Science.gov

Sample records for bacterial dna viruses

  1. A structural DNA binding protein of African swine fever virus with similarity to bacterial histone-like proteins.

    PubMed

    Borca, M V; Irusta, P M; Kutish, G F; Carillo, C; Afonso, C L; Burrage, A T; Neilan, J G; Rock, D L

    1996-01-01

    Here we describe an African swine fever virus (ASFV) protein encoded by the open reading frame 5-AR that shares structural and functional similarities with the family of bacterial histone-like proteins which include histone-like DNA binding proteins, integration host factor, and Bacillus phage SPO1 transcription factor, TF1. The ASFV 5-AR gene was cloned by PCR and expressed in E. coli. Monospecific antiserum prepared to the 5-AR bacterial expression product specifically immunoprecipitated a protein of approximately 11.6 kDa from ASFV infected swine macrophages at late times post infection. Additionally, the 5-AR expression product was strongly recognized by ASFV convalescent pig serum, indicating its antigenicity during natural infection. Cloned p11.6 bound both double and single stranded DNA-cellulose columns. Consistent with a DNA binding function, immunoelectronmicroscopy localized p11.6 to the virion nucleoid, To our knowledge, p11.6 is the first bacterial histone-like DNA-binding protein found in an animal virus or eukaryotic cell system. PMID:8634022

  2. Cloning human herpes virus 6A genome into bacterial artificial chromosomes and study of DNA replication intermediates

    PubMed Central

    Borenstein, Ronen; Frenkel, Niza

    2009-01-01

    Cloning of large viral genomes into bacterial artificial chromosomes (BACs) facilitates analyses of viral functions and molecular mutagenesis. Previous derivations of viral BACs involved laborious recombinations within infected cells. We describe a single-step production of viral BACs by direct cloning of unit length genomes, derived from circular or head-to-tail concatemeric DNA replication intermediates. The BAC cloning is independent of intracellular recombinations and DNA packaging constraints. We introduced the 160-kb human herpes virus 6A (HHV-6A) genome into BACs by digesting the viral DNA replicative intermediates with the Sfil enzyme that cleaves the viral genome in a single site. The recombinant BACs contained also the puromycin selection gene, GFP, and LoxP sites flanking the BAC sequences. The HHV-6A-BAC vectors were retained stably in puromycin selected 293T cells. In the presence of irradiated helper virus, supplying most likely proteins enhancing gene expression they expressed early and late genes in SupT1 T cells. The method is especially attractive for viruses that replicate inefficiently and for viruses propagated in suspension cells. We have used the fact that the BAC cloning “freezes” the viral DNA replication intermediates to analyze their structure. The results revealed that HHV-6A-BACs contained a single direct repeat (DR) rather than a DR-DR sequence, predicted to arise by circularization of parental genomes with a DR at each terminus. HHV-6A DNA molecules prepared from the infected cells also contained DNA molecules with a single DR. Such forms were not previously described for HHV-6 DNA. PMID:19858479

  3. 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

  4. Ribonucleotides in Bacterial DNA

    PubMed Central

    Schroeder, Jeremy W.; Randall, Justin R.; Matthews, Lindsay A.; Simmons, Lyle A.

    2014-01-01

    In all living cells, DNA is the storage medium for genetic information. Being quite stable, DNA is well-suited for its role in storage and propagation of information, but RNA is also covalently included in DNA through various mechanisms. Recent studies also demonstrate useful aspects of including ribonucleotides in the genome during repair. Therefore, our understanding of the consequences of RNA inclusion into bacterial genomic DNA is just beginning, but with its high frequency of occurrence the consequences and potential benefits are likely to be numerous and diverse. In this review, we discuss the processes that cause ribonucleotide inclusion in genomic DNA, the pathways important for ribonucleotide removal and the consequences that arise should ribonucleotides remain nested in genomic DNA. PMID:25387798

  5. Novel ssDNA virus recovered from estuarine Mollusc (Amphibola crenata) whose replication associated protein (Rep) shares similarities with Rep-like sequences of bacterial origin.

    PubMed

    Dayaram, Anisha; Goldstien, Sharyn; Zawar-Reza, Peyman; Gomez, Christopher; Harding, Jon S; Varsani, Arvind

    2013-05-01

    Over the past couple of years highly diverse novel ssDNA viruses have been discovered. Here, we present the first ssDNA virus, Gastropod-associated circular ssDNA virus (GaCSV), recovered from a mollusc Amphibola crenata Martyn 1784, which is a deposit feeder that grazes micro-organisms and organic detritus on the surface of tidal mudflats. The GaCSV (2351 nt) genome contains two large bidirectionally transcribed ORFs. The smaller ORF (874 nt) has similarities to viral replication-associated protein (Rep) sequences of some bacteria and circoviruses, whereas the larger ORF (955 nt) does not relate to any sequences in public databases and we presume it potentially encodes the capsid protein. Phylogenetic analysis shows that the GaCSV Rep clusters with Rep-like sequences of bacterial origin, highlighting the role of ssDNA viruses in horizontal gene transfer. The occurrence of previously unknown viruses in organisms associated with human pollution is a relatively unexplored field. PMID:23364192

  6. Tumorigenic DNA viruses

    SciTech Connect

    Klein, G.

    1989-01-01

    The eighth volume of Advances in Viral Oncology focuses on the three major DNA virus groups with a postulated or proven tumorigenic potential: papillomaviruses, animal hepatitis viruses, and the Epstein-Bar virus. In the opening chapters, the contributors analyze the evidence that papillomaviruses and animal hepatitis viruses are involved in tumorigenesis and describe the mechanisms that trigger virus-host cell interactions. A detailed section on the Epstein-Barr virus (EBV) - comprising more than half the book - examines the transcription and mRNA processing patterns of the virus genome; the mechanisms by which EBV infects lymphoid and epithelial cells; the immunological aspects of the virus; the actions of EBV in hosts with Acquired Immune Deficiency Syndrome; and the involvement of EBV in the etiology of Burkitt's lymphoma.

  7. Isolation and Characterization of Bacterial DNA.

    ERIC Educational Resources Information Center

    Wilson, W. David; Davidson, Michael W.

    1979-01-01

    An inexpensive DNA preparation is presented which starts with commercially available frozen packed bacterial cells. Methods for analyzing the DNA are also presented, along with physical chemical experiments which can be done using the purified DNA. (BB)

  8. Structure of a Bacterial Virus DNA-Injection Protein Complex Reveals a Decameric Assembly with a Constricted Molecular Channel.

    PubMed

    Zhao, Haiyan; Speir, Jeffrey A; Matsui, Tsutomu; Lin, Zihan; Liang, Lingfei; Lynn, Anna Y; Varnado, Brittany; Weiss, Thomas M; Tang, Liang

    2016-01-01

    The multi-layered cell envelope structure of Gram-negative bacteria represents significant physical and chemical barriers for short-tailed phages to inject phage DNA into the host cytoplasm. Here we show that a DNA-injection protein of bacteriophage Sf6, gp12, forms a 465-kDa, decameric assembly in vitro. The electron microscopic structure of the gp12 assembly shows a ~150-Å, mushroom-like architecture consisting of a crown domain and a tube-like domain, which embraces a 25-Å-wide channel that could precisely accommodate dsDNA. The constricted channel suggests that gp12 mediates rapid, uni-directional injection of phage DNA into host cells by providing a molecular conduit for DNA translocation. The assembly exhibits a 10-fold symmetry, which may be a common feature among DNA-injection proteins of P22-like phages and may suggest a symmetry mismatch with respect to the 6-fold symmetric phage tail. The gp12 monomer is highly flexible in solution, supporting a mechanism for translocation of the protein through the conduit of the phage tail toward the host cell envelope, where it assembles into a DNA-injection device. PMID:26882199

  9. Structure of a bacterial virus DNA-injection protein complex reveals a decameric assembly with a constricted molecular channel

    DOE PAGESBeta

    Zhao, Haiyan; Speir, Jeffrey A.; Matsui, Tsutomu; Lin, Zihan; Liang, Lingfei; Lynn, Anna Y.; Varnado, Brittany; Weiss, Thomas M.; Tang, Liang; Schuch, Raymond

    2016-02-16

    The multi-layered cell envelope structure of Gram-negative bacteria represents significant physical and chemical barriers for short-tailed phages to inject phage DNA into the host cytoplasm. Here we show that a DNA-injection protein of bacteriophage Sf6, gp12, forms a 465-kDa, decameric assembly in vitro. The electron microscopic structure of the gp12 assembly shows a ~150-Å, mushroom-like architecture consisting of a crown domain and a tube-like domain, which embraces a 25-Å-wide channel that could precisely accommodate dsDNA. The constricted channel suggests that gp12 mediates rapid, uni-directional injection of phage DNA into host cells by providing a molecular conduit for DNA translocation. Themore » assembly exhibits a 10-fold symmetry, which may be a common feature among DNA-injection proteins of P22-like phages and may suggest a symmetry mismatch with respect to the 6-fold symmetric phage tail. As a result, the gp12 monomer is highly flexible in solution, supporting a mechanism for translocation of the protein through the conduit of the phage tail toward the host cell envelope, where it assembles into a DNA-injection device.« less

  10. Structure of a Bacterial Virus DNA-Injection Protein Complex Reveals a Decameric Assembly with a Constricted Molecular Channel

    PubMed Central

    Zhao, Haiyan; Speir, Jeffrey A.; Matsui, Tsutomu; Lin, Zihan; Liang, Lingfei; Lynn, Anna Y.; Varnado, Brittany; Weiss, Thomas M.; Tang, Liang

    2016-01-01

    The multi-layered cell envelope structure of Gram-negative bacteria represents significant physical and chemical barriers for short-tailed phages to inject phage DNA into the host cytoplasm. Here we show that a DNA-injection protein of bacteriophage Sf6, gp12, forms a 465-kDa, decameric assembly in vitro. The electron microscopic structure of the gp12 assembly shows a ~150-Å, mushroom-like architecture consisting of a crown domain and a tube-like domain, which embraces a 25-Å-wide channel that could precisely accommodate dsDNA. The constricted channel suggests that gp12 mediates rapid, uni-directional injection of phage DNA into host cells by providing a molecular conduit for DNA translocation. The assembly exhibits a 10-fold symmetry, which may be a common feature among DNA-injection proteins of P22-like phages and may suggest a symmetry mismatch with respect to the 6-fold symmetric phage tail. The gp12 monomer is highly flexible in solution, supporting a mechanism for translocation of the protein through the conduit of the phage tail toward the host cell envelope, where it assembles into a DNA-injection device. PMID:26882199

  11. Nuclear entry of DNA viruses

    PubMed Central

    Fay, Nikta; Panté, Nelly

    2015-01-01

    DNA viruses undertake their replication within the cell nucleus, and therefore they must first deliver their genome into the nucleus of their host cells. Thus, trafficking across the nuclear envelope is at the basis of DNA virus infections. Nuclear transport of molecules with diameters up to 39 nm is a tightly regulated process that occurs through the nuclear pore complex (NPC). Due to the enormous diversity of virus size and structure, each virus has developed its own strategy for entering the nucleus of their host cells, with no two strategies alike. For example, baculoviruses target their DNA-containing capsid to the NPC and subsequently enter the nucleus intact, while the hepatitis B virus capsid crosses the NPC but disassembles at the nuclear side of the NPC. For other viruses such as herpes simplex virus and adenovirus, although both dock at the NPC, they have each developed a distinct mechanism for the subsequent delivery of their genome into the nucleus. Remarkably, other DNA viruses, such as parvoviruses and human papillomaviruses, access the nucleus through an NPC-independent mechanism. This review discusses our current understanding of the mechanisms used by DNA viruses to deliver their genome into the nucleus, and further presents the experimental evidence for such mechanisms. PMID:26029198

  12. Immobilization of plasmid DNA in bacterial ghosts.

    PubMed

    Mayrhofer, Peter; Tabrizi, Chakameh Azimpour; Walcher, Petra; Haidinger, Wolfgang; Jechlinger, Wolfgang; Lubitz, Werner

    2005-02-16

    The development of novel delivery vehicles is crucial for the improvement of DNA vaccine efficiency. In this report, we describe a new platform technology, which is based on the immobilization of plasmid DNA in the cytoplasmic membrane of a bacterial carrier. This technology retains plasmid DNA (Self-Immobilizing Plasmid, pSIP) in the host envelope complex due to a specific protein/DNA interaction during and after protein E-mediated lysis. The resulting bacterial ghosts (empty bacterial envelopes) loaded with pDNA were analyzed in detail by real time PCR assays. We could verify that pSIP plasmids were retained in the pellets of lysed Escherichia coli cultures indicating that they are efficiently anchored in the inner membrane of bacterial ghosts. In contrast, a high percentage of control plasmids that lack essential features of the self-immobilization system were expelled in the culture broth during the lysis process. We believe that the combination of this plasmid immobilization procedure and the protein E-mediated lysis technology represents an efficient in vivo technique for the production of non-living DNA carrier vehicles. In conclusion, we present a "self-loading", non-living bacterial DNA delivery vector for vaccination endowed with intrinsic adjuvant properties of the Gram-negative bacterial cell envelope. PMID:15681093

  13. DNA supercoiling and bacterial gene expression.

    PubMed

    Dorman, Charles J

    2006-01-01

    DNA in bacterial cells is maintained in a negatively supercoiled state. This contributes to the organization of the bacterial nucleoid and also influences the global gene expression pattern in the cell through modulatory effects on transcription. Supercoiling arises as a result of changes to the linking number of the relaxed double-stranded DNA molecule and is set and reset by the action of DNA topoisomerases. This process is subject to a multitude of influences that are usually summarized as environmental stress. Responsiveness of linking number change to stress offers the promise of a mechanism for the wholesale adjustment of the transcription programme of the cell as the bacterium experiences different environments. Recent data from DNA microarray experiments support this proposition. The emerging picture is one of DNA supercoiling acting at or near the apex of a regulatory hierarchy where it collaborates with nucleoid-associated proteins and transcription factors to determine the gene expression profile of the cell. PMID:17338437

  14. Translocation of DNA across bacterial membranes.

    PubMed Central

    Dreiseikelmann, B

    1994-01-01

    DNA translocation across bacterial membranes occurs during the biological processes of infection by bacteriophages, conjugative DNA transfer of plasmids, T-DNA transfer, and genetic transformation. The mechanism of DNA translocation in these systems is not fully understood, but during the last few years extensive data about genes and gene products involved in the translocation processes have accumulated. One reason for the increasing interest in this topic is the discussion about horizontal gene transfer and transkingdom sex. Analyses of genes and gene products involved in DNA transfer suggest that DNA is transferred through a protein channel spanning the bacterial envelope. No common model exists for DNA translocation during phage infection. Perhaps various mechanisms are necessary as a result of the different morphologies of bacteriophages. The DNA translocation processes during conjugation, T-DNA transfer, and transformation are more consistent and may even be compared to the excretion of some proteins. On the basis of analogies and homologies between the proteins involved in DNA translocation and protein secretion, a common basic model for these processes is presented. PMID:7968916

  15. A DNA Virus of Drosophila

    PubMed Central

    Unckless, Robert L.

    2011-01-01

    Little is known about the viruses infecting most species. Even in groups as well-studied as Drosophila, only a handful of viruses have been well-characterized. A viral metagenomic approach was used to explore viral diversity in 83 wild-caught Drosophila innubila, a mushroom feeding member of the quinaria group. A single fly that was injected with, and died from, Drosophila C Virus (DCV) was added to the sample as a control. Two-thirds of reads in the infected sample had DCV as the best BLAST hit, suggesting that the protocol developed is highly sensitive. In addition to the DCV hits, several sequences had Oryctes rhinoceros Nudivirus, a double-stranded DNA virus, as a best BLAST hit. The virus associated with these sequences was termed Drosophila innubila Nudivirus (DiNV). PCR screens of natural populations showed that DiNV was both common and widespread taxonomically and geographically. Electron microscopy confirms the presence of virions in fly fecal material similar in structure to other described Nudiviruses. In 2 species, D. innubila and D. falleni, the virus is associated with a severe (∼80–90%) loss of fecundity and significantly decreased lifespan. PMID:22053195

  16. Comparing Bacterial DNA Microarray Fingerprints

    SciTech Connect

    Willse, Alan R.; Chandler, Darrell P.; White, Amanda M.; Protic, Miroslava; Daly, Don S.; Wunschel, Sharon C.

    2005-08-15

    Detecting subtle genetic differences between microorganisms is an important problem in molecular epidemiology and microbial forensics. In a typical investigation, gel electrophoresis is used to compare randomly amplified DNA fragments between microbial strains, where the patterns of DNA fragment sizes are proxies for a microbe's genotype. The limited genomic sample captured on a gel is often insufficient to discriminate nearly identical strains. This paper examines the application of microarray technology to DNA fingerprinting as a high-resolution alternative to gel-based methods. The so-called universal microarray, which uses short oligonucleotide probes that do not target specific genes or species, is intended to be applicable to all microorganisms because it does not require prior knowledge of genomic sequence. In principle, closely related strains can be distinguished if the number of probes on the microarray is sufficiently large, i.e., if the genome is sufficiently sampled. In practice, we confront noisy data, imperfectly matched hybridizations, and a high-dimensional inference problem. We describe the statistical problems of microarray fingerprinting, outline similarities with and differences from more conventional microarray applications, and illustrate the statistical fingerprinting problem for 10 closely related strains from three Bacillus species, and 3 strains from non-Bacillus species.

  17. Cloning of a very virulent plus, 686 strain of Marek’s disease virus as a bacterial artificial chromosome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial artificial chromosome (BAC) vectors were first developed to facilitate propagation and manipulation of large DNA fragments. This technology was later used to clone full-length genomes of large DNA viruses to study viral gene function. Marek’s disease virus (MDV) is a highly oncogenic herpe...

  18. Recombination in Eukaryotic Single Stranded DNA Viruses

    PubMed Central

    Martin, Darren P.; Biagini, Philippe; Lefeuvre, Pierre; Golden, Michael; Roumagnac, Philippe; Varsani, Arvind

    2011-01-01

    Although single stranded (ss) DNA viruses that infect humans and their domesticated animals do not generally cause major diseases, the arthropod borne ssDNA viruses of plants do, and as a result seriously constrain food production in most temperate regions of the world. Besides the well known plant and animal-infecting ssDNA viruses, it has recently become apparent through metagenomic surveys of ssDNA molecules that there also exist large numbers of other diverse ssDNA viruses within almost all terrestrial and aquatic environments. The host ranges of these viruses probably span the tree of life and they are likely to be important components of global ecosystems. Various lines of evidence suggest that a pivotal evolutionary process during the generation of this global ssDNA virus diversity has probably been genetic recombination. High rates of homologous recombination, non-homologous recombination and genome component reassortment are known to occur within and between various different ssDNA virus species and we look here at the various roles that these different types of recombination may play, both in the day-to-day biology, and in the longer term evolution, of these viruses. We specifically focus on the ecological, biochemical and selective factors underlying patterns of genetic exchange detectable amongst the ssDNA viruses and discuss how these should all be considered when assessing the adaptive value of recombination during ssDNA virus evolution. PMID:21994803

  19. Does Blood of Healthy Subjects Contain Bacterial Ribosomal DNA?

    PubMed Central

    Nikkari, Simo; McLaughlin, Ian J.; Bi, Wanli; Dodge, Deborah E.; Relman, David A.

    2001-01-01

    Real-time PCR methods with primers and a probe targeting conserved regions of the bacterial 16S ribosomal DNA (rDNA) revealed a larger amount of rDNA in blood specimens from healthy individuals than in matched reagent controls. However, the origins and identities of these blood-associated bacterial rDNA sequences remain obscure. PMID:11326021

  20. Bacterial Respiratory Infections Complicating Human Immunodeficiency Virus.

    PubMed

    Feldman, Charles; Anderson, Ronald

    2016-04-01

    Opportunistic bacterial and fungal infections of the lower respiratory tract, most commonly those caused by Streptococcus pneumoniae (the pneumococcus), Mycobacterium tuberculosis, and Pneumocystis jirovecii, remain the major causes of mortality in those infected with human immunodeficiency virus (HIV). Bacterial respiratory pathogens most prevalent in those infected with HIV, other than M. tuberculosis, represent the primary focus of the current review with particular emphasis on the pneumococcus, the leading cause of mortality due to HIV infection in the developed world. Additional themes include (1) risk factors; (2) the predisposing effects of HIV-mediated suppression on pulmonary host defenses, possibly intensified by smoking; (3) clinical and laboratory diagnosis, encompassing assessment of disease severity and outcome; and (4) antibiotic therapy. The final section addresses current recommendations with respect to pneumococcal immunization in the context of HIV infection, including an overview of the rationale underpinning the current "prime-boost" immunization strategy based on sequential administration of pneumococcal conjugate vaccine 13 and pneumococcal polysaccharide vaccine 23. PMID:26974299

  1. Structure of large dsDNA viruses

    PubMed Central

    Klose, Thomas; Rossmann, Michael G.

    2015-01-01

    Nucleocytoplasmic large dsDNA viruses (NCLDVs) encompass an ever-increasing group of large eukaryotic viruses, infecting a wide variety of organisms. The set of core genes shared by all these viruses includes a major capsid protein with a double jelly-roll fold forming an icosahedral capsid, which surrounds a double layer membrane that contains the viral genome. Furthermore, some of these viruses, such as the members of the Mimiviridae and Phycodnaviridae have a unique vertex that is used during infection to transport DNA into the host. PMID:25003382

  2. Virulent Marek's Disease Virus Generated from Infectious Bacterial Artificial Chromosome Clones with Complete DNA Sequence and Implication of Viral Genetic Homogeniety in Pathogenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic homogeneity of a test population is essential to precisely associate a viral genome sequence and its phenotype at the nucleotide level. However, homogeneity is not easy to achieve for Marek’s disease virus (MDV) due to its strictly cell-associated replication. To address this problem, two vi...

  3. Bacterial DNA findings in ruptured and unruptured intracranial aneurysms.

    PubMed

    Pyysalo, Mikko J; Pyysalo, Liisa M; Pessi, Tanja; Karhunen, Pekka J; Lehtimäki, Terho; Oksala, Niku; Öhman, Juha E

    2016-05-01

    Objective Chronic inflammation has earlier been detected in ruptured intracranial aneurysms. A previous study detected both dental bacterial DNA and bacterial-driven inflammation in ruptured intracranial aneurysm walls. The aim of this study was to compare the presence of oral and pharyngeal bacterial DNA in ruptured and unruptured intracranial aneurysms. The hypothesis was that oral bacterial DNA findings would be more common and the amount of bacterial DNA would be higher in ruptured aneurysm walls than in unruptured aneurysm walls. Materials and methods A total of 70 ruptured (n = 42) and unruptured (n = 28) intracranial aneurysm specimens were obtained perioperatively in aneurysm clipping operations. Aneurysmal sac tissue was analysed using a real-time quantitative polymerase chain reaction to detect bacterial DNA from several oral species. Both histologically non-atherosclerotic healthy vessel wall obtained from cardiac by-pass operations (LITA) and arterial blood samples obtained from each aneurysm patient were used as control samples. Results Bacterial DNA was detected in 49/70 (70%) of the specimens. A total of 29/42 (69%) of the ruptured and 20/28 (71%) of the unruptured aneurysm samples contained bacterial DNA of oral origin. Both ruptured and unruptured aneurysm tissue samples contained significantly more bacterial DNA than the LITA control samples (p-values 0.003 and 0.001, respectively). There was no significant difference in the amount of bacterial DNA between the ruptured and unruptured samples. Conclusion Dental bacterial DNA can be found using a quantitative polymerase chain reaction in both ruptured and unruptured aneurysm walls, suggesting that bacterial DNA plays a role in the pathogenesis of cerebral aneurysms in general, rather than only in ruptured aneurysms. PMID:26777430

  4. Temperate bacterial viruses as double-edged swords in bacterial warfare.

    PubMed

    Gama, João Alves; Reis, Ana Maria; Domingues, Iolanda; Mendes-Soares, Helena; Matos, Ana Margarida; Dionisio, Francisco

    2013-01-01

    It has been argued that bacterial cells may use their temperate viruses as biological weapons. For instance, a few bacterial cells among a population of lysogenic cells could release the virus and kill susceptible non-lysogenic competitors, while their clone mates would be immune. Because viruses replicate inside their victims upon infection, this process would amplify their number in the arena. Sometimes, however, temperate viruses spare recipient cells from death by establishing themselves in a dormant state inside cells. This phenomenon is called lysogenization and, for some viruses such as the λ virus, the probability of lysogenization increases with the multiplicity of infection. Therefore, the amplification of viruses leads to conflicting predictions about the efficacy of temperate viruses as biological weapons: amplification can increase the relative advantage of clone mates of lysogens but also the likelihood of saving susceptible cells from death, because the probability of lysogenization is higher. To test the usefulness of viruses as biological weapons, we performed competition experiments between lysogenic Escherichia coli cells carrying the λ virus and susceptible λ-free E. coli cells, either in a structured or unstructured habitat. In structured and sometimes in unstructured habitats, the λ virus qualitatively behaved as a "replicating toxin". However, such toxic effect of λ viruses ceased after a few days of competition. This was due to the fact that many of initially susceptible cells became lysogenic. Massive lysogenization of susceptible cells occurred precisely under the conditions where the amplification of the virus was substantial. From then on, these cells and their descendants became immune to the λ virus. In conclusion, if at short term bacterial cells may use temperate viruses as biological weapons, after a few days only the classical view of temperate bacterial viruses as parasitic agents prevails. PMID:23536852

  5. Temperate Bacterial Viruses as Double-Edged Swords in Bacterial Warfare

    PubMed Central

    Gama, João Alves; Reis, Ana Maria; Domingues, Iolanda; Mendes-Soares, Helena; Matos, Ana Margarida; Dionisio, Francisco

    2013-01-01

    It has been argued that bacterial cells may use their temperate viruses as biological weapons. For instance, a few bacterial cells among a population of lysogenic cells could release the virus and kill susceptible non-lysogenic competitors, while their clone mates would be immune. Because viruses replicate inside their victims upon infection, this process would amplify their number in the arena. Sometimes, however, temperate viruses spare recipient cells from death by establishing themselves in a dormant state inside cells. This phenomenon is called lysogenization and, for some viruses such as the λ virus, the probability of lysogenization increases with the multiplicity of infection. Therefore, the amplification of viruses leads to conflicting predictions about the efficacy of temperate viruses as biological weapons: amplification can increase the relative advantage of clone mates of lysogens but also the likelihood of saving susceptible cells from death, because the probability of lysogenization is higher. To test the usefulness of viruses as biological weapons, we performed competition experiments between lysogenic Escherichia coli cells carrying the λ virus and susceptible λ-free E. coli cells, either in a structured or unstructured habitat. In structured and sometimes in unstructured habitats, the λ virus qualitatively behaved as a “replicating toxin”. However, such toxic effect of λ viruses ceased after a few days of competition. This was due to the fact that many of initially susceptible cells became lysogenic. Massive lysogenization of susceptible cells occurred precisely under the conditions where the amplification of the virus was substantial. From then on, these cells and their descendants became immune to the λ virus. In conclusion, if at short term bacterial cells may use temperate viruses as biological weapons, after a few days only the classical view of temperate bacterial viruses as parasitic agents prevails. PMID:23536852

  6. Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

    PubMed Central

    Yun, Sang-Im; Song, Byung-Hak; Kim, Jin-Kyoung; Lee, Young-Min

    2015-01-01

    Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of >10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase. PMID:26780115

  7. Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses.

    PubMed

    Yun, Sang-Im; Song, Byung-Hak; Kim, Jin-Kyoung; Lee, Young-Min

    2015-01-01

    Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of >10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase. PMID:26780115

  8. The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes

    PubMed Central

    Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

    2016-01-01

    Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity. We identified and analyzed all major functional groups of DNA replication proteins in all available proteomes of dsDNA viruses. Our results show that some proteins are common to viruses infecting all domains of life and likely represent components of the ancestral core set. These include B-family polymerases, SF3 helicases, archaeo-eukaryotic primases, clamps and clamp loaders of the archaeo-eukaryotic type, RNase H and ATP-dependent DNA ligases. We also discovered a clear correlation between genome size and self-sufficiency of viral DNA replication, the unanticipated dominance of replicative helicases and pervasive functional associations among certain groups of DNA replication proteins. Altogether, our results provide a comprehensive view on the diversity and evolution of replication systems in the DNA virome and uncover fundamental principles underlying the orchestration of viral DNA replication. PMID:27112572

  9. The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes.

    PubMed

    Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

    2016-06-01

    Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity. We identified and analyzed all major functional groups of DNA replication proteins in all available proteomes of dsDNA viruses. Our results show that some proteins are common to viruses infecting all domains of life and likely represent components of the ancestral core set. These include B-family polymerases, SF3 helicases, archaeo-eukaryotic primases, clamps and clamp loaders of the archaeo-eukaryotic type, RNase H and ATP-dependent DNA ligases. We also discovered a clear correlation between genome size and self-sufficiency of viral DNA replication, the unanticipated dominance of replicative helicases and pervasive functional associations among certain groups of DNA replication proteins. Altogether, our results provide a comprehensive view on the diversity and evolution of replication systems in the DNA virome and uncover fundamental principles underlying the orchestration of viral DNA replication. PMID:27112572

  10. DNA Tumor Viruses and Cell Metabolism

    PubMed Central

    Mushtaq, Muhammad; Darekar, Suhas

    2016-01-01

    Viruses play an important role in cancerogenesis. It is estimated that approximately 20% of all cancers are linked to infectious agents. The viral genes modulate the physiological machinery of infected cells that lead to cell transformation and development of cancer. One of the important adoptive responses by the cancer cells is their metabolic change to cope up with continuous requirement of cell survival and proliferation. In this review we will focus on how DNA viruses alter the glucose metabolism of transformed cells. Tumor DNA viruses enhance “aerobic” glycolysis upon virus-induced cell transformation, supporting rapid cell proliferation and showing the Warburg effect. Moreover, viral proteins enhance glucose uptake and controls tumor microenvironment, promoting metastasizing of the tumor cells. PMID:27034740

  11. Crystal Structure of a Bacterial Type IB DNA Topoisomerase Reveals a Preassembled Active Site in the Absence of DNA

    SciTech Connect

    Patel, Asmita; Shuman, Stewart; Mondragon, Alfonso

    2010-03-08

    Type IB DNA topoisomerases are found in all eukarya, two families of eukaryotic viruses (poxviruses and mimivirus), and many genera of bacteria. They alter DNA topology by cleaving and resealing one strand of duplex DNA via a covalent DNA-(3-phosphotyrosyl)-enzyme intermediate. Bacterial type IB enzymes were discovered recently and are described as poxvirus-like with respect to their small size, primary structures, and bipartite domain organization. Here we report the 1.75-{angstrom} crystal structure of Deinococcus radiodurans topoisomerase IB (DraTopIB), a prototype of the bacterial clade. DraTopIB consists of an amino-terminal (N) {beta}-sheet domain (amino acids 1-90) and a predominantly {alpha}-helical carboxyl-terminal (C) domain (amino acids 91-346) that closely resemble the corresponding domains of vaccinia virus topoisomerase IB. The five amino acids of DraTopIB that comprise the catalytic pentad (Arg-137, Lys-174, Arg-239, Asn-280, and Tyr-289) are preassembled into the active site in the absence of DNA in a manner nearly identical to the pentad configuration in human topoisomerase I bound to DNA. This contrasts with the apoenzyme of vaccinia topoisomerase, in which three of the active site constituents are either displaced or disordered. The N and C domains of DraTopIB are splayed apart in an 'open' conformation, in which the surface of the catalytic domain containing the active site is exposed for DNA binding. A comparison with the human topoisomerase I-DNA cocrystal structure suggests how viral and bacterial topoisomerase IB enzymes might bind DNA circumferentially via movement of the N domain into the major groove and clamping of a disordered loop of the C domain around the helix.

  12. The Autonomous Glycosylation of Large DNA Viruses.

    PubMed

    Piacente, Francesco; Gaglianone, Matteo; Laugieri, Maria Elena; Tonetti, Michela G

    2015-01-01

    Glycosylation of surface molecules is a key feature of several eukaryotic viruses, which use the host endoplasmic reticulum/Golgi apparatus to add carbohydrates to their nascent glycoproteins. In recent years, a newly discovered group of eukaryotic viruses, belonging to the Nucleo-Cytoplasmic Large DNA Virus (NCLDV) group, was shown to have several features that are typical of cellular organisms, including the presence of components of the glycosylation machinery. Starting from initial observations with the chlorovirus PBCV-1, enzymes for glycan biosynthesis have been later identified in other viruses; in particular in members of the Mimiviridae family. They include both the glycosyltransferases and other carbohydrate-modifying enzymes and the pathways for the biosynthesis of the rare monosaccharides that are found in the viral glycan structures. These findings, together with genome analysis of the newly-identified giant DNA viruses, indicate that the presence of glycogenes is widespread in several NCLDV families. The identification of autonomous viral glycosylation machinery leads to many questions about the origin of these pathways, the mechanisms of glycan production, and eventually their function in the viral replication cycle. The scope of this review is to highlight some of the recent results that have been obtained on the glycosylation systems of the large DNA viruses, with a special focus on the enzymes involved in nucleotide-sugar production. PMID:26690138

  13. The Autonomous Glycosylation of Large DNA Viruses

    PubMed Central

    Piacente, Francesco; Gaglianone, Matteo; Laugieri, Maria Elena; Tonetti, Michela G.

