacterial host. PMID:18547525

  5. The DNA ejection process in bacteriophage lambda

    NASA Astrophysics Data System (ADS)

    Grayson, Paul

    Bacteriophages have long served as model systems through which the nature of life may be explored. From a physical or mechanical point of view, phages are excellent examples of natural nanotechnology: they are nanometer-scale systems which depend critically on forces, pressures, velocities, and other fundamentally physical quantities for their biological functions. The study of the physical properties of phages has therefore provided an arena for application of physics to biology. In particular, recent studies of the motor responsible for packaging a phage gnome into a capsid showed a buildup of pressure within the capsid of tens of atmospheres. This thesis reports a combined theoretical and experimental study on various aspects of the genome ejection process, so that a comparison may be drawn with the packaging experiments. In particular, we examine various theoretical models of the forces within a phage capsid, deriving formulas both for the force driving genome ejection and for the velocity at which the genome is translocated into a host cell. We describe an experiment in which the force was measured as a function of the amount of genome within the phage capsid, and another where the genome ejection velocity was measured for single phages under the microscope. We make direct quantitative comparisons between the theory and experiments, stringently testing the extent to which we are able to model the genome ejection process.

  6. Evidence for bacteriophage T7 tail extension during DNA injection

    PubMed Central

    Serwer, Philip; Wright, Elena T; Hakala, Kevin W; Weintraub, Susan T

    2008-01-01

    Background Electron micrographs of bacteriophage T7 reveal a tail shorter than needed to reach host cytoplasm during infection-initiating injection of a T7 DNA molecule through the tail and cell envelope. However, recent data indicate that internal T7 proteins are injected before the DNA molecule is injected. Thus, bacteriophage/host adsorption potentially causes internal proteins to become external and lengthen the tail for DNA injection. But, the proposed adsorption-induced tail lengthening has never been visualized. Findings In the present study, electron microscopy of particles in T7 lysates reveals a needle-like capsid extension that attaches partially emptied bacteriophage T7 capsids to non-capsid vesicles and sometimes enters an attached vesicle. This extension is 40–55 nm long, 1.7–2.4× longer than the T7 tail and likely to be the proposed lengthened tail. The extension is 8–11 nm in diameter, thinner than most of the tail, with an axial hole 3–4 nm in diameter. Though the bound vesicles are not identified by microscopy, these vesicles resemble the major vesicles in T7 lysates, found to be E. coli outer membrane vesicles by non-denaturing agarose gel electrophoresis, followed by mass spectrometry. Conclusion The observed lengthened tail is long enough to reach host cytoplasm during DNA injection. Its channel is wide enough to be a conduit for DNA injection and narrow enough to clamp DNA during a previously observed stalling/re-starting of injection. However, its outer diameter is too large to explain formation by passing of an intact assembly through any known capsid hole unless the hole is widened. PMID:18710489

  7. REDOR NMR Characterization of DNA Packaging in Bacteriophage T4

    PubMed Central

    Yu, Tsyr-Yan; Schaefer, Jacob

    2008-01-01

    Bacteriophage T4 is a large-tailed E. coli virus whose capsid is 120 × 86 nm. ATP-driven DNA packaging of the T4 capsid results in the loading of a 171-kb genome in less than 5 minutes during viral infection. We have isolated 50-mg quantities of uniform 15N and [ε-15N]lysine-labeled bacteriophage T4. We have also introduced 15NH4+ into filled, unlabeled capsids from synthetic medium by exchange. We have examined lyo- and cryoprotected lyophilized T4 using 15N{31P} and 31P{15N} rotational-echo double resonance. The results of these experiments have shown that: (i) packaged DNA is in an unperturbed duplex B-form conformation; (ii) the DNA phosphate negative charge is balanced by lysyl amines (3.2%), polyamines (5.8%), and monovalent cations (40%); and (iii) 11% of lysyl amines, 40% of –NH2 groups of polyamines, and 80% of monovalent cations within the lyophilized T4 capsid, are involved in the DNA charge balance. The NMR evidence suggests that DNA enters the T4 capsid in a charge-unbalanced state. We propose that electrostatic interactions may provide free energy to supplement the nanomotor-driven T4 DNA packaging. PMID:18703073

  8. Inhibition of DNA ejection from bacteriophage by Mg+2 counterions

    NASA Astrophysics Data System (ADS)

    Lee, Sell; Tran, C. V.; Nguyen, T. T.

    2011-03-01

    The problem of inhibiting viral DNA ejection from bacteriophages by multivalent counterions, specifically Mg+2 counterions, is studied. Experimentally, it is known that MgSO4 salt has a strong and nonmonotonic effect on the amount of DNA ejected. There exists an optimal concentration at which the minimum amount of DNA is ejected from the virus. At lower or higher concentrations, more DNA is ejected from the capsid. We propose that this phenomenon is the result of DNA overcharging by Mg+2 multivalent counterions. As Mg+2 concentration increases from zero, the net charge of DNA changes from negative to positive. The optimal inhibition corresponds to the Mg+2 concentration where DNA is neutral. At lower/higher concentrations, DNA genome is charged. It prefers to be in solution to lower its electrostatic self-energy, which consequently leads to an increase in DNA ejection. By fitting our theory to available experimental data, the strength of DNA-DNA short range attraction energies, mediated by Mg+2, is found to be -0.004 kBT per nucleotide base. This and other fitted parameters agree well with known values from other experiments and computer simulations. The parameters are also in agreement qualitatively with values for tri- and tetravalent counterions.

  9. DNA Packaging Motor Assembly Intermediate of Bacteriophage ϕ29

    PubMed Central

    Koti, Jaya S.; Morais, Marc C.; Rajagopal, Raj; Owen, Barbara A. L.; McMurray, Cynthia T.; Anderson, Dwight L.

    2008-01-01

    Unraveling the structure and assembly of the DNA packaging ATPases of the tailed, double-stranded DNA bacteriophages is integral to understanding the mechanism of DNA translocation. Here the bacteriophage ϕ29 packaging ATPase, gene product gp16 (gp16), was over-expressed in soluble form in Bacillus subtilis (pSAC), purified to near homogeneity, and assembled to the ϕ29 precursor capsid (prohead) to produce a packaging motor intermediate that was fully active in in vitro DNA packaging. The formation of higher oligomers of the gp16 from monomers was concentration dependent and was characterized by analytical ultracentrifugation, gel filtration and electron microscopy. The binding of multiple copies of gp16 to the prohead was dependent upon the presence of an oligomer of 174- or 120-base prohead RNA (pRNA) fixed to the head-tail connector at the unique portal vertex of the prohead. The use of mutant pRNAs demonstrated that gp16 bound specifically to the A-helix of pRNA, and ribonuclease footprinting of gp16 on pRNA showed that gp16 protected the CC residues of the CCA bulge (residues 18–20) of the A-helix. The binding of gp16 to the prohead/pRNA to constitute the complete and active packaging motor was confirmed by cryo-electron microscopy three-dimensional reconstruction of the prohead/pRNA/gp16 complex. The complex was capable of supercoiling DNA-gp3 as observed previously for gp16 alone, and therefore the binding of gp16 to the prohead, rather than first to DNA-gp3, represents an alternative packaging motor assembly pathway. PMID:18674782

  10. Cloning and characterization of bacteriophage-like DNA from Haemophilus somnus homologous to phages P2 and HP1.

    PubMed Central

    Pontarollo, R A; Rioux, C R; Potter, A A

    1997-01-01

    In an attempt to identify and characterize components of a heme uptake system of Haemophilus somnus, an Escherichia coli cosmid library of H. somnus genomic DNA was screened for the ability to bind hemin (Hmb+). The Hmb+ phenotype was associated with a 7,814-bp HindIII fragment of H. somnus DNA that was subcloned and sequenced. Thirteen open reading frames (orfs) were identified, all transcribed in one direction, and transposon mutagenesis identified orf7 as the gene associated with the Hmb+ phenotype. Orf7 (178 amino acids) has extensive homology with the lysozymes of bacteriophages P-A2, P21, P22, PZA, phi-29, phi-vML3, T4, or HP1. The orf7 gene complemented the lytic function of the K gene of phage P2 and the R gene of phage lambda. A lysozyme assay using supernatants from whole-cell lysates of E. coli cultures harboring plasmid pRAP501 or pGCH2 (both of which express the orf7 gene product) exhibited significant levels of lysozyme activity. The orf6 gene upstream of orf7 has the dual start motif common to the holins encoded by lambdoid S genes, and the orf6 gene product has significant homology to the holins of phages HP1 and P21. When expressed from a tac promoter, the orf6 gene product caused immediate cell death without lysis, while cultures expressing the orf7 gene product grew at normal rates but lysed immediately after the addition of chloroform. Based on this data, we concluded that the Hmb+ phenotype was an artifact resulting from the expression of cloned lysis genes which were detrimental to the E. coli host. The DNA flanking the cloned lysis genes contains orfs that are similar to structural and DNA packaging genes of phage P2. Polyclonal antiserum against Orf2, which is homologous to the major capsid precursor protein (gpN) of phage P2, detected a 40,000-M(r) protein expressed from pRAP401 but did not detect Orf2 in H. somnus, lysates. The phage-like DNA was detected in the serum-susceptible preputial strains HS-124P and HS-127P but was absent from

  11. A promiscuous DNA packaging machine from bacteriophage T4.

    PubMed

    Zhang, Zhihong; Kottadiel, Vishal I; Vafabakhsh, Reza; Dai, Li; Chemla, Yann R; Ha, Taekjip; Rao, Venigalla B

    2011-01-01

    Complex viruses are assembled from simple protein subunits by sequential and irreversible assembly. During genome packaging in bacteriophages, a powerful molecular motor assembles at the special portal vertex of an empty prohead to initiate packaging. The capsid expands after about 10%-25% of the genome is packaged. When the head is full, the motor cuts the concatemeric DNA and dissociates from the head. Conformational changes, particularly in the portal, are thought to drive these sequential transitions. We found that the phage T4 packaging machine is highly promiscuous, translocating DNA into finished phage heads as well as into proheads. Optical tweezers experiments show that single motors can force exogenous DNA into phage heads at the same rate as into proheads. Single molecule fluorescence measurements demonstrate that phage heads undergo repeated initiations, packaging multiple DNA molecules into the same head. These results suggest that the phage DNA packaging machine has unusual conformational plasticity, powering DNA into an apparently passive capsid receptacle, including the highly stable virus shell, until it is full. These features probably led to the evolution of viral genomes that fit capsid volume, a strikingly common phenomenon in double-stranded DNA viruses, and will potentially allow design of a novel class of nanocapsid delivery vehicles. PMID:21358801

  12. Length quantization of DNA partially expelled from heads of a bacteriophage T3 mutant

    SciTech Connect

    Serwer, Philip; Wright, Elena T.; Liu, Zheng; Jiang, Wen

    2014-05-15

    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. - Graphical abstract: Highlights: • We implement directed evolution- and DNA-sequencing-based phage assembly genetics. • We purify stable, mutant phage heads with a partially leaked mature DNA molecule. • Native gels and DNase-protection show leaked DNA segments to have quantized lengths. • Native gels after DNase I-removal of leaked DNA reveal the capsids to vary in radius. • Thus, we hypothesize leaked DNA quantization via variably quantized capsid radius.

  13. DNA damage under simulated extraterrestrial conditions in bacteriophage T7

    NASA Astrophysics Data System (ADS)

    Fekete, A.; Kovács, G.; Hegedüs, M.; Módos, K.; Rontó, Gy.; Lammer, H.; Panitz, C.

    The experiment ``Phage and uracil response'' (PUR) will be accommodated in the EXPOSE facility of the ISS aiming to examine and quantify the effect of specific space conditions on bacteriophage T7 and isolated T7 DNA thin films. To achieve this new method was elaborated for the preparation of DNA and nucleoprotein thin films (1). During the EXPOSE Experiment Verification Tests (EVT) the samples were exposed to vacuum (10 -6 Pa), to monochromatic (254 nm) and polychromatic (200-400 nm) UV radiation in air as well in simulated space vacuum. Using neutral density (ND) filters dose-effect curves were performed in order to define the maximum doses tolerated, and we also studied the effect of temperature in vacuum as well as the influence of temperature fluctuations. We obtained substantial evidence that DNA lesions (e.g. strand breaks, DNA-protein cross-links, DNA-DNA cross-links) accumulate throughout exposure. DNA damage was determined by quantitative PCR using 555 bp and 3826 bp fragments of T7 DNA (2) and by neutral and alkaline agarose gel electrophoresis; the structural/chemical effects were analyzed by spectroscopic and microscopical methods. Characteristic changes in the absorption spectrum, in the electrophoretic pattern of DNA and the decrease of the amount of the PCR products have been detected indicating the damage of isolated and intraphage DNA. Preliminary results suggest a synergistic action of space vacuum and UV radiation with DNA being the critical target. Fekete et al. J. Luminescence 102-103, 469-475, 2003 Hegedüs et al. Photochem. Photobiol. 78, 213-219, 2003

  14. New physical map of bacteriophage T5 DNA.

    PubMed Central

    Rhoades, M

    1982-01-01

    The locations of 103 cleavage sites, produced by 13 restriction endonucleases, were mapped on the DNA of bacteriophage T5. Single- and double-digest fragment sizes were determined by agarose gel electrophoresis, using restriction fragments of phi X174 DNA and lambda DNA as molecular weight standards. Map coordinates were determined by a computer-based least-squares procedures (J. Schroeder and F. Blattner, Gene [Amst] 4:167-174, 1978). The fragment sizes predicted by the final map are all within 2% of the measured values. Based on this analysis, T5st(+) DNA contains 121,300 base pairs (Mr, 80.3 X 10(6) and has a terminal repetition of 10,160 base pairs (Mr, 6.7 X 10(6)). Restriction endonuclease analysis after treatment with exonuclease III and a single-strand-specific endonuclease allowed precise localization of five of the natural single-chain interruptions in T5 DNA. Revised locations for several T5 deletions were also determined. Images PMID:6287031

  15. DNA damage under simulated extraterrestrial conditions in bacteriophage T7

    NASA Astrophysics Data System (ADS)

    Fekete, A.; Módos, K.; Hegedüs, M.; Kovács, G.; Rontó, Gy.; Péter, Á.; Lammer, H.; Panitz, C.

    The experiment "Phage and Uracil response" will be accommodated in the EXPOSE facility of the International Space Station. Its objective is to examine and quantify the effect of specific space conditions on nucleic acid models, especially on bacteriophage T7 and isolated T7 DNA thin films. In order to define the environmental and technical requirements of the EXPOSE, the samples were subjected to the experiment verification test (EVT). During EVT, the samples were exposed to vacuum (10 -4-10 -6 Pa) and polychromatic UV-radiation (200-400 nm) in air, in inert atmosphere, as well as in simulated space vacuum. The effect of extreme temperature in vacuum and the influence of temperature fluctuations around 0 °C were also studied. The total intraphage/isolated DNA damage was determined by quantitative PCR using 555 and 3826 bp fragments of T7 DNA. The type of the damage was resolved using a combination of enzymatic probes and neutral and alkaline agarose gel electrophoresis; the structural/chemical effects were analyzed by spectroscopic and microscopical methods. We obtained substantial evidence that DNA lesions accumulate throughout exposure, but the amount of damage depends on the thickness of the layers. According to our preliminary results, the damages by exposure to conditions of dehydration and UV-irradiation are larger than the sum of vacuum alone, or radiation alone case, suggesting a synergistic action of space vacuum and UV radiation with DNA being the critical target.

  16. Dynamics of the T4 Bacteriophage DNA Packasome Motor

    PubMed Central

    Dixit, Aparna; Ray, Krishanu; Lakowicz, Joseph R.; Black, Lindsay W.

    2011-01-01

    Conserved bacteriophage ATP-based DNA translocation motors consist of a multimeric packaging terminase docked onto a unique procapsid vertex containing a portal ring. DNA is translocated into the empty procapsid through the portal ring channel to high density. In vivo the T4 phage packaging motor deals with Y- or X-structures in the replicative concatemer substrate by employing a portal-bound Holliday junction resolvase that trims and releases these DNA roadblocks to packaging. Here using dye-labeled packaging anchored 3.7-kb Y-DNAs or linear DNAs, we demonstrate FRET between the dye-labeled substrates and GFP portal-containing procapsids and between GFP portal and single dye-labeled terminases. We show using FRET-fluorescence correlation spectroscopy that purified T4 gp49 endonuclease VII resolvase can release DNA compression in vitro in prohead portal packaging motor anchored and arrested Y-DNA substrates. In addition, using active terminases labeled at the N- and C-terminal ends with a single dye molecule, we show by FRET distance of the N-terminal GFP-labeled portal protein containing prohead at 6.9 nm from the N terminus and at 5.7 nm from the C terminus of the terminase. Packaging with a C-terminal fluorescent terminase on a GFP portal prohead, FRET shows a reduction in distance to the GFP portal of 0.6 nm in the arrested Y-DNA as compared with linear DNA; the reduction is reversed by resolvase treatment. Conformational changes in both the motor proteins and the DNA substrate itself that are associated with the power stroke of the motor are consistent with a proposed linear motor employing a terminal-to-portal DNA grip-and-release mechanism. PMID:21454482

  17. The Viral DNA Packaging Motor of Bacteriophage Lambda

    NASA Astrophysics Data System (ADS)

    Catalano, Carlos E.

    2007-03-01

    Terminase enzymes are common to both eukaryotic and prokaryotic double-stranded DNA viruses. These enzymes, which serve as molecular motors that selectively ``package'' viral DNA into a pre-formed procapsid structure, are among the most powerful biological motors characterized to date. Bacteriophage lambda terminase is a heteroligomer composed of gpA and gpNu1 subunits. The smaller gpNu1 subunit is required for specific recognition of viral DNA, a process that is modulated by ATP. The gpA subunit possesses site-specific nuclease and helicase activities that ``mature'' the viral genome prior to packaging. The subunit further possesses a DNA translocase activity that is central to the packaging motor complex. Discrete ATPase sites in gpA modulate the DNA maturation reactions and fuel the DNA packaging reaction. Kinetic characterization of lambda terminase indicates significant interaction between the multiple catalytic sites of the enzyme and has led to a minimal kinetic model describing the assembly of a catalytically-competent packaging motor complex. Biophysical studies demonstrate that purified lambda terminase forms a homogenous, heterotrimeric structure consisting of one gpA subunit in association with two gpNu1 proteins. Four heterotrimers further assemble into a ring-like structure of sufficient size to encircle duplex DNA. The ensemble of data suggests that the ring tetramer represents the biologically relevant, catalytically-competent motor complex responsible for genome processing and packaging reactions. We present a model for the functional DNA packaging motor complex that finds general utility in our global understanding of the enzymology of virus assembly.

  18. Double-stranded DNA organization in bacteriophage heads: An alternative toroid-based model

    SciTech Connect

    Hud, N.V.

    1995-10-01

    Studies of the organization of double-stranded DNA within bacteriophage heads during the past four decades have produced a wealth of data. However, despite the presentation of numerous models, the true organization of DNA within phage heads remains unresolved. The observations of toroidal DNA structures in electron micrographs of phage lysates have long been cited as support for the organization of DNA in a spool-like fashion. This particular model, like all other models, has not been found to be consistent with all available data. Recently, the authors proposed that DNA within toroidal condensates produced in vitro is organized in a manner significantly different from that suggested by the spool model. This new toroid model has allowed the development of an alternative model for DNA organization within bacteriophage heads that is consistent with a wide range of biophysical data. Here the authors propose that bacteriophage DNA is packaged in a toroid that is folded into a highly compact structure.

  19. Single molecule studies of DNA packaging by bacteriophages

    NASA Astrophysics Data System (ADS)

    Fuller, Derek Nathan

    The DNA packaging dynamics of bacteriophages φ29, gamma, and T4 were studied at the single molecule level using a dual trap optical tweezers. Also, a method for producing long DNA molecules by PCR for optical tweezers studies of protein DNA interactions is presented and thoroughly characterized. This DNA preparation technique provided DNA samples for the φ29 and T4 studies. In the studies of φ29, the role of charge was investigated by varying the ionic conditions of the packaging buffer. Ionic conditions in which the DNA charge was highly screened due to divalent and trivalent cations showed the lowest resistance to packaging of the DNA to high density. This confirmed the importance of counterions in shielding the DNA interstrand repulsion when packaged to high density. While the ionic nature of the packaging buffer had a strong effect on packaging velocities, there was no clear trend between the counterion-screened charge of the DNA and the maximum packaging velocity. The packaging studies of lambda and T4 served as systems for comparative studies with φ29. Each system showed similarities to the φ29 system and unique differences. Both the lambda and T4 packaging motors were capable of generating forces in excess of 50 pN and showed remarkably high processivity, similar to φ29. However, dynamic structural transitions were observed with lambda that are not observed with φ29. The packaging of the lambda genome showed capsid expansion at approximately 30 percent of the genome packaged and capsid rupture at 90 percent of the genome packaged in the absence of capsid stabilizing protein gpD. Unique to the T4 packaging motor, packaging dynamics showed a remarkable amount of variability in velocities. This variability was seen both within individual packaging phages and from one phage to the next. This is possibly due to different conformational states of the packaging machinery. Additionally, lambda and T4 had average packaging velocities under minimal load of 600

  20. Novel DNA packaging recognition in the unusual bacteriophage N15

    SciTech Connect

    Feiss, Michael; Geyer, Henriette; Klingberg, Franco; Moreno, Norma; Forystek, Amanda; Maluf, Nasib Karl; Sippy, Jean

    2015-08-15

    Phage lambda's cosB packaging recognition site is tripartite, consisting of 3 TerS binding sites, called R sequences. TerS binding to the critical R3 site positions the TerL endonuclease for nicking cosN to generate cohesive ends. The N15 cos (cos{sup N15}) is closely related to cos{sup λ}, but whereas the cosB{sup N15} subsite has R3, it lacks the R2 and R1 sites and the IHF binding site of cosB{sup λ}. A bioinformatic study of N15-like phages indicates that cosB{sup N15} also has an accessory, remote rR2 site, which is proposed to increase packaging efficiency, like R2 and R1 of lambda. N15 plus five prophages all have the rR2 sequence, which is located in the TerS-encoding 1 gene, approximately 200 bp distal to R3. An additional set of four highly related prophages, exemplified by Monarch, has R3 sequence, but also has R2 and R1 sequences characteristic of cosB–λ. The DNA binding domain of TerS-N15 is a dimer. - Highlights: • There are two classes of DNA packaging signals in N15-related phages. • Phage N15's TerS binding site: a critical site and a possible remote accessory site. • Viral DNA recognition signals by the λ-like bacteriophages: the odd case of N15.

  1. Packaging of DNA by bacteriophage epsilon15: structure, forces, and thermodynamics.

    PubMed

    Petrov, Anton S; Lim-Hing, Krista; Harvey, Stephen C

    2007-07-01

    The structure of bacteriophage epsilon15 has recently been determined by 3D reconstruction of single particle cryo-electron microscopy images. Although this study revealed that the viral genome inside the bacteriophage is on average coaxially spooled, individual DNA conformations inside the capsid could not be determined. In the current study, we present the results of 40 independent simulations of DNA packaging into epsilon15 using the previously described low-resolution model for bacteriophages. In addition to coaxially spooled conformations, we also observe a number of folded-toroidal patterns, but the density averaged over all conformations closely resembles the experimental density. Thermodynamic analysis of the simulations predicts that a force of approximately 125 pN would be required to package DNA into epsilon15. We also show that the origin of this force is predominantly due to electrostatic and entropic contributions. However, the DNA conformation is determined primarily by the need to minimize the DNA bending energy. PMID:17637341

  2. Visualization of Bacteriophage T3 Capsids with DNA Incompletely Packaged In Vivo

    PubMed Central

    Fang, Ping-An; Wright, Elena T.; Weintraub, Susan T.; Hakala, Kevin; Wu, Weimin; Serwer, Philip; Jiang, Wen

    2009-01-01

    The tightly packaged 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 ∼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 ∼10-4 of packaged DNA in all particles) and are initially detected by non-denaturing 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 (cryo-EM) 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 (∼15 Å), 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) forms 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 like those seen

  3. Evidence for non-equilibrium dynamics in viral DNA packaging from optical tweezers measurements

    NASA Astrophysics Data System (ADS)

    Berndsen, Zachary T.; Keller, Nicholas; Smith, Douglas E.

    2013-09-01

    In many viruses molecular motors generate large forces to package DNA to high densities. The dynamics and energetics of this process is a subject of wide debate and is of interest as a model for studying confined polymer physics in general. Here we present preliminary results showing that DNA in bacteriophage phi29 undergoes nonequilibrium conformational dynamics during packaging with a relaxation time >60,000x longer than for free DNA and >3000x longer than reported for DNA confined in nanochannels. Nonequilibrium dynamics significantly increases the load on the motor, causes heterogeneity in the rates of packaging, and causes frequent pausing in motor translocation.

  4. Magic-angle spinning NMR of intact bacteriophages: Insights into the capsid, DNA and their interface

    NASA Astrophysics Data System (ADS)

    Abramov, Gili; Morag, Omry; Goldbourt, Amir

    2015-04-01

    Bacteriophages are viruses that infect bacteria. They are complex macromolecular assemblies, which are composed of multiple protein subunits that protect genomic material and deliver it to specific hosts. Various biophysical techniques have been used to characterize their structure in order to unravel phage morphogenesis. Yet, most bacteriophages are non-crystalline and have very high molecular weights, in the order of tens of MegaDaltons. Therefore, complete atomic-resolution characterization on such systems that encompass both capsid and DNA is scarce. In this perspective article we demonstrate how magic-angle spinning solid-state NMR has and is used to characterize in detail bacteriophage viruses, including filamentous and icosahedral phage. We discuss the process of sample preparation, spectral assignment of both capsid and DNA and the use of chemical shifts and dipolar couplings to probe the capsid-DNA interface, describe capsid structure and dynamics and extract structural differences between viruses.

  5. Magic-angle spinning NMR of intact bacteriophages: insights into the capsid, DNA and their interface.

    PubMed

    Abramov, Gili; Morag, Omry; Goldbourt, Amir

    2015-04-01

    Bacteriophages are viruses that infect bacteria. They are complex macromolecular assemblies, which are composed of multiple protein subunits that protect genomic material and deliver it to specific hosts. Various biophysical techniques have been used to characterize their structure in order to unravel phage morphogenesis. Yet, most bacteriophages are non-crystalline and have very high molecular weights, in the order of tens of MegaDaltons. Therefore, complete atomic-resolution characterization on such systems that encompass both capsid and DNA is scarce. In this perspective article we demonstrate how magic-angle spinning solid-state NMR has and is used to characterize in detail bacteriophage viruses, including filamentous and icosahedral phage. We discuss the process of sample preparation, spectral assignment of both capsid and DNA and the use of chemical shifts and dipolar couplings to probe the capsid-DNA interface, describe capsid structure and dynamics and extract structural differences between viruses. PMID:25797007

  6. Initiation of bacteriophage Φ29 DNA packaging studied by optical tweezers manipulation of single DNA molecules

    NASA Astrophysics Data System (ADS)

    Rickgauer, John Peter; Fuller, Derek N.; Hu, Bo; Grimes, Shelley; Jardine, Paul J.; Anderson, Dwight L.; Smith, Douglas E.

    2006-08-01

    A key step in the life cycle of many viruses, including bacteriophages, adenoviruses, and herpesviruses, is the packaging of replicated viral genomes into pre-assembled proheads by the action of ATP-dependent portal motor complexes. Here we present a method that allows the initiation of packaging by single complexes to be studied using optical tweezers. A procedure is developed for assembling phage Φ29 prohead-motor complexes, which are demonstrated to bind and begin translocation of a target DNA molecule within only a few seconds. We show that the Φ29 DNA terminal protein (gene product 3), which functions to prime DNA replication, also has a dramatic effect on packaging. The DNA tether length measured immediately after binding varied from ~30-100% of the full length, yet shortened monotonically, indicating that packaging does not strictly begin at the terminal end of the DNA. Removal of the terminal protein eliminated this variability, causing packaging to initiate at or very near the end of the DNA. These findings, taken together with electron microscopy data, suggest that rather than simply threading into the portal, the motor captures and dynamically tensions a DNA loop, and that the function of the terminal protein is to load DNA segments on both sides of the loop junction onto separate DNA translocating units.

  7. DNA buckling in bacteriophage cavities as a mechanism to aid virus assembly.

    PubMed

    Hirsh, Andrew D; Perkins, N C

    2015-03-01

    While relatively simple biologically, bacteriophages are sophisticated biochemical machines that execute a precise sequence of events during virus assembly, DNA packaging, and ejection. These stages of the viral life cycle require intricate coordination of viral components whose structures are being revealed by single molecule experiments and high resolution (cryo-electron microscopy) reconstructions. For example, during packaging, bacteriophages employ some of the strongest known molecular motors to package DNA against increasing pressure within the viral capsid shell. Located upstream of the motor is an elaborate portal system through which DNA is threaded. A high resolution reconstruction of the portal system for bacteriophage ϕ29 reveals that DNA buckles inside a small cavity under large compressive forces. In this study, we demonstrate that DNA can also buckle in other bacteriophages including T7 and P22. Using a computational rod model for DNA, we demonstrate that a DNA buckle can initiate and grow within the small confines of a cavity under biologically-attainable force levels. The forces of DNA-cavity contact and DNA-DNA electrostatic repulsion ultimately limit cavity filling. Despite conforming to very different cavity geometries, the buckled DNA within T7 and P22 exhibits near equal volumetric energy density (∼1kT/nm(3)) and energetic cost of packaging (∼22kT). We hypothesize that a DNA buckle creates large forces on the cavity interior to signal the conformational changes to end packaging. In addition, a DNA buckle may help retain the genome prior to tail assembly through significantly increased contact area with the portal. PMID:25613203

  8. Incorporation of viral DNA packaging motor channel in lipid bilayers for real-time, single-molecule sensing of chemicals and double-stranded DNA

    PubMed Central

    Haque, Farzin; Geng, Jia; Montemagno, Carlo; Guo, Peixuan

    2013-01-01

    Over the past decade, nanopores have rapidly emerged as stochastic biosensors. This protocol describes the cloning, expression, and purification of the channel of bacteriophage phi29 DNA packaging nanomotor and its subsequent incorporation into lipid membranes for single-pore sensing of dsDNA and chemicals. The membrane-embedded phi29 nanochannels remain functional and structurally intact under a range of conditions. When ions and macromolecules translocate through these nanochannels, reliable fingerprint changes in conductance are observed. Compared with other well studied biological pores, the phi29 nanochannel has a larger cross-sectional area, which enables the translocation of dsDNA. Furthermore, specific amino acids can be introduced by site-directed mutagenesis within the large cavity of the channel to conjugate receptors that are able to bind specific ligands or analytes for desired applications. The lipid membrane embedded nanochannel system has immense potential nanotechnological and biomedical applications in bioreactors, environmental sensing, drug monitoring, controlled drug delivery, early disease diagnosis, and high-throughput DNA sequencing. The total time required for completing one round of this protocol is around one month. PMID:23348364

  9. Evolution of double-stranded DNA viruses of eukaryotes: from bacteriophages to transposons to giant viruses

    PubMed Central

    Koonin, Eugene V; Krupovic, Mart; Yutin, Natalya

    2015-01-01

    Diverse eukaryotes including animals and protists are hosts to a broad variety of viruses with double-stranded (ds) DNA genomes, from the largest known viruses, such as pandoraviruses and mimiviruses, to tiny polyomaviruses. Recent comparative genomic analyses have revealed many evolutionary connections between dsDNA viruses of eukaryotes, bacteriophages, transposable elements, and linear DNA plasmids. These findings provide an evolutionary scenario that derives several major groups of eukaryotic dsDNA viruses, including the proposed order “Megavirales,” adenoviruses, and virophages from a group of large virus-like transposons known as Polintons (Mavericks). The Polintons have been recently shown to encode two capsid proteins, suggesting that these elements lead a dual lifestyle with both a transposon and a viral phase and should perhaps more appropriately be named polintoviruses. Here, we describe the recently identified evolutionary relationships between bacteriophages of the family Tectiviridae, polintoviruses, adenoviruses, virophages, large and giant DNA viruses of eukaryotes of the proposed order “Megavirales,” and linear mitochondrial and cytoplasmic plasmids. We outline an evolutionary scenario under which the polintoviruses were the first group of eukaryotic dsDNA viruses that evolved from bacteriophages and became the ancestors of most large DNA viruses of eukaryotes and a variety of other selfish elements. Distinct lines of origin are detectable only for herpesviruses (from a different bacteriophage root) and polyoma/papillomaviruses (from single-stranded DNA viruses and ultimately from plasmids). Phylogenomic analysis of giant viruses provides compelling evidence of their independent origins from smaller members of the putative order “Megavirales,” refuting the speculations on the evolution of these viruses from an extinct fourth domain of cellular life. PMID:25727355

  10. A bacteriophage T4 in vitro system to clone long DNA molecules. Final report, June 1, 1990--January 31, 1996

    SciTech Connect

    Rao, V.B.

