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Sample records for plasmid induces anti-cd40

  1. Antitumor effects of anti-CD40/CpG immunotherapy combined with gemcitabine or 5-fluorouracil chemotherapy in the B16 melanoma model.

    PubMed

    Qu, Xiaoyi; Felder, Mildred A R; Perez Horta, Zulmarie; Sondel, Paul M; Rakhmilevich, Alexander L

    2013-12-01

    Our previous studies demonstrated that anti-CD40 mAb (anti-CD40) can synergize with CpG oligodeoxynucleotides (CpG) to mediate antitumor effects by activating myeloid cells, such as macrophages in tumor-bearing mice. Separate teams have shown that chemotherapy with gemcitabine (GEM) or 5-fluorouracil (5-FU) can reduce tumor-induced myeloid-derived suppressor cells (MDSC) in mice. In this study we asked if the same chemotherapy regimens with GEM or 5-FU will enhance the antitumor effect of anti-CD40 and CpG. Using the model of B16 melanoma growing intraperitoneally in syngeneic C57BL/6 mice, we show that these GEM or 5-FU treatment regimens reduced MDSC in the peritoneal cavity of tumor-bearing mice. Treatment of mice with GEM or 5-FU did not significantly affect the antitumor function of macrophages as assessed in vitro. In vivo, treatment with these GEM or 5-FU regimens followed by anti-CD40/CpG resulted in antitumor effects similar to those of anti-CD40/CpG in the absence of GEM or 5-FU. Likewise, reduction of MDSC by in vivo anti-Gr-1 mAb treatment did not significantly affect anti-CD40/CpG antitumor responses. Together, the results show that the GEM or 5-FU chemotherapy regimens did not substantially affect the antitumor effects induced by anti-CD40/CpG immunotherapy.

  2. Synergy of anti-CD40, CpG and MPL in activation of mouse macrophages.

    PubMed

    Shi, Yongyu; Felder, Mildred A R; Sondel, Paul M; Rakhmilevich, Alexander L

    2015-08-01

    Activation of macrophages is a prerequisite for their antitumor effects. Several reagents, including agonistic anti-CD40 monoclonal antibody (anti-CD40), CpG oligodeoxynucleotides (CpG) and monophosphoryl lipid A (MPL), can stimulate activation of macrophages. Our previous studies showed synergy between anti-CD40 and CpG and between anti-CD40 and MPL in macrophage activation and antitumor efficacy in mice. In the present study, we asked whether there was synergy among these three reagents. The activation of adherent peritoneal exudate cells (PEC) obtained from mice injected with anti-CD40 and then treated with CpG and/or MPL in vitro was determined by their ability to suppress proliferation of tumor cells and to produce various cytokines and chemokines in vitro. Cell sorting and histology followed by functional testing showed that macrophages were the main cell population in PEC activated by CD40 ligation in vivo. A combination of anti-CD40, CpG or MPL activated PEC to suppress proliferation of B16 cells and produce nitric oxide far greater than the single reagents or any of the double combinations of these reagents. In addition, the combination of all three reagents activated PEC to secrete IL-12, IFN-γ and MCP-1 to a greater degree than any single reagent or any two combined reagents. These results demonstrate that macrophages can be synergistically activated by anti-CD40, CpG and MPL, suggesting that this novel combined approach might be further investigated as potential cancer therapy.

  3. Anti-CD40 ligand monoclonal antibody delays the progression of murine autoimmune cholangitis.

    PubMed

    Tanaka, H; Yang, G-X; Iwakoshi, N; Knechtle, S J; Kawata, K; Tsuneyama, K; Leung, P; Coppel, R L; Ansari, A A; Joh, T; Bowlus, C; Gershwin, M E

    2013-12-01

    While there have been significant advances in our understanding of the autoimmune responses and the molecular nature of the target autoantigens in primary biliary cirrhosis (PBC), unfortunately these data have yet to be translated into new therapeutic agents. We have taken advantage of a unique murine model of autoimmune cholangitis in which mice expressing a dominant negative form of transforming growth factor β receptor II (dnTGFβRII), under the control of the CD4 promoter, develop an intense autoimmune cholangitis associated with serological features similar to human PBC. CD40-CD40 ligand (CD40L) is a major receptor-ligand pair that provides key signals between cells of the adaptive immune system, prompting us to determine the therapeutic potential of treating autoimmune cholangitis with anti-CD40L antibody (anti-CD40L; MR-1). Four-week-old dnTGFβRII mice were injected intraperitoneally with either anti-CD40L or control immunoglobulin (Ig)G at days 0, 2, 4 and 7 and then weekly until 12 or 24 weeks of age and monitored for the progress of serological and histological features of PBC, including rigorous definition of liver cellular infiltrates and cytokine production. Administration of anti-CD40L reduced liver inflammation significantly to 12 weeks of age. In addition, anti-CD40L initially lowered the levels of anti-mitochondrial autoantibodies (AMA), but these reductions were not sustained. These data indicate that anti-CD40L delays autoimmune cholangitis, but the effect wanes over time. Further dissection of the mechanisms involved, and defining the events that lead to the reduction in therapeutic effectiveness will be critical to determining whether such efforts can be applied to PBC.

  4. Characterization of a Broadly Reactive Anti-CD40 Agonistic Monoclonal Antibody for Potential Use as an Adjuvant

    PubMed Central

    Waghela, Suryakant D.; Lokhandwala, Shehnaz; Ambrus, Andy; Bray, Jocelyn; Vuong, Christina; Vinodkumar, Vanitha; Dominowski, Paul J.; Rai, Sharath; Mwangi, Duncan; Foss, Dennis L.; Mwangi, Waithaka

    2017-01-01

    Lack of safe and effective adjuvants is a major hindrance to the development of efficacious vaccines. Signaling via CD40 pathway leads to enhanced antigen processing and presentation, nitric oxide expression, pro-inflammatory cytokine expression by antigen presenting cells, and stimulation of B-cells to undergo somatic hypermutation, immunoglobulin class switching, and proliferation. Agonistic anti-CD40 antibodies have shown promising adjuvant qualities in human and mouse vaccine studies. An anti-CD40 monoclonal antibody (mAb), designated 2E4E4, was identified and shown to have strong agonistic effects on primary cells from multiple livestock species. The mAb recognize swine, bovine, caprine, and ovine CD40, and evoked 25-fold or greater proliferation of peripheral blood mononuclear cells (PBMCs) from these species relative to cells incubated with an isotype control (p<0.001). In addition, the mAb induced significant nitric oxide (p<0.0001) release by bovine macrophages. Furthermore, the mAb upregulated the expression of MHC-II by PBMCs, and stimulated significant (p<0.0001) IL-1α, IL6, IL-8, and TNF-α expression by PBMCs. These results suggest that the mAb 2E4E4 can target and stimulate cells from multiple livestock species and thus, it is a potential candidate for adjuvant development. This is the first study to report an anti-swine CD40 agonistic mAb that is also broadly reactive against multiple species. PMID:28107431

  5. The antileukemia activity of a human anti-CD40 antagonist antibody, HCD122, on human chronic lymphocytic leukemia cells

    PubMed Central

    Klabunde, Sha; Lin, Karen; Georgakis, Georgios V.; Cherukuri, Anu; Holash, Jocelyn; Goldbeck, Cheryl; Xu, Xiaomei; Kadel, Edward E.; Lee, Sang Hoon; Aukerman, Sharon Lea; Jallal, Bahija; Aziz, Natasha; Weng, Wen-Kai; Wierda, William; O'Brien, Susan; Younes, Anas

    2008-01-01

    B-cell chronic lymphocytic leukemia (B-CLL) is a lymphoproliferative disorder characterized by the surface expression of CD20, CD5 antigens, as well as the receptor CD40. Activation of CD40 by its ligand (CD40L) induces proliferation and rescues the cells from spontaneous and chemotherapy-induced apoptosis. CD40 activation also induces secretion of cytokines, such as IL-6, IL-10, TNF-α, IL-8, and GM-CSF, which are involved in tumor cell survival, migration, and interaction with cells in the tumor microenvironment. Here we demonstrate that in primary B-CLL tumor cells, the novel antagonist anti-CD40 monoclonal antibody, HCD122, inhibits CD40L-induced activation of signaling pathways, proliferation and survival, and secretion of cytokines. Furthermore, HCD122 is also a potent mediator of antibody-dependent cellular cytotoxicity (ADCC), lysing B-CLL cells more efficiently than rituximab in vitro, despite a significantly higher number of cell surface CD20 binding sites compared with CD40. Unlike rituximab, however, HCD122 (formerly CHIR-12.12) does not internalize upon binding to the cells. Our data suggest that HCD122 may inhibit B-CLL growth by blocking CD40 signaling and by ADCC-mediated cell lysis. PMID:18497318

  6. The antileukemia activity of a human anti-CD40 antagonist antibody, HCD122, on human chronic lymphocytic leukemia cells.

    PubMed

    Luqman, Mohammad; Klabunde, Sha; Lin, Karen; Georgakis, Georgios V; Cherukuri, Anu; Holash, Jocelyn; Goldbeck, Cheryl; Xu, Xiaomei; Kadel, Edward E; Lee, Sang Hoon; Aukerman, Sharon Lea; Jallal, Bahija; Aziz, Natasha; Weng, Wen-Kai; Wierda, William; O'Brien, Susan; Younes, Anas

    2008-08-01

    B-cell chronic lymphocytic leukemia (B-CLL) is a lymphoproliferative disorder characterized by the surface expression of CD20, CD5 antigens, as well as the receptor CD40. Activation of CD40 by its ligand (CD40L) induces proliferation and rescues the cells from spontaneous and chemotherapy-induced apoptosis. CD40 activation also induces secretion of cytokines, such as IL-6, IL-10, TNF-alpha, IL-8, and GM-CSF, which are involved in tumor cell survival, migration, and interaction with cells in the tumor microenvironment. Here we demonstrate that in primary B-CLL tumor cells, the novel antagonist anti-CD40 monoclonal antibody, HCD122, inhibits CD40L-induced activation of signaling pathways, proliferation and survival, and secretion of cytokines. Furthermore, HCD122 is also a potent mediator of antibody-dependent cellular cytotoxicity (ADCC), lysing B-CLL cells more efficiently than rituximab in vitro, despite a significantly higher number of cell surface CD20 binding sites compared with CD40. Unlike rituximab, however, HCD122 (formerly CHIR-12.12) does not internalize upon binding to the cells. Our data suggest that HCD122 may inhibit B-CLL growth by blocking CD40 signaling and by ADCC-mediated cell lysis.

  7. IN VITRO TESTING OF AN ANTI-CD40 MONOCLONAL ANTIBODY, CLONE 2C10, IN PRIMATES AND PIGS

    PubMed Central

    Lee, Whayoung; Satyananda, Vikas; Iwase, Hayato; Tanaka, Takayuki; Miyagawa, Yuko; Long, Cassandra; Ayares, David; Cooper, David KC; Hara, Hidetaka

    2015-01-01

    Background The CD40/CD154 and CD28/B7 pathways are important in allo- and xeno-transplantation. Owing to the thrombotic complications of anti-CD154mAb, anti-CD40mAb has emerged as a promising inhibitor of costimulation. Various clones of anti-CD40mAb have been developed against primate species, e.g., clone 2C10 against rhesus monkeys. We have compared the in vitro efficacy of 2C10 to prevent a T cell response in primates and pigs. Methods The binding of 2C10 to antigen-presenting cells (PBMCs [B cells]) of humans, rhesus and cynomolgus monkeys, baboons, and pigs was measured by flow cytometry, and was also tested indirectly by a blocking assay. The functional capacity of 2C10 was tested by mixed lymphocyte reaction (MLR) with polyclonal stimulation by phytohemagglutinin (PHA) and also with wild-type pig aortic endothelial cells (pAECs) as stimulators. Results There was a significant reduction in binding of 2C10 to baboon PBMCs compared to rhesus, cynomolgus, and human PBMCs, and minimal binding to pig PBMCs. The blocking assay confirmed that the binding of 2C10 was significantly lower to baboon PBMCs when compared to the other primate species tested. The functional assay with PHA showed significantly reduced inhibition of PBMC proliferation in humans, cynomolgus monkeys, and baboons compared to rhesus monkeys, which was confirmed on MLR with pAECs. Conclusions Since both the binding and functional activity of 2C10 in the baboon is lower than in rhesus monkeys, in vivo treatment using 2C10 in the baboon might require a higher dose or more frequent administration in comparison to rhesus monkeys. It may also be beneficial to develop species-specific clones of anti-CD40mAb. PMID:26458513

  8. Effects of gene transfer CTLA4Ig and anti-CD40L monoclonal antibody on islet xenograft rejection in mice.

    PubMed

    Zhang, J; Li, H; Jiang, N; Zhang, Q; Wang, G-S; Yi, H-M; Fu, B-S; Wang, G-Y; Yang, Y; Chen, G-H

    2010-06-01

    Blockade of a costimulatory pathway by adenovirus-mediated cytotoxic T lymphocyte associated antigen 4 immunoglobulin (CTLA4-Ig) gene transfer and anti-CD40L mAb(MR1) have been reported to enhance graft survival in several experimental transplantation models. In this study, we investigated the effects of gene transfer of CTLA4Ig and MR1 on islet xenograft rejection in mice. Recombinant adenovirus AdCTLA4Ig was constructed to express CTLA4Ig. Islet grafts from adult male DA rats transferred with AdCTLA4Ig were transplanted to streptozocin-induced diabetic Balb/c mice. The diabetic mice were treated with MR1 after transplantation. We evaluated the islet xenograft mean survival time as well as changes in interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-alpha) levels in transplanted mice. The mean survival of islet xenografts in the MR1 treatment group was 34.9 +/- 5.62 days, in the AdCTLA4Ig treatment group it was 56.5 +/- 10.64 days, and in the AdCTLA4Ig plus MR1 treatment group it was 112.9 +/- 19.26 days, all significantly prolonged compared with an untreated group (8.1 +/- 0.83 days). Within 1 week after transplantation the levels of IL-2 and TNF-alpha showed sharp increases in the untreated group, being significantly higher than those observed prior to transplantation. In conclusion, using both AdCTLA4Ig and MR1 can improve the islet xenograft survival. The beneficial effects of the combined use of the 2 reagents were superior to either 1 alone, possibly related to down-regulated expression of Th1 cell-related cytokines.