    2015-01-01

    Glycosylation of surface molecules is a key feature of several eukaryotic viruses, which use the host endoplasmic reticulum/Golgi apparatus to add carbohydrates to their nascent glycoproteins. In recent years, a newly discovered group of eukaryotic viruses, belonging to the Nucleo-Cytoplasmic Large DNA Virus (NCLDV) group, was shown to have several features that are typical of cellular organisms, including the presence of components of the glycosylation machinery. Starting from initial observations with the chlorovirus PBCV-1, enzymes for glycan biosynthesis have been later identified in other viruses; in particular in members of the Mimiviridae family. They include both the glycosyltransferases and other carbohydrate-modifying enzymes and the pathways for the biosynthesis of the rare monosaccharides that are found in the viral glycan structures. These findings, together with genome analysis of the newly-identified giant DNA viruses, indicate that the presence of glycogenes is widespread in several NCLDV families. The identification of autonomous viral glycosylation machinery leads to many questions about the origin of these pathways, the mechanisms of glycan production, and eventually their function in the viral replication cycle. The scope of this review is to highlight some of the recent results that have been obtained on the glycosylation systems of the large DNA viruses, with a special focus on the enzymes involved in nucleotide-sugar production. PMID:26690138

  14. Multifaceted roles of extracellular DNA in bacterial physiology.

    PubMed

    Vorkapic, Dina; Pressler, Katharina; Schild, Stefan

    2016-02-01

    In textbooks, DNA is generally defined as the universal storage material for genetic information in all branches of life. Beyond this important intracellular role, DNA can also be present outside of living cells and is an abundant biopolymer in aquatic and terrestrial ecosystems. The origin of extracellular DNA in such ecological niches is diverse: it can be actively secreted or released by prokaryotic and eukaryotic cells by means of autolysis, apoptosis, necrosis, bacterial secretion systems or found in association with extracellular bacterial membrane vesicles. Especially for bacteria, extracellular DNA represents a significant and convenient element that can be enzymatically modulated and utilized for multiple purposes. Herein, we discuss briefly the main origins of extracellular DNA and the most relevant roles for the bacterial physiology, such as biofilm formation, nutrient source, antimicrobial means and horizontal gene transfer. PMID:26328805

  15. Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome

    PubMed Central

    Almazán, Fernando; González, José M.; Pénzes, Zoltan; Izeta, Ander; Calvo, Enrique; Plana-Durán, Juan; Enjuanes, Luis

    2000-01-01

    The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. Herein, we show that the application of two strategies, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules. A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome. The rescued coronavirus conserved all of the genetic markers introduced throughout the sequence and showed a standard mRNA pattern and the antigenic characteristics expected for the synthetic virus. The cDNA was transcribed within the nucleus, and the RNA translocated to the cytoplasm. Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed. The cDNA was derived from an attenuated isolate that replicates exclusively in the respiratory tract of swine. During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate. The synthetic virus replicated abundantly in the enteric tract and was fully virulent, demonstrating that the tropism and virulence of the recovered coronavirus can be modified. This demonstration opens up the possibility of employing this infectious cDNA as a vector for vaccine development in human, porcine, canine, and feline species susceptible to group 1 coronaviruses. PMID:10805807

  16. DNA Topology and the Initiation of Virus DNA Packaging.

    PubMed

    Oh, Choon Seok; Sippy, Jean; Charbonneau, Bridget; Crow Hutchinson, Jennifer; Mejia-Romero, Olga Esther; Barton, Michael; Patel, Priyal; Sippy, Rachel; Feiss, Michael

    2016-01-01

    During progeny assembly, viruses selectively package virion genomes from a nucleic acid pool that includes host nucleic acids. For large dsDNA viruses, including tailed bacteriophages and herpesviruses, immature viral DNA is recognized and translocated into a preformed icosahedral shell, the prohead. Recognition involves specific interactions between the viral packaging enzyme, terminase, and viral DNA recognition sites. Generally, viral DNA is recognized by terminase's small subunit (TerS). The large terminase subunit (TerL) contains translocation ATPase and endonuclease domains. In phage lambda, TerS binds a sequence repeated three times in cosB, the recognition site. TerS binding to cosB positions TerL to cut the concatemeric DNA at the adjacent nicking site, cosN. TerL introduces staggered nicks in cosN, generating twelve bp cohesive ends. Terminase separates the cohesive ends and remains bound to the cosB-containing end, in a nucleoprotein structure called Complex I. Complex I docks on the prohead's portal vertex and translocation ensues. DNA topology plays a role in the TerSλ-cosBλ interaction. Here we show that a site, I2, located between cosN and cosB, is critically important for an early DNA packaging step. I2 contains a complex static bend. I2 mutations block DNA packaging. I2 mutant DNA is cut by terminase at cosN in vitro, but in vivo, no cos cleavage is detected, nor is there evidence for Complex I. Models for what packaging step might be blocked by I2 mutations are presented. PMID:27144448

  17. DNA Topology and the Initiation of Virus DNA Packaging

    PubMed Central

    Oh, Choon Seok; Sippy, Jean; Charbonneau, Bridget; Crow Hutchinson, Jennifer; Mejia-Romero, Olga Esther; Barton, Michael; Patel, Priyal; Sippy, Rachel; Feiss, Michael

    2016-01-01

    During progeny assembly, viruses selectively package virion genomes from a nucleic acid pool that includes host nucleic acids. For large dsDNA viruses, including tailed bacteriophages and herpesviruses, immature viral DNA is recognized and translocated into a preformed icosahedral shell, the prohead. Recognition involves specific interactions between the viral packaging enzyme, terminase, and viral DNA recognition sites. Generally, viral DNA is recognized by terminase’s small subunit (TerS). The large terminase subunit (TerL) contains translocation ATPase and endonuclease domains. In phage lambda, TerS binds a sequence repeated three times in cosB, the recognition site. TerS binding to cosB positions TerL to cut the concatemeric DNA at the adjacent nicking site, cosN. TerL introduces staggered nicks in cosN, generating twelve bp cohesive ends. Terminase separates the cohesive ends and remains bound to the cosB-containing end, in a nucleoprotein structure called Complex I. Complex I docks on the prohead’s portal vertex and translocation ensues. DNA topology plays a role in the TerSλ-cosBλ interaction. Here we show that a site, I2, located between cosN and cosB, is critically important for an early DNA packaging step. I2 contains a complex static bend. I2 mutations block DNA packaging. I2 mutant DNA is cut by terminase at cosN in vitro, but in vivo, no cos cleavage is detected, nor is there evidence for Complex I. Models for what packaging step might be blocked by I2 mutations are presented. PMID:27144448

  18. Evolutionary genomics of nucleo-cytoplasmic large DNA viruses.

    PubMed

    Iyer, Lakshminarayan M; Balaji, S; Koonin, Eugene V; Aravind, L

    2006-04-01

    (primarily, endosymbionts and parasites). Amongst the expansions, there are multiple families of predicted virus-specific signaling and regulatory domains. Most NCLDVs have also acquired large arrays of genes related to ubiquitin signaling, and the animal viruses in particular have independently evolved several defenses against apoptosis and immune response, including growth factors and potential inhibitors of cytokine signaling. The mimivirus displays an enormous array of genes of bacterial provenance, including a representative of a new class of predicted papain-like peptidases. It is further demonstrated that a significant number of genes found in NCLDVs also have homologs in bacteriophages, although a vertical relationship between the NCLDVs and a particular bacteriophage group could not be established. On the basis of these observations, two alternative scenarios for the origin of the NCLDVs and other groups of large DNA viruses of eukaryotes are considered. One of these scenarios posits an early assembly of an already large DNA virus precursor from which various large DNA viruses diverged through an ongoing process of displacement of the original genes by xenologous or non-orthologous genes from various sources. The second scenario posits convergent emergence, on multiple occasions, of large DNA viruses from small plasmid-like precursors through independent accretion of similar sets of genes due to strong selective pressures imposed by their life cycles and hosts. PMID:16494962

  19. Viruses as Predisposing Factors to Bacterial Pneumonia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The etiology of respiratory disease is complex. Multifactorial in origin, respiratory disease results from the interaction of stress, multiple viruses, and multiple bacteria. A wide variety of different stressors and agents can be involved in the disease process. Healthy ruminants can carry one or...

  20. DNA Microarray for Detection of Gastrointestinal Viruses

    PubMed Central

    Martínez, Miguel A.; Soto-del Río, María de los Dolores; Gutiérrez, Rosa María; Chiu, Charles Y.; Greninger, Alexander L.; Contreras, Juan Francisco; López, Susana; Arias, Carlos F.

    2014-01-01

    Gastroenteritis is a clinical illness of humans and other animals that is characterized by vomiting and diarrhea and caused by a variety of pathogens, including viruses. An increasing number of viral species have been associated with gastroenteritis or have been found in stool samples as new molecular tools have been developed. In this work, a DNA microarray capable in theory of parallel detection of more than 100 viral species was developed and tested. Initial validation was done with 10 different virus species, and an additional 5 species were validated using clinical samples. Detection limits of 1 × 103 virus particles of Human adenovirus C (HAdV), Human astrovirus (HAstV), and group A Rotavirus (RV-A) were established. Furthermore, when exogenous RNA was added, the limit for RV-A detection decreased by one log. In a small group of clinical samples from children with gastroenteritis (n = 76), the microarray detected at least one viral species in 92% of the samples. Single infection was identified in 63 samples (83%), and coinfection with more than one virus was identified in 7 samples (9%). The most abundant virus species were RV-A (58%), followed by Anellovirus (15.8%), HAstV (6.6%), HAdV (5.3%), Norwalk virus (6.6%), Human enterovirus (HEV) (9.2%), Human parechovirus (1.3%), Sapporo virus (1.3%), and Human bocavirus (1.3%). To further test the specificity and sensitivity of the microarray, the results were verified by reverse transcription-PCR (RT-PCR) detection of 5 gastrointestinal viruses. The RT-PCR assay detected a virus in 59 samples (78%). The microarray showed good performance for detection of RV-A, HAstV, and calicivirus, while the sensitivity for HAdV and HEV was low. Furthermore, some discrepancies in detection of mixed infections were observed and were addressed by reverse transcription-quantitative PCR (RT-qPCR) of the viruses involved. It was observed that differences in the amount of genetic material favored the detection of the most abundant

  1. Cryo-electron tomography of bacterial viruses

    SciTech Connect

    Guerrero-Ferreira, Ricardo C.; Wright, Elizabeth R.

    2013-01-05

    Bacteriophage particles contain both simple and complex macromolecular assemblages and machines that enable them to regulate the infection process under diverse environmental conditions with a broad range of bacterial hosts. Recent developments in cryo-electron tomography (cryo-ET) make it possible to observe the interactions of bacteriophages with their host cells under native-state conditions at unprecedented resolution and in three-dimensions. This review describes the application of cryo-ET to studies of bacteriophage attachment, genome ejection, assembly and egress. Current topics of investigation and future directions in the field are also discussed.

  2. Bacterial vaginosis and human immunodeficiency virus infection

    PubMed Central

    Spear, Gregory T; St John, Elizabeth; Zariffard, M Reza

    2007-01-01

    Epidemiologic studies indicate that bacterial vaginosis (BV), a common alteration of lower genital tract flora in women, is associated with increased susceptibility to HIV infection. Other recent studies show that HIV is detected more frequently and at higher levels in the lower genital tract of HIV-seropositive women with BV. In vitro studies show that genital tract secretions from women with BV or flora associated with BV induce HIV expression in infected cells. The increased HIV expression appears to be due at least in part to activation through Toll-like receptors (TLR), specifically TLR2. Further research is needed to elucidate how BV contributes to HIV acquisition and transmission. PMID:17953761

  3. Horizontal transfer of DNA methylation patterns into bacterial chromosomes

    PubMed Central

    Shin, Jung-Eun; Lin, Chris; Lim, Han N.

    2016-01-01

    Horizontal gene transfer (HGT) is the non-inherited acquisition of novel DNA sequences. HGT is common and important in bacteria because it enables the rapid generation of new phenotypes such as antibiotic resistance. Here we show that in vivo and in vitro DNA methylation patterns can be horizontally transferred into bacterial chromosomes to program cell phenotypes. The experiments were performed using a synthetic system in Escherichia coli where different DNA methylation patterns within the cis-regulatory sequence of the agn43 gene turn on or off a fluorescent reporter (CFP). With this system we demonstrated that DNA methylation patterns not only accompany the horizontal transfer of genes into the bacterial cytoplasm but can be transferred into chromosomes by: (i) bacteriophage P1 transduction; and (ii) transformation of extracellular synthetic DNA. We also modified the experimental system by replacing CFP with the SgrS small RNA, which regulates glucose and methyl α-D-glucoside uptake, and showed that horizontally acquired DNA methylation patterns can increase or decrease cell fitness. That is, horizontally acquired DNA methylation patterns can result in the selection for and against cells that have HGT. Findings from these proof-of-concept experiments have applications in synthetic biology and potentially broad implications for bacterial adaptation and evolution. PMID:27084942

  4. Horizontal transfer of DNA methylation patterns into bacterial chromosomes.

    PubMed

    Shin, Jung-Eun; Lin, Chris; Lim, Han N

    2016-05-19

    Horizontal gene transfer (HGT) is the non-inherited acquisition of novel DNA sequences. HGT is common and important in bacteria because it enables the rapid generation of new phenotypes such as antibiotic resistance. Here we show that in vivo and in vitro DNA methylation patterns can be horizontally transferred into bacterial chromosomes to program cell phenotypes. The experiments were performed using a synthetic system in Escherichia coli where different DNA methylation patterns within the cis-regulatory sequence of the agn43 gene turn on or off a fluorescent reporter (CFP). With this system we demonstrated that DNA methylation patterns not only accompany the horizontal transfer of genes into the bacterial cytoplasm but can be transferred into chromosomes by: (i) bacteriophage P1 transduction; and (ii) transformation of extracellular synthetic DNA. We also modified the experimental system by replacing CFP with the SgrS small RNA, which regulates glucose and methyl α-D-glucoside uptake, and showed that horizontally acquired DNA methylation patterns can increase or decrease cell fitness. That is, horizontally acquired DNA methylation patterns can result in the selection for and against cells that have HGT. Findings from these proof-of-concept experiments have applications in synthetic biology and potentially broad implications for bacterial adaptation and evolution. PMID:27084942

  5. Small DNA Tumor Viruses: Large Contributors to Biomedical Sciences

    PubMed Central

    Howley, Peter M.; Livingston, David M.

    2009-01-01

    Studies of the small DNA tumor viruses (the polyomaviruses, the adenoviruses and the papillomaviruses) have led to fundamental discoveries that have advanced our understanding of basic mammalian cell molecular biology processes such as transcription and DNA replication, uncovered pathways and genes often perturbed in human cancer, and identified bona fide human cancer viruses. In this article we examine the many contributions that have come from the small DNA tumor virus field and provide a recounting of some of the major landmark. PMID:19136134

  6. The bacterial DnaA-trio replication origin element specifies single-stranded DNA initiator binding.

    PubMed

    Richardson, Tomas T; Harran, Omar; Murray, Heath

    2016-06-16

    DNA replication is tightly controlled to ensure accurate inheritance of genetic information. In all organisms, initiator proteins possessing AAA+ (ATPases associated with various cellular activities) domains bind replication origins to license new rounds of DNA synthesis. In bacteria the master initiator protein, DnaA, is highly conserved and has two crucial DNA binding activities. DnaA monomers recognize the replication origin (oriC) by binding double-stranded DNA sequences (DnaA-boxes); subsequently, DnaA filaments assemble and promote duplex unwinding by engaging and stretching a single DNA strand. While the specificity for duplex DnaA-boxes by DnaA has been appreciated for over 30 years, the sequence specificity for single-strand DNA binding has remained unknown. Here we identify a new indispensable bacterial replication origin element composed of a repeating trinucleotide motif that we term the DnaA-trio. We show that the function of the DnaA-trio is to stabilize DnaA filaments on a single DNA strand, thus providing essential precision to this binding mechanism. Bioinformatic analysis detects DnaA-trios in replication origins throughout the bacterial kingdom, indicating that this element is part of the core oriC structure. The discovery and characterization of the novel DnaA-trio extends our fundamental understanding of bacterial DNA replication initiation, and because of the conserved structure of AAA+ initiator proteins these findings raise the possibility of specific recognition motifs within replication origins of higher organisms. PMID:27281207

  7. DNA repair in bacterial cultures and plasmid DNA exposed to infrared laser for treatment of pain

    NASA Astrophysics Data System (ADS)

    Canuto, K. S.; Sergio, L. P. S.; Marciano, R. S.; Guimarães, O. R.; Polignano, G. A. C.; Geller, M.; Paoli, F.; Fonseca, A. S.

    2013-06-01

    Biostimulation of tissues by low intensity lasers has been described on a photobiological basis and clinical protocols are recommended for treatment of various diseases, but their effects on DNA are controversial. The objective of this work was to evaluate effects of low intensity infrared laser exposure on survival and bacterial filamentation in Escherichia coli cultures, and induction of DNA lesions in bacterial plasmids. In E. coli cultures and plasmids exposed to an infrared laser at fluences used to treat pain, bacterial survival and filamentation and DNA lesions in plasmids were evaluated by electrophoretic profile. Data indicate that the infrared laser (i) increases survival of E. coli wild type in 24 h of stationary growth phase, (ii) induces bacterial filamentation, (iii) does not alter topological forms of plasmids and (iv) does not alter the electrophoretic profile of plasmids incubated with exonuclease III or formamidopyrimidine DNA glycosylase. A low intensity infrared laser at the therapeutic fluences used to treat pain can alter survival of E. coli wild type, induce filamentation in bacterial cells, depending on physiologic conditions and DNA repair, and induce DNA lesions other than single or double DNA strand breaks or alkali-labile sites, which are not targeted by exonuclease III or formamidopyrimidine DNA glycosylase.

  8. Detection of DNA viruses in prostate cancer

    PubMed Central

    Smelov, Vitaly; Bzhalava, Davit; Arroyo Mühr, Laila Sara; Eklund, Carina; Komyakov, Boris; Gorelov, Andrey; Dillner, Joakim; Hultin, Emilie

    2016-01-01

    We tested prostatic secretions from men with and without prostate cancer (13 cases and 13 matched controls) or prostatitis (18 cases and 18 matched controls) with metagenomic sequencing. A large number (>200) of viral reads was only detected among four prostate cancer cases (1 patient each positive for Merkel cell polyomavirus, JC polyomavirus and Human Papillomavirus types 89 or 40, respectively). Lower numbers of reads from a large variety of viruses were detected in all patient groups. Our knowledge of the biology of the prostate may be furthered by the fact that DNA viruses are commonly shed from the prostate and can be readily detected by metagenomic sequencing of expressed prostate secretions. PMID:27121729

  9. Genomics of Bacterial and Archaeal Viruses: Dynamics within the Prokaryotic Virosphere

    PubMed Central

    Krupovic, Mart; Prangishvili, David; Hendrix, Roger W.; Bamford, Dennis H.

    2011-01-01

    Summary: Prokaryotes, bacteria and archaea, are the most abundant cellular organisms among those sharing the planet Earth with human beings (among others). However, numerous ecological studies have revealed that it is actually prokaryotic viruses that predominate on our planet and outnumber their hosts by at least an order of magnitude. An understanding of how this viral domain is organized and what are the mechanisms governing its evolution is therefore of great interest and importance. The vast majority of characterized prokaryotic viruses belong to the order Caudovirales, double-stranded DNA (dsDNA) bacteriophages with tails. Consequently, these viruses have been studied (and reviewed) extensively from both genomic and functional perspectives. However, albeit numerous, tailed phages represent only a minor fraction of the prokaryotic virus diversity. Therefore, the knowledge which has been generated for this viral system does not offer a comprehensive view of the prokaryotic virosphere. In this review, we discuss all families of bacterial and archaeal viruses that contain more than one characterized member and for which evolutionary conclusions can be attempted by use of comparative genomic analysis. We focus on the molecular mechanisms of their genome evolution as well as on the relationships between different viral groups and plasmids. It becomes clear that evolutionary mechanisms shaping the genomes of prokaryotic viruses vary between different families and depend on the type of the nucleic acid, characteristics of the virion structure, as well as the mode of the life cycle. We also point out that horizontal gene transfer is not equally prevalent in different virus families and is not uniformly unrestricted for diverse viral functions. PMID:22126996

  10. Recovery and identification of bacterial DNA from illicit drugs.

    PubMed

    Cho, Kaymann T; Richardson, Michelle M; Kirkbride, K Paul; McNevin, Dennis; Nelson, Michelle; Pianca, Dennis; Roffey, Paul; Gahan, Michelle E

    2014-02-01

    Bacterial infections, including Bacillus anthracis (anthrax), are a common risk associated with illicit drug use, particularly among injecting drug users. There is, therefore, an urgent need to survey illicit drugs used for injection for the presence of bacteria and provide valuable information to health and forensic authorities. The objectives of this study were to develop a method for the extraction of bacterial DNA from illicit drugs and conduct a metagenomic survey of heroin and methamphetamine seized in the Australian Capital Territory during 2002-2011 for the presence of pathogens. Trends or patterns in drug contamination and their health implications for injecting drug users were also investigated. Methods based on the ChargeSwitch(®)gDNA mini kit (Invitrogen), QIAamp DNA extraction mini kit (QIAGEN) with and without bead-beating, and an organic phenol/chloroform extraction with ethanol precipitation were assessed for the recovery efficiency of both free and cellular bacterial DNA. Bacteria were identified using polymerase chain reaction and electrospray ionization-mass spectrometry (PCR/ESI-MS). An isopropanol pre-wash to remove traces of the drug and diluents, followed by a modified ChargeSwitch(®) method, was found to efficiently lyse cells and extract free and cellular DNA from Gram-positive and Gram-negative bacteria in heroin and methamphetamine which could then be identified by PCR/ESI-MS. Analysis of 12 heroin samples revealed the presence of DNA from species of Comamonas, Weissella, Bacillus, Streptococcus and Arthrobacter. No organisms were detected in the nine methamphetamine samples analysed. This study develops a method to extract and identify Gram-positive and Gram-negative bacteria from illicit drugs and demonstrates the presence of a range of bacterial pathogens in seized drug samples. These results will prove valuable for future work investigating trends or patterns in drug contamination and their health implications for injecting drug

  11. Viral hemorrhagic fevers of animals caused by DNA viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here we outline serious diseases of food and fiber animals that cause damaging economic effect on products all over the world. The only vector-borne DNA virus is included here, such as African swine fever virus, and the herpes viruses discussed have a complex epidemiology characterized by outbreak...

  12. Structures of giant icosahedral eukaryotic dsDNA viruses

    PubMed Central

    Xiao, Chuan; Rossmann, Michael G.

    2011-01-01

    In the last twenty years, numerous giant, dsDNA, icosahedral viruses have been discovered and assigned to the nucleocytoplasmic large dsDNA virus (NCLDV) clade. The major capsid proteins of these viruses consist of two consecutive jelly-roll domains, assembled into trimers, with pseudo 6-fold symmetry. The capsomers are assembled into arrays that have either p6 (as in Paramecium bursaria Chlorella virus-1) or p3 symmetry (as in Mimivirus). Most of the NCLDV viruses have a membrane that separates the nucleocapsid from the external capsid. PMID:21909343

  13. Inhibition of virus DNA replication by artificial zinc finger proteins.

    PubMed

    Sera, Takashi

    2005-02-01

    Prevention of virus infections is a major objective in agriculture and human health. One attractive approach to the prevention is inhibition of virus replication. To demonstrate this concept in vivo, an artificial zinc finger protein (AZP) targeting the replication origin of the Beet severe curly top virus (BSCTV), a model DNA virus, was created. In vitro DNA binding assays indicated that the AZP efficiently blocked binding of the viral replication protein (Rep), which initiates virus replication, to the replication origin. All of the transgenic Arabidopsis plants expressing the AZP showed phenotypes strongly resistant to virus infection, and 84% of the transgenic plants showed no symptom. Southern blot analysis demonstrated that BSCTV replication was completely suppressed in the transgenic plants. Since the mechanism of viral DNA replication is well conserved among plants and mammals, this approach could be applied not only to agricultural crop protection but also to the prevention of virus infections in humans. PMID:15681461

  14. How Can Plant DNA Viruses Evade siRNA-Directed DNA Methylation and Silencing?

    PubMed Central

    Pooggin, Mikhail M.

    2013-01-01

    Plants infected with DNA viruses produce massive quantities of virus-derived, 24-nucleotide short interfering RNAs (siRNAs), which can potentially direct viral DNA methylation and transcriptional silencing. However, growing evidence indicates that the circular double-stranded DNA accumulating in the nucleus for Pol II-mediated transcription of viral genes is not methylated. Hence, DNA viruses most likely evade or suppress RNA-directed DNA methylation. This review describes the specialized mechanisms of replication and silencing evasion evolved by geminiviruses and pararetoviruses, which rescue viral DNA from repressive methylation and interfere with transcriptional and post-transcriptional silencing of viral genes. PMID:23887650

  15. Sensing Viruses by Mechanical Tension of DNA in Responsive Hydrogels

    NASA Astrophysics Data System (ADS)

    Shin, Jaeoh; Cherstvy, Andrey G.; Metzler, Ralf

    2014-04-01

    The rapid worldwide spread of severe viral infections, often involving novel mutations of viruses, poses major challenges to our health-care systems. This means that tools that can efficiently and specifically diagnose viruses are much needed. To be relevant for broad applications in local health-care centers, such tools should be relatively cheap and easy to use. In this paper, we discuss the biophysical potential for the macroscopic detection of viruses based on the induction of a mechanical stress in a bundle of prestretched DNA molecules upon binding of viruses to the DNA. We show that the affinity of the DNA to the charged virus surface induces a local melting of the double helix into two single-stranded DNA. This process effects a mechanical stress along the DNA chains leading to an overall contraction of the DNA. Our results suggest that when such DNA bundles are incorporated in a supporting matrix such as a responsive hydrogel, the presence of viruses may indeed lead to a significant, macroscopic mechanical deformation of the matrix. We discuss the biophysical basis for this effect and characterize the physical properties of the associated DNA melting transition. In particular, we reveal several scaling relations between the relevant physical parameters of the system. We promote this DNA-based assay as a possible tool for efficient and specific virus screening.

  16. Solid-to-fluid – like DNA transition in viruses facilitates infection

    SciTech Connect

    Liu, Ting; Sae-Ueng, Udom; Li, Dong; Lander, Gabriel C.; Zuo, Xiaobing; Jonsson, Bengt; Rau, Donald; Shefer, Ivetta; Evilevitch, Alex

    2014-10-14

    Releasing the packaged viral DNA into the host cell is an essential process to initiate viral infection. In many double-stranded DNA bacterial viruses and herpesviruses, the tightly packaged genome is hexagonally ordered and stressed in the protein shell, called the capsid. DNA condensed in this state inside viral capsids has been shown to be trapped in a glassy state, with restricted molecular motion in vitro. This limited intracapsid DNA mobility is caused by the sliding friction between closely packaged DNA strands, as a result of the repulsive interactions between the negative charges on the DNA helices. It had been unclear how this rigid crystalline structure of the viral genome rapidly ejects from the capsid, reaching rates of 60,000 bp/s. Through a combination of single- molecule and bulk techniques, we determined how the structure and energy of the encapsidated DNA in phage λ regulates the mobility required for its ejection. Our data show that packaged λ -DNA undergoes a solid-to-fluid – like disordering transition as a function of temperature, resultin g locally in less densely packed DNA, reducing DNADNA repulsions. This p rocess leads to a sig- nificant increase in genome mobility or fluidity, which facilitates genome release at temperatures close to that of viral infection (37 °C), suggesting a remarkab le physical adaptation of bac- terial viruses to the environment of Escherichia coli cells in a human host.

  17. At a crossroads: human DNA tumor viruses and the host DNA damage response.

    PubMed

    Nikitin, Pavel A; Luftig, Micah A

    2011-07-01

    Human DNA tumor viruses induce host cell proliferation in order to establish the necessary cellular milieu to replicate viral DNA. The consequence of such viral-programmed induction of proliferation coupled with the introduction of foreign replicating DNA structures makes these viruses particularly sensitive to the host DNA damage response machinery. In fact, sensors of DNA damage are often activated and modulated by DNA tumor viruses in both latent and lytic infection. This article focuses on the role of the DNA damage response during the life cycle of human DNA tumor viruses, with a particular emphasis on recent advances in our understanding of the role of the DNA damage response in EBV, Kaposi's sarcoma-associated herpesvirus and human papillomavirus infection. PMID:21927617

  18. Viral Carcinogenesis: Factors Inducing DNA Damage and Virus Integration

    PubMed Central

    Chen, Yan; Williams, Vonetta; Filippova, Maria; Filippov, Valery; Duerksen-Hughes, Penelope

    2014-01-01

    Viruses are the causative agents of 10%–15% of human cancers worldwide. The most common outcome for virus-induced reprogramming is genomic instability, including accumulation of mutations, aberrations and DNA damage. Although each virus has its own specific mechanism for promoting carcinogenesis, the majority of DNA oncogenic viruses encode oncogenes that transform infected cells, frequently by targeting p53 and pRB. In addition, integration of viral DNA into the human genome can also play an important role in promoting tumor development for several viruses, including HBV and HPV. Because viral integration requires the breakage of both the viral and the host DNA, the integration rate is believed to be linked to the levels of DNA damage. DNA damage can be caused by both endogenous and exogenous factors, including inflammation induced by either the virus itself or by co-infections with other agents, environmental agents and other factors. Typically, cancer develops years to decades following the initial infection. A better understanding of virus-mediated carcinogenesis, the networking of pathways involved in transformation and the relevant risk factors, particularly in those cases where tumorigenesis proceeds by way of virus integration, will help to suggest prophylactic and therapeutic strategies to reduce the risk of virus-mediated cancer. PMID:25340830

  19. Amyloid-DNA composites of bacterial biofilms stimulate autoimmunity

    PubMed Central

    Gallo, Paul M.; Rapsinski, Glenn J.; Wilson, R. Paul; Oppong, Gertrude O.; Sriram, Uma; Goulian, Mark; Buttaro, Bettina; Caricchio, Roberto; Gallucci, Stefania; Tükel, Çagla

    2015-01-01

    SUMMARY Research on the human microbiome has established that commensal and pathogenic bacteria can influence obesity, cancer, and autoimmunity through mechanisms mostly unknown. We found that a component of bacterial biofilms, the amyloid protein curli, irreversibly formed fibers with bacterial DNA during biofilm formation. This interaction accelerated amyloid polymerization and created potent immunogenic complexes that activated immune cells, including dendritic cells, to produce cytokines such as Type I interferons, which are pathogenic in systemic lupus erythematosus (SLE). When given systemically, curli-DNA composites triggered immune activation and production of autoantibodies in lupus-prone and wild-type mice. We also found that the infection of lupus-prone mice with curli-producing bacteria triggered higher autoantibody titers compared to curli-deficient bacteria. These data provide a mechanism by which the microbiome and biofilm-producing enteric infections may contribute to the progression of SLE and point to a potential molecular target for treatment of autoimmunity. PMID:26084027

  20. Bacterial Genotoxins: Merging the DNA Damage Response into Infection Biology.

    PubMed

    Grasso, Francesca; Frisan, Teresa

    2015-01-01

    Bacterial genotoxins are unique among bacterial toxins as their molecular target is DNA. The consequence of intoxication or infection is induction of DNA breaks that, if not properly repaired, results in irreversible cell cycle arrest (senescence) or death of the target cells. At present, only three bacterial genotoxins have been identified. Two are protein toxins: the cytolethal distending toxin (CDT) family produced by a number of Gram-negative bacteria and the typhoid toxin produced by Salmonella enterica serovar Typhi. The third member, colibactin, is a peptide-polyketide genotoxin, produced by strains belonging to the phylogenetic group B2 of Escherichia coli. This review will present the cellular effects of acute and chronic intoxication or infection with the genotoxins-producing bacteria. The carcinogenic properties and the role of these effectors in the context of the host-microbe interaction will be discussed. We will further highlight the open questions that remain to be solved regarding the biology of this unusual family of bacterial toxins. PMID:26270677

  1. Bacterial Genotoxins: Merging the DNA Damage Response into Infection Biology

    PubMed Central

    Grasso, Francesca; Frisan, Teresa

    2015-01-01

    Bacterial genotoxins are unique among bacterial toxins as their molecular target is DNA. The consequence of intoxication or infection is induction of DNA breaks that, if not properly repaired, results in irreversible cell cycle arrest (senescence) or death of the target cells. At present, only three bacterial genotoxins have been identified. Two are protein toxins: the cytolethal distending toxin (CDT) family produced by a number of Gram-negative bacteria and the typhoid toxin produced by Salmonella enterica serovar Typhi. The third member, colibactin, is a peptide-polyketide genotoxin, produced by strains belonging to the phylogenetic group B2 of Escherichia coli. This review will present the cellular effects of acute and chronic intoxication or infection with the genotoxins-producing bacteria. The carcinogenic properties and the role of these effectors in the context of the host-microbe interaction will be discussed. We will further highlight the open questions that remain to be solved regarding the biology of this unusual family of bacterial toxins. PMID:26270677

  2. A DNA polymerase activity is associated with Cauliflower Mosaic Virus.

    PubMed Central

    Menissier, J; Laquel, P; Lebeurier, G; Hirth, L

    1984-01-01

    A DNA polymerase activity is found within the Cauliflower Mosaic Virus (CaMV) particle. Analysis of the reaction product reveals that the linear form of the virion DNA is preferentially labelled. The molecular weight of the DNA polymerase as determined on an "activity gel" is 76 kDa. Images PMID:6514573

  3. Targeting CTCF to Control Virus Gene Expression: A Common Theme amongst Diverse DNA Viruses

    PubMed Central

    Pentland, Ieisha; Parish, Joanna L.

    2015-01-01

    All viruses target host cell factors for successful life cycle completion. Transcriptional control of DNA viruses by host cell factors is important in the temporal and spatial regulation of virus gene expression. Many of these factors are recruited to enhance virus gene expression and thereby increase virus production, but host cell factors can also restrict virus gene expression and productivity of infection. CCCTC binding factor (CTCF) is a host cell DNA binding protein important for the regulation of genomic chromatin boundaries, transcriptional control and enhancer element usage. CTCF also functions in RNA polymerase II regulation and in doing so can influence co-transcriptional splicing events. Several DNA viruses, including Kaposi’s sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and human papillomavirus (HPV) utilize CTCF to control virus gene expression and many studies have highlighted a role for CTCF in the persistence of these diverse oncogenic viruses. CTCF can both enhance and repress virus gene expression and in some cases CTCF increases the complexity of alternatively spliced transcripts. This review article will discuss the function of CTCF in the life cycle of DNA viruses in the context of known host cell CTCF functions. PMID:26154016

  4. Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

    SciTech Connect

    Kennedy, Edward M.; Cullen, Bryan R.

    2015-05-15

    CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

  5. Chimeric viruses blur the borders between the major groups of eukaryotic single-stranded DNA viruses.

    PubMed

    Roux, Simon; Enault, François; Bronner, Gisèle; Vaulot, Daniel; Forterre, Patrick; Krupovic, Mart

    2013-01-01

    Metagenomic studies have uncovered an astonishing diversity of ssDNA viruses encoding replication proteins (Reps) related to those of eukaryotic Circoviridae, Geminiviridae or Nanoviridae; however, exact evolutionary relationships among these viruses remain obscure. Recently, a unique chimeric virus (CHIV) genome, which has apparently emerged via recombination between ssRNA and ssDNA viruses, has been discovered. Here we report on the assembly of 13 new CHIV genomes recovered from various environments. Our results indicate a single event of capsid protein (CP) gene capture from an RNA virus in the history of this virus group. The domestication of the CP gene was followed by an unprecedented recurrent replacement of the Rep genes in CHIVs with distant counterparts from diverse ssDNA viruses. We suggest that parasitic and symbiotic interactions between unicellular eukaryotes were central for the emergence of CHIVs and that such turbulent evolution was primarily dictated by incongruence between the CP and Rep proteins. PMID:24193254

  6. The bacterial DnaC helicase loader is a DnaB ring breaker

    PubMed Central

    Arias-Palomo, Ernesto; O’Shea, Valerie L.; Hood, Iris V.; Berger, James M.

    2013-01-01

    Summary Dedicated AAA+ ATPases help deposit hexameric ring-shaped helicases onto DNA to promote replication in cellular organisms. To understand how loading occurs, we used negative-stain electron microscopy and small-angle X-ray scattering to determine the ATP-bound structure of the intact E. coli DnaB•DnaC helicase/loader complex. The 480 kDa dodecamer forms a three-tiered assembly, in which DnaC adopts a spiral configuration that remodels N-terminal scaffolding and C-terminal motor regions of DnaB to produce a clear break in the helicase ring. Surprisingly, DnaC’s AAA+ fold is dispensable for ring remodeling, as the isolated helicase-binding domain of DnaC can both load DnaB onto DNA and increase the efficiency by which the helicase acts on substrates in vitro. Our data demonstrate that DnaC opens DnaB by a mechanism akin to that of polymerase clamp loaders, and indicate that bacterial replicative helicases, like their eukaryotic counterparts, possess auto-regulatory elements that influence how the hexameric motor domains are loaded onto and unwind DNA. PMID:23562643

  7. Activation of the DNA Damage Response by RNA Viruses.

    PubMed

    Ryan, Ellis L; Hollingworth, Robert; Grand, Roger J

    2016-01-01

    RNA viruses are a genetically diverse group of pathogens that are responsible for some of the most prevalent and lethal human diseases. Numerous viruses introduce DNA damage and genetic instability in host cells during their lifecycles and some species also manipulate components of the DNA damage response (DDR), a complex and sophisticated series of cellular pathways that have evolved to detect and repair DNA lesions. Activation and manipulation of the DDR by DNA viruses has been extensively studied. It is apparent, however, that many RNA viruses can also induce significant DNA damage, even in cases where viral replication takes place exclusively in the cytoplasm. DNA damage can contribute to the pathogenesis of RNA viruses through the triggering of apoptosis, stimulation of inflammatory immune responses and the introduction of deleterious mutations that can increase the risk of tumorigenesis. In addition, activation of DDR pathways can contribute positively to replication of viral RNA genomes. Elucidation of the interactions between RNA viruses and the DDR has provided important insights into modulation of host cell functions by these pathogens. This review summarises the current literature regarding activation and manipulation of the DDR by several medically important RNA viruses. PMID:26751489

  8. Activation of the DNA Damage Response by RNA Viruses

    PubMed Central

    Ryan, Ellis L.; Hollingworth, Robert; Grand, Roger J.