    1997-09-01

    A summary is presented of the following objectives: development of a bacteriophage T4 in vitro system, and techniques to clone long segments of foreign DNA; development of a giant prohead DNA packaging system that could potentially be used to clone even a megabase size DNA; and development of techniques to rapidly map the cloned DNA inserts.

  11. Bacteriophage T4 DNA Topoisomerase Mediates Illegitimate Recombination in vitro

    NASA Astrophysics Data System (ADS)

    Ikeda, Hideo

    1986-02-01

    We have found that purified T4 DNA topoisomerase promotes recombination between two phage λ DNA molecules in an in vitro system. In this cross, T4 DNA topoisomerase alone is able to catalyze the recombination and produce a linear monomer recombinant DNA that can be packaged in vitro. ATP is not required for this recombination, while oxolinic acid stimulates it. The recombinant DNA molecules contain duplications or deletions, and the crossovers take place between nonhomologous and nonspecific sequences of λ DNA. Therefore, the recombination mediated by the T4 DNA topoisomerase is an illegitimate recombination that is similar to that mediated by Escherichia coli DNA gyrase. A model was proposed previously in which DNA gyrase molecules that are bound to DNA associate with each other and lead to the exchange of DNA strands through the exchange of DNA gyrase subunits. This model is also applicable to the recombination mediated by T4 DNA topoisomerase.

  12. Elastic Properties and Heterogeneous Stiffness of the Phi29 Motor Connector Channel

    PubMed Central

    Kumar, Rajendra; Grubmüller, Helmut

    2014-01-01

    The DNA packaging motor of the bacteriophage ϕ29, comprising head-tail connector, ATPase, and pRNA, transports the viral DNA inside the procapsid against pressure differences of up to ∼60 atm during replication. Several models for the DNA packaging mechanism have been proposed, which attribute different roles to the connector, and require specific mechanical properties of the connector. To characterize these properties at the atomic level, and to understand how the connector withstands this large pressure, we have carried out molecular dynamics simulations of the whole connector both in equilibrium and under mechanical stress. The simulations revealed a quite heterogeneous distribution of stiff and soft regions, resembling that of typical composite materials that are also optimized to resist mechanical stress. In particular, the conserved middle α-helical region is found to be remarkably stiff, similar only to structural proteins forming viral shell, silk, or collagen. In contrast, large parts of the peripheral interface to the ϕ29 procapsid turned out to be rather soft. Force probe and umbrella sampling simulations showed that large connector deformations are remarkably reversible, and served to calculate the free energies required for these deformations. In particular, for an untwisting deformation by 12°, as postulated by the untwist-twist model, more than four times’ larger energy is required than is available from hydrolysis of one ATP molecule. Combined with previous experiments, this result is incompatible with the untwist-twist model. In contrast, our simulations support the recently proposed one-way revolution model and suggest in structural terms how the connector blocks DNA leakage. In particular, conserved loops at the rim of the central channel, which are in direct contact with the DNA, are found to be rather flexible and tightly anchored to the rigid central region. These findings suggest a check-valve mechanism, with the flexible loops

  13. Charting the Structure and Energetics of Packaged DNA in Bacteriophages

    NASA Astrophysics Data System (ADS)

    Qiu, Xiangyun; Rau, Donald C.; Parsegian, V. Adrian; Fang, Li Tai; Knobler, Charles M.; Gelbart, William M.

    2009-03-01

    Many bacterial viruses resort to pressure in order to infect bacteria, e.g., lambda phage stores its dsDNA genome at surprisingly high pressure and then uses this pressure to drive delivery of the genome. We report on a biophysical interrogation of the DNA configuration and pressure in lambda phage by combining structural and thermodynamic measurements with theoretical modeling. Changes in DNA organization in the capsid are monitored using solution small angle x-ray scattering (SAXS). We vary the DNA-DNA repulsion and DNA bending contributions to the capsid pressure by changing salt concentrations and packaged length, and augment SAXS data with osmotic stress measurements to elicit the evolving structure and energetics of the packaged DNA.

  14. Simulation experiments of the effect of space environment on bacteriophage and DNA thin films

    NASA Technical Reports Server (NTRS)

    Fekete, A.; Ronto, Gy; Hegedus, M.; Modos, K.; Berces, A.; Kovacs, G.; Lammer, H.; Panitz, C.

    2004-01-01

    The main goal of PUR experiment (phage and uracil response) is to examine and quantify the effect of specific space conditions on nucleic acid models. To achieve this an improved method was elaborated for the preparation of DNA and bacteriophage thin films. The homogeneity of the films was controlled by UV spectroscopy and microscopy. To provide experimental evidence for the hypothesis that interplanetary transfer of the genetic material is possible, phage T7 and isolated T7 DNA thin films have been exposed to selected space conditions: intense UVC radiation (lambda=254 nm) and high vacuum (10(-4) Pa). The effects of DNA hydration, conformation and packing on UV radiation damage were examined. Characteristic changes in the absorption spectrum, in the electrophoretic pattern of DNA and the decrease of the amount of PCR products have been detected indicating the photodamage of isolated and intraphage DNA. c2004 COSPAR. Published by Elsevier Ltd. All rights reserved.

  15. A novel DNA-binding protein from Campylobacter jejuni bacteriophage NCTC12673.

    PubMed

    Arutyunov, Denis; Szymanski, Christine M

    2015-11-01

    We previously suggested that the double-stranded genomic DNA of Campylobacter jejuni bacteriophage NCTC12673 was complexed with proteins. Mass spectrometry of peptides obtained from tryptic digests of purified phage DNA indicated that phage protein Gp001 co-purified with the DNA. Gp001 is an acidic protein that lacks any obvious homology or conserved domains found in known DNA-binding proteins. The DNA-binding ability of recombinant Gp001 was examined using an electrophoretic mobility shift assay. Slow DNA-Gp001 complex formation was observed at pH 5.5, but not at neutral or basic pH. This nucleoprotein complex had difficulty entering agarose gels used in the assay while proteinase K pretreatment released the DNA from the complex. No mobility shift was observed when the DNA was immediately subjected to electrophoresis after mixing with Gp001, even if both components were separately pre-incubated at pH 5.5. The complexed DNA was unable to transform chemically competent Escherichia coli cells and was less susceptible to degradation by nucleases. The formation of Gp001-DNA complexes at low pH may provide a mechanism for maintaining DNA integrity while the phage pursues its host through the gastrointestinal tract. Also, this feature can potentially be used to improve DNA delivery protocols applied in gene therapy. PMID:26363017

  16. In vitro synthesis of large peptide molecules using glucosylated single-stranded bacteriophage T4D DNA template.

    PubMed Central

    Hulen, C; Legault-Demare, J

    1975-01-01

    Denatured Bacteriophage T4D DNA is able to stimulate aminoacid incorporation into TCA-precipitable material in an in vitro protein synthesis system according to base DNA sequences. Newly synthesized polypeptides remain associated with ribosomes and have a molecular weight in range of 15,000 to 45,000 Daltons. PMID:1052527

  17. Fluctuation Pressure Assisted Ejection of DNA From Bacteriophage

    NASA Astrophysics Data System (ADS)

    Harrison, Michael J.

    2011-03-01

    The role of thermal pressure fluctuations excited within tightly packaged DNA while it is ejected from protein capsid shells is discussed in a model calculation. At equilibrium before ejection we assume the DNA is folded many times into a bundle of parallel segments that forms an equilibrium conformation at minimum free energy, which presses tightly against capsid walls. Using a canonical ensemble at temperature T we calculate internal pressure fluctuations against a slowly moving or static capsid mantle for an elastic continuum model of the folded DNA bundle. It is found that fluctuating pressures on the capsid from thermal excitation of longitudinal acoustic vibrations in the bundle whose wavelengths are exceeded by the bend persistence length may have root-mean-square values that are several tens of atmospheres for typically small phage dimensions. Comparisons are given with measured data on three mutants of lambda phage with different base pair lengths and total genome ejection pressures.

  18. Can Changes in Temperature or Ionic Conditions Modify the DNA Organization in the Full Bacteriophage Capsid?

    PubMed

    Frutos, Marta de; Leforestier, Amélie; Degrouard, Jéril; Zambrano, Nebraska; Wien, Frank; Boulanger, Pascale; Brasilès, Sandrine; Renouard, Madalena; Durand, Dominique; Livolant, Françoise

    2016-07-01

    We compared four bacteriophage species, T5, λ, T7, and Φ29, to explore the possibilities of DNA reorganization in the capsid where the chain is highly concentrated and confined. First, we did not detect any change in DNA organization as a function of temperature between 20 to 40 °C. Second, the presence of spermine (4+) induces a significant enlargement of the typical size of the hexagonal domains in all phages. We interpret these changes as a reorganization of DNA by slight movements of defects in the structure, triggered by a partial screening of repulsive interactions. We did not detect any signal characteristic of a long-range chiral organization of the encapsidated DNA in the presence and in the absence of spermine. PMID:27152667

  19. Identification of a protein bound to the termini of bacteriophage PRD1 DNA.

    PubMed Central

    Bamford, D; McGraw, T; MacKenzie, G; Mindich, L

    1983-01-01

    Lipid-containing bacteriophage PRD1 has a double-stranded DNA genome of about 14,500 nucleotide base pairs. The phage can infect Escherichia coli and Salmonella typhimurium as well as other gram-negative bacteria harboring an appropriate plasmid. [35S]methionine label is incorporated into the DNA band early in infection. The label remains associated with DNA through phenol extraction and boiling with sodium dodecyl sulfate. Nuclease treatment of the genome released a protein which migrated as an early phage-specific protein (P8). This protein is also necessary for phage DNA replication. By restriction enzyme analysis it was shown that protein was associated with the terminal restriction fragments. Extracts of infected cells catalyzed the labeling of protein P8 with [alpha-32P]dGTP. Images PMID:6620455

  20. DNA compaction by the bacteriophage protein Cox studied on the single DNA molecule level using nanofluidic channels.

    PubMed

    Frykholm, Karolin; Berntsson, Ronnie Per-Arne; Claesson, Magnus; de Battice, Laura; Odegrip, Richard; Stenmark, Pål; Westerlund, Fredrik

    2016-09-01

    The Cox protein from bacteriophage P2 forms oligomeric filaments and it has been proposed that DNA can be wound up around these filaments, similar to how histones condense DNA. We here use fluorescence microscopy to study single DNA-Cox complexes in nanofluidic channels and compare how the Cox homologs from phages P2 and WΦ affect DNA. By measuring the extension of nanoconfined DNA in absence and presence of Cox we show that the protein compacts DNA and that the binding is highly cooperative, in agreement with the model of a Cox filament around which DNA is wrapped. Furthermore, comparing microscopy images for the wild-type P2 Cox protein and two mutants allows us to discriminate between compaction due to filament formation and compaction by monomeric Cox. P2 and WΦ Cox have similar effects on the physical properties of DNA and the subtle, but significant, differences in DNA binding are due to differences in binding affinity rather than binding mode. The presented work highlights the use of single DNA molecule studies to confirm structural predictions from X-ray crystallography. It also shows how a small protein by oligomerization can have great impact on the organization of DNA and thereby fulfill multiple regulatory functions. PMID:27131370

  1. Host DNA replication forks are not preferred targets for bacteriophage Mu transposition.

    PubMed Central

    Nakai, H; Taylor, A L

    1985-01-01

    Bacteriophage Mu DNA integration in Escherichia coli strains infected after alignment of chromosomal replication was analyzed by a sandwich hybridization assay. The results indicated that Mu integrated into chromosomal segments at various distances from oriC with similar kinetics. In an extension of these studies, various Hfr strains were infected after alignment of chromosomal replication, and Mu transposition was shut down early after infection. The positions of integrated Mu copies were inferred from the transfer kinetics of Mu to an F- strain. Our analysis indicated that the location of Mu DNA in the host chromosome was not dependent on the positions of host replication forks at the time of infection. However, the procedure for aligning chromosomal replication affected DNA transfer by various Hfr strains differently, and this effect could account for prior results suggesting preferential integration of Mu at host replication forks. Images PMID:3159718

  2. Translesion synthesis is the main component of SOS repair in bacteriophage lambda DNA.

    PubMed Central

    Defais, M; Lesca, C; Monsarrat, B; Hanawalt, P

    1989-01-01

    Agents that interfere with DNA replication in Escherichia coli induce physiological adaptations that increase the probability of survival after DNA damage and the frequency of mutants among the survivors (the SOS response). Such agents also increase the survival rate and mutation frequency of irradiated bacteriophage after infection of treated bacteria, a phenomenon known as Weigle reactivation. In UV-irradiated single-stranded DNA phage, Weigle reactivation is thought to occur via induced, error-prone replication through template lesions (translesion synthesis [P. Caillet-Fauquet, M: Defais, and M. Radman, J. Mol. Biol. 117:95-112, 1977]). Weigle reactivation occurs with higher efficiency in double-stranded DNA phages such as lambda, and we therefore asked if another process, recombination between partially replicated daughter molecules, plays a major role in this case. To distinguish between translesion synthesis and recombinational repair, we studied the early replication of UV-irradiated bacteriophage lambda in SOS-induced and uninduced bacteria. To avoid complications arising from excision of UV lesions, we used bacterial uvrA mutants, in which such excision does not occur. Our evidence suggests that translesion synthesis is the primary component of Weigle reactivation of lambda phage in the absence of excision repair. The greater efficiency in Weigle reactivation of double-stranded DNA phage could thus be attributed to some inducible excision repair unable to occur on single-stranded DNA. In addition, after irradiation, lambda phage replication seems to switch prematurely from the theta mode to the rolling circle mode. Images PMID:2527845

  3. Conformational Dynamics of Bacteriophage T7 DNA Polymerase and its Processivity Factor, Escherichia coli thioredoxin

    SciTech Connect

    Akabayov, B.; Akabayov, S; Lee , S; Tabor, S; Kulczyk , A; Richardson, C

    2010-01-01

    Gene 5 of bacteriophage T7 encodes a DNA polymerase (gp5) responsible for the replication of the phage DNA. Gp5 polymerizes nucleotides with low processivity, dissociating after the incorporation of 1 to 50 nucleotides. Thioredoxin (trx) of Escherichia coli binds tightly (Kd = 5 nM) to a unique segment in the thumb subdomain of gp5 and increases processivity. We have probed the molecular basis for the increase in processivity. A single-molecule experiment reveals differences in rates of enzymatic activity and processivity between gp5 and gp5/trx. Small angle X-ray scattering studies combined with nuclease footprinting reveal two conformations of gp5, one in the free state and one upon binding to trx. Comparative analysis of the DNA binding clefts of DNA polymerases and DNA binding proteins show that the binding surface contains more hydrophobic residues than other DNA binding proteins. The balanced composition between hydrophobic and charged residues of the binding site allows for efficient sliding of gp5/trx on the DNA. We propose a model for trx-induced conformational changes in gp5 that enhance the processivity by increasing the interaction of gp5 with DNA.

  4. Kinetics of Mismatch Formation opposite Lesions by the Replicative DNA Polymerase from Bacteriophage RB69

    SciTech Connect

    Hogg, Matthew; Rudnicki, Jean; Midkiff, John; Reha-Krantz, Linda; Doubli, Sylvie; Wallace, Susan S.

    2010-04-12

    The fidelity of DNA replication is under constant threat from the formation of lesions within the genome. Oxidation of DNA bases leads to the formation of altered DNA bases such as 8-oxo-7,8-dihydroguanine, commonly called 8-oxoG, and 2-hydroxyadenenine, or 2-OHA. In this work we have examined the incorporation kinetics opposite these two oxidatively derived lesions as well as an abasic site analogue by the replicative DNA polymerase from bacteriophage RB69. We compared the kinetic parameters for both wild type and the low fidelity L561A variant. While nucleotide incorporation rates (k{sub pol}) were generally higher for the variant, the presence of a lesion in the templating position reduced the ability of both the wild-type and variant DNA polymerases to form ternary enzyme-DNA-dNTP complexes. Thus, the L561A substitution does not significantly affect the ability of the RB69 DNA polymerase to recognize damaged DNA; instead, the mutation increases the probability that nucleotide incorporation will occur. We have also solved the crystal structure of the L561A variant forming an 8-oxoG {center_dot} dATP mispair and show that the propensity for forming this mispair depends on an enlarged polymerase active site.

  5. DNA compaction by the bacteriophage protein Cox studied on the single DNA molecule level using nanofluidic channels

    PubMed Central

    Frykholm, Karolin; Berntsson, Ronnie Per-Arne; Claesson, Magnus; de Battice, Laura; Odegrip, Richard; Stenmark, Pål; Westerlund, Fredrik

    2016-01-01

    The Cox protein from bacteriophage P2 forms oligomeric filaments and it has been proposed that DNA can be wound up around these filaments, similar to how histones condense DNA. We here use fluorescence microscopy to study single DNA–Cox complexes in nanofluidic channels and compare how the Cox homologs from phages P2 and WΦ affect DNA. By measuring the extension of nanoconfined DNA in absence and presence of Cox we show that the protein compacts DNA and that the binding is highly cooperative, in agreement with the model of a Cox filament around which DNA is wrapped. Furthermore, comparing microscopy images for the wild-type P2 Cox protein and two mutants allows us to discriminate between compaction due to filament formation and compaction by monomeric Cox. P2 and WΦ Cox have similar effects on the physical properties of DNA and the subtle, but significant, differences in DNA binding are due to differences in binding affinity rather than binding mode. The presented work highlights the use of single DNA molecule studies to confirm structural predictions from X-ray crystallography. It also shows how a small protein by oligomerization can have great impact on the organization of DNA and thereby fulfill multiple regulatory functions. PMID:27131370

  6. Novel DNA packaging recognition in the unusual bacteriophage N15.

    PubMed

    Feiss, Michael; Geyer, Henriette; Klingberg, Franco; Moreno, Norma; Forystek, Amanda; Maluf, Nasib Karl; Sippy, Jean

    2015-08-01

    Phage lambda's cosB packaging recognition site is tripartite, consisting of 3 TerS binding sites, called R sequences. TerS binding to the critical R3 site positions the TerL endonuclease for nicking cosN to generate cohesive ends. The N15 cos (cos(N15)) is closely related to cos(λ), but whereas the cosB(N15) subsite has R3, it lacks the R2 and R1 sites and the IHF binding site of cosB(λ). A bioinformatic study of N15-like phages indicates that cosB(N15) also has an accessory, remote rR2 site, which is proposed to increase packaging efficiency, like R2 and R1 of lambda. N15 plus five prophages all have the rR2 sequence, which is located in the TerS-encoding 1 gene, approximately 200 bp distal to R3. An additional set of four highly related prophages, exemplified by Monarch, has R3 sequence, but also has R2 and R1 sequences characteristic of cosB-λ. The DNA binding domain of TerS-N15 is a dimer. PMID:25956737

  7. Novel DNA Packaging Recognition in the Unusual Bacteriophage N15

    PubMed Central

    Feiss, Michael; Geyer, Henriette; Klingberg, Franco; Moreno, Norma; Sippy, Jean

    2015-01-01

    Phage lambda’s cosB packaging recognition site is tripartite, consisting of 3 TerS binding sites, called R sequences. TerS binding to the critical R3 site positions the TerL endonuclease for nicking cosN to generate cohesive ends. The N15 cos (cos-N15) is closely related to cos-λ, but whereas the cosB-N15 subsite has R3, it lacks the R2 and R1 sites and the IHF binding site of cosB-λ. A bioinformatic study of N15-like phages indicates that cosB-N15 also has an accessory, remote rR2 site, which is proposed to increase packaging efficiency, like R2 and R1 of lambda. N15 plus five prophages all have the rR2 sequence, which is located in the TerS-encoding 1 gene, approx. 200 bp distal to R3. An additional set of four highly related prophages, exemplified by Monarch, has R3 sequence, but also has R2 and R1 sequences characteristic of cosB-lambda. The DNA binding domain of TerS-N15 is a dimer. PMID:25956737

  8. Fabrication of massive sheets of single layer patterned arrays using lipid directed reengineered phi29 motor dodecamer.

    PubMed

    Xiao, Feng; Sun, Jinchuan; Coban, Oana; Schoen, Peter; Wang, Joseph Che-Yen; Cheng, R Holland; Guo, Peixuan

    2009-01-27

    The bottom-up assembly of patterned arrays is an exciting and important area in current nanotechnology. Arrays can be engineered to serve as components in chips for a virtually inexhaustible list of applications ranging from disease diagnosis to ultra-high-density data storage. Phi29 motor dodecamer has been reported to form elegant multilayer tetragonal arrays. However, multilayer protein arrays are of limited use for nanotechnological applications which demand nanoreplica or coating technologies. The ability to produce a single layer array of biological structures with high replication fidelity represents a significant advance in the area of nanomimetics. In this paper, we report on the assembly of single layer sheets of reengineered phi29 motor dodecamer. A thin lipid monolayer was used to direct the assembly of massive sheets of single layer patterned arrays of the reengineered motor dodecamer. Uniform, clean and highly ordered arrays were constructed as shown by both transmission electron microscopy and atomic force microscopy imaging. PMID:19206255

  9. Evidence for an electrostatic mechanism of force generation by the bacteriophage T4 DNA packaging motor

    NASA Astrophysics Data System (ADS)

    Migliori, Amy D.; Keller, Nicholas; Alam, Tanfis I.; Mahalingam, Marthandan; Rao, Venigalla B.; Arya, Gaurav; Smith, Douglas E.

    2014-06-01

    How viral packaging motors generate enormous forces to translocate DNA into viral capsids remains unknown. Recent structural studies of the bacteriophage T4 packaging motor have led to a proposed mechanism wherein the gp17 motor protein translocates DNA by transitioning between extended and compact states, orchestrated by electrostatic interactions between complimentarily charged residues across the interface between the N- and C-terminal subdomains. Here we show that site-directed alterations in these residues cause force dependent impairments of motor function including lower translocation velocity, lower stall force and higher frequency of pauses and slips. We further show that the measured impairments correlate with computed changes in free-energy differences between the two states. These findings support the proposed structural mechanism and further suggest an energy landscape model of motor activity that couples the free-energy profile of motor conformational states with that of the ATP hydrolysis cycle.

  10. The role of bacteriophage T7 gene 2 protein in DNA replication.

    PubMed Central

    Mooney, P Q; North, R; Molineux, I J

    1980-01-01

    The in vivo function of the gene 2 protein of bacteriophage T7 has been examined. The gene 2 protein appears to modulate the activity of the gene 3 endonuclease in order to prevent the premature degradation of any newly-formed DNA concatemers. This modulation is not however a direct interacton between the two proteins. In single-burst experiments rifamycin can substitute for the gene 2 protein, allowing formation of fast-sedimenting replicative DNA intermediates and progeny phage production. This suggests that the sole function of the gene 2 protein is inhibition of the host RNA polymerase and that the latter enzyme directs or promotes the endonucleolytic action of the gene 3 protein. PMID:7001361

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

    PubMed Central

    Leavitt, Justin C.; Gogokhia, Lasha; Gilcrease, Eddie B.; Bhardwaj, Anshul; Cingolani, Gino; Casjens, Sherwood R.

    2013-01-01

    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. PMID:23951045

  12. Salt-Dependent DNA-DNA Spacings in Intact Bacteriophage λ Reflect Relative Importance of DNA Self-Repulsion and Bending Energies

    NASA Astrophysics Data System (ADS)

    Qiu, Xiangyun; Rau, Donald C.; Parsegian, V. Adrian; Fang, Li Tai; Knobler, Charles M.; Gelbart, William M.

    2011-01-01

    Using solution synchrotron x-ray scattering, we measure the variation of DNA-DNA d spacings in bacteriophage λ with mono-, di-, and polyvalent salt concentrations, for wild-type [48.5×103 base pairs (bp)] and short-genome-mutant (37.8 kbp) strains. From the decrease in d spacings with increasing salt, we deduce the relative contributions of DNA self-repulsion and bending to the energetics of packaged phage genomes. We quantify the DNA-DNA interaction energies within the intact phage by combining the measured d spacings in the capsid with measurements of osmotic pressure in DNA assemblies under the same salt conditions in bulk solution. In the commonly used Tris-Mg buffer, the DNA-DNA interaction energies inside the phage capsids are shown to be about 1kT/bp, an order of magnitude larger than the bending energies.

  13. Salt-Dependent DNA-DNA Spacings in Intact Bacteriophage lambda Reflect Relative Importance of DNA Self-Repulsion and Bending Energies

    SciTech Connect

    X Qiu; D Rau; V Parsegian; L Fang; C Knobler; W Gelbart

    2011-12-31

    Using solution synchrotron x-ray scattering, we measure the variation of DNA-DNA d spacings in bacteriophage {lambda} with mono-, di-, and polyvalent salt concentrations, for wild-type [48.5 x 10{sup 3} base pairs (bp)] and short-genome-mutant (37.8 kbp) strains. From the decrease in d spacings with increasing salt, we deduce the relative contributions of DNA self-repulsion and bending to the energetics of packaged phage genomes. We quantify the DNA-DNA interaction energies within the intact phage by combining the measured d spacings in the capsid with measurements of osmotic pressure in DNA assemblies under the same salt conditions in bulk solution. In the commonly used Tris-Mg buffer, the DNA-DNA interaction energies inside the phage capsids are shown to be about 1 kT/bp, an order of magnitude larger than the bending energies.

  14. Exclusion of small terminase mediated DNA threading models for genome packaging in bacteriophage T4.

    PubMed

    Gao, Song; Zhang, Liang; Rao, Venigalla B

    2016-05-19

    Tailed bacteriophages and herpes viruses use powerful molecular machines to package their genomes. The packaging machine consists of three components: portal, motor (large terminase; TerL) and regulator (small terminase; TerS). Portal, a dodecamer, and motor, a pentamer, form two concentric rings at the special five-fold vertex of the icosahedral capsid. Powered by ATPase, the motor ratchets DNA into the capsid through the portal channel. TerS is essential for packaging, particularly for genome recognition, but its mechanism is unknown and controversial. Structures of gear-shaped TerS rings inspired models that invoke DNA threading through the central channel. Here, we report that mutations of basic residues that line phage T4 TerS (gp16) channel do not disrupt DNA binding. Even deletion of the entire channel helix retained DNA binding and produced progeny phage in vivo On the other hand, large oligomers of TerS (11-mers/12-mers), but not small oligomers (trimers to hexamers), bind DNA. These results suggest that TerS oligomerization creates a large outer surface, which, but not the interior of the channel, is critical for function, probably to wrap viral genome around the ring during packaging initiation. Hence, models involving TerS-mediated DNA threading may be excluded as an essential mechanism for viral genome packaging. PMID:26984529

  15. Exclusion of small terminase mediated DNA threading models for genome packaging in bacteriophage T4

    PubMed Central

    Gao, Song; Zhang, Liang; Rao, Venigalla B.

    2016-01-01

    Tailed bacteriophages and herpes viruses use powerful molecular machines to package their genomes. The packaging machine consists of three components: portal, motor (large terminase; TerL) and regulator (small terminase; TerS). Portal, a dodecamer, and motor, a pentamer, form two concentric rings at the special five-fold vertex of the icosahedral capsid. Powered by ATPase, the motor ratchets DNA into the capsid through the portal channel. TerS is essential for packaging, particularly for genome recognition, but its mechanism is unknown and controversial. Structures of gear-shaped TerS rings inspired models that invoke DNA threading through the central channel. Here, we report that mutations of basic residues that line phage T4 TerS (gp16) channel do not disrupt DNA binding. Even deletion of the entire channel helix retained DNA binding and produced progeny phage in vivo. On the other hand, large oligomers of TerS (11-mers/12-mers), but not small oligomers (trimers to hexamers), bind DNA. These results suggest that TerS oligomerization creates a large outer surface, which, but not the interior of the channel, is critical for function, probably to wrap viral genome around the ring during packaging initiation. Hence, models involving TerS-mediated DNA threading may be excluded as an essential mechanism for viral genome packaging. PMID:26984529

  16. Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9

    PubMed Central

    Bryson, Alexandra L.; Hwang, Young; Sherrill-Mix, Scott; Wu, Gary D.; Lewis, James D.; Black, Lindsay; Clark, Tyson A.