  9. Earlier low-dose TBI or DST overcomes CD8+ T-cell-mediated alloresistance to allogeneic marrow in recipients of anti-CD40L.

    PubMed

    Takeuchi, Yasuo; Ito, Hiroshi; Kurtz, Josef; Wekerle, Thomas; Ho, Leon; Sykes, Megan

    2004-01-01

    Treatment with a single injection of anti-CD40L (CD154) monoclonal antibody (mAb) and fully mismatched allogeneic bone marrow transplant (BMT) allows rapid tolerization of CD4+ T cells to the donor. The addition of in vivo CD8 T-cell depletion leads to permanent mixed hematopoietic chimerism and tolerance. We now describe two approaches that obviate the requirement for CD8 T-cell depletion by rapidly tolerizing recipient CD8 T cells in addition to CD4 cells. Administration of donor-specific transfusion (DST) to mice receiving 3 Gy total body irradiation (TBI), BMT and anti-CD40L mAb on day 0 uniformly led to permanent mixed chimerism and tolerance, compared with only 40% of mice receiving similar treatment without DST. In the absence of DST, moving the timing of 3 Gy TBI to day -1 or day -2 instead of day 0 led to rapid (by 2 weeks) induction of CD8+ cell tolerance, and also permitted uniform achievement of permanent mixed chimerism and donor-specific tolerance in recipients of anti-CD40L and BMT on day 0. These nontoxic regimens overcome CD8+ and CD4+ T-cell-mediated alloresistance without requiring host T-cell depletion, permitting the induction of permanent mixed chimerism and tolerance.

  10. Macrophages and Fc-receptor interactions contribute to the antitumour activities of the anti-CD40 antibody SGN-40.

    PubMed

    Oflazoglu, E; Stone, I J; Brown, L; Gordon, K A; van Rooijen, N; Jonas, M; Law, C-L; Grewal, I S; Gerber, H-P

    2009-01-13

    SGN-40 is a therapeutic antibody targeting CD40, which induces potent anti-lymphoma activities via direct apoptotic signalling cells and by cell-mediated cytotoxicity. Here we show antibody-dependent cellular phagocytosis (ADCP) by macrophages to contribute significantly to the therapeutic activities and that the antitumour effects of SGN-40 depend on Fc interactions.

  11. Plasmid DNA damage induced by helium atmospheric pressure plasma jet

    NASA Astrophysics Data System (ADS)

    Han, Xu; Cantrell, William A.; Escobar, Erika E.; Ptasinska, Sylwia

    2014-03-01

    A helium atmospheric pressure plasma jet (APPJ) is applied to induce damage to aqueous plasmid DNA. The resulting fractions of the DNA conformers, which indicate intact molecules or DNA with single- or double-strand breaks, are determined using agarose gel electrophoresis. The DNA strand breaks increase with a decrease in the distance between the APPJ and DNA samples under two working conditions of the plasma source with different parameters of applied electric pulses. The damage level induced in the plasmid DNA is also enhanced with increased plasma irradiation time. The reactive species generated in the APPJ are characterized by optical emission spectra, and their roles in possible DNA damage processes occurring in an aqueous environment are also discussed.

  12. GFP plasmid-induced defects in Salmonella invasion depend on plasmid architecture, not protein expression.

    PubMed

    Clark, Leann; Martinez-Argudo, Isabel; Humphrey, Tom J; Jepson, Mark A

    2009-02-01

    We have investigated the impact of plasmids and GFP expression on invasion of cultured epithelial cells by Salmonella enterica Typhimurium strain SL1344. The invasiveness of SL1344 carrying plasmids derived from pBR322, encoding promoterless GFP or constitutively expressed rpsM-GFP, was compared under optimal growth conditions with that of SL1344(pBR322), unmodified SL1344 and a strain with chromosome-integrated rpsM-GFP. The strain carrying pBR322 exhibited normal invasion, but the presence of modified plasmids impaired invasiveness, and impairment was exacerbated by plasmid-encoded chloramphenicol resistance (CmR). Using a different antibiotic resistance marker, kanamycin (KmR), did not impair invasiveness. Despite the effect of plasmid-encoded CmR, the strain containing chromosomally encoded GFP, also carrying a CmR gene, was as invasive as the wild-type. To investigate the mechanism by which plasmid carriage decreases invasion, we monitored SPI-1 gene expression using prgH promoter activity as an index of SPI-1 activity. An SL1344 strain with a chromosome-integrated prgH::gfp reporter construct exhibited lower GFP expression during exponential phase when carrying plasmids incorporating CmR or gfp, mirroring invasion data. These data provide evidence that suppression of SPI-1 gene expression is a major factor in the loss of invasiveness associated with plasmid carriage. Our findings also indicate that some plasmids, especially those carrying CmR, should be used with caution, as virulence traits and gene expression may be affected by their presence. Integration of reporter proteins into the bacterial chromosome, however, appears to circumvent the adverse effects observed with plasmids.

  13. Inducible expression of photoacoustic reporter gene tyrosinase in cells using a single plasmid

    NASA Astrophysics Data System (ADS)

    Paproski, Robert J.; Zemp, Roger J.

    2012-02-01

    We have previously demonstrated that tyrosinase is a reporter gene for photoacoustic imaging since tyrosinase is the rate-limiting step in the synthesis of melanin, a pigment capable of producing strong photoacoustic signals. We previously created a cell line capable of inducible tyrosinase expression (important due to toxicity of melanin) by stably transfecting tyrosinase in MCF-7 Tet-OnR cell line (Clontech) which expresses a doxycycline-controlled transactivator. Unfortunately, Clontech provides few Tet-On Advanced cell lines making it difficult to have inducible tyrosinase expression in cell lines not provided by Clontech. In order to simplify the creation of cell lines with inducible expression of tyrosinase, we created a single plasmid that encodes both the transactivator as well as tyrosinase. PCR was used to amplify both the transactivator and tyrosinase from the Tet-OnR Advanced and pTRE-Tight-TYR plasmids, respectively. Both PCR products were cloned into the pEGFP-N1 plasmid and the newly created plasmid was transfected into ZR-75-1, MCF-7, and MIA PaCa-1 cells using lipofectamine. After several days, brown melanin was only observed in cells incubated with doxycycline, suggesting that the newly created single plasmid allowed inducible tyrosinase expression in many different cells lines.

  14. Transcription of ColE1Ap mbeC induced by conjugative plasmids from twelve different incompatibility groups.

    PubMed Central

    Selvaratnam, S; Gealt, M A

    1993-01-01

    Although nonconjugative mobilizable plasmids require helping functions of conjugative plasmids in order to be mobilized into recipients, at least some genes from the nonconjugative plasmids may be induced to assist in the DNA transfer process. Conjugative plasmids from 12 different incompatibility groups mobilized the nonconjugative plasmid ColE1Ap between Escherichia coli strains. Introduction of any of the conjugative plasmids into the ColE1Ap-containing strain resulted in an induction of mbeC, the product of which is a component of the mobilization relaxation complex. Each of the conjugative plasmids caused protein to bind specifically to mbe promoter DNA, suggesting a direct regulatory interaction. Images PMID:8226641

  15. Plasmid-determined inducible efflux is responsible for resistance to cadmium, zinc, and cobalt in Alcaligenes eutrophus.

    PubMed Central

    Nies, D H; Silver, S

    1989-01-01

    In Alcaligenes eutrophus CH34, resistance to chromate is plasmid determined, inducible, and based on decreased net accumulation of the metal anion. Plasmid-encoded resistances to zinc, cadmium, cobalt, and nickel are resulting from inducible, energy-dependent cation efflux systems. PMID:2914875

  16. Combination of monoclonal antibodies with DST inhibits accelerated rejection mediated by memory T cells to induce long-lived heart allograft acceptance in mice.

    PubMed

    Shao, Wei; Chen, Jibing; Dai, Helong; Peng, Yuanzheng; Wang, Feng; Xia, Junjie; Thorlacius, Henrik; Zhu, Qi; Qi, Zhongquan

    2011-08-30

    Donor-reactive memory T cells mediated accelerated rejection is known as a barrier to the survival of transplanted organs. We investigated the combination of different monoclonal antibodies (mAbs) and donor-specific transfusion (DST) in memory T cells-based adoptive mice model. In the presence of donor-reactive memory T cells, the mean survival time (MST) of grafts in the anti-CD40L/LFA-1/DST group was 49.8d. Adding anti-CD44/CD70 mAbs to anti-CD40L/LFA-1/DST treatment. The MST was more than 100 d (MST>100 d). Compared with anti-CD40L/LFA-1/DST group, anti-CD40L/LFA-1/CD44/CD70/DST group notably reduced the expansion of memory T cells, enhanced the proportion of CD4+Foxp3+ regulatory T cells (Tregs) and suppressed donor-specific responses. Our data suggest that anti-CD40L/LFA-1/CD44/CD70mAbs and DST can synergistically inhibit accelerated rejection mediated by memory T cells to induce long-lived heart allograft acceptance in mice.

  17. Kinds and spectrum of mutations induced by 1-nitrosopyrene adducts during plasmid replication in human cells.

    PubMed Central

    Yang, J L; Maher, V M; McCormick, J J

    1988-01-01

    1-Nitropyrene has been shown in bacterial assays to be the principal mutagenic agent in diesel emission particulates. It has also been shown to be mutagenic in human fibroblasts and carcinogenic in animals. To investigate the kinds of mutations induced by this carcinogen and compare them with those induced by a structurally related carcinogen, (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetra-hydrobenzo [a]pyrene (BPDE) (J.-L. Yang, V. M. Maher, and J. J. McCormick, Proc. Natl. Acad. Sci. USA 84:3787-3791, 1987), we treated a shuttle vector with tritiated 1-nitrosopyrene (1-NOP), a carcinogenic mutagenic intermediate metabolite of 1-nitropyrene which forms the same DNA adduct as the parent compound, and introduced the plasmids into a human embryonic kidney cell line, 293, for DNA replication to take place. The treated plasmid, pZ189, carrying a bacterial suppressor tRNA target gene, supF, was allowed 48 h to replicate in the human cells. Progeny plasmids were then rescued, purified, and introduced into bacteria carrying an amber mutation in the beta-galactosidase gene in order to detect those carrying mutations in the supF gene. The frequency of mutants increased in direct proportion to the number of DNA-1-NOP adducts formed per plasmid. At the highest level of adduct formation tested, the frequency of supF mutants was 26 times higher than the background frequency of 1.4 X 10(-4). DNA sequencing of 60 unequivocally independent mutant derived from 1-NOP-treated plasmids indicated that 80% contained a single base substitution, 5% had two base substitutions, 4% had small insertions or deletions (1 or 2 base pairs), and 11% showed a deletion or insertion of 4 or more base pairs. Sequence data from 25 supF mutants derived from untreated plasmids showed that 64% contained deletions of 4 or more base pairs. The majority (83%) of the base substitution in mutants from 1-NOP-treated plasmids were transversions, with 73% of these being G . C --> T . A. This

  18. Plasmid-deficient Chlamydia muridarum fail to induce immune pathology and protect against oviduct disease.

    PubMed

    O'Connell, Catherine M; Ingalls, Robin R; Andrews, Charles W; Scurlock, Amy M; Darville, Toni

    2007-09-15

    Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection in the world. In women, genital infection can cause endometritis and pelvic inflammatory disease with the severe sequelae of ectopic pregnancy or infertility. Chlamydia sp. do not damage tissues directly, but induce an injurious host inflammatory response at the infected site. In the murine model of genital disease with Chlamydia muridarum, TLR2 plays a role in both early production of inflammatory mediators and development of chronic oviduct pathology. We report the results of studies with plasmid-cured C. muridarum mutants that retain the ability to infect the murine genital tract, but fail to cause disease in the oviduct. These mutants do not stimulate TLR2-dependent cytokine production in mice, nor in innate immune cells or epithelial cells in vitro. They induce an effective Th1 immune response, with no evidence for Th1-immune-mediated collateral tissue damage. Furthermore, mice previously infected with the plasmid-deficient strains are protected against oviduct disease upon challenge with virulent C. muridarum. If plasmid-cured derivatives of human C. trachomatis biovars exhibit similar phenotypic characteristics, they have the potential to serve as vaccines to prevent human disease.