    2016-01-01

    RNA viruses are a genetically diverse group of pathogens that are responsible for some of the most prevalent and lethal human diseases. Numerous viruses introduce DNA damage and genetic instability in host cells during their lifecycles and some species also manipulate components of the DNA damage response (DDR), a complex and sophisticated series of cellular pathways that have evolved to detect and repair DNA lesions. Activation and manipulation of the DDR by DNA viruses has been extensively studied. It is apparent, however, that many RNA viruses can also induce significant DNA damage, even in cases where viral replication takes place exclusively in the cytoplasm. DNA damage can contribute to the pathogenesis of RNA viruses through the triggering of apoptosis, stimulation of inflammatory immune responses and the introduction of deleterious mutations that can increase the risk of tumorigenesis. In addition, activation of DDR pathways can contribute positively to replication of viral RNA genomes. Elucidation of the interactions between RNA viruses and the DDR has provided important insights into modulation of host cell functions by these pathogens. This review summarises the current literature regarding activation and manipulation of the DDR by several medically important RNA viruses. PMID:26751489

  9. Virus and Bacterial Cell Chemical Analysis by NanoSIMS

    SciTech Connect

    Weber, P; Holt, J

    2008-07-28

    In past work for the Department of Homeland Security, the LLNL NanoSIMS team has succeeded in extracting quantitative elemental composition at sub-micron resolution from bacterial spores using nanometer-scale secondary ion mass spectrometry (NanoSIMS). The purpose of this task is to test our NanoSIMS capabilities on viruses and bacterial cells. This initial work has proven successful. We imaged Tobacco Mosaic Virus (TMV) and Bacillus anthracis Sterne cells using scanning electron microscopy (SEM) and then analyzed those samples by NanoSIMS. We were able resolve individual viral particles ({approx}18 nm by 300 nm) in the SEM and extract correlated elemental composition in the NanoSIMS. The phosphorous/carbon ratio observed in TMV is comparable to that seen in bacterial spores (0.033), as was the chlorine/carbon ratio (0.11). TMV elemental composition is consistent from spot to spot, and TMV is readily distinguished from debris by NanoSIMS analysis. Bacterial cells were readily identified in the SEM and relocated in the NanoSIMS for elemental analysis. The Ba Sterne cells were observed to have a measurably lower phosphorous/carbon ratio (0.005), as compared to the spores produced in the same run (0.02). The chlorine/carbon ratio was approximately 2.5X larger in the cells (0.2) versus the spores (0.08), while the fluorine/carbon ratio was approximately 10X lower in the cells (0.008) than the spores (0.08). Silicon/carbon ratios for both cells and spores encompassed a comparable range. The initial data in this study suggest that high resolution analysis is useful because it allows the target agent to be analyzed separate from particulates and other debris. High resolution analysis would also be useful for trace sample analysis. The next step in this work is to determine the potential utility of elemental signatures in these kinds of samples. We recommend bulk analyses of media and agent samples to determine the range of media compositions in use, and to determine how

  10. Characterisation of DNA forms associated with cassava latent virus infection.

    PubMed Central

    Stanley, J; Townsend, R

    1985-01-01

    In addition to the major encapsidated DNA species found in preparations of cassava latent virus (genomic DNAs 1 and 2) there are minor DNA populations of twice (dimeric) and approximately half genome length. Both minor species resemble the genomic DNAs in that they are composed of predominantly circular single-stranded DNA. All of these size groups have a corresponding covalently-closed circular double-stranded DNA form in infected tissue. Infectivity studies using cloned DNAs 1 and 2 show that dimeric DNA routinely appears, suggesting it to be an intermediate in the DNA replicative cycle that can be encapsidated at low efficiency. In contrast, half unit length DNA has not yet been detected after multiple passaging of virus derived from the cloned DNA inoculum. Half unit length DNAs appear to be derived exclusively from DNA 2 and consist of a population of molecules exhibiting a relatively specific deletion. As they have an inhibitory effect on virus multiplication, their encapsidated forms are analogous to defective interfering particles associated with other eukaryotic DNA containing viruses. Small primer molecules associated with the genomic single-stranded DNAs, as reported for another geminivirus, have not been detected in CLV. Images PMID:4000956

  11. Resistance of Aerosolized Bacterial Viruses to Relative Humidity and Temperature.

    PubMed

    Verreault, Daniel; Marcoux-Voiselle, Mélissa; Turgeon, Nathalie; Moineau, Sylvain; Duchaine, Caroline

    2015-10-01

    The use of aerosolized bacteriophages as surrogates for hazardous viruses might simplify and accelerate the discovery of links between viral components and their persistence in the airborne state under diverse environmental conditions. In this study, four structurally distinct lytic phages, MS2 (single-stranded RNA [ssRNA]), ϕ6 (double-stranded RNA [dsRNA]), ϕX174 (single-stranded DNA [ssDNA]), and PR772 (double-stranded DNA [dsDNA]), were nebulized into a rotating chamber and exposed to various levels of relative humidity (RH) and temperature as well as to germicidal UV radiation. The aerosolized viral particles were allowed to remain airborne for up to 14 h before being sampled for analysis by plaque assays and quantitative PCRs. Phages ϕ6 and MS2 were the most resistant at low levels of relative humidity, while ϕX174 was more resistant at 80% RH. Phage ϕ6 lost its infectivity immediately after exposure to 30°C and 80% RH. The infectivity of all tested phages rapidly declined as a function of the exposure time to UVC radiation, phage MS2 being the most resistant. Taken altogether, our data indicate that these aerosolized phages behave differently under various environmental conditions and highlight the necessity of carefully selecting viral simulants in bioaerosol studies. PMID:26253683

  12. Resistance of Aerosolized Bacterial Viruses to Relative Humidity and Temperature

    PubMed Central

    Verreault, Daniel; Marcoux-Voiselle, Mélissa; Turgeon, Nathalie; Moineau, Sylvain

    2015-01-01

    The use of aerosolized bacteriophages as surrogates for hazardous viruses might simplify and accelerate the discovery of links between viral components and their persistence in the airborne state under diverse environmental conditions. In this study, four structurally distinct lytic phages, MS2 (single-stranded RNA [ssRNA]), ϕ6 (double-stranded RNA [dsRNA]), ϕX174 (single-stranded DNA [ssDNA]), and PR772 (double-stranded DNA [dsDNA]), were nebulized into a rotating chamber and exposed to various levels of relative humidity (RH) and temperature as well as to germicidal UV radiation. The aerosolized viral particles were allowed to remain airborne for up to 14 h before being sampled for analysis by plaque assays and quantitative PCRs. Phages ϕ6 and MS2 were the most resistant at low levels of relative humidity, while ϕX174 was more resistant at 80% RH. Phage ϕ6 lost its infectivity immediately after exposure to 30°C and 80% RH. The infectivity of all tested phages rapidly declined as a function of the exposure time to UVC radiation, phage MS2 being the most resistant. Taken altogether, our data indicate that these aerosolized phages behave differently under various environmental conditions and highlight the necessity of carefully selecting viral simulants in bioaerosol studies. PMID:26253683

  13. The dynamic DNA methylomes of double-stranded DNA viruses associated with human cancer

    PubMed Central

    Fernandez, Agustin F.; Rosales, Cecilia; Lopez-Nieva, Pilar; Graña, Osvaldo; Ballestar, Esteban; Ropero, Santiago; Espada, Jesus; Melo, Sonia A.; Lujambio, Amaia; Fraga, Mario F.; Pino, Irene; Javierre, Biola; Carmona, Francisco J.; Acquadro, Francesco; Steenbergen, Renske D.M.; Snijders, Peter J.F.; Meijer, Chris J.; Pineau, Pascal; Dejean, Anne; Lloveras, Belen; Capella, Gabriel; Quer, Josep; Buti, Maria; Esteban, Juan-Ignacio; Allende, Helena; Rodriguez-Frias, Francisco; Castellsague, Xavier; Minarovits, Janos; Ponce, Jordi; Capello, Daniela; Gaidano, Gianluca; Cigudosa, Juan Cruz; Gomez-Lopez, Gonzalo; Pisano, David G.; Valencia, Alfonso; Piris, Miguel Angel; Bosch, Francesc X.; Cahir-McFarland, Ellen; Kieff, Elliott; Esteller, Manel

    2009-01-01

    The natural history of cancers associated with virus exposure is intriguing, since only a minority of human tissues infected with these viruses inevitably progress to cancer. However, the molecular reasons why the infection is controlled or instead progresses to subsequent stages of tumorigenesis are largely unknown. In this article, we provide the first complete DNA methylomes of double-stranded DNA viruses associated with human cancer that might provide important clues to help us understand the described process. Using bisulfite genomic sequencing of multiple clones, we have obtained the DNA methylation status of every CpG dinucleotide in the genome of the Human Papilloma Viruses 16 and 18 and Human Hepatitis B Virus, and in all the transcription start sites of the Epstein-Barr Virus. These viruses are associated with infectious diseases (such as hepatitis B and infectious mononucleosis) and the development of human tumors (cervical, hepatic, and nasopharyngeal cancers, and lymphoma), and are responsible for 1 million deaths worldwide every year. The DNA methylomes presented provide evidence of the dynamic nature of the epigenome in contrast to the genome. We observed that the DNA methylome of these viruses evolves from an unmethylated to a highly methylated genome in association with the progression of the disease, from asymptomatic healthy carriers, through chronically infected tissues and pre-malignant lesions, to the full-blown invasive tumor. The observed DNA methylation changes have a major functional impact on the biological behavior of the viruses. PMID:19208682

  14. 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

  15. The role of respiratory viruses in the etiology of bacterial pneumonia

    PubMed Central

    Lee, Kyu Han; Gordon, Aubree; Foxman, Betsy

    2016-01-01

    Pneumonia is the leading cause of death among children less than 5 years old worldwide. A wide range of viral, bacterial and fungal agents can cause pneumonia: although viruses are the most common etiologic agent, the severity of clinical symptoms associated with bacterial pneumonia and increasing antibiotic resistance makes bacterial pneumonia a major public health concern. Bacterial pneumonia can follow upper respiratory viral infection and complicate lower respiratory viral infection. Secondary bacterial pneumonia is a major cause of influenza-related deaths. In this review, we evaluate the following hypotheses: (i) respiratory viruses influence the etiology of pneumonia by altering bacterial community structure in the upper respiratory tract (URT) and (ii) respiratory viruses promote or inhibit colonization of the lower respiratory tract (LRT) by certain bacterial species residing in the URT. We conducted a systematic review of the literature to examine temporal associations between respiratory viruses and bacteria and a targeted review to identify potential mechanisms of interactions. We conclude that viruses both alter the bacterial community in the URT and promote bacterial colonization of the LRT. However, it is uncertain whether changes in the URT bacterial community play a substantial role in pneumonia etiology. The exception is Streptococcus pneumoniae where a strong link between viral co-infection, increased carriage and pneumococcal pneumonia has been established. PMID:26884414

  16. The role of respiratory viruses in the etiology of bacterial pneumonia: An ecological perspective.

    PubMed

    Lee, Kyu Han; Gordon, Aubree; Foxman, Betsy

    2016-01-01

    Pneumonia is the leading cause of death among children less than 5 years old worldwide. A wide range of viral, bacterial and fungal agents can cause pneumonia: although viruses are the most common etiologic agent, the severity of clinical symptoms associated with bacterial pneumonia and increasing antibiotic resistance makes bacterial pneumonia a major public health concern. Bacterial pneumonia can follow upper respiratory viral infection and complicate lower respiratory viral infection. Secondary bacterial pneumonia is a major cause of influenza-related deaths. In this review, we evaluate the following hypotheses: (i) respiratory viruses influence the etiology of pneumonia by altering bacterial community structure in the upper respiratory tract (URT) and (ii) respiratory viruses promote or inhibit colonization of the lower respiratory tract (LRT) by certain bacterial species residing in the URT. We conducted a systematic review of the literature to examine temporal associations between respiratory viruses and bacteria and a targeted review to identify potential mechanisms of interactions. We conclude that viruses both alter the bacterial community in the URT and promote bacterial colonization of the LRT. However, it is uncertain whether changes in the URT bacterial community play a substantial role in pneumonia etiology. The exception is Streptococcus pneumoniae where a strong link between viral co-infection, increased carriage and pneumococcal pneumonia has been established. PMID:26884414

  17. Frog Virus 3 DNA Replication Occurs in Two Stages

    PubMed Central

    Goorha, R.

    1982-01-01

    Viral DNA synthesis in frog virus 3 (FV3)-infected cells occurs both in the nucleus and in the cytoplasm (Goorha et al., Virology 84:32-51, 1978). Relationships between viral DNA molecules synthesized in these two compartments and their role in the virus replication were examined. The data presented here suggest that (i) FV3 DNA replicated in two stages and (ii) nucleus and cytoplasm were the sites of stages 1 and 2 of DNA replication, respectively. Stages 1 and 2 were further distinguished by their temporal appearance during infection and by the sizes of the replicating DNA as determined by sedimentation in neutral sucrose gradients. In stage 1, replicating molecules, between the size of unit and twice the unit length, were produced early in infection (2 h postinfection). In contrast, stage 2 of DNA replication occurred only after 3 h postinfection, and replicating molecules were large concatemers. Results of pulse-chase experiments showed that the concatemeric DNA served as the precursor for the production of mature FV3 DNA. Denaturation of concatemeric DNA with alkali or digestion with S1 nuclease reduced it to less than genome size molecules, indicating the presence of extensive single-stranded regions. Analysis of replicating DNA by equilibrium centrifugation in CsCl gradients after a pulse-chase suggested that these single-stranded regions were subsequently repaired. Based on these and previous data, a scheme of FV3 replication is presented. According to this scheme, FV3 utilizes the nucleus for early transcription and stage 1 of DNA replication. The viral DNA is then transported to the cytoplasm, where it participates in stage 2 DNA replication to form a concatemeric replication complex. The processing of concatemers to produce mature viral DNA and virus assembly also occurs in the cytoplasm. This mode of replication is strikingly different from any other known DNA virus. PMID:7109033

  18. Cloning of a very virulent plus, 686 strain of Marek's disease virus as a bacterial artificial chromosome.

    PubMed

    Reddy, Sanjay M; Sun, Aijun; Khan, Owais A; Lee, Lucy F; Lupiani, Blanca

    2013-06-01

    Bacterial artificial chromosome (BAC) vectors were first developed to facilitate propagation and manipulation of large DNA fragments. This technology was later used to clone full-length genomes of large DNA viruses to study viral gene function. Marek's disease virus (MDV) is a highly oncogenic herpesvirus that causes rapid induction of T-cell lymphomas in chickens. Based on the virus's ability to cause disease in vaccinated chickens, MDV strains are classified into pathotypes, with the most virulent strains belonging to the very virulent plus (vv+) pathotype. Here we report the construction of BAC clones of 686 (686-BAC), a vv+ strain of MDV. Transfection of DNA isolated from two independent clones into duck embryo fibroblasts resulted in recovery of infectious virus. Pathogenesis studies showed that the BAC-derived 686 viruses were more virulent than Md5, a vv strain of MDV. With the use of a two-step red-mediated mutagenesis process, both copies of viral interleukin 8 (vIL-8) were deleted from the MDV genome, showing that 686-BACs were amenable to mutagenesis techniques. The generation of BAC clones from a vv+ strain of MDV is a significant step toward understanding molecular basis of MDV pathogenesis. PMID:23901763

  19. Human cytomegalovirus induces JC virus DNA replication in human fibroblasts.

    PubMed Central

    Heilbronn, R; Albrecht, I; Stephan, S; Bürkle, A; zur Hausen, H

    1993-01-01

    JC virus, a human papovavirus, is the causative agent of the demyelinating brain disease progressive multifocal leucoencephalopathy (PML). PML is a rare but fatal disease which develops as a complication of severe immunosuppression. Latent JC virus is harbored by many asymptomatic carriers and is transiently reactivated from the latent state upon immunosuppression. JC virus has a very restricted host range, with human glial cells being the only tissue in which it can replicate at reasonable efficiency. Evidence that latent human cytomegalovirus is harbored in the kidney similar to latent JC virus led to the speculation that during episodes of impaired immunocompetence, cytomegalovirus might serve as helper virus for JC virus replication in otherwise nonpermissive cells. We show here that cytomegalovirus infection indeed leads to considerable JC virus DNA replication in cultured human fibroblasts that are nonpermissive for the replication of JC virus alone. Cytomegalovirus-mediated JC virus replication is dependent on the JC virus origin of replication and T antigen. Ganciclovir-induced inhibition of cytomegalovirus replication is associated with a concomitant inhibition of JC virus replication. These results suggest that reactivation of cytomegalovirus during episodes of immunosuppression might lead to activation of latent JC virus, which would enhance the probability of subsequent PML development. Ganciclovir-induced repression of both cytomegalovirus and JC virus replication may form the rational basis for the development of an approach toward treatment or prevention of PML. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8248262

  20. Paleozoic origin of insect large dsDNA viruses.

    PubMed

    Thézé, Julien; Bézier, Annie; Periquet, Georges; Drezen, Jean-Michel; Herniou, Elisabeth A

    2011-09-20

    To understand how extant viruses interact with their hosts, we need a historical framework of their evolutionary association. Akin to retrovirus or hepadnavirus viral fossils present in eukaryotic genomes, bracoviruses are integrated in braconid wasp genomes and are transmitted by Mendelian inheritance. However, unlike viral genomic fossils, they have retained functional machineries homologous to those of large dsDNA viruses pathogenic to arthropods. Using a phylogenomic approach, we resolved the relationships between bracoviruses and their closest free relatives: baculoviruses and nudiviruses. The phylogeny showed that bracoviruses are nested within the nudivirus clade. Bracoviruses establish a bridge between the virus and animal worlds. Their inclusion in a virus phylogeny allowed us to relate free viruses to fossils. The ages of the wasps were used to calibrate the virus phylogeny. Bayesian analyses revealed that insect dsDNA viruses first evolved at ∼310 Mya in the Paleozoic Era during the Carboniferous Period with the first insects. Furthermore the virus diversification time frame during the Mesozoic Era appears linked to the diversification of insect orders; baculoviruses that infect larvae evolved at the same period as holometabolous insects. These results imply ancient coevolution by resource tracking between several insect dsDNA virus families and their hosts, dating back to 310 Mya. PMID:21911395

  1. Comparison of commercial DNA extraction kits for isolation and purification of bacterial and eukaryotic DNA from PAH-contaminated soils.

    PubMed

    Mahmoudi, Nagissa; Slater, Greg F; Fulthorpe, Roberta R

    2011-08-01

    Molecular characterization of the microbial populations of soils and sediments contaminated with polycyclic aromatic hydrocarbons (PAHs) is often a first step in assessing intrinsic biodegradation potential. However, soils are problematic for molecular analysis owing to the presence of organic matter, such as humic acids. Furthermore, the presence of contaminants, such as PAHs, can cause further challenges to DNA extraction, quantification, and amplification. The goal of our study was to compare the effectiveness of four commercial soil DNA extraction kits (UltraClean Soil DNA Isolation kit, PowerSoil DNA Isolation kit, PowerMax Soil DNA Isolation kit, and FastDNA SPIN kit) to extract pure, high-quality bacterial and eukaryotic DNA from PAH-contaminated soils. Six different contaminated soils were used to determine if there were any biases among the kits due to soil properties or level of contamination. Extracted DNA was used as a template for bacterial 16S rDNA and eukaryotic 18S rDNA amplifications, and PCR products were subsequently analyzed using denaturing gel gradient electrophoresis (DGGE). We found that the FastDNA SPIN kit provided significantly higher DNA yields for all soils; however, it also resulted in the highest levels of humic acid contamination. Soil texture and organic carbon content of the soil did not affect the DNA yield of any kit. Moreover, a liquid-liquid extraction of the DNA extracts found no residual PAHs, indicating that all kits were effective at removing contaminants in the extraction process. Although the PowerSoil DNA Isolation kit gave relatively low DNA yields, it provided the highest quality DNA based on successful amplification of both bacterial and eukaryotic DNA for all six soils. DGGE fingerprints among the kits were dramatically different for both bacterial and eukaryotic DNA. The PowerSoil DNA Isolation kit revealed multiple bands for each soil and provided the most consistent DGGE profiles among replicates for both

  2. The Role of Bacterial Chaperones in the Circulative Transmission of Plant Viruses by Insect Vectors

    PubMed Central

    Kliot, Adi; Ghanim, Murad

    2013-01-01

    Persistent circulative transmission of plant viruses involves complex interactions between the transmitted virus and its insect vector. Several studies have shown that insect vector proteins are involved in the passage and the transmission of the virus. Interestingly, proteins expressed by bacterial endosymbionts that reside in the insect vector, were also shown to influence the transmission of these viruses. Thus far, the transmission of two plant viruses that belong to different virus genera was shown to be facilitated by a bacterial chaperone protein called GroEL. This protein was shown to be implicated in the transmission of Potato leafroll virus (PLRV) by the green peach aphid Myzus persicae, and the transmission of Tomato yellow leaf curl virus (TYLCV) by the sweetpotato whitefly Bemisia tabaci. These tri-trophic levels of interactions and their possible evolutionary implications are reviewed. PMID:23783810

  3. Regulation of bacterial metabolic activity by dissolved organic carbon and viruses

    NASA Astrophysics Data System (ADS)

    Xu, Jie; Jing, Hongmei; Sun, Mingming; Harrison, Paul J.; Liu, Hongbin

    2013-12-01

    regulation of bacterial metabolic activity by viruses and dissolved organic carbon (DOC) was examined using natural microbial communities in three treatments (active viruses, inactive viruses, and virus free) at two contrasting coastal sites (pristine vs. eutrophic) with substantial differences in environmental conditions during the wet and dry seasons. Our results showed that net growth rates and production of bacterioplankton were reduced primarily by viruses via repressing metabolically active bacteria with high nucleic acid (HNA) content which had a high capacity for incorporating carbon, while bacterial respiration was primarily regulated by DOC lability. The quality of organic matter played a more important role in regulating bacterial growth efficiency (BGE) than the supply of organic matter in eutrophic coastal waters. The lack of HMW-DOC and high carbon demand in the virus-free treatment resulted in a significant increase in cell-specific bacterial respiration, which was responsible for the lowest bacterial growth efficiency among the three treatments. The presence of viruses did not necessarily lower bacterial growth efficiency since virus-induced mortality alleviated bacterial carbon demand and enhanced carbon cycling. Virus-induced mortality was greater in relatively pristine waters than eutrophic waters, likely since the high supply of substrates alleviated the pressure of viral infection, through extracellular proteases produced by bacteria, which might result in the hydrolytic destruction or modification of viral capsids. An important implication of our results was that the input of riverine DOC and nutrients improved bacterial metabolic activity by alleviating virus-induced mortality of bacteria in estuarine and coastal waters.

  4. Examination of Bacterial Inhibition Using a Catalytic DNA

    PubMed Central

    Qu, Long; Ali, M. Monsur; Aguirre, Sergio D.; Liu, Hongxia; Jiang, Yuyang; Li, Yingfu

    2014-01-01

    Determination of accurate dosage of existing antibiotics and discovery of new antimicrobials or probiotics entail simple but effective methods that can conveniently track bacteria growth and inhibition. Here we explore the application of a previously reported fluorogenic E. coli-specific DNAzyme (catalytic DNA), RFD-EC1, as a molecular probe for monitoring bacterial inhibition exerted by antibiotics and for studying bacterial competition as a result of cohabitation. Because the DNAzyme method provides a convenient way to monitor the growth of E. coli, it is capable of determining the minimal inhibitory concentration (MIC) of antibiotics much faster than the conventional optical density (OD) method. In addition, since the target for RFD-EC1 is an extracellular protein molecule from E. coli, RFD-EC1 is able to identify pore-forming antibiotics or compounds that can cause membrane leakage. Finally, RFD-EC1 can be used to analyse the competition of cohabitating bacteria, specifically the inhibition of growth of E. coli by Bacillus subtilis. The current work represents the first exploration of a catalytic DNA for microbiological applications and showcases the utility of bacteria-sensing fluorogenic DNAzymes as simple molecular probes to facilitate antibiotic and probiotic research. PMID:25531274

  5. The role of variable DNA tandem repeats in bacterial adaptation.

    PubMed

    Zhou, Kai; Aertsen, Abram; Michiels, Chris W

    2014-01-01

    DNA tandem repeats (TRs), also designated as satellite DNA, are inter- or intragenic nucleotide sequences that are repeated two or more times in a head-to-tail manner. Because TR tracts are prone to strand-slippage replication and recombination events that cause the TR copy number to increase or decrease, loci containing TRs are hypermutable. An increasing number of examples illustrate that bacteria can exploit this instability of TRs to reversibly shut down or modulate the function of specific genes, allowing them to adapt to changing environments on short evolutionary time scales without an increased overall mutation rate. In this review, we discuss the prevalence and distribution of inter- and intragenic TRs in bacteria and the mechanisms of their instability. In addition, we review evidence demonstrating a role of TR variations in bacterial adaptation strategies, ranging from immune evasion and tissue tropism to the modulation of environmental stress tolerance. Nevertheless, while bioinformatic analysis reveals that most bacterial genomes contain a few up to several dozens of intra- and intergenic TRs, only a small fraction of these have been functionally studied to date. PMID:23927439

  6. Examination of bacterial inhibition using a catalytic DNA.

    PubMed

    Qu, Long; Ali, M Monsur; Aguirre, Sergio D; Liu, Hongxia; Jiang, Yuyang; Li, Yingfu

    2014-01-01

    Determination of accurate dosage of existing antibiotics and discovery of new antimicrobials or probiotics entail simple but effective methods that can conveniently track bacteria growth and inhibition. Here we explore the application of a previously reported fluorogenic E. coli-specific DNAzyme (catalytic DNA), RFD-EC1, as a molecular probe for monitoring bacterial inhibition exerted by antibiotics and for studying bacterial competition as a result of cohabitation. Because the DNAzyme method provides a convenient way to monitor the growth of E. coli, it is capable of determining the minimal inhibitory concentration (MIC) of antibiotics much faster than the conventional optical density (OD) method. In addition, since the target for RFD-EC1 is an extracellular protein molecule from E. coli, RFD-EC1 is able to identify pore-forming antibiotics or compounds that can cause membrane leakage. Finally, RFD-EC1 can be used to analyse the competition of cohabitating bacteria, specifically the inhibition of growth of E. coli by Bacillus subtilis. The current work represents the first exploration of a catalytic DNA for microbiological applications and showcases the utility of bacteria-sensing fluorogenic DNAzymes as simple molecular probes to facilitate antibiotic and probiotic research. PMID:25531274

  7. DNA confinement drives uncoating of the HIV Virus

    NASA Astrophysics Data System (ADS)

    Rouzina, I.; Bruinsma, R.

    2014-09-01

    The enzyme reverse transcriptase converts single-stranded RNA molecules into double-stranded DNA molecules inside mature HIV viral capsids. We present a model for the uncoating of the HIV virus where the capsid uncoating process is driven by the confinement force exerted on the capsid wall porduced to the double-stranded DNA generated by reverse transcriptase.

  8. Infectious Maize rayado fino virus from cloned cDNA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize rayado fino virus (MRFV) is the type member of the marafiviruses within the family Tymoviridae. A cDNA clone from which infectious RNA can be transcribed was produced from a US isolate of MRFV (MRFV-US). Infectivity of transcripts derived from cDNA clones was demonstrated by infection of mai...

  9. Identification of a novel circular DNA virus in pig feces

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Metagenomic analysis of fecal samples collected from a swine with diarrhea detected sequences encoding a replicase (Rep) protein typically found in small circular Rep-encoding ssDNA (CRESS-DNA) viruses. The complete 3,062 nucleotide genome was generated and found to encode two bi-directionally trans...

  10. Taxonomic distribution of large DNA viruses in the sea

    PubMed Central

    Monier, Adam; Claverie, Jean-Michel; Ogata, Hiroyuki

    2008-01-01

    Background Viruses are ubiquitous and the most abundant biological entities in marine environments. Metagenomics studies are increasingly revealing the huge genetic diversity of marine viruses. In this study, we used a new approach - 'phylogenetic mapping' - to obtain a comprehensive picture of the taxonomic distribution of large DNA viruses represented in the Sorcerer II Global Ocean Sampling Expedition metagenomic data set. Results Using DNA polymerase genes as a taxonomic marker, we identified 811 homologous sequences of likely viral origin. As expected, most of these sequences corresponded to phages. Interestingly, the second largest viral group corresponded to that containing mimivirus and three related algal viruses. We also identified several DNA polymerase homologs closely related to Asfarviridae, a viral family poorly represented among isolated viruses and, until now, limited to terrestrial animal hosts. Finally, our approach allowed the identification of a new combination of genes in 'viral-like' sequences. Conclusion Albeit only recently discovered, giant viruses of the Mimiviridae family appear to constitute a diverse, quantitatively important and ubiquitous component of the population of large eukaryotic DNA viruses in the sea. PMID:18598358

  11. STATIC AND KINETIC SITE-SPECIFIC PROTEIN-DNA PHOTOCROSSLINKING: ANALYSIS OF BACTERIAL TRANSCRIPTION INITIATION COMPLEXES

    PubMed Central

    Naryshkin, Nikolai; Druzhinin, Sergei; Revyakin, Andrei; Kim, Younggyu; Mekler, Vladimir; Ebright, Richard H.

    2009-01-01

    Static site-specific protein-DNA photocrosslinking permits identification of protein-DNA interactions within multiprotein-DNA complexes. Kinetic site-specific protein-DNA photocrosslinking--involving rapid-quench-flow mixing and pulsed-laser irradiation--permits elucidation of pathways and kinetics of formation of protein-DNA interactions within multiprotein-DNA complexes. We present detailed protocols for application of static and kinetic site-specific protein-DNA photocrosslinking to bacterial transcription initiation complexes. PMID:19378179

  12. Rescue of recombinant Newcastle disease virus from cDNA.

    PubMed

    Ayllon, Juan; García-Sastre, Adolfo; Martínez-Sobrido, Luis

    2013-01-01

    Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae(1), is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA(2-5). Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs. PMID:24145366

  13. Rescue of Recombinant Newcastle Disease Virus from cDNA

    PubMed Central

    Ayllon, Juan; García-Sastre, Adolfo; Martínez-Sobrido, Luis

    2013-01-01

    Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs. PMID:24145366

  14. Generation of an infectious Negev virus cDNA clone

    PubMed Central

    Gorchakov, Rodion V.; Tesh, Robert B.; Weaver, Scott C.

    2014-01-01

    The genus Negevirus consists of insect-only viruses isolated from mosquitoes and sandflies. Here, we report the successful construction of a full-length infectious cDNA clone of Negev virus (NEGV) strain M30957. Viral RNA was transcribed in vitro and virus was readily rescued with or without the use of a cap analogue. These results strongly suggest that NEGV, and likely other members within the genus, is a non-segmented, single-stranded, positive-sense RNA virus. PMID:24878640

  15. Origin of giant viruses from smaller DNA viruses not from a fourth domain of cellular life

    PubMed Central

    Yutin, Natalya; Wolf, Yuri I.; Koonin, Eugene V.

    2015-01-01

    The numerous and diverse eukaryotic viruses with large double-stranded DNA genomes that at least partially reproduce in the cytoplasm of infected cells apparently evolved from a single virus ancestor. This major group of viruses is known as Nucleocytoplasmic Large DNA Viruses (NCLDV) or the proposed order Megavirales. Among the “Megavirales”, there are three groups of giant viruses with genomes exceeding 500 kb, namely Mimiviruses, Pithoviruses, and Pandoraviruses that hold the current record of viral genome size, about 2.5 Mb. Phylogenetic analysis of conserved, ancestral NLCDV genes clearly shows that these three groups of giant viruses have three distinct origins within the “Megavirales”. The Mimiviruses constitute a distinct family that is distantly related to Phycodnaviridae, Pandoraviruses originate from a common ancestor with Coccolithoviruses within the Phycodnaviridae family, and Pithoviruses are related to Iridoviridae and Marseilleviridae. Maximum likelihood reconstruction of gene gain and loss events during the evolution of the “Megavirales” indicates that each group of giant viruses evolved from viruses with substantially smaller and simpler gene repertoires. Initial phylogenetic analysis of universal genes, such as translation system components, encoded by some giant viruses, in particular Mimiviruses, has led to the hypothesis that giant viruses descend from a fourth, probably extinct domain of cellular life. The results of our comprehensive phylogenomic analysis of giant viruses refute the fourth domain hypothesis and instead indicate that the universal genes have been independently acquired by different giant viruses from their eukaryotic hosts. PMID:25042053

  16. Frequency of varicella zoster virus DNA in human adrenal glands.

    PubMed

    Badani, Hussain; White, Teresa; Schulick, Nicole; Raeburn, Christopher D; Topkaya, Ibrahim; Gilden, Don; Nagel, Maria A

    2016-06-01

    Varicella zoster virus (VZV) becomes latent in ganglionic neurons derived from neural crest cells. Because the adrenal gland also contains medullary chromaffin cells of neural crest origin, we examined human adrenal glands and medullary chromaffin cell tumors (pheochromocytomas) for VZV and herpes simplex virus type 1 (HSV-1). We found VZV, but not HSV-1, DNA in 4/63 (6 %) normal adrenal glands. No VZV transcripts or antigens were detected in the 4 VZV DNA-positive samples. No VZV or HSV-1 DNA was found in 21 pheochromocytomas. PMID:26843382

  17. Replication of herpes simplex virus DNA: localization of replication recognition signals within defective virus genomes.