    2015-01-01

    ABSTRACT The genomic DNAs of tailed bacteriophages are commonly modified by the attachment of chemical groups. Some forms of DNA modification are known to protect phage DNA from cleavage by restriction enzymes, but others are of unknown function. Recently, the CRISPR-Cas nuclease complexes were shown to mediate bacterial adaptive immunity by RNA-guided target recognition, raising the question of whether phage DNA modifications may also block attack by CRISPR-Cas9. We investigated phage T4 as a model system, where cytosine is replaced with glucosyl-hydroxymethylcytosine (glc-HMC). We first quantified the extent and distribution of covalent modifications in T4 DNA by single-molecule DNA sequencing and enzymatic probing. We then designed CRISPR spacer sequences targeting T4 and found that wild-type T4 containing glc-HMC was insensitive to attack by CRISPR-Cas9 but mutants with unmodified cytosine were sensitive. Phage with HMC showed only intermediate sensitivity. While this work was in progress, another group reported examples of heavily engineered CRISRP-Cas9 complexes that could, in fact, overcome the effects of T4 DNA modification, indicating that modifications can inhibit but do not always fully block attack. PMID:26081634

  17. Structure and physical map of Rhodopseudomonas sphaeroides bacteriophage RS1 DNA.

    PubMed Central

    Donohue, T J; Chory, J; Goldsand, T E; Lynn, S P; Kaplan, S

    1985-01-01

    We analyzed, by restriction endonuclease mapping and electron microscopy, the genome of the lytic Rhodopseudomonas sphaeroides-specific bacteriophage RS1 and characterized it as a linear molecule of approximately 60 to 65 kilobases. When the DNA from purified phage particles was examined by several independent methods, considerable size heterogeneity was apparent in the RS1 DNA. This size heterogeneity was concluded to be of biological origin, was independent of the specific host strain used to propagate virus, and was not due to the presence of host DNA within or nonspecifically associated with purified virions. In addition, treatment of RS1 DNA with either BAL 31 nuclease or DNA polymerase I Klenow fragment revealed that several distinct regions exist within the viral chromosome which contain free 3' hydroxyl groups. A restriction endonuclease map of the RS1 genome was constructed by using the restriction endonucleases EcoRI, ClaI, KpnI, BamHI, MluI, SmaI, and BclI; thereby allowing the positioning of some 40 restriction sites within the viral genome. The results are discussed in terms of the significance and the possible biological origin of the unique features discovered within the phage RS1 DNA. Images PMID:2989552

  18. Further Studies on Bacteriophage T4 DNA Synthesis in Sucrose-Plasmolyzed Cells

    PubMed Central

    Stafford, Mary E.; Reddy, G. Prem Veer; Mathews, Christopher K.

    1977-01-01

    This paper describes several technical improvements in the sucrose-plasmolyzed cell system used in earlier experiments on DNA synthesis in situ with Escherichia coli infected by DNA-defective mutants of bacteriophage T4 (W. L. Collinsworth and C. K. Mathews, J. Virol. 13:908-915, 1974). Using this system, which is based primarily on that of M. G. Wovcha et al. (Proc. Natl. Acad. Sci. U.S.A. 70:2196-2200, 1973), we reinvestigated the properties of mutants bearing lesions in genes 1, 41, and 62, and we resolved some disagreements with data reported from that laboratory. We also asked whether the DNA-delay phenotype of T4 mutants is related to possible early leakage of DNA precursors from infected cells. Such cells display defective DNA synthesis in situ, even when ample DNA precursors are made available. Thus, the lesions associated with these mutations seem to manifest themselves at the level of macromolecular metabolism. Similarly, we examined an E. coli mutant defective in its ability to support T4 production, apparently because of a lesion affecting DNA synthesis (L. Simon et al., Nature [London] 252:451-455). In the plasmolyzed cell system, reduced nucleotide incorporation is seen, indicating also that the genetic defect does not involve DNA precursor synthesis. The plasmolyzed cell system incorporates deoxynucleotide 5′-monophosphates into DNA severalfold more rapidly than the corresponding 5′-triphosphates. This is consistent with the idea that DNA precursor-synthesizing enzymes are functionally organized to shuttle substrates to their sites of utilization. PMID:328926

  19. Architecture of the bacteriophage T4 activator MotA/promoter DNA interaction during sigma appropriation.

    PubMed

    Hsieh, Meng-Lun; James, Tamara D; Knipling, Leslie; Waddell, M Brett; White, Stephen; Hinton, Deborah M

    2013-09-20

    Gene expression can be regulated through factors that direct RNA polymerase to the correct promoter sequence at the correct time. Bacteriophage T4 controls its development in this way using phage proteins that interact with host RNA polymerase. Using a process called σ appropriation, the T4 co-activator AsiA structurally remodels the σ(70) subunit of host RNA polymerase, while a T4 activator, MotA, engages the C terminus of σ(70) and binds to a DNA promoter element, the MotA box. Structures for the N-terminal (NTD) and C-terminal (CTD) domains of MotA are available, but no structure exists for MotA with or without DNA. We report the first molecular map of the MotA/DNA interaction within the σ-appropriated complex, which we obtained by using the cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). We conjugated surface-exposed, single cysteines in MotA with FeBABE and performed cleavage reactions in the context of stable transcription complexes. The DNA cleavage sites were analyzed using ICM Molsoft software and three-dimensional physical models of MotA(NTD), MotA(CTD), and the DNA to investigate shape complementarity between the protein and the DNA and to position MotA on the DNA. We found that the unusual "double wing" motif present within MotA(CTD) resides in the major groove of the MotA box. In addition, we have used surface plasmon resonance to show that MotA alone is in a very dynamic equilibrium with the MotA element. Our results demonstrate the utility of fine resolution FeBABE mapping to determine the architecture of protein-DNA complexes that have been recalcitrant to traditional structure analyses. PMID:23902794

  20. Assembly Architecture and DNA Binding of the Bacteriophage P22 Terminase Small Subunit

    PubMed Central

    Němeček, Daniel; Lander, Gabriel C.; Johnson, John E.; Casjens, Sherwood R.; Thomas, George J.

    2008-01-01

    Summary Morphogenesis of bacteriophage P22 involves the packaging of double-stranded DNA into a preassembled procapsid. DNA is translocated by a powerful virally-encoded molecular motor called terminase, which comprises large (gp2, 499 residues) and small (gp3, 162 residues) subunits. While gp2 contains the phosphohydrolase and endonuclease activities of terminase, the function of gp3 may be to regulate specific and nonspecific modes of DNA recognition as well as the enzymatic activities of gp2. Electron microscopy shows that wildtype gp3 self-assembles into a stable and monodisperse nonameric ring. A three-dimensional reconstruction at 18 Å resolution provides the first glimpse of P22 terminase architecture and implies two distinct modes of interaction with DNA – involving a central channel of 20 Å diameter and radial spikes separated by 34 Å. Electromobility shift assays indicate that the gp3 ring binds dsDNA nonspecifically in vitro via electrostatic interactions between the positively charged C-terminus of gp3 (residues 143–152) and phosphates of the DNA backbone. Raman spectra show that nonameric rings formed by subunits truncated at residue 142 retain the subunit fold, despite the loss of DNA-binding activity. Difference density maps between gp3 rings containing full-length and C-terminally truncated subunits are consistent with localization of residues 143–152 along the central channel of the nonameric ring. The results suggest a plausible molecular mechanism for gp3 function in DNA recognition and translocation. PMID:18775728

  1. Subunit Conformations and Assembly States of a DNA Translocating Motor: The Terminase of Bacteriophage P22

    PubMed Central

    Němeček, Daniel; Gilcrease, Eddie B.; Kang, Sebyung; Prevelige, Peter E.; Casjens, Sherwood; Thomas, George J.

    2007-01-01

    Bacteriophage P22, a podovirus infecting strains of Salmonella typhimurium, packages a 42 kbp genome using a headful mechanism. DNA translocation is accomplished by the phage terminase, a powerful molecular motor consisting of large and small subunits. Although many of the structural proteins of the P22 virion have been well characterized, little is known about the terminase subunits and their molecular mechanism of DNA translocation. We report here structural and assembly properties of ectopically expressed and highly purified terminase large and small subunits. The large subunit (gp2), which contains the nuclease and ATPase activities of terminase, exists as a stable monomer with an α/β fold. The small subunit (gp3), which recognizes DNA for packaging and may regulate gp2 activity, exhibits a highly α-helical secondary structure and self-associates to form a stable oligomeric ring in solution. For wildtype gp3, the ring contains nine subunits, as demonstrated by hydrodynamic measurements, electron microscopy and native mass spectrometry. We have also characterized a gp3 mutant (Ala 112 → Thr) that forms a ten subunit ring, despite a subunit fold indistinguishable from wildtype. Both the nonameric and decameric gp3 rings exhibit nonspecific DNA binding activity, and gp2 is able to bind strongly to the DNA/gp3 complex but not to DNA alone. We propose a scheme for the roles of P22 terminase large and small subunits in the recruitment and packaging of viral DNA and discuss the model in relation to proposals for terminase-driven DNA translocation in other phages. PMID:17945256

  2. Nonessential region of bacteriophage P4: DNA sequence, transcription, gene products, and functions.

    PubMed Central

    Ghisotti, D; Finkel, S; Halling, C; Dehò, G; Sironi, G; Calendar, R

    1990-01-01

    We sequenced the leftmost 2,640 base pairs of bacteriophage P4 DNA, thus completing the sequence of the 11,627-base-pair P4 genome. The newly sequenced region encodes three nonessential genes, which are called gop, beta, and cII (in order, from left to right). The gop gene product kills Escherichia coli when the beta protein is absent; the gop and beta genes are transcribed rightward from the same promoter. The cII gene is transcribed leftward to a rho-independent terminator. Mutation of this terminator creates a temperature-sensitive phenotype, presumably owing to a defect in expression of the beta gene. Images PMID:2403440

  3. Gp15 and gp16 cooperate in translocating bacteriophage T7 DNA into the infected cell

    PubMed Central

    Chang, Chung-Yu; Kemp, Priscilla; Molineux, Ian J.

    2010-01-01

    Loss of up to four amino acids from the C terminus of the 1318 residue bacteriophage T7 gp16 allows plaque formation at normal efficiencies. Loss of five residues results in non-infective virions, and loss of twelve prevents assembly of stable particles. However, replacing the C-terminal seven with nineteen non-native residues allows assembly of non-infective virions. The latter adsorb and eject internal core proteins into the cell envelope but no phage DNA enters the cytoplasm. Extragenic suppressors of the defective gene 16 lie in gene 15; the mutant gp15 proteins not only re-establish infectivity, they fully restore the kinetics of genome internalization to those exhibited by wild-type phage. After ejection from the infecting particle, gp15 and gp16 thus function together in ratcheting the leading end of the T7 genome into the cytoplasm of the infected cell. PMID:20036409

  4. Identification of a New Motif in Family B DNA Polymerases by Mutational Analyses of the Bacteriophage T4 DNA Polymerase

    PubMed Central

    Li, Vincent; Hogg, Matthew; Reha-Krantz, Linda J.

    2011-01-01

    Structure-based protein sequence alignments of family B DNA polymerases revealed a conserved motif that is formed from interacting residues between loops from the N-terminal and palm domains and between the N-terminal loop and a conserved proline residue. The importance of the motif for function of the bacteriophage T4 DNA polymerase was revealed by suppressor analysis. T4 DNA polymerases that form weak replicating complexes cannot replicate DNA when the dGTP pool is reduced. The conditional lethality provides the means to identify amino acid substitutions that restore replication activity under low dGTP conditions by either correcting the defect produced by the first amino acid substitution or by generally increasing the stability of polymerase complexes; the second type are global suppressors that can effectively counter the reduced stability caused by a variety of amino acid substitutions. Some amino acid substitutions that increase the stability of polymerase complexes produce a new phenotype - sensitivity to the antiviral drug phosphonoacetic acid. Amino acid substitutions that confer decreased ability to replicate DNA under low dGTP conditions or drug sensitivity were identified in the new motif, which suggests that the motif functions in regulating the stability of polymerase complexes. Additional suppressor analyses revealed an apparent network of interactions that link the new motif to the fingers domain and to two patches of conserved residues that bind DNA. The collection of mutant T4 DNA polymerases provides a foundation for future biochemical studies to determine how DNA polymerases remain stably associated with DNA while waiting for the next available dNTP, how DNA polymerases translocate, and the biochemical basis for sensitivity to antiviral drugs. PMID:20493878

  5. Dynamic Measurements of the Position, Orientation, and DNA Content of Individual Unlabeled Bacteriophages.

    PubMed

    Goldfain, Aaron M; Garmann, Rees F; Jin, Yan; Lahini, Yoav; Manoharan, Vinothan N

    2016-07-01

    A complete understanding of the cellular pathways involved in viral infections will ultimately require a diverse arsenal of experimental techniques, including methods for tracking individual viruses and their interactions with the host. Here we demonstrate the use of holographic microscopy to track the position, orientation, and DNA content of unlabeled bacteriophages (phages) in solution near a planar, functionalized glass surface. We simultaneously track over 100 individual λ phages at a rate of 100 Hz across a 33 μm × 33 μm portion of the surface. The technique determines the in-plane motion of the phage to nanometer precision, and the height of the phage above the surface to 100 nm precision. Additionally, we track the DNA content of individual phages as they eject their genome following the addition of detergent-solubilized LamB receptor. The technique determines the fraction of DNA remaining in the phage to within 10% of the total 48.5 kilobase pairs. Analysis of the data reveals that under certain conditions, λ phages move along the surface with their heads down and intermittently stick to the surface by their tails, causing them to stand up. Furthermore, we find that in buffer containing high concentrations of both monovalent and divalent salts, λ phages eject their entire DNA in about 7 s. Taken together, these measurements highlight the potential of holographic microscopy to resolve the fast kinetics of the early stages of phage infection. PMID:27063451

  6. Structure of the RNA claw of the DNA packaging motor of bacteriophage Φ29.

    PubMed

    Harjes, Elena; Kitamura, Aya; Zhao, Wei; Morais, Marc C; Jardine, Paul J; Grimes, Shelley; Matsuo, Hiroshi

    2012-10-01

    Bacteriophage DNA packaging motors translocate their genomic DNA into viral heads, compacting it to near-crystalline density. The Bacillus subtilis phage 29 has a unique ring of RNA (pRNA) that is an essential component of its motor, serving as a scaffold for the packaging ATPase. Previously, deletion of a three-base bulge (18-CCA-20) in the pRNA A-helix was shown to abolish packaging activity. Here, we solved the structure of this crucial bulge by nuclear magnetic resonance (NMR) using a 27mer RNA fragment containing the bulge (27b). The bulge actually involves five nucleotides (17-UCCA-20 and A100), as U17 and A100 are not base paired as predicted. Mutational analysis showed these newly identified bulge residues are important for DNA packaging. The bulge introduces a 33-35° bend in the helical axis, and inter-helical motion around this bend appears to be restricted. A model of the functional 120b pRNA was generated using a 27b NMR structure and the crystal structure of the 66b prohead-binding domain. Fitting this model into a cryo-EM map generated a pentameric pRNA structure; five helices projecting from the pRNA ring resemble an RNA claw. Biochemical analysis suggested that this shape is important for coordinated motor action required for DNA translocation. PMID:22879380

  7. Bacteriophage T4 Mutants Hypersensitive to an Antitumor Agent That Induces Topoisomerase-DNA Cleavage Complexes

    PubMed Central

    Woodworth, D. L.; Kreuzer, K. N.

    1996-01-01

    Many antitumor agents and antibiotics affect cells by interacting with type II topoisomerases, stabilizing a covalent enzyme-DNA complex. A pathway of recombination can apparently repair this DNA damage. In this study, transposon mutagenesis was used to identify possible components of the repair pathway in bacteriophage T4. Substantial increases in sensitivity to the antitumor agent m-AMSA [4'-(9-acridinyl-amino) methanesulfon-m-anisidide] were found with transposon insertion mutations that inactivate any of six T4-encoded proteins: UvsY (DNA synaptase accessory protein), UvsW (unknown function), Rnh (RNase H and 5' to 3' DNA exonuclease), α-gt (α-glucosyl transferase), gp47.1 (uncharacterized), and NrdB (β subunit of ribonucleotide reductase). The role of the rnh gene in drug sensitivity was further characterized. First, an in-frame rnh deletion mutation was constructed and analyzed, providing evidence that the absence of Rnh protein causes hypersensitivity to m-AMSA. Second, the m-AMSA sensitivity of the rnh-deletion mutant was shown to require a drug-sensitive T4 topoisomerase. Third, analysis of double mutants suggested that uvsW and rnh mutations impair a common step in the recombinational repair pathway for m-AMSA-induced damage. Finally, the rnh-deletion mutant was found to be hypersensitive to UV, implicating Rnh in recombinational repair of UV-induced damage. PMID:8807283

  8. RNA Nanoparticles Derived from Three-Way Junction of Phi29 Motor pRNA Are Resistant to I-125 and Cs-131 Radiation

    PubMed Central

    Li, Hui; Rychahou, Piotr G.; Cui, Zheng; Pi, Fengmei; Evers, B. Mark; Shu, Dan

    2015-01-01

    Radiation reagents that specifically target tumors are in high demand for the treatment of cancer. The emerging field of RNA nanotechnology might provide new opportunities for targeted radiation therapy. This study investigates whether chemically modified RNA nanoparticles derived from the packaging RNA (pRNA) three-way junction (3WJ) of phi29 DNA-packaging motor are resistant to potent I-125 and Cs-131 radiation, which is a prerequisite for utilizing these RNA nanoparticles as carriers for targeted radiation therapy. pRNA 3WJ nanoparticles were constructed and characterized, and the stability of these nanoparticles under I-125 and Cs-131 irradiation with clinically relevant doses was examined. RNA nanoparticles derived from the pRNA 3WJ targeted tumors specifically and they were stable under irradiation of I-125 and Cs-131 with clinically relevant doses ranging from 1 to 90 Gy over a significantly long time up to 20 days, while control plasmid DNA was damaged at 20 Gy or higher. PMID:26017686

  9. DNA driven self-assembly of micron-sized rods using DNA-grafted bacteriophage fd virions

    NASA Astrophysics Data System (ADS)

    Unwin, R. R.; Cabanas, R. A.; Yanagishima, T.; Blower, T. R.; Takahashi, H.; Salmond, G. P. C.; Edwardson, J. M.; Fraden, S.; Eiser, E.

    We have functionalized the sides of fd bacteriophage virions with oligonucleotides to induce DNA hybridization driven self-assembly of high aspect ratio filamentous particles. Potential impacts of this new structure range from an entirely new building block in DNA origami structures, inclusion of virions in DNA nanostructures and nanomachines, to a new means of adding thermotropic control to lyotropic liquid crystal systems. A protocol for producing the virions in bulk is reviewed. Thiolated oligonucleotides are attached to the viral capsid using a heterobifunctional chemical linker. A commonly used system is utilized, where a sticky, single-stranded DNA strand is connected to an inert double-stranded spacer to increase inter-particle connectivity. Solutions of fd virions carrying complementary strands are mixed, annealed, and their aggregation is studied using dynamic light scattering (DLS), fluorescence microscopy, and atomic force microscopy (AFM). Aggregation is clearly observed on cooling, with some degree of local order, and is reversible when temperature is cycled through the DNA hybridization transition.

  10. DNA driven self-assembly of micron-sized rods using DNA-grafted bacteriophage fd virions.

    PubMed

    Unwin, R R; Cabanas, R A; Yanagishima, T; Blower, T R; Takahashi, H; Salmond, G P C; Edwardson, J M; Fraden, S; Eiser, E

    2015-03-28

    We have functionalized the sides of fd bacteriophage virions with oligonucleotides to induce DNA hybridization driven self-assembly of high aspect ratio filamentous particles. Potential impacts of this new structure range from an entirely new building block in DNA origami structures, inclusion of virions in DNA nanostructures and nanomachines, to a new means of adding thermotropic control to lyotropic liquid crystal systems. A protocol for producing the virions in bulk is reviewed. Thiolated oligonucleotides are attached to the viral capsid using a heterobifunctional chemical linker. A commonly used system is utilized, where a sticky, single-stranded DNA strand is connected to an inert double-stranded spacer to increase inter-particle connectivity. Solutions of fd virions carrying complementary strands are mixed, annealed, and their aggregation is studied using dynamic light scattering (DLS), fluorescence microscopy, and atomic force microscopy (AFM). Aggregation is clearly observed on cooling, with some degree of local order, and is reversible when temperature is cycled through the DNA hybridization transition. PMID:25732957

  11. Replication of linear duplex DNA in vitro with bacteriophage T5 DNA polymerase

    SciTech Connect

    Fujimura, R. K.; Das, S. K.; Allison, D. P.; Roop, B. C.

    1980-01-01

    Two sets of experiments are presented that attempt to contribute to understanding the mechanisms of DNA replication. The specific areas discussed are fidelity of DNA replication and initiation of replication of duplex DNA. (ACR)

  12. DNA Recognition by the DNA Primase of Bacteriophage T7: A Structure Function Study of the Zinc-Binding Domain

    SciTech Connect

    Akabayov, B.; Lee, S; Akabayov, S; Rekhi, S; Zhu, B; Richardson, C

    2009-01-01

    Synthesis of oligoribonucleotide primers for lagging-strand DNA synthesis in the DNA replication system of bacteriophage T7 is catalyzed by the primase domain of the gene 4 helicase-primase. The primase consists of a zinc-binding domain (ZBD) and an RNA polymerase (RPD) domain. The ZBD is responsible for recognition of a specific sequence in the ssDNA template whereas catalytic activity resides in the RPD. The ZBD contains a zinc ion coordinated with four cysteine residues. We have examined the ligation state of the zinc ion by X-ray absorption spectroscopy and biochemical analysis of genetically altered primases. The ZBD of primase engaged in catalysis exhibits considerable asymmetry in coordination to zinc, as evidenced by a gradual increase in electron density of the zinc together with elongation of the zinc-sulfur bonds. Both wild-type primase and primase reconstituted from purified ZBD and RPD have a similar electronic change in the level of the zinc ion as well as the configuration of the ZBD. Single amino acid replacements in the ZBD (H33A and C36S) result in the loss of both zinc binding and its structural integrity. Thus the zinc in the ZBD may act as a charge modulation indicator for the surrounding sulfur atoms necessary for recognition of specific DNA sequences.

  13. DNA Packaging Specificity of Bacteriophage N15 with an Excursion into the Genetics of a Cohesive End Mismatch

    PubMed Central

    Feiss, Michael; Young Min, Jea; Sultana, Sawsan; Patel, Priyal; Sippy, Jean

    2015-01-01

    During DNA replication by the λ-like bacteriophages, immature concatemeric DNA is produced by rolling circle replication. The concatemers are processed into mature chromosomes with cohesive ends, and packaged into prohead shells, during virion assembly. Cohesive ends are generated by the viral enzyme terminase, which introduces staggered nicks at cos, an approx. 200 bp-long sequence containing subsites cosQ, cosN and cosB. Interactions of cos subsites of immature concatemeric DNA with terminase orchestrate DNA processing and packaging. To initiate DNA packaging, terminase interacts with cosB and nicks cosN. The cohesive ends of N15 DNA differ from those of λ at 2/12 positions. Genetic experiments show that phages with chromosomes containing mismatched cohesive ends are functional. In at least some infections, the cohesive end mismatch persists through cyclization and replication, so that progeny phages of both allelic types are produced in the infected cell. N15 possesses an asymmetric packaging specificity: N15 DNA is not packaged by phages λ or 21, but surprisingly, N15-specific terminase packages λ DNA. Implications for genetic interactions among λ-like bacteriophages are discussed. PMID:26633301

  14. DNA sequences at the ends of the genome of bacteriophage Mu essential for transposition.

    PubMed Central

    Groenen, M A; Timmers, E; van de Putte, P

    1985-01-01

    We have determined the minimal DNA sequences at the ends of the genome of bacteriophage Mu that are required for its transposition. A mini-Mu was constructed on a multicopy plasmid that enabled the manipulation of the DNA sequences at its ends without affecting the genes essential for transposition. The genes A and B, which were cloned outside the ends of the mini-Mu on the same plasmid, were both needed for optimal transposition. In our experimental system the predominant end products of the transposition are cointegrates both in the presence and in the absence of B. Two regions ending approximately 25 and 160 bp from the left end and one ending approximately 50 bp from the right end appear to be essential for optimal transposition. Overlapping with these regions, a 22-base-pair sequence was recognized with the consensus Y-G-T-T-C-A-Y-T-N-N-A-A-R-Y-R-C-G-A-A-A-A, where Y and R represent any pyrimidine and purine, respectively. At the left end these sequences occur as direct repeats; at the right end this sequence is inverted with respect to those at the left end. PMID:2984681

  15. Bacteriophage orphan DNA methyltransferases: insights from their bacterial origin, function, and occurrence.

    PubMed

    Murphy, James; Mahony, Jennifer; Ainsworth, Stuart; Nauta, Arjen; van Sinderen, Douwe

    2013-12-01

    Type II DNA methyltransferases (MTases) are enzymes found ubiquitously in the prokaryotic world, where they play important roles in several cellular processes, such as host protection and epigenetic regulation. Three classes of type II MTases have been identified thus far in bacteria which function in transferring a methyl group from S-adenosyl-l-methionine (SAM) to a target nucleotide base, forming N-6-methyladenine (class I), N-4-methylcytosine (class II), or C-5-methylcytosine (class III). Often, these MTases are associated with a cognate restriction endonuclease (REase) to form a restriction-modification (R-M) system protecting bacterial cells from invasion by foreign DNA. When MTases exist alone, which are then termed orphan MTases, they are believed to be mainly involved in regulatory activities in the bacterial cell. Genomes of various lytic and lysogenic phages have been shown to encode multi- and mono-specific orphan MTases that have the ability to confer protection from restriction endonucleases of their bacterial host(s). The ability of a phage to overcome R-M and other phage-targeting resistance systems can be detrimental to particular biotechnological processes such as dairy fermentations. Conversely, as phages may also be beneficial in certain areas such as phage therapy, phages with additional resistance to host defenses may prolong the effectiveness of the therapy. This minireview will focus on bacteriophage-encoded MTases, their prevalence and diversity, as well as their potential origin and function. PMID:24123737

  16. Function of DNA polymerase I in RNA-primed synthesis of bacteriophage M-13 duplex DNA.

    PubMed Central

    Schneck, P K; Staudenbauer, W L; Hofschneider, P H

    1976-01-01

    Cell-free extracts from Escherichia coli contain a DNA polymerase activity resistant to SH-blocking agents, which is capable of synthesizing complementary strand DNA on a circular M-13 DNA template by extension of RNA primers. This activity is considered to be identical with DNA polymerase I (or some altered form of this enzyme) since it is missing in extracts from po1A- cells. DNA synthesis in the presence of SH-blocking agents occurs at a reduced rate as compared to untreated controls and leads to the formation of DNA chains of defined size (0.4-0.5 genome's length). It is concluded that efficient M-13 duplex DNA synthesis requires the cooperation of both DNA polymerase I and III. PMID:1272793

  17. DNA-Binding Proteins Essential for Protein-Primed Bacteriophage Φ29 DNA Replication

    PubMed Central

    Salas, Margarita; Holguera, Isabel; Redrejo-Rodríguez, Modesto; de Vega, Miguel

    2016-01-01

    Bacillus subtilis phage Φ29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5′ ends of the DNA. This protein, called terminal protein (TP), is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The DNA polymerase further elongates the nascent DNA chain in a processive manner, coupling strand displacement with elongation. The viral protein p5 is a single-stranded DNA binding protein (SSB) that binds to the single strands generated by strand displacement during the elongation process. Viral protein p6 is a double-stranded DNA binding protein (DBP) that preferentially binds to the origins of replication at the Φ29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for Φ29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in Φ29 DNA replication both in vitro and in vivo, as well as on the implication of several B. subtilis DNA-binding proteins in different processes of the viral cycle. We will revise the enzymatic activities of the Φ29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3′–5′ exonucleolysis and pyrophosphorolysis. The resolution of the Φ29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made Φ29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of Φ29 TP revealed the existence of three domains: the priming domain, where the primer residue Ser232, as well as Phe230, involved in the determination of the initiating nucleotide, are located, the intermediate domain, involved in DNA polymerase binding, and the N-terminal domain, responsible for DNA binding and

  18. DNA-Binding Proteins Essential for Protein-Primed Bacteriophage Φ29 DNA Replication.

    PubMed

    Salas, Margarita; Holguera, Isabel; Redrejo-Rodríguez, Modesto; de Vega, Miguel

    2016-01-01

    Bacillus subtilis phage Φ29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5' ends of the DNA. This protein, called terminal protein (TP), is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The DNA polymerase further elongates the nascent DNA chain in a processive manner, coupling strand displacement with elongation. The viral protein p5 is a single-stranded DNA binding protein (SSB) that binds to the single strands generated by strand displacement during the elongation process. Viral protein p6 is a double-stranded DNA binding protein (DBP) that preferentially binds to the origins of replication at the Φ29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for Φ29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in Φ29 DNA replication both in vitro and in vivo, as well as on the implication of several B. subtilis DNA-binding proteins in different processes of the viral cycle. We will revise the enzymatic activities of the Φ29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3'-5' exonucleolysis and pyrophosphorolysis. The resolution of the Φ29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made Φ29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of Φ29 TP revealed the existence of three domains: the priming domain, where the primer residue Ser232, as well as Phe230, involved in the determination of the initiating nucleotide, are located, the intermediate domain, involved in DNA polymerase binding, and the N-terminal domain, responsible for DNA binding and localization of the

  19. Is the In Vitro Ejection of Bacteriophage DNA Quasistatic? A Bulk to Single Virus Study

    PubMed Central

    Chiaruttini, N.; de Frutos, M.; Augarde, E.; Boulanger, P.; Letellier, L.; Viasnoff, V.

    2010-01-01

    Abstract Bacteriophage T5 DNA ejection is a complex process that occurs on several timescales in vitro. By using a combination of bulk and single phage measurements, we quantitatively study the three steps of the ejection—binding to the host receptor, channel-opening, and DNA release. Each step is separately addressed and its kinetics parameters evaluated. We reconstruct the bulk kinetics from the distribution of single phage events by following individual DNA molecules with unprecedented time resolution. We show that, at the single phage level, the ejection kinetics of the DNA happens by rapid transient bursts that are not correlated to any genome sequence defects. We speculate that these transient pauses are due to local phase transitions of the DNA inside the capsid. We predict that such pauses should be seen for other phages with similar DNA packing ratios. PMID:20643062

  20. The tight linkage between DNA replication and double-strand break repair in bacteriophage T4

    PubMed Central

    George, James W.; Stohr, Bradley A.; Tomso, Daniel J.; Kreuzer, Kenneth N.