  19. Spermine and spermidine protection of plasmid DNA against single-strand breaks induced by singlet oxygen.

    PubMed Central

    Khan, A U; Di Mascio, P; Medeiros, M H; Wilson, T

    1992-01-01

    Oxidative damage to DNA induced by singlet molecular oxygen (1O2*) includes single-strand breaks, which the biologically occurring 1O2* quenchers spermine and spermidine are shown to prevent. These polyamines at a physiological concentration (10 mM) reduce the percentage of the open circular form of pBR322 plasmid DNA, which is generated at the expense of the native supercoiled form when the plasmids are incubated with a chemical source of 1O2*, the water-soluble endoperoxide of 3,3'-(1,4-naphthylidene)dipropionate. Spermine and spermidine can be expected to protect DNA against other damaging effects of 1O2*. Images PMID:1454831

  20. Plasmid-Based Generation of Induced Neural Stem Cells from Adult Human Fibroblasts

    PubMed Central

    Capetian, Philipp; Azmitia, Luis; Pauly, Martje G.; Krajka, Victor; Stengel, Felix; Bernhardi, Eva-Maria; Klett, Mariana; Meier, Britta; Seibler, Philip; Stanslowsky, Nancy; Moser, Andreas; Knopp, Andreas; Gillessen-Kaesbach, Gabriele; Nikkhah, Guido; Wegner, Florian; Döbrössy, Máté; Klein, Christine

    2016-01-01

    Direct reprogramming from somatic to neural cell types has become an alternative to induced pluripotent stem cells. Most protocols employ viral expression systems, posing the risk of random genomic integration. Recent developments led to plasmid-based protocols, lowering this risk. However, these protocols either relied on continuous presence of a variety of small molecules or were only able to reprogram murine cells. We therefore established a reprogramming protocol based on vectors containing the Epstein-Barr virus (EBV)-derived oriP/EBNA1 as well as the defined expression factors Oct3/4, Sox2, Klf4, L-myc, Lin28, and a small hairpin directed against p53. We employed a defined neural medium in combination with the neurotrophins bFGF, EGF and FGF4 for cultivation without the addition of small molecules. After reprogramming, cells demonstrated a temporary increase in the expression of endogenous Oct3/4. We obtained induced neural stem cells (iNSC) 30 days after transfection. In contrast to previous results, plasmid vectors as well as a residual expression of reprogramming factors remained detectable in all cell lines. Cells showed a robust differentiation into neuronal (72%) and glial cells (9% astrocytes, 6% oligodendrocytes). Despite the temporary increase of pluripotency-associated Oct3/4 expression during reprogramming, we did not detect pluripotent stem cells or non-neural cells in culture (except occasional residual fibroblasts). Neurons showed electrical activity and functional glutamatergic synapses. Our results demonstrate that reprogramming adult human fibroblasts to iNSC by plasmid vectors and basic neural medium without small molecules is possible and feasible. However, a full set of pluripotency-associated transcription factors may indeed result in the acquisition of a transient (at least partial) pluripotent intermediate during reprogramming. In contrast to previous reports, the EBV-based plasmid system remained present and active inside the cells at

  1. Low energy electron induced damage to plasmid DNA pQE30

    SciTech Connect

    Kumar, S. V. K.; Pota, Tasneem; Peri, Dinakar; Dongre, Anushka D.; Rao, Basuthkar J.

    2012-07-28

    Low energy electrons (LEEs) are produced in copious amounts by the primary radiation used in radiation therapy. The damage caused to the DNA by these secondary electrons in the energy range 5-22 eV has been studied to understand their possible role in radiation induced damage. Electrons are irradiated on dried films of plasmid DNA (pQE30) and analysed using agarose gel electrophoresis. Single strand breaks (SSBs) induced by LEE to supercoiled plasmid DNA show resonance structures at 7, 12, and 15 eV for low doses and 6, 10, and {approx}18 eV at saturation doses. The present measurements have an overall agreement with the literature that LEEs resonantly induce SSBs in DNA. Resonant peaks in the SSBs induced by LEEs at 7, 12, and 15 eV with the lowest employed dose in the current study are somewhat different from those reported earlier by two groups. The observed differences are perhaps related to the irradiation dose, conditions and the nature of DNA employed, which is further elaborated.

  2. Low energy electron induced damage to plasmid DNA pQE30

    NASA Astrophysics Data System (ADS)

    Kumar, S. V. K.; Pota, Tasneem; Peri, Dinakar; Dongre, Anushka D.; Rao, Basuthkar J.

    2012-07-01

    Low energy electrons (LEEs) are produced in copious amounts by the primary radiation used in radiation therapy. The damage caused to the DNA by these secondary electrons in the energy range 5-22 eV has been studied to understand their possible role in radiation induced damage. Electrons are irradiated on dried films of plasmid DNA (pQE30) and analysed using agarose gel electrophoresis. Single strand breaks (SSBs) induced by LEE to supercoiled plasmid DNA show resonance structures at 7, 12, and 15 eV for low doses and 6, 10, and ˜18 eV at saturation doses. The present measurements have an overall agreement with the literature that LEEs resonantly induce SSBs in DNA. Resonant peaks in the SSBs induced by LEEs at 7, 12, and 15 eV with the lowest employed dose in the current study are somewhat different from those reported earlier by two groups. The observed differences are perhaps related to the irradiation dose, conditions and the nature of DNA employed, which is further elaborated.

  3. Comprehensive set of integrative plasmid vectors for copper-inducible gene expression in Myxococcus xanthus.

    PubMed

    Gómez-Santos, Nuria; Treuner-Lange, Anke; Moraleda-Muñoz, Aurelio; García-Bravo, Elena; García-Hernández, Raquel; Martínez-Cayuela, Marina; Pérez, Juana; Søgaard-Andersen, Lotte; Muñoz-Dorado, José

    2012-04-01

    Myxococcus xanthus is widely used as a model system for studying gliding motility, multicellular development, and cellular differentiation. Moreover, M. xanthus is a rich source of novel secondary metabolites. The analysis of these processes has been hampered by the limited set of tools for inducible gene expression. Here we report the construction of a set of plasmid vectors to allow copper-inducible gene expression in M. xanthus. Analysis of the effect of copper on strain DK1622 revealed that copper concentrations of up to 500 μM during growth and 60 μM during development do not affect physiological processes such as cell viability, motility, or aggregation into fruiting bodies. Of the copper-responsive promoters in M. xanthus reported so far, the multicopper oxidase cuoA promoter was used to construct expression vectors, because no basal expression is observed in the absence of copper and induction linearly depends on the copper concentration in the culture medium. Four different plasmid vectors have been constructed, with different marker selection genes and sites of integration in the M. xanthus chromosome. The vectors have been tested and gene expression quantified using the lacZ gene. Moreover, we demonstrate the functional complementation of the motility defect caused by lack of PilB by the copper-induced expression of the pilB gene. These versatile vectors are likely to deepen our understanding of the biology of M. xanthus and may also have biotechnological applications.

  4. Induced mutagenesis of plasmid and chromosomal genes inserted into the plasmid DNA. II. Mutagenic action of chemical factors

    SciTech Connect

    Esipova, V.V.; Vedunova, S.L.; Kriviskii, A.S.

    1986-02-01

    Following the study of the mutagenic action of UV and ..gamma..-radiation on plasmid DNA in vitro, they investigated the induction of mutations under the influence of chemical mutagens on the same DNA of plasmid RSF2124, determining the synthesis of colicine E1 and resistance to ampicillin. The inactivating action of the mutagen was assessed from the yield of transformants resistant to the antibiotic and the mutagenic effect from the loss by colonies of transformants that were capable of releasing colicine into the external medium. In these experiments they mainly used chemical compounds whose mutagenic effect if well known in other systems (transforming and transfecting DNA, microbial viruses). As a result all mutagens tested for their activity were divided into four groups: first group, those exceeding the level of mutagenesis by more than 100-fold above the spontaneous background (hydroxylamine, O-methylhydroxylamine); second group, those exceeding it by a factor of 10 (UV radiation (lambda = 254 nm), W-mutagenesis, ionizing radiation, nitrous acid, mitomycin C); third group, those exceeding it by a factor of <10 (indirect UV mutagenesis, nitrous acid, ..beta..-chloroethyldiethylamine hydrochloride, nitrosoguanidine); fourth group, no mutagenic effect (acridine orange, ethyl methane sulfonate, sodium azide, 0-..beta..-diethylaminoethylhydroxylamine).

  5. Damage to dry plasmid DNA induced by nanosecond XUV-laser pulses

    NASA Astrophysics Data System (ADS)

    Nováková, Eva; Davídková, Marie; Vyšín, Ludék; Burian, Tomáš; Grisham, Michael E.; Heinbuch, Scott; Rocca, Jorge J.; Juha, Libor

    2011-06-01

    Ionizing radiation induces a variety of DNA damages including single-strand breaks (SSBs), double-strand breaks (DSBs), abasic sites, modified sugar and bases. Most theoretical and experimental studies have been focused on DNA strand scissions, in particular production of DNA double-strand breaks. DSBs have been proven to be a key damage at a molecular level responsible for the formation of chromosomal aberrations, leading often to cell death. The complexity of lesions produced in DNA by ionizing radiations is thought to depend on the amount of energy deposited at the site of each lesion. We have studied the nature of DNA damage induced directly by the pulsed 46.9 nm radiation provided by a capillary-discharge Ne-like Ar laser (CDL). Different surface doses were delivered with a repetition rate of a few Hz and an average pulse energy ~ 1 μJ. A simple model DNA molecule, i.e., dried closed-circular plasmid DNA (pBR322), was irradiated. The agarose gel electrophoresis method was used for determination of both SSB and DSB yields. Results are compared with a previous study of plasmid DNA irradiated with a single sub-nanosecond 1-keV X-ray pulse produced by a large-scale, double-stream gas puff target, illuminated by sub-kJ, near-infrared (NIR) focused laser pulses at the PALS facility (Prague Asterix Laser System).

  6. Metal-Induced Stabilization and Activation of Plasmid Replication Initiator RepB

    PubMed Central

    Ruiz-Masó, José A.; Bordanaba-Ruiseco, Lorena; Sanz, Marta; Menéndez, Margarita; del Solar, Gloria

    2016-01-01

    Initiation of plasmid rolling circle replication (RCR) is catalyzed by a plasmid-encoded Rep protein that performs a Tyr- and metal-dependent site-specific cleavage of one DNA strand within the double-strand origin (dso) of replication. The crystal structure of RepB, the initiator protein of the streptococcal plasmid pMV158, constitutes the first example of a Rep protein structure from RCR plasmids. It forms a toroidal homohexameric ring where each RepB protomer consists of two domains: the C-terminal domain involved in oligomerization and the N-terminal domain containing the DNA-binding and endonuclease activities. Binding of Mn2+ to the active site is essential for the catalytic activity of RepB. In this work, we have studied the effects of metal binding on the structure and thermostability of full-length hexameric RepB and each of its separate domains by using different biophysical approaches. The analysis of the temperature-induced changes in RepB shows that the first thermal transition, which occurs at a range of temperatures physiologically relevant for the pMV158 pneumococcal host, represents an irreversible conformational change that affects the secondary and tertiary structure of the protein, which becomes prone to self-associate. This transition, which is also shown to result in loss of DNA binding capacity and catalytic activity of RepB, is confined to its N-terminal domain. Mn2+ protects the protein from undergoing this detrimental conformational change and the observed protection correlates well with the high-affinity binding of the cation to the active site, as substituting one of the metal-ligands at this site impairs both the protein affinity for Mn2+and the Mn2+-driven thermostabilization effect. The level of catalytic activity of the protein, especially in the case of full-length RepB, cannot be explained based only on the high-affinity binding of Mn2+ at the active site and suggests the existence of additional, lower-affinity metal binding site

  7. Heavy ion induced damage to plasmid DNA: plateau region vs. spread out Bragg-peak

    NASA Astrophysics Data System (ADS)

    Dang, H. M.; van Goethem, M. J.; van der Graaf, E. R.; Brandenburg, S.; Hoekstra, R.; Schlathölter, T.