    PubMed Central

    Vlazny, D A; Frenkel, N

    1981-01-01

    Serially passaged herpes simplex virus type 1 (HSV-1) strain Justin was previously shown to contain defective virus genomes consisting of head-to-tail reiterations of sequences derived from the end of the S component of the standard virus DNA. Cotransfection of purified monomeric defective genome repeat units with foster helper virus DNAs onto rabbit skin cells resulted in regeneration and replication of concatemeric defective DNA molecules which were successfully encapsidated. Thus, defective HSV-1 (Justin) genomes contain, within their limited DNA sequences, a sufficient set of recognition sites required for HSV DNA replication and packaging. The arrangement of repeat units within the regenerated defective virus genomes was consistent with their replication by a rolling circle mechanism in which a single repeat unit served as the circularized template. This replication occurred most actively late after infection and could be shown to be inhibited by low concentrations of phosphonoacetate known to inhibit the HSV-specified viral DNA polymerase selectively. The resultant concatemers were shown to be cleaved to Mr 100 X 10(6) DNA molecules which were terminated at one end with the proper ac end sequence of the parental standard virus DNA. Images PMID:6262768

  18. Compiling Multicopy Single-Stranded DNA Sequences from Bacterial Genome Sequences

    PubMed Central

    Yoo, Wonseok; Lim, Dongbin

    2016-01-01

    A retron is a bacterial retroelement that encodes an RNA gene and a reverse transcriptase (RT). The former, once transcribed, works as a template primer for reverse transcription by the latter. The resulting DNA is covalently linked to the upstream part of the RNA; this chimera is called multicopy single-stranded DNA (msDNA), which is extrachromosomal DNA found in many bacterial species. Based on the conserved features in the eight known msDNA sequences, we developed a detection method and applied it to scan National Center for Biotechnology Information (NCBI) RefSeq bacterial genome sequences. Among 16,844 bacterial sequences possessing a retron-type RT domain, we identified 48 unique types of msDNA. Currently, the biological role of msDNA is not well understood. Our work will be a useful tool in studying the distribution, evolution, and physiological role of msDNA. PMID:27103888

  19. Shared catalysis in virus entry and bacterial cell wall depolymerization.

    PubMed

    Cohen, Daniel N; Sham, Yuk Y; Haugstad, Greg D; Xiang, Ye; Rossmann, Michael G; Anderson, Dwight L; Popham, David L

    2009-04-01

    Bacterial virus entry and cell wall depolymerization require the breakdown of peptidoglycan (PG), the peptide-cross-linked polysaccharide matrix that surrounds bacterial cells. Structural studies of lysostaphin, a PG lytic enzyme (autolysin), have suggested that residues in the active site facilitate hydrolysis, but a clear mechanism for this reaction has remained unsolved. The active-site residues and a structural pattern of beta-sheets are conserved among lysostaphin homologs (such as LytM of Staphylococcus aureus) and the C-terminal domain of gene product 13 (gp13), a protein at the tail tip of the Bacillus subtilis bacteriophage varphi29. gp13 activity on PG and muropeptides was assayed using high-performance liquid chromatography, and gp13 was found to be a d,d-endopeptidase that cleaved the peptide cross-link. Computational modeling of the B. subtilis cross-linked peptide into the gp13 active site suggested that Asp195 may facilitate scissile-bond activation and that His247 is oriented to mediate nucleophile generation. To our knowledge, this is the first model of a Zn(2)(+) metallopeptidase and its substrate. Residue Asp195 of gp13 was found to be critical for Zn(2)(+) binding and catalysis by substitution mutagenesis with Ala or Cys. Circular dichroism and particle-induced X-ray emission spectroscopy showed that the general protein folding and Zn(2)(+) binding were maintained in the Cys mutant but reduced in the Ala mutant. These findings together support a model in which the Asp195 and His247 in gp13 and homologous residues in the LytM and lysostaphin active sites facilitate hydrolysis of the peptide substrate that cross-links PG. Thus, these autolysins and phage-entry enzymes have a shared chemical mechanism of action. PMID:19361422

  20. A virus of hyperthermophilic archaea with a unique architecture among DNA viruses.

    PubMed

    Rensen, Elena Ilka; Mochizuki, Tomohiro; Quemin, Emmanuelle; Schouten, Stefan; Krupovic, Mart; Prangishvili, David

    2016-03-01

    Viruses package their genetic material in diverse ways. Most known strategies include encapsulation of nucleic acids into spherical or filamentous virions with icosahedral or helical symmetry, respectively. Filamentous viruses with dsDNA genomes are currently associated exclusively with Archaea. Here, we describe a filamentous hyperthermophilic archaeal virus, Pyrobaculum filamentous virus 1 (PFV1), with a type of virion organization not previously observed in DNA viruses. The PFV1 virion, 400 ± 20 × 32 ± 3 nm, contains an envelope and an inner core consisting of two structural units: a rod-shaped helical nucleocapsid formed of two 14-kDa major virion proteins and a nucleocapsid-encompassing protein sheath composed of a single major virion protein of 18 kDa. The virion organization of PFV1 is superficially similar to that of negative-sense RNA viruses of the family Filoviridae, including Ebola virus and Marburg virus. The linear dsDNA of PFV1 carries 17,714 bp, including 60-bp-long terminal inverted repeats, and contains 39 predicted ORFs, most of which do not show similarities to sequences in public databases. PFV1 is a lytic virus that completely disrupts the host cell membrane at the end of the infection cycle. PMID:26884161

  1. A virus of hyperthermophilic archaea with a unique architecture among DNA viruses

    PubMed Central

    Rensen, Elena Ilka; Mochizuki, Tomohiro; Quemin, Emmanuelle; Schouten, Stefan; Krupovic, Mart; Prangishvili, David

    2016-01-01

    Viruses package their genetic material in diverse ways. Most known strategies include encapsulation of nucleic acids into spherical or filamentous virions with icosahedral or helical symmetry, respectively. Filamentous viruses with dsDNA genomes are currently associated exclusively with Archaea. Here, we describe a filamentous hyperthermophilic archaeal virus, Pyrobaculum filamentous virus 1 (PFV1), with a type of virion organization not previously observed in DNA viruses. The PFV1 virion, 400 ± 20 × 32 ± 3 nm, contains an envelope and an inner core consisting of two structural units: a rod-shaped helical nucleocapsid formed of two 14-kDa major virion proteins and a nucleocapsid-encompassing protein sheath composed of a single major virion protein of 18 kDa. The virion organization of PFV1 is superficially similar to that of negative-sense RNA viruses of the family Filoviridae, including Ebola virus and Marburg virus. The linear dsDNA of PFV1 carries 17,714 bp, including 60-bp-long terminal inverted repeats, and contains 39 predicted ORFs, most of which do not show similarities to sequences in public databases. PFV1 is a lytic virus that completely disrupts the host cell membrane at the end of the infection cycle. PMID:26884161

  2. Subversion and piracy: DNA viruses and immune evasion.

    PubMed

    Haig, D M

    2001-06-01

    During the co-evolution of viruses with their vertebrate hosts, the DNA viruses have acquired an impressive array of immunomodulatory genes to combat host immune responses and their hosts have developed a sophisticated immune system to contain virus infections. In order to replicate, the viruses have evolved mechanisms to inhibit key host anti-virus responses that include apoptosis, interferon production, chemokine production, inflammatory cytokine production, and the activity of cytotoxic T-cells, natural killer cells and antibody. In addition, some of the viruses encode cytokine or chemokine homologues that recruit or expand cell numbers for infection or that subvert the host cellular response from a protective response to a benign one. The specificity of the viral immunomodulatory molecules reflects the life cycle and the pathogenesis of the viruses. Herpesviruses achieve latency in host cells by inducing cell survival and protecting infected cells from immune recognition. This involves interference with cell signal transduction pathways. Many of the viral immunomodulatory proteins are homologues of host proteins that appear to have been pirated from the host and reassorted in the virus genomes. Some of these have unique functions and indicate novel or important aspects of both viral pathogenesis and host immunity to viruses. The specific example of orf virus infection of sheep is described. PMID:11676616

  3. SERE, a widely dispersed bacterial repetitive DNA element.

    PubMed

    Rajashekara, G; Koeuth, T; Nevile, S; Back, A; Nagaraja, K V; Lupski, J R; Kapur, V

    1998-06-01

    The presence of a Salmonella serotype Enteritidis repeat element (SERE) located within the upstream regulatory region of the sefABCD operon encoding fimbrial proteins is reported. DNA dot-blot hybridisation analyses and computerised searches of genetic databases indicate that SERE is well conserved and widely distributed throughout the bacterial and archaeal kingdoms. A SERE-based polymerase chain reaction (SERE-PCR) assay was developed to fingerprint 54 isolates of Enteritidis representing nine distinct phage types and 54 isolates of other Salmonella serotypes. SERE-PCR identified five distinct fingerprint profiles among the 54 Enteritidis isolates; no correlation between phage types and SERE-PCR fingerprint patterns was noticed. SERE-PCR was reproducible, rapid and easy to perform. The results of this investigation suggest that the limited heterogeneity of SERE-PCR fingerprint patterns can be utilised to develop serotype- and serogroup-specific fingerprint patterns for isolates of Enteritidis. PMID:9879967

  4. DNA Viruses: The Really Big Ones (Giruses)

    PubMed Central

    Van Etten, James L.; Lane, Leslie C.; Dunigan, David D.

    2010-01-01

    Viruses with genomes greater than 300 kb and up to 1200 kb are being discovered with increasing frequency. These large viruses (often called giruses) can encode up to 900 proteins and also many tRNAs. Consequently, these viruses have more protein-encoding genes than many bacteria, and the concept of small particle/small genome that once defined viruses is no longer valid. Giruses infect bacteria and animals although most of the recently discovered ones infect protists. Thus, genome gigantism is not restricted to a specific host or phylogenetic clade. To date, most of the giruses are associated with aqueous environments. Many of these large viruses (phycodnaviruses and Mimiviruses) probably have a common evolutionary ancestor with the poxviruses, iridoviruses, asfarviruses, ascoviruses, and a recently discovered Marseillevirus. One issue that is perhaps not appreciated by the microbiology community is that large viruses, even ones classified in the same family, can differ significantly in morphology, lifestyle, and genome structure. This review focuses on some of these differences rather than provides extensive details about individual viruses. PMID:20690825

  5. Interactions of Dnd proteins involved in bacterial DNA phosphorothioate modification

    PubMed Central

    Xiong, Wei; Zhao, Gong; Yu, Hao; He, Xinyi

    2015-01-01

    DNA phosphorothioation (PT) is the first discovered physiological DNA backbone modification, in which a non-bridging oxygen atom of the phosphodiester bond is replaced with a sulfur atom in Rp (rectus for plane) configuration. PT modification is governed by a highly conserved gene cluster dndA/iscS-dndBCDE that is widespread across bacterial and archaeal species. However, little is known about how these proteins coordinately react with each other to perform oxygen–sulfur swap. We here demonstrated that IscS, DndC, DndD and DndE form a protein complex of which the molecular ratio for four proteins in the complex is approximate 1:1:1:1. DndB here displayed little or weak affinity to the complex and the constructs harboring dndACDE can confer the host in vivo PT modification. Using co-purification and pull down strategy, we demonstrated that the four proteins assemble into a pipeline in collinear to its gene organization, namely, IscS binding to DndC, DndC binding to DndD, and DndD binding to DndE. Moreover, weak interactions between DndE and IscS, DndE and DndC were also identified. PMID:26539172

  6. Detection of bacterial pathogens in environmental samples using DNA microarrays.

    PubMed

    Call, Douglas R; Borucki, Monica K; Loge, Frank J

    2003-05-01

    Polymerase chain reaction (PCR) is an important tool for pathogen detection, but historically, it has not been possible to accurately identify PCR products without sequencing, Southern blots, or dot-blots. Microarrays can be coupled with PCR where they serve as a set of parallel dot-blots to enhance product detection and identification. Microarrays are composed of many discretely located probes on a solid substrate such as glass. Each probe is composed of a sequence that is complimentary to a pathogen-specific gene sequence. PCR is used to amplify one or more genes and the products are then hybridized to the array to identify species-specific polymorphism within one or more genes. We illustrate this type of array using 16S rDNA probes suitable for distinguishing between several salmonid pathogens. We also describe the use of microarrays for direct detection of either RNA or DNA without the aid of PCR, although the sensitivity of these systems currently limits their application for pathogen detection. Finally, microarrays can also be used to "fingerprint" bacterial isolates and they can be used to identify diagnostic markers suitable for developing new PCR-based detection assays. We illustrate this type of array for subtyping an important food-borne pathogen, Listeria monocytogenes. PMID:12654494

  7. Adenosine triphosphatases of thermophilic archaeal double-stranded DNA viruses

    PubMed Central

    2014-01-01

    Adenosine triphosphatases (ATPases) of double-stranded (ds) DNA archaeal viruses are structurally related to the AAA+ hexameric helicases and translocases. These ATPases have been implicated in viral life cycle functions such as DNA entry into the host, and viral genome packaging into preformed procapsids. We summarize bioinformatical analyses of a wide range of archaeal ATPases, and review the biochemical and structural properties of those archaeal ATPases that have measurable ATPase activity. We discuss their potential roles in genome delivery into the host, virus assembly and genome packaging in comparison to hexameric helicases and packaging motors from bacteriophages. PMID:25105011

  8. Next generation sequencing of DNA-launched Chikungunya vaccine virus.

    PubMed

    Hidajat, Rachmat; Nickols, Brian; Forrester, Naomi; Tretyakova, Irina; Weaver, Scott; Pushko, Peter

    2016-03-01

    Chikungunya virus (CHIKV) represents a pandemic threat with no approved vaccine available. Recently, we described a novel vaccination strategy based on iDNA® infectious clone designed to launch a live-attenuated CHIKV vaccine from plasmid DNA in vitro or in vivo. As a proof of concept, we prepared iDNA plasmid pCHIKV-7 encoding the full-length cDNA of the 181/25 vaccine. The DNA-launched CHIKV-7 virus was prepared and compared to the 181/25 virus. Illumina HiSeq2000 sequencing revealed that with the exception of the 3' untranslated region, CHIKV-7 viral RNA consistently showed a lower frequency of single-nucleotide polymorphisms than the 181/25 RNA including at the E2-12 and E2-82 residues previously identified as attenuating mutations. In the CHIKV-7, frequencies of reversions at E2-12 and E2-82 were 0.064% and 0.086%, while in the 181/25, frequencies were 0.179% and 0.133%, respectively. We conclude that the DNA-launched virus has a reduced probability of reversion mutations, thereby enhancing vaccine safety. PMID:26855330

  9. Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

    PubMed Central

    Kennedy, Edward M.; Cullen, Bryan R.

    2015-01-01

    CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

  10. Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment.

    PubMed

    Kennedy, Edward M; Cullen, Bryan R

    2015-05-01

    CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

  11. Reconstruction of a Bacterial Genome from DNA Cassettes

    SciTech Connect

    Christopher Dupont; John Glass; Laura Sheahan; Shibu Yooseph; Lisa Zeigler Allen; Mathangi Thiagarajan; Andrew Allen; Robert Friedman; J. Craig Venter

    2011-12-31

    This basic research program comprised two major areas: (1) acquisition and analysis of marine microbial metagenomic data and development of genomic analysis tools for broad, external community use; (2) development of a minimal bacterial genome. Our Marine Metagenomic Diversity effort generated and analyzed shotgun sequencing data from microbial communities sampled from over 250 sites around the world. About 40% of the 26 Gbp of sequence data has been made publicly available to date with a complete release anticipated in six months. Our results and those mining the deposited data have revealed a vast diversity of genes coding for critical metabolic processes whose phylogenetic and geographic distributions will enable a deeper understanding of carbon and nutrient cycling, microbial ecology, and rapid rate evolutionary processes such as horizontal gene transfer by viruses and plasmids. A global assembly of the generated dataset resulted in a massive set (5Gbp) of genome fragments that provide context to the majority of the generated data that originated from uncultivated organisms. Our Synthetic Biology team has made significant progress towards the goal of synthesizing a minimal mycoplasma genome that will have all of the machinery for independent life. This project, once completed, will provide fundamentally new knowledge about requirements for microbial life and help to lay a basic research foundation for developing microbiological approaches to bioenergy.

  12. Herpes simplex virus amplicon: effect of size on replication of constructed defective genomes containing eucaryotic DNA sequences.

    PubMed Central

    Kwong, A D; Frenkel, N

    1984-01-01

    Previous studies (R. R. Spaete and N. Frenkel, Cell 30:295-304, 1982) have documented the potential use of defective virus vectors (amplicons) derived from herpes simplex virus for the efficient introduction of foreign DNA sequences into eucaryotic cells. Specifically, cotransfection of cells with helper virus DNA and cloned amplicons (8 to 10 kilobases [kb]) containing bacterial plasmid DNA sequences linked to a set of herpes simplex virus cis-acting propagation signals (a replication origin and a cleavage-packaging signal) resulted in the generation of virus stocks containing packaged defective genomes that consisted of uniform head-to-tail reiterations of the chimeric seed amplicon sequences. The chimeric defective genomes could be stably propagated in virus stocks and could thus be used to efficiently infect cells. We now report on additional studies designed to propagate relatively large sets of eucaryotic DNA sequences within chimeric packaged defective genomes. These studies have utilized a 12-kb chicken DNA sequence encoding the chicken ovalbumin gene and cloned by Lai et al. (Proc. Natl. Acad. Sci. U.S.A. 77:244-248, 1980) in the plasmid pOV12. Virus stocks derived from cells cotransfected with helper virus DNA and chimeric amplicons (overall size of 19.8 kb, of which 12 kb corresponded to the chicken DNA) contained defective genomes composed of reiterations of the 19.8-kb seed amplicon sequences. However, in addition to the authentically sized repeat units, defective genomes in the derivative virus stocks contained smaller repeat units representing deleted versions of the seed 19.8-kb amplicons. The recombinational events leading to the formation of deleted repeats did not appear to occur at unique sites, as shown by comparative analyses of multiple, independently generated virus series propagated from separate transfections. In contast, seed amplicons ranging in size from 11 to 15 kb and containing subsets of the 12-kb chicken DNA sequences replicated

  13. Influence of Calcium in Extracellular DNA Mediated Bacterial Aggregation and Biofilm Formation

    PubMed Central

    Koop, Leena; Wong, Yie Kuan; Ahmed, Safia; Siddiqui, Khawar Sohail; Manefield, Mike

    2014-01-01

    Calcium (Ca2+) has an important structural role in guaranteeing the integrity of the outer lipopolysaccharide layer and cell walls of bacterial cells. Extracellular DNA (eDNA) being part of the slimy matrix produced by bacteria promotes biofilm formation through enhanced structural integrity of the matrix. Here, the concurrent role of Ca2+ and eDNA in mediating bacterial aggregation and biofilm formation was studied for the first time using a variety of bacterial strains and the thermodynamics of DNA to Ca2+ binding. It was found that the eDNA concentrations under both planktonic and biofilm growth conditions were different among bacterial strains. Whilst Ca2+ had no influence on eDNA release, presence of eDNA by itself favours bacterial aggregation via attractive acid-base interactions in addition, its binding with Ca2+ at biologically relevant concentrations was shown further increase in bacterial aggregation via cationic bridging. Negative Gibbs free energy (ΔG) values in iTC data confirmed that the interaction between DNA and Ca2+ is thermodynamically favourable and that the binding process is spontaneous and exothermic owing to its highly negative enthalpy. Removal of eDNA through DNase I treatment revealed that Ca2+ alone did not enhance cell aggregation and biofilm formation. This discovery signifies the importance of eDNA and concludes that existence of eDNA on bacterial cell surfaces is a key facilitator in binding of Ca2+ to eDNA thereby mediating bacterial aggregation and biofilm formation. PMID:24651318

  14. Herpes simplex virus induces the replication of foreign DNA.

    PubMed Central

    Danovich, R M; Frenkel, N

    1988-01-01

    Plasmids containing the simian virus 40 (SV40) DNA replication origin and the large T gene are replicated efficiently in Vero monkey cells but not in rabbit skin cells. Efficient replication of the plasmids was observed in rabbit skin cells infected with herpes simplex virus type 1 (HSV-1) and HSV-2. The HSV-induced replication required the large T antigen and the SV40 replication origin. However, it produced concatemeric molecules resembling replicative intermediates of HSV DNA and was sensitive to phosphonoacetate at concentrations known to inhibit the HSV DNA polymerase. Therefore, it involved the HSV DNA polymerase itself or a viral gene product(s) which was expressed following the replication of HSV DNA. Analyses of test plasmids lacking SV40 or HSV DNA sequences showed that, under some conditions, HSV also induced low-level replication of test plasmids containing no known eucaryotic replication origins. Together, these results show that HSV induces a DNA replicative activity which amplifies foreign DNA. The relevance of these findings to the putative transforming potential of HSV is discussed. Images PMID:2850486

  15. Herpes simplex virus induces the replication of foreign DNA

    SciTech Connect

    Danovich, R.M.; Frenkel, N.

    1988-08-01

    Plasmids containing the simian virus 40 (SV40) DNA replication origin and the large T gene are replicated in Vero monkey cells but not in rabbit skin cells. Efficient replication of the plasmids was observed in rabbit cells infected with herpes simplex virus type 1 (HSV-1) and HSV-2. The HSV-induced replication required the large T antigen and the SV40 replication origin. However, it produced concatemeric molecules resembling replicative intermediates of HSV DNA and was sensitive to phosphonoacetate at concentrations known to inhibit the HSV DNA polymerase. Therefore, it involved the HSV DNA polymerase itself or a viral gene product(s) which was expressed following the replication of HSV DNA. Analyses of test plasmids lacking SV40 or HSV DNA sequences showed that, under some conditions. HSV also induced low-level replication of test plasmids containing no known eucaryotic replication origins. Together, these results show that HSV induces a DNA replicative activity which amplifies foreign DNA. The relevance of these findings to the putative transforming potential of HSV is discussed.

  16. Biodiversity and distribution of polar freshwater DNA viruses

    PubMed Central

    Aguirre de Cárcer, Daniel; López-Bueno, Alberto; Pearce, David A.; Alcamí, Antonio

    2015-01-01

    Viruses constitute the most abundant biological entities and a large reservoir of genetic diversity on Earth. Despite the recent surge in their study, our knowledge on their actual biodiversity and distribution remains sparse. We report the first metagenomic analysis of Arctic freshwater viral DNA communities and a comparative analysis with other freshwater environments. Arctic viromes are dominated by unknown and single-stranded DNA viruses with no close relatives in the database. These unique viral DNA communities mostly relate to each other and present some minor genetic overlap with other environments studied, including an Arctic Ocean virome. Despite common environmental conditions in polar ecosystems, the Arctic and Antarctic DNA viromes differ at the fine-grain genetic level while sharing a similar taxonomic composition. The study uncovers some viral lineages with a bipolar distribution, suggesting a global dispersal capacity for viruses, and seemingly indicates that viruses do not follow the latitudinal diversity gradient known for macroorganisms. Our study sheds light into the global biogeography and connectivity of viral communities. PMID:26601189

  17. Biodiversity and distribution of polar freshwater DNA viruses.

    PubMed

    Aguirre de Cárcer, Daniel; López-Bueno, Alberto; Pearce, David A; Alcamí, Antonio

    2015-06-01

    Viruses constitute the most abundant biological entities and a large reservoir of genetic diversity on Earth. Despite the recent surge in their study, our knowledge on their actual biodiversity and distribution remains sparse. We report the first metagenomic analysis of Arctic freshwater viral DNA communities and a comparative analysis with other freshwater environments. Arctic viromes are dominated by unknown and single-stranded DNA viruses with no close relatives in the database. These unique viral DNA communities mostly relate to each other and present some minor genetic overlap with other environments studied, including an Arctic Ocean virome. Despite common environmental conditions in polar ecosystems, the Arctic and Antarctic DNA viromes differ at the fine-grain genetic level while sharing a similar taxonomic composition. The study uncovers some viral lineages with a bipolar distribution, suggesting a global dispersal capacity for viruses, and seemingly indicates that viruses do not follow the latitudinal diversity gradient known for macroorganisms. Our study sheds light into the global biogeography and connectivity of viral communities. PMID:26601189

  18. Terminal region sequence variations in variola virus DNA.

    PubMed

    Massung, R F; Loparev, V N; Knight, J C; Totmenin, A V; Chizhikov, V E; Parsons, J M; Safronov, P F; Gutorov, V V; Shchelkunov, S N; Esposito, J J

    1996-07-15

    Genome DNA terminal region sequences were determined for a Brazilian alastrim variola minor virus strain Garcia-1966 that was associated with an 0.8% case-fatality rate and African smallpox strains Congo-1970 and Somalia-1977 associated with variola major (9.6%) and minor (0.4%) mortality rates, respectively. A base sequence identity of > or = 98.8% was determined after aligning 30 kb of the left- or right-end region sequences with cognate sequences previously determined for Asian variola major strains India-1967 (31% death rate) and Bangladesh-1975 (18.5% death rate). The deduced amino acid sequences of putative proteins of > or = 65 amino acids also showed relatively high identity, although the Asian and African viruses were clearly more related to each other than to alastrim virus. Alastrim virus contained only 10 of 70 proteins that were 100% identical to homologs in Asian strains, and 7 alastrim-specific proteins were noted. PMID:8661439

  19. Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA glycosylase

    SciTech Connect

    Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert; Chattopadhyay, Debasish

    2015-06-02

    Background: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. Results: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This also represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. In comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. Conclusion: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.

  20. Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA glycosylase

    DOE PAGESBeta

    Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert; Chattopadhyay, Debasish

    2015-06-02

    Background: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. Results: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This alsomore » represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. In comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. Conclusion: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.« less

  1. Bovine Leukemia Virus DNA in Human Breast Tissue

    PubMed Central

    Shen, Hua Min; Jensen, Hanne M.; Choi, K. Yeon; Sun, Dejun; Nuovo, Gerard

    2014-01-01

    Bovine leukemia virus (BLV), a deltaretrovirus, causes B-cell leukemia/lymphoma in cattle and is prevalent in herds globally. A previous finding of antibodies against BLV in humans led us to examine the possibility of human infection with BLV. We focused on breast tissue because, in cattle, BLV DNA and protein have been found to be more abundant in mammary epithelium than in lymphocytes. In human breast tissue specimens, we identified BLV DNA by using nested liquid-phase PCR and DNA sequencing. Variations from the bovine reference sequence were infrequent and limited to base substitutions. In situ PCR and immunohistochemical testing localized BLV to the secretory epithelium of the breast. Our finding of BLV in human tissues indicates a risk for the acquisition and proliferation of this virus in humans. Further research is needed to determine whether BLV may play a direct role in human disease. PMID:24750974

  2. Cleavage of influenza A virus H1 hemagglutinin by swine respiratory bacterial proteases.

    PubMed Central

    Callan, R J; Hartmann, F A; West, S E; Hinshaw, V S

    1997-01-01

    Cleavage of influenza A virus hemagglutinin (HA) is required for expression of fusion activity and virus entry into cells. Extracellular proteases are responsible for the proteolytic cleavage activation of avirulent avian and mammalian influenza viruses and contribute to pathogenicity and tissue tropism. The relative contributions of host and microbial proteases to cleavage activation in natural infection remain to be established. We examined 23 respiratory bacterial pathogens and 150 aerobic bacterial isolates cultured from the nasal cavities of pigs for proteolytic activity. No evidence of secreted proteases was found for the bacterial pathogens, including Haemophilus parasuis, Pasteurella multocida, Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, and Streptococcus suis. Proteolytic bacteria were isolated from 7 of 11 swine nasal samples and included Staphylococcus chromogenes, Staphylococcus hyicus, Aeromonas caviae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Enterococcus sp. Only P. aeruginosa secreted a protease, elastase, that cleaved influenza virus HA. However, compared to trypsin, the site of cleavage by elastase was shifted one amino acid in the carboxy-terminal direction and resulted in inactivation of the virus. Under the conditions of this study, we identified several bacterial isolates from the respiratory tracts of pigs that secrete proteases in vitro. However, none of these proteolytic isolates demonstrated direct cleavage activation of influenza virus HA. PMID:9311838

  3. Bacterial DNA Content in the Intestinal Wall from Infants with Necrotizing Enterocolitis

    PubMed Central

    Bucher, Brian T.; McDuffie, Lucas A.; Shaikh, Nurmohammad; Tarr, Phillip I.; Warner, Barbara B.; Hamvas, Aaron; White, Francis V.; Erwin, Christopher R.; Warner, Brad W.

    2011-01-01

    PURPOSE The objectives of our study were to quantify mucosal bacterial DNA within specimens from neonates undergoing small bowel resection for necrotizing enterocolitis (NEC). METHODS We obtained clinical information and pathologic specimens from all infants diagnosed with NEC who underwent surgical treatment at our institution from 1999–2008. Bacterial and human DNA were isolated from paraffin-embedded surgical specimens and real-time PCR was used to amplify bacterial and human genes. Linear regression was used to quantify the amount of human and bacterial DNA in our specimens. RESULTS From a cohort of fifty infants, we identified twenty-three infants who underwent both surgical resection and subsequent intestinal reanastomosis. Thirteen (59%) of the neonates had Bell's Stage III NEC, and nine (41%) had Stage II. There was significantly more bacterial DNA in the resection specimens than in the reanastomosis specimens. This corresponds to a median (IQR) increase of 1.81 (1.11–4.69) fold bacterial DNA in the resection specimen compared to the reanastomosis specimen (p<0.05). CONCLUSION There is more bacterial DNA in infants with acute NEC compared with the same infants after the NEC had clinically resolved. These findings underscore the potential relevance of adherent or invasive bacteria across the bowel wall in the pathogenesis of NEC. PMID:21683193

  4. Molecular cloning and characterization of human papilloma virus DNA derived from a laryngeal papilloma.

    PubMed Central

    Gissmann, L; Diehl, V; Schultz-Coulon, H J; zur Hausen, H

    1982-01-01

    Papilloma virus DNA from a laryngeal papilloma was cloned in phage lambda L 47 and characterized after cleavage with different restriction enzymes. Hybridization with the DNAs of human papilloma virus types 1, 2, 3, 4, 5, and 8 showed no homology under stringent hybridization conditions. Human papilloma virus type 6 DNA, however, was partially identical to laryngeal papilloma virus DNA; different restriction enzyme fragments hybridizing with the other DNA were identified on each genome. The degree of homology was determined by reassociation kinetics to be 25%. According to the present nomenclature, laryngeal papilloma virus therefore represents a different type of human papilloma virus and is tentatively designated as human papilloma virus type 11. Sequences homologous to laryngeal papilloma virus DNA were also found in four of nine additional laryngeal papillomas. Attempt to detect homologous DNA in 12 carcinomas of the larynx were negative. Images PMID:6292500

  5. T-antigen-DNA polymerase alpha complex implicated in simian virus 40 DNA replication.

    PubMed Central

    Smale, S T; Tjian, R

    1986-01-01

    We have combined in vitro DNA replication reactions and immunological techniques to analyze biochemical interactions between simian virus (SV40) large T antigen and components of the cellular replication apparatus. First, in vitro SV40 DNA replication was characterized with specific origin mutants. Next, monoclonal antibodies were used to demonstrate that a specific domain of T antigen formed a complex with cellular DNA polymerase alpha. Several antibodies were identified that coprecipitated T antigen and DNA polymerase alpha, while others were found to selectively prevent this interaction and concomitantly inhibit DNA replication. DNA polymerase alpha also bound efficiently to a T-antigen affinity column, confirming the immunoprecipitation results and providing a useful method for purification of the complete protein complex. Taken together, these results suggest that the T-antigen-polymerase association may be a key step in the initiation of SV40 DNA replication. Images PMID:3025630

  6. Effects of viruses on bacterial functions under contrasting nutritional conditions for four species of bacteria isolated from Hong Kong waters

    NASA Astrophysics Data System (ADS)

    Liu, Hao; Yuan, Xiangcheng; Xu, Jie; Harrison, Paul J.; He, Lei; Yin, Kedong

    2015-09-01

    Free living viruses are ubiquitous in marine waters and concentrations are usually several times higher than the bacterial abundance. These viruses are capable of lysing host bacteria and therefore, play an important role in the microbial loop in oligotrophic waters. However, few studies have been conducted to compare the role of viruses in regulating bacterial abundance and heterotrophic activities between natural oligotrophic waters and anthropogenic influenced eutrophic waters. In this study, we examined viral effects on bacterial functions of four single bacterial species incubated with natural viral assemblages in seawater samples from eutrophic and oligotrophic waters. The viral-lysis of bacteria was significantly higher in eutrophic than oligotrophic waters. This suggests that viruses were capable of controlling bacterial abundance, respiration and production in the eutrophic waters. Cellular bacterial respiration and production was higher with viruses than without viruses, which was more evident in the oligotrophic waters. These results indicate that viruses can slow down bacterial consumption of oxygen and reduce bacteria-induced eutrophication effects in anthropogenic eutrophic waters, but switch to the role of sustaining the bacterial population when nutrients are limiting. There were bacterial species differences in resisting viral attack, which can influence the dominance and biodiversity of bacterial species in coastal waters.

  7. Effects of viruses on bacterial functions under contrasting nutritional conditions for four species of bacteria isolated from Hong Kong waters

    PubMed Central

    Liu, Hao; Yuan, Xiangcheng; Xu, Jie; Harrison, Paul J.; He, Lei; Yin, Kedong

    2015-01-01

    Free living viruses are ubiquitous in marine waters and concentrations are usually several times higher than the bacterial abundance. These viruses are capable of lysing host bacteria and therefore, play an important role in the microbial loop in oligotrophic waters. However, few studies have been conducted to compare the role of viruses in regulating bacterial abundance and heterotrophic activities between natural oligotrophic waters and anthropogenic influenced eutrophic waters. In this study, we examined viral effects on bacterial functions of four single bacterial species incubated with natural viral assemblages in seawater samples from eutrophic and oligotrophic waters. The viral-lysis of bacteria was significantly higher in eutrophic than oligotrophic waters. This suggests that viruses were capable of controlling bacterial abundance, respiration and production in the eutrophic waters. Cellular bacterial respiration and production was higher with viruses than without viruses, which was more evident in the oligotrophic waters. These results indicate that viruses can slow down bacterial consumption of oxygen and reduce bacteria-induced eutrophication effects in anthropogenic eutrophic waters, but switch to the role of sustaining the bacterial population when nutrients are limiting. There were bacterial species differences in resisting viral attack, which can influence the dominance and biodiversity of bacterial species in coastal waters. PMID:26404394

  8. Structures of herpes simplex virus type 1 genes required for replication of virus DNA.

    PubMed Central

    McGeoch, D J; Dalrymple, M A; Dolan, A; McNab, D; Perry, L J; Taylor, P; Challberg, M D

    1988-01-01

    Recently, a method has been developed to identify regions in the genome of herpes simplex virus type 1 (HSV-1) which contain genes required for DNA synthesis from an HSV-1 origin of DNA replication, and seven genomic loci have been identified as representing the necessary and sufficient gene set for such replication (C. A. Wu, N. J. Nelson, D. J. McGeoch, and M. D. Challberg, J. Virol. 62:435-443, 1988). Two of the loci represent the well-known genes for DNA polymerase and major DNA-binding protein, but the remainder had little or no previous characterization. In this report we present the DNA sequences of the five newly identified genes and their deduced transcript organizations and encoded amino acid sequences. These genes were designated UL5, UL8, UL9, UL42, and UL52 and were predicted to encode proteins with molecular weights of, respectively, 99,000, 80,000, 94,000, 51,000, and 114,000. All of these genes had clear counterparts in the genome of the related alphaherpesvirus varicella-zoster virus, but only UL5 and UL52 were detectably conserved in the distantly related gammaherpesvirus Epstein-Barr virus, as judged by amino acid sequence similarity. The sequence of the UL5 protein, and of its counterparts in the other viruses, contained a region closely resembling known ATP-binding sites; this could be indicative, for instance, of a helicase or primase activity. PMID:2826807

  9. cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication.

    PubMed Central

    Hartl, M; Willnow, T; Fanning, E

    1990-01-01

    Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of the plasmid in TC7 monkey cells, although the plasmids expressed replication-competent T antigen. Rat cells were transformed with linearized wild-type SV40 plasmid DNA with or without fragments of the mouse DNA in cis. Although all of the rat cell lines expressed approximately equal amounts of T antigen and p53, transformants carrying SV40 DNA linked to either of the same two replication suppressor fragments produced significantly less free SV40 DNA after fusion with permissive cells than those transformed by SV40 DNA without a cellular insert or with a cellular insert lacking suppressor activity. The results suggest that two independent segments of cellular DNA act in cis to suppress SV40 replication in vivo, either as a plasmid or integrated in chromosomal DNA. Images PMID:2159549

  10. Properties of the DNA-binding domain of the simian virus 40 large T antigen.

    PubMed Central

    McVey, D; Strauss, M; Gluzman, Y

    1989-01-01

    T antigen (Tag) from simian virus 40 binds specifically to two distinct sites in the viral origin of replication and to single-stranded DNA. Analysis of the protein domain responsible for these activities revealed the following. (i) The C-terminal boundary of the origin-specific and single-strand-specific DNA-binding domain is at or near amino acid 246; furthermore, the maximum of these DNA-binding activities coincides with a narrow C-terminal boundary, spanning 4 amino acids (246 to 249) and declines sharply in proteins with C termini which differ by a few (4 to 10) amino acids; (ii) a polypeptide spanning residues 132 to 246 of Tag is an independent domain responsible for origin-specific DNA binding and presumably for single-stranded DNA binding; and (iii) a comparison of identical N-terminal fragments of Tag purified from mammalian and bacterial cells revealed differential specificity and levels of activity between the two sources of protein. A role for posttranslational modification (phosphorylation) in controlling the DNA-binding activity of Tag is discussed. Images PMID:2555700

  11. Low-Resolution Structure of Vaccinia Virus DNA Replication Machinery

    PubMed Central

    Sèle, Céleste; Gabel, Frank; Gutsche, Irina; Ivanov, Ivan; Burmeister, Wim P.