    2001-01-01

    Double-strand break (DSB) repair and DNA replication are tightly linked in the life cycle of bacteriophage T4. Indeed, the major mode of phage DNA replication depends on recombination proteins and can be stimulated by DSBs. DSB-stimulated DNA replication is dramatically demonstrated when T4 infects cells carrying two plasmids that share homology. A DSB on one plasmid triggered extensive replication of the second plasmid, providing a useful model for T4 recombination-dependent replication (RDR). This system also provides a view of DSB repair in T4-infected cells and revealed that the DSB repair products had been replicated in their entirety by the T4 replication machinery. We analyzed the detailed structure of these products, which do not fit the simple predictions of any of three models for DSB repair. We also present evidence that the T4 RDR system functions to restart stalled or inactivated replication forks. First, we review experiments involving antitumor drug-stabilized topoisomerase cleavage complexes. The results suggest that forks blocked at cleavage complexes are resolved by recombinational repair, likely involving RDR. Second, we show here that the presence of a T4 replication origin on one plasmid substantially stimulated recombination events between it and a homologous second plasmid that did not contain a T4 origin. Furthermore, replication of the second plasmid was increased when the first plasmid contained the T4 origin. Our interpretation is that origin-initiated forks become inactivated at some frequency during replication of the first plasmid and are then restarted via RDR on the second plasmid. PMID:11459966

  1. Structure-function analysis of the DNA translocating portal of the bacteriophage T4 packaging machine.

    PubMed

    Padilla-Sanchez, Victor; Gao, Song; Kim, Hyung Rae; Kihara, Daisuke; Sun, Lei; Rossmann, Michael G; Rao, Venigalla B

    2014-03-01

    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. PMID:24126213

  2. Insight into DNA and Protein Transport in Double-stranded DNA Viruses: The Structure of Bacteriophage N4

    PubMed Central

    Choi, Kyung H.; McPartland, Jennifer; Kaganman, Irene; Bowman, Valorie D.; Rothman-Denes, Lucia B.; Rossmann, Michael G.

    2008-01-01

    SUMMARY Bacteriophage N4 encapsidates a 3,500 amino acid-long DNA-dependent RNA polymerase (vRNAP), which is injected into the host along with the N4 genome upon infection. The three-dimensional structures of wild-type and mutant N4 viruses lacking gp17, gp50, or gp65 were determined by cryo-electron microscopy. The virion has an icosahedral capsid with T = 9 quasi-symmetry that encapsidates well-organized dsDNA and vRNAP. The tail, attached at a unique pentameric vertex of the head, consists of a neck, twelve appendages, and six ribbons that constitute a non-contractile sheath around a central tail tube. Comparison of wild-type and mutant virus structures in conjunction with bioinformatics established the identity and virion locations of the major capsid protein (gp56), a decorating protein (gp17), the vRNAP (gp50), the tail sheath (gp65), the appendages (gp66), and the portal protein (gp59). The N4 virion organization provides insights into its assembly, and suggests a mechanism for genome and vRNAP transport strategies utilized by this unique system. PMID:18374942

  3. Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages

    SciTech Connect

    Sun, Siyang; Gao, Song; Kondabagil, Kiran; Xiang, Ye; Rossmann, Michael G.; Rao, Venigalla B.

    2012-04-04

    Tailed DNA bacteriophages assemble empty procapsids that are subsequently filled with the viral genome by means of a DNA packaging machine situated at a special fivefold vertex. The packaging machine consists of a 'small terminase' and a 'large terminase' component. One of the functions of the small terminase is to initiate packaging of the viral genome, whereas the large terminase is responsible for the ATP-powered translocation of DNA. The small terminase subunit has three domains, an N-terminal DNA-binding domain, a central oligomerization domain, and a C-terminal domain for interacting with the large terminase. Here we report structures of the central domain in two different oligomerization states for a small terminase from the T4 family of phages. In addition, we report biochemical studies that establish the function for each of the small terminase domains. On the basis of the structural and biochemical information, we propose a model for DNA packaging initiation.

  4. The Scrunchworm Hypothesis: Transitions Between A-DNA and B-DNA Provide the Driving Force for Genome Packaging in Double-Stranded DNA Bacteriophages

    PubMed Central

    Harvey, Stephen C.

    2015-01-01

    Double-stranded DNA bacteriophages have motors that drive the genome into preformed capsids, using the energy releas ed by hydrolysis of ATP to overcome the forces opposing DNA packaging. Viral packaging motors are the strongest of all biological motors, but it is not known how they generate these forces. Several models for the process of mechanochemical force generation have been put forward, but there is no consensus on which, if any, of these is correct. All the existing models assume that protein-generated forces drive the DNA forward. The scrunchworm hypothesis proposes that the DNA molecule is the active force-generating core of the motor, not simply a substrate on which the motor operates. The protein components of the motor dehydrate a section of the DNA, converting it from the B form to the A form and shortening it by about 23%. The proteins then rehydrate the DNA, which converts back to the B form. Other regions of the motor grip and release the DNA to capture the shortening-lengthening motions of the B→A→B cycle (“scrunching”), so that DNA is pulled into the motor and pushed forward into the capsid. This DNA-centric mechanism provides a quantitative physical explanation for the magnitude of the forces generated by viral packaging motors. It also provides a simple explanation for the fact that each of the steps in the burst cycle advances the DNA by 2.5 base pairs. The scrunchworm hypothesis is consistent with a large body of published data, and it makes four experimentally testable predictions. PMID:25486612

  5. In vitro construction of bacteriophage lambda carrying segments of the Escherichia coli chromosome: selection of hybrids containing the gene for DNA ligase.

    PubMed Central

    Cameron, J R; Panasenko, S M; Lehman, I R; Davis, R W

    1975-01-01

    DNA from lambdagt-lambdaB bacteriophage was cleaved with EcoRI endonuclease and fragments from EcoRI-digested E. coli DNA were inserted. This DNA was used to infect E. coli, and phages containing the gene for DNA ligase were isolated by genetic selection. Two different hybrids were found with the same E. coli segment inserted in opposite orientations. Both hybrids produced similar levels of ligase as measured in crude extracts of infected cells. Images PMID:1103146

  6. Structure and Energetics of Encapsidated DNA in Bacteriophage HK97 Studied by Scanning Calorimetry and Cryo-electron Microscopy

    PubMed Central

    Duda, Robert L.; Ross, Philip D.; Cheng, Naiqian; Firek, Brian A.; Hendrix, Roger W.; Conway, James F.; Steven, Alasdair C.

    2009-01-01

    Encapsidation of duplex DNA by bacteriophages represents an extreme case of genome condensation, reaching near-crystalline concentrations of DNA. The HK97 system is well suited to study this phenomenon in view of detailed knowledge of its capsid structure. To characterize the interactions involved, we combined calorimetry with cryo-EM and native gel electrophoresis. We found that, as in other phages, HK97 DNA is organized in coaxially wound nested shells. When scanned in buffer containing 1mM [Mg++], filled capsids exhibit a complex thermal profile between 82° and 96°, to which DNA melting and capsid denaturation both contribute. In the absence of (unbound) [Mg++], DNA melting shifts to lower temperatures and the two events are resolved. Filled capsids release their DNA at temperatures well below the onset of DNA melting or capsid denaturation. On heating, the internal pressure increases, causing the DNA to exit – probably, via the portal vertex; the capsid, although largely intact, sustains local damage that leads to an earlier onset of thermal denaturation. Filled capsids differ structurally from empty capsids in the curvature of their protein shell, a change attributable to outwards pressure exerted by the DNA. We propose that this transition is sensed by the portal which is embedded in the capsid wall, whereupon the portal's structure and its interactions with terminase, the packaging enzyme, are altered, thus signaling that packaging is at or approaching completion. PMID:19540242

  7. Effects of the T4 bacteriophage gene 32 product on the efficiency and fidelity of DNA amplification using T4 DNA polymerase.

    PubMed

    Sandhu, D K; Keohavong, P

    1994-06-24

    Two bacteriophage DNA polymerases (Pol), T4 Pol and modified T7 Pol, were used to catalyze DNA amplification in vitro by PCR, and their efficiency and fidelity in DNA amplification were examined in the presence and absence of the T4 bacteriophage gene 32-encoded protein (SSB32). The SSB32 protein significantly improved the efficiency of amplification by T4 Pol. Examination of the amplified DNA by denaturing gradient gel electrophoresis (DGGE) revealed that the protein also reduced the rates of error produced by T4 Pol during PCR, from 6.3 x 10(-6) to 2.0 x 10(-6) errors per base duplication after 10(11)-fold amplification. This protein also improved, but to a lesser extent, the fidelity of modified T7 Pol, from 1.80 x 10(-5) to 1.15 x 10(-5) errors per base duplication. High fidelity polymerase chain reaction (hifi-PCR) is needed for studies requiring isolation of mutant sequences present as only a small fraction of the wild type in the amplified DNA. Although several thermostable Pol are currently available for use in automated PCR, their fidelity was found to be significantly lower than that of the thermosensitive T4 Pol. Therefore, T4 Pol is useful for studies requiring hifi-PCR, although this enzyme needs to be added in the reaction mixture during every cycle of PCR. PMID:8026758

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

    PubMed Central

    2014-01-01

    Background Double-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. Results Single-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

  9. Single molecule analysis of DNA wrapping and looping by a circular 14mer wheel of the bacteriophage 186 CI repressor.

    PubMed

    Wang, Haowei; Dodd, Ian B; Dunlap, David D; Shearwin, Keith E; Finzi, Laura

    2013-06-01

    The lytic-lysogenic decision in bacteriophage 186 is governed by the 186 CI repressor protein in a unique way. The 186 CI is proposed to form a wheel-like oligomer that can mediate either wrapped or looped nucleoprotein complexes to provide the cooperative and competitive interactions needed for regulation. Although consistent with structural, biochemical and gene expression data, many aspects of this model are based on inference. Here, we use atomic force microscopy (AFM) to reveal the various predicted wrapped and looped species, and new ones, for CI regulation of lytic and lysogenic transcription. Automated AFM analysis showed CI particles of the predicted dimensions on the DNA, with CI multimerization favoured by DNA binding. Measurement of the length of the wrapped DNA segments indicated that CI may move on the DNA, wrapping or releasing DNA on either side of the wheel. Tethered particle motion experiments were consistent with wrapping and looping of DNA by CI in solution, where in contrast to λ repressor, the looped species were exceptionally stable. The CI regulatory system provides an intriguing comparison with that of nucleosomes, which share the ability to wrap and release similar sized segments of DNA. PMID:23620280

  10. Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: gp32 monomer binding.

    PubMed

    Jose, Davis; Weitzel, Steven E; Baase, Walter A; von Hippel, Peter H

    2015-10-30

    Combining biophysical measurements on T4 bacteriophage replication complexes with detailed structural information can illuminate the molecular mechanisms of these 'macromolecular machines'. Here we use the low energy circular dichroism (CD) and fluorescent properties of site-specifically introduced base analogues to map and quantify the equilibrium binding interactions of short (8 nts) ssDNA oligomers with gp32 monomers at single nucleotide resolution. We show that single gp32 molecules interact most directly and specifically near the 3'-end of these ssDNA oligomers, thus defining the polarity of gp32 binding with respect to the ssDNA lattice, and that only 2-3 nts are directly involved in this tight binding interaction. The loss of exciton coupling in the CD spectra of dimer 2-AP (2-aminopurine) probes at various positions in the ssDNA constructs, together with increases in fluorescence intensity, suggest that gp32 binding directly extends the sugar-phosphate backbone of this ssDNA oligomer, particularly at the 3'-end and facilitates base unstacking along the entire 8-mer lattice. These results provide a model (and 'DNA map') for the isolated gp32 binding to ssDNA targets, which serves as the nucleation step for the cooperative binding that occurs at transiently exposed ssDNA sequences within the functioning T4 DNA replication complex. PMID:26275775

  11. Pyrovanadolysis: a Pyrophosphorolysis-like Reaction Mediated by Pyrovanadate MN2plus and DNA Polymerase of Bacteriophage T7

    SciTech Connect

    B Akabayov; A Kulczyk; S Akabayov; C Thiele; L McLaughlin; B Beauchamp; C Richardson

    2011-12-31

    DNA polymerases catalyze the 3'-5'-pyrophosphorolysis of a DNA primer annealed to a DNA template in the presence of pyrophosphate (PP{sub i}). In this reversal of the polymerization reaction, deoxynucleotides in DNA are converted to deoxynucleoside 5'-triphosphates. Based on the charge, size, and geometry of the oxygen connecting the two phosphorus atoms of PP{sub i}, a variety of compounds was examined for their ability to carry out a reaction similar to pyrophosphorolysis. We describe a manganese-mediated pyrophosphorolysis-like activity using pyrovanadate (VV) catalyzed by the DNA polymerase of bacteriophage T7. We designate this reaction pyrovanadolysis. X-ray absorption spectroscopy reveals a shorter Mn-V distance of the polymerase-VV complex than the Mn-P distance of the polymerase-PP{sub i} complex. This structural arrangement at the active site accounts for the enzymatic activation by Mn-VV. We propose that the Mn{sup 2+}, larger than Mg{sup 2+}, fits the polymerase active site to mediate binding of VV into the active site of the polymerase. Our results may be the first documentation that vanadium can substitute for phosphorus in biological processes.

  12. Localization and DNA sequence analysis of the C gene of bacteriophage Mu, the positive regulator of Mu late transcription.

    PubMed Central

    Margolin, W; Howe, M M

    1986-01-01

    The C gene of bacteriophage Mu, required for transcription of the phage late genes, was localized by construction and analysis of a series of deleted derivatives of pKN50, a plasmid containing a 9.4 kb Mu DNA fragment which complements Mu C amber mutant phages for growth. One such deleted derivative, pWM10, containing only 0.5 kb of Mu DNA, complements C amber phages and transactivates the mom gene, one of the Mu late genes dependent on C for activation. The DNA sequence of the 0.5 kb fragment predicts a single long open reading frame coding for a 140 amino acid protein. Sequence analysis of DNA containing a C amber mutation located the base change to the second codon of this reading frame. Generation of a frameshift mutation by filling in a BglII site spanning codon 114 of this reading frame resulted in the loss of C complementation and transactivation activity. These results indicate that this open reading frame encodes the Mu C gene product. Comparison of the predicted amino acid sequence of the C protein with those of other transcriptional regulatory proteins revealed some similarity to a region highly conserved among bacterial sigma factors. Images PMID:3014438

  13. Binding of Mn-deoxyribonucleoside Triphosphates to the Active Site of the DNA Polymerase of Bacteriophage T7

    SciTech Connect

    B Akabayov; C Richardson

    2011-12-31

    Divalent metal ions are crucial as cofactors for a variety of intracellular enzymatic activities. Mg{sup 2+}, as an example, mediates binding of deoxyribonucleoside 5'-triphosphates followed by their hydrolysis in the active site of DNA polymerase. It is difficult to study the binding of Mg{sup 2+} to an active site because Mg{sup 2+} is spectroscopically silent and Mg{sup 2+} binds with low affinity to the active site of an enzyme. Therefore, we substituted Mg{sup 2+} with Mn{sup 2+}:Mn{sup 2+} that is not only visible spectroscopically but also provides full activity of the DNA polymerase of bacteriophage T7. In order to demonstrate that the majority of Mn{sup 2+} is bound to the enzyme, we have applied site-directed titration analysis of T7 DNA polymerase using X-ray near edge spectroscopy. Here we show how X-ray near edge spectroscopy can be used to distinguish between signal originating from Mn{sup 2+} that is free in solution and Mn{sup 2+} bound to the active site of T7 DNA polymerase. This method can be applied to other enzymes that use divalent metal ions as a cofactor.

  14. Determinants of affinity and mode of DNA binding at the carboxy terminus of the bacteriophage SPO1-encoded type II DNA-binding protein, TF1.

    PubMed Central

    Andera, L; Geiduschek, E P

    1994-01-01

    The role of the carboxy-terminal amino acids of the bacteriophage SPO1-encoded type II DNA-binding protein, TF1, in DNA binding was analyzed. Chain-terminating mutations truncating the normally 99-amino-acid TF1 at amino acids 96, 97, and 98 were constructed, as were missense mutations substituting cysteine, arginine, and serine for phenylalanine at amino acid 97 and tryptophan for lysine at amino acid 99. The binding of the resulting proteins to a synthetic 44-bp binding site in 5-(hydroxymethyl)uracil DNA, to binding sites in larger SPO1 [5-(hydroxymethyl)uracil-containing] DNA fragments, and to thymine-containing homologous DNA was analyzed by gel retardation and also by DNase I and hydroxy radical footprinting. We conclude that the C tail up to and including phenylalanine at amino acid 97 is essential for DNA binding and that the two C-terminal amino acids, 98 and 99, are involved in protein-protein interactions between TF1 dimers bound to DNA. Images PMID:8113176

  15. The 29 DNA Polymerase: Protein-Primer Structure Suggests a Model of the Initiation to Elongation Transition

    SciTech Connect

    Kamtekar,S.; Berman, A.; Wang, J.; Lazaro, J.; Vega, M.; Blanco, L.; Salas, M.; Steitz, T.

    2006-01-01

    The absolute requirement for primers in the initiation of DNA synthesis poses a problem for replicating the ends of linear chromosomes. The DNA polymerase of bacteriophage {phi}29 solves this problem by using a serine hydroxyl of terminal protein to prime replication. The 3.0 Angstroms resolution structure shows one domain of terminal protein making no interactions, a second binding the polymerase and a third domain containing the priming serine occupying the same binding cleft in the polymerase as duplex DNA does during elongation. Thus, the progressively elongating DNA duplex product must displace this priming domain. Further, this heterodimer of polymerase and terminal protein cannot accommodate upstream template DNA, thereby explaining its specificity for initiating DNA synthesis only at the ends of the bacteriophage genome. We propose a model for the transition from the initiation to the elongation phases in which the priming domain of terminal protein moves out of the active site as polymerase elongates the primer strand. The model indicates that terminal protein should dissociate from polymerase after the incorporation of approximately six nucleotides.

  16. Location of the unique integration site on an Escherichia coli chromosome by bacteriophage lambda DNA in vivo.

    PubMed

    Tal, Asaf; Arbel-Goren, Rinat; Costantino, Nina; Court, Donald L; Stavans, Joel

    2014-05-20

    The search for specific sequences on long genomes is a key process in many biological contexts. How can specific target sequences be located with high efficiency, within physiologically relevant times? We addressed this question for viral integration, a fundamental mechanism of horizontal gene transfer driving prokaryotic evolution, using the infection of Escherichia coli bacteria with bacteriophage λ and following the establishment of a lysogenic state. Following the targeting process in individual live E. coli cells in real time revealed that λ DNA remains confined near the entry point of a cell following infection. The encounter between the 15-bp-long target sequence on the chromosome and the recombination site on the viral genome is facilitated by the directed motion of bacterial DNA generated during chromosome replication, in conjunction with constrained diffusion of phage DNA. Moving the native bacterial integration site to different locations on the genome and measuring the integration frequency in these strains reveals that the frequencies of the native site and a site symmetric to it relative to the origin are similar, whereas both are significantly higher than when the integration site is moved near the terminus, consistent with the replication-driven mechanism we propose. This novel search mechanism is yet another example of the exquisite coevolution of λ with its host. PMID:24799672

  17. In vivo and in vitro phosphorylation of DNA-dependent RNA polymerase of Escherichia coli by bacteriophage-T7-induced protein kinase.

    PubMed Central

    Zillig, W; Fujiki, H; Blum, W; Janeković, D; Schweiger, M; Rahmsdorf, H; Ponta, H; Hirsch-Kauffmann, M

    1975-01-01

    After infection with bacteriophage T7 the beta' and to a lesser extent the beta subunits of E. coli DNA-dependent RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) are phosphorylated by a phage-gene-encoded protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). The phosphorylation occurs on threonine residues and appears site-specific. It is probably the molecular basis of the early transcriptional control. Images PMID:1101258

  18. Requirement of E. coli DNA synthesis functions for the lytic replication of bacteriophage P1.

    PubMed

    Hay, N; Cohen, G

    1983-11-01

    P1 lytic growth was examined in a number of different temperature sensitive mutants of E. coli that affect chromosomal replication. Growth was analyzed by measurements of phage burst sizes and specific DNA synthesis. Efficient P1 growth required each of the bacterial elongation functions dnaE (polC), dnaZ (sub units of E. coli polymerase III holoenzyme), and dnaG (primase) but was not dependent on the elongation function dnaB (mobile promoter). Of two initiation functions tested the dnaA function was found to be dispensable for normal growth whereas the dnaC function was essential. Temperature shift experiments with different dnaC mutants showed that the initiation component of the dnaC function was needed continuously throughout at least the first half of the lytic cycle, while the dnaC elongation activity was probably required during the entire cycle for normal phage yields. In two respects the dependence of P1 lytic growth on E. coli DNA synthesis functions was significantly different from that reported for P1 plasmid replication (Scott and Vapnek, 1980). Thus, lytic replication was far more dependent on a functional polC gene product than was plasmid replication and did not require the bacterial dnaB product. PMID:6359668

  19. Development of a New Method for Detection and Identification of Oenococcus oeni Bacteriophages Based on Endolysin Gene Sequence and Randomly Amplified Polymorphic DNA

    PubMed Central

    Doria, Francesca; Napoli, Chiara; Costantini, Antonella; Berta, Graziella; Saiz, Juan-Carlos

    2013-01-01

    Malolactic fermentation (MLF) is a biochemical transformation conducted by lactic acid bacteria (LAB) that occurs in wine at the end of alcoholic fermentation. Oenococcus oeni is the main species responsible for MLF in most wines. As in other fermented foods, where bacteriophages represent a potential risk for the fermentative process, O. oeni bacteriophages have been reported to be a possible cause of unsuccessful MLF in wine. Thus, preparation of commercial starters that take into account the different sensitivities of O. oeni strains to different phages would be advisable. However, currently, no methods have been described to identify phages infecting O. oeni. In this study, two factors are addressed: detection and typing of bacteriophages. First, a simple PCR method was devised targeting a conserved region of the endolysin (lys) gene to detect temperate O. oeni bacteriophages. For this purpose, 37 O. oeni strains isolated from Italian wines during different phases of the vinification process were analyzed by PCR for the presence of the lys gene, and 25 strains gave a band of the expected size (1,160 bp). This is the first method to be developed that allows identification of lysogenic O. oeni strains without the need for time-consuming phage bacterial-lysis induction methods. Moreover, a phylogenetic analysis was conducted to type bacteriophages. After the treatment of bacteria with UV light, lysis was obtained for 15 strains, and the 15 phage DNAs isolated were subjected to two randomly amplified polymorphic DNA (RAPD)-PCRs. By combining the RAPD profiles and lys sequences, 12 different O. oeni phages were clearly distinguished. PMID:23728816

  20. Development of a new method for detection and identification of Oenococcus oeni bacteriophages based on endolysin gene sequence and randomly amplified polymorphic DNA.

    PubMed

    Doria, Francesca; Napoli, Chiara; Costantini, Antonella; Berta, Graziella; Saiz, Juan-Carlos; Garcia-Moruno, Emilia

    2013-08-01

    Malolactic fermentation (MLF) is a biochemical transformation conducted by lactic acid bacteria (LAB) that occurs in wine at the end of alcoholic fermentation. Oenococcus oeni is the main species responsible for MLF in most wines. As in other fermented foods, where bacteriophages represent a potential risk for the fermentative process, O. oeni bacteriophages have been reported to be a possible cause of unsuccessful MLF in wine. Thus, preparation of commercial starters that take into account the different sensitivities of O. oeni strains to different phages would be advisable. However, currently, no methods have been described to identify phages infecting O. oeni. In this study, two factors are addressed: detection and typing of bacteriophages. First, a simple PCR method was devised targeting a conserved region of the endolysin (lys) gene to detect temperate O. oeni bacteriophages. For this purpose, 37 O. oeni strains isolated from Italian wines during different phases of the vinification process were analyzed by PCR for the presence of the lys gene, and 25 strains gave a band of the expected size (1,160 bp). This is the first method to be developed that allows identification of lysogenic O. oeni strains without the need for time-consuming phage bacterial-lysis induction methods. Moreover, a phylogenetic analysis was conducted to type bacteriophages. After the treatment of bacteria with UV light, lysis was obtained for 15 strains, and the 15 phage DNAs isolated were subjected to two randomly amplified polymorphic DNA (RAPD)-PCRs. By combining the RAPD profiles and lys sequences, 12 different O. oeni phages were clearly distinguished. PMID:23728816

  1. Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase

    PubMed Central

    Morin, José A.; Cao, Francisco J.; Lázaro, José M.; Arias-Gonzalez, J. Ricardo; Valpuesta, José M.; Carrascosa, José L.; Salas, Margarita; Ibarra, Borja

    2015-01-01

    During DNA replication replicative polymerases move in discrete mechanical steps along the DNA template. To address how the chemical cycle is coupled to mechanical motion of the enzyme, here we use optical tweezers to study the translocation mechanism of individual bacteriophage Phi29 DNA polymerases during processive DNA replication. We determine the main kinetic parameters of the nucleotide incorporation cycle and their dependence on external load and nucleotide (dNTP) concentration. The data is inconsistent with power stroke models for translocation, instead supports a loose-coupling mechanism between chemical catalysis and mechanical translocation during DNA replication. According to this mechanism the DNA polymerase works by alternating between a dNTP/PPi-free state, which diffuses thermally between pre- and post-translocated states, and a dNTP/PPi-bound state where dNTP binding stabilizes the post-translocated state. We show how this thermal ratchet mechanism is used by the polymerase to generate work against large opposing loads (∼50 pN). PMID:25800740

  2. Development of a simple cell lysis method for recombinant DNA using bacteriophage lambda lysis genes.

    PubMed

    Jang, Boyun; Jung, Yuna; Lim, Dongbin

    2007-12-01

    In this study, we describe the development of a simple and efficient method for cell lysis via the insertion of a bacteriophage lambda lysis gene cluster into the pET22b expression vector in the following order; the T7 promoter, a gene for a target protein intended for production, Sam7 and R. This insertion of R and Sam7 into pET22b exerted no detrimental effects on cellular growth or the production of a target protein. The induction of the T7 promoter did not in itself result in the autolysis of cells in culture but the harvested cells were readily broken by freezing and thawing. We compared the efficiency of the cell lysis technique by freezing and thawing to that observed with sonication, and determined that both methods completely disintegrated the cells and released proteins into the solution. With our modification of pET22b, the lysis of cells became quite simple, efficient, and reliable. This strategy may prove useful for a broad variety of applications, particularly in experiments requiring extensive cell breakage, including library screening and culture condition exploration, in addition to protein purification. PMID:18176547

  3. Mechanism of termination of bacteriophage DNA packaging investigated with optical tweezers

    NASA Astrophysics Data System (ADS)

    delToro, Damian J.; Smith, Douglas E.

    2012-10-01

    The genomes of many dsDNA viruses are replicated by a mechanism that produces a long concatemer of multiple genomes. These viruses utilize multifunctional molecular motor complexes referred to as "terminases" that can excise a unit genome length of DNA and package it into preformed viral shells. Remarkably, the terminase motor can initiate packaging at the appropriate start point, translocate DNA, sense when a sufficient length has been packaged, and then switch into a mode where it arrests and cleaves the DNA to release a filled virus particle. We have recently developed an improved method to measure single phage lambda DNA packaging using dual-trap optical tweezers and pre-stalled motor-DNA-procapsid complexes. We are applying this method to test proposed mechanisms for the sensor that triggers termination; specifically a velocity-monitor model vs. energy-monitor model vs. capsid-filling monitor model.

  4. Structure of the connector of bacteriophage T7 at 8A resolution: structural homologies of a basic component of a DNA translocating machinery.

    PubMed

    Agirrezabala, Xabier; Martín-Benito, Jaime; Valle, Mikel; González, José M; Valencia, Alfonso; Valpuesta, José María; Carrascosa, José L

    2005-04-15

    The three-dimensional structure of the bacteriophage T7 head-to-tail connector has been obtained at 8A resolution using cryo-electron microscopy and single-particle analysis from purified recombinant connectors. The general morphology of the T7 connector is that of a 12-folded toroidal homopolymer with a channel that runs along the longitudinal axis of the particle. The structure of the T7 connector reveals many structural similarities with the connectors from other bacteriophages. Docking of the atomic structure of the varphi29 connector into the three-dimensional reconstruction of T7 connector reveals that the narrow, distal region of the two oligomers are almost identical. This region of the varphi29 connector has been suggested to be involved in DNA translocation, and is composed of an alpha-beta-alpha-beta-beta-alpha motif. A search for alpha-helices in the same region of the T7 three-dimensional map has located three alpha-helices in approximately the same position as those of the varphi29 connector. A comparison of the predicted secondary structure of several bacteriophage connectors, including among others T7, varphi29, P22 and SPP1, reveals that, despite the lack of sequence homology, they seem to contain the same alpha-beta-alpha-beta-beta-alpha motif as that present in the varphi29 connector. These results allow us to suggest a common architecture related to a basic component of the DNA translocating machinery for several viruses. PMID:15784250

  5. The annotated complete DNA sequence of Enterococcus faecalis bacteriophage φEf11 and its comparison with all available phage and predicted prophage genomes.