    2011-08-01

    We have investigated the damage of synthetic plasmid pBR322 DNA in dilute aqueous solutions induced by fast carbon ions. The relative contribution of indirect damage and direct damage to the DNA itself is expected to vary with linear energy transfer along the ion track, with the direct damage contribution increasing towards the Bragg peak. Therefore, 12C ions at the spread-out Bragg peak (dose averaged LET∞ = 189 ± 15 keV/ μm) and in the plateau region of the Bragg curve (LET = 40 keV/ μm) were employed and the radical scavenger concentration in the plasmid solution was varied to quantify the indirect effect. In order to minimize the influence of 12C break-up fragments, a relatively low initial energy of 90 MeV/nucleon was employed for the carbon ions. DNA damage has been quantified by subsequent electrophoresis on agarose gels. We find that strand breaks due to both indirect and direct effects are systematically higher in the plateau region as compared to the Bragg peak region with the difference being smallest at high scavenging capacities. In view of the fact that the relative biological effectiveness for many biological endpoints is maximum at the Bragg peak our findings imply that DNA damage at the Bragg peak is qualitatively most severe.

  8. The Salmonella virulence plasmid enhances Salmonella-induced lysis of macrophages and influences inflammatory responses.

    PubMed Central

    Guilloteau, L A; Wallis, T S; Gautier, A V; MacIntyre, S; Platt, D J; Lax, A J

    1996-01-01

    The Salmonella dublin virulence plasmid mediates systemic infection in mice and cattle. Here, we analyze the interaction between wild-type and plasmid-cured Salmonella strains with phagocytes in vitro and in vivo. The intracellular recovery of S. dublin from murine peritoneal and bovine alveolar macrophages cultured in the presence of gentamicin in vitro was not related to virulence plasmid carriage. However, the virulence plasmid increased the lytic activity of S. dublin, Salmonella typhimurium, and Salmonella choleraesuis for resident or activated mouse peritoneal macrophages. Lysis was not mediated by spv genes and was abolished by cytochalasin D treatment. Peritoneal and splenic macrophages were isolated from mice 4 days after intraperitoneal infection with wild-type or plasmid-cured S. dublin strains. The wild-type strain was recovered in significantly higher numbers than the plasmid-cured strain. However, the intracellular killing rates of such cells cultured in vitro for both S. dublin strains were not significantly different. Four days after infection, there was a lower increase of phagocyte numbers in the peritoneal cavities and spleens of mice infected with the wild-type strain compared with the plasmid-cured strain. The virulence plasmid influenced the survival of macrophages in vitro following infection in vivo as assessed by microscopy. Cells from mice infected with the plasmid-cured strain survived better than those from mice infected with the wild-type strain. This is the first report demonstrating an effect of the virulence plasmid on the interaction of Salmonella strains with macrophages. Plasmid-mediated macrophage dysfunction could influence the recruitment and/or the activation of phagocytic cells and consequently the net growth of Salmonella strains during infection. PMID:8757880

  9. Yields of strand breaks and base lesions induced by soft X-rays in plasmid DNA.

    PubMed

    Yokoya, A; Fujii, K; Ushigome, T; Shikazono, N; Urushibara, A; Watanabe, R

    2006-01-01

    The yields of soft-X-ray-induced DNA damages have been measured by using closed-circular plasmid DNA. Several DNA solutions with three kinds of radical scavenger capacity and also fully hydrated DNA samples were irradiated to compare the contribution by indirect reaction of diffusible water radicals, such as OH*, with those by direct action of secondary electrons. The yields of prompt single- (SSBs) and double-strand breaks (DSBs) decrease with increasing scavenging capacity. The SSB yields for soft X-rays are approximately midway those between gamma-ray and ultrasoft X-ray data previously reported. Heat labile sites are observed only in the low scavenger condition. The yields of the base lesions revealed by post irradiation treatment with base excision repair enzymes showed a similar value for Nth and Fpg protein except in the hydrated sample. These results indicate that the direct effect of soft X-rays induces the damages with different efficiency from those by indirect effect.

  10. Iteron Plasmids.

    PubMed

    Konieczny, Igor; Bury, Katarzyna; Wawrzycka, Aleksandra; Wegrzyn, Katarzyna

    2014-12-01

    Iteron-containing plasmids are model systems for studying the metabolism of extrachromosomal genetic elements in bacterial cells. Here we describe the current knowledge and understanding of the structure of iteron-containing replicons, the structure of the iteron plasmid encoded replication initiation proteins, and the molecular mechanisms for iteron plasmid DNA replication initiation. We also discuss the current understanding of control mechanisms affecting the plasmid copy number and how host chaperone proteins and proteases can affect plasmid maintenance in bacterial cells.

  11. Single- and double-strand breaks induced in plasmid DNA irradiated by ultra-soft X-rays

    NASA Astrophysics Data System (ADS)

    Fayard, B.; Touati, A.; Sage, E.; Abel, F.; Champion, C.; Chetoui, A.

    1999-01-01

    In order to investigate the molecular consequences of a carbon K photo-ionization located on DNA, dry pBS plasmid samples were irradiated with ultra-soft X-rays at energies below and above the carbon K-threshold (E_K=278 eV). Single- and double-strand breaks (ssb and dsb) were quantified after resolution of the three plasmid forms (supercoiled, relaxed circular, linear) by gel electrophoresis. A factor of 1.2 was found between the doses required at 250 eV and 380 eV to induce the same number of dsb per plasmid. Dans le but d'étudier les conséquences à l'échelle moléculaire d'une photo- ionisation en couche K du carbone de l'ADN, des dépots de plasmides ont été irradiés à sec par des X ultra-mous d'énergies situées de part et d'autre du seuil d'ionisation en couche interne du carbone (E_K=278 eV). Les taux de cassures simple- et double-brin (ssb et dsb) ont été quantifiées après résolution des trois formes de plasmide (surenroulé, circulaire relaché, linéaire) par électrophorèse. Un facteur de 1.2 a été mesuré entre les doses nécessaires à 250 eV et 380 eV pour produire le même nombre de dsb par plasmide.

  12. Beneficial effects of intramyocardial mesenchymal stem cells and VEGF165 plasmid injection in rats with furazolidone induced dilated cardiomyopathy.

    PubMed

    Yu, Qin; Fang, Weiyi; Zhu, Ning; Zheng, Xiaoqun; Na, Rongmei; Liu, Baiting; Meng, Lili; Li, Zhu; Li, Qianxiao; Li, Xiaofei

    2015-08-01

    To explore the impact of myocardial injection of mesenchymal stem cells (MSCs) and specific recombinant human VEGF165 (hVEGF165 ) plasmid on collagen remodelling in rats with furazolidone induced dilated cardiomyopathy (DCM). DCM was induced by furazolidone (0.3 mg/bodyweight (g)/day per gavage for 8 weeks). Rats were then divided into four groups: (i) PBS group (n = 18): rats received equal volume myocardial PBS injection; (ii) MSCs group (n = 17): 100 μl culture medium containing 10(5) MSCs were injected into four sites of left ventricular free wall (25 μl per site); (iii) GENE group (n = 18): pCMVen-MLC2v-EGFP-VEGF165 plasmid [5 × 10(9) pfu (0.2 ml)] were injected into four sites of left ventricular free wall (0.05 ml per site)] and (iv) MSCs+GENE group (n = 17): rats received both myocardial MSCs and pCMVen-MLC2v-EGFP-VEGF165 plasmid injections. After 4 weeks, cardiac function was evaluated by echocardiography. Myocardial mRNA expressions of type I, type III collagen and transforming growth factor (TGF)-β1 were detected by RT-PCR. The protein expression of hVEGF165 was determined by Western blot. Myocardial protein expression of hVEGF165 was demonstrated in GENE and MSCs+GENE groups. Cardiac function was improved in MSCs, GENE and MSCs+GENE groups. Collagen volume fraction was significantly reduced and myocardial TGF-β1 mRNA expression significantly down-regulated in both GENE and MSCs+GENE groups, collagen type I/III ratio reduction was more significant in MSCs+GENE group than in MSCs or GENE group. Myocardial MSCs and hVEGF165 plasmid injection improves cardiac function possibly through down-regulating myocardial TGF-β1 expression and reducing the type I/III collagen ratio in this DCM rat model.

  13. Plasmid-Encoded Pgp3 Is a Major Virulence Factor for Chlamydia muridarum To Induce Hydrosalpinx in Mice

    PubMed Central

    Liu, Yuanjun; Huang, Yumeng; Yang, Zhangsheng; Sun, Yina; Gong, Siqi; Hou, Shuping; Chen, Chaoqun; Li, Zhongyu; Liu, Quanzhong; Wu, Yimou; Baseman, Joel

    2014-01-01

    Hydrosalpinx induction in mice by Chlamydia muridarum infection, a model that has been used to study C. trachomatis pathogenesis in women, is known to depend on the cryptic plasmid that encodes eight genes designated pgp1 to pgp8. To identify the plasmid-encoded pathogenic determinants, we evaluated C. muridarum transformants deficient in the plasmid-borne gene pgp3, -4, or -7 for induction of hydrosalpinx. C. muridarum transformants with an in-frame deletion of either pgp3 or -4 but not -7 failed to induce hydrosalpinx. The deletion mutant phenotype was reproduced by using transformants with premature termination codon insertions in the corresponding pgp genes (to minimize polar effects inherent in the deletion mutants). Pgp4 is known to regulate pgp3 expression, while lack of Pgp3 does not significantly affect Pgp4 function. Thus, we conclude that Pgp3 is an effector virulence factor and that lack of Pgp3 may be responsible for the attenuation in C. muridarum pathogenicity described above. This attenuated pathogenicity was further correlated with a rapid decrease in chlamydial survival in the lower genital tract and reduced ascension to the upper genital tract in mice infected with C. muridarum deficient in Pgp3 but not Pgp7. The Pgp3-deficient C. muridarum organisms were also less invasive when delivered directly to the oviduct on day 7 after inoculation. These observations demonstrate that plasmid-encoded Pgp3 is required for C. muridarum survival in the mouse genital tract and represents a major virulence factor in C. muridarum pathogenesis in mice. PMID:25287930

  14. Novel synthetic plasmid and Doggybone™ DNA vaccines induce neutralizing antibodies and provide protection from lethal influenza challenge in mice

    PubMed Central

    Scott, Veronica L; Patel, Ami; Villarreal, Daniel O; Hensley, Scott E; Ragwan, Edwin; Yan, Jian; Sardesai, Niranjan Y; Rothwell, Paul J; Extance, Jonathan P; Caproni, Lisa J; Weiner, David B

    2015-01-01

    Nucleic acid-based vaccines (NAVs) are a promising alternative to conventional influenza vaccines with the potential to increase influenza vaccine availability due to their simplicity in design and rapid speed of production. NAVs can also target multiple influenza antigens and control flu variants. Traditionally NAVs have been DNA plasmids however, we are continuing to explore new methods that may enhance vaccine efficacy. Recently new focus has been on RNA cassettes as NAVs. RNA vaccines combine conceptual advantages in that they focus on delivery of only the coding cassette. However, RNA vaccines have a short half-life and cause interferon-induced fevers. Here we describe a new NAV approach where we study delivery of a linear DNA cassette [Doggybone™ linear closed DNA [(dbDNA™)] produced by an enzymatic process that yields an antigen expression cassette comprising a promoter, DNA antigen, poly A tail, and telomeric ends. This focused approach has many of the advantages of plasmid DNA as well as a minimal cassette size similar to RNA strategies. For this study, we characterized the specific CD4+ and CD8+ T cell responses and determined the hemagglutination inhibition (HI) titers induced by dbDNA™ and compared the responses with those of an optimized plasmid DNA (pDNA) vaccine encoding the same H1N1 influenza A/PR/8/34 HA gene. Immunizations with the constructs resulted in similar humoral and cellular immune responses. Both constructs induced high-titer HI antibodies and fully protected animals from lethal viral challenge. The data obtained from this study provides important validation for further development of novel vector approaches. PMID:26091432

  15. Episomal plasmid-based generation of induced pluripotent stem cells from fetal femur-derived human mesenchymal stromal cells.