    2013-01-01

    Smallpox caused by the poxvirus variola virus is a highly lethal disease that marked human history and was eradicated in 1979 thanks to a worldwide mass vaccination campaign. This virus remains a significant threat for public health due to its potential use as a bioterrorism agent and requires further development of antiviral drugs. The viral genome replication machinery appears to be an ideal target, although very little is known about its structure. Vaccinia virus is the prototypic virus of the Orthopoxvirus genus and shares more than 97% amino acid sequence identity with variola virus. Here we studied four essential viral proteins of the replication machinery: the DNA polymerase E9, the processivity factor A20, the uracil-DNA glycosylase D4, and the helicase-primase D5. We present the recombinant expression and biochemical and biophysical characterizations of these proteins and the complexes they form. We show that the A20D4 polymerase cofactor binds to E9 with high affinity, leading to the formation of the A20D4E9 holoenzyme. Small-angle X-ray scattering yielded envelopes for E9, A20D4, and A20D4E9. They showed the elongated shape of the A20D4 cofactor, leading to a 150-Å separation between the polymerase active site of E9 and the DNA-binding site of D4. Electron microscopy showed a 6-fold rotational symmetry of the helicase-primase D5, as observed for other SF3 helicases. These results favor a rolling-circle mechanism of vaccinia virus genome replication similar to the one suggested for tailed bacteriophages. PMID:23175373

  12. Construction of infectious cDNA clones for RNA viruses: Turnip crinkle virus.

    PubMed

    Ryabov, Eugene V

    2008-01-01

    Reverse genetic approach is widely used in virology as it makes possible direct identification of viral gene function and uses RNA genomes as vectors. Production of infectious cDNA clones is an essential step in developing a reverse genetic system for an RNA virus. Here, we present rapid method for generation of infectious cDNA clone for Turnip crinkle virus (TCV). The infectious cDNA clone could be used for production of in vitro transcripts with the T7 RNA polymerase which could be used for infection of plants or plant cell protoplasts. The procedure described here includes purification of TCV, viral RNA extraction, reverse transcription, PCR amplification of the full-length cDNA copy of TCV linked to a T7 RNA polymerase promoter, cloning into a plasmid vector, in vitro transcription, and selection of infectious clones. PMID:18370276

  13. Sequence of figwort mosaic virus DNA (caulimovirus group).

    PubMed

    Richins, R D; Scholthof, H B; Shepherd, R J

    1987-10-26

    The nucleotide sequence of an infectious clone of figwort mosaic virus (FMV) was determined using the dideoxynucleotide chain termination method. The double-stranded DNA genome (7743 base pairs) contained eight open reading frames (ORFs), seven of which corresponded approximately in size and location to the ORFs found in the genome of cauliflower mosaic virus (CaMV) and carnation etched ring virus (CERV). ORFs I and V of FMV demonstrated the highest degrees of nucleotide and amino acid sequence homology with the equivalent coding regions of CaMV and CERV. Regions II, III and IV showed somewhat less homology with the analogous regions of CaMV and CERV, and ORF VI showed homology with the corresponding gene of CaMV and CERV in only a short segment near the middle of the putative gene product. A 16 nucleotide sequence, complementary to the 3' terminus of methionine initiator tRNA (tRNAimet) and presumed to be the primer binding site for initiation of reverse transcription to produce minus strand DNA, was found in the FMV genome near the discontinuity in the minus strand. Sequences near the three interruptions in the plus strand of FMV DNA bear strong resemblance to similarly located sequences of 3 other caulimoviruses and are inferred to be initiation sites for second strand DNA synthesis. Additional conserved sequences in the small and large intergenic regions are pointed out including a highly conserved 35 bp sequence that occurs in the latter region. PMID:3671088

  14. Sequence of figwort mosaic virus DNA (caulimovirus group).

    PubMed Central

    Richins, R D; Scholthof, H B; Shepherd, R J

    1987-01-01

    The nucleotide sequence of an infectious clone of figwort mosaic virus (FMV) was determined using the dideoxynucleotide chain termination method. The double-stranded DNA genome (7743 base pairs) contained eight open reading frames (ORFs), seven of which corresponded approximately in size and location to the ORFs found in the genome of cauliflower mosaic virus (CaMV) and carnation etched ring virus (CERV). ORFs I and V of FMV demonstrated the highest degrees of nucleotide and amino acid sequence homology with the equivalent coding regions of CaMV and CERV. Regions II, III and IV showed somewhat less homology with the analogous regions of CaMV and CERV, and ORF VI showed homology with the corresponding gene of CaMV and CERV in only a short segment near the middle of the putative gene product. A 16 nucleotide sequence, complementary to the 3' terminus of methionine initiator tRNA (tRNAimet) and presumed to be the primer binding site for initiation of reverse transcription to produce minus strand DNA, was found in the FMV genome near the discontinuity in the minus strand. Sequences near the three interruptions in the plus strand of FMV DNA bear strong resemblance to similarly located sequences of 3 other caulimoviruses and are inferred to be initiation sites for second strand DNA synthesis. Additional conserved sequences in the small and large intergenic regions are pointed out including a highly conserved 35 bp sequence that occurs in the latter region. PMID:3671088

  15. Bacterial lipopolysaccharide binding enhances virion stability and promotes environmental fitness of an enteric virus

    PubMed Central

    Robinson, Christopher M.; Jesudhasan, Palmy R.; Pfeiffer, Julie K.

    2014-01-01

    Summary Enteric viruses, including poliovirus and reovirus, encounter a vast microbial community in the mammalian gastrointestinal tract, which has been shown to promote virus replication and pathogenesis. Investigating the underlying mechanisms, we find that poliovirus binds bacterial surface polysaccharides, which enhances virion stability and cell attachment by increasing binding to the viral receptor. Additionally, we identified a poliovirus mutant, VP1-T99K, with reduced lipopolysaccharide (LPS) binding. Although T99K and WT poliovirus cell attachment, replication and pathogenesis in mice are equivalent, following peroral inoculation of mice, VP1-T99K poliovirus was unstable in feces. Consequently, the ratio of mutant virus in feces is reduced following additional cycles of infection in mice. Thus, the mutant virus incurs a fitness cost when environmental stability is a factor. These data suggest that poliovirus binds bacterial surface polysaccharides, enhancing cell attachment and environmental stability, potentially promoting transmission to a new host. PMID:24439896

  16. The adeno-associated virus rep gene suppresses herpes simplex virus-induced DNA amplification.

    PubMed Central

    Heilbronn, R; Bürkle, A; Stephan, S; zur Hausen, H

    1990-01-01

    Herpes simplex virus (HSV) induces within the host cell genome DNA amplification which can be suppressed by coinfection with adeno-associated virus (AAV). To characterize the AAV functions mediating this effect, cloned AAV type 2 wild-type or mutant genomes were transfected into simian virus 40 (SV40)-transformed hamster cells together with the six HSV replication genes (encoding UL5, UL8, major DNA-binding protein, DNA polymerase, UL42, and UL52) which together are necessary and sufficient for the induction of SV40 DNA amplification (R. Heilbronn and H. zur Hausen, J. Virol. 63:3683-3692, 1989). The AAV rep gene was identified as being responsible for the complete inhibition of HSV-induced SV40 DNA amplification. Likewise, rep inhibited origin-dependent HSV replication. rep neither killed the transfected host cells nor interfered with gene expression from the cotransfected amplification genes. This points to a specific interference with HSV-induced DNA amplification. Images PMID:2159559

  17. Genomic DNA fingerprint analysis of biotype 1 Gardnerella vaginalis from patients with and without bacterial vaginosis.

    PubMed Central

    Wu, S R; Hillier, S L; Nath, K

    1996-01-01

    Of the 20 biotype 1 Gardnerella vaginalis isolates analyzed, 10 from patients with bacterial vaginosis and 10 from patients without bacterial vaginosis, none shared the same DNA fingerprint. However, a 1.18-kb HindIII fragment was common among 18 of the 20 biotype 1 isolates in a restriction fragment length polymorphism analysis with a 7.9-kb G. vaginalis DNA probe. PMID:8748302

  18. Crystal Structure of the Vaccinia Virus Uracil-DNA Glycosylase in Complex with DNA.

    PubMed

    Burmeister, Wim P; Tarbouriech, Nicolas; Fender, Pascal; Contesto-Richefeu, Céline; Peyrefitte, Christophe N; Iseni, Frédéric

    2015-07-17

    Vaccinia virus polymerase holoenzyme is composed of the DNA polymerase catalytic subunit E9 associated with its heterodimeric co-factor A20·D4 required for processive genome synthesis. Although A20 has no known enzymatic activity, D4 is an active uracil-DNA glycosylase (UNG). The presence of a repair enzyme as a component of the viral replication machinery suggests that, for poxviruses, DNA synthesis and base excision repair is coupled. We present the 2.7 Å crystal structure of the complex formed by D4 and the first 50 amino acids of A20 (D4·A201-50) bound to a 10-mer DNA duplex containing an abasic site resulting from the cleavage of a uracil base. Comparison of the viral complex with its human counterpart revealed major divergences in the contacts between protein and DNA and in the enzyme orientation on the DNA. However, the conformation of the dsDNA within both structures is very similar, suggesting a dominant role of the DNA conformation for UNG function. In contrast to human UNG, D4 appears rigid, and we do not observe a conformational change upon DNA binding. We also studied the interaction of D4·A201-50 with different DNA oligomers by surface plasmon resonance. D4 binds weakly to nonspecific DNA and to uracil-containing substrates but binds abasic sites with a Kd of <1.4 μm. This second DNA complex structure of a family I UNG gives new insight into the role of D4 as a co-factor of vaccinia virus DNA polymerase and allows a better understanding of the structural determinants required for UNG action. PMID:26045555

  19. Blocking primers reduce co-amplification of plant DNA when studying bacterial endophyte communities.

    PubMed

    Arenz, Brett E; Schlatter, Dan C; Bradeen, James M; Kinkel, Linda L

    2015-10-01

    A blocking primer set based on the technique described by Vestheim and Jarman (2008) was developed to reduce amplification of non-target plant DNA when conducting metagenomic studies on bacterial endophyte communities. Bacterial amplification efficiency was increased 300-fold compared to standard PCR in an Illumina-based study of Sorghastrum nutans leaves. PMID:26159909

  20. Negatively supercoiled simian virus 40 DNA contains Z-DNA segments within transcriptional enhancer sequences

    NASA Technical Reports Server (NTRS)

    Nordheim, A.; Rich, A.

    1983-01-01

    Three 8-base pair (bp) segments of alternating purine-pyrimidine from the simian virus 40 enhancer region form Z-DNA on negative supercoiling; minichromosome DNase I-hypersensitive sites determined by others bracket these three segments. A survey of transcriptional enhancer sequences reveals a pattern of potential Z-DNA-forming regions which occur in pairs 50-80 bp apart. This may influence local chromatin structure and may be related to transcriptional activation.

  1. Viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lytic bacteriophages, viruses which infect and lyse bacterial cells, can provide a natural method to reduce bacterial pathogens on produce commodities. The use of multi-phage cocktails is most likely to be effective against bacterial pathogens on produce commodities, and minimize the development of...

  2. Low-energy plasma immersion ion implantation to induce DNA transfer into bacterial E. coli

    NASA Astrophysics Data System (ADS)

    Sangwijit, K.; Yu, L. D.; Sarapirom, S.; Pitakrattananukool, S.; Anuntalabhochai, S.

    2015-12-01

    Plasma immersion ion implantation (PIII) at low energy was for the first time applied as a novel biotechnology to induce DNA transfer into bacterial cells. Argon or nitrogen PIII at low bias voltages of 2.5, 5 and 10 kV and fluences ranging from 1 × 1012 to 1 × 1017 ions/cm2 treated cells of Escherichia coli (E. coli). Subsequently, DNA transfer was operated by mixing the PIII-treated cells with DNA. Successes in PIII-induced DNA transfer were demonstrated by marker gene expressions. The induction of DNA transfer was ion-energy, fluence and DNA-size dependent. The DNA transferred in the cells was confirmed functioning. Mechanisms of the PIII-induced DNA transfer were investigated and discussed in terms of the E. coli cell envelope anatomy. Compared with conventional ion-beam-induced DNA transfer, PIII-induced DNA transfer was simpler with lower cost but higher efficiency.

  3. Relationships Between Environmental Factors, Bacterial Indicators, and the Occurrence of Enteric Viruses in Estuarine Sediments

    PubMed Central

    LaBelle, Raymond L.; Gerba, Charles P.; Goyal, Sagar M.; Melnick, Joseph L.; Cech, Irina; Bogdan, Gregory F.

    1980-01-01

    Current standards for evaluation of the public health safety of recreational and shellfish-harvesting waters are based upon bacteriological analysis, but do not include an evaluation of the number of viruses. The objective of this study was to determine the occurrence of enteric viruses in estuarine sediments and to find a relationship, if any, between the presence of viruses in seawater or sediment or both and various biological and physicochemical characteristics of the environment. Viruses were found in greater numbers in sediment than in overlying seawater on a volume basis. Several types of enteroviruses were isolated: coxsackievirus types A16, B1, and B5, echovirus type 1, and poliovirus type 2. On several occasions, viruses were isolated from sediments when overlying seawaters met bacteriological water quality standards for recreational use. Statistical analysis of the relationship between viruses in seawater or in sediment and other variables measured yielded only one significant association: the number of viruses in sediment was found to be positively correlated with the number of fecal coliforms in sediment. No other physical, chemical, or biological characteristic of seawater or sediment that was measured showed statistically significant association with viral numbers. No correlation was found between bacterial indicators and virus in the overlying waters. The data indicated that evaluation of the presence of bacteria and viruses in sediment may provide additional insight into long-term water quality conditions and that indicator bacteria in water are not reflective of the concentration of enteric viruses in marine waters. PMID:6247974

  4. Carriage of λ Latent Virus Is Costly for Its Bacterial Host due to Frequent Reactivation in Monoxenic Mouse Intestine.

    PubMed

    De Paepe, Marianne; Tournier, Laurent; Moncaut, Elisabeth; Son, Olivier; Langella, Philippe; Petit, Marie-Agnès

    2016-02-01

    Temperate phages, the bacterial viruses able to enter in a dormant prophage state in bacterial genomes, are present in the majority of bacterial strains for which the genome sequence is available. Although these prophages are generally considered to increase their hosts' fitness by bringing beneficial genes, studies demonstrating such effects in ecologically relevant environments are relatively limited to few bacterial species. Here, we investigated the impact of prophage carriage in the gastrointestinal tract of monoxenic mice. Combined with mathematical modelling, these experimental results provided a quantitative estimation of key parameters governing phage-bacteria interactions within this model ecosystem. We used wild-type and mutant strains of the best known host/phage pair, Escherichia coli and phage λ. Unexpectedly, λ prophage caused a significant fitness cost for its carrier, due to an induction rate 50-fold higher than in vitro, with 1 to 2% of the prophage being induced. However, when prophage carriers were in competition with isogenic phage susceptible bacteria, the prophage indirectly benefited its carrier by killing competitors: infection of susceptible bacteria led to phage lytic development in about 80% of cases. The remaining infected bacteria were lysogenized, resulting overall in the rapid lysogenization of the susceptible lineage. Moreover, our setup enabled to demonstrate that rare events of phage gene capture by homologous recombination occurred in the intestine of monoxenic mice. To our knowledge, this study constitutes the first quantitative characterization of temperate phage-bacteria interactions in a simplified gut environment. The high prophage induction rate detected reveals DNA damage-mediated SOS response in monoxenic mouse intestine. We propose that the mammalian gut, the most densely populated bacterial ecosystem on earth, might foster bacterial evolution through high temperate phage activity. PMID:26871586

  5. Carriage of λ Latent Virus Is Costly for Its Bacterial Host due to Frequent Reactivation in Monoxenic Mouse Intestine

    PubMed Central

    De Paepe, Marianne; Tournier, Laurent; Moncaut, Elisabeth; Son, Olivier; Langella, Philippe; Petit, Marie-Agnès

    2016-01-01

    Temperate phages, the bacterial viruses able to enter in a dormant prophage state in bacterial genomes, are present in the majority of bacterial strains for which the genome sequence is available. Although these prophages are generally considered to increase their hosts’ fitness by bringing beneficial genes, studies demonstrating such effects in ecologically relevant environments are relatively limited to few bacterial species. Here, we investigated the impact of prophage carriage in the gastrointestinal tract of monoxenic mice. Combined with mathematical modelling, these experimental results provided a quantitative estimation of key parameters governing phage-bacteria interactions within this model ecosystem. We used wild-type and mutant strains of the best known host/phage pair, Escherichia coli and phage λ. Unexpectedly, λ prophage caused a significant fitness cost for its carrier, due to an induction rate 50-fold higher than in vitro, with 1 to 2% of the prophage being induced. However, when prophage carriers were in competition with isogenic phage susceptible bacteria, the prophage indirectly benefited its carrier by killing competitors: infection of susceptible bacteria led to phage lytic development in about 80% of cases. The remaining infected bacteria were lysogenized, resulting overall in the rapid lysogenization of the susceptible lineage. Moreover, our setup enabled to demonstrate that rare events of phage gene capture by homologous recombination occurred in the intestine of monoxenic mice. To our knowledge, this study constitutes the first quantitative characterization of temperate phage-bacteria interactions in a simplified gut environment. The high prophage induction rate detected reveals DNA damage-mediated SOS response in monoxenic mouse intestine. We propose that the mammalian gut, the most densely populated bacterial ecosystem on earth, might foster bacterial evolution through high temperate phage activity. PMID:26871586

  6. Recombinant expression and purification of "virus-like" bacterial encapsulin protein cages.

    PubMed

    Rurup, W Frederik; Cornelissen, Jeroen J L M; Koay, Melissa S T

    2015-01-01

    Ultracentrifugation, particularly the use of sucrose or cesium chloride density gradients, is a highly reliable and efficient technique for the purification of virus-like particles and protein cages. Since virus-like particles and protein cages have a unique size compared to cellular macromolecules and organelles, the rate of migration can be used as a tool for purification. Here we describe a detailed protocol for the purification of recently discovered virus-like assemblies called bacterial encapsulins from Thermotoga maritima and Brevibacterium linens. PMID:25358773

  7. Lateral Gene Transfer of Family A DNA Polymerases between Thermophilic Viruses, Aquificae, and Apicomplexa

    PubMed Central

    Schoenfeld, Thomas W.; Murugapiran, Senthil K.; Dodsworth, Jeremy A.; Floyd, Sally; Lodes, Michael; Mead, David A.; Hedlund, Brian P.

    2013-01-01

    Bioinformatics and functional screens identified a group of Family A-type DNA Polymerase (polA) genes encoded by viruses inhabiting circumneutral and alkaline hot springs in Yellowstone National Park and the US Great Basin. The proteins encoded by these viral polA genes (PolAs) shared no significant sequence similarity with any known viral proteins but were remarkably similar to PolAs encoded by two of three families of the bacterial phylum Aquificae and by several apicoplast-targeted PolA-like proteins found in the eukaryotic phylum Apicomplexa, which includes the obligate parasites Plasmodium, Babesia, and Toxoplasma. The viral gene products share signature elements previously associated only with Aquificae and Apicomplexa PolA-like proteins and were similar to proteins encoded by prophage elements of a variety of otherwise unrelated Bacteria, each of which additionally encoded a prototypical bacterial PolA. Unique among known viral DNA polymerases, the viral PolA proteins of this study share with the Apicomplexa proteins large amino-terminal domains with putative helicase/primase elements but low primary sequence similarity. The genomic context and distribution, phylogeny, and biochemistry of these PolA proteins suggest that thermophilic viruses transferred polA genes to the Apicomplexa, likely through secondary endosymbiosis of a virus-infected proto-apicoplast, and to the common ancestor of two of three Aquificae families, where they displaced the orthologous cellular polA gene. On the basis of biochemical activity, gene structure, and sequence similarity, we speculate that the xenologous viral-type polA genes may have functions associated with diversity-generating recombination in both Bacteria and Apicomplexa. PMID:23608703

  8. Comparison of different methods for isolation of bacterial DNA from retail oyster tissues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oysters are filter-feeders that bio-accumulate bacteria in water while feeding. To evaluate the bacterial genomic DNA extracted from retail oyster tissues, including the gills and digestive glands, four isolation methods were used. Genomic DNA extraction was performed using the Allmag™ Blood Genomic...

  9. Discovery, Prevalence, and Persistence of Novel Circular Single-Stranded DNA Viruses in the Ctenophores Mnemiopsis leidyi and Beroe ovata.

    PubMed

    Breitbart, Mya; Benner, Bayleigh E; Jernigan, Parker E; Rosario, Karyna; Birsa, Laura M; Harbeitner, Rachel C; Fulford, Sidney; Graham, Carina; Walters, Anna; Goldsmith, Dawn B; Berger, Stella A; Nejstgaard, Jens C

    2015-01-01

    Gelatinous zooplankton, such as ctenophores and jellyfish, are important components of marine and brackish ecosystems and play critical roles in aquatic biogeochemistry. As voracious predators of plankton, ctenophores have key positions in aquatic food webs and are often successful invaders when introduced to new areas. Gelatinous zooplankton have strong impacts on ecosystem services, particularly in coastal environments. However, little is known about the factors responsible for regulating population dynamics of gelatinous organisms, including biological interactions that may contribute to bloom demise. Ctenophores are known to contain specific bacterial communities and a variety of invertebrate parasites and symbionts; however, no previous studies have examined the presence of viruses in these organisms. Building upon recent studies demonstrating a diversity of single-stranded DNA viruses that encode a replication initiator protein (Rep) in aquatic invertebrates, this study explored the presence of circular, Rep-encoding single-stranded DNA (CRESS-DNA) viruses in the ctenophores Mnemiopsis leidyi and Beroe ovata collected from the Skidaway River Estuary and Savannah River in Georgia, USA. Using rolling circle amplification followed by restriction enzyme digestion, this study provides the first evidence of viruses in ctenophores. Investigation of four CRESS-DNA viruses over an 8-month period using PCR demonstrated temporal trends in viral prevalence and indicated that some of the viruses may persist in ctenophore populations throughout the year. Although future work needs to examine the ecological roles of these ctenophore-associated viruses, this study indicates that viral infection may play a role in population dynamics of gelatinous zooplankton. PMID:26733971

  10. Discovery, Prevalence, and Persistence of Novel Circular Single-Stranded DNA Viruses in the Ctenophores Mnemiopsis leidyi and Beroe ovata

    PubMed Central

    Breitbart, Mya; Benner, Bayleigh E.; Jernigan, Parker E.; Rosario, Karyna; Birsa, Laura M.; Harbeitner, Rachel C.; Fulford, Sidney; Graham, Carina; Walters, Anna; Goldsmith, Dawn B.; Berger, Stella A.; Nejstgaard, Jens C.

    2015-01-01

    Gelatinous zooplankton, such as ctenophores and jellyfish, are important components of marine and brackish ecosystems and play critical roles in aquatic biogeochemistry. As voracious predators of plankton, ctenophores have key positions in aquatic food webs and are often successful invaders when introduced to new areas. Gelatinous zooplankton have strong impacts on ecosystem services, particularly in coastal environments. However, little is known about the factors responsible for regulating population dynamics of gelatinous organisms, including biological interactions that may contribute to bloom demise. Ctenophores are known to contain specific bacterial communities and a variety of invertebrate parasites and symbionts; however, no previous studies have examined the presence of viruses in these organisms. Building upon recent studies demonstrating a diversity of single-stranded DNA viruses that encode a replication initiator protein (Rep) in aquatic invertebrates, this study explored the presence of circular, Rep-encoding single-stranded DNA (CRESS-DNA) viruses in the ctenophores Mnemiopsis leidyi and Beroe ovata collected from the Skidaway River Estuary and Savannah River in Georgia, USA. Using rolling circle amplification followed by restriction enzyme digestion, this study provides the first evidence of viruses in ctenophores. Investigation of four CRESS-DNA viruses over an 8-month period using PCR demonstrated temporal trends in viral prevalence and indicated that some of the viruses may persist in ctenophore populations throughout the year. Although future work needs to examine the ecological roles of these ctenophore-associated viruses, this study indicates that viral infection may play a role in population dynamics of gelatinous zooplankton. PMID:26733971

  11. DNA-sensing inflammasomes: regulation of bacterial host defense and the gut microbiota.

    PubMed

    Man, Si Ming; Karki, Rajendra; Kanneganti, Thirumala-Devi

    2016-06-01

    DNA sensors are formidable immune guardians of the host. At least 14 cytoplasmic DNA sensors have been identified in recent years, each with specialized roles in driving inflammation and/or cell death. Of these, AIM2 is a sensor of dsDNA, and forms an inflammasome complex to activate the cysteine protease caspase-1, mediates the release of the proinflammatory cytokines IL-1β and IL-18, and induces pyroptosis. The inflammasome sensor NLRP3 can also respond to DNA in the forms of oxidized mitochondrial DNA and the DNA derivative RNA:DNA hybrids produced by bacteria, whereas the putative inflammasome sensor IFI16 responds to viral DNA in the nucleus. Although inflammasomes provoke inflammation for anti-microbial host defense, they must also maintain homeostasis with commensal microbiota. Here, we outline recent advances highlighting the complex relationship between DNA-sensing inflammasomes, bacterial host defense and the gut microbiota. PMID:27056948

  12. Short bacterial DNA fragments: detection in dialysate and induction of cytokines.

    PubMed

    Schindler, Ralf; Beck, Werner; Deppisch, Reinhold; Aussieker, Mario; Wilde, Adelheid; Göhl, Hermann; Frei, Ulrich

    2004-12-01

    A number of bacterial cytokine-inducing substances (CIS) such as lipopolysaccharides (LPS) and exotoxins have been detected in dialysate and may contribute to inflammation in hemodialysis patients. Short DNA fragments, oligodeoxynucleotides (ODN) of 6 to 20 nucleotides, are able to bind to Toll-like receptors and are stimulatory on immune cells. ODN induce natural killer cell activity and induce IFN-gamma, TNF-alpha, and IL-6 from mononuclear cells. The presence of ODN in dialysate samples and bacterial cultures was investigated. ODN were extracted from fluids by adsorption to reverse-phase columns. ODN were detected in 18 of 20 investigated dialysate samples, in eight of 10 reverse-osmosis water samples, and in all cultures from various bacterial strains. The presence of bacterial DNA in dialysate was confirmed by PCR specific for bacterial tRNA gene sequences. Saline for intravenous use contained 0.02 +/- 0.01 microg/ml DNA, dialysate samples contained 0.28 +/- 0.02 microg/ml, and Pseudomonas cultures contained 1.0 +/- 0.03 microg/ml DNA. ODN from bacterial cultures were only partially removed by ultrafiltration and were able to diffuse through regular high-flux dialyzer membranes. Synthetic cytosine-guanosine dinucleotide-containing ODN were able to induce IL-6 in human mononuclear cells. It is concluded that short bacterial-derived DNA fragments are present in clinically used fluids, e.g., dialysate. These fragments are of sufficient small size to pass through dialyzer membranes. Bacterial DNA fragments may be an overlooked factor contributing to inflammation in hemodialysis patients. PMID:15579524

  13. Unraveling the Interaction of Silver Nanoparticles with Mammalian and Bacterial DNA.

    PubMed

    Pramanik, Srikrishna; Chatterjee, Sabyasachi; Saha, Arindam; Devi, Parukuttyamma Sujatha; Suresh Kumar, Gopinatha

    2016-06-23

    The focus of this study was to understand and unravel the interaction of silver nanoparticles (AgNPs) with different types of Deoxyribonucleic acid (DNA), mammalian and bacterial, having different base pair compositions. Binding of spherical silver nanoparticles (AgNPs) to Calf thymus (CT) DNA, Escherichia coli (EC) DNA and Micrococcus lysodeikticus (ML) DNA has been studied to gain insights into their mode of interaction and specificity. Interaction of AgNPs with synthetic DNA has also been carried out. On the basis of absorption, thermal melting, isothermal calorimetry and viscosity studies, we could establish the mode of binding and specificity of the synthesized silver nanoparticles with mammalian and bacterial DNA. Thermal melting (Tm) studies indicated a decrease in the Tm of all the DNAs, confirming the destabilization of DNA stacks on interaction with AgNPs. Comparative interaction studies with single stranded (ss) and double stranded (ds) DNAs further confirmed the specificity of the particles toward ds DNA. On the basis of the results we could confirm that the synthesized AgNPs could be used for selective detection of DNA through their DNA binding mechanism. In addition, the AgNPs-DNA complexes exhibited distinct differences in the SERS spectra making it an interesting SERS platform for identifying ds DNA. The optical and physical properties of AgNPs help in differentiating the DNAs of different base pair compositions through their binding affinity and specificity. PMID:27294883

  14. Protection against respiratory syncytial virus infection by DNA immunization.

    PubMed

    Li, X; Sambhara, S; Li, C X; Ewasyshyn, M; Parrington, M; Caterini, J; James, O; Cates, G; Du, R P; Klein, M

    1998-08-17

    Respiratory syncytial virus (RSV) remains a major cause of morbidity and mortality in infants and the elderly and is a continuing challenge for vaccine development. A murine T helper cell (Th) type 2 response associates with enhanced lung pathology, which has been observed in past infant trials using formalin-inactivated RSV vaccine. In this study, we have engineered an optimized plasmid DNA vector expressing the RSV fusion (F) protein (DNA-F). DNA-F was as effective as live RSV in mice at inducing neutralizing antibody and cytotoxic T lymphocyte responses, protection against infection, and high mRNA expression of lung interferon gamma after viral challenge. Furthermore, a DNA-F boost could switch a preestablished anti-RSV Th2 response towards a Th1 response. Critical elements for the optimization of the plasmid constructs included expression of a secretory form of the F protein and the presence of the rabbit beta-globin intron II sequence upstream of the F-encoding sequence. In addition, anti-F systemic immune response profile could be modulated by the route of DNA-F delivery: intramuscular immunization resulted in balanced responses, whereas intradermal immunization resulted in a Th2 type of response. Thus, DNA-F immunization may provide a novel and promising RSV vaccination strategy. PMID:9705950

  15. Plasma Levels of Bacterial DNA Correlate with Immune Activation and the Magnitude of Immune Restoration in Persons with Antiretroviral-Treated HIV Infection

    PubMed Central

    Jiang, Wei; Lederman, Michael M.; Hunt, Peter; Sieg, Scott F.; Haley, Kathryn; Rodriguez, Benigno; Landay, Alan; Martin, Jeffrey; Sinclair, Elizabeth; Asher, Ava I.; Deeks, Steven G.; Douek, Daniel C.; Brenchley, Jason M.

    2009-01-01

    The significance of elevated plasma levels of bacterial lipopolysaccharide (LPS) in persons with chronic HIV infection remains undefined. We measured LPS levels by use of limulus lysate assay, and DNA sequences encoding bacterial ribosomal 16S RNA (16S rDNA) were assessed by quantitative polymerase chain reactions in plasma samples obtained from 242 donors. Plasma levels of 16S rDNA were significantly higher in human immunodeficiency virus (HIV)–infected subjects than in uninfected subjects, and they correlated with LPS levels. Higher levels of 16S rDNA were associated with higher levels of T cell activation and with lower levels of CD4 T cell restoration during antiretroviral therapy. Antiretroviral therapy reduces but does not fully normalize plasma levels of bacterial 16S rDNA, an index of microbial translocation from the gastrointestinal tract. High levels of 16S rDNA during therapy are strongly associated with reduced increases in the CD4+ T lymphocyte count, irrespective of plasma HIV RNA levels. These findings are consistent with the importance of microbial translocation in immunodeficiency and T cell homeostasis in chronic HIV infection. PMID:19265479

  16. Influenza A virus and secondary bacterial infection in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Influenza A virus (IAV) infection alone causes significant disease characterized by respiratory distress and poor growth in pigs. Endemic strains of IAV in North America pigs consist of the subtypes H1N1, H1N2, and H3N2. These circulating strains contain the triple reassortant internal gene (TRIG) c...

  17. Purification of DNA for bacterial productivity estimates. [Escherichia coli

    SciTech Connect

    Burnison, B.K.; Nuttley, D.J. )

    1990-02-01

    (methyl-{sup 3}H)thymidine-labeled DNA from natural populations of aquatic bacteria was completely separated from RNA and protein by hydroxylapatite chromatography. The procedure was validated by monitoring increases in Escherichia coli cell count, A{sup 550}, DNA concentration, and thymidine incorporation into DNA isolated by the proposed technique. The procedure can be used in the field and does not rely on the use of acid-base hydrolysis or volatile organic solvents.

  18. The bacterial DNA repair protein Mfd confers resistance to the host nitrogen immune response.

    PubMed

    Guillemet, Elisabeth; Leréec, Alain; Tran, Seav-Ly; Royer, Corinne; Barbosa, Isabelle; Sansonetti, Philippe; Lereclus, Didier; Ramarao, Nalini

    2016-01-01

    Production of reactive nitrogen species (NO) is a key step in the immune response following infections. NO induces lesions to bacterial DNA, thus limiting bacterial growth within hosts. Using two pathogenic bacteria, Bacillus cereus and Shigella flexneri, we show that the DNA-repair protein Mfd (Mutation-Frequency-Decline) is required for bacterial resistance to the host-NO-response. In both species, a mutant deficient for mfd does not survive to NO, produced in vitro or by phagocytic cells. In vivo, the ∆mfd mutant is avirulent and unable to survive the NO-stress. Moreover, NO induces DNA-double-strand-breaks and point mutations in the Δmfd mutant. In overall, these observations demonstrate that NO damages bacterial DNA and that Mfd is required to maintain bacterial genomic integrity. This unexpected discovery reveals that Mfd, a typical housekeeping gene, turns out to be a true virulence factor allowing survival and growth of the pathogen in its host, due to its capacity to protect the bacterium against NO, a key molecule of the innate immune defense. As Mfd is widely conserved in the bacterial kingdom, these data highlight a mechanism that may be used by a large spectrum of bacteria to overcome the host immune response and especially the mutagenic properties of NO. PMID:27435260

  19. The bacterial DNA repair protein Mfd confers resistance to the host nitrogen immune response

    PubMed Central

    Guillemet, Elisabeth; Leréec, Alain; Tran, Seav-Ly; Royer, Corinne; Barbosa, Isabelle; Sansonetti, Philippe; Lereclus, Didier; Ramarao, Nalini

    2016-01-01

    Production of reactive nitrogen species (NO) is a key step in the immune response following infections. NO induces lesions to bacterial DNA, thus limiting bacterial growth within hosts. Using two pathogenic bacteria, Bacillus cereus and Shigella flexneri, we show that the DNA-repair protein Mfd (Mutation-Frequency-Decline) is required for bacterial resistance to the host-NO-response. In both species, a mutant deficient for mfd does not survive to NO, produced in vitro or by phagocytic cells. In vivo, the ∆mfd mutant is avirulent and unable to survive the NO-stress. Moreover, NO induces DNA-double-strand-breaks and point mutations in the Δmfd mutant. In overall, these observations demonstrate that NO damages bacterial DNA and that Mfd is required to maintain bacterial genomic integrity. This unexpected discovery reveals that Mfd, a typical housekeeping gene, turns out to be a true virulence factor allowing survival and growth of the pathogen in its host, due to its capacity to protect the bacterium against NO, a key molecule of the innate immune defense. As Mfd is widely conserved in the bacterial kingdom, these data highlight a mechanism that may be used by a large spectrum of bacteria to overcome the host immune response and especially the mutagenic properties of NO. PMID:27435260

  20. Genomoviridae: a new family of widespread single-stranded DNA viruses.

    PubMed

    Krupovic, Mart; Ghabrial, Said A; Jiang, Daohong; Varsani, Arvind

    2016-09-01

    Here, we introduce a new family of eukaryote-infecting single-stranded (ss) DNA viruses that was created recently by the International Committee on Taxonomy of Viruses (ICTV). The family, named Genomoviridae, contains a single genus, Gemycircularvirus, which currently has one recognized virus species, Sclerotinia gemycircularvirus 1. Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) is currently the sole representative isolate of the family; however, a great number of SsHADV-1-like ssDNA virus genomes has been sequenced from various environmental, plant- and animal-associated samples, indicating that members of family Genomoviridae are widespread and abundant in the environment. PMID:27343045

  1. The inflammasome as a target of modulation by DNA viruses

    PubMed Central

    DeFilippis, Victor

    2014-01-01

    The cellular innate immune response represents the initial reaction of a host against infecting pathogens. Host cells detect incoming microbes by way of a large and expanding array of receptors that react with evolutionarily conserved molecular patterns exhibited by microbial intruders. These receptors are responsible for initiating signaling that leads to both transcriptional activation of immunologically important genes as well as protease-dependent processing of cellular proteins. The inflammasome refers to a protein complex that functions as an activation platform for the cysteine protease caspase-1, which then processes inflammatory molecules such as IL-1β and IL-18 into functional forms. Assembly of this complex is triggered following receptor-mediated detection of pathogen-associated molecules. Receptors have been identified that are essential to inflammasome activation in response to numerous molecular patterns including virus-associated molecules such as DNA. In fact, the importance of cytoplasmic DNA as an immune stimulus is exemplified by the existence of at least nine distinct cellular receptors capable of initiating innate reactivity in response to this molecule. Viruses that employ DNA as genomic material include herpesviruses, poxviruses and adenoviruses. Each has been described as capable of inducing inflammasome-mediated activity. Interestingly, however, the cellular molecules responsible for these responses appear to vary according to host species, cell type and even viral strain. Secretion of IL-1β and IL-18 are important components of antimicrobial immunity and, as a result, pathogens have evolved factors to evade or counteract this response. This includes DNA-based viruses, many of which encode multiple redundant counteractive molecules. However, it is clear that such phenotypes are only beginning to be uncovered. The purpose of this review is to describe what is known regarding the activation of inflammasome-mediated processes in response to

  2. The influenza virus nucleoprotein synthesized from cloned DNA in a simian virus 40 vector is detected in the nucleus.

    PubMed Central

    Lin, B C; Lai, C J

    1983-01-01

    We obtained DNA sequences coding for the nucleoprotein (NP) of an influenza A virus by reverse transcription of virion RNA with synthetic oligonucleotide primers. Terminal sequence analysis showed that the cloned gene contained a full-length copy of the virion RNA segment. The NP-specific DNA was inserted into the late region of a simian virus 40 vector, and the DNA recombinant was propagated in the presence of an early simian virus 40 temperature-sensitive mutant helper. Infection of African green monkey kidney cells with the recombinant produced a polypeptide immunoprecipitable with NP-specific antisera. The polypeptide product had a molecular weight of 56,000, identical to that of the nucleoprotein of influenza virus as estimated on polyacrylamide gels. The putative NP was detected in the nucleus of infected primate cells by an immunofluorescence assay. This nuclear localization of NP from recombinant DNA was similar to that seen during influenza virus infection. Images PMID:6296449

  3. Bones hold the key to DNA virus history and epidemiology.

    PubMed

    Toppinen, M; Perdomo, M F; Palo, J U; Simmonds, P; Lycett, S J; Söderlund-Venermo, M; Sajantila, A; Hedman, K

    2015-01-01

    DNA in human skeletal remains represents an important historical source of host genomic information and potentially of infecting viruses. However, little is known about viral persistence in bone. We searched ca. 70-year-old long bones of putative Finnish casualties from World War II for parvovirus B19 (B19V) DNA, and found a remarkable prevalence of 45%. The viral sequences were exclusively of genotypes 2 (n = 41), which disappeared from circulation in 1970´s, or genotype 3 (n = 2), which has never been reported in Northern Europe. Based on mitochondrial and Y-chromosome profiling, the two individuals carrying B19V genotype 3 were likely from the Soviet Red Army. The most recent common ancestor for all genotypes was estimated at early 1800s. This work demonstrates the forms of B19V that circulated in the first half of the 20(th) century and provides the first evidence of the suitability of bone for exploration of DNA viruses. PMID:26611279

  4. Bones hold the key to DNA virus history and epidemiology

    PubMed Central

    Toppinen, M.; Perdomo, M. F.; Palo, J. U.; Simmonds, P.; Lycett, S. J.; Söderlund-Venermo, M.; Sajantila, A.; Hedman, K.