    PubMed

    Stevens, Roy H; Ektefaie, Mahmoud R; Fouts, Derrick E

    2011-04-01

    φEf11 is a temperate Siphoviridae bacteriophage isolated by induction from a lysogenic Enterococcus faecalis strain. The φEf11 DNA was completely sequenced and found to be 42,822 bp in length, with a G+C mol% of 34.4%. Genome analysis revealed 65 ORFs, accounting for 92.8% of the DNA content. All except for seven of the ORFs displayed sequence similarities to previously characterized proteins. The genes were arranged in functional modules, organized similar to that of several other phages of low GC Gram-positive bacteria; however, the number and arrangement of lysis-related genes were atypical of these bacteriophages. A 159 bp noncoding region between predicted cI and cro genes is highly similar to the functionally characterized early promoter region of lactococcal temperate phage TP901-1, and possessed a predicted stem-loop structure in between predicted P(L) and P(R) promoters, suggesting a novel mechanism of repression of these two bacteriophages from the λ paradigm. Comparison with all available phage and predicted prophage genomes revealed that the φEf11 genome displays unique features, suggesting that φEf11 may be a novel member of a larger family of temperate prophages that also includes lactococcal phages. Trees based on the blast score ratio grouped this family by tail fiber similarity, suggesting that these trees are useful for identifying phages with similar tail fibers. PMID:21204936

  6. The mechanism of transcriptional activation by the topologically DNA-linked sliding clamp of bacteriophage T4.

    PubMed

    Kolesky, Scott E; Ouhammouch, Mohamed; Geiduschek, E Peter

    2002-08-30

    Three viral proteins participate directly in transcription of bacteriophage T4 late genes: the sigma-family protein gp55 provides promoter recognition, gp33 is the co-activator, and gp45 is the activator of transcription; gp33 also represses transcription in the absence of gp45. Transcriptional activation by gp45, the toroidal sliding clamp of the T4 DNA polymerase holoenzyme, requires assembly at primer-template junctions by its clamp loader. The mechanism of transcriptional activation has been analyzed by examining rates of formation of open promoter complexes. The basal gp55-RNA polymerase holoenzyme is only weakly held in its initially formed closed promoter complex, which subsequently opens very slowly. Activation ( approximately 320-fold in this work) increases affinity in the closed complex and accelerates promoter opening. Promoter opening by gp55 is also thermo-irreversible: the T4 late promoter does not open at 0 degrees C, but once opened at 30 degrees C remains open upon shift to the lower temperature. At a hybrid promoter for sigma(70) and gp55-holoenzymes, only gp55 confers thermo-irreversibility of promoter opening. Interaction of gp45 with a C-terminal epitope of gp33 is essential for the co-activator function of gp33. PMID:12206760

  7. Synergistic effect of heat and solar UV on DNA damage and water disinfection of E. coli and bacteriophage MS2.

    PubMed

    Theitler, Dana Jennifer; Nasser, Abid; Gerchman, Yoram; Kribus, Abraham; Mamane, Hadas

    2012-12-01

    The response of a representative virus and indicator bacteria to heating, solar irradiation, or their combination, was investigated in a controlled solar simulator and under real sun conditions. Heating showed higher inactivation of Escherichia coli compared to the bacteriophage MS2. Heating combined with natural or simulated solar irradiation demonstrated a synergistic effect on the inactivation of E. coli, with up to 3-log difference for 50 °C and natural sun insolation of 2,000 kJ m(-2) (compared to the sum of the separate treatments). Similar synergistic effect was also evident when solar-UV induced DNA damage to E. coli was assessed using the endonuclease sensitive site assay (ESS). MS2 was found to be highly resistant to irradiation and heat, with a slightly synergistic effect observed only at 59 °C and natural sun insolation of 5,580 kJ m(-2). Heat treatment also hindered light-dependent recovery of E. coli making the treatment much more effective. PMID:23165717

  8. Recombinational Repair of DNA Damage in Escherichia coli and Bacteriophage λ

    PubMed Central

    Kuzminov, Andrei

    1999-01-01

    Although homologous recombination and DNA repair phenomena in bacteria were initially extensively studied without regard to any relationship between the two, it is now appreciated that DNA repair and homologous recombination are related through DNA replication. In Escherichia coli, two-strand DNA damage, generated mostly during replication on a template DNA containing one-strand damage, is repaired by recombination with a homologous intact duplex, usually the sister chromosome. The two major types of two-strand DNA lesions are channeled into two distinct pathways of recombinational repair: daughter-strand gaps are closed by the RecF pathway, while disintegrated replication forks are reestablished by the RecBCD pathway. The phage λ recombination system is simpler in that its major reaction is to link two double-stranded DNA ends by using overlapping homologous sequences. The remarkable progress in understanding the mechanisms of recombinational repair in E. coli over the last decade is due to the in vitro characterization of the activities of individual recombination proteins. Putting our knowledge about recombinational repair in the broader context of DNA replication will guide future experimentation. PMID:10585965

  9. Old, New, and Widely True: the Bacteriophage T4 DNA Packaging Mechanism

    PubMed Central

    Black, Lindsay W.

    2015-01-01

    DNA packaging into empty viral procapsids by ATP-driven motor proteins applies widely among viruses. Recent fluorescence studies of phage T4 reveal: 1) the small terminase subunit (TerS) synapses pac homologs by a two ring mechanism to gauge DNA maturation and allow packaging by the large terminase subunit (TerL); 2) translocation of linear DNA is efficient by TerL acting alone; expansion of the procapsid is controlled by the portal-terminase assembly; 3) both ends of the packaged DNA are held at the portal, showing a loop of DNA is packaged; 4) transient spring-like compression of B form to A form-like DNA accompanies translocation; 5) the C-terminal domain of TerL is docked to the portal and moves toward it when stalled; 6) a portal bound resolvase can release stalled Y-DNA compression and allow translocation in vitro; and 7) ATP powered translocation on A form dsDNA is supported by recent hexameric helicase studies. PMID:25728298

  10. Molecular Characterization of a Novel Temperate Sinorhizobium Bacteriophage, ФLM21, Encoding DNA Methyltransferase with CcrM-Like Specificity

    PubMed Central

    Dziewit, Lukasz; Oscik, Karolina; Bartosik, Dariusz

    2014-01-01

    ABSTRACT ΦLM21 is a temperate phage isolated from Sinorhizobium sp. strain LM21 (Alphaproteobacteria). Genomic analysis and electron microscopy suggested that ΦLM21 is a member of the family Siphoviridae. The phage has an isometric head and a long noncontractile tail. The genome of ΦLM21 has 50,827 bp of linear double-stranded DNA encoding 72 putative proteins, including proteins responsible for the assembly of the phage particles, DNA packaging, transcription, replication, and lysis. Virion proteins were characterized using mass spectrometry, leading to the identification of the major capsid and tail components, tape measure, and a putative portal protein. We have confirmed the activity of two gene products, a lytic enzyme (a putative chitinase) and a DNA methyltransferase, sharing sequence specificity with the cell cycle-regulating methyltransferase (CcrM) of the bacterial host. Interestingly, the genome of Sinorhizobium phage ΦLM21 shows very limited similarity to other known phage genome sequences and is thus considered unique. IMPORTANCE Prophages are known to play an important role in the genomic diversification of bacteria via horizontal gene transfer. The influence of prophages on pathogenic bacteria is very well documented. However, our knowledge of the overall impact of prophages on the survival of their lysogenic, nonpathogenic bacterial hosts is still limited. In particular, information on prophages of the agronomically important Sinorhizobium species is scarce. In this study, we describe the isolation and molecular characterization of a novel temperate bacteriophage, ΦLM21, of Sinorhizobium sp. LM21. Since we have not found any similar sequences, we propose that this bacteriophage is a novel species. We conducted a functional analysis of selected proteins. We have demonstrated that the phage DNA methyltransferase has the same sequence specificity as the cell cycle-regulating methyltransferase CcrM of its host. We point out that this phenomenon of

  11. Structural and Molecular Basis for Coordination in a Viral DNA Packaging Motor

    PubMed Central

    Reyes-Aldrete, Emilio; Sherman, Michael B.; Woodson, Michael; Atz, Rockney; Grimes, Shelley; Jardine, Paul J.; Morais, Marc C.

    2016-01-01

    SUMMARY Ring NTPases are a class of ubiquitous molecular motors involved in basic biological partitioning processes. dsDNA viruses encode ring ATPases that translocate their genomes to near-crystalline densities within pre-assembled viral capsids. Here, X-ray crystallography, cryoEM, and biochemical analyses of the dsDNA packaging motor in bacteriophage phi29 show how individual subunits are arranged in a pentameric ATPase ring, and suggest how their activities are coordinated to translocate dsDNA. The resulting pseudo-atomic structure of the motor and accompanying functional analyses show how ATP is bound in the ATPase active site; identify two DNA contacts, including a potential DNA translocating loop; demonstrate that a trans-acting arginine finger is involved in coordinating hydrolysis around the ring; and suggest a functional coupling between the arginine finger and the DNA translocating loop. The ability to visualize the motor in action illuminates how the different motor components interact with each other and with their DNA substrate. PMID:26904950

  12. Structural and Molecular Basis for Coordination in a Viral DNA Packaging Motor.

    PubMed

    Mao, Huzhang; Saha, Mitul; Reyes-Aldrete, Emilio; Sherman, Michael B; Woodson, Michael; Atz, Rockney; Grimes, Shelley; Jardine, Paul J; Morais, Marc C

    2016-03-01

    Ring NTPases are a class of ubiquitous molecular motors involved in basic biological partitioning processes. dsDNA viruses encode ring ATPases that translocate their genomes to near-crystalline densities within pre-assembled viral capsids. Here, X-ray crystallography, cryoEM, and biochemical analyses of the dsDNA packaging motor in bacteriophage phi29 show how individual subunits are arranged in a pentameric ATPase ring and suggest how their activities are coordinated to translocate dsDNA. The resulting pseudo-atomic structure of the motor and accompanying functional analyses show how ATP is bound in the ATPase active site; identify two DNA contacts, including a potential DNA translocating loop; demonstrate that a trans-acting arginine finger is involved in coordinating hydrolysis around the ring; and suggest a functional coupling between the arginine finger and the DNA translocating loop. The ability to visualize the motor in action illuminates how the different motor components interact with each other and with their DNA substrate. PMID:26904950

  13. Structural Basis for DNA-Hairpin Promoter Recognition by the Bacteriophage N4 Virion RNA Polymerase

    SciTech Connect

    Gleghorn, M.; Davydova, E; Rothman-Denes, L; Murakami, K

    2008-01-01

    Coliphage N4 virion-encapsidated RNA polymerase (vRNAP) is a member of the phage T7-like single-subunit RNA polymerase (RNAP) family. Its central domain (mini-vRNAP) contains all RNAP functions of the full-length vRNAP, which recognizes a 5 to 7 base pair stem and 3 nucleotide loop hairpin DNA promoter. Here, we report the X-ray crystal structures of mini-vRNAP bound to promoters. Mini-vRNAP uses four structural motifs to recognize DNA sequences at the hairpin loop and stem and to unwind DNA. Despite their low sequence similarity, three out of four motifs are shared with T7 RNAP that recognizes a double-stranded DNA promoter. The binary complex structure and results of engineered disulfide linkage experiments reveal that the plug and motif B loop, which block the access of template DNA to the active site in the apo-form mini-vRNAP, undergo a large-scale conformational change upon promoter binding, explaining the restricted promoter specificity that is critical for N4 phage early transcription.

  14. Designing disordered materials using DNA-coated colloids of bacteriophage fd and gold.

    PubMed

    Ruff, Z; Nathan, S H; Unwin, R R; Zupkauskas, M; Joshi, D; Salmond, G P C; Grey, C P; Eiser, E

    2016-04-12

    DNA has emerged as an exciting binding agent for programmable colloidal self-assembly. Its popularity derives from its unique properties: it provides highly specific short-ranged interactions and at the same time it acts as a steric stabilizer against non-specific van der Waals and Coulomb interactions. Because complementary DNA strands are linked only via hydrogen bonds, DNA-mediated binding is thermally reversible: it provides an effective attraction that can be switched off by raising the temperature only by a few degrees. In this article we introduce a new binary system made of DNA-functionalized filamentous fd viruses of ∼880 nm length with an aspect ratio of ∼100, and 50 nm gold nanoparticles (gold NPs) coated with the complementary DNA strands. When quenching mixtures below the melt temperature Tm, at which the attraction is switched on, we observe aggregation. Conversely, above Tm the system melts into a homogenous particulate 'gas'. We present the aggregation behavior of three different gold NP to virus ratios and compare them to a gel made solely of gold NPs. In particular, we have investigated the aggregate structures as a function of cooling rate and determine how they evolve as function of time for given quench depths, employing fluorescence microscopy. Structural information was extracted in the form of an effective structure factor and chord length distributions. Rapid cooling rates lead to open aggregates, while slower controlled cooling rates closer to equilibrium DNA hybridization lead to more fine-stranded gels. Despite the different structures we find that for both cooling rates the quench into the two-phase region leads to initial spinodal decomposition, which becomes arrested. Surprisingly, although the fine-stranded gel is disordered, the overall structure and the corresponding length scale distributions in the system are remarkably reproducible. Such highly porous systems can be developed into new functional materials. PMID:26864018

  15. Genetics of Critical Contacts and Clashes in the DNA Packaging Specificities of Bacteriophages λ and 21

    PubMed Central

    Sippy, Jean; Patel, Priyal; Vahanian, Nicole; Sippy, Rachel; Feiss, Michael

    2016-01-01

    The cos sites in λ and 21 chromosomes contain binding sites that recruit terminase to initiate DNA packaging. The small subunits of terminase, gpNu1 (λ) and gp1 (21), have winged helix-turn-helix DNA binding domains, where the recognition helixes differ in four of nine residues. To initiate packaging, the small subunit binds three R sequences in the cosB subsite. λ and 21 cannot package each other's DNA, due to recognition helix and R sequence differences. In λ and 21 cosBs, two bp, tri1 and tri2, are conserved in the R sequences yet differ between the phages; they are proposed to play a role in phage-specific packaging by λ and 21. Genetic experiments done with mixed and matched terminase and cosB alleles show packaging specificity depends on favorable contacts and clashes. These interactions indicate that the recognition helixes orient with residues 20 and 24 proximal to tri2 and tri1, respectively. PMID:25543962

  16. The Fitness Effects of Random Mutations in Single-Stranded DNA and RNA Bacteriophages

    PubMed Central

    Domingo-Calap, Pilar; Cuevas, José M.; Sanjuán, Rafael

    2009-01-01

    Mutational fitness effects can be measured with relatively high accuracy in viruses due to their small genome size, which facilitates full-length sequencing and genetic manipulation. Previous work has shown that animal and plant RNA viruses are very sensitive to mutation. Here, we characterize mutational fitness effects in single-stranded (ss) DNA and ssRNA bacterial viruses. First, we performed a mutation-accumulation experiment in which we subjected three ssDNA (ΦX174, G4, F1) and three ssRNA phages (Qβ, MS2, and SP) to plaque-to-plaque transfers and chemical mutagenesis. Genome sequencing and growth assays indicated that the average fitness effect of the accumulated mutations was similar in the two groups. Second, we used site-directed mutagenesis to obtain 45 clones of ΦX174 and 42 clones of Qβ carrying random single-nucleotide substitutions and assayed them for fitness. In ΦX174, 20% of such mutations were lethal, whereas viable ones reduced fitness by 13% on average. In Qβ, these figures were 29% and 10%, respectively. It seems therefore that high mutational sensitivity is a general property of viruses with small genomes, including those infecting animals, plants, and bacteria. Mutational fitness effects are important for understanding processes of fitness decline, but also of neutral evolution and adaptation. As such, these findings can contribute to explain the evolution of ssDNA and ssRNA viruses. PMID:19956760

  17. Structure-specific DNA binding by bacteriophage T5 5'-->3' exonuclease.

    PubMed Central

    Garforth, S J; Sayers, J R

    1997-01-01

    Phage T5 exonuclease is a 5'-->3'exodeoxyribonuclease that also exhibits endonucleolytic activity on flap structures (branched duplex DNA containing a free single-stranded 5'-end). Oligonucleotides were used to construct duplexes with either blunt ends, 5'-overhangs, 3'-overhangs, a flap or a forked end (pseudo-Y). The binding of T5 exonuclease to various structures was investigated using native electrophoretic mobility shift assays (EMSA) in the absence of the essential divalent metal cofactor. Binding of T5 exonuclease to either blunt-ended duplexes or single-stranded oligonucleotides could not be detected by EMSA. However, duplexes with 5'-overhangs, flaps and pseudo-Y structures showed decreased mobility with added T5 exonuclease. On binding to DNA the wild-type enzyme was rendered partially resistant to proteolysis, yielding a biologically active 31.5 kDa fragment. However, the protein-DNA complex remained susceptible to inactivation by p-hydroxymercuribenzoate (PHMB, a cysteine-specific modifying agent), suggesting that neither cysteine is intimately associated with substrate binding. Replacement of both cysteine residues of the molecule with serine did not greatly alter the catalytic or binding characteristics of the protein but did render it highly resistant to inhibition by PHMB. PMID:9380501

  18. Primary structure and functional analysis of the lysis genes of Lactobacillus gasseri bacteriophage phi adh.

    PubMed

    Henrich, B; Binishofer, B; Bläsi, U

    1995-02-01

    The lysis genes of the Lactobacillus gasseri bacteriophage phi adh were isolated by complementation of a lambda Sam mutation in Escherichia coli. Nucleotide sequencing of a 1,735-bp DNA fragment revealed two adjacent coding regions of 342 bp (hol) and 951 bp (lys) in the same reading frame which appear to belong to a common transcriptional unit. Proteins corresponding to the predicted gene products, holin (12.9 kDa) and lysin (34.7 kDa), were identified by in vitro and in vivo expression of the cloned genes. The phi adh holin is a membrane-bound protein with structural similarity to lysis proteins of other phage, known to be required for the transit of murein hydrolases through the cytoplasmic membrane. The phi adh lysin shows homology with mureinolytic enzymes encoded by the Lactobacillus bulgaricus phage mv4, the Streptococcus pneumoniae phage Cp-1, Cp-7, and Cp-9, and the Lactococcus lactis phage phi LC3. Significant homology with the N termini of known muramidases suggests that phi adh lysin acts by a similar catalytic mechanism. In E. coli, the phi adh lysin seems to be associated with the total membrane fraction, from which it can be extracted with lauryl sarcosinate. Either one of the phi adh lysis proteins provoked lysis of E. coli when expressed along with holins or lysins of phage lambda or Bacillus subtilis phage phi 29. Concomitant expression of the combined holin and lysin functions of phi adh in E. coli, however, did not result in efficient cell lysis. PMID:7836307

  19. Biological Physics Prize talk: Grabbing the Cat by the Tail: Studies of DNA Packaging by Single φ 29 Bacteriophage Particles Using Optical Tweezers

    NASA Astrophysics Data System (ADS)

    Bustamante, Carlos

    2002-03-01

    I will present our recent results on the packaging of DNA by the connector motor at the base of the head of bacteriophage φ 29. As part of their infection cycle, many viruses must package their newly replicated genomes inside a protein capsid to insure its proper transport and delivery to other host cells. Bacteriophage φ 29 packages its 6.6 mm long double-stranded DNA into a 42 nm dia. x 54 nm high capsid via a portal complex that hydrolyses ATP. This process is remarkable because entropic, electrostatic, and bending energies of the DNA must be overcome to package the DNA to near-crystalline density. We have used optical tweezers to pull on single DNA molecules as they are packaged, thus demonstrating that the portal complex is a force generating motor. We find that this motor can work against loads of up to ~57 picoNewtons on average, making it one of the strongest molecular motors ever reported. Movements of over 5 mm are observed, indicating high processivity. Pauses and slips also occur, particularly at higher forces. We establish the force-velocity relationship of the motor and find that the rate-limiting step of the motor's cycle is force dependent even at low loads. Interestingly, the packaging rate decreases as the prohead is filled, indicating that an internal pressure builds up due to DNA compression. We estimate that at the end of the packaging the capsid pressure is ~15 MegaPascals, corresponding to an internal force of ~50 pN acting on the motor. The biological implications of this internal pressure and the mechano-chemical efficiency of the engine are discussed.

  20. Forces from the Portal Govern the Late-Stage DNA Transport in a Viral DNA Packaging Nanomotor.

    PubMed

    Jing, Peng; Burris, Benjamin; Zhang, Rong

    2016-07-12

    In the Phi29 bacteriophage, the DNA packaging nanomotor packs its double-stranded DNA genome into the virus capsid. At the late stage of DNA packaging, the negatively charged genome is increasingly compacted at a higher density in the capsid with a higher internal pressure. During the process, two Donnan effects, osmotic pressure and Donnan equilibrium potentials, are significantly amplified, which, in turn, affect the channel activity of the portal protein, GP10, embedded in the semipermeable capsid shell. In the research, planar lipid bilayer experiments were used to study the channel activities of the viral protein. The Donnan effect on the conformational changes of the viral protein was discovered, indicating GP10 may not be a static channel at the late stage of DNA packaging. Due to the conformational changes, GP10 may generate electrostatic forces that govern the DNA transport. For the section of the genome DNA that remains outside of the connector channel, a strong repulsive force from the viral protein would be generated against the DNA entry; however, for the section of the genome DNA within the channel, the portal protein would become a Brownian motor, which adopts the flash Brownian ratchet mechanism to pump the DNA against the increasingly built-up internal pressure (up to 20 atm) in the capsid. Therefore, the DNA transport in the nanoscale viral channel at the late stage of DNA packaging could be a consequence of Brownian movement of the genomic DNA, which would be rectified and harnessed by the forces from the interior wall of the viral channel under the influence of the Donnan effect. PMID:27410744

  1. Bacteriophage Resistance of a ΔthyA Mutant of Lactococcus lactis Blocked in DNA Replication

    PubMed Central

    Pedersen, Martin B.; Jensen, Peter R.; Janzen, Thomas; Nilsson, Dan

    2002-01-01

    The thyA gene, which encodes thymidylate synthase (TS), of Lactococcus lactis CHCC373 was sequenced, including the upstream and downstream regions. We then deleted part of thyA by gene replacement. The resulting strain, MBP71 ΔthyA, was devoid of TS activity, and in media without thymidine, such as milk, there was no detectable dTTP pool in the cells. Hence, DNA replication was abolished, and acidification by MBP71 was completely unaffected by the presence of nine different phages tested at a multiplicity of infection (MOI) of 0.1. Nonreplicating MBP71 must be inoculated at a higher level than CHCC373 to achieve a certain pH within a specified time. For a pH of 5.2 to be reached in 6 h, the inoculation level of MBP71 must be 17-fold higher than for CHCC373. However, by adding a limiting amount of thymidine this could be lowered to just 5-fold the normal amount, while acidification was unaffected with MBP71 up to an MOI of 0.01. It was found that nonreplicating MBP71 produced largely the same products as CHCC373, though the acetaldehyde production of the former was higher. PMID:12039762

  2. Cytoplasmic bacteriophage display system

    DOEpatents

    Studier, F.W.; Rosenberg, A.H.

    1998-06-16

    Disclosed are display vectors comprising DNA encoding a portion of a structural protein from a cytoplasmic bacteriophage, joined covalently to a protein or peptide of interest. Exemplified are display vectors wherein the structural protein is the T7 bacteriophage capsid protein. More specifically, in the exemplified display vectors the C-terminal amino acid residue of the portion of the capsid protein is joined to the N-terminal residue of the protein or peptide of interest. The portion of the T7 capsid protein exemplified comprises an N-terminal portion corresponding to form 10B of the T7 capsid protein. The display vectors are useful for high copy number display or lower copy number display (with larger fusion). Compositions of the type described herein are useful in connection with methods for producing a virus displaying a protein or peptide of interest. 1 fig.

  3. Cytoplasmic bacteriophage display system

    DOEpatents

    Studier, F. William; Rosenberg, Alan H.

    1998-06-16

    Disclosed are display vectors comprising DNA encoding a portion of a structural protein from a cytoplasmic bacteriophage, joined covalently to a protein or peptide of interest. Exemplified are display vectors wherein the structural protein is the T7 bacteriophage capsid protein. More specifically, in the exemplified display vectors the C-terminal amino acid residue of the portion of the capsid protein is joined to the N-terminal residue of the protein or peptide of interest. The portion of the T7 capsid protein exemplified comprises an N-terminal portion corresponding to form 10B of the T7 capsid protein. The display vectors are useful for high copy number display or lower copy number display (with larger fusion). Compositions of the type described herein are useful in connection with methods for producing a virus displaying a protein or peptide of interest.

  4. The MotA transcription factor from bacteriophage T4 contains a novel DNA-binding domain : the 'double wing' motif.

    SciTech Connect

    Li, N.; Sickmier, E. A.; Zhang, R.; Joachimiak, A.; White, S. W.; Biosciences Division; St. Jude Children's Research Hospital; Univ. of Tennessee Health Science Center; Corixa Inc.

    2002-01-01

    MotA is a transcription factor from bacteriophage T4 that helps adapt the host Escherichia coli transcription apparatus to T4 middle promoters. We have determined the crystal structure of the C-terminal DNA-binding domain of MotA (MotCF) to 1.6 A resolution using multiwavelength, anomalous diffraction methods. The structure reveals a novel DNA-binding alpha/beta motif that contains an exposed beta-sheet surface that mediates interactions with the DNA. Independent biochemical experiments have shown that MotCF binds to one surface of a single turn of DNA through interactions in adjacent major and minor grooves. We present a model of the interaction in which beta-ribbons at opposite corners of the six-stranded beta-sheet penetrate the DNA grooves, and call the motif a 'double wing' to emphasize similarities to the 'winged-helix' motif. The model is consistent with data on how MotA functions at middle promoters, and provides an explanation for why MotA can form non-specific multimers on DNA.

  5. Single Molecule Photobleaching (SMPB) Technology for Counting of RNA, DNA, Protein and Other Molecules in Nanoparticles and Biological Complexes by TIRF Instrumentation

    PubMed Central

    Zhang, Hui; Guo, Peixuan

    2014-01-01

    Direct counting of biomolecules within biological complexes or nanomachines is demanding. Single molecule counting using optical microscopy is challenging due to the diffraction limit. The Single Molecule Photobleaching (SMPB) technology for direct counting developed by our team (Shu et al, EMBO J, 2007, 26:527; Zhang et al, RNA, 2007, 13:1793) offers a simple and straightforward method to determine the stoichiometry of molecules or subunits within biocomplexes or nanomachines at nanometer scales. Stoichiometry is determined by real-time observation of the number of descending steps resulted from the photobleaching of individual fluorophore. This technology has now been used extensively for single molecule counting of protein, RNA, and other macromolecules in a variety of complexes or nanostructures. Here, we elucidate the SMPB technology, using the counting of RNA molecules within a bacteriophage phi29 DNA-packaging biomotor as an example. The method described here can be applied to the single molecule counting of other molecules in other systems. The construction of a concise, simple and economical single molecule total internal reflection fluorescence (TIRF) microscope combining prism-type and objective-type TIRF is described. The imaging system contains a deep-cooled sensitive EMCCD camera with single fluorophore detection sensitivity, a laser combiner for simultaneous dual-color excitation, and a Dual-View™ imager to split the multiple outcome signals to different detector channels based on their wavelengths. Methodology of the single molecule photobleaching assay used to elucidate the stoichiometry of RNA on phi29 DNA packaging motor and the mechanism of protein/RNA interaction are described. Different methods for single fluorophore labeling of RNA molecules are reviewed. The process of statistical modeling to reveal the true copy number of the biomolecules based on binomial distribution is also described. PMID:24440482

  6. Cellstat--A continuous culture system of a bacteriophage for the study of the mutation rate and the selection process at the DNA level

    NASA Astrophysics Data System (ADS)

    Husimi, Yuzuru; Nishigaki, Koichi; Kinoshita, Yasunori; Tanaka, Toyosuke

    1982-04-01

    A bacteriophage is continuously cultured in the flow of the host bacterial cell under the control of a minicomputer. In the culture, the population of the noninfected cell is kept constant by the endogeneous regulation mechanism, so it is called the ''cellstat'' culture. Due to the high dilution rate of the host cell, the mutant cell cannot be selected in the cellstat. Therefore, the cellstat is suitable for the study of the mutation rate and the selection process of a bacteriophage under well-defined environmental conditions (including physiological condition of the host cell) without being interfered by host-cell mutations. Applications to coliphage fd, a secretion type phage, are shown as a measurement example. A chimera between fd and a plasmid pBR322 is cultured more than 100 h. The process of population changeovers by deletion mutants indicates that the deletion hot spots exist in this cloning vector and that this apparatus can be used also for testing instability of a recombinant DNA.

  7. Nucleotide-type chemical shift assignment of the encapsulated 40 kbp dsDNA in intact bacteriophage T7 by MAS solid-state NMR.

    PubMed

    Abramov, Gili; Goldbourt, Amir

    2014-08-01

    The icosahedral bacteriophage T7 is a 50 MDa double-stranded DNA (dsDNA) virus that infects Escherichia coli. Although there is substantial information on the physical and morphological properties of T7, structural information, based mostly on Raman spectroscopy and cryo-electron microscopy, is limited. Here, we apply the magic-angle spinning (MAS) solid-state NMR (SSNMR) technique to study a uniformly (13)C and (15)N labeled wild-type T7 phage. We describe the details of the large-scale preparation and purification of an isotopically enriched phage sample under fully hydrated conditions, and show a complete (13)C and a near-complete (15)N nucleotide-type specific assignment of the sugar and base moieties in the 40 kbp dsDNA of T7 using two-dimensional (13)C-(13)C and (15)N-(13)C correlation experiments. The chemical shifts are interpreted as reporters of a B-form conformation of the encapsulated dsDNA. While MAS SSNMR was found to be extremely useful in determining the structures of proteins in native-like environments, its application to nucleic acids has lagged behind, leaving a missing (13)C and (15)N chemical shift database. This work therefore expands the (13)C and (15)N database of real B-form DNA systems, and opens routes to characterize more complex nucleic acid systems by SSNMR. PMID:24875850

  8. Bacteriophage T4D Head Morphogenesis. VIII. DNA-Protein Associations in Intermediate Head Structures That Accumulate in Gene 49− Mutant-Infected Cells

    PubMed Central

    Glinert, Susan J.; Luftig, Ronald B.

    1977-01-01

    We have utilized the gene 49− mutant-infected cells of bacteriophage T4D to accumulate large numbers of nucleic acid-protein intermediate head structures. These heads were used as substrates for experiments in the investigations of the mechanism of DNA packaging. Specifically, we have examined: (i) the susceptibility of the DNA in these structures to digestion by a variety of nucleases after a series of increasing temperature pulses from 25 to 100°C, (ii) the physicochemical characteristics of the DNA inside these heads, and (iii) the mechanism by which proteins are displaced from the interior of the head after treatment with basic proteins. We isolated DNA from these gene 49− heads by use of gradient centrifugation procedures. The DNA had a molecular weight of 8 × 106 and a density of 1.697 ± 0.005 g/cm3, and it contained a short resistant fraction (SRF) which, when associated with the gene 49− heads, exhibited AT-protected regions that were not susceptible to micrococcal nuclease digestion. Such a fraction may contain pieces which are important in the initial association of the DNA with the prohead. Exposure of the gene 49− intermediate capsid structures to basic proteins, such as bovine trypsin inhibitor, lysozyme, and l-polylysine-70, caused a displacement of an amorphous-appearing structure which may be a complex of the gene 49− DNA and interior components of the capsid (e.g., internal proteins, polyamines). Our general conclusion is that in the gene 49− intermediate head structures which are only partly filled with DNA, this DNA is held inside the head by strong electrostatic linkages with interior polypeptides and polyamines. Images PMID:875137

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

    PubMed Central

    Chiaruttini, Nicolas; Letellier, Lucienne; Viasnoff, Virgile

    2013-01-01

    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. PMID:24376806

  10. Characterization of the bacteriophage lambda excisionase (Xis) protein: the C-terminus is required for Xis-integrase cooperativity but not for DNA binding.