    PubMed

    Megges, Matthias; Oreffo, Richard O C; Adjaye, James

    2016-01-01

    Human bone mesenchymal stromal cells derived from fetal femur 55 days post-conception were reprogrammed to induced pluripotent stem cells using episomal plasmid-based expression of OCT4, SOX2, NANOG, LIN28, SV40LT, KLF4 and c-MYC and supplemented with the following pathway inhibitors - TGFβ receptor inhibitor (A-83-01), MEK inhibitor (PD325901), GSK3β inhibitor (CHIR99021) and ROCK inhibitor (HA-100). Successful induction of pluripotency in two iPS-cell lines was demonstrated in vitro and by the Pluritest.

  16. Plasmid Biopharmaceuticals.

    PubMed

    Prazeres, Duarte Miguel F; Monteiro, Gabriel A

    2014-12-01

    Plasmids are currently an indispensable molecular tool in life science research and a central asset for the modern biotechnology industry, supporting its mission to produce pharmaceutical proteins, antibodies, vaccines, industrial enzymes, and molecular diagnostics, to name a few key products. Furthermore, plasmids have gradually stepped up in the past 20 years as useful biopharmaceuticals in the context of gene therapy and DNA vaccination interventions. This review provides a concise coverage of the scientific progress that has been made since the emergence of what are called today plasmid biopharmaceuticals. The most relevant topics are discussed to provide researchers with an updated overview of the field. A brief outline of the initial breakthroughs and innovations is followed by a discussion of the motivation behind the medical uses of plasmids in the context of therapeutic and prophylactic interventions. The molecular characteristics and rationale underlying the design of plasmid vectors as gene transfer agents are described and a description of the most important methods used to deliver plasmid biopharmaceuticals in vivo (gene gun, electroporation, cationic lipids and polymers, and micro- and nanoparticles) is provided. The major safety issues (integration and autoimmunity) surrounding the use of plasmid biopharmaceuticals is discussed next. Aspects related to the large-scale manufacturing are also covered, and reference is made to the plasmid products that have received marketing authorization as of today.

  17. The Agrobacterium Ti Plasmids.

    PubMed

    Christie, Peter J; Gordon, Jay E

    2014-12-01

    Agrobacterium tumefaciens is a plant pathogen with the capacity to deliver a segment of oncogenic DNA carried on a large plasmid called the tumor-inducing or Ti plasmid to susceptible plant cells. A. tumefaciens belongs to the class Alphaproteobacteria, whose members include other plant pathogens (Agrobacterium rhizogenes), plant and insect symbionts (Rhizobium spp. and Wolbachia spp., respectively), human pathogens (Brucella spp., Bartonella spp., Rickettsia spp.), and nonpathogens (Caulobacter crescentus, Rhodobacter sphaeroides). Many species of Alphaproteobacteria carry large plasmids ranging in size from ∼100 kb to nearly 2 Mb. These large replicons typically code for functions essential for cell physiology, pathogenesis, or symbiosis. Most of these elements rely on a conserved gene cassette termed repABC for replication and partitioning, and maintenance at only one or a few copies per cell. The subject of this review is the ∼200-kb Ti plasmids carried by infectious strains of A. tumefaciens. We will summarize the features of this plasmid as a representative of the repABC family of megaplasmids. We will also describe novel features of this plasmid that enable A. tumefaciens cells to incite tumor formation in plants, sense and respond to an array of plant host and bacterial signal molecules, and maintain and disseminate the plasmid among populations of agrobacteria. At the end of this review, we will describe how this natural genetic engineer has been adapted to spawn an entire industry of plant biotechnology and review its potential for use in future therapeutic applications of plant and nonplant species.

  18. Lethality induced by a single site-specific double-strand break in a dispensable yeast plasmid.

    PubMed Central

    Bennett, C B; Lewis, A L; Baldwin, K K; Resnick, M A

    1993-01-01

    Cells of the yeast Saccharomyces cerevisiae are delayed in the G2 phase of the cell cycle following chromosomal DNA damage. This arrest is RAD9-dependent and suggests a signaling mechanism(s) between chromosomal lesions and cell cycling. We examined the global nature of growth inhibition caused by an HO endonuclease-induced double-strand break (DSB) at a 45-bp YZ sequence (from MAT YZ) in a non-yeast region of a dispensable single-copy plasmid. The presence of an unrepaired DSB results in cellular death even though the plasmid is dispensable. Loss of cell viability is partially dependent on the RAD9 gene product. Following induction of the DSB, 40% of RAD+ and 49% of rad9 delta cells [including both unbudded (G1) and budded (S plus G2) cells] did not progress further in the cell cycle. The remaining RAD+ cells progressed to form microcolonies (< 30 cells) containing aberrantly shaped inviable cells. For the rad9 delta mutant, the majority of the remaining cells produced viable colonies accounting for the greater survival of the rad9 delta strain. Based on the profound effects of a single nonchromosomal DNA lesion, this system provides a convenient means for studying the signaling effects of a DNA lesion, as well as for designing strategies for modulating cell proliferation. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8516308

  19. Chemical adjuvants for plasmid DNA vaccines.

    PubMed

    Greenland, John R; Letvin, Norman L

    2007-05-10

    Plasmid DNA vaccines are a promising modality for immunization against a variety of human pathogens. Immunization via multiple routes with plasmid DNA can elicit potent cellular immune responses, and these immunogens can be administered repeatedly without inducing anti-vector immunity. Nonetheless, the immunogenicity of plasmid DNA vaccines has been limited by problems associated with delivery. A number of adjuvants have been designed to improve plasmid DNA immunogenicity, either by directly stimulating the immune system or by enhancing plasmid DNA expression. Chemical adjuvants for enhancing plasmid DNA expression include liposomes, polymers, and microparticles, all of which have shown promise for enhancing the expression and immunogenicity of plasmid DNA vaccines in animal models. Micro- and nanoparticles have not been shown to enhance immune responses to plasmid DNA vaccines. However, formulation of plasmid DNA with some non-particulate polymeric adjuvants has led to a statistically significant enhancement of immune responses. Further development of these technologies will significantly improve the utility of plasmid DNA vaccination.

  20. Transfer of the pheromone-inducible plasmid pCF10 among Enterococcus faecalis microorganisms colonizing the intestine of mini-pigs.

    PubMed

    Licht, Tine Rask; Laugesen, Dorthe; Jensen, Lars Bogø; Jacobsen, Bodil Lund

    2002-01-01

    A new animal model, the streptomycin-treated mini-pig, was developed in order to allow colonization of defined strains of Enterococcus faecalis in numbers sufficient to study plasmid transfer. Transfer of the pheromone-inducible pCF10 plasmid between streptomycin-resistant strains of E. faecalis OG1 was investigated in the model. The plasmid encodes resistance to tetracycline. Numbers of recipient, donor, and transconjugant bacteria were monitored by selective plating of fecal samples, and transconjugants were subsequently verified by PCR. After being ingested by the mini-pigs, the recipient strain persisted in the intestine at levels between 10(6) and 10(7) CFU per g of feces throughout the experiment. The donor strain, which carried different resistance markers but was otherwise chromosomally isogenic to the recipient strain, was given to the pigs 3 weeks after the recipient strain. The donor cells were initially present in high numbers (10(6) CFU per g) in feces, but they did not persist in the intestine at detectable levels. Immediately after introduction of the donor bacteria, transconjugant cells appeared and persisted in fecal samples at levels between 10(3) and 10(4) CFU per g until the end of the experiment. These observations showed that even in the absence of selective tetracycline pressure, plasmid pCF10 was transferred from ingested E. faecalis cells to other E. faecalis organisms already present in the intestinal environment and that the plasmid subsequently persisted in the intestine.

  1. Plasmid DNA immunization with Trypanosoma cruzi genes induces cardiac and clinical protection against Chagas disease in the canine model

    PubMed Central

    2012-01-01

    The only existing preventive measure against American trypanosomosis, or Chagas disease, is the control of the transmitting insect, which has only been effective in a few South American regions. Currently, there is no vaccine available to prevent this disease. Here, we present the clinical and cardiac levels of protection induced by expression to Trypanosoma cruzi genes encoding the TcSP and TcSSP4 proteins in the canine model. Physical examination, diagnostic chagasic serology, and serial electrocardiograms were performed before and after immunization, as well as after experimental infection. We found that immunization with recombinant plasmids prevented hyperthermia in the acute phase of experimental infection and produced lymphadenomegaly as an immunological response against the parasite and additionally prevented heart rate elevation (tachycardia) in the acute and/or chronic stages of infection. Immunization with T. cruzi genes encoding the TcSP and TcSSP4 antigens diminished the quality and quantity of the electrocardiographic abnormalities, thereby avoiding progression to more severe developments such as right bundle branch block or ventricular premature complexes in a greater number of dogs. PMID:23148870

  2. Selective ploidy ablation, a high-throughput plasmid transfer protocol, identifies new genes affecting topoisomerase I–induced DNA damage

    PubMed Central

    Reid, Robert J.D.; González-Barrera, Sergio; Sunjevaric, Ivana; Alvaro, David; Ciccone, Samantha; Wagner, Marisa; Rothstein, Rodney

    2011-01-01

    We have streamlined the process of transferring plasmids into any yeast strain library by developing a novel mating-based, high-throughput method called selective ploidy ablation (SPA). SPA uses a universal plasmid donor strain that contains conditional centromeres on every chromosome. The plasmid-bearing donor is mated to a recipient, followed by removal of all donor-strain chromosomes, producing a haploid strain containing the transferred plasmid. As proof of principle, we used SPA to transfer plasmids containing wild-type and mutant alleles of DNA topoisomerase I (TOP1) into the haploid yeast gene-disruption library. Overexpression of Top1 identified only one sensitive mutation, rpa34, while overexpression of top1-T722A allele, a camptothecin mimetic, identified 190 sensitive gene-disruption strains along with rpa34. In addition to known camptothecin-sensitive strains, this set contained mutations in genes involved in the Rpd3 histone deacetylase complex, the kinetochore, and vesicle trafficking. We further show that mutations in several ESCRT vesicle trafficking components increase Top1 levels, which is dependent on SUMO modification. These findings demonstrate the utility of the SPA technique to introduce plasmids into the haploid gene-disruption library to discover new interacting pathways. PMID:21173034

  3. The gene therapy of collagen-induced arthritis in rats by intramuscular administration of the plasmid encoding TNF-binding domain of variola virus CrmB protein.

    PubMed

    Shchelkunov, S N; Taranov, O S; Tregubchak, T V; Maksyutov, R A; Silkov, A N; Nesterov, A E; Sennikov, S V

    2016-07-01

    Wistar rats with collagen-induced arthritis were intramuscularly injected with the recombinant plasmid pcDNA/sTNF-BD encoding the sequence of the TNF-binding protein domain of variola virus CrmB protein (VARV sTNF-BD) or the pcDNA3.1 vector. Quantitative analysis showed that the histopathological changes in the hind-limb joints of rats were most severe in the animals injected with pcDNA3.1 and much less severe in the group of rats injected with pcDNA/sTNF-BD, which indicates that gene therapy of rheumatoid arthritis is promising in the case of local administration of plasmids governing the synthesis of VARV immunomodulatory proteins.

  4. Plasmids in diatom species.

    PubMed Central

    Hildebrand, M; Corey, D K; Ludwig, J R; Kukel, A; Feng, T Y; Volcani, B E

    1991-01-01

    We have discovered plasmids in 5 of 18 diatom species surveyed. In several species, more than one type of plasmid is present. Several of the plasmids show similarity by hybridization previously characterized plasmids in Cylindrotheca fusiformis (J. D. Jacobs et al., unpublished data). Additionally, there is similarity between the plasmids found in C. fusiformis and chloroplast DNA in three diatom species. These results add to the evidence that the plasmids have features of mobile genetic elements. Images PMID:1885558

  5. Polyamine-induced Z-DNA conformation in plasmids containing (dA-dC)n.(dG-dT)n inserts and increased binding of lupus autoantibodies to the Z-DNA form of plasmids.