    2015-01-01

    DNA in human skeletal remains represents an important historical source of host genomic information and potentially of infecting viruses. However, little is known about viral persistence in bone. We searched ca. 70-year-old long bones of putative Finnish casualties from World War II for parvovirus B19 (B19V) DNA, and found a remarkable prevalence of 45%. The viral sequences were exclusively of genotypes 2 (n = 41), which disappeared from circulation in 1970´s, or genotype 3 (n = 2), which has never been reported in Northern Europe. Based on mitochondrial and Y-chromosome profiling, the two individuals carrying B19V genotype 3 were likely from the Soviet Red Army. The most recent common ancestor for all genotypes was estimated at early 1800s. This work demonstrates the forms of B19V that circulated in the first half of the 20th century and provides the first evidence of the suitability of bone for exploration of DNA viruses. PMID:26611279

  5. Novel linear DNA vaccines induce protective immune responses against lethal infection with influenza virus type A/H5N1.

    PubMed

    Kendirgi, Frédéric; Yun, Nadezda E; Linde, Nathaniel S; Zacks, Michele A; Smith, Jeanon N; Smith, Jennifer K; McMicken, Harilyn; Chen, Yin; Paessler, Slobodan

    2008-01-01

    Vaccine development for possible influenza pandemics has been challenging. Conventional vaccines such as inactivated and live attenuated virus preparations are limited in terms of production speed and capacity. DNA vaccination has emerged as a potential alternative to conventional vaccines against influenza pandemics. In this study, we use a novel, cell-free DNA manufacturing process (synDNA) to produce prototype linear DNA vaccines against the influenza virus type A/H5N1. This synDNA process does not require bacterial fermentation, so it avoids the use of antibiotic resistance genes and other nucleic acid sequences unrelated to the antigen gene expression in the actual therapeutic DNA construct. The efficacy of various vaccines expressing the hemagglutinin and neuraminidase proteins (H5N1 synDNA), hemagglutinin alone (H5 synDNA) or neuraminidase alone (N1 synDNA) was evaluated in mice. Two of the constructs (H5 synDNA and H5N1 synDNA) induced a robust protective immune response with up to 93% of treated mice surviving a lethal challenge of a virulent influenza A/Vietnam/1203/04 H5N1 isolate. In combination with a potent biological activity and simplified production footprint, these characteristics make DNA vaccines prepared with our synDNA process highly suitable as alternatives to other vaccine preparations. PMID:18443425

  6. DNA recovery from a single bacterial cell using charge-reversible magnetic nanoparticles.

    PubMed

    Maeda, Yoshiaki; Toyoda, Takahiro; Mogi, Takeyuki; Taguchi, Tomoyuki; Tanaami, Takeo; Yoshino, Tomoko; Matsunaga, Tadashi; Tanaka, Tsuyoshi

    2016-03-01

    Highly efficient DNA recovery from a single bacterial cell was performed by means of imidazole-modified magnetic nanoparticles (Imi-MNPs). The modification by imidazole was confirmed by Fourier transform infrared spectroscopy. The Imi-MNPs were highly efficient at DNA extraction owing to the charge-reversible properties of Imi-MNPs, whereby DNA is attached to the particles at low pH and eluted at high pH because of electrostatic interactions. The DNA recovery ratio was determined by real-time PCR, and it revealed that complete recovery was guaranteed at ≥10(3) genome copies of Bacillus subtilis. Extraction of DNA from single bacterial cells was followed by PCR amplification of 16S rDNA and capillary electrophoresis. We achieved detection of single bacterial cells with a detection rate of 80%. We believe that our DNA recovery strategy may serve as a powerful tool for efficient DNA extraction and should be useful for quality control of cosmetics, foods, and pharmaceutical products. PMID:26704992

  7. Altered Virome and Bacterial Microbiome in Human Immunodeficiency Virus-Associated Acquired Immunodeficiency Syndrome.

    PubMed

    Monaco, Cynthia L; Gootenberg, David B; Zhao, Guoyan; Handley, Scott A; Ghebremichael, Musie S; Lim, Efrem S; Lankowski, Alex; Baldridge, Megan T; Wilen, Craig B; Flagg, Meaghan; Norman, Jason M; Keller, Brian C; Luévano, Jesús Mario; Wang, David; Boum, Yap; Martin, Jeffrey N; Hunt, Peter W; Bangsberg, David R; Siedner, Mark J; Kwon, Douglas S; Virgin, Herbert W

    2016-03-01

    Human immunodeficiency virus (HIV) infection is associated with increased intestinal translocation of microbial products and enteropathy as well as alterations in gut bacterial communities. However, whether the enteric virome contributes to this infection and resulting immunodeficiency remains unknown. We characterized the enteric virome and bacterial microbiome in a cohort of Ugandan patients, including HIV-uninfected or HIV-infected subjects and those either treated with anti-retroviral therapy (ART) or untreated. Low peripheral CD4 T cell counts were associated with an expansion of enteric adenovirus sequences and this increase was independent of ART treatment. Additionally, the enteric bacterial microbiome of patients with lower CD4 T counts exhibited reduced phylogenetic diversity and richness with specific bacteria showing differential abundance, including increases in Enterobacteriaceae, which have been associated with inflammation. Thus, immunodeficiency in progressive HIV infection is associated with alterations in the enteric virome and bacterial microbiome, which may contribute to AIDS-associated enteropathy and disease progression. PMID:26962942

  8. Detection of hepatitis A virus in seeded estuarine samples by hybridization with cDNA probes

    SciTech Connect

    Jiang, X.; Estes, M.K.; Metcalf, T.G.; Melnick, J.L

    1986-10-01

    The development and trials of a nucleic acid hybridization test for the detection of hepatitis A virus (HAV) in estuarine samples within 48 h are described. Approximately 10/sup 4/ physical particlels of HAV per dot could be detected. Test sensitivity was optimized by the consideration of hydbridization stringency, /sup 32/P energy level, probe concentration, and nucleic acid binding to filters. Test specificity was shown by a lack of cross-hybridization with other enteroviruses and unrelated nucleic acids. Potential false-positive reactions between bacterial DNA in samples and residual vector DNA contamination of purified nucleotide sequences in probes were eliminated by DNase treatment of samples. Humic acid at concentrations of up to 100 mg/liter caused only insignificant decreases in test sensitivity. Interference with hybridization by organic components of virus-containing eluates was removed by proteinase K digestion followed by phenol extraction and ethanol precipitation. The test is suitable for detecting naturally occurring HAV in samples from polluted estuarine environments.

  9. Mechanisms of ion-bombardment-induced DNA transfer into bacterial E. coli cells

    NASA Astrophysics Data System (ADS)

    Yu, L. D.; Sangwijit, K.; Prakrajang, K.; Phanchaisri, B.; Thongkumkoon, P.; Thopan, P.; Singkarat, S.; Anuntalabhochai, S.

    2014-05-01

    As a useful ion beam biotechnology, ion-bombardment-induced DNA transfer into bacterial Escherichia coli (E. coli) cells has been successfully operated using argon ions. In the process ion bombardment of the bacterial cells modifies the cell envelope materials to favor the exogenous DNA molecules to pass through the envelope to enter the cell. The occurrence of the DNA transfer induction was found ion energy and fluence dependent in a complex manner. At ion energy of a few keV and a few tens of keV to moderate fluences the DNA transfer could be induced by ion bombardment of the bacterial cells, while at the same ion energy but to high fluences DNA transfer could not be induced. On the other hand, when the ion energy was medium, about 10-20 keV, the DNA transfer could not be induced by ion bombardment of the cells. The complexity of the experimental results indicated a complex mechanism which should be related to the complex structure of the bacterial E. coli cell envelope. A phase diagram was proposed to interpret different mechanisms involved as functions of the ion energy and fluence.

  10. Elasticity-mediated nematiclike bacterial organization in model extracellular DNA matrix.

    PubMed

    Smalyukh, Ivan I; Butler, John; Shrout, Joshua D; Parsek, Matthew R; Wong, Gerard C L

    2008-09-01

    DNA is a common extracellular matrix component of bacterial biofilms. We find that bacteria can spontaneously order in a matrix of aligned concentrated DNA, in which rod-shaped cells of Pseudomonas aeruginosa follow the orientation of extended DNA chains. The alignment of bacteria is ensured by elasticity and liquid crystalline properties of the DNA matrix. These findings show how behavior of planktonic bacteria may be modified in extracellular polymeric substances of biofilms and illustrate the potential of using complex fluids to manipulate embedded nanosized and microsized active particles. PMID:18850984

  11. Guide DNA technique in bacterial ribonuclease P reaction for effective processing of tRNA precursor.

    PubMed

    Tanaka, Terumichi; Hori, Yoshiaki; Kikuchi, Yo

    2002-10-01

    Previously, we found that a small (approx. 20-mer) DNA hybridizing to the 5'-leader region of a tRNA precursor enhances the cleavage efficiency in bacterial ribonuclease P reaction. We named this technique the 'guide DNA technique'. Detailed analyses showed that the length of the guide DNA, concentration of the guide DNA and the hybridizing position affected the cleavage efficiency: for an effective cleavage reaction, guide DNA should be designed to hybridize to the region on the cleavage site, should be 20 bases or more in length and should be of high concentration. The presence of a 5'-flanking region in the DNA did not affect the cleavage reaction. The guide DNA technique is a useful tool for effective preparation of mature tRNA molecules in vitro. PMID:12241548

  12. Detection of herpes simplex virus-specific DNA sequences in latently infected mice and in humans.

    PubMed Central

    Efstathiou, S; Minson, A C; Field, H J; Anderson, J R; Wildy, P

    1986-01-01

    Herpes simplex virus-specific DNA sequences have been detected by Southern hybridization analysis in both central and peripheral nervous system tissues of latently infected mice. We have detected virus-specific sequences corresponding to the junction fragment but not the genomic termini, an observation first made by Rock and Fraser (Nature [London] 302:523-525, 1983). This "endless" herpes simplex virus DNA is both qualitatively and quantitatively stable in mouse neural tissue analyzed over a 4-month period. In addition, examination of DNA extracted from human trigeminal ganglia has shown herpes simplex virus DNA to be present in an "endless" form similar to that found in the mouse model system. Further restriction enzyme analysis of latently infected mouse brainstem and human trigeminal DNA has shown that this "endless" herpes simplex virus DNA is present in all four isomeric configurations. Images PMID:3003377

  13. Immunization of Pigs with a Particle-Mediated DNA Vaccine to Influenza A Virus Protects against Challenge with Homologous Virus

    PubMed Central

    Macklin, Michael D.; McCabe, Dennis; McGregor, Martha W.; Neumann, Veronica; Meyer, Todd; Callan, Robert; Hinshaw, Virginia S.; Swain, William F.

    1998-01-01

    Particle-mediated delivery of a DNA expression vector encoding the hemagglutinin (HA) of an H1N1 influenza virus (A/Swine/Indiana/1726/88) to porcine epidermis elicits a humoral immune response and accelerates the clearance of virus in pigs following a homotypic challenge. Mucosal administration of the HA expression plasmid elicits an immune response that is qualitatively different than that elicited by the epidermal vaccination in terms of inhibition of the initial virus infection. In contrast, delivery of a plasmid encoding an influenza virus nucleoprotein from A/PR/8/34 (H1N1) to the epidermis elicits a strong humoral response but no detectable protection in terms of nasal virus shed. The efficacy of the HA DNA vaccine was compared with that of a commercially available inactivated whole-virus vaccine as well as with the level of immunity afforded by previous infection. The HA DNA and inactivated viral vaccines elicited similar protection in that initial infection was not prevented, but subsequent amplification of the infection is limited, resulting in early clearance of the virus. Convalescent animals which recovered from exposure to virulent swine influenza virus were completely resistant to infection when challenged. The porcine influenza A virus system is a relevant preclinical model for humans in terms of both disease and gene transfer to the epidermis and thus provides a basis for advancing the development of DNA-based vaccines. PMID:9445052

  14. Targeting hepatitis B virus cccDNA using CRISPR/Cas9.

    PubMed

    Kennedy, Edward M; Kornepati, Anand V R; Cullen, Bryan R

    2015-11-01

    Despite the existence of an excellent prophylactic vaccine and the development of highly effective inhibitors of the viral polymerase, chronic hepatitis B virus (HBV) infection remains a major source of morbidity and mortality, especially in Africa and Asia. A significant problem is that, while polymerase inhibitors can effectively prevent the production of viral genomic DNA from pre-genomic RNA transcripts, they do not prevent the transcription and translation of viral mRNAs from the covalently closed circular DNA (cccDNA) templates present in the nuclei of infected cells. Moreover, because these cccDNAs are highly stable, chronic HBV infections are only very rarely cured by the use of polymerase inhibitors and these drugs clearly cannot entirely prevent the subsequent development of HBV-related morbidities such as cirrhosis and hepatocellular carcinoma. As a result, there has been considerable interest in the possibility of developing treatment approaches that directly target cccDNA for elimination. Here, we discuss recent publications that analyze the ability of the bacterial CRISPR/Cas DNA editing machinery to be repurposed as a tool for the specific cleavage and destruction of HBV cccDNAs in the nuclei of infected cells and consider which steps will be necessary to make CRISPR/Cas targeting of HBV DNA a clinically feasible approach to the treatment of chronic infections in humans. This article forms part of a symposium in Antiviral Research on "An unfinished story: from the discovery of the Australia antigen to the development of new curative therapies for hepatitis B." PMID:26476375

  15. Purification of transfection-grade plasmid DNA from bacterial cells with superparamagnetic nanoparticles

    NASA Astrophysics Data System (ADS)

    Chiang, Chen-Li; Sung, Ching-Shan

    2006-07-01

    The functionalized magnetic nanobeads were used to develop a rapid protocol for extracting and purifying transfection-grade plasmid DNA from bacterial culture. Nanosized superparamagnetic nanoparticles (Fe 3O 4) were prepared by chemical coprecipitation method using Fe 2+, Fe 3+ salt, and ammonium hydroxide under a nitrogen atmosphere. The surface of Fe 3O 4 nanoparticles was modified by coating with the multivalent cationic agent, polyethylenimine (PEI). The PEI-modified magnetic nanobeads were employed to simplify the purification of plasmid DNA from bacterial cells. We demonstrated a useful plasmid, pRSETB-EGFP, encoding the green fluorescent protein with T7 promoter, was amplified in DE3 strain of Escherichia coli. The loaded nanobeads are recovered by magnetically driven separation and regenerated by exposure to the elution buffer with optimal ionic strength (1.25 M) and pH (9.0). Up to approximately 819 μg of high-purity (A 260/A 280 ratio=1.86) plasmid DNA was isolated from 100 ml of overnight bacterial culture. The eluted plasmid DNA was used directly for restriction enzyme digestion, bacterial cell transformation and animal cell transfection applications with success. The PEI-modified magnetic nanobead delivers significant time-savings, overall higher yields and better transfection efficiencies compared to anion-exchange and other methods. The results presented in this report show that PEI-modified magnetic nanobeads are suitable for isolation and purification of transfection-grade plasmid DNA.

  16. Facile, High Quality Sequencing of Bacterial Genomes from Small Amounts of DNA

    PubMed Central

    Vuyisich, Momchilo; Arefin, Ayesha; Davenport, Karen; Feng, Shihai; Gleasner, Cheryl; McMurry, Kim; Parson-Quintana, Beverly; Price, Jennifer; Scholz, Matthew; Chain, Patrick

    2014-01-01

    Sequencing bacterial genomes has traditionally required large amounts of genomic DNA (~1 μg). There have been few studies to determine the effects of the input DNA amount or library preparation method on the quality of sequencing data. Several new commercially available library preparation methods enable shotgun sequencing from as little as 1 ng of input DNA. In this study, we evaluated the NEBNext Ultra library preparation reagents for sequencing bacterial genomes. We have evaluated the utility of NEBNext Ultra for resequencing and de novo assembly of four bacterial genomes and compared its performance with the TruSeq library preparation kit. The NEBNext Ultra reagents enable high quality resequencing and de novo assembly of a variety of bacterial genomes when using 100 ng of input genomic DNA. For the two most challenging genomes (Burkholderia spp.), which have the highest GC content and are the longest, we also show that the quality of both resequencing and de novo assembly is not decreased when only 10 ng of input genomic DNA is used. PMID:25478564

  17. 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

  18. Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency

    PubMed Central

    Tu, Qiang; Yin, Jia; Fu, Jun; Herrmann, Jennifer; Li, Yuezhong; Yin, Yulong; Stewart, A. Francis; Müller, Rolf; Zhang, Youming

    2016-01-01

    Bacterial competent cells are essential for cloning, construction of DNA libraries, and mutagenesis in every molecular biology laboratory. Among various transformation methods, electroporation is found to own the best transformation efficiency. Previous electroporation methods are based on washing and electroporating the bacterial cells in ice-cold condition that make them fragile and prone to death. Here we present simple temperature shift based methods that improve DNA transformation and recombineering efficiency in E. coli and several other gram-negative bacteria thereby economizing time and cost. Increased transformation efficiency of large DNA molecules is a significant advantage that might facilitate the cloning of large fragments from genomic DNA preparations and metagenomics samples. PMID:27095488

  19. Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency.

    PubMed

    Tu, Qiang; Yin, Jia; Fu, Jun; Herrmann, Jennifer; Li, Yuezhong; Yin, Yulong; Stewart, A Francis; Müller, Rolf; Zhang, Youming

    2016-01-01

    Bacterial competent cells are essential for cloning, construction of DNA libraries, and mutagenesis in every molecular biology laboratory. Among various transformation methods, electroporation is found to own the best transformation efficiency. Previous electroporation methods are based on washing and electroporating the bacterial cells in ice-cold condition that make them fragile and prone to death. Here we present simple temperature shift based methods that improve DNA transformation and recombineering efficiency in E. coli and several other gram-negative bacteria thereby economizing time and cost. Increased transformation efficiency of large DNA molecules is a significant advantage that might facilitate the cloning of large fragments from genomic DNA preparations and metagenomics samples. PMID:27095488

  20. Construction and characterization of bacterial artificial chromosomes (BACs) containing herpes simplex virus full-length genomes.

    PubMed

    Nagel, Claus-Henning; Pohlmann, Anja; Sodeik, Beate

    2014-01-01

    Bacterial artificial chromosomes (BACs) are suitable vectors not only to maintain the large genomes of herpesviruses in Escherichia coli but also to enable the traceless introduction of any mutation using modern tools of bacterial genetics. To clone a herpes simplex virus genome, a BAC replication origin is first introduced into the viral genome by homologous recombination in eukaryotic host cells. As part of their nuclear replication cycle, genomes of herpesviruses circularize and these replication intermediates are then used to transform bacteria. After cloning, the integrity of the recombinant viral genomes is confirmed by restriction length polymorphism analysis and sequencing. The BACs may then be used to design virus mutants. Upon transfection into eukaryotic cells new herpesvirus strains harboring the desired mutations can be recovered and used for experiments in cultured cells as well as in animal infection models. PMID:24671676

  1. Bacterial DNA Sifted from the Trichoplax adhaerens (Animalia: Placozoa) Genome Project Reveals a Putative Rickettsial Endosymbiont

    PubMed Central

    Driscoll, Timothy; Gillespie, Joseph J.; Nordberg, Eric K.; Azad, Abdu F.; Sobral, Bruno W.

    2013-01-01

    Eukaryotic genome sequencing projects often yield bacterial DNA sequences, data typically considered as microbial contamination. However, these sequences may also indicate either symbiont genes or lateral gene transfer (LGT) to host genomes. These bacterial sequences can provide clues about eukaryote–microbe interactions. Here, we used the genome of the primitive animal Trichoplax adhaerens (Metazoa: Placozoa), which is known to harbor an uncharacterized Gram-negative endosymbiont, to search for the presence of bacterial DNA sequences. Bioinformatic and phylogenomic analyses of extracted data from the genome assembly (181 bacterial coding sequences [CDS]) and trace read archive (16S rDNA) revealed a dominant proteobacterial profile strongly skewed to Rickettsiales (Alphaproteobacteria) genomes. By way of phylogenetic analysis of 16S rDNA and 113 proteins conserved across proteobacterial genomes, as well as identification of 27 rickettsial signature genes, we propose a Rickettsiales endosymbiont of T. adhaerens (RETA). The majority (93%) of the identified bacterial CDS belongs to small scaffolds containing prokaryotic-like genes; however, 12 CDS were identified on large scaffolds comprised of eukaryotic-like genes, suggesting that T. adhaerens might have recently acquired bacterial genes. These putative LGTs may coincide with the placozoan’s aquatic niche and symbiosis with RETA. This work underscores the rich, and relatively untapped, resource of eukaryotic genome projects for harboring data pertinent to host–microbial interactions. The nature of unknown (or poorly characterized) bacterial species may only emerge via analysis of host genome sequencing projects, particularly if these species are resistant to cell culturing, as are many obligate intracellular microbes. Our work provides methodological insight for such an approach. PMID:23475938

  2. Fragment-based discovery of 6-azaindazoles as inhibitors of bacterial DNA ligase.

    PubMed

    Howard, Steven; Amin, Nader; Benowitz, Andrew B; Chiarparin, Elisabetta; Cui, Haifeng; Deng, Xiaodong; Heightman, Tom D; Holmes, David J; Hopkins, Anna; Huang, Jianzhong; Jin, Qi; Kreatsoulas, Constantine; Martin, Agnes C L; Massey, Frances; McCloskey, Lynn; Mortenson, Paul N; Pathuri, Puja; Tisi, Dominic; Williams, Pamela A

    2013-12-12

    Herein we describe the application of fragment-based drug design to bacterial DNA ligase. X-ray crystallography was used to guide structure-based optimization of a fragment-screening hit to give novel, nanomolar, AMP-competitive inhibitors. The lead compound 13 showed antibacterial activity across a range of pathogens. Data to demonstrate mode of action was provided using a strain of S. aureus, engineered to overexpress DNA ligase. PMID:24900632

  3. Fragment-Based Discovery of 6-Azaindazoles As Inhibitors of Bacterial DNA Ligase

    PubMed Central

    2013-01-01

    Herein we describe the application of fragment-based drug design to bacterial DNA ligase. X-ray crystallography was used to guide structure-based optimization of a fragment-screening hit to give novel, nanomolar, AMP-competitive inhibitors. The lead compound 13 showed antibacterial activity across a range of pathogens. Data to demonstrate mode of action was provided using a strain of S. aureus, engineered to overexpress DNA ligase. PMID:24900632

  4. Artifically inserting a reticuloendotheliosis virus long terminal repeat into a bacterial artificial chromosome clone of Marek's disease virus (MDV) alters expression of nearby MDV genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The long terminal repeat (LTR) sequence of reticuloendotheliosis virus (REV) was inserted into the very virulent Marek’s disease virus (MDV) Md5 bacterial artificial chromosome clone. The insertion site was nearly identical to the REV LTR that was naturally inserted into the JM/102W strain of MDV fo...

  5. p53-Mediated Cellular Response to DNA Damage in Cells with Replicative Hepatitis B Virus

    NASA Astrophysics Data System (ADS)

    Puisieux, Alain; Ji, Jingwei; Guillot, Celine; Legros, Yann; Soussi, Thierry; Isselbacher, Kurt; Ozturk, Mehmet

    1995-02-01

    Wild-type p53 acts as a tumor suppressor gene by protecting cells from deleterious effects of genotoxic agents through the induction of a G_1/S arrest or apoptosis as a response to DNA damage. Transforming proteins of several oncogenic DNA viruses inactivate tumor suppressor activity of p53 by blocking this cellular response. To test whether hepatitis B virus displays a similar effect, we studied the p53-mediated cellular response to DNA damage in 2215 hepatoma cells with replicative hepatitis B virus. We demonstrate that hepatitis B virus replication does not interfere with known cellular functions of p53 protein.

  6. Comparative study of HOCl-inflicted damage to bacterial DNA ex vivo and within cells.

    PubMed

    Suquet, Christine; Warren, Jeffrey J; Seth, Nimulrith; Hurst, James K

    2010-01-15

    The prospects for using bacterial DNA as an intrinsic probe for HOCl and secondary oxidants/chlorinating agents associated with it has been evaluated using both in vitro and in vivo studies. Single-strand and double-strand breaks occurred in bare plasmid DNA that had been exposed to high levels of HOCl, although these reactions were very inefficient compared to polynucleotide chain cleavage caused by the OH.-generating reagent, peroxynitrite. Plasmid nicking was not increased when intact Escherichia coli were exposed to HOCl; rather, the amount of recoverable plasmid diminished in a dose-dependent manner. At concentration levels of HOCl exceeding lethal doses, genomic bacterial DNA underwent extensive fragmentation and the amount of precipitable DNA-protein complexes increased several-fold. The 5-chlorocytosine content of plasmid and genomic DNA isolated from HOCl-exposed E. coli was also slightly elevated above controls, as measured by mass spectrometry of the deaminated product, 5-chlorouracil. However, the yields were not dose-dependent over the bactericidal concentration range. Genomic DNA recovered from E. coli that had been subjected to phagocytosis by human neutrophils occasionally showed small increases in 5-chlorocytosine content when compared to analogous cellular reactions where myeloperoxidase activity was inhibited by azide ion. Overall, the amount of isolable 5-chlorouracil from the HOCl-exposed bacterial cells was far less than the damage manifested in polynucleotide bond cleavage and cross-linking. PMID:19850004

  7. Gyramides Prevent Bacterial Growth by Inhibiting DNA Gyrase and Altering Chromosome Topology

    PubMed Central

    2015-01-01

    Antibiotics targeting DNA gyrase have been a clinical success story for the past half-century, and the emergence of bacterial resistance has fueled the search for new gyrase inhibitors. In this paper we demonstrate that a new class of gyrase inhibitors, the gyramides, are bacteriostatic agents that competitively inhibit the ATPase activity of Escherichia coli gyrase and produce supercoiled DNA in vivo. E. coli cells treated with gyramide A have abnormally localized, condensed chromosomes that blocks DNA replication and interrupts chromosome segregation. The resulting alterations in DNA topology inhibit cell division through a mechanism that involves the SOS pathway. Importantly, gyramide A is a specific inhibitor of gyrase and does not inhibit the closely related E. coli enzyme topoisomerase IV. E. coli mutants with reduced susceptibility to gyramide A do not display cross-resistance to ciprofloxacin and novobiocin. The results demonstrate that the gyramides prevent bacterial growth by a mechanism in which the topological state of chromosomes is altered and halts DNA replication and segregation. The specificity and activity of the gyramides for inhibiting gyrase makes these compounds important chemical tools for studying the mechanism of gyrase and the connection between DNA topology and bacterial cell division. PMID:24712739

  8. UV Radiation Damage and Bacterial DNA Repair Systems

    ERIC Educational Resources Information Center

    Zion, Michal; Guy, Daniel; Yarom, Ruth; Slesak, Michaela

    2006-01-01

    This paper reports on a simple hands-on laboratory procedure for high school students in studying both radiation damage and DNA repair systems in bacteria. The sensitivity to ultra-violet (UV) radiation of both "Escherichia coli" and "Serratia marcescens" is tested by radiating them for varying time periods. Two growth temperatures are used in…

  9. DNA condensates organized by the capsid protein VP15 in White Spot Syndrome Virus

    SciTech Connect

    Liu Yingjie; Wu Jinlu; Chen Hu; Hew, Choy Leong; Yan Jie

    2010-12-20

    The White Spot Syndrome Virus (WSSV) has a large circular double-stranded DNA genome of around 300 kb and it replicates in the nucleus of the host cells. The machinery of how the viral DNA is packaged has been remained unclear. VP15, a highly basic protein, is one of the major capsid proteins found in the virus. Previously, it was shown to be a DNA binding protein and was hypothesized to participate in the viral DNA packaging process. Using Atomic Force Microscopy imaging, we show that the viral DNA is associated with a (or more) capsid proteins. The organized viral DNA qualitatively resembles the conformations of VP15 induced DNA condensates in vitro. Furthermore, single-DNA manipulation experiments revealed that VP15 is able to condense single DNA against forces of a few pico Newtons. Our results suggest that VP15 may aid in the viral DNA packaging process by directly condensing DNA.

  10. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes.

    PubMed Central

    Versalovic, J; Koeuth, T; Lupski, J R

    1991-01-01

    Dispersed repetitive DNA sequences have been described recently in eubacteria. To assess the distribution and evolutionary conservation of two distinct prokaryotic repetitive elements, consensus oligonucleotides were used in polymerase chain reaction [PCR] amplification and slot blot hybridization experiments with genomic DNA from diverse eubacterial species. Oligonucleotides matching Repetitive Extragenic Palindromic [REP] elements and Enterobacterial Repetitive Intergenic Consensus [ERIC] sequences were synthesized and tested as opposing PCR primers in the amplification of eubacterial genomic DNA. REP and ERIC consensus oligonucleotides produced clearly resolvable bands by agarose gel electrophoresis following PCR amplification. These band patterns provided unambiguous DNA fingerprints of different eubacterial species and strains. Both REP and ERIC probes hybridized preferentially to genomic DNA from Gram-negative enteric bacteria and related species. Widespread distribution of these repetitive DNA elements in the genomes of various microorganisms should enable rapid identification of bacterial species and strains, and be useful for the analysis of prokaryotic genomes. Images PMID:1762913

  11. Preparation of high molecular weight gDNA and bacterial artificial chromosome (BAC) libraries in plants.

    PubMed

    Biradar, Siddanagouda S; Nie, Xiaojun; Feng, Kewei; Weining, Song

    2014-01-01

    Bacterial artificial chromosome (BAC) libraries are extremely valuable large-insert DNA libraries for physical mapping, positional cloning, comparative genomic analysis, complete genome sequencing, and evolutionary studies. Due to their stability and relative simplicity BAC libraries are most preferred over other approaches for cloning large genomic DNA fragments for large-insert libraries. Isolation of intact high molecular weight (HMW) DNA is a critical step underlying the success of large-insert genomic DNA library construction. It requires the isolation of purified nuclei, embedding them into LMP agarose plugs, restriction digestion of the plugs, and quite often size selection using PFGE and electro-elution of insert DNA. The construction of BAC libraries is complex and challenging for most molecular laboratories. To facilitate the construction of BAC libraries, we present a step-by-step protocol for isolation of HMW DNA and construction of plant BAC libraries. PMID:24243195

  12. Effects of Bacterial Microflora of the Lower Digestive Tract of Free-Range Waterfowl on Influenza Virus Activation ▿

    PubMed Central

    King, Marcus D.; Guentzel, M. Neal; Arulanandam, Bernard P.; Bodour, Adria A.; Brahmakshatriya, Vinayak; Lupiani, Blanca; Chambers, James P.

    2011-01-01

    Proteolytic cleavage activation of influenza virus hemagglutinin (HA0) is required for cell entry via receptor-mediated endocytosis. Despite numerous studies describing bacterial protease-mediated influenza A viral activation in mammals, very little is known about the role of intestinal bacterial flora of birds in hemagglutinin cleavage/activation. Therefore, the cloaca of wild waterfowl was examined for (i) representative bacterial types and (ii) their ability to cleave in a “trypsin-like” manner the precursor viral hemagglutinin molecule (HA0). Using radiolabeled HA0, bacterial secretion-mediated trypsin-like conversion of HA0 to HA1 and HA2 peptide products was observed to various degrees in 42 of 44 bacterial isolates suggestive of influenza virus activation in the cloaca of wild waterfowl. However, treatment of uncleaved virus with all bacterial isolates gave rise to substantially reduced emergent virus progeny compared with what was expected. Examination of two isolates exhibiting pronounced trypsin-like conversion of HA0 to HA1 and HA2 peptide products and low infectivity revealed lipase activity to be present. Because influenza virus possesses a complex lipid envelope, the presence of lipid hydrolase activity could in part account for the observed less-than-expected level of viable progeny. A thorough characterization of respective isolate protease HA0 hydrolysis products as well as other resident activities (i.e., lipase) is ongoing such that the role of these respective contributors in virus activation/inactivation can be firmly established. PMID:21531837

  13. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... recommendations as specified under 40 CFR part 792, subpart J the following specific information should be... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.”...

  14. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... recommendations as specified under 40 CFR part 792, subpart J the following specific information should be... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.”...

  15. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... recommendations as specified under 40 CFR part 792, subpart J the following specific information should be... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.”...

  16. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... recommendations as specified under 40 CFR part 792, subpart J the following specific information should be... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.”...

  17. 40 CFR 798.5500 - Differential growth inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... recommendations as specified under 40 CFR part 792, subpart J the following specific information should be... repair proficient and repair deficient bacteria: âBacterial DNA damage or repair tests.â 798.5500 Section... inhibition of repair proficient and repair deficient bacteria: “Bacterial DNA damage or repair tests.”...

  18. Construction and gene expression analysis of a single-stranded DNA minivector based on an inverted terminal repeat of adeno-associated virus.