    PubMed Central

    Numrych, T E; Gumport, R I; Gardner, J F

    1992-01-01

    We have performed a mutational analysis of the xis gene of bacteriophage lambda. The Xis protein is 72 amino acids in length and required for excisive recombination. Twenty-six mutants of Xis were isolated that were impaired or deficient in lambda excision. Mutant proteins that contained amino acid substitutions in the N-terminal 49 amino acids of Xis were defective in excisive recombination and were unable to bind DNA. In contrast, one mutant protein containing a leucine to proline substitution at position 60 and two truncated proteins containing either the N-terminal 53 or 64 amino acids continued to bind lambda DNA, interact cooperatively with FIS and promote excision. However, these three mutants were unable to bind DNA cooperatively with Int. Cooperativity between wild-type Xis and Int required the presence of FIS, but not the Int core-type binding sites. This study shows that Xis has at least two functional domains and also demonstrates the importance of the cooperativity in DNA binding of FIS, Xis and Int in lambda excision. Images PMID:1396573

  11. Chlamydia bacteriophages.

    PubMed

    Śliwa-Dominiak, Joanna; Suszyńska, Ewa; Pawlikowska, Małgorzata; Deptuła, Wiesław

    2013-11-01

    Phages are called "good viruses" due to their ability to infect and kill pathogenic bacteria. Chlamydia are small, Gram-negative (G-) microbes that can be dangerous to human and animals. In humans, these bacteria are etiological agents of diseases such as psittacosis or respiratory tract diseases, while in animals, the infection may result in enteritis in cattle and chronic bowel diseases, as well as miscarriages in sheep. The first-known representative of chlamydiaphages was Chp1. It was discovered in Chlamydia psittaci isolates. Since then, four more species of chlamydiaphages have been identified [Chp2, Chp3, φCPG1 φCPAR39 (φCpn1) and Chp4]. All of them were shown to infect Chlamydia species. This paper described all known chlamydiaphages. They were characterised in terms of origin, host range, and their molecular structure. The review concerns the characterisation of bacteriophages that infects pathogenic and dangerous bacteria with unusual, intracellular life cycles that are pathogenic. In the era of antibiotic resistance, it is difficult to cure chlamydophilosis. Those bacteriophages can be an alternative to antibiotics, but before this happens, we need to get to know chlamydiaphages better. PMID:23903989

  12. 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

    NASA Astrophysics Data System (ADS)

    Petrov, Anton S.; Douglas, Scott S.; Harvey, Stephen C.

    2013-03-01

    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.

  13. Construction and properties of a temperature-sensitive mutation in the gene for the bacteriophage SPO1 DNA-binding protein TF1.

    PubMed Central

    Sayre, M H; Geiduschek, E P

    1990-01-01

    The Bacillus subtilis bacteriophage SPO1 encodes the DNA-binding protein TF1, a homolog of the ubiquitous type II DNA-binding proteins that are components of bacterial chromatin. The known three-dimensional structure of a related protein was used in devising a scheme of site-directed mutagenesis that led to the creation of a temperature-sensitive mutation in the TF1 gene. At the nonpermissive temperature, this mutation disrupted the temporal regulation of viral protein synthesis and processing, altered the kinetics of accumulation of at least one viral transcript, and prohibited the production of infective progeny phage. We suggest that TF1 function is required to shut off the expression of several early-middle and middle viral genes and that TF1 plays a role in phage head morphogenesis. Spontaneous second-site mutations of the temperature-sensitive mutant TF1 allele that suppressed its associated phenotypes were analyzed. These suppressor mutations conferred greater amino acid sequence homology with the type II DNA-binding protein from the thermophile Bacillus stearothermophilus. Images PMID:2115873

  14. Bacteriophage GIL01 gp7 interacts with host LexA repressor to enhance DNA binding and inhibit RecA-mediated auto-cleavage.

    PubMed

    Fornelos, Nadine; Butala, Matej; Hodnik, Vesna; Anderluh, Gregor; Bamford, Jaana K; Salas, Margarita

    2015-09-01

    The SOS response in Eubacteria is a global response to DNA damage and its activation is increasingly associated with the movement of mobile genetic elements. The temperate phage GIL01 is induced into lytic growth using the host's SOS response to genomic stress. LexA, the SOS transcription factor, represses bacteriophage transcription by binding to a set of SOS boxes in the lysogenic promoter P1. However, LexA is unable to efficiently repress GIL01 transcription unless the small phage-encoded protein gp7 is also present. We found that gp7 forms a stable complex with LexA that enhances LexA binding to phage and cellular SOS sites and interferes with RecA-mediated auto-cleavage of LexA, the key step in the initiation of the SOS response. Gp7 did not bind DNA, alone or when complexed with LexA. Our findings suggest that gp7 induces a LexA conformation that favors DNA binding but disfavors LexA auto-cleavage, thereby altering the dynamics of the cellular SOS response. This is the first account of an accessory factor interacting with LexA to regulate transcription. PMID:26138485

  15. Bacteriophage GIL01 gp7 interacts with host LexA repressor to enhance DNA binding and inhibit RecA-mediated auto-cleavage

    PubMed Central

    Fornelos, Nadine; Butala, Matej; Hodnik, Vesna; Anderluh, Gregor; Bamford, Jaana K.; Salas, Margarita

    2015-01-01

    The SOS response in Eubacteria is a global response to DNA damage and its activation is increasingly associated with the movement of mobile genetic elements. The temperate phage GIL01 is induced into lytic growth using the host's SOS response to genomic stress. LexA, the SOS transcription factor, represses bacteriophage transcription by binding to a set of SOS boxes in the lysogenic promoter P1. However, LexA is unable to efficiently repress GIL01 transcription unless the small phage-encoded protein gp7 is also present. We found that gp7 forms a stable complex with LexA that enhances LexA binding to phage and cellular SOS sites and interferes with RecA-mediated auto-cleavage of LexA, the key step in the initiation of the SOS response. Gp7 did not bind DNA, alone or when complexed with LexA. Our findings suggest that gp7 induces a LexA conformation that favors DNA binding but disfavors LexA auto-cleavage, thereby altering the dynamics of the cellular SOS response. This is the first account of an accessory factor interacting with LexA to regulate transcription. PMID:26138485

  16. Selective inhibition by methoxyamine of the apurinic/apyrimidinic endonuclease activity associated with pyrimidine dimer-DNA glycosylases from Micrococcus luteus and bacteriophage T4

    SciTech Connect

    Liuzzi, M.; Weinfeld, M.; Paterson, M.C.

    1987-06-16

    The UV endonucleases from Micrococcus luteus and bacteriophage T4 possess two catalytic activities specific for the site of cyclobutane pyrimidine dimers in UV-irradiated DNA: a DNA glycosylase that cleaves the 5'-glycosyl bond of the dimerized pyrimidines and an apurinic/apyrimidinic (AP) endonuclease that thereupon incises the phosphodiester bond 3' to the resulting apyrimidinic site. The authors have explored the potential use of methoxyamine, a chemical that reacts at neutral pH with AP sites in DNA, as a selective inhibitor of the AP endonuclease activities residing in the M. luteus and T4 enzymes. The presence of 50 mM methoxyamine during incubation of UV-treated, (/sup 3/H)thymine-labeled poly(dA) x poly(dT) with either enzyme preparation was found to protect completely the irradiated copolymer from endonucleolytic attack at dimer sites, as assayed by yield of acid-soluble radioactivity. In contrast, the dimer-DNA glycosylase activity of each enzyme remained fully functional, as monitored retrospectively by release of free thymine after either photochemical-(5 kJ/m/sup 2/, 254 nm) or photoenzymic- (Escherichia coli photolyase plus visible light) induced reversal of pyrimidine dimers in the UV-damaged substrate. The data demonstrate that the inhibition of the strand-incision reaction arises because of chemical modification of the AP sites and is not due to inactivation of the enzyme by methoxyamine. The results, combined with earlier findings for 5'-acting AP endonucleases, strongly suggest that methoxyamine is a highly specific inhibitor of virtually all AP endonucleases, irrespective of their modes of action, and may therefore prove useful in a wide variety of DNA repair studies.

  17. Crystallographic Insights into the Autocatalytic Assembly Mechanism of a Bacteriophage Tail Spike

    SciTech Connect

    Xiang, Ye; Leiman, Petr G.; Li, Long; Grimes, Shelley; Anderson, Dwight L.; Rossmann, Michael G.

    2010-02-03

    The tailed bacteriophage phi29 has 12 'appendages' (gene product 12, gp12) attached to its neck region that participate in host cell recognition and entry. In the cell, monomeric gp12 undergoes proteolytic processing that releases the C-terminal domain during assembly into trimers. We report here crystal structures of the protein before and after catalytic processing and show that the C-terminal domain of gp12 is an 'autochaperone' that aids trimerization. We also show that autocleavage of the C-terminal domain is a posttrimerization event that is followed by a unique ATP-dependent release. The posttranslationally modified N-terminal part has three domains that function to attach the appendages to the phage, digest the cell wall teichoic acids, and bind irreversibly to the host, respectively. Structural and sequence comparisons suggest that some eukaryotic and bacterial viruses as well as bacterial adhesins might have a similar maturation mechanism as is performed by phi29 gp12 for Bacillus subtilis.

  18. Measurement of steady-state kinetic parameters for DNA unwinding by the bacteriophage T4 Dda helicase: use of peptide nucleic acids to trap single-stranded DNA products of helicase reactions

    PubMed Central

    Nanduri, Bindu; Eoff, Robert L.; Tackett, Alan J.; Raney, Kevin D.

    2001-01-01

    Measurement of steady-state rates of unwinding of double-stranded oligonucleotides by helicases is hampered due to rapid reannealing of the single-stranded DNA products. Including an oligonucleotide in the reaction mixture which can hybridize with one of the single strands can prevent reannealing. However, helicases bind to single-stranded DNA, therefore the additional oligonucleotide can sequester the enzyme, leading to slower observed rates for unwinding. To circumvent this problem, the oligonucleotide that serves as a trap was replaced with a strand of peptide nucleic acid (PNA). Fluorescence polarization was used to determine that a 15mer PNA strand does not bind to the bacteriophage T4 Dda helicase. Steady-state kinetic parameters of unwinding catalyzed by Dda were determined by using PNA as a trapping strand. The substrate consisted of a partial duplex with 15 nt of single-stranded DNA and 15 bp. In the presence of 250 nM substrate and 1 nM Dda, the rate of unwinding in the presence of the DNA trapping strand was 0.30 nM s–1 whereas the rate was 1.34 nM s–1 in the presence of the PNA trapping strand. PNA prevents reannealing of single-stranded DNA products, but does not sequester the helicase. This assay will prove useful in defining the complete kinetic mechanism for unwinding of oligonucleotide substrates by this helicase. PMID:11433029

  19. Two recombination-dependent DNA replication pathways of bacteriophage T4, and their roles in mutagenesis and horizontal gene transfer

    PubMed Central

    Mosig, Gisela; Gewin, John; Luder, Andreas; Colowick, Nancy; Vo, Daniel

    2001-01-01

    Two major pathways of recombination-dependent DNA replication, “join-copy” and “join-cut-copy,” can be distinguished in phage T4: join-copy requires only early and middle genes, but two late proteins, endonuclease VII and terminase, are uniquely important in the join-cut-copy pathway. In wild-type T4, timing of these pathways is integrated with the developmental program and related to transcription and packaging of DNA. In primase mutants, which are defective in origin-dependent lagging-strand DNA synthesis, the late pathway can bypass the lack of primers for lagging-strand DNA synthesis. The exquisitely regulated synthesis of endo VII, and of two proteins from its gene, explains the delay of recombination-dependent DNA replication in primase (as well as topoisomerase) mutants, and the temperature-dependence of the delay. Other proteins (e.g., the single-stranded DNA binding protein and the products of genes 46 and 47) are important in all recombination pathways, but they interact differently with other proteins in different pathways. These homologous recombination pathways contribute to evolution because they facilitate acquisition of any foreign DNA with limited sequence homology during horizontal gene transfer, without requiring transposition or site-specific recombination functions. Partial heteroduplex repair can generate what appears to be multiple mutations from a single recombinational intermediate. The resulting sequence divergence generates barriers to formation of viable recombinants. The multiple sequence changes can also lead to erroneous estimates in phylogenetic analyses. PMID:11459968

  20. Partial replication of UV-irradiated T4 bacteriophage DNA results in amplification of specific genetic areas

    SciTech Connect

    Ling, S.; Vogelbacker, H.H.; Restifo, L.L.; Mattson, T.; Kozinski, A.W.

    1981-11-01

    Upon infection of Escherichia coli with bormodeoxyuridine-labeled T4 phage that had received 10 lethal hits of UV irradiation, a sizable amount of phage DNA was synthesized (approximately 36 phage equivalent units of DNA per infected bacterium), although very little multiplicity reactivation occurs. This progeny DNA was isolated and analyzed. This DNA was biased in its genetic representation, as shown by hybridization to cloned segments of the T4 genome immobilized on nitrocellulose filters. Preferentially amplified areas corresponded to regions containing origins of T4 DNA replication. The size of the progeny DNA increased with time after infection, possibly due to recombination between partial replicas and nonreplicated subunits or due to the gradual overcoming of the UV damage. As the size of the progeny DNA increased, all of the genes were more equally represented, resulting in a decrease in the genetic bias. Amplification of specific genetic areas was also observed upon infection with UV-irradiated, non-bromo-deoxyuridine-substituted (light) phage. However, the genetic bias observed in this case was not as great as that observed with bromodeoxyuridine-substituted phage. This is most likely due to the higher efficiency of multiplicity reactivation of the light phage.

  1. Bacteriophage biocontrol of foodborne pathogens.

    PubMed

    Kazi, Mustafa; Annapure, Uday S

    2016-03-01

    Bacteriophages are viruses that only infect bacterial cells. Phages are categorized based on the type of their life cycle, the lytic cycle cause lysis of the bacterium with the release of multiple phage particles where as in lysogenic phase the phage DNA is incorporated into the bacterial genome. Lysogeny does not result in lysis of the host. Lytic phages have several potential applications in the food industry as biocontrol agents, biopreservatives and as tools for detecting pathogens. They have also been proposed as alternatives to antibiotics in animal health. Two unique features of phage relevant for food safety are that they are harmless to mammalian cells and high host specificity, keeping the natural microbiota undisturbed. However, the recent approval of bacteriophages as food additives has opened the discussion about 'edible viruses'. This article reviews in detail the application of phages for the control of foodborne pathogens in a process known as "biocontrol". PMID:27570260

  2. Semiconservative DNA replication is initiated at a single site in recombination-deficient gene 32 mutants of bacteriophage T4.

    PubMed Central

    Dannenberg, R; Mosig, G

    1981-01-01

    We have investigated, by electron microscopy, replicative intermediate produced early after infection of Escherichia coli with two phage T4 gene 32 mutants (amA453 and tsG26) which replicate their parental DNA but are defective in secondary replications and in moderating the activities of recombination nucleases. Under conditions completely restrictive for progeny production, both of these mutant produced replicative intermediates, each containing a single internal loop. Both branches of these loops were double stranded; i.e., both leading and lagging strands were synthesized. The replicative intermediates of these mutants qualitatively and quantitatively resembled early replicating wild-type T4 chromosomes after solitary infection of E. coli. However, in contrast to intracellular wild-type T4 DNA isolated from multiple infection, the mutant DNAs showed neither multiple branches nor multiple tandem loops. These results demonstrate that a truncated gene 32 protein which consists of less than one-third of the wild-type T4 helix-destabilizing protein can facilitate the functions of T4 replication proteins, specifically those of T4 DNA polymerase and priming proteins. Our results also support the hypothesis that the generation of multiple tandem loops or branches in vegetative T4 DNA depends on recombination (Mosig et al., in B. Alberts, ed., Mechanistic Studies of DNA Replication and Genetic Recombination, p. 527-543, Academic Press, Inc., New York, 1980). Images PMID:7321104

  3. Bacteriophage T5 transfer RNA

    SciTech Connect

    Hunt, C.; Desai, S.M.; Vaughan, J.; Weiss, S.B.

    1980-04-10

    Previous studies from this laboratory have provided a high resolution map for 16 tRNA genes located on the continuous heavy DNA strand of bacteriophage T5 DNA. All of the T5 tRNA genes were located in three clusters within the DNA C segment, except for tRNA/sup Arg/, which mapped on the left end of the DNA D segment. In this report, we present evidence for the presence of eight additional T5 tRNA species, five of which are located in two new loci within the DNA C segment. We also describe a two-dimensional gel electrophoresis system for the separation and isolation of T5 tRNA species from crude infected RNA preparations. The gel electrophoresis system separates tRNA isoacceptors specific for different amino acids; evidence is presented that the isoacceptors for isoleucine, histidine, and serine are coded by different T5 genes.

  4. Models for the Binary Complex of Bacteriophage T4 Gp59 Helicase Loading Protein. GP32 Single-Stranded DNA-Binding Protein and Ternary Complex with Pseudo-Y Junction DNA

    SciTech Connect

    Hinerman, Jennifer M.; Dignam, J. David; Mueser, Timothy C.

    2012-04-05

    The bacteriophage T4 gp59 helicase assembly protein (gp59) is required for loading of gp41 replicative helicase onto DNA protected by gp32 single-stranded DNA-binding protein. The gp59 protein recognizes branched DNA structures found at replication and recombination sites. Binding of gp32 protein (full-length and deletion constructs) to gp59 protein measured by isothermal titration calorimetry demonstrates that the gp32 protein C-terminal A-domain is essential for protein-protein interaction in the absence of DNA. Sedimentation velocity experiments with gp59 protein and gp32ΔB protein (an N-terminal B-domain deletion) show that these proteins are monomers but form a 1:1 complex with a dissociation constant comparable with that determined by isothermal titration calorimetry. Small angle x-ray scattering (SAXS) studies indicate that the gp59 protein is a prolate monomer, consistent with the crystal structure and hydrodynamic properties determined from sedimentation velocity experiments. SAXS experiments also demonstrate that gp32ΔB protein is a prolate monomer with an elongated A-domain protruding from the core. Moreover, fitting structures of gp59 protein and the gp32 core into the SAXS-derived molecular envelope supports a model for the gp59 protein-gp32ΔB protein complex. Our earlier work demonstrated that gp59 protein attracts full-length gp32 protein to pseudo-Y junctions. A model of the gp59 protein-DNA complex, modified to accommodate new SAXS data for the binary complex together with mutational analysis of gp59 protein, is presented in the accompanying article (Dolezal, D., Jones, C. E., Lai, X., Brister, J. R., Mueser, T. C., Nossal, N. G., and Hinton, D. M. (2012) J. Biol. Chem. 287, 18596–18607).

  5. The bof gene of bacteriophage P1: DNA sequence and evidence for roles in regulation of phage c1 and ref genes.

    PubMed Central

    Schaefer, T S; Hays, J B

    1990-01-01

    The C1 repressor of bacteriophage P1 acts via 14 or more distinct operators. This repressor represses its own synthesis as well as the synthesis of other gene products. Previously, mutation of an auxiliary regulatory gene, bof, has been shown to increase expression of some C1-regulated P1 genes (e.g., ref) but to decrease expression of others (e.g., ban). In this study the bof gene was isolated on the basis of its ability to depress stimulation of Escherichia coli chromosomal recombination by the P1 ref gene, if and only if a source of C1 was present. C1 alone, but not Bof alone, was partially effective. The bofDNA sequence encodes an 82-codon reading frame that begins with a TTG codon and includes the sites of the bof-1(Am) mutation and a bof::Tn5 null mutation. Expression of ref::lacZ and cl::lacZ fusion genes was partially repressed in trans by a P1 bof-1 prophage or by plasmid-encoded C1 alone, which was in agreement with effects on Ref-stimulated recombination and with previous indirect evidence for c1 autoregulation. Repression of both fusion genes by plasmid-encoded C1 plus Bof or by a P1 bof+ prophage was more complete. When the C1 source also included a 0.7-kilobase region upstream from C1 which encodes the coi gene, repression of both c1::lacZ and ref::lacZ by C1 alone or by C1 plus Bof was much less effective, as if Coi interfered with C1 repressor function. PMID:2345146

  6. Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: polynucleotide binding and cooperativity

    PubMed Central

    Jose, Davis; Weitzel, Steven E.; Baase, Walter A.; Michael, Miya M.; von Hippel, Peter H.

    2015-01-01

    We here use our site-specific base analog mapping approach to study the interactions and binding equilibria of cooperatively-bound clusters of the single-stranded DNA binding protein (gp32) of the T4 DNA replication complex with longer ssDNA (and dsDNA) lattices. We show that in cooperatively bound clusters the binding free energy appears to be equi-partitioned between the gp32 monomers of the cluster, so that all bind to the ssDNA lattice with comparable affinity, but also that the outer domains of the gp32 monomers at the ends of the cluster can fluctuate on and off the lattice and that the clusters of gp32 monomers can slide along the ssDNA. We also show that at very low binding densities gp32 monomers bind to the ssDNA lattice at random, but that cooperatively bound gp32 clusters bind preferentially at the 5′-end of the ssDNA lattice. We use these results and the gp32 monomer-binding results of the companion paper to propose a detailed model for how gp32 might bind to and interact with ssDNA lattices in its various binding modes, and also consider how these clusters might interact with other components of the T4 DNA replication complex. PMID:26275774

  7. Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: polynucleotide binding and cooperativity.

    PubMed

    Jose, Davis; Weitzel, Steven E; Baase, Walter A; Michael, Miya M; von Hippel, Peter H

    2015-10-30

    We here use our site-specific base analog mapping approach to study the interactions and binding equilibria of cooperatively-bound clusters of the single-stranded DNA binding protein (gp32) of the T4 DNA replication complex with longer ssDNA (and dsDNA) lattices. We show that in cooperatively bound clusters the binding free energy appears to be equi-partitioned between the gp32 monomers of the cluster, so that all bind to the ssDNA lattice with comparable affinity, but also that the outer domains of the gp32 monomers at the ends of the cluster can fluctuate on and off the lattice and that the clusters of gp32 monomers can slide along the ssDNA. We also show that at very low binding densities gp32 monomers bind to the ssDNA lattice at random, but that cooperatively bound gp32 clusters bind preferentially at the 5'-end of the ssDNA lattice. We use these results and the gp32 monomer-binding results of the companion paper to propose a detailed model for how gp32 might bind to and interact with ssDNA lattices in its various binding modes, and also consider how these clusters might interact with other components of the T4 DNA replication complex. PMID:26275774

  8. Thirteen Virulent and Temperate Bacteriophages of Lactobacillus bulgaricus and Lactobacillus lactis Belong to a Single DNA Homology Group

    PubMed Central

    Mata, Mireille; Trautwetter, Annie; Luthaud, Gisèle; Ritzenthaler, Paul

    1986-01-01

    Thirteen virulent phages and two temperate phages of two closely related bacterial species (Lactobacillus lactis and L. bulgaricus) were compared for their protein composition, their antigenic properties, their restriction endonuclease patterns, and their DNA homology. The immunoblotting studies and the DNA-DNA hybridizations showed that the phages could be differentiated into two groups. One group contained 2 temperate phages of L. bulgaricus and 11 virulent phages of L. lactis. Inside each group, at least two common proteins of identical sizes could be detected for each phage. These proteins were able to cross-react in immunoblotting experiments with an antiserum raised against one phage of the same group. Temperate phage DNAs showed partial homology with DNAs from some virulent phages. These homologies seem to be located on the region coding for the structural proteins since recombinant plasmids coding for one of the major phage proteins of one phage were able to hybridize with the DNAs from phages of the same group. These results suggest that temperate and virulent phages may be related to one another. Images PMID:16347174

  9. BACTERIOPHAGE: BIOLOGY AND GENETICS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophage are viruses that infect bacteria. Bacteriophage are very small and made up of a protein coat with an inner core containing their genetic material. They infect bacterium, by attaching to the bacterial cell and injecting their nucleic acids into the bacteria. The phages then use the bac...

  10. Bacteriophages Infecting Propionibacterium acnes

    PubMed Central

    2013-01-01

    Viruses specifically infecting bacteria, or bacteriophages, are the most common biological entity in the biosphere. As such, they greatly influence bacteria, both in terms of enhancing their virulence and in terms of killing them. Since the first identification of bacteriophages in the beginning of the 20th century, researchers have been fascinated by these microorganisms and their ability to eradicate bacteria. In this review, we will cover the history of the Propionibacterium acnes bacteriophage research and point out how bacteriophage research has been an important part of the research on P. acnes itself. We will further discuss recent findings from phage genome sequencing and the identification of phage sequence signatures in clustered regularly interspaced short palindromic repeats (CRISPRs). Finally, the potential to use P. acnes bacteriophages as a therapeutic strategy to combat P. acnes-associated diseases will be discussed. PMID:23691509

  11. Biochemical analysis of the substrate specificity and sequence preference of endonuclease IV from bacteriophage T4, a dC-specific endonuclease implicated in restriction of dC-substituted T4 DNA synthesis

    PubMed Central

    Hirano, Nobutaka; Ohshima, Hiroyuki; Takahashi, Hideo

    2006-01-01

    Endonuclease IV encoded by denB of bacteriophage T4 is implicated in restriction of deoxycytidine (dC)-containing DNA in the host Escherichia coli. The enzyme was synthesized with the use of a wheat germ cell-free protein synthesis system, given a lethal effect of its expression in E.coli cells, and was purified to homogeneity. The purified enzyme showed high activity with single-stranded (ss) DNA and denatured dC-substituted T4 genomic double-stranded (ds) DNA but exhibited no activity with dsDNA, ssRNA or denatured T4 genomic dsDNA containing glucosylated deoxyhydroxymethylcytidine. Characterization of Endo IV activity revealed that the enzyme catalyzed specific endonucleolytic cleavage of the 5′ phosphodiester bond of dC in ssDNA with an efficiency markedly dependent on the surrounding nucleotide sequence. The enzyme preferentially targeted 5′-dTdCdA-3′ but tolerated various combinations of individual nucleotides flanking this trinucleotide sequence. These results suggest that Endo IV preferentially recognizes short nucleotide sequences containing 5′-dTdCdA-3′, which likely accounts for the limited digestion of ssDNA by the enzyme and may be responsible in part for the indispensability of a deficiency in denB for stable synthesis of dC-substituted T4 genomic DNA. PMID:16971463

  12. Tandem pentuplication of a DNA segment in a derivative of bacteriophage P2: its use in the study of the mechanism of DNA annealing.

    PubMed Central

    Bertani, G; Chattoraj, D K

    1980-01-01

    From a tandem duplication mutant of phage P2, triplication, quadruplication and pentuplication forms were derived. They were recognized by decreased virion heat stability resulting from the increase in DNA content, and were confirmed by electron microscope heteroduplex mapping. These forms of partially repeated DNA are quite stable in P2 because of the low level of recombination typical of this phage. Under conditions normally employed for full DNA renaturation, these high order repeat chromosomes gave often incomplete renaturation over the repeated segments. Based on current models for DNA renaturation, several predictions were made and tested. The results, although not quantitatively exhaustive, indicated that base pairing proceeding from a nucleation site was sufficiently slow to allow a second nucleation to occur with a fair probability over a length of a few thousand base pairs. Images PMID:7433123

  13. Two novel temperate bacteriophages co-existing in Aeromonas sp. ARM81 - characterization of their genomes, proteomes and DNA methyltransferases.

    PubMed

    Dziewit, Lukasz; Radlinska, Monika

    2016-08-01

    Aeromonas species are causative agents of a wide spectrum of diseases in animals and humans. Although these bacteria are commonly found in various environments, little is known about their phages. Thus far, only one temperate Aeromonas phage has been characterized. Whole-genome sequencing of an Aeromonas sp. strain ARM81 revealed the presence of two prophage clusters. One of them is integrated into the chromosome and the other was maintained as an extrachromosomal, linear plasmid-like prophage encoding a protelomerase. Both prophages were artificially and spontaneously inducible. We separately isolated both phages and compared their genomes with other known viruses. The novel phages show no similarity to the previously characterized Aeromonas phages and might represent new evolutionary lineages of viruses infecting Aeromonadaceae. Apart from the comparative genomic analyses of these phages, complemented with their structural and molecular characterization, a functional analysis of four DNA methyltransferases encoded by these viruses was conducted. One of the investigated N6-adenine-modifying enzymes shares sequence specificity with a Dam-like methyltransferase of its bacterial host, while another one is non-specific, as it catalyzes adenine methylation in various sequence contexts. The presented results shed new light on the diversity of Aeromonas temperate phages. PMID:27184451

  14. Solid-State and Biological Nanopore for Real-Time Sensing of Single Chemical and Sequencing of DNA

    PubMed Central

    Haque, Farzin; Li, Jinghong; Wu, Hai-Chen; Liang, Xing-Jie; Guo, Peixuan

    2013-01-01

    Sensitivity and specificity are two most important factors to take into account for molecule sensing, chemical detection and disease diagnosis. A perfect sensitivity is to reach the level where a single molecule can be detected. An ideal specificity is to reach the level where the substance can be detected in the presence of many contaminants. The rapidly progressing nanopore technology is approaching this threshold. A wide assortment of biomotors and cellular pores in living organisms perform diverse biological functions. The elegant design of these transportation machineries has inspired the development of single molecule detection based on modulations of the individual current blockage events. The dynamic growth of nanotechnology and nanobiotechnology has stimulated rapid advances in the study of nanopore based instrumentation over the last decade, and inspired great interest in sensing of single molecules including ions, nucleotides, enantiomers, drugs, and polymers such as PEG, RNA, DNA, and polypeptides. This sensing technology has been extended to medical diagnostics and third generation high throughput DNA sequencing. This review covers current nanopore detection platforms including both biological pores and solid state counterparts. Several biological nanopores have been studied over the years, but this review will focus on the three best characterized systems including α-hemolysin and MspA, both containing a smaller channel for the detection of single-strand DNA, as well as bacteriophage phi29 DNA packaging motor connector that contains a larger channel for the passing of double stranded DNA. The advantage and disadvantage of each system are compared; their current and potential applications in nanomedicine, biotechnology, and nanotechnology are discussed. PMID:23504223

  15. Solid-State and Biological Nanopore for Real-Time Sensing of Single Chemical and Sequencing of DNA.

    PubMed

    Haque, Farzin; Li, Jinghong; Wu, Hai-Chen; Liang, Xing-Jie; Guo, Peixuan

    2013-02-01

    Sensitivity and specificity are two most important factors to take into account for molecule sensing, chemical detection and disease diagnosis. A perfect sensitivity is to reach the level where a single molecule can be detected. An ideal specificity is to reach the level where the substance can be detected in the presence of many contaminants. The rapidly progressing nanopore technology is approaching this threshold. A wide assortment of biomotors and cellular pores in living organisms perform diverse biological functions. The elegant design of these transportation machineries has inspired the development of single molecule detection based on modulations of the individual current blockage events. The dynamic growth of nanotechnology and nanobiotechnology has stimulated rapid advances in the study of nanopore based instrumentation over the last decade, and inspired great interest in sensing of single molecules including ions, nucleotides, enantiomers, drugs, and polymers such as PEG, RNA, DNA, and polypeptides. This sensing technology has been extended to medical diagnostics and third generation high throughput DNA sequencing. This review covers current nanopore detection platforms including both biological pores and solid state counterparts. Several biological nanopores have been studied over the years, but this review will focus on the three best characterized systems including α-hemolysin and MspA, both containing a smaller channel for the detection of single-strand DNA, as well as bacteriophage phi29 DNA packaging motor connector that contains a larger channel for the passing of double stranded DNA. The advantage and disadvantage of each system are compared; their current and potential applications in nanomedicine, biotechnology, and nanotechnology are discussed. PMID:23504223

  16. Genome Sequences of Two Bacillus cereus Group Bacteriophages, Eyuki and AvesoBmore

    PubMed Central

    Erill, Ivan

    2015-01-01

    The genomes of two double-stranded DNA (dsDNA) bacteriophages isolated on Bacillus thuringiensis show similarity to previously sequenced phages and provide evidence of the mosaicism of phage genomes. PMID:26472840

  17. Antiviral effect of cationic compounds on bacteriophages

    PubMed Central

    Ly-Chatain, Mai H.; Moussaoui, Saliha; Vera, Annabelle; Rigobello, Véronique; Demarigny, Yann

    2013-01-01

    The antiviral activity of several cationic compounds – cetyltrimethylammonium bromide (CTAB), chitosan, nisin, and lysozyme – was investigated on the bacteriophage c2 (DNA head and non-contractile tail) infecting Lactococcus strains and the bacteriophage MS2 (F-specific RNA) infecting E. coli. Firstly, these activities were evaluated in a phosphate buffer pH 7 – 10 mM. The CTAB had a virucidal effect on the Lactococcus bacteriophages, but not on the MS2. After 1 min of contact with 0.125 mM CTAB, the c2 population was reduced from 6 to 1.5 log(pfu)/mL and completely deactivated at 1 mM. On the contrary, chitosan inhibited the MS2 more than it did the bacteriophages c2. No antiviral effect was observed for the nisin or the lysozyme on bacteriophages after 1 min of treatment. A 1 and 2.5 log reduction was respectively observed for nisin and lysozyme when the treatment time increased (5 or 10 min). These results showed that the antiviral effect depended both on the virus and structure of the antimicrobial compounds. The antiviral activity of these compounds was also evaluated in different physico-chemical conditions and in complex matrices. The antiviral activity of CTAB was impaired in acid pH and with an increase of the ionic strength. These results might be explained by the electrostatic interactions between cationic compounds and negatively charged particles such as bacteriophages or other compounds in a matrix. Milk proved to be protective suggesting the components of food could interfere with antimicrobial compounds. PMID:23487495

  18. Template reporter bacteriophage platform and multiple bacterial detection assays based thereon

    NASA Technical Reports Server (NTRS)

    Goodridge, Lawrence (Inventor)

    2007-01-01

    The invention is a method for the development of assays for the simultaneous detection of multiple bacteria. A bacteria of interest is selected. A host bacteria containing plasmid DNA from a T even bacteriophage that infects the bacteria of interest is infected with T4 reporter bacteriophage. After infection, the progeny bacteriophage are plating onto the bacteria of interest. The invention also includes single-tube, fast and sensitive assays which utilize the novel method.