    PubMed Central

    Thomas, T J; Thomas, T

    1994-01-01

    Blocks of potential Z-DNA-forming (dA-dC)n.(dG-dT)n sequences are ubiquitous in eukaryotic genomes. We examined whether naturally occurring polyamines, putrescine, spermidine and spermine, could provoke the Z-DNA conformation in plasmids pDHf2 and pDHf14 with 23 and 60 bp inserts respectively of (dA-dC)n.(dG-dT)n sequences using an e.l.i.s.a. Spermidine and spermine could provoke Z-DNA conformation in these plasmids, but putrescine was ineffective. For pDHf2 and pDHf14, the concentration of spermidine at the midpoint of B-DNA to Z-DNA transition was 25 microM, whereas that of spermine was 16 microM. Polyamine structural specificity was evident in the ability of spermidine homologues to induce Z-DNA. Inorganic cations, Co(NH3)6(3+) and Ru(NH3)6(3+), were ineffective. Our experiments also showed increased binding of anti-DNA autoantibodies from lupus patients as well as autoimmune MRL-lpr/lpr mice to pDHf2 and pDHf14 in the presence of polyamines. These data demonstrate that small blocks of (dA-dC)n.(dG-dT)n sequences could assume the Z-DNA conformation in the presence of natural polyamines. Increased concentrations of polyamines in the sera of lupus patients might facilitate immune complex-formation involving circulating DNA and anti-Z-DNA antibodies. PMID:8135759

  6. Inhibition of gamma-radiation induced DNA damage in plasmid pBR322 by TMG, a water-soluble derivative of vitamin E.

    PubMed

    Rajagopalan, Rema; Wani, Khalida; Huilgol, Nagaraj G; Kagiya, Tsutomu V; Nair, Cherupally K Krishnan

    2002-06-01

    Alpha-tocopherol monoglucoside (TMG), a water-soluble derivative of alpha-tocopherol, has been examined for its ability to protect DNA against radiation-induced strand breaks. Gamma radiation, up to a dose of 6 Gy (dose rate, 0.7 Gy/minute), induced a dose-dependent increase in single strand breaks (SSBs) in plasmid pBR322 DNA. TMG inhibited the formation of gamma-radiation induced DNA single strand breaks (SSBs) in a concentration-dependent manner; 500 microM of TMG protected the single strand breaks completely. It also protected thymine glycol formation induced by gamma-radiation in a dose-dependent manner, based on an estimation of thymine glycol by HPLC.

  7. Plasmid DNA manufacturing technology.

    PubMed

    Carnes, Aaron E; Williams, James A

    2007-01-01

    Today, plasmid DNA is becoming increasingly important as the next generation of biotechnology products (gene medicines and DNA vaccines) make their way into clinical trials, and eventually into the pharmaceutical marketplace. This review summarizes recent patents and patent applications relating to plasmid manufacturing, in the context of a comprehensive description of the plasmid manufacturing intellectual property landscape. Strategies for plasmid manufacturers to develop or in-license key plasmid manufacturing technologies are described with the endpoint of efficiently producing kg quantities of plasmid DNA of a quality that meets anticipated European and FDA quality specifications for commercial plasmid products.

  8. Plasmids from Euryarchaeota.

    PubMed

    Forterre, Patrick; Krupovic, Mart; Raymann, Kasie; Soler, Nicolas

    2014-12-01

    Many plasmids have been described in Euryarchaeota, one of the three major archaeal phyla, most of them in salt-loving haloarchaea and hyperthermophilic Thermococcales. These plasmids resemble bacterial plasmids in terms of size (from small plasmids encoding only one gene up to large megaplasmids) and replication mechanisms (rolling circle or theta). Some of them are related to viral genomes and form a more or less continuous sequence space including many integrated elements. Plasmids from Euryarchaeota have been useful for designing efficient genetic tools for these microorganisms. In addition, they have also been used to probe the topological state of plasmids in species with or without DNA gyrase and/or reverse gyrase. Plasmids from Euryarchaeota encode both DNA replication proteins recruited from their hosts and novel families of DNA replication proteins. Euryarchaeota form an interesting playground to test evolutionary hypotheses on the origin and evolution of viruses and plasmids, since a robust phylogeny is available for this phylum. Preliminary studies have shown that for different plasmid families, plasmids share a common gene pool and coevolve with their hosts. They are involved in gene transfer, mostly between plasmids and viruses present in closely related species, but rarely between cells from distantly related archaeal lineages. With few exceptions (e.g., plasmids carrying gas vesicle genes), most archaeal plasmids seem to be cryptic. Interestingly, plasmids and viral genomes have been detected in extracellular membrane vesicles produced by Thermococcales, suggesting that these vesicles could be involved in the transfer of viruses and plasmids between cells.

  9. The mechanism of plasmid curing in bacteria.

    PubMed

    Spengler, Gabriella; Molnár, Annamária; Schelz, Zsuzsanna; Amaral, Leonard; Sharples, Derek; Molnár, Joseph

    2006-07-01

    plasmids. The inhibition of conjugational transfer of antibiotic resistance plasmid can be exploited to reduce the spread of antibiotic resistance plasmid in the ecosystem. Inhibition of plasmid replication at various stages, as shown in the "rolling circle" model (replication, partition, conjugal transfer) may also be the theoretical basis for the elimination of bacterial virulence in the case of plasmid mediated pathogenicity and antibiotic resistance. The large number of compounds tested for antiplasmid effects provides opportunities for QSAR studies in order to find a correlation between the antiplasmid effect and the supramolecular chemistry of these plasmid curing compounds. Plasmid elimination in vitro provides a method of isolating plasmid free bacteria for biotechnology without any risk of inducing mutations.

  10. Non-DSB clustered DNA lesions induced by ionizing radiation are largely responsible for the loss of plasmid DNA functionality in the presence of cisplatin.

    PubMed

    Kouass Sahbani, S; Rezaee, M; Cloutier, P; Sanche, L; Hunting, D J

    2014-06-25

    The combination of cisplatin and ionizing radiation (IR) increases cell toxicity by both enhancing DNA damage and inhibiting repair mechanisms. Although the formation of cluster DNA lesions, particularly double-strand breaks (DSB) at the site of cisplatin-DNA-adducts has been reported to induce cell death, the contribution of DSB and non-DSB cluster lesions to the cellular toxicity is still unknown. Although both lesions are toxic, it is not always possible to measure their frequency and cell survival in the same model system. To overcome this problem, here, we investigate the effect of cisplatin-adducts on the induction of DSB and non-DSB cluster DNA lesions by IR and determine the impact of such lesions on plasmid functionality. Cluster lesions are two or more lesions on opposite DNA strands with a short distance such that error free repair is difficult or impossible. At a ratio of two cisplatin per plasmid, irradiation of platinated DNA in solution with (137)Cs γ-rays shows enhancements in the formation of DNA DSB and non-DSB cluster lesions by factors of 2.6 and 2.1, respectively, compared to unmodified DNA. However, in absolute terms, the yield for non-DSB cluster lesions is far larger than that for DSB, by a factor of 26. Unmodified and cisplatin-modified DNA were irradiated and subsequently transformed into Escherichia coli to give survival curves representing the functionality of the plasmid DNA as a function of radiation dose. Our results demonstrate that non-DSB cluster lesions are the only toxic lesions present at a sufficient frequency to account for the loss of DNA functionality. Our data also show that Frank-DSB lesions are simply too infrequent to account for the loss of DNA functionality. In conclusion, non-DSB cluster DNA damage is known to be difficult to repair and is probably the lesion responsible for the loss of functionality of DNA modified by cisplatin.

  11. N-ICE plasmids for generating N-terminal 3 × FLAG tagged genes that allow inducible, constitutive or endogenous expression in Saccharomyces cerevisiae.

    PubMed

    Zhang, Yueping; Serratore, Nina D; Briggs, Scott D

    2016-12-12

    PCR-mediated homologous recombination is a powerful approach to introduce epitope tags into the chromosomal loci at the N-terminus or the C-terminus of targeted genes. Although strategies of C-terminal epitope tagging of target genes at their loci are simple and widely used in yeast, C-terminal epitope tagging is not practical for all proteins. For example, a C-terminal tag may affect protein function or a protein may get cleaved or processed, resulting in the loss of the epitope tag. Therefore, N-terminal epitope tagging may be necessary to resolve these problems. In some cases, an epitope tagging strategy is used to introduce a heterologous promoter with the epitope tag at the N-terminus of a gene of interest. The potential issue with this strategy is that the tagged gene is not expressed at the endogenous level. Another strategy after integration is to excise the selection marker, using the Cre-LoxP system, leaving the epitope tagged gene expressed from the endogenous promoter. However, N-terminal epitope tagging of essential genes using this strategy requires a diploid strain followed by tetrad dissection. Here we present 14 new plasmids for N-terminal tagging, which combines two previous strategies for epitope tagging in a haploid strain. These 'N-ICE' plasmids were constructed so that non-essential and essential genes can be N-terminally 3 × FLAG tagged and expressed from an inducible promoter (GAL1), constitutive promoters (CYC1 or PYK1) or the endogenous promoter. We have validated the N-ICE plasmid system by N-terminal tagging two non-essential genes (SET1 and SET2) and two essential genes (ERG11 and PKC1). Copyright © 2016 John Wiley & Sons, Ltd.

  12. Plasmid diversity in neisseriae.

    PubMed

    van Passel, Mark W J; van der Ende, Arie; Bart, Aldert

    2006-08-01

    Horizontal gene transfer constitutes an important force in prokaryotic genome evolution, and it is well-known that plasmids are vehicles for DNA transfer. Chromosomal DNA is frequently exchanged between pathogenic and commensal neisseriae, but relatively little is known about plasmid diversity and prevalence among these nasopharyngeal inhabitants. We investigated the plasmid contents of 18 Neisseria lactamica isolates and 20 nasopharyngeal Neisseria meningitidis isolates. Of 18 N. lactamica strains, 9 harbored one or more plasmids, whereas only one N. meningitidis isolate contained a plasmid. Twelve plasmids were completely sequenced, while five plasmid sequences from the public databases were also included in the analyses. On the basis of nucleic acid sequences, mobilization, and replicase protein alignments, we distinguish six different plasmid groups (I to VI). Three plasmids from N. lactamica appeared to be highly similar on the nucleotide level to the meningococcal plasmids pJS-A (>99%) and pJS-B (>75%). The genetic organizations of two plasmids show a striking resemblance with that of the recently identified meningococcal disease-associated (MDA) phage, while four putative proteins encoded by these plasmids show 25% to 39% protein identity to those encoded by the MDA phage. The putative promoter of the gene encoding the replicase on these plasmids contains a polycytidine tract, suggesting that replication is subjected to phase variation. In conclusion, extensive plasmid diversity is encountered among commensal neisseriae. Members of three plasmid groups are found in both pathogenic and commensal neisseriae, indicating plasmid exchange between these species. Resemblance between plasmids and MDA phage may be indicative of dissemination of phage-related sequences among pathogenic and commensal neisseriae.

  13. Immunogenicity and Protective Response Induced by Recombinant Plasmids Based on the BAB1_0267 and BAB1_0270 Open Reading Frames of Brucella abortus 2308 in BALB/c Mice

    PubMed Central

    Gómez, Leonardo A.; Alvarez, Francisco I.; Fernández, Pablo A.; Flores, Manuel R.; Molina, Raúl E.; Coloma, Roberto F.; Oñate, Angel A.