    PubMed

    Ping, Han; Liu, Xiaomei; Zhu, Dongqin; Li, Taiming; Zhang, Chun

    2015-04-01

    The plasmid vectors currently used for nonviral gene transfer have the disadvantage of carrying a bacterial backbone and an antibiotic resistance gene, which may cause side effects. The adeno-associated virus (AAV) genome is a linear single-stranded DNA (ssDNA) molecule with palindromic inverted terminal repeat (ITR) sequences forming double-stranded DNA (dsDNA) hairpin (HP) structures at each end. Based on the AAV genome, we constructed an AAV-ITR ssDNA minivector that consists of a GFP expression cassette flanked by both ITR sequences of 125 nucleotides. The minivectors were produced by digestion of the parental plasmids followed by denaturation. The self-complementary inverted T-shaped HP structure of the minivector was automatically formed. The HEK 293T cells were transfected with the AAV-ITR ssDNA minivector, plasmid, and dsDNA expression cassette. The results showed that AAV-ITR ssDNA minivector had relatively low gene expression efficiency in vitro. However, we found that the GFP expression efficiency of the D sequence-deleted AAV-ITR ssDNA minivector was significantly increased and was similar to those obtained with the plasmid and dsDNA expression cassette. Our data suggest that the AAV-ITR ssDNA minivector may be a new type of gene expression vector for gene therapy besides the virus and plasmid. PMID:25555376

  19. Discovery of a novel circular DNA virus in the Forbes sea star, Asterias forbesi.

    PubMed

    Fahsbender, Elizabeth; Hewson, Ian; Rosario, Karyna; Tuttle, Allison D; Varsani, Arvind; Breitbart, Mya

    2015-09-01

    A single-stranded DNA (ssDNA) virus, Asterias forbesi-associated circular virus (AfaCV), was discovered in a Forbes sea star displaying symptoms of sea star wasting disease (SSWD). The AfaCV genome organization is typical of circular Rep-encoding ssDNA (CRESS-DNA) viruses and is similar to that of members of the family Circoviridae. PCR-based surveys indicate that AfaCV is not clearly associated with SSWD, whereas the sea star-associated densovirus (SSaDV), recently implicated in SSWD in the Pacific, was prevalent in symptomatic specimens. AfaCV represents the first CRESS-DNA virus detected in echinoderms, adding to the growing diversity of these viruses recently recovered from invertebrates. PMID:26112764

  20. Use of Quantitative 16S Ribosomal DNA Detection for Diagnosis of Central Vascular Catheter-Associated Bacterial Infection

    PubMed Central

    Warwick, S.; Wilks, M.; Hennessy, E.; Powell-Tuck, J.; Small, M.; Sharp, J.; Millar, M. R.

    2004-01-01

    Many central vascular catheters (CVCs) are removed unnecessarily because current diagnostic methods for CVC-associated infection are unreliable. A quantitative PCR assay using primers and probe targeted to bacterial 16S ribosomal DNA was used to measure the levels of bacterial DNA in blood samples drawn through the CVC in a population of patients receiving intravenous nutrition. Bacterial DNA concentrations were raised in 16 of 16 blood samples taken during episodes of probable bacterial CVC-associated infection. Bacterial DNA concentrations were raised in 4 of 29 episodes in which bacterial CVC-associated infection was unlikely. The use of this technique has the potential to substantially reduce the unnecessary removal of CVCs. PMID:15070980

  1. Application of silica magnetite nanocomposites to the isolation of ultrapure plasmid DNA from bacterial cells

    NASA Astrophysics Data System (ADS)

    Chiang, Chen-Li; Sung, Ching-Shan; Chen, Chuh-Yean

    2006-10-01

    The aim of this study was to develop a simple and rapid method for purification of ultrapure plasmid DNA with high yields from bacterial cultures. Nanosized superparamagnetic nanoparticles (Fe 3O 4) were prepared by chemical precipitation method using Fe 2+, Fe 3+ salt, and ammonium hydroxide under a nitrogen atmosphere. Silica-magnetite nanocomposites were prepared by the method of acid hydrolysis of tetraethoxysilane (TEOS) to coat the silica onto magnetite nanoparticles. DNA was adsorbed to the support under high salt conditions, and recovered directly in water for immediate downstream application, without the need for precipitation. We demonstrated that a useful plasmid, pRSETB-EGFP, encoding for the green fluorescent protein with T7 promoter, could be amplified in Escherichia coli of DE3 strain. Up to approximately 43 μg of high-purity ( A260/ A280 ratio=1.75) plasmid DNA was isolated from 3 ml of an overnight bacterial culture. The eluted plasmid DNA was used directly for restriction enzyme digestion, bacterial cell transformation and polymerase chain reaction (PCR) amplification with success. The protocol, starting from the preparation of bacterial lysate and ending with purified plasmid takes less than 8 min. The silica-magnetite nanocomposites deliver significant time-savings, overall higher yields, lower RNA contamination, and better PCR amplification compared to commercial available silica-based and other methods.

  2. 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

  3. Assays for Hepatitis B Virus DNA-and RNA-Dependent DNA Polymerase Activities.

    PubMed

    Shaw, T; Locarnini, S A

    2000-01-01

    Genomes of the hepatitis B viruses (HBVs) consist of approx 3.2 kb of partly double-stranded DNA containing three or four overlapping open reading frames, the largest of which encodes the viral polymerase (Pol) protein. After entry into the cell and uncoating, the viral genome is transported to the nucleus where it is converted into a covalently closed circular (CCC) or supercoiled molecule by cellular repair mechanisms. The viral CCC DNA is transcribed, presumably by host cell RNA polymerase II, into unspliced, capped polyadenylated mRNA species from which viral proteins are transcribed. In addition, terminally redundant 3.5-kb RNA transcripts, which function as pregenomes, are produced and exported to the cytoplasm where they are packaged into viral core particles in which reverse transcription, pregenome degradation, and duplication occurs, reproducing the partly double-stranded HBV genome (for recent review, see ref. 1). Besides its essential role in HBV genome replication, HBV Pol is also involved in virus assembly, and because hepadnaviruses do not encode enzymes functionally equivalent to deoxynucleoside kinases (2), functions associated with HBV Pol are probably the only virus-specific targets for antiviral activity of nucleoside analogs. In vitro assays for inhibition of HBV Pol functions by deoxynucleoside triphosphate (dNTP) analogs are useful indicators but, because of restrictions imposed by hepatocyte enzymology, provide no guarantee of potential anti-HBV activity of the parent (deoxy)nucleoside analogs in intact cells (2). PMID:21331902

  4. Immunogenicity of varicella zoster virus glycoprotein E DNA vaccine

    PubMed Central

    BAO, LIDAO; WEI, GUOMIN; GAN, HONGMEI; REN, XIANHUA; MA, RUILIAN; WANG, YI; LV, HAIJUN

    2016-01-01

    In the present study a eukaryotic expression vector of varicella zoster virus (VZV) glycoprotein E (gE) was constructed and enabled to express in COS7 cells. Furthermore, a specific immune response against the VZV gE eukaryotic expression plasmid was induced in BALB/c mice. The VZV gE gene was amplified using polymerase chain reaction (PCR) and cloned into a eukaryotic expression vector, pcDNA3.1. The recombinant vector was subsequently transfected into COS7 cells using a liposome transfection reagent. The recombinant protein was instantaneously expressed by the transfected cells, as detected by immunohistochemistry, and the recombinant pcDNA-VZV gE plasmid was subsequently used to immunize mice. Tissue expression levels were analyzed by reverse transcription-PCR. In addition, the levels of serum antibodies and spleen lymphocyte proliferation activity were investigated. The amplified target gene included the full-length gE gene (~2.7 kb), and the recombinant expression vector induced gE expression in COS7 cells. In addition, the expression plasmid induced sustained expression in vivo following immunization of mice. Furthermore, the plasmid was capable of inducing specific antibody production and effectively stimulating T cell proliferation. Effective humoral and cellular immunity was triggered in the mice immunized with the VZV gE eukaryotic expression vector. The results of the present study laid the foundation for future research into a VZV DNA vaccine. PMID:27168804

  5. Stabilising the Herpes Simplex Virus capsid by DNA packaging

    NASA Astrophysics Data System (ADS)

    Wuite, Gijs; Radtke, Kerstin; Sodeik, Beate; Roos, Wouter

    2009-03-01

    Three different types of Herpes Simplex Virus type 1 (HSV-1) nuclear capsids can be distinguished, A, B and C capsids. These capsids types are, respectively, empty, contain scaffold proteins, or hold DNA. We investigate the physical properties of these three capsids by combining biochemical and nanoindentation techniques. Atomic Force Microscopy (AFM) experiments show that A and C capsids are mechanically indistinguishable whereas B capsids already break at much lower forces. By extracting the pentamers with 2.0 M GuHCl or 6.0 M Urea we demonstrate an increased flexibility of all three capsid types. Remarkably, the breaking force of the B capsids without pentamers does not change, while the modified A and C capsids show a large drop in their breaking force to approximately the value of the B capsids. This result indicates that upon DNA packaging a structural change at or near the pentamers occurs which mechanically reinforces the capsids structure. The reported binding of proteins UL17/UL25 to the pentamers of the A and C capsids seems the most likely candidate for such capsids strengthening. Finally, the data supports the view that initiation of DNA packaging triggers the maturation of HSV-1 capsids.

  6. Synthesis of hybrid bacterial plasmids containing highly repeated satellite DNA.

    PubMed

    Brutlag, D; Fry, K; Nelson, T; Hung, P

    1977-03-01

    Hybrid plasmid molecules containing tandemly repeated Drosophila satellite DNA were constructed using a modification of the (dA)-(dT) homopolymer procedure of Lobban and Kaiser (1973). Recombinant plasmids recovered after transformation of recA bacteria contained 10% of the amount of satellite DNA present in the transforming molecules. The cloned plasmids were not homogenous in size. Recombinant plasmids isolated from a single colony contained populations of circular molecules which varied both in the length of the satellite region and in the poly(dA)-(dt) regions linking satellite and vector. While subcloning reduced the heterogeneity of these plasmid populations, continued cell growth caused further variations in the size of the repeated regions. Two different simple sequence satellites of Drosophila melanogaster (1.672 and 1.705 g/cm3) were unstable in both recA and recBC hosts and in both pSC101 and pCR1 vectors. We propose that this recA-independent instability of tandemly repeated sequences is due to unequal intramolecular recombination events in replicating DNA molecules, a mechanism analogous to sister chromatid exchange in eucaryotes. PMID:403010

  7. Multiple Layers of Chimerism in a Single-Stranded DNA Virus Discovered by Deep Sequencing

    PubMed Central

    Krupovic, Mart; Zhi, Ning; Li, Jungang; Hu, Gangqing; Koonin, Eugene V.; Wong, Susan; Shevchenko, Sofiya; Zhao, Keji; Young, Neal S.

    2015-01-01

    Viruses with single-stranded (ss) DNA genomes infect hosts in all three domains of life and include many medically, ecologically, and economically important pathogens. Recently, a new group of ssDNA viruses with chimeric genomes has been discovered through viral metagenomics. These chimeric viruses combine capsid protein genes and replicative protein genes that, respectively, appear to have been inherited from viruses with positive-strand RNA genomes, such as tombusviruses, and ssDNA genomes, such as circoviruses, nanoviruses or geminiviruses. Here, we describe the genome sequence of a new representative of this virus group and reveal an additional layer of chimerism among ssDNA viruses. We show that not only do these viruses encompass genes for capsid proteins and replicative proteins that have distinct evolutionary histories, but also the replicative genes themselves are chimeras of functional domains inherited from viruses of different families. Our results underscore the importance of horizontal gene transfer in the evolution of ssDNA viruses and the role of genetic recombination in the emergence of novel virus groups. PMID:25840414

  8. Functional characterization and inhibition of the type II DNA topoisomerase coded by African swine fever virus.

    PubMed

    Coelho, João; Ferreira, Fernando; Martins, Carlos; Leitão, Alexandre

    2016-06-01

    DNA topoisomerases are essential for DNA metabolism and while their role is well studied in prokaryotes and eukaryotes, it is less known for virally-encoded topoisomerases. African swine fever virus (ASFV) is a nucleo-cytoplasmic large DNA virus that infects Ornithodoros ticks and all members of the family Suidae, representing a global threat for pig husbandry with no effective vaccine nor treatment. It was recently demonstrated that ASFV codes for a type II topoisomerase, highlighting a possible target for control of the virus. In this work, the ASFV DNA topoisomerase II was expressed in Saccharomyces cerevisiae and found to efficiently decatenate kDNA and to processively relax supercoiled DNA. Optimal conditions for its activity were determined and its sensitivity to a panel of topoisomerase poisons and inhibitors was evaluated. Overall, our results provide new knowledge on viral topoisomerases and on ASFV, as well as a possible target for the control of this virus. PMID:27060564

  9. Circulating Bacterial-Derived DNA Fragment Level Is a Strong Predictor of Cardiovascular Disease in Peritoneal Dialysis Patients

    PubMed Central

    Szeto, Cheuk-Chun; Kwan, Bonnie Ching-Ha; Chow, Kai-Ming; Kwok, Jeffrey Sung-Shing; Lai, Ka-Bik; Cheng, Phyllis Mei-Shan; Pang, Wing-Fai; Ng, Jack Kit-Chung; Chan, Michael Ho-Ming; Lit, Lydia Choi-Wan; Leung, Chi-Bon; Li, Philip Kam-Tao

    2015-01-01

    Background Circulating bacterial DNA fragment is related to systemic inflammatory state in peritoneal dialysis (PD) patients. We hypothesize that plasma bacterial DNA level predicts cardiovascular events in new PD patients. Methods We measured plasma bacterial DNA level in 191 new PD patients, who were then followed for at least a year for the development of cardiovascular event, hospitalization, and patient survival. Results The average age was 59.3 ± 11.8 years; plasma bacterial DNA level 34.9 ± 1.5 cycles; average follow up 23.2 ± 9.7 months. At 24 months, the event-free survival was 86.1%, 69.8%, 55.4% and 30.8% for plasma bacterial DNA level quartiles I, II, III and IV, respectively (p < 0.0001). After adjusting for confounders, plasma bacterial DNA level, baseline residual renal function and malnutrition-inflammation score were independent predictors of composite cardiovascular end-point; each doubling in plasma bacterial DNA level confers a 26.9% (95% confidence interval, 13.0 – 42.5%) excess in risk. Plasma bacterial DNA also correlated with the number of hospital admission (r = -0.379, p < 0.0001) and duration of hospitalization for cardiovascular reasons (r = -0.386, p < 0.0001). Plasma bacterial DNA level did not correlate with baseline arterial pulse wave velocity (PWV), but with the change in carotid-radial PWV in one year (r = -0.238, p = 0.005). Conclusions Circulating bacterial DNA fragment level is a strong predictor of cardiovascular event, need of hospitalization, as well as the progressive change in arterial stiffness in new PD patients. PMID:26010741

  10. A novel virus genome discovered in an extreme environment suggests recombination between unrelated groups of RNA and DNA viruses

    PubMed Central

    2012-01-01

    Background Viruses are known to be the most abundant organisms on earth, yet little is known about their collective origin and evolutionary history. With exceptionally high rates of genetic mutation and mosaicism, it is not currently possible to resolve deep evolutionary histories of the known major virus groups. Metagenomics offers a potential means of establishing a more comprehensive view of viral evolution as vast amounts of new sequence data becomes available for comparative analysis. Results Bioinformatic analysis of viral metagenomic sequences derived from a hot, acidic lake revealed a circular, putatively single-stranded DNA virus encoding a major capsid protein similar to those found only in single-stranded RNA viruses. The presence and circular configuration of the complete virus genome was confirmed by inverse PCR amplification from native DNA extracted from lake sediment. The virus genome appears to be the result of a RNA-DNA recombination event between two ostensibly unrelated virus groups. Environmental sequence databases were examined for homologous genes arranged in similar configurations and three similar putative virus genomes from marine environments were identified. This result indicates the existence of a widespread but previously undetected group of viruses. Conclusions This unique viral genome carries implications for theories of virus emergence and evolution, as no mechanism for interviral RNA-DNA recombination has yet been identified, and only scant evidence exists that genetic exchange occurs between such distinct virus lineages. Reviewers This article was reviewed by EK, MK (nominated by PF) and AM. For the full reviews, please go to the Reviewers' comments section. PMID:22515485

  11. Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes

    PubMed Central

    Schmeisser, Falko; Weir, Jerry P

    2007-01-01

    Background Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC) technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2) BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. While the BAC mutagenesis procedure is a powerful method to generate HSV-2 recombinants, particularly in the absence of selective marker in eukaryotic culture, the mutagenesis procedure is still difficult and cumbersome. Results Here we describe the incorporation of a phage lambda recombination system into an allele replacement vector. This strategy enables any DNA fragment containing the phage attL recombination sites to be efficiently inserted into the attR sites of the allele replacement vector using phage lambda clonase. We also describe how the incorporation of EGFP into the allele replacement vector can facilitate the selection of the desired cross-over recombinant BACs when the allele replacement reaction is a viral gene deletion. Finally, we incorporate the lambda phage recombination sites directly into an HSV-2 BAC vector for direct recombination of gene cassettes using the phage lambda clonase-driven recombination reaction. Conclusion Together, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors. PMID:17501993

  12. A Novel AT-Rich DNA Recognition Mechanism for Bacterial Xenogeneic Silencer MvaT

    PubMed Central

    Ding, Pengfei; McFarland, Kirsty A.; Jin, Shujuan; Tong, Grace; Duan, Bo; Yang, Ally; Hughes, Timothy R.; Liu, Jun; Dove, Simon L.; Navarre, William Wiley; Xia, Bin

    2015-01-01

    Bacterial xenogeneic silencing proteins selectively bind to and silence expression from many AT rich regions of the chromosome. They serve as master regulators of horizontally acquired DNA, including a large number of virulence genes. To date, three distinct families of xenogeneic silencers have been identified: H-NS of Proteobacteria, Lsr2 of the Actinomycetes, and MvaT of Pseudomonas sp. Although H-NS and Lsr2 family proteins are structurally different, they all recognize the AT-rich DNA minor groove through a common AT-hook-like motif, which is absent in the MvaT family. Thus, the DNA binding mechanism of MvaT has not been determined. Here, we report the characteristics of DNA sequences targeted by MvaT with protein binding microarrays, which indicates that MvaT prefers binding flexible DNA sequences with multiple TpA steps. We demonstrate that there are clear differences in sequence preferences between MvaT and the other two xenogeneic silencer families. We also determined the structure of the DNA-binding domain of MvaT in complex with a high affinity DNA dodecamer using solution NMR. This is the first experimental structure of a xenogeneic silencer in complex with DNA, which reveals that MvaT recognizes the AT-rich DNA both through base readout by an “AT-pincer” motif inserted into the minor groove and through shape readout by multiple lysine side chains interacting with the DNA sugar-phosphate backbone. Mutations of key MvaT residues for DNA binding confirm their importance with both in vitro and in vivo assays. This novel DNA binding mode enables MvaT to better tolerate GC-base pair interruptions in the binding site and less prefer A tract DNA when compared to H-NS and Lsr2. Comparison of MvaT with other bacterial xenogeneic silencers provides a clear picture that nature has evolved unique solutions for different bacterial genera to distinguish foreign from self DNA. PMID:26068099

  13. Super-Resolution Microscopy and Tracking of DNA-Binding Proteins in Bacterial Cells

    PubMed Central

    Uphoff, Stephan

    2016-01-01

    Summary The ability to detect individual fluorescent molecules inside living cells has enabled a range of powerful microscopy techniques that resolve biological processes on the molecular scale. These methods have also transformed the study of bacterial cell biology, which was previously obstructed by the limited spatial resolution of conventional microscopy. In the case of DNA-binding proteins, super-resolution microscopy can visualize the detailed spatial organization of DNA replication, transcription, and repair processes by reconstructing a map of single-molecule localizations. Furthermore, DNA binding activities can be observed directly by tracking protein movement in real time. This allows identifying subpopulations of DNA-bound and diffusing proteins, and can be used to measure DNA-binding times in vivo. This chapter provides a detailed protocol for super-resolution microscopy and tracking of DNA-binding proteins in Escherichia coli cells. The protocol covers the construction of cell strains and describes data acquisition and analysis procedures, such as super-resolution image reconstruction, mapping single-molecule tracks, computing diffusion coefficients to identify molecular subpopulations with different mobility, and analysis of DNA-binding kinetics. While the focus is on the study of bacterial chromosome biology, these approaches are generally applicable to other molecular processes and cell types. PMID:27283312

  14. Bacterial origin recognition complexes direct assembly of higher-order DnaA oligomeric structures.

    PubMed

    Miller, Diana T; Grimwade, Julia E; Betteridge, Thu; Rozgaja, Tania; Torgue, Julien J-C; Leonard, Alan C

    2009-11-01

    Eukaryotic initiator proteins form origin recognition complexes (ORCs) that bind to replication origins during most of the cell cycle and direct assembly of prereplication complexes (pre-RCs) before the onset of S phase. In the eubacterium Escherichia coli, there is a temporally similar nucleoprotein complex comprising the initiator protein DnaA bound to three high-affinity recognition sites in the unique origin of replication, oriC. At the time of initiation, this high-affinity DnaA-oriC complex (the bacterial ORC) accumulates additional DnaA that interacts with lower-affinity sites in oriC, forming a pre-RC. In this paper, we investigate the functional role of the bacterial ORC and examine whether it mediates low-affinity DnaA-oriC interactions during pre-RC assembly. We report that E. coli ORC is essential for DnaA occupation of low-affinity sites. The assistance given by ORC is directed primarily to proximal weak sites and requires oligomerization-proficient DnaA. We propose that in bacteria, DnaA oligomers of limited length and stability emerge from single high-affinity sites and extend toward weak sites to facilitate their loading as a key stage of prokaryotic pre-RC assembly. PMID:19833870

  15. Plasma bacterial and mitochondrial DNA distinguish bacterial sepsis from sterile systemic inflammatory response syndrome and quantify inflammatory tissue injury in nonhuman primates.

    PubMed

    Sursal, Tolga; Stearns-Kurosawa, Deborah J; Itagaki, Kiyoshi; Oh, Sun-Young; Sun, Shiqin; Kurosawa, Shinichiro; Hauser, Carl J

    2013-01-01

    Systemic inflammatory response syndrome (SIRS) is a fundamental host response common to bacterial infection and sterile tissue injury. Systemic inflammatory response syndrome can cause organ dysfunction and death, but its mechanisms are incompletely understood. Moreover, SIRS can progress to organ failure or death despite being sterile or after control of the inciting infection. Biomarkers discriminating between sepsis, sterile SIRS, and postinfective SIRS would therefore help direct care. Circulating mitochondrial DNA (mtDNA) is a damage-associated molecular pattern reflecting cellular injury. Circulating bacterial 16S DNA (bDNA) is a pathogen-associated pattern (PAMP) reflecting ongoing infection. We developed quantitative polymerase chain reaction assays to quantify these markers, and predicting their plasma levels might help distinguish sterile injury from infection. To study these events in primates, we assayed banked serum from Papio baboons that had undergone a brief challenge of intravenous Bacillus anthracis delta Sterne (modified to remove toxins) followed by antibiotics (anthrax) that causes organ failure and death. To investigate the progression of sepsis to "severe" sepsis and death, we studied animals where anthrax was pretreated with drotrecogin alfa (activated protein C), which attenuates sepsis in baboons. We also contrasted lethal anthrax bacteremia against nonlethal E. coli bacteremia and against sterile tissue injury from Shiga-like toxin 1. Bacterial DNA and mtDNA levels in timed samples were correlated with blood culture results and assays of organ function. Sterile injury by Shiga-like toxin 1 increased mtDNA, but bDNA was undetectable: consistent with the absence of infection. The bacterial challenges caused parallel early bDNA and mtDNA increases, but bDNA detected pathogens even after bacteria were undetectable by culture. Sublethal E. coli challenge only caused transient rises in mtDNA consistent with a self-limited injury. In lethal

  16. Distinct Circular Single-Stranded DNA Viruses Exist in Different Soil Types

    PubMed Central

    Swanson, Maud M.; Cock, Peter J. A.; Dawson, Lorna; Freitag, Thomas E.; Singh, Brajesh K.; Torrance, Lesley; Mushegian, Arcady R.

    2015-01-01

    The potential dependence of virus populations on soil types was examined by electron microscopy, and the total abundance of virus particles in four soil types was similar to that previously observed in soil samples. The four soil types examined differed in the relative abundances of four morphological groups of viruses. Machair, a unique type of coastal soil in western Scotland and Ireland, differed from the others tested in having a higher proportion of tailed bacteriophages. The other soils examined contained predominantly spherical and thin filamentous virus particles, but the Machair soil had a more even distribution of the virus types. As the first step in looking at differences in populations in detail, virus sequences from Machair and brown earth (agricultural pasture) soils were examined by metagenomic sequencing after enriching for circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) virus genomes. Sequences from the family Microviridae (icosahedral viruses mainly infecting bacteria) of CRESS-DNA viruses were predominant in both soils. Phylogenetic analysis of Microviridae major coat protein sequences from the Machair viruses showed that they spanned most of the diversity of the subfamily Gokushovirinae, whose members mainly infect obligate intracellular parasites. The brown earth soil had a higher proportion of sequences that matched the morphologically similar family Circoviridae in BLAST searches. However, analysis of putative replicase proteins that were similar to those of viruses in the Circoviridae showed that they are a novel clade of Circoviridae-related CRESS-DNA viruses distinct from known Circoviridae genera. Different soils have substantially different taxonomic biodiversities even within ssDNA viruses, which may be driven by physicochemical factors. PMID:25841004

  17. Distinct circular single-stranded DNA viruses exist in different soil types.

    PubMed

    Reavy, Brian; Swanson, Maud M; Cock, Peter J A; Dawson, Lorna; Freitag, Thomas E; Singh, Brajesh K; Torrance, Lesley; Mushegian, Arcady R; Taliansky, Michael

    2015-06-15

    The potential dependence of virus populations on soil types was examined by electron microscopy, and the total abundance of virus particles in four soil types was similar to that previously observed in soil samples. The four soil types examined differed in the relative abundances of four morphological groups of viruses. Machair, a unique type of coastal soil in western Scotland and Ireland, differed from the others tested in having a higher proportion of tailed bacteriophages. The other soils examined contained predominantly spherical and thin filamentous virus particles, but the Machair soil had a more even distribution of the virus types. As the first step in looking at differences in populations in detail, virus sequences from Machair and brown earth (agricultural pasture) soils were examined by metagenomic sequencing after enriching for circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) virus genomes. Sequences from the family Microviridae (icosahedral viruses mainly infecting bacteria) of CRESS-DNA viruses were predominant in both soils. Phylogenetic analysis of Microviridae major coat protein sequences from the Machair viruses showed that they spanned most of the diversity of the subfamily Gokushovirinae, whose members mainly infect obligate intracellular parasites. The brown earth soil had a higher proportion of sequences that matched the morphologically similar family Circoviridae in BLAST searches. However, analysis of putative replicase proteins that were similar to those of viruses in the Circoviridae showed that they are a novel clade of Circoviridae-related CRESS-DNA viruses distinct from known Circoviridae genera. Different soils have substantially different taxonomic biodiversities even within ssDNA viruses, which may be driven by physicochemical factors. PMID:25841004

  18. DNA-crosslinker cisplatin eradicates bacterial persister cells.

    PubMed

    Chowdhury, Nityananda; Wood, Thammajun L; Martínez-Vázquez, Mariano; García-Contreras, Rodolfo; Wood, Thomas K

    2016-09-01

    For all bacteria, nearly every antimicrobial fails since a subpopulation of the bacteria enter a dormant state known as persistence, in which the antimicrobials are rendered ineffective due to the lack of metabolism. This tolerance to antibiotics makes microbial infections the leading cause of death worldwide and makes treating chronic infections, including those of wounds problematic. Here, we show that the FDA-approved anti-cancer drug cisplatin [cis-diamminodichloroplatinum(II)], which mainly forms intra-strand DNA crosslinks, eradicates Escherichia coli K-12 persister cells through a growth-independent mechanism. Additionally, cisplatin is more effective at killing Pseudomonas aeruginosa persister cells than mitomycin C, which forms inter-strand DNA crosslinks, and cisplatin eradicates the persister cells of several pathogens including enterohemorrhagic E. coli, Staphylococcus aureus, and P. aeruginosa. Cisplatin was also highly effective against clinical isolates of S. aureus and P. aeruginosa. Therefore, cisplatin has broad spectrum activity against persister cells. Biotechnol. Bioeng. 2016;113: 1984-1992. © 2016 Wiley Periodicals, Inc. PMID:26914280

  19. Comparison of DNA preservation methods for environmental bacterial community samples

    USGS Publications Warehouse

    Gray, Michael A.; Pratte, Zoe A.; Kellogg, Christina A.

    2013-01-01

    Field collections of environmental samples, for example corals, for molecular microbial analyses present distinct challenges. The lack of laboratory facilities in remote locations is common, and preservation of microbial community DNA for later study is critical. A particular challenge is keeping samples frozen in transit. Five nucleic acid preservation methods that do not require cold storage were compared for effectiveness over time and ease of use. Mixed microbial communities of known composition were created and preserved by DNAgard™, RNAlater®, DMSO–EDTA–salt (DESS), FTA® cards, and FTA Elute® cards. Automated ribosomal intergenic spacer analysis and clone libraries were used to detect specific changes in the faux communities over weeks and months of storage. A previously known bias in FTA® cards that results in lower recovery of pure cultures of Gram-positive bacteria was also detected in mixed community samples. There appears to be a uniform bias across all five preservation methods against microorganisms with high G + C DNA. Overall, the liquid-based preservatives (DNAgard™, RNAlater®, and DESS) outperformed the card-based methods. No single liquid method clearly outperformed the others, leaving method choice to be based on experimental design, field facilities, shipping constraints, and allowable cost.

  20. Synthesis, integration, and restriction and modification of mycoplasma virus L2 DNA

    SciTech Connect

    Dybvig, K.

    1981-01-01

    Mycoplasma virus L2 is an enveloped, nonlytic virus containing double-stranded, superhelical DNA. The L2 virion contains about 7 to 8 major proteins identified by SDS-polyacrylamide gel electrophoresis, but the virion has no discernible capsid structure. It has been suggested that the L2 virion is a DNA-protein condensation surrounded by a lipid-protein membrane. The host for mycoplasma virus L2 is Acholeplasma laidlawii. A. laidlawii has no cell wall and contains a small genome, 1 x 10/sup 9/ daltons, which is two to three times smaller than that of most bacteria. Infection of A. laidlawii by L2 is nonlytic. The studies in this thesis show that L2 DNA synthesis begins at about 1 hour of infection and lasts throughout the infection. Viral DNA synthesis is inhibited by chloramphenicol, streptomycin, and novobiocin. Packaging of L2 DNA into progeny virus is also inhibited by chloramphenicol and novobiocin. It is concluded that protein synthesis and probably DNA gyrase activity are required for L2 DNA synthesis, and for packaging of L2 DNA into progeny virus. DNA-DNA hybridization studies demonstrate that L2 DNA integrates into the host cell during infection, and subsequent to infection the cells are mycoplasma virus L2 lysogens. The viral site of integration has been roughly mapped. L2 virus is restricted and modified by A. laidlawii strains JA1 and K2. The nature of the modification in strain K2 has been elucidated. Two L2 variants containing insertions in the viral DNA were identified in these studies. Restriction endonuclease cleavage maps of these variants have been determined. DNA from L2 and another isolate of L2, MV-Lg-L 172, are compared in these studies. 74 references, 33 figures, 6 tables. (ACR)

  1. Protocols for Developing Novel Chikungunya Virus DNA Vaccines.

    PubMed

    Chung, Christopher; Ugen, Kenneth E; Sardesai, Niranjan Y; Weiner, David B; Muthumani, Kar

    2016-01-01

    To date, there have been several million infections by the Chikungunya virus (CHIKV), a mosquito-transmitted emerging pathogen that is considered to be taxonomically an Old World RNA virus. Although original CHIKV outbreaks were restricted to India, East Asian countries, Northern Italy, and France, a recent sharp rise had been identified in 41 countries or territories in the Caribbean, Central America, South America, and North America. A total of 1,012,347 suspected and 22,579 laboratory-confirmed CHIKV cases have been reported from these areas, which signals an increasing risk to the US mainland. Unlike past epidemics that were usually associated with Ae. aegypti transmission, the Caribbean outbreak was associated with Ae. albopictus transmission as the principal mosquito vector. In addition, the substantial increase in the number of deaths during this epidemic, as well as incidence of neurologic disease, suggests that CHIKV may have become more virulent. Currently, there are no licensed vaccines or therapeutics available for CHIKV or its associated disease pathologies. Therefore, development of new vaccines and therapies that could confer immunity and/or treat clinical symptoms of CHIKV is greatly desired. This chapter describes the use of entirely cutting edge technologies/methodologies developed by our group for the development and evaluation of novel DNA vaccines against CHIKV. PMID:27233283

  2. Previously unknown and highly divergent ssDNA viruses populate the oceans

    PubMed Central

    Labonté, Jessica M; Suttle, Curtis A

    2013-01-01

    Single-stranded DNA (ssDNA) viruses are economically important pathogens of plants and animals, and are widespread in oceans; yet, the diversity and evolutionary relationships among marine ssDNA viruses remain largely unknown. Here we present the results from a metagenomic study of composite samples from temperate (Saanich Inlet, 11 samples; Strait of Georgia, 85 samples) and subtropical (46 samples, Gulf of Mexico) seawater. Most sequences (84%) had no evident similarity to sequenced viruses. In total, 608 putative complete genomes of ssDNA viruses were assembled, almost doubling the number of ssDNA viral genomes in databases. These comprised 129 genetically distinct groups, each represented by at least one complete genome that had no recognizable similarity to each other or to other virus sequences. Given that the seven recognized families of ssDNA viruses have considerable sequence homology within them, this suggests that many of these genetic groups may represent new viral families. Moreover, nearly 70% of the sequences were similar to one of these genomes, indicating that most of the sequences could be assigned to a genetically distinct group. Most sequences fell within 11 well-defined gene groups, each sharing a common gene. Some of these encoded putative replication and coat proteins that had similarity to sequences from viruses infecting eukaryotes, suggesting that these were likely from viruses infecting eukaryotic phytoplankton and zooplankton. PMID:23842650

  3. Previously unknown and highly divergent ssDNA viruses populate the oceans.

    PubMed

    Labonté, Jessica M; Suttle, Curtis A

    2013-11-01

    Single-stranded DNA (ssDNA) viruses are economically important pathogens of plants and animals, and are widespread in oceans; yet, the diversity and evolutionary relationships among marine ssDNA viruses remain largely unknown. Here we present the results from a metagenomic study of composite samples from temperate (Saanich Inlet, 11 samples; Strait of Georgia, 85 samples) and subtropical (46 samples, Gulf of Mexico) seawater. Most sequences (84%) had no evident similarity to sequenced viruses. In total, 608 putative complete genomes of ssDNA viruses were assembled, almost doubling the number of ssDNA viral genomes in databases. These comprised 129 genetically distinct groups, each represented by at least one complete genome that had no recognizable similarity to each other or to other virus sequences. Given that the seven recognized families of ssDNA viruses have considerable sequence homology within them, this suggests that many of these genetic groups may represent new viral families. Moreover, nearly 70% of the sequences were similar to one of these genomes, indicating that most of the sequences could be assigned to a genetically distinct group. Most sequences fell within 11 well-defined gene groups, each sharing a common gene. Some of these encoded putative replication and coat proteins that had similarity to sequences from viruses infecting eukaryotes, suggesting that these were likely from viruses infecting eukaryotic phytoplankton and zooplankton. PMID:23842650

  4. Bacterial-mediated DNA delivery to tumour associated phagocytic cells.

    PubMed

    Byrne, W L; Murphy, C T; Cronin, M; Wirth, T; Tangney, M

    2014-12-28

    Phagocytic cells including macrophages, dendritic cells and neutrophils are now recognised as playing a negative role in many disease settings including cancer. In particular, macrophages are known to play a pathophysiological role in multiple diseases and present a valid and ubiquitous therapeutic target. The technology to target these phagocytic cells in situ, both selectively and efficiently, is required in order to translate novel therapeutic modalities into clinical reality. We present a novel delivery strategy using non-pathogenic bacteria to effect gene delivery specifically to tumour-associated phagocytic cells. Non-invasive bacteria lack the ability to actively enter host cells, except for phagocytic cells. We exploit this natural property to effect 'passive transfection' of tumour-associated phagocytic cells following direct administration of transgene-loaded bacteria to tumour regions. Using an in vitro-differentiated human monocyte cell line and two in vivo mouse models (an ovarian cancer ascites and a solid colon tumour model) proof of delivery is demonstrated with bacteria carrying reporter constructs. The results confirm that the delivery strategy is specific for phagocytic cells and that the bacterial vector itself recruits more phagocytic cells to the tumour. While proof of delivery to phagocytic cells is demonstrated in vivo for solid and ascites tumour models, this strategy may be applied to other settings, including non-cancer related disease. PMID:25466954