  19. Bacteria vs. Bacteriophages: Parallel Evolution of Immune Arsenals

    PubMed Central

    Shabbir, Muhammad A. B.; Hao, Haihong; Shabbir, Muhammad Z.; Wu, Qin; Sattar, Adeel; Yuan, Zonghui

    2016-01-01

    Bacteriophages are the most common entities on earth and represent a constant challenge to bacterial populations. To fend off bacteriophage infection, bacteria evolved immune systems to avert phage adsorption and block invader DNA entry. They developed restriction–modification systems and mechanisms to abort infection and interfere with virion assembly, as well as newly recognized clustered regularly interspaced short palindromic repeats (CRISPR). In response to bacterial immune systems, bacteriophages synchronously evolved resistance mechanisms, such as the anti-CRISPR systems to counterattack bacterial CRISPR-cas systems, in a continuing evolutionary arms race between virus and host. In turn, it is fundamental to the survival of the bacterial cell to evolve a system to combat bacteriophage immune strategies. PMID:27582740

  20. Bacteria vs. Bacteriophages: Parallel Evolution of Immune Arsenals.

    PubMed

    Shabbir, Muhammad A B; Hao, Haihong; Shabbir, Muhammad Z; Wu, Qin; Sattar, Adeel; Yuan, Zonghui

    2016-01-01

    Bacteriophages are the most common entities on earth and represent a constant challenge to bacterial populations. To fend off bacteriophage infection, bacteria evolved immune systems to avert phage adsorption and block invader DNA entry. They developed restriction-modification systems and mechanisms to abort infection and interfere with virion assembly, as well as newly recognized clustered regularly interspaced short palindromic repeats (CRISPR). In response to bacterial immune systems, bacteriophages synchronously evolved resistance mechanisms, such as the anti-CRISPR systems to counterattack bacterial CRISPR-cas systems, in a continuing evolutionary arms race between virus and host. In turn, it is fundamental to the survival of the bacterial cell to evolve a system to combat bacteriophage immune strategies. PMID:27582740

  1. Helicase assembly protein Gp59 of bacteriophage T4: fluorescence anisotropy and sedimentation studies of complexes formed with derivatives of Gp32, the phage ssDNA binding protein.

    PubMed

    Xu, H; Wang, Y; Bleuit, J S; Morrical, S W

    2001-06-26

    The gene 59 protein (gp59) of bacteriophage T4 performs a vital function in phage DNA replication by directing the assembly of gp41, the DNA helicase component of the T4 primosome, onto lagging strand ssDNA at nascent replication forks. The helicase assembly activity of gp59 is required for optimum efficiency of helicase acquisition by the replication fork during strand displacement DNA synthesis and is essential for helicase and primosome assembly during T4 recombination-dependent DNA replication transactions. Of central importance is the ability of gp59 to load the gp41 helicase onto ssDNA previously coated with cooperatively bound molecules of gp32, the T4 ssDNA binding protein. Gp59 heteroassociations with ssDNA, gp32, and gp41 all appear to be essential for this loading reaction. Previous studies demonstrated that a tripartite complex containing gp59 and gp32 simultaneously cooccupying ssDNA is an essential intermediate in gp59-dependent helicase loading; however, the biochemical and structural parameters of gp59-gp32 complexes with or without ssDNA are currently unknown. To better understand gp59-gp32 interactions, we performed fluorescence anisotropy and analytical ultracentrifugation experiments employing native or rhodamine-labeled gp59 species in combination with altered forms of gp32, allowing us to determine their binding parameters, shape parameters, and other hydrodynamic properties. Two truncated forms of gp32 were used: gp32-B, which lacks the N-terminal B-domain required for cooperative binding to ssDNA and for stable self-association, and A-domain fragment, which is the C-terminal peptide of gp32 lacking ssDNA binding ability. Results indicate that gp59 binds with high affinity to either gp32 derivative to form a 1:1 heterodimer. In both cases, heterodimer formation is accompanied by a conformational change in gp59 which correlates with decreased gp59-DNA binding affinity. Hydrodynamic modeling suggests an asymmetric prolate ellipsoid shape for gp

  2. Common mechanisms of DNA translocation motors in bacteria and viruses using one-way revolution mechanism without rotation.

    PubMed

    Guo, Peixuan; Zhao, Zhengyi; Haak, Jeannie; Wang, Shaoying; Wu, Dong; Meng, Bing; Weitao, Tao

    2014-01-01

    Biomotors were once described into two categories: linear motor and rotation motor. Recently, a third type of biomotor with revolution mechanism without rotation has been discovered. By analogy, rotation resembles the Earth rotating on its axis in a complete cycle every 24h, while revolution resembles the Earth revolving around the Sun one circle per 365 days (see animations http://nanobio.uky.edu/movie.html). The action of revolution that enables a motor free of coiling and torque has solved many puzzles and debates that have occurred throughout the history of viral DNA packaging motor studies. It also settles the discrepancies concerning the structure, stoichiometry, and functioning of DNA translocation motors. This review uses bacteriophages Phi29, HK97, SPP1, P22, T4, and T7 as well as bacterial DNA translocase FtsK and SpoIIIE or the large eukaryotic dsDNA viruses such as mimivirus and vaccinia virus as examples to elucidate the puzzles. These motors use ATPase, some of which have been confirmed to be a hexamer, to revolve around the dsDNA sequentially. ATP binding induces conformational change and possibly an entropy alteration in ATPase to a high affinity toward dsDNA; but ATP hydrolysis triggers another entropic and conformational change in ATPase to a low affinity for DNA, by which dsDNA is pushed toward an adjacent ATPase subunit. The rotation and revolution mechanisms can be distinguished by the size of channel: the channels of rotation motors are equal to or smaller than 2 nm, that is the size of dsDNA, whereas channels of revolution motors are larger than 3 nm. Rotation motors use parallel threads to operate with a right-handed channel, while revolution motors use a left-handed channel to drive the right-handed DNA in an anti-chiral arrangement. Coordination of several vector factors in the same direction makes viral DNA-packaging motors unusually powerful and effective. Revolution mechanism that avoids DNA coiling in translocating the lengthy genomic

  3. Bacteriophage therapy against Enterobacteriaceae.

    PubMed

    Xu, Youqiang; Liu, Yong; Liu, Yang; Pei, Jiangsen; Yao, Su; Cheng, Chi

    2015-02-01

    The Enterobacteriaceae are a class of gram-negative facultative anaerobic rods, which can cause a variety of diseases, such as bacteremia, septic arthritis, endocarditis, osteomyelitis, lower respiratory tract infections, skin and soft-tissue infections, urinary tract infections, intra-abdominal infections and ophthalmic infections, in humans, poultry, animals and fish. Disease caused by Enterobacteriaceae cause the deaths of millions of people every year, resulting in enormous economic loss. Drug treatment is a useful and efficient way to control Enterobacteriaceae infections. However, with the abuse of antibiotics, drug resistance has been found in growing number of Enterobacteriaceae infections and, as such, there is an urgent need to find new methods of control. Bacteriophage therapy is an efficient alternative to antibiotics as it employs a different antibacterial mechanism. This paper summarizes the history of bacteriophage therapy, its bacterial lytic mechanisms, and the studies that have focused on Enterobacteriaceae and bacteriophage therapy. PMID:25662887

  4. Hyperexpansion of RNA Bacteriophage Diversity

    PubMed Central

    Krishnamurthy, Siddharth R.; Janowski, Andrew B.; Zhao, Guoyan; Barouch, Dan; Wang, David

    2016-01-01

    Bacteriophage modulation of microbial populations impacts critical processes in ocean, soil, and animal ecosystems. However, the role of bacteriophages with RNA genomes (RNA bacteriophages) in these processes is poorly understood, in part because of the limited number of known RNA bacteriophage species. Here, we identify partial genome sequences of 122 RNA bacteriophage phylotypes that are highly divergent from each other and from previously described RNA bacteriophages. These novel RNA bacteriophage sequences were present in samples collected from a range of ecological niches worldwide, including invertebrates and extreme microbial sediment, demonstrating that they are more widely distributed than previously recognized. Genomic analyses of these novel bacteriophages yielded multiple novel genome organizations. Furthermore, one RNA bacteriophage was detected in the transcriptome of a pure culture of Streptomyces avermitilis, suggesting for the first time that the known tropism of RNA bacteriophages may include gram-positive bacteria. Finally, reverse transcription PCR (RT-PCR)-based screening for two specific RNA bacteriophages in stool samples from a longitudinal cohort of macaques suggested that they are generally acutely present rather than persistent. PMID:27010970

  5. Hyperexpansion of RNA Bacteriophage Diversity.

    PubMed

    Krishnamurthy, Siddharth R; Janowski, Andrew B; Zhao, Guoyan; Barouch, Dan; Wang, David

    2016-03-01

    Bacteriophage modulation of microbial populations impacts critical processes in ocean, soil, and animal ecosystems. However, the role of bacteriophages with RNA genomes (RNA bacteriophages) in these processes is poorly understood, in part because of the limited number of known RNA bacteriophage species. Here, we identify partial genome sequences of 122 RNA bacteriophage phylotypes that are highly divergent from each other and from previously described RNA bacteriophages. These novel RNA bacteriophage sequences were present in samples collected from a range of ecological niches worldwide, including invertebrates and extreme microbial sediment, demonstrating that they are more widely distributed than previously recognized. Genomic analyses of these novel bacteriophages yielded multiple novel genome organizations. Furthermore, one RNA bacteriophage was detected in the transcriptome of a pure culture of Streptomyces avermitilis, suggesting for the first time that the known tropism of RNA bacteriophages may include gram-positive bacteria. Finally, reverse transcription PCR (RT-PCR)-based screening for two specific RNA bacteriophages in stool samples from a longitudinal cohort of macaques suggested that they are generally acutely present rather than persistent. PMID:27010970

  6. Common Mechanisms of DNA translocation motors in Bacteria and Viruses Using One-way Revolution Mechanism without Rotation

    PubMed Central

    Guo, Peixuan; Zhao, Zhengyi; Haak, Jeannie; Wang, Shaoying; Weitao, Tao

    2014-01-01

    Biomotors were once classified into two categories: linear motor and rotation motor. For decades, the viral DNA-packaging motor has been popularly believed to be a five-fold rotation motor. Recently, a third type of biomotor with revolution mechanism without rotation has been discovered. By analogy, rotation resembles the Earth rotating on its axis in a complete cycle every 24 hours, while revolution resembles the Earth revolving around the Sun one circle per 365 days (see animations http://nanobio.uky.edu/movie.html). The action of revolution that enables a motor free of coiling and torque has solved many puzzles and debates that have occurred throughout the history of viral DNA packaging motor studies. It also settles the discrepancies concerning the structure, stoichiometry, and functioning of DNA translocation motors. This review uses bacteriophages Phi29, HK97, SPP1, P22, T4, T7 as well as bacterial DNA translocase FtsK and SpoIIIE as examples to elucidate the puzzles. These motors use a ATPase, some of which have been confirmed to be a hexamer, to revolve around the dsDNA sequentially. ATP binding induces conformational change and possibly an entropy alteration in ATPase to a high affinity toward dsDNA; but ATP hydrolysis triggers another entropic and conformational change in ATPase to a low affinity for DNA, by which dsDNA is pushed toward an adjacent ATPase subunit. The rotation and revolution mechanisms can be distinguished by the size of channel: the channels of rotation motors are equal to or smaller than 2 nm, whereas channels of revolution motors are larger than 3 nm. Rotation motors use parallel threads to operate with a right-handed channel, while revolution motors use a left-handed channel to drive the right-handed DNA in an anti-parallel arrangement. Coordination of several vector factors in the same direction makes viral DNA-packaging motors unusually powerful and effective. Revolution mechanism avoids DNA coiling in translocating the lengthy

  7. Bacteriophages of Clostridium perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The specific aims of the book chapter are to: (1) Briefly review the nomenclature of bacteriophages and how these agents are classified. (2) Discuss the problems associated with addition/removal of antibiotics in commercial animal feeds. (3) Provide a brief overview of Clostridium perfringens biolog...

  8. BACTERIOPHAGE THERAPY AND CAMPYLOBACTER

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The book chapter reports efforts to exploit Campylobacter-specific bacteriophages to reduce the numbers of Campylobacter jejuni and C. coli colonizing poultry and contaminating poultry meat products. Controlling campylobacters in poultry represents one of the greatest challenges to the agriculture a...

  9. Multiple roles of genome-attached bacteriophage terminal proteins

    SciTech Connect

    Redrejo-Rodríguez, Modesto; Salas, Margarita

    2014-11-15

    Protein-primed replication constitutes a generalized mechanism to initiate DNA or RNA synthesis in linear genomes, including viruses, gram-positive bacteria, linear plasmids and mobile elements. By this mechanism a specific amino acid primes replication and becomes covalently linked to the genome ends. Despite the fact that TPs lack sequence homology, they share a similar structural arrangement, with the priming residue in the C-terminal half of the protein and an accumulation of positively charged residues at the N-terminal end. In addition, various bacteriophage TPs have been shown to have DNA-binding capacity that targets TPs and their attached genomes to the host nucleoid. Furthermore, a number of bacteriophage TPs from different viral families and with diverse hosts also contain putative nuclear localization signals and localize in the eukaryotic nucleus, which could lead to the transport of the attached DNA. This suggests a possible role of bacteriophage TPs in prokaryote-to-eukaryote horizontal gene transfer. - Highlights: • Protein-primed genome replication constitutes a strategy to initiate DNA or RNA synthesis in linear genomes. • Bacteriophage terminal proteins (TPs) are covalently attached to viral genomes by their primary function priming DNA replication. • TPs are also DNA-binding proteins and target phage genomes to the host nucleoid. • TPs can also localize in the eukaryotic nucleus and may have a role in phage-mediated interkingdom gene transfer.

  10. Isolation and characterization of a bacteriophage lytic for Desulfovibrio salexigens, a salt-requiring, sulfate-reducing bacterium

    SciTech Connect

    Kamimura, Kazuo; Araki, Michio )

    1989-03-01

    A bacteriophage that lysed Desulfovibrio salexigens cells was isolated from marine sediments and preliminarily characterized by electron microscopy and electrophoretic analysis of structural proteins and genomic nucleic acid. The bacteriophage had an icosahedral head and a long flexible tail, and the buoyant density of the bacteriophage particles was 1.468 g/ml in cesium chloride. The particles consisted of a double-stranded DNA molecule about 33 kilobase pairs long and at least 11 structural proteins.

  11. Complete Genome Sequences of Lytic Bacteriophages of Xanthomonas arboricola pv. Juglandis

    PubMed Central

    Vasquez, Ignacio; Santos, Leonardo; Segovia, Cristopher; Ayala, Manuel; Alvarado, Romina; Nuñez, Pablo

    2016-01-01

    Three bacteriophages, f20-Xaj, f29-Xaj, and f30-Xaj, with lytic activity against Xanthomonas arboricola pv. juglandis were isolated from walnut trees (VIII Bío Bío Region, Chile). These lytic bacteriophages have double-stranded DNA (dsDNA) genomes of 43,851 bp, 41,865 bp, and 44,262 bp, respectively. These are the first described bacteriophages with lytic activity against X. arboricola pv. juglandis that can be utilized as biocontrol agents. PMID:27257210

  12. Complete Genome Sequences of Lytic Bacteriophages of Xanthomonas arboricola pv. juglandis.

    PubMed

    Retamales, Julio; Vasquez, Ignacio; Santos, Leonardo; Segovia, Cristopher; Ayala, Manuel; Alvarado, Romina; Nuñez, Pablo; Santander, Javier

    2016-01-01

    Three bacteriophages, f20-Xaj, f29-Xaj, and f30-Xaj, with lytic activity against Xanthomonas arboricola pv. juglandis were isolated from walnut trees (VIII Bío Bío Region, Chile). These lytic bacteriophages have double-stranded DNA (dsDNA) genomes of 43,851 bp, 41,865 bp, and 44,262 bp, respectively. These are the first described bacteriophages with lytic activity against X. arboricola pv. juglandis that can be utilized as biocontrol agents. PMID:27257210

  13. Evolution and the complexity of bacteriophages

    PubMed Central

    Serwer, Philip

    2007-01-01

    Background The genomes of both long-genome (> 200 Kb) bacteriophages and long-genome eukaryotic viruses have cellular gene homologs whose selective advantage is not explained. These homologs add genomic and possibly biochemical complexity. Understanding their significance requires a definition of complexity that is more biochemically oriented than past empirically based definitions. Hypothesis Initially, I propose two biochemistry-oriented definitions of complexity: either decreased randomness or increased encoded information that does not serve immediate needs. Then, I make the assumption that these two definitions are equivalent. This assumption and recent data lead to the following four-part hypothesis that explains the presence of cellular gene homologs in long bacteriophage genomes and also provides a pathway for complexity increases in prokaryotic cells: (1) Prokaryotes underwent evolutionary increases in biochemical complexity after the eukaryote/prokaryote splits. (2) Some of the complexity increases occurred via multi-step, weak selection that was both protected from strong selection and accelerated by embedding evolving cellular genes in the genomes of bacteriophages and, presumably, also archaeal viruses (first tier selection). (3) The mechanisms for retaining cellular genes in viral genomes evolved under additional, longer-term selection that was stronger (second tier selection). (4) The second tier selection was based on increased access by prokaryotic cells to improved biochemical systems. This access was achieved when DNA transfer moved to prokaryotic cells both the more evolved genes and their more competitive and complex biochemical systems. Testing the hypothesis I propose testing this hypothesis by controlled evolution in microbial communities to (1) determine the effects of deleting individual cellular gene homologs on the growth and evolution of long genome bacteriophages and hosts, (2) find the environmental conditions that select for the

  14. Binding of the N-terminal domain of the lactococcal bacteriophage TP901-1 CI repressor to its target DNA: a crystallography, small angle scattering, and nuclear magnetic resonance study.

    PubMed

    Frandsen, Kristian H; Rasmussen, Kim K; Jensen, Malene Ringkjøbing; Hammer, Karin; Pedersen, Margit; Poulsen, Jens-Christian N; Arleth, Lise; Lo Leggio, Leila

    2013-10-01

    In most temperate bacteriophages, regulation of the choice of lysogenic or lytic life cycle is controlled by a CI repressor protein. Inhibition of transcription is dependent on a helix-turn-helix motif, often located in the N-terminal domain (NTD), which binds to specific DNA sequences (operator sites). Here the crystal structure of the NTD of the CI repressor from phage TP901-1 has been determined at 1.6 Å resolution, and at 2.6 Å resolution in complex with a 9 bp double-stranded DNA fragment that constitutes a half-site of the OL operator. This N-terminal construct, comprising residues 2-74 of the CI repressor, is monomeric in solution as shown by nuclear magnetic resonance (NMR), small angle X-ray scattering, and gel filtration and is monomeric in the crystal structures. The binding interface between the NTD and the half-site in the crystal is very similar to the interface that can be mapped by NMR in solution with a full palindromic site. The interactions seen in the complexes (in the crystal and in solution) explain the observed affinity for the OR site that is lower than that for the OL site and the specificity for the recognized DNA sequence in comparison to that for other repressors. Compared with many well-studied phage repressor systems, the NTD from TP901-1 CI has a longer extended scaffolding helix that, interestingly, is strongly conserved in putative repressors of Gram-positive pathogens. On the basis of sequence comparisons, we suggest that these bacteria also possess repressor/antirepressor systems similar to that found in phage TP901-1. PMID:24047404

  15. Existence of lysogenic bacteriophages in Bacillus thuringiensis type strains.

    PubMed

    Roh, Jong Yul; Park, Jong Bin; Liu, Qin; Kim, Song Eun; Tao, Xueying; Choi, Tae Woong; Choi, Jae Young; Kim, Woo Jin; Jin, Byung Rae; Je, Yeon Ho

    2013-07-01

    We screened the existence of bacteriophages in 67 Bacillus thuringiensis type strains by phage DNA extraction and PCR using phage terminase small subunit (TerS)-specific primers to the supernatants and the precipitated pellets of Bt cultures, and by transmission electron microscopy. The various bacteriophages were observed from the supernatants of 22 type strains. Ten type strains showed the extracted phage DNAs and the amplified fragment by TerS PCR but 12 type strains showed only the phage DNAs. Their morphological characteristic suggests that they belong to Family Siphoviridae which had a long tail and symmetrical head. PMID:23632013

  16. Precipitation of filamentous bacteriophages for their selective recovery in primary purification.

    PubMed

    Branston, Steven; Stanley, Emma; Keshavarz-Moore, Eli; Ward, John

    2012-01-01

    Filamentous bacteriophages and their derivatives are showing great promise as a whole new class of industrial agents, such as biologically based nano-materials and viral vectors. This raises challenges for their large-scale manufacture, principally due to the lack of bioprocessing knowledge. This article addresses what will be a potentially important option in the primary purification of the bacteriophages. Polyethylene glycol (PEG)-salt dual precipitants, calcium ions, spermidine, and isoelectric precipitation were first examined for their potential suitability for bacteriophage concentration under both pure and broth conditions. Successful precipitants were further studied on the basis of their selective purification ability from DNA and protein contaminants in a clarified broth system. Both PEG-based and isoelectric precipitations resulted in bacteriophage purity improvements, and PEG-based precipitations offered the highest selectivities. This work shows that precipitation of bacteriophages can be an effective primary purification step in a large-scale bioprocess. PMID:21905275

  17. M13 Bacteriophage Based Protein Sensors

    NASA Astrophysics Data System (ADS)

    Lee, Ju Hun

    Despite significant progress in biotechnology and biosensing, early detection and disease diagnosis remains a critical issue for improving patient survival rates and well-being. Many of the typical detection schemes currently used possess issues such as low sensitivity and accuracy and are also time consuming to run and expensive. In addition, multiplexed detection remains difficult to achieve. Therefore, developing advanced approaches for reliable, simple, quantitative analysis of multiple markers in solution that also are highly sensitive are still in demand. In recent years, much of the research has primarily focused on improving two key components of biosensors: the bio-recognition agent (bio-receptor) and the transducer. Particular bio-receptors that have been used include antibodies, aptamers, molecular imprinted polymers, and small affinity peptides. In terms of transducing agents, nanomaterials have been considered as attractive candidates due to their inherent nanoscale size, durability and unique chemical and physical properties. The key focus of this thesis is the design of a protein detection and identification system that is based on chemically engineered M13 bacteriophage coupled with nanomaterials. The first chapter provides an introduction of biosensors and M13 bacteriophage in general, where the advantages of each are provided. In chapter 2, an efficient and enzyme-free sensor is demonstrated from modified M13 bacteriophage to generate highly sensitive colorimetric signals from gold nanocrystals. In chapter 3, DNA conjugated M13 were used to enable facile and rapid detection of antigens in solution that also provides modalities for identification. Lastly, high DNA loadings per phage was achieved via hydrozone chemistry and these were applied in conjunction with Raman active DNA-gold/silver core/shell nanoparticles toward highly sensitive SERS sensing.

  18. Mutations in Nu1, the gene encoding the small subunit of bacteriophage lambda terminase, suppress the postcleavage DNA packaging defect of cosB mutations.

    PubMed Central

    Cai, Z H; Hwang, Y; Cue, D; Catalano, C; Feiss, M

    1997-01-01

    The linear double-stranded DNA molecules in lambda virions are generated by nicking of concatemeric intracellular DNA by terminase, the lambda DNA packaging enzyme. Staggered nicks are introduced at cosN to generate the cohesive ends of virion DNA. After nicking, the cohesive ends are separated by terminase; terminase bound to the left end of the DNA to be packaged then binds the empty protein shell, i.e., the prohead, and translocation of DNA into the prohead occurs. cosB, a site adjacent to cosN, is a terminase binding site. cosB facilitates the rate and fidelity of the cosN cleavage reaction by serving as an anchoring point for gpNu1, the small subunit of terminase. cosB is also crucial for the formation of a stable terminase-DNA complex, called complex I, formed after cosN cleavage. The role of complex I is to bind the prohead. Mutations in cosB affect both cosB functions, causing mild defects in cosN cleavage and severe packaging defects. The lethal cosB R3- R2- R1- mutation contains a transition mutation in each of the three gpNu1 binding sites of cosB. Pseudorevertants of lambda cosB R3- R2- R1- DNA contain suppressor mutations affecting gpNu1. Results of experiments that show that two such suppressors, Nu1ms1 and Nu1ms3, do not suppress the mild cosN cleavage defect caused by the cosB R3- R2- R1- mutation but strongly suppress the DNA packaging defect are presented. It is proposed that the suppressing terminases, unlike the wild-type enzyme, are able to assemble a stable complex I with cosB R3- R2- R1- DNA. Observations on the adenosine triphosphatase activities and protease susceptibilities of gpNu1 of the Nu1ms1 and Nu1ms3 terminases indicate that the conformation of gpNu1 is altered in the suppressing terminases. PMID:9098042

  19. Identification of the Block in the Intracellular Replication of Single-Stranded DNA of Photodynamically Inactivated Bacteriophage φX174

    PubMed Central

    Chaudhuri, Utpal C.; Poddar, Ramendra K.

    1973-01-01

    32P-labeled single-stranded DNA phage φX174 was photodynamically inactivated by irradiation in air with visible light in the presence of the acridine dye, proflavine sulfate. The inactivated phages could adsorb to the host cells but failed to lyse them. Formation of intracellular mature phages was almost completely inhibited. Photodynamic lesions in φX174 DNA caused intracellular formation of defective double-stranded replicative form molecules which ultimately reverted to the single-stranded configuration. PMID:4570924

  20. Excision repair and patch size in UV-irradiated bacteriophage T4

    SciTech Connect

    Yarosh, D.B.; Rosenstein, B.S.; Setlow, R.B.

    1981-11-01

    We determined the average size of excision repair patches in repair of UV lesions in bacteriophage T4 by measuring the photolysis of bromodeoxyuridine incorporated during repair. The average patch was small, approximately four nucleotides long. In control experiments with the denV1 excision-deficient mutant, we encountered an artifact, a protein(s) which remained bound to phenol-extracted DNA and prevented nicking by the UV-specific endonucleases of Micrococcus luteus and bacteriophage T4.

  1. Excision repair and patch size in UV-irradiated bacteriophage T4

    SciTech Connect

    Yarosh, D.B.; Rosenstein, B.S.; Setlow, R.B.

    1981-11-01

    We determined the average size of excision repair patches in repair of UV lesions in bacteriophage T4 by measuring the photolysis of bromodeoxyuridine incorporated during repair. The average patch was small, approximately four nucleotides long. In control, experiments with the denV/sub 1/ excision-deificient mutant, we encountered an artifact, a protein(s) which remained bound to phenol-extracted DNA and prevented nicking by the UV-specific endonucleases of Micrococcus luteus and bacteriophage T4.

  2. Genetically modified bacteriophages.

    PubMed

    Sagona, Antonia P; Grigonyte, Aurelija M; MacDonald, Paul R; Jaramillo, Alfonso

    2016-04-18

    Phages or bacteriophages, viruses that infect and replicate inside bacteria, are the most abundant microorganisms on earth. The realization that antibiotic resistance poses a substantial risk to the world's health and global economy is revitalizing phage therapy as a potential solution. The increasing ease by which phage genomes can be modified, owing to the influx of new technologies, has led to an expansion of their natural capabilities, and a reduced dependence on phage isolation from environmental sources. This review will discuss the way synthetic biology has accelerated the construction of genetically modified phages and will describe the wide range of their applications. It will further provide insight into the societal and economic benefits that derive from the use of recombinant phages in various sectors, from health to biodetection, biocontrol and the food industry. PMID:26906932

  3. Immunodetection and N-terminal sequencing of DNA replication proteins of bacteriophage BFK20 - lytic phage of Brevibacterium flavum.