    2016-01-01

    Immunogenicity induced by recombinant plasmids based on the BAB1_0267 and BAB1_0270 open reading frames (ORFs) of Brucella abortus 2308 was evaluated. Bioinformatics analyses indicate that the BAB1_0267 and BAB1_0270 ORFs encode a protein with a SH3 domain and a Zn-dependent metalloproteinase, respectively. Both ORFs have important effects on intracellular survival and replication of B. abortus 2308, mediated via professional and non-professional phagocytic cells. Our results show that immunization with the recombinant plasmid based on the BAB1_0267 ORF significantly increases the production of IgG1, levels of IFN-γ and the lymphoproliferative response of splenocytes. However, BAB1_0267 did not provide significant levels of protection. The plasmid based on the BAB1_0270 significantly increased IgG2a production, levels of IFN-γ and TNF-α, and the lymphoproliferative response of splenocytes. These results demonstrate that immunization with the BAB1_0270 derived recombinant plasmid induce a Th1-type immune response, correlated with a heightened resistance to B. abortus 2308 infection in mice. It is concluded that the Th1-type immune response against bacterial Zn-dependent metalloproteinase induces a protective response in mice, and that pV270 recombinant plasmid is an effective candidate microbicide against brucellosis. PMID:27747197

  14. Designing plasmid vectors.

    PubMed

    Tolmachov, Oleg

    2009-01-01

    Nonviral gene therapy vectors are commonly based on recombinant bacterial plasmids or their derivatives. The plasmids are propagated in bacteria, so, in addition to their therapeutic cargo, they necessarily contain a bacterial replication origin and a selection marker, usually a gene conferring antibiotic resistance. Structural and maintenance plasmid stability in bacteria is required for the plasmid DNA production and can be achieved by carefully choosing a combination of the therapeutic DNA sequences, replication origin, selection marker, and bacterial strain. The use of appropriate promoters, other regulatory elements, and mammalian maintenance devices ensures that the therapeutic gene or genes are adequately expressed in target human cells. Optimal immune response to the plasmid vectors can be modulated via inclusion or exclusion of DNA sequences containing immunostimulatory CpG sequence motifs. DNA fragments facilitating construction of plasmid vectors should also be considered for inclusion in the design of plasmid vectors. Techniques relying on site-specific or homologous recombination are preferred for construction of large plasmids (>15 kb), while digestion of DNA by restriction enzymes with subsequent ligation of the resulting DNA fragments continues to be the mainstream approach for generation of small- and medium-size plasmids. Rapid selection of a desired recombinant plasmid against a background of other plasmids continues to be a challenge. In this chapter, the emphasis is placed on efficient and flexible versions of DNA cloning protocols using selection of recombinant plasmids by restriction endonucleases directly in the ligation mixture.

  15. Broad host range plasmids.

    PubMed

    Jain, Aayushi; Srivastava, Preeti

    2013-11-01

    Plasmids are and will remain important cloning vehicles for biotechnology. They have also been associated with the spread of a number of diseases and therefore are a subject of environmental concern. With the advent of sequencing technologies, the database of plasmids is increasing. It will be of immense importance to identify the various bacterial hosts in which the plasmid can replicate. The present review article describes the features that confer broad host range to the plasmids, the molecular basis of plasmid host range evolution, and applications in recombinant DNA technology and environment.

  16. Genome-Based Genetic Tool Development for Bacillus methanolicus: Theta- and Rolling Circle-Replicating Plasmids for Inducible Gene Expression and Application to Methanol-Based Cadaverine Production

    PubMed Central

    Irla, Marta; Heggeset, Tonje M. B.; Nærdal, Ingemar; Paul, Lidia; Haugen, Tone; Le, Simone B.; Brautaset, Trygve; Wendisch, Volker F.

    2016-01-01

    Bacillus methanolicus is a thermophilic methylotroph able to overproduce amino acids from methanol, a substrate not used for human or animal nutrition. Based on our previous RNA-seq analysis a mannitol inducible promoter and a putative mannitol activator gene mtlR were identified. The mannitol inducible promoter was applied for controlled gene expression using fluorescent reporter proteins and a flow cytometry analysis, and improved by changing the -35 promoter region and by co-expression of the mtlR regulator gene. For independent complementary gene expression control, the heterologous xylose-inducible system from B. megaterium was employed and a two-plasmid gene expression system was developed. Four different replicons for expression vectors were compared with respect to their copy number and stability. As an application example, methanol-based production of cadaverine was shown to be improved from 11.3 to 17.5 g/L when a heterologous lysine decarboxylase gene cadA was expressed from a theta-replicating rather than a rolling-circle replicating vector. The current work on inducible promoter systems and compatible theta- or rolling circle-replicating vectors is an important extension of the poorly developed B. methanolicus genetic toolbox, valuable for genetic engineering and further exploration of this bacterium. PMID:27713731

  17. Genome-Based Genetic Tool Development for Bacillus methanolicus: Theta- and Rolling Circle-Replicating Plasmids for Inducible Gene Expression and Application to Methanol-Based Cadaverine Production.

    PubMed

    Irla, Marta; Heggeset, Tonje M B; Nærdal, Ingemar; Paul, Lidia; Haugen, Tone; Le, Simone B; Brautaset, Trygve; Wendisch, Volker F

    2016-01-01

    Bacillus methanolicus is a thermophilic methylotroph able to overproduce amino acids from methanol, a substrate not used for human or animal nutrition. Based on our previous RNA-seq analysis a mannitol inducible promoter and a putative mannitol activator gene mtlR were identified. The mannitol inducible promoter was applied for controlled gene expression using fluorescent reporter proteins and a flow cytometry analysis, and improved by changing the -35 promoter region and by co-expression of the mtlR regulator gene. For independent complementary gene expression control, the heterologous xylose-inducible system from B. megaterium was employed and a two-plasmid gene expression system was developed. Four different replicons for expression vectors were compared with respect to their copy number and stability. As an application example, methanol-based production of cadaverine was shown to be improved from 11.3 to 17.5 g/L when a heterologous lysine decarboxylase gene cadA was expressed from a theta-replicating rather than a rolling-circle replicating vector. The current work on inducible promoter systems and compatible theta- or rolling circle-replicating vectors is an important extension of the poorly developed B. methanolicus genetic toolbox, valuable for genetic engineering and further exploration of this bacterium.

  18. Degradative plasmids from sphingomonads.

    PubMed

    Stolz, Andreas

    2014-01-01

    Large plasmids ('megaplasmids') are commonly found in members of the Alphaproteobacterial family Sphingomonadaceae ('sphingomonads'). These plasmids contribute to the extraordinary catabolic flexibility of this group of organisms, which degrade a broad range of recalcitrant xenobiotic compounds. The genomes of several sphingomonads have been sequenced during the last years. In the course of these studies, also the sequences of several plasmids have been determined. The analysis of the published information and the sequences deposited in the public databases allowed a first classification of these plasmids into a restricted number of groups according to the proteins involved in the initiation of replication, plasmid partition and conjugation. The sequence comparisons demonstrated that the plasmids from sphingomonads encode for four main groups of replication initiation (Rep) proteins. These Rep proteins belong to the protein superfamilies RepA_C (Pfam 04796), Rep_3 (Pfam 01051), RPA (Pfam 10134) and HTH-36 (Pfam 13730). The 'degradative megaplasmids' pNL2, pCAR3, pSWIT02, pCHQ1, pISP0, and pISP1, which code for genes involved in the degradation of aromatic hydrocarbons, carbazole, dibenzo-p-dioxin and γ-hexachlorocyclohexane, carry Rep proteins which either belong to the RepA_C- (plasmids pNL2, pCAR3, pSWIT02), Rep-3- (plasmids pCHQ1, pISP0) or RPA-superfamily (pISP1). The classification of these 'degradative megaplasmids' into three groups is also supported by sequence comparisons of the proteins involved in plasmid partition (ParAB) and the organization of the three genes on the respective plasmids. All analysed 'degradative megaplasmids' carry genes, which might allow a conjugative transfer of the plasmids. Sequence comparisons of these genes suggest the presence of at least two types of transfer functions, which either are closer related to the tra- or vir-genes previously described for plasmids from other sources.

  19. Choosing and using Schizosaccharomyces pombe plasmids.

    PubMed

    Siam, Rania; Dolan, William P; Forsburg, Susan L

    2004-07-01

    A wide range of plasmids has been developed for molecular studies in the fission yeast Schizosaccharomyces pombe. This includes general purpose episomes, expression vectors, epitope tagging plasmids, and integration vectors. This review describes the typical features of S. pombe vectors, including replication origins, positive and negative selection markers, and constitutive and inducible promoter systems. We will also discuss vectors with epitope tags and how these can be used to modify episomal or endogenous gene sequences. Considerations for choosing and using a plasmid are presented and specialized methods are described.

  20. Plasmid Partition Mechanisms.

    PubMed

    Baxter, Jamie C; Funnell, Barbara E

    2014-12-01

    The stable maintenance of low-copy-number plasmids in bacteria is actively driven by partition mechanisms that are responsible for the positioning of plasmids inside the cell. Partition systems are ubiquitous in the microbial world and are encoded by many bacterial chromosomes as well as plasmids. These systems, although different in sequence and mechanism, typically consist of two proteins and a DNA partition site, or prokaryotic centromere, on the plasmid or chromosome. One protein binds site-specifically to the centromere to form a partition complex, and the other protein uses the energy of nucleotide binding and hydrolysis to transport the plasmid, via interactions with this partition complex inside the cell. For plasmids, this minimal cassette is sufficient to direct proper segregation in bacterial cells. There has been significant progress in the last several years in our understanding of partition mechanisms. Two general areas that have developed are (i) the structural biology of partition proteins and their interactions with DNA and (ii) the action and dynamics of the partition ATPases that drive the process. In addition, systems that use tubulin-like GTPases to partition plasmids have recently been identified. In this chapter, we concentrate on these recent developments and the molecular details of plasmid partition mechanisms.

  1. Yeast DNA plasmids.

    PubMed

    Gunge, N

    1983-01-01

    The study of yeast DNA plasmids has been initiated with the discovery of the 2-micron DNA in Saccharomyces cerevisiae. This multiple copy plasmid, organized into chromatin structure in vivo, probably exists in the nucleus and provides a good system to obtain information on eukaryotic DNA replication. Yeast transformation with the 2-micron DNA or artificially constructed chimeric plasmids had contributed significantly to the study of the molecular biology of yeast and eukaryotes, allowing the isolation and characterization of various genes, ars, centromeres, and telomeres, and also serving as a tool to study the expression of various heterologous genes. Encouraged by these fruitful results, new yeast plasmids have been screened among phylogenetically distant yeasts. The linear DNA plasmids (pGKl1 and pGKl2) from Kluyveromyces lactis are the first case of yeast plasmids associated with biological function (killer phenotype). This plasmid system would be ideal as a model to study the structure and function of eukaryotic linear chromosomes. The extracellular secretion of protein toxin suggests the plasmids to be an excellent candidate for a secretion vector. The importance of yeasts as suitable materials for the study of eukaryotic cell biology would be much enhanced by the advent of new transformation systems with diverse host yeasts of genetically and phylogenetically distinct properties.

  2. Mobility of plasmids.

    PubMed

    Smillie, Chris; Garcillán-Barcia, M Pilar; Francia, M Victoria; Rocha, Eduardo P C; de la Cruz, Fernando

    2010-09-01

    Plasmids are key vectors of horizontal gene transfer and essential genetic engineering tools. They code for genes involved in many aspects of microbial biology, including detoxication, virulence, ecological interactions, and antibiotic resistance. While many studies have decorticated the mechanisms of mobility in model plasmids, the identification and characterization of plasmid mobility from genome data are unexplored. By reviewing the available data and literature, we established a computational protocol to identify and classify conjugation and mobilization genetic modules in 1,730 plasmids. This allowed the accurate classification of proteobacterial conjugative or mobilizable systems in a combination of four mating pair formation and six relaxase families. The available evidence suggests that half of the plasmids are nonmobilizable and that half of the remaining plasmids are conjugative. Some conjugative systems are much more abundant than others and preferably associated with some clades or plasmid sizes. Most very large plasmids are nonmobilizable, with evidence of ongoing domestication into secondary chromosomes. The evolution of conjugation elements shows ancient divergence between mobility systems, with relaxases and type IV coupling proteins (T4CPs) often following separate paths from type IV secretion systems. Phylogenetic patterns of mobility proteins are consistent with the phylogeny of the host prokaryotes, suggesting that plasmid mobility is in general circumscribed within large clades. Our survey suggests the existence of unsuspected new relaxases in archaea and new conjugation systems in cyanobacteria and actinobacteria. Few genes, e.g., T4CPs, relaxases, and VirB4, are at the core of plasmid conjugation, and together with accessory genes, they have evolved into specific systems adapted to specific physiological and ecological contexts.

  3. Prime-boost vaccination with plasmid DNA followed by recombinant vaccinia virus expressing BgGARP induced a partial protective immunity to inhibit Babesia gibsoni proliferation in dogs.

    PubMed

    Cao, Shinuo; Mousa, Ahmed Abdelmoniem; Aboge, Gabriel Oluga; Kamyingkird, Ketsarin; Zhou, Mo; Moumouni, Paul Franck Adjou; Terkawi, Mohamad Alaa; Masatani, Tatsunori; Nishikawa, Yoshifumi; Suzuki, Hiroshi; Fukumoto, Shinya; Xuan, Xuenan

    2013-12-01

    A heterologous prime-boost vaccination regime with DNA and recombinant vaccinia virus (rvv) vectors expressing relevant antigens has been shown to induce effective immune responses against several infectious pathogens. In this study, we describe the effectiveness of the prime-boost strategy by immunizing dogs with a recombinant plasmid followed by vaccinia virus, both of which expressed the glutamic acid-rich protein (BgGARP) of Babesia gibsoni. The dogs immunized with the prime-boost regime developed a significantly high level of specific antibodies against BgGARP when compared with the control groups. The antibody level was strongly increased after a booster immunization with a recombinant vaccinia virus. Two weeks after the booster immunization with a recombinant vaccinia virus expressing BgGARP, the dogs were challenged with B. gibsoni parasite. The dogs immunized with the prime-boost regime showed partial protection, manifested as a significantly low level of parasitemia. These results indicated that this type of DNA/rvv prime-boost immunization approach may have use against B. gibsoni infection in dogs.

  4. Chlamydial plasmids and bacteriophages.

    PubMed

    Pawlikowska-Warych, Małgorzata; Śliwa-Dominiak, Joanna; Deptuła, Wiesław

    2015-01-01

    Chlamydia are absolute pathogens of humans and animals; despite being rather well recognised, they are still open for discovery. One such discovery is the occurrence of extrachromosomal carriers of genetic information. In prokaryotes, such carriers include plasmids and bacteriophages, which are present only among some Chlamydia species. Plasmids were found exclusively in Chlamydia (C.) trachomatis, C. psittaci, C. pneumoniae, C. suis, C. felis, C. muridarum and C. caviae. In prokaryotic organisms, plasmids usually code for genes that facilitate survival of the bacteria in the environment (although they are not essential). In chlamydia, their role has not been definitely recognised, apart from the fact that they participate in the synthesis of glycogen and encode proteins responsible for their virulence. Furthermore, in C. suis it was evidenced that the plasmid is integrated in a genomic island and contains the tetracycline-resistance gene. Bacteriophages specific for chlamydia (chlamydiaphages) were detected only in six species: C. psittaci, C. abortus, C. felis, C. caviae C. pecorum and C. pneumoniae. These chlamydiaphages cause inhibition of the developmental cycle, and delay transformation of reticulate bodies (RBs) into elementary bodies (EBs), thus reducing the possibility of infecting other cells in time. Plasmids and bacteriophages can be used in the diagnostics of chlamydioses; although especially in the case of plasmids, they are already used for detection of chlamydial infections. In addition, bacteriophages could be used as therapeutic agents to replace antibiotics, potentially addressing the problem of increasing antibiotic-resistance among chlamydia.

  5. Inducing humoral and cellular responses to multiple sporozoite and liver-stage malaria antigens using exogenous plasmid DNA.

    PubMed

    Ferraro, B; Talbott, K T; Balakrishnan, A; Cisper, N; Morrow, M P; Hutnick, N A; Myles, D J; Shedlock, D J; Obeng-Adjei, N; Yan, J; Kayatani, A K K; Richie, N; Cabrera, W; Shiver, R; Khan, A S; Brown, A S; Yang, M; Wille-Reece, U; Birkett, A J; Sardesai, N Y; Weiner, D B

    2013-10-01

    A vaccine candidate that elicits humoral and cellular responses to multiple sporozoite and liver-stage antigens may be able to confer protection against Plasmodium falciparum malaria; however, a technology for formulating and delivering such a vaccine has remained elusive. Here, we report the preclinical assessment of an optimized DNA vaccine approach that targets four P. falciparum antigens: circumsporozoite protein (CSP), liver stage antigen 1 (LSA1), thrombospondin-related anonymous protein (TRAP), and cell-traversal protein for ookinetes and sporozoites (CelTOS). Synthetic DNA sequences were designed for each antigen with modifications to improve expression and were delivered using in vivo electroporation (EP). Immunogenicity was evaluated in mice and nonhuman primates (NHPs) and assessed by enzyme-linked immunosorbent assay (ELISA), gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assay, and flow cytometry. In mice, DNA with EP delivery induced antigen-specific IFN-γ production, as measured by ELISpot assay and IgG seroconversion against all antigens. Sustained production of IFN-γ, interleukin-2, and tumor necrosis factor alpha was elicited in both the CD4(+) and CD8(+) T cell compartments. Furthermore, hepatic CD8(+) lymphocytes produced LSA1-specific IFN-γ. The immune responses conferred to mice by this approach translated to the NHP model, which showed cellular responses by ELISpot assay and intracellular cytokine staining. Notably, antigen-specific CD8(+) granzyme B(+) T cells were observed in NHPs. Collectively, the data demonstrate that delivery of gene sequences by DNA/EP encoding malaria parasite antigens is immunogenic in animal models and can harness both the humoral and cellular arms of the immune system.

  6. Plasmid partitioning systems of conjugative plasmids from Clostridium perfringens.

    PubMed

    Adams, Vicki; Watts, Thomas D; Bulach, Dieter M; Lyras, Dena; Rood, Julian I

    2015-07-01

    Many pathogenic strains of Clostridium perfringens carry several highly similar toxin or antibiotic resistance plasmids that have 35 to 40 kb of very closely related syntenous sequences, including regions that carry the genes encoding conjugative transfer, plasmid replication and plasmid maintenance functions. Key questions are how are these closely related plasmids stably maintained in the same cell and what is the basis for plasmid incompatibility in C. perfringens. Comparative analysis of the Rep proteins encoded by these plasmids suggested that this protein was not the basis for plasmid incompatibility since plasmids carried in a single strain often encoded an almost identical Rep protein. These plasmids all carried a similar, but not identical, parMRC plasmid partitioning locus. Phylogenetic analysis of the deduced ParM proteins revealed that these proteins could be divided into ten separate groups. Importantly, in every strain that carried more than one of these plasmids, the respective ParM proteins were from different phylogenetic groups. Similar observations were made from the analysis of phylogenetic trees of the ParR proteins and the parC loci. These findings provide evidence that the basis for plasmid incompatibility in the conjugative toxin and resistance plasmid family from C. perfringens resides in subtle differences in the parMRC plasmid partitioning loci carried by these plasmids.

  7. SMV1 virus-induced CRISPR spacer acquisition from the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2

    PubMed Central

    Erdmann, Susanne; Shah, Shiraz A.; Garrett, Roger A.

    2013-01-01

    Organisms of the crenarchaeal order Sulfolobales carry complex CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune systems. These systems are modular and show extensive structural and functional diversity, especially in their interference complexes. The primary targets are an exceptional range of diverse viruses, many of which propagate stably within cells and follow lytic life cycles without producing cell lysis. These properties are consistent with the difficulty of activating CRISPR spacer uptake in the laboratory, but appear to conflict with the high complexity and diversity of the CRISPR immune systems that are found among the Sulfolobales. In the present article, we re-examine the first successful induction of archaeal spacer acquisition in our laboratory that occurred exclusively for the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2 that was co-infected with the virus SMV1 (Sulfolobus monocaudavirus 1). Although we reaffirm that protospacer selection is essentially a random process with respect to the pMGB1 genome, we identified single spacer sequences specific for each of CRISPR loci C, D and E that, exceptionally, occurred in many sequenced clones. Moreover, the same sequence was reproducibly acquired for a given locus in independent experiments, consistent with it being the first protospacer to be selected. There was also a small protospacer bias (1.6:1) to the antisense strand of protein genes. In addition, new experiments demonstrated that spacer acquisition in the previously inactive CRISPR locus A could be induced on freeze–thawing of the infected cells, suggesting that environmental stress can facilitate activation. Coincidentally with spacer acquisition, a mobile OrfB element was deleted from pMGB1, suggesting that interplay can occur between spacer acquisition and transposition. PMID:24256236

  8. SMV1 virus-induced CRISPR spacer acquisition from the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2.

    PubMed

    Erdmann, Susanne; Shah, Shiraz A; Garrett, Roger A

    2013-12-01

    Organisms of the crenarchaeal order Sulfolobales carry complex CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune systems. These systems are modular and show extensive structural and functional diversity, especially in their interference complexes. The primary targets are an exceptional range of diverse viruses, many of which propagate stably within cells and follow lytic life cycles without producing cell lysis. These properties are consistent with the difficulty of activating CRISPR spacer uptake in the laboratory, but appear to conflict with the high complexity and diversity of the CRISPR immune systems that are found among the Sulfolobales. In the present article, we re-examine the first successful induction of archaeal spacer acquisition in our laboratory that occurred exclusively for the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2 that was co-infected with the virus SMV1 (Sulfolobus monocaudavirus 1). Although we reaffirm that protospacer selection is essentially a random process with respect to the pMGB1 genome, we identified single spacer sequences specific for each of CRISPR loci C, D and E that, exceptionally, occurred in many sequenced clones. Moreover, the same sequence was reproducibly acquired for a given locus in independent experiments, consistent with it being the first protospacer to be selected. There was also a small protospacer bias (1.6:1) to the antisense strand of protein genes. In addition, new experiments demonstrated that spacer acquisition in the previously inactive CRISPR locus A could be induced on freeze-thawing of the infected cells, suggesting that environmental stress can facilitate activation. Coincidentally with spacer acquisition, a mobile OrfB element was deleted from pMGB1, suggesting that interplay can occur between spacer acquisition and transposition.

  9. Coimmunization with an optimized IL15 plasmid adjuvant enhances humoral immunity via stimulating B cells induced by genetically engineered DNA vaccines expressing consensus JEV and WNV E DIII.

    PubMed

    Ramanathan, Mathura P; Kutzler, Michele A; Kuo, Yuan-Chia; Yan, Jian; Liu, Harrison; Shah, Vidhi; Bawa, Amrit; Selling, Bernard; Sardesai, Niranjan Y; Kim, J Joseph; Weiner, David B

    2009-07-09

    The Japanese encephalitis virus (JEV) and West Nile virus (WNV) are responsible for a large proportion of viral encephalitis in humans. Currently, there is no FDA approved specific treatment for either, though there are attempts to develop vaccines against both viruses. In this study, we proposed novel genetically engineered DNA vaccines against these two neurotrophic flaviviruses. The structural domain III (DIII) of E protein from these viruses is reported to carry dominant epitopes that induce neutralizing antibodies. Therefore we created consensus sequence of DIII domain across numerous strains of JEV and WNV. Based on the consensus amino acid sequence, synthetic codon and RNA optimized DIII-expressing DNA vaccine constructs with an efficient leader sequence were synthesized for immunization studies. In addition, we also constructed a genetically engineered IL15 DNA vaccine molecular adjuvant for co-stimulating the immune response against DIII clones. Vaccine constructs were delivered into BALB/C mice intramuscularly followed by electroporation using the CELLECTRA in vivo electroporator. We have observed that the combined delivery of both WNV DIII and IL15-ECRO DNA vaccine constructs resulted in not only the highest level of antibody against DIII, but also enhanced cross reactivity with two other antigens tested. Also, coimmunization with IL15 plasmid further increased the immune response by four- to five-fold. Importantly, we have shown that IL15 coimmunization adjuvanted humoral responses against DIII antigens by elevating the level of antibody secreting B cells. Such a DNA vaccine approach may better help to control potential travel related infectious agents such as JEV.

  10. Microwave effects on plasmid DNA.

    PubMed

    Sagripanti, J L; Swicord, M L; Davis, C C

    1987-05-01

    The exposure of purified plasmid DNA to microwave radiation at nonthermal levels in the frequency range from 2.00 to 8.75 GHz produces single- and double-strand breaks that are detected by agarose gel electrophoresis. Microwave-induced damage to DNA depends on the presence of small amounts of copper. This effect is dependent upon both the microwave power and the duration of the exposure. Cuprous, but not cupric, ions were able to mimic the effects produced by microwaves on DNA.

  11. Microwave effects on plasmid DNA

    SciTech Connect

    Sagripanti, J.L.; Swicord, M.L.; Davis, C.C.

    1987-05-01

    The exposure of purified plasmid DNA to microwave radiation at nonthermal levels in the frequency range from 2.00 to 8.75 GHz produces single- and double-strand breaks that are detected by agarose gel electrophoresis. Microwave-induced damage to DNA depends on the presence of small amounts of copper. This effect is dependent upon both the microwave power and the duration of the exposure. Cuprous, but not cupric, ions were able to mimic the effects produced by microwaves on DNA.

  12. Cationized gelatin hydrogels mixed with plasmid DNA induce stronger and more sustained gene expression than atelocollagen at calvarial bone defects in vivo.

    PubMed

    Komatsu, K; Shibata, T; Shimada, A; Ideno, H; Nakashima, K; Tabata, Y; Nifuji, A

    2016-01-01

    Gene transduction of exogenous factors at local sites in vivo is