  5. Architecture and Conservation of the Bacterial DNA Replication Machinery, an Underexploited Drug Target

    PubMed Central

    Robinson, Andrew; Causer, Rebecca J; Dixon, Nicholas E

    2012-01-01

    New antibiotics with novel modes of action are required to combat the growing threat posed by multi-drug resistant bacteria. Over the last decade, genome sequencing and other high-throughput techniques have provided tremendous insight into the molecular processes underlying cellular functions in a wide range of bacterial species. We can now use these data to assess the degree of conservation of certain aspects of bacterial physiology, to help choose the best cellular targets for development of new broad-spectrum antibacterials. DNA replication is a conserved and essential process, and the large number of proteins that interact to replicate DNA in bacteria are distinct from those in eukaryotes and archaea; yet none of the antibiotics in current clinical use acts directly on the replication machinery. Bacterial DNA synthesis thus appears to be an underexploited drug target. However, before this system can be targeted for drug design, it is important to understand which parts are conserved and which are not, as this will have implications for the spectrum of activity of any new inhibitors against bacterial species, as well as the potential for development of drug resistance. In this review we assess similarities and differences in replication components and mechanisms across the bacteria, highlight current progress towards the discovery of novel replication inhibitors, and suggest those aspects of the replication machinery that have the greatest potential as drug targets. PMID:22206257

  6. Improving soil bacterial taxa–area relationships assessment using DNA meta-barcoding

    PubMed Central

    Terrat, S; Dequiedt, S; Horrigue, W; Lelievre, M; Cruaud, C; Saby, N P A; Jolivet, C; Arrouays, D; Maron, P-A; Ranjard, L; Chemidlin Prévost-Bouré, N

    2015-01-01

    The evaluation of the taxa–area relationship (TAR) with molecular fingerprinting data demonstrated the spatial structuration of soil microorganisms and provided insights into the processes shaping their diversity. The increasing use of massive sequencing technologies in biodiversity investigations has now raised the question of the advantages of such technologies over the fingerprinting approach for elucidation of the determinism of soil microbial community assembly in broad-scale biogeographic studies. Our objectives in this study were to compare DNA fingerprinting and meta-barcoding approaches for evaluating soil bacterial TAR and the determinism of soil bacterial community assembly on a broad scale. This comparison was performed on 392 soil samples from four French geographic regions with different levels of environmental heterogeneity. Both molecular approaches demonstrated a TAR with a significant slope but, because of its more sensitive description of soil bacterial community richness, meta-barcoding provided significantly higher and more accurate estimates of turnover rates. Both approaches were useful in evidencing the processes shaping bacterial diversity variations on a broad scale. When different taxonomic resolutions were considered for meta-barcoding data, they significantly influenced the estimation of turnover rates but not the relative importance of each component process. Altogether, DNA meta-barcoding provides a more accurate evaluation of the TAR and may lead to re-examination of the processes shaping soil bacterial community assembly. This should provide new insights into soil microbial ecology in the context of sustainable use of soil resources. PMID:25293875

  7. Can VHS Virus Bypass the Protective Immunity Induced by DNA Vaccination in Rainbow Trout?

    PubMed Central

    Sepúlveda, Dagoberto; Lorenzen, Niels

    2016-01-01

    DNA vaccines encoding viral glycoproteins have been very successful for induction of protective immunity against diseases caused by rhabdoviruses in cultured fish species. However, the vaccine concept is based on a single viral gene and since RNA viruses are known to possess high variability and adaptation capacity, this work aimed at evaluating whether viral haemorrhagic septicaemia virus (VHSV), an RNA virus and member of Rhabdoviridae family, was able to evade the protective immune response induced by the DNA vaccination of rainbow trout. The experiments comprised repeated passages of a highly pathogenic VHSV isolate in a fish cell line in the presence of neutralizing fish serum (in vitro approach), and in rainbow trout immunized with the VHS DNA vaccine (in vivo approach). For the in vitro approach, the virus collected from the last passage (passaged virus) was as sensitive as the parental virus to serum neutralization, suggesting that the passaging did not promote the selection of virus populations able to bypass the neutralization by serum antibodies. Also, in the in vivo approach, where virus was passaged several times in vaccinated fish, no increased virulence nor increased persistence in vaccinated fish was observed in comparison with the parental virus. However, some of the vaccinated fish did get infected and could transmit the infection to naïve cohabitant fish. The results demonstrated that the DNA vaccine induced a robust protection, but also that the immunity was non-sterile. It is consequently important not to consider vaccinated fish as virus free in veterinary terms. PMID:27054895

  8. C-terminal region of bacterial Ku controls DNA bridging, DNA threading and recruitment of DNA ligase D for double strand breaks repair

    PubMed Central

    McGovern, Stephen; Baconnais, Sonia; Roblin, Pierre; Nicolas, Pierre; Drevet, Pascal; Simonson, Héloïse; Piétrement, Olivier; Charbonnier, Jean-Baptiste; Le Cam, Eric; Noirot, Philippe; Lecointe, François

    2016-01-01

    Non-homologous end joining is a ligation process repairing DNA double strand breaks in eukaryotes and many prokaryotes. The ring structured eukaryotic Ku binds DNA ends and recruits other factors which can access DNA ends through the threading of Ku inward the DNA, making this protein a key ingredient for the scaffolding of the NHEJ machinery. However, this threading ability seems unevenly conserved among bacterial Ku. As bacterial Ku differ mainly by their C-terminus, we evaluate the role of this region in the loading and the threading abilities of Bacillus subtilis Ku and the stimulation of the DNA ligase LigD. We identify two distinct sub-regions: a ubiquitous minimal C-terminal region and a frequent basic C-terminal extension. We show that truncation of one or both of these sub-regions in Bacillus subtilis Ku impairs the stimulation of the LigD end joining activity in vitro. We further demonstrate that the minimal C-terminus is required for the Ku-LigD interaction, whereas the basic extension controls the threading and DNA bridging abilities of Ku. We propose that the Ku basic C-terminal extension increases the concentration of Ku near DNA ends, favoring the recruitment of LigD at the break, thanks to the minimal C-terminal sub-region. PMID:26961308

  9. Inhibition of Cellular DNA Synthesis in Cells Infected with Infectious Pancreatic Necrosis Virus

    PubMed Central

    Lothrop, David; Nicholson, Bruce L.

    1974-01-01

    In asynchronous RTG-2 cell cultures infected with infectious pancreatic necrosis (IPN) virus, inhibition of cellular DNA synthesis, but not protein synthesis, was detected 5 to 6 h postinfection and was 80 to 90% complete by 7 to 8 h. Inhibition of DNA synthesis was largely abolished by UV irradiation of the virus. Sedimentation analyses of phenol-extracted DNA indicated that native cellular DNA was not degraded during infection. Sedimentation on alkaline sucrose gradients of DNA from cells pulsed with radioactive thymidine for varying periods indicated that elongation of nascent DNA chains proceeded normally in infected cells. These and previous results suggest that IPN virus infection results in a reduction of the number of chromosomal sites active in DNA synthesis but does not affect the rate of polymerization at active sites. Cells synchronized with excess thymidine and hydroxyurea and infected with virus at the time of release from the block demonstrated an inhibition of DNA synthesis 3 h postinfection. Cells infected 4 h prior to release continued to synthesize normal amounts of DNA for 1 to 2 h after release. These results indicated that DNA synthesis in early synthetic phase is relatively insensitive to inhibition by IPN virus. PMID:4852469

  10. Identification of bacterial DNA markers for the detection of human fecal pollution in water.

    PubMed

    Shanks, Orin C; Domingo, Jorge W Santo; Lu, Jingrang; Kelty, Catherine A; Graham, James E

    2007-04-01

    We used genome fragment enrichment and bioinformatics to identify several microbial DNA sequences with high potential for use as markers in PCR assays for detection of human fecal contamination in water. Following competitive solution-phase hybridization of total DNA from human and pig fecal samples, 351 plasmid clones were sequenced and were determined to define 289 different genomic DNA regions. These putative human-specific fecal bacterial DNA sequences were then analyzed by dot blot hybridization, which confirmed that 98% were present in the source human fecal microbial community and absent from the original pig fecal DNA extract. Comparative sequence analyses of these sequences suggested that a large number (43.5%) were predicted to encode bacterial secreted or surface-associated proteins. Deoxyoligonucleotide primers capable of annealing to a subset of 26 of the candidate sequences predicted to encode factors involved in interactions with host cells were then used in the PCR and did not amplify markers in DNA from any additional pig fecal specimens. These 26 PCR assays exhibited a range of specificity in tests with 11 other animal sources, with more than half amplifying markers only in specimens from dogs or cats. Four assays were more specific, detecting markers only in specimens from humans, including those from 18 different human populations examined. We then demonstrated the potential utility of these assays by using them to detect human fecal contamination in several impacted watersheds. PMID:17209067

  11. Identification of Bacterial DNA Markers for the Detection of Human Fecal Pollution in Water▿ †

    PubMed Central

    Shanks, Orin C.; Domingo, Jorge W. Santo; Lu, Jingrang; Kelty, Catherine A.; Graham, James E.

    2007-01-01

    We used genome fragment enrichment and bioinformatics to identify several microbial DNA sequences with high potential for use as markers in PCR assays for detection of human fecal contamination in water. Following competitive solution-phase hybridization of total DNA from human and pig fecal samples, 351 plasmid clones were sequenced and were determined to define 289 different genomic DNA regions. These putative human-specific fecal bacterial DNA sequences were then analyzed by dot blot hybridization, which confirmed that 98% were present in the source human fecal microbial community and absent from the original pig fecal DNA extract. Comparative sequence analyses of these sequences suggested that a large number (43.5%) were predicted to encode bacterial secreted or surface-associated proteins. Deoxyoligonucleotide primers capable of annealing to a subset of 26 of the candidate sequences predicted to encode factors involved in interactions with host cells were then used in the PCR and did not amplify markers in DNA from any additional pig fecal specimens. These 26 PCR assays exhibited a range of specificity in tests with 11 other animal sources, with more than half amplifying markers only in specimens from dogs or cats. Four assays were more specific, detecting markers only in specimens from humans, including those from 18 different human populations examined. We then demonstrated the potential utility of these assays by using them to detect human fecal contamination in several impacted watersheds. PMID:17209067

  12. The effects of a low-intensity red laser on bacterial growth, filamentation and plasmid DNA

    NASA Astrophysics Data System (ADS)

    Roos, C.; Santos, J. N.; Guimarães, O. R.; Geller, M.; Paoli, F.; Fonseca, A. S.

    2013-07-01

    Exposure of nonphotosynthesizing microorganisms to light could increase cell division in cultures, a phenomenon denominated as biostimulation. However, data concerning the importance of the genetic characteristics of cells on this effect are as yet scarce. The aim of this work was to evaluate the effects of a low-intensity red laser on the growth, filamentation and plasmids in Escherichia coli cells proficient and deficient in DNA repair. E. coli cultures were exposed to a laser (658 nm, 10 mW, 1 and 8 J cm-2) to study bacterial growth and filamentation. Also, bacterial cultures hosting pBSK plasmids were exposed to the laser to study DNA topological forms from the electrophoretic profile in agarose gels. Data indicate the low-intensity red laser: (i) had no effect on the growth of E. coli wild type and exonuclease III deficient cells; (ii) induced bacterial filamentation, (iii) led to no alteration in the electrophoretic profile of plasmids from exonuclease III deficient cells, but plasmids from wild type cells were altered. A low-intensity red laser at the low fluences used in phototherapy has no effect on growth, but induces filamentation and alters the topological forms of plasmid DNA in E. coli cultures depending on the DNA repair mechanisms.

  13. Conformational diversity of single-stranded DNA from bacterial repetitive extragenic palindromes: Implications for the DNA recognition elements of transposases.

    PubMed

    Charnavets, Tatsiana; Nunvar, Jaroslav; Nečasová, Iva; Völker, Jens; Breslauer, Kenneth J; Schneider, Bohdan

    2015-10-01

    Repetitive extragenic palindrome (REP)-associated tyrosine transposase enzymes (RAYTs) bind REP DNA domains and catalyze their cleavage. Genomic sequence analyses identify potential noncoding REP sequences associated with RAYT-encoding genes. To probe the conformational space of potential RAYT DNA binding domains, we report here spectroscopic and calorimetric measurements that detect and partially characterize the solution conformational heterogeneity of REP oligonucleotides from six bacterial species. Our data reveal most of these REP oligonucleotides adopt multiple conformations, suggesting that RAYTs confront a landscape of potential DNA substrates in dynamic equilibrium that could be selected, enriched, and/or induced via differential binding. Thus, the transposase-bound DNA motif may not be the predominant conformation of the isolated REP domain. Intriguingly, for several REPs, the circular dichroism spectra suggest guanine tetraplexes as potential alternative or additional RAYT recognition elements, an observation consistent with these REP domains being highly nonrandom, with tetraplex-favoring 5'-G and 3'-C-rich segments. In fact, the conformational heterogeneity of REP domains detected and reported here, including the formation of noncanonical DNA secondary structures, may reflect a general feature required for recognition by RAYT transposases. Based on our biophysical data, we propose guanine tetraplexes as an additional DNA recognition element for binding by RAYT transposase enzymes. PMID:25951997

  14. Higher prevalence of Epstein-Barr virus DNA in deeper periodontal pockets of chronic periodontitis in Japanese patients.

    PubMed

    Kato, Ayako; Imai, Kenichi; Ochiai, Kuniyasu; Ogata, Yorimasa

    2013-01-01

    Periodontitis, a complex chronic inflammatory disease caused by subgingival infection, is among the most prevalent microbial diseases in humans. Although traditional microbiological research on periodontitis has focused on putative bacteria such as Porphyromonas gingivalis, the herpes virus is proposed to be involved in the pathogenesis of periodontitis because bacterial etiology alone does not adequately explain various clinical aspects. In this study, we established for the first time, more Epstein-Barr virus (EBV) DNA is found deeper in periodontal pockets of chronic periodontitis in Japanese patients. Subgingival samples were collected from 85 patients with chronic periodontitis having two periodontal sites with probing depths (PD) of ≤ 3 mm (shallow) or ≥ 5 mm (deep) and were subjected to a nested polymerase chain reaction. EBV DNA was more frequently detected in patients with deeper PD sites (66%) than in those with shallow PD sites (48%) or healthy controls (45%). Coexistence of EBV DNA and P. gingivalis was significantly higher in patients with deeper PD sites (40%) than in those with shallow PD sites (14%) or healthy controls (13%). Although no difference in clinical index for periodontitis, the odds ratio of EBV DNA in patients with deeper PD sites was 2.36, which was 2.07-fold higher than that in those with shallow PD sites. Interestingly, the odds of acquiring chronic periodontitis (PD ≥ 5 mm) were higher in the presence of both EBV DNA and P. gingivalis compared with either EBV DNA or P. gingivalis only. In addition, we also observed that EBV-encoded small RNA (EBER) in positive cells of human gingival tissues. These results would suggest that EBV DNA may serve as a pathogenic factor leading to chronic periodontitis among Japanese patients. PMID:23991022

  15. Lyophilization to improve the sensitivity of qPCR for bacterial DNA detection in serum: the Q fever paradigm.

    PubMed

    Edouard, Sophie; Raoult, Didier

    2016-06-01

    Quantitative real-time PCR (qPCR) on serum provides significant added value to the diagnosis of Q fever, mainly at the acute stage of the disease in seronegative patients and in patients with endocarditis. We evaluated the benefits of Coxiella burnetii DNA concentration in serum by lyophilization to improve qPCR sensitivity. The detection limit of qPCR was determined by comparing six 10-fold dilutions of serum (calibrated with 104 bacteria ml-1) with and without lyophilization. We also tested, after lyophilization, 73 sera from patients with acute Q fever and 10 sera from patients with endocarditis for which specific qPCR for C. burnetii performed under our usual conditions remained negative. Lyophilization of DNA was found to improve sensitivity of the qPCR; the limit of detection of C. burnetii DNA by qPCR was 100-fold lower in lyophilized sera (1 bacterium ml-1) than in non-lyophilized sera (102 bacteria ml-1). Among the 73 sera from patients with acute Q fever, 26 (36 %) were positive after lyophilization, demonstrating a sensitivity gain of 44 % for early negative sera and 30 % for positive sera compared to our usual qPCR conditions. Sensitivity was also higher in sera from patients with endocarditis for which 8/10 (80 %) were positive after lyophilization. Our results serve as a proof of concept that lyophilization increases the sensitivity of qPCR in serum by concentrating bacterial DNA. This technique may be applied for the earlier diagnosis of other fastidious bacteria or viruses and extended to other clinical samples. PMID:27008653

  16. The structure of a thermophilic archaeal virus shows a dsDNA viral capsid type that spans all domains of life

    SciTech Connect

    G. Rice; L. Tang; K. Stedman; F. Roberto; J. Spuhler; E. Gillitzer; J. E. Johnson; T. Douglas; M. Young

    2004-05-01

    Of the three domains of life (Eukarya, Bacteria, and Archaea), the least understood is Archaea and its associated viruses. Many Archaea are extremophiles, with species that are capable of growth at some of the highest temperatures and extremes of pH of all known organisms. Phylogenetic rRNA-encoding DNA analysis places many of the hyperthermophilic Archaea (species with an optimum growth >80°C) at the base of the universal tree of life, suggesting that thermophiles were among the first forms of life on earth. Very few viruses have been identified from Archaea as compared to Bacteria and Eukarya. We report here the structure of a hyperthermophilic virus isolated from an archaeal host found in hot springs in Yellowstone National Park. The sequence of the circular double-stranded DNA viral genome shows that it shares little similarity to other known genes in viruses or other organisms. By comparing the tertiary and quaternary structures of the coat protein of this virus with those of a bacterial and an animal virus, we find conformational relationships among all three, suggesting that some viruses may have a common ancestor that precedes the division into three domains of life >3 billion years ago.

  17. The structure of a thermophilic archaeal virus shows a double-stranded DNA viral capsid type that spans all domains of life.

    PubMed

    Rice, George; Tang, Liang; Stedman, Kenneth; Roberto, Francisco; Spuhler, Josh; Gillitzer, Eric; Johnson, John E; Douglas, Trevor; Young, Mark

    2004-05-18

    Of the three domains of life (Eukarya, Bacteria, and Archaea), the least understood is Archaea and its associated viruses. Many Archaea are extremophiles, with species that are capable of growth at some of the highest temperatures and extremes of pH of all known organisms. Phylogenetic rRNA-encoding DNA analysis places many of the hyperthermophilic Archaea (species with an optimum growth > or = 80 degrees C) at the base of the universal tree of life, suggesting that thermophiles were among the first forms of life on earth. Very few viruses have been identified from Archaea as compared to Bacteria and Eukarya. We report here the structure of a hyperthermophilic virus isolated from an archaeal host found in hot springs in Yellowstone National Park. The sequence of the circular double-stranded DNA viral genome shows that it shares little similarity to other known genes in viruses or other organisms. By comparing the tertiary and quaternary structures of the coat protein of this virus with those of a bacterial and an animal virus, we find conformational relationships among all three, suggesting that some viruses may have a common ancestor that precedes the division into three domains of life >3 billion years ago. PMID:15123802

  18. Construction of infectious cDNA clone derived from a classical swine fever virus field isolate in BAC vector using in vitro overlap extension PCR and recombination.

    PubMed

    Kamboj, Aman; Saini, Mohini; Rajan, Lekshmi S; Patel, Chhabi Lal; Chaturvedi, V K; Gupta, Praveen K

    2015-12-15

    To develop reverse genetics system of RNA viruses, cloning of full-length viral genome is required which is often challenging due to many steps involved. In this study, we report cloning of full-length cDNA from an Indian field isolate (CSFV/IVRI/VB-131) of classical swine fever virus (CSFV) using in vitro overlap extension PCR and recombination which drastically reduced the number of cloning steps. The genome of CSFV was amplified in six overlapping cDNA fragments, linked by overlap extension PCR and cloned in a bacterial artificial chromosome (BAC) vector using in vitro recombination method to generate full-length cDNA clone. The full-length CSFV cDNA clone was found stable in E. coli Stellar and DH10B cells. The full-length RNA was transcribed in vitro using T7 RNA polymerase and transfected in PK15 cells using Neon-tip electroporator to rescue infectious CSFV. The progeny CSFV was propagated in PK15 cells and found indistinguishable from the parent virus. The expression of CSFV proteins were detected in cytoplasm of PK15 cells infected with progeny CSFV at 72 h post-infection. We concluded that the in vitro overlap extension PCR and recombination method is useful to construct stable full-length cDNA clone of RNA virus in BAC vector. PMID:26478540

  19. The PYRIN domain-only protein POP3 inhibits ALR inflammasomes and regulates responses to infection with DNA viruses.

    PubMed

    Khare, Sonal; Ratsimandresy, Rojo A; de Almeida, Lúcia; Cuda, Carla M; Rellick, Stephanie L; Misharin, Alexander V; Wallin, Melissa C; Gangopadhyay, Anu; Forte, Eleonora; Gottwein, Eva; Perlman, Harris; Reed, John C; Greaves, David R; Dorfleutner, Andrea; Stehlik, Christian

    2014-04-01

    The innate immune system responds to infection and tissue damage by activating cytosolic sensory complexes called 'inflammasomes'. Cytosolic DNA is sensed by AIM2-like receptors (ALRs) during bacterial and viral infections and in autoimmune diseases. Subsequently, recruitment of the inflammasome adaptor ASC links ALRs to the activation of caspase-1. A controlled immune response is crucial for maintaining homeostasis, but the regulation of ALR inflammasomes is poorly understood. Here we identified the PYRIN domain (PYD)-only protein POP3, which competes with ASC for recruitment to ALRs, as an inhibitor of DNA virus-induced activation of ALR inflammasomes in vivo. Data obtained with a mouse model with macrophage-specific POP3 expression emphasize the importance of the regulation of ALR inflammasomes in monocytes and macrophages. PMID:24531343

  20. Long-term persistence and bacterial transformation potential of transplastomic plant DNA in soil.

    PubMed

    Pontiroli, Alessandra; Ceccherini, Maria-Teresa; Poté, John; Wildi, Walter; Kay, Elisabeth; Nannipieri, Paolo; Vogel, Timothy M; Simonet, Pascal; Monier, Jean-Michel

    2010-06-01

    The long-term physical persistence and biological activity of transplastomic plant DNA (transgenes contained in the chloroplast genome) either purified and added to soil or naturally released by decaying tobacco leaves in soil was determined. Soil microcosms were amended with transplastomic tobacco leaves or purified plant DNA and incubated for up to 4 years. Total DNA was extracted from soil and the number of transgenes (aadA, which confers resistance to both spectinomycin and streptomycin) was quantified by quantitative PCR. The biological activity of these transgenes was assessed by transformation in the bacterial strain Acinetobacter sp. BD413(pBAB2) in vitro. While the proportion of transgenes recovered increased with the increasing amount of transplastomic DNA added, plant DNA was rapidly degraded over time. The number of transgenes recovered decreased about 10,000 fold within 2 weeks. Data reveal, however, that a small fraction of the plant DNA escaped degradation. Transgene sequences were still detected after 4 years and transformation assays showed that extracted DNA remained biologically active and could still transform competent cells of Acinetobacter sp. BD413(pBAB2). The approach presented here quantified the number of transgenes (based on quantitative PCR of 50% of the gene) released and persisting in the environment over time and provided new insights into the fate of transgenic plant DNA in soil. PMID:20493252

  1. Structure of Bacterial LigD -phosphoesterase Unveils a DNA Repair Superfamily

    SciTech Connect

    Nair, P.; Smith, P; Shuman, S

    2010-01-01

    The DNA ligase D (LigD) 3{prime}-phosphoesterase (PE) module is a conserved component of the bacterial nonhomologous end-joining (NHEJ) apparatus that performs 3{prime} end-healing reactions at DNA double-strand breaks. Here we report the 1.9 {angstrom} crystal structure of Pseudomonas aeruginosa PE, which reveals that PE exemplifies a unique class of DNA repair enzyme. PE has a distinctive fold in which an eight stranded {beta} barrel with a hydrophobic interior supports a crescent-shaped hydrophilic active site on its outer surface. Six essential side chains coordinate manganese and a sulfate mimetic of the scissile phosphate. The PE active site and mechanism are unique vis a vis other end-healing enzymes. We find PE homologs in archaeal and eukaryal proteomes, signifying that PEs comprise a DNA repair superfamily.

  2. Isolation of cis-acting vaccinia virus DNA fragments promoting the expression of herpes simplex virus thymidine kinase by recombinant viruses.

    PubMed Central

    Vassef, A; Mars, M; Dru, A; Plucienniczak, A; Streeck, R E; Beaud, G

    1985-01-01

    Recombinant TK- vaccinia viruses containing the pBR322 sequence inserted in either orientation within the coding sequence of the viral thymidine kinase gene were constructed. They were characterized by genomic analysis, hybridization studies, reversion to wild-type virus by in vivo recombination, and rescue from their genomes of plasmids which contained all or parts of the pBR322 sequence. TK- cells were infected with one of these recombinant viruses and then transfected with pools of chimeric plasmids composed of a cloned herpes simplex virus thymidine kinase gene which contained upstream inserts of different vaccinia DNA fragments prepared by restriction or sonication. Recombination between homologous pBR322 sequences within infected cells generated selectable recombinant viruses in which expression of the herpes simplex virus thymidine kinase gene was promoted by the upstream vaccinia insert. These viruses were characterized by genomic analysis, hybridization, and in vivo or in vitro phosphorylation of (5-[125I]deoxycytidine as a specific assay for the expressed herpes simplex virus thymidine kinase. Vaccinia DNA inserts were isolated conveniently for transfer to bacteria by rescuing appropriate plasmids from the genome of recombinant viruses. The sequence of 100 nucleotides adjacent to the upstream region of the herpes simplex virus gene was determined in nine different inserts measuring 0.17 to 1.07 kilobase pairs. Images PMID:2989553

  3. Electrochemical direct immobilization of DNA sequences for label-free herpes virus detection

    NASA Astrophysics Data System (ADS)

    Tam, Phuong Dinh; Trung, Tran; Tuan, Mai Anh; Chien, Nguyen Duc

    2009-09-01

    DNA sequences/bio-macromolecules of herpes virus (5'-AT CAC CGA CCC GGA GAG GGA C-3') were directly immobilized into polypyrrole matrix by using the cyclic voltammetry method, and grafted onto arrays of interdigitated platinum microelectrodes. The morphology surface of the obtained PPy/DNA of herpes virus composite films was investigated by a FESEM Hitachi-S 4800. Fourier transform infrared spectroscopy (FTIR) was used to characterize the PPy/DNA film and to study the specific interactions that may exist between DNA biomacromolecules and PPy chains. Attempts are made to use these PPy/DNA composite films for label-free herpes virus detection revealed a response time of 60 s in solutions containing as low as 2 nM DNA concentration, and self life of six months when immerged in double distilled water and kept refrigerated.

  4. Rapid generation of pandemic influenza virus vaccine candidate strains using synthetic DNA

    PubMed Central

    Verity, Erin E.; Camuglia, Sarina; Agius, Catherine T.; Ong, Chi; Shaw, Robert; Barr, Ian; Middleton, Deborah; Rockman, Steven

    2011-01-01

    Please cite this paper as: Verity et al. (2011) Rapid generation of pandemic influenza virus vaccine candidate strains using synthetic DNA. Influenza and Other Respiratory Viruses DOI:10.1111/j.1750‐2659.2011.00273.x. Background  Vaccination is considered the most effective means of reducing influenza burden. The emergence of H5N1 and pandemic spread of novel H1N1/2009 viruses reinforces the need to have strategies in place to rapidly develop seed viruses for vaccine manufacture. Methods  Candidate pandemic vaccine strains consisting of the circulating strain haemagglutinin (HA) and neuraminidase (NA) in an A/PR/8/34 backbone were generated using alternative synthetic DNA approaches, including site‐directed mutagenesis of DNA encoding related virus strains, and rapid generation of virus using synthetic DNA cloned into plasmid vectors. Results  Firstly, synthetic A/Bar Headed Goose/Qinghai/1A/2005 (H5N1) virus was generated from an A/Vietnam/1194/2004 template using site‐directed mutagenesis. Secondly, A/Whooper Swan/Mongolia/244/2005 (H5N1) and A/California/04/09 (H1N1) viruses were generated using synthetic DNA encoding the viral HA and NA genes. Replication and antigenicity of the synthetic viruses were comparable to that of the corresponding non‐synthetic viruses. Conclusions  In the event of an influenza pandemic, the use of these approaches may significantly reduce the time required to generate and distribute the vaccine seed virus and vaccine manufacture. These approaches also offer the advantage of not needing to handle wild‐type virus, potentially diminishing biocontainment requirements. PMID:21771285

  5. Exposure to West Nile Virus Increases Bacterial Diversity and Immune Gene Expression in Culex pipiens

    PubMed Central

    Zink, Steven D.; Van Slyke, Greta A.; Palumbo, Michael J.; Kramer, Laura D.; Ciota, Alexander T.

    2015-01-01

    Complex interactions between microbial residents of mosquitoes and arboviruses are likely to influence many aspects of vectorial capacity and could potentially have profound effects on patterns of arbovirus transmission. Such interactions have not been well studied for West Nile virus (WNV; Flaviviridae, Flavivirus) and Culex spp. mosquitoes. We utilized next-generation sequencing of 16S ribosomal RNA bacterial genes derived from Culex pipiens Linnaeus following WNV exposure and/or infection and compared bacterial populations and broad immune responses to unexposed mosquitoes. Our results demonstrate that WNV infection increases the diversity of bacterial populations and is associated with up-regulation of classical invertebrate immune pathways including RNA interference (RNAi), Toll, and Jak-STAT (Janus kinase-Signal Transducer and Activator of Transcription). In addition, WNV exposure alone, without the establishment of infection, results in similar alterations to microbial and immune signatures, although to a lesser extent. Multiple bacterial genera were found in greater abundance in WNV-exposed and/or infected mosquitoes, yet the most consistent and notable was the genus Serratia. PMID:26516902

  6. Viral terminal protein directs early organization of phage DNA replication at the bacterial nucleoid

    PubMed Central

    Muñoz-Espín, Daniel; Holguera, Isabel; Ballesteros-Plaza, David; Carballido-López, Rut; Salas, Margarita

    2010-01-01

    The mechanism leading to protein-primed DNA replication has been studied extensively in vitro. However, little is known about the in vivo organization of the proteins involved in this fundamental process. Here we show that the terminal proteins (TPs) of phages ϕ29 and PRD1, infecting the distantly related bacteria Bacillus subtilis and Escherichia coli, respectively, associate with the host bacterial nucleoid independently of other viral-encoded proteins. Analyses of phage ϕ29 revealed that the TP N-terminal domain (residues 1–73) possesses sequence-independent DNA-binding capacity and is responsible for its nucleoid association. Importantly, we show that in the absence of the TP N-terminal domain the efficiency of ϕ29 DNA replication is severely affected. Moreover, the TP recruits the phage DNA polymerase to the bacterial nucleoid, and both proteins later are redistributed to enlarged helix-like structures in an MreB cytoskeleton-dependent way. These data disclose a key function for the TP in vivo: organizing the early viral DNA replication machinery at the cell nucleoid. PMID:20823229

  7. Exploiting bacterial DNA gyrase as a drug target: current state and perspectives.

    PubMed

    Collin, Frédéric; Karkare, Shantanu; Maxwell, Anthony

    2011-11-01

    DNA gyrase is a type II topoisomerase that can introduce negative supercoils into DNA at the expense of ATP hydrolysis. It is essential in all bacteria but absent from higher eukaryotes, making it an attractive target for antibacterials. The fluoroquinolones are examples of very successful gyrase-targeted drugs, but the rise in bacterial resistance to these agents means that we not only need to seek new compounds, but also new modes of inhibition of this enzyme. We review known gyrase-specific drugs and toxins and assess the prospects for developing new antibacterials targeted to this enzyme. PMID:21904817

  8. The action of the bacterial toxin, microcin B17, on DNA gyrase.

    PubMed

    Parks, William M; Bottrill, Andrew R; Pierrat, Olivier A; Durrant, Marcus C; Maxwell, Anthony

    2007-04-01

    Microcin B17 (MccB17) is a peptide-based bacterial toxin that targets DNA gyrase, the bacterial enzyme that introduces supercoils into DNA. The site and mode of action of MccB17 on gyrase are unclear. We review what is currently known about MccB17-gyrase interactions and summarise approaches to understanding its mode of action that involve modification of the toxin. We describe experiments in which treatment of the toxin at high pH leads to the deamidation of two asparagine residues to aspartates. The modified toxin was found to be inactive in vivo and in vitro, suggesting that the Asn residues are essential for activity. Following on from these studies we have used molecular modelling to suggest a 3D structure for microcin B17. We discuss the implications of this model for MccB17 action and investigate the possibility that it binds metal ions. PMID:17276574

  9. Cloacal aerobic bacterial flora and absence of viruses in free-living slow worms (Anguis fragilis), grass snakes (Natrix natrix) and European Adders (Vipera berus) from Germany.

    PubMed

    Schmidt, Volker; Mock, Ronja; Burgkhardt, Eileen; Junghanns, Anja; Ortlieb, Falk; Szabo, Istvan; Marschang, Rachel; Blindow, Irmgard; Krautwald-Junghanns, Maria-Elisabeth

    2014-12-01

    Disease problems caused by viral or bacterial pathogens are common in reptiles kept in captivity. There is no information available on the incidence of viral pathogens or the physiological cloacal bacterial flora of common free-living reptiles in Germany. Therefore, 56 free-living reptiles including 23 European adders (Vipera berus), 12 grass snakes (Natrix natrix) and 21 slow worms (Anguis fragilis) were investigated on the island Hiddensee in northeastern Germany. Pharyngeal and cloacal swabs were taken immediately after capture. Bacteriological examination was performed from the cloacal swabs to study the aerobic cloacal flora. Molecular biological examination included amplification of DNA or RNA from adeno-, rana- and ferlaviruses as well as culturing on Russell's viper heart cells for virus isolation. Salmonella spp. were isolated from European adders but not from the other reptiles examined. The minimal inhibitory concentration was determined from the isolated Salmonella spp. However, some potentially human pathogenic bacteria, such as Proteus vulgaris, Aeromonas hydrophila, Klebsiella pneumoniae and Escherichia coli were isolated. Viruses were not detected in any of the examined reptiles. To the authors' best knowledge, the present study is the first survey of viral pathogens in free-living snakes and slow worms in Germany and the first survey of cloacal aerobic bacterial flora of slow worms. PMID:24866333

  10. Identification of novel Bromus- and Trifolium-associated circular DNA viruses.

    PubMed

    Kraberger, Simona; Farkas, Kata; Bernardo, Pauline; Booker, Cameron; Argüello-Astorga, Gerardo R; Mesléard, François; Martin, Darren P; Roumagnac, Philippe; Varsani, Arvind

    2015-05-01

    The genomes of a large number of highly diverse novel circular DNA viruses from a wide range of sources have been characterised in recent years, including circular single-stranded DNA (ssDNA) viruses that share similarities with plant-infecting ssDNA viruses of the family Geminiviridae. Here, we describe six novel circular DNA viral genomes that encode replication-associated (Rep) proteins that are most closely related to those of either geminiviruses or gemycircularviruses (a new group of ssDNA viruses that are closely related to geminiviruses). Four possible viral genomes were recovered from Bromus hordeaceus sampled in New Zealand, and two were recovered from B. hordeaceus and Trifolium resupinatum sampled in France. Two of the viral genomes from New Zealand (one from the North Island and one from the South Island each) share >99 % sequence identity, and two genomes recovered from B. hordeaceus and T. resupinatum sampled in France share 74 % identity. All of the viral genomes that were recovered were found to have a major open reading frame on both their complementary and virion-sense strands, one of which likely encodes a Rep and the other a capsid protein. Although future infectivity studies are needed to identify the host range of these viruses, this is the first report of circular DNA viruses associated with grasses in New Zealand. PMID:25701210