    PubMed

    Bukovská, G; Halgašová, N; Hromadová, L; Koščová, H; Bukovský, M

    2014-01-01

    Phages are excellent models for studying the mechanism of DNA replication in prokaryotes. Identification of phage proteins involved in phage DNA replication is the first prerequisite for elucidation of the phage replication module. We focused on replication proteins gp41 (a putative helicase from SF2 superfamily), gp43 (a RepA-like protein), and gp44 (a putative DNA polymerase A) of phage BFK20 grown in Brevibacterium flavum. To identify them in the phage-host system, we prepared antibodies to these proteins which were cloned and expressed in Escherichia coli as his-tagged recombinant proteins. After purification to homogeneity the recombinant proteins served for raising specific polyclonal antibodies in mice. Using these antibodies in Western blot analysis the phage proteins gp41, gp43 and gp44 were detected during the phage growth cycle. The proteins gp41 and gp43, prepared from cell lysate by ammonium sulphate precipitation, were N-terminally sequenced and found to contain the sequences N-SVKPRELR-C and N-MLGSTML-C, respectively. This means that gp41 starts with serine but not with common methionine. We consider these findings an initial but important step towards more thorough characterization of replication proteins of phage BFK20. PMID:24957720

  4. IMPORTANCE OF THE DYNAMICS OF BACTERIOPHAGE-HOST INTERACTIONS TO BACTERIAL ABUNDANCE AND GENETIC DIVERSITY IN AQUATIC ENVIRONMENTS (RESEARCH BRIEF)

    EPA Science Inventory

    Using Pseudomonas aeruginosa and its bacteriophages as a model system, we have clearly demonstrated a significant potential for viral-mediated gene transfer (transduction) of both plasmid and chromosomal DNA in freshwater microbial populations. These investigations have predicted...

  5. DNA Nanoparticles for Improved Protein Synthesis In Vitro

    PubMed Central

    Galinis, Robertas; Stonyte, Greta; Kiseliovas, Vaidotas; Zilionis, Rapolas; Studer, Sabine; Hilvert, Donald; Janulaitis, Arvydas

    2016-01-01

    Abstract The amplification and digital quantification of single DNA molecules are important in biomedicine and diagnostics. Beyond quantifying DNA molecules in a sample, the ability to express proteins from the amplified DNA would open even broader applications in synthetic biology, directed evolution, and proteomics. Herein, a microfluidic approach is reported for the production of condensed DNA nanoparticles that can serve as efficient templates for in vitro protein synthesis. Using phi29DNA polymerase and a multiple displacement amplification reaction, single DNA molecules were converted into DNA nanoparticles containing up to about 104 clonal gene copies of the starting template. DNA nanoparticle formation was triggered by accumulation of inorganic pyrophosphate (produced during DNA synthesis) and magnesium ions from the buffer. Transcription–translation reactions performed in vitro showed that individual DNA nanoparticles can serve as efficient templates for protein synthesis in vitro. PMID:26821778

  6. Isolation and Characterization of Bacteriophages Against Pseudomonas syringae pv. actinidiae Causing Bacterial Canker Disease in Kiwifruit.

    PubMed

    Yu, Ji-Gang; Lim, Jeong-A; Song, Yu-Rim; Heu, Sunggi; Kim, Gyoung Hee; Koh, Young Jin; Oh, Chang-Sik

    2016-02-01

    Pseudomonas syringae pv. actinidiae causes bacterial canker disease in kiwifruit. Owing to the prohibition of agricultural antibiotic use in major kiwifruit-cultivating countries, alternative methods need to be developed to manage this disease. Bacteriophages are viruses that specifically infect target bacteria and have recently been reconsidered as potential biological control agents for bacterial pathogens owing to their specificity in terms of host range. In this study, we isolated bacteriophages against P. syringae pv. actinidiae from soils collected from kiwifruit orchards in Korea and selected seven bacteriophages for further characterization based on restriction enzyme digestion patterns of genomic DNA. Among the studied bacteriophages, two belong to the Myoviridae family and three belong to the Podoviridae family, based on morphology observed by transmission electron microscopy. The host range of the selected bacteriophages was confirmed using 18 strains of P. syringae pv. actinidiae, including the Psa2 and Psa3 groups, and some were also effective against other P. syringae pathovars. Lytic activity of the selected bacteriophages was sustained in vitro until 80 h, and their activity remained stable up to 50°C, at pH 11, and under UV-B light. These results indicate that the isolated bacteriophages are specific to P. syringae species and are resistant to various environmental factors, implying their potential use in control of bacterial canker disease in kiwifruits. PMID:26628254

  7. Bacteriophage lysis: mechanism and regulation.

    PubMed Central

    Young, R

    1992-01-01

    Bacteriophage lysis involves at least two fundamentally different strategies. Most phages elaborate at least two proteins, one of which is a murein hydrolase, or lysin, and the other is a membrane protein, which is given the designation holin in this review. The function of the holin is to create a lesion in the cytoplasmic membrane through which the murein hydrolase passes to gain access to the murein layer. This is necessary because phage-encoded lysins never have secretory signal sequences and are thus incapable of unassisted escape from the cytoplasm. The holins, whose prototype is the lambda S protein, share a common organization in terms of the arrangement of charged and hydrophobic residues, and they may all contain at least two transmembrane helical domains. The available evidence suggests that holins oligomerize to form nonspecific holes and that this hole-forming step is the regulated step in phage lysis. The correct scheduling of the lysis event is as much an essential feature of holin function as is the hole formation itself. In the second strategy of lysis, used by the small single-stranded DNA phage phi X174 and the single-stranded RNA phage MS2, no murein hydrolase activity is synthesized. Instead, there is a single species of small membrane protein, unlike the holins in primary structure, which somehow causes disruption of the envelope. These lysis proteins function by activation of cellular autolysins. A host locus is required for the lytic function of the phi X174 lysis gene E. Images PMID:1406491

  8. How long can bacteriophage λ change its mind?

    PubMed Central

    Semsey, Szabolcs; Campion, Christopher; Mohamed, Abdu; Svenningsen, Sine Lo

    2015-01-01

    A key event in the lifecycle of a temperate bacteriophage is the choice between lysis and lysogeny upon infection of a susceptible host cell. In a recent paper, we showed that a prolonged period exists after the decision to lysogenize, during which bacteriophage λ can abandon the initial decision, and instead develop lytically, as a response to the accumulation of the late lytic regulatory protein Q. Here, we present evidence that expression of Q does not induce replication of λ DNA, suggesting that the DNA to be packaged into the resulting phage progeny was already present at the time of the initial decision to lysogenize. We summarize our findings in a working model of the key determinants of the duration of the post-decision period during which it is possible for the infected cell to switch from the lysogeny decision to successful lytic development. PMID:26459429

  9. Bacteriophage recombination systems and biotechnical applications.

    PubMed

    Nafissi, Nafiseh; Slavcev, Roderick

    2014-04-01

    Bacteriophage recombination systems have been widely used in biotechnology for modifying prokaryotic species, for creating transgenic animals and plants, and more recently, for human cell gene manipulation. In contrast to homologous recombination, which benefits from the endogenous recombination machinery of the cell, site-specific recombination requires an exogenous source of recombinase in mammalian cells. The mechanism of bacteriophage evolution and their coexistence with bacterial cells has become a point of interest ever since bacterial viruses' life cycles were first explored. Phage recombinases have already been exploited as valuable genetic tools and new phage enzymes, and their potential application to genetic engineering and genome manipulation, vectorology, and generation of new transgene delivery vectors, and cell therapy are attractive areas of research that continue to be investigated. The significance and role of phage recombination systems in biotechnology is reviewed in this paper, with specific focus on homologous and site-specific recombination conferred by the coli phages, λ, and N15, the integrase from the Streptomyces phage, ΦC31, the recombination system of phage P1, and the recently characterized recombination functions of Yersinia phage, PY54. Key steps of the molecular mechanisms involving phage recombination functions and their application to molecular engineering, our novel exploitations of the PY54-derived recombination system, and its application to the development of new DNA vectors are discussed. PMID:24442504

  10. The gene for type A streptococcal exotoxin (erythrogenic toxin) is located in bacteriophage T12.

    PubMed Central

    Weeks, C R; Ferretti, J J

    1984-01-01

    The infection of Streptococcus pyogenes T25(3) with the temperate bacteriophage T12 results in the conversion of the nontoxigenic strain to type A streptococcal exotoxin (erythrogenic toxin) production. Although previous research has established that integration of the bacteriophage genome into the host chromosome is not essential for exotoxin production, the location of the gene on the bacteriophage or bacterial chromosome had not been determined. In the present investigation, recombinant DNA techniques were used to determine whether the gene specifying type A streptococcal exotoxin (speA) production is located on the bacteriophage chromosome. Bacteriophage T12 was obtained from S. pyogenes T25(3)(T12) by induction with mitomycin C, and after isolation of bacteriophage DNA by phenol-chloroform extraction, the DNA was digested with restriction enzymes and ligated with Escherichia coli plasmid pHP34 or the Streptococcus-E. coli shuttle vector pSA3. Transformation of E. coli HB101 with the recombinant molecules allowed selection of E. coli clones containing bacteriophage T12 genes. Immunological assays with specific antibody revealed the presence of type A streptococcal exotoxin in sonicates of E. coli transformants. Subcloning experiments localized the speA gene to a 1.7-kilobase segment of the bacteriophage T12 genome flanked by SalI and HindIII sites. Introduction of the pSA3 vector containing the speA gene into Streptococcus sanguis (Challis) resulted in transformants that secreted the type A exotoxin. Immunological analysis showed that the type A streptococcal exotoxin produced by E. coli and S. sanguis transformants was identical to the type A exotoxin produced by S. pyogenes T25(3)(T12). Southern blot hybridizations with the cloned fragment confirmed its presence in the bacteriophage T12 genome and its absence in the T25(3) nonlysogen. Therefore, the gene for type A streptococcal exotoxin is located in the bacteriophage genome, and conversion of S. pyogenes T

  11. MUTATIONAL SPECTRUM AND RECOMBINOGENIC EFFECTS INDUCED BY AMINOFLUORENE ADDUCTS IN BACTERIOPHAGE M13 (JOURNAL VERSION)

    EPA Science Inventory

    Double stranded replicative form (RFI) DNA of bacteriophage M13mp10 has been modified in vitro to various extents with N-hydroxy-2-aminofluorene (N-OH-AF) and then transfected into E. coli cells. HPLC analysis of the modified DNA shows that only dG-C8-AF adducts are formed. Appro...

  12. T4 bacteriophage as a phage display platform.

    PubMed

    Gamkrelidze, Mariam; Dąbrowska, Krystyna

    2014-07-01

    Analysis of molecular events in T4-infected Escherichia coli has revealed some of the most important principles of biology, including relationships between structures of genes and their products, virus-induced acquisition of metabolic function, and morphogenesis of complex structures through sequential gene product interaction rather than sequential gene activation. T4 bacteriophages and related strains were applied in the first formulations of many fundamental biological concepts. These include the unambiguous recognition of nucleic acids as the genetic material, the definition of the gene by fine-structure mutation, recombinational and functional analyses, the demonstration that the genetic code is triplet, the discovery of mRNA, the importance of recombination and DNA replications, light-dependent and light-independent DNA repair mechanisms, restriction and modification of DNA, self-splicing of intron/exon arrangement in prokaryotes, translation bypassing and others. Bacteriophage T4 possesses unique features that make it a good tool for a multicomponent vaccine platform. Hoc/Soc-fused antigens can be assembled on the T4 capsid in vitro and in vivo. T4-based phage display combined with affinity chromatography can be applied as a new method for bacteriophage purification. The T4 phage display system can also be used as an attractive approach for cancer therapy. The data show the efficient display of both single and multiple HIV antigens on the phage T4 capsid and offer insights for designing novel particulate HIV or other vaccines that have not been demonstrated by other vector systems. PMID:24828789

  13. Lysogenic bacteriophage isolated from acidophilium

    DOEpatents

    Ward, Thomas W.; Bruhn, Debby F.; Bulmer, Deborah K.

    1992-01-01

    A bacteriophage identified as .phi.Ac1 capable of infecting acidophilic heterotropic bacteria (such as Acidiphilium sp.) and processes for genetically engineering acidophilic bacteria for biomining or sulfur removal from coal are disclosed. The bacteriophage is capable of growth in cells existing at pH at or below 3.0. Lytic forms of the phage introduced into areas experiencing acid drainage kill the bacteria causing such drainage. Lysogenic forms of the phase having genes for selective removal of metallic or nonmetallic elements can be introduced into acidophilic bacteria to effect removal of the desired element form ore or coal.

  14. Bacteriophage therapy: a regulatory perspective.

    PubMed

    Pelfrene, Eric; Willebrand, Elsa; Cavaleiro Sanches, Ana; Sebris, Zigmars; Cavaleri, Marco

    2016-08-01

    Despite the recognized problem of antibiotic multidrug resistance, very few antibacterial agents with new mechanisms of action are under development. Bacteriophage therapy could offer one alternative strategy to mitigate this challenge. Although widely used throughout the 20th century in Eastern Europe and the former Soviet Union, this potential therapy has not yet been investigated according to rigorous scientific standards. This paper reports on a multistakeholder meeting held at the EMA, which outlined the existing regulatory framework to which such therapy should adhere and reviewed the current obstacles and shortcomings in scientific development for bacteriophage therapy. PMID:27068400

  15. Structure and function of bacteriophage T4

    PubMed Central

    Yap, Moh Lan; Rossmann, Michael G

    2014-01-01

    Bacteriophage T4 is the most well-studied member of Myoviridae, the most complex family of tailed phages. T4 assembly is divided into three independent pathways: the head, the tail and the long tail fibers. The prolate head encapsidates a 172 kbp concatemeric dsDNA genome. The 925 Å-long tail is surrounded by the contractile sheath and ends with a hexagonal baseplate. Six long tail fibers are attached to the baseplate’s periphery and are the host cell’s recognition sensors. The sheath and the baseplate undergo large conformational changes during infection. X-ray crystallography and cryo-electron microscopy have provided structural information on protein–protein and protein–nucleic acid interactions that regulate conformational changes during assembly and infection of Escherichia coli cells. PMID:25517898

  16. Bacteriophage lambda: Early pioneer and still relevant.

    PubMed

    Casjens, Sherwood R; Hendrix, Roger W

    2015-05-01

    Molecular genetic research on bacteriophage lambda carried out during its golden age from the mid-1950s to mid-1980s was critically important in the attainment of our current understanding of the sophisticated and complex mechanisms by which the expression of genes is controlled, of DNA virus assembly and of the molecular nature of lysogeny. The development of molecular cloning techniques, ironically instigated largely by phage lambda researchers, allowed many phage workers to switch their efforts to other biological systems. Nonetheless, since that time the ongoing study of lambda and its relatives has continued to give important new insights. In this review we give some relevant early history and describe recent developments in understanding the molecular biology of lambda's life cycle. PMID:25742714

  17. Bacteriophage lambda: early pioneer and still relevant

    PubMed Central

    Casjens, Sherwood R.; Hendrix, Roger W.

    2015-01-01

    Molecular genetic research on bacteriophage lambda carried out during its golden age from the mid 1950's to mid 1980's was critically important in the attainment of our current understanding of the sophisticated and complex mechanisms by which the expression of genes is controlled, of DNA virus assembly and of the molecular nature of lysogeny. The development of molecular cloning techniques, ironically instigated largely by phage lambda researchers, allowed many phage workers to switch their efforts to other biological systems. Nonetheless, since that time the ongoing study of lambda and its relatives have continued to give important new insights. In this review we give some relevant early history and describe recent developments in understanding the molecular biology of lambda's life cycle. PMID:25742714

  18. Bacteriophage therapy for safeguarding animal and human health: a review.

    PubMed

    Tiwari, Ruchi; Dhama, Kuldeep; Kumar, Amit; Rahal, Anu; Kapoor, Sanjay

    2014-02-01

    Since the discovery of bacteriophages at the beginning of the 19th century their contribution to bacterial evolution and ecology and use in a variety of applications in biotechnology and medicine has been recognized and understood. Bacteriophages are natural bacterial killers, proven as best biocontrol agents due to their ability to lyse host bacterial cells specifically thereby helping in disease prevention and control. The requirement of such therapeutic approach is straight away required in view of the global emergence of Multidrug Resistant (MDR) strains of bacteria and rapidly developing resistance to antibiotics in both animals and humans along with increasing food safety concerns including of residual antibiotic toxicities. Phage typing is a popular tool to differentiate bacterial isolates and to identify and characterize outbreak-associated strains of Salmonella, Campylobacter, Escherichia and Listeria. Numerous methods viz. plaque morphology, ultracentrifugation in the density gradient of CsCl2, and random amplified polymorphic DNA (RAPD) have been found to be effective in detection of various phages. Bacteriophages have been isolated and recovered from samples of animal waste products of different livestock farms. High titer cocktails of broad spectrum lytic bacteriophages are usually used for clinical trial for assessing their therapeutic efficacy against antibiotic unresponsive infections in different animals. Bacteriophage therapy also helps to fight various bacterial infections of poultry viz. colibacillosis, salmonellosis and listeriosis. Moreover, the utility of phages concerning biosafety has raised the importance to explore and popularize the therapeutic dimension of this promising novel therapy which forms the topic of discussion of the present review. PMID:24897784

  19. Bacteriophage endolysins as novel antimicrobials

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Endolysins are enzymes used by bacteriophages at the end of their replication cycle to degrade the peptidoglycan of the bacterial host from within, resulting in cell lysis and release of progeny virions. Due to the absence of an outer membrane in the Gram-positive bacterial cell wall, endolysins can...

  20. Bacteriophage therapy in animal production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Concerns over the consequences of bacterial resistance to antibiotics with the use of antibiotics in animal production have led to an increase in research on alternatives to antibiotics. Bacteriophages kill bacteria, are natural, safe, plentiful, self replicating, self limiting, can be used to spec...

  1. Bacteriophages and their implications on future biotechnology: a review

    PubMed Central

    2012-01-01

    Recently it has been recognized that bacteriophages, the natural predators of bacteria can be used efficiently in modern biotechnology. They have been proposed as alternatives to antibiotics for many antibiotic resistant bacterial strains. Phages can be used as biocontrol agents in agriculture and petroleum industry. Moreover phages are used as vehicles for vaccines both DNA and protein, for the detection of pathogenic bacterial strain, as display system for many proteins and antibodies. Bacteriophages are diverse group of viruses which are easily manipulated and therefore they have potential uses in biotechnology, research, and therapeutics. The aim of this review article is to enable the wide range of researchers, scientists, and biotechnologist who are putting phages into practice, to accelerate the progress and development in the field of biotechnology. PMID:22234269

  2. My Life with Bacteriophage φ29

    PubMed Central

    Salas, Margarita

    2012-01-01

    This article is a survey of my scientific work over 52 years. During my postdoctoral stay in Severo Ochoa's laboratory, I determined the direction of reading of the genetic message, and I discovered two proteins that I showed to be involved in the initiation of protein synthesis. The work I have done in Spain with bacteriophage φ29 for 45 years has been very rewarding. I can say that I was lucky because I did not expect that φ29 would give so many interesting results, but I worked hard, with a lot of dedication and enthusiasm, and I was there when the luck arrived. I would like to emphasize our work on the control of φ29 DNA transcription and, in particular, the finding for the first time of a protein covalently linked to the 5′-ends of φ29 DNA that we later showed to be the primer for the initiation of phage DNA replication. Very relevant was the discovery of the φ29 DNA polymerase, with its properties of extremely high processivity and strand displacement capacity, together with its high fidelity. The φ29 DNA polymerase has become an ideal enzyme for DNA amplification, both rolling-circle and whole-genome linear amplification. I am also very proud of the many brilliant students and collaborators with whom I have worked over the years and who have become excellent scientists. This Reflections article is not intended to be the end of my scientific career. I expect to work for many years to come. PMID:23124207

  3. Genome of Bacteriophage P1†

    PubMed Central

    Łobocka, Małgorzata B.; Rose, Debra J.; Plunkett, Guy; Rusin, Marek; Samojedny, Arkadiusz; Lehnherr, Hansjörg; Yarmolinsky, Michael B.; Blattner, Frederick R.

    2004-01-01

    P1 is a bacteriophage of Escherichia coli and other enteric bacteria. It lysogenizes its hosts as a circular, low-copy-number plasmid. We have determined the complete nucleotide sequences of two strains of a P1 thermoinducible mutant, P1 c1-100. The P1 genome (93,601 bp) contains at least 117 genes, of which almost two-thirds had not been sequenced previously and 49 have no homologs in other organisms. Protein-coding genes occupy 92% of the genome and are organized in 45 operons, of which four are decisive for the choice between lysis and lysogeny. Four others ensure plasmid maintenance. The majority of the remaining 37 operons are involved in lytic development. Seventeen operons are transcribed from σ70 promoters directly controlled by the master phage repressor C1. Late operons are transcribed from promoters recognized by the E. coli RNA polymerase holoenzyme in the presence of the Lpa protein, the product of a C1-controlled P1 gene. Three species of P1-encoded tRNAs provide differential controls of translation, and a P1-encoded DNA methyltransferase with putative bifunctionality influences transcription, replication, and DNA packaging. The genome is particularly rich in Chi recombinogenic sites. The base content and distribution in P1 DNA indicate that replication of P1 from its plasmid origin had more impact on the base compositional asymmetries of the P1 genome than replication from the lytic origin of replication. PMID:15489417

  4. Response of bacteriophage T7 biological dosimeter to dehydration and extraterrestrial solar UV radiation

    NASA Astrophysics Data System (ADS)

    Hegedüs, M.; Fekete, A.; Módos, K.; Kovács, G.; Rontó, Gy.; Lammer, H.; Panitz, C.

    2007-02-01

    The experiment "Phage and uracil response" (PUR) will be accommodated in the EXPOSE facility of the ISS. Bacteriophage T7/isolated T7 DNA will be exposed to different subsets of extreme environmental parameters in space, in order to study the Responses of Organisms to the Space Environment (ROSE). Launch into orbit is preceded by EXPOSE Experiment Verification Tests (EVT) to optimize the methods and the evaluation. Bacteriophage T7/isolated T7 DNA thin layers were exposed to vacuum ( 10-6Pa), to monochromatic (254 nm) and polychromatic (200-400 nm) UV radiation in air as well as in simulated space vacuum. Using neutral density (ND) filters dose-effect curves were performed in order to define the maximum doses tolerated. The effect of temperature fluctuation in vacuum was also studied. The structural/chemical effects on bacteriophage T7/isolated T7 DNA were analyzed by spectroscopic and microscopical methods. Characteristic changes in the absorption spectrum and in the electrophoretic pattern of phage/DNA have been detected indicating the damage of isolated and intraphage DNA. DNA damage was also determined by quantitative PCR (QPCR) using 555 and 3826 bp fragments of T7 DNA. We obtained substantial evidence that DNA lesions (e.g. strand breaks, DNA-protein cross-links, cyclobutane pirimidine dimers (CPDs) etc.) accumulate throughout exposure. Preliminary results suggest a synergistic action of space vacuum and UV radiation with DNA being the critical target.

  5. Functional eukaryotic nuclear localization signals are widespread in terminal proteins of bacteriophages

    PubMed Central

    Redrejo-Rodríguez, Modesto; Muñoz-Espín, Daniel; Holguera, Isabel; Mencía, Mario; Salas, Margarita

    2012-01-01

    A number of prokaryotic proteins have been shown to contain nuclear localization signals (NLSs), although its biological role remains sometimes unclear. Terminal proteins (TPs) of bacteriophages prime DNA replication and become covalently linked to the genome ends. We predicted NLSs within the TPs of bacteriophages from diverse families and hosts and, indeed, the TPs of Φ29, Nf, PRD1, Bam35, and Cp-1, out of seven TPs tested, were found to localize to the nucleus when expressed in mammalian cells. Detailed analysis of Φ29 TP led us to identify a bona fide NLS within residues 1–37. Importantly, gene delivery into the eukaryotic nucleus is enhanced by the presence of Φ29 TP attached to the 5′ DNA ends. These findings show a common feature of TPs from diverse bacteriophages targeting the eukaryotic nucleus and suggest a possible common function by facilitating the horizontal transfer of genes between prokaryotes and eukaryotes. PMID:23091024

  6. Bacteriophage Procurement for Therapeutic Purposes

    PubMed Central

    Weber-Dąbrowska, Beata; Jończyk-Matysiak, Ewa; Żaczek, Maciej; Łobocka, Małgorzata; Łusiak-Szelachowska, Marzanna; Górski, Andrzej

    2016-01-01

    Bacteriophages (phages), discovered 100 years ago, are able to infect and destroy only bacterial cells. In the current crisis of antibiotic efficacy, phage therapy is considered as a supplementary or even alternative therapeutic approach. Evolution of multidrug-resistant and pandrug-resistant bacterial strains poses a real threat, so it is extremely important to have the possibility to isolate new phages for therapeutic purposes. Our phage laboratory and therapy center has extensive experience with phage isolation, characterization, and therapeutic application. In this article we present current progress in bacteriophages isolation and use for therapeutic purposes, our experience in this field and its practical implications for phage therapy. We attempt to summarize the state of the art: properties of phages, the methods for their isolation, criteria of phage selection for therapeutic purposes and limitations of their use. Perspectives for the use of genetically engineered phages to specifically target bacterial virulence-associated genes are also briefly presented. PMID:27570518

  7. Bacteriophage Procurement for Therapeutic Purposes.

    PubMed

    Weber-Dąbrowska, Beata; Jończyk-Matysiak, Ewa; Żaczek, Maciej; Łobocka, Małgorzata; Łusiak-Szelachowska, Marzanna; Górski, Andrzej

    2016-01-01

    Bacteriophages (phages), discovered 100 years ago, are able to infect and destroy only bacterial cells. In the current crisis of antibiotic efficacy, phage therapy is considered as a supplementary or even alternative therapeutic approach. Evolution of multidrug-resistant and pandrug-resistant bacterial strains poses a real threat, so it is extremely important to have the possibility to isolate new phages for therapeutic purposes. Our phage laboratory and therapy center has extensive experience with phage isolation, characterization, and therapeutic application. In this article we present current progress in bacteriophages isolation and use for therapeutic purposes, our experience in this field and its practical implications for phage therapy. We attempt to summarize the state of the art: properties of phages, the methods for their isolation, criteria of phage selection for therapeutic purposes and limitations of their use. Perspectives for the use of genetically engineered phages to specifically target bacterial virulence-associated genes are also briefly presented. PMID:27570518

  8. Bacteriophage biocontrol in wastewater treatment.

    PubMed

    Jassim, Sabah A A; Limoges, Richard G; El-Cheikh, Hassan

    2016-04-01

    Waterborne bacterial pathogens in wastewater remains an important public health concern, not only because of the environmental damage, morbidity and mortality that they cause, but also due to the high cost of disinfecting wastewater by using physical and chemical methods in treatment plants. Bacteriophages are proposed as bacterial pathogen indicators and as an alternative biological method for wastewater treatment. Phage biocontrol in large scale treatment requires adaptive and aggressive phages that are able to overcome the environmental forces that interfere with phage-host interactions while targeting unwanted bacterial pathogens and preventing biofilms and foaming. This review will shed light on aspects of using bacteriophage programming technology in wastewater plants to rapidly target and reduce undesirable bacteria without harming the useful bacteria needed for biodegradation. PMID:26941243

  9. Novel Podoviridae Family Bacteriophage Infecting Weissella cibaria Isolated from Kimchi

    PubMed Central

    Holo, Helge; Jeon, Sang-Rok; Nes, Ingolf F.; Yoon, Sung-Sik

    2012-01-01

    The first complete genome sequence of a phage infecting Weissella cibaria (Weissella kimchii) is presented. The bacteriophage ϕYS61 was isolated from kimchi, a Korean fermented vegetable dish. Bacteriophages are recognized as a serious problem in industrial fermentations; however, ϕYS61 differed from many virulent phages associated with food fermentations since it was difficult to propagate and was very susceptible to resistance development. Sequence analysis revealed that ϕYS61 resembles Podoviridae of the subfamily Picovirinae. Within the subfamily Picovirinae, the ϕ29-like phages have been extensively studied, and their terminal protein-primed DNA replication is well characterized. Our data strongly suggest that ϕYS61 also replicates by a protein-primed mechanism. Weissella phage ϕYS61 is, however, markedly different from members of the Picovirinae with respect to genome size and morphology. Picovirinae are characterized by small (approximately 20-kb) genomes which contrasts with the 33,594-bp genome of ϕYS61. Based on electron microscopy analysis, ϕYS61 was classified as a member of the Podoviridae of morphotype C2, similar to the ϕ29-like phages, but its capsid dimensions are significantly larger than those reported for these phages. The novelty of ϕYS61 was also emphasized by the low number of open reading frames (ORFs) showing significant similarity to database sequences. We propose that the bacteriophage ϕYS61 should represent a new subfamily within the family Podoviridae. PMID:22885743

  10. Novel bacteriophages containing a genome of another bacteriophage within their genomes.

    PubMed

    Swanson, Maud M; Reavy, Brian; Makarova, Kira S; Cock, Peter J; Hopkins, David W; Torrance, Lesley; Koonin, Eugene V; Taliansky, Michael

    2012-01-01

    A novel bacteriophage infecting Staphylococus pasteuri was isolated during a screen for phages in Antarctic soils. The phage named SpaA1 is morphologically similar to phages of the family Siphoviridae. The 42,784 bp genome of SpaA1 is a linear, double-stranded DNA molecule with 3' protruding cohesive ends. The SpaA1 genome encompasses 63 predicted protein-coding genes which cluster within three regions of the genome, each of apparently different origin, in a mosaic pattern. In two of these regions, the gene sets resemble those in prophages of Bacillus thuringiensis kurstaki str. T03a001 (genes involved in DNA replication/transcription, cell entry and exit) and B. cereus AH676 (additional regulatory and recombination genes), respectively. The third region represents an almost complete genome (except for the short terminal segments) of a distinct bacteriophage, MZTP02. Nearly the same gene module was identified in prophages of B. thuringiensis serovar monterrey BGSC 4AJ1 and B. cereus Rock4-2. These findings suggest that MZTP02 can be shuttled between genomes of other bacteriophages and prophages, leading to the formation of chimeric genomes. The presence of a complete phage genome in the genome of other phages apparently has not been described previously and might represent a 'fast track' route of virus evolution and horizontal gene transfer. Another phage (BceA1) nearly identical in sequence to SpaA1, and also including the almost complete MZTP02 genome within its own genome, was isolated from a bacterium of the B. cereus/B. thuringiensis group. Remarkably, both SpaA1 and BceA1 phages can infect B. cereus and B. thuringiensis, but only one of them, SpaA1, can infect S. pasteuri. This finding is best compatible with a scenario in which MZTP02 was originally contained in BceA1 infecting Bacillus spp, the common hosts for these two phages, followed by emergence of SpaA1 infecting S. pasteuri. PMID:22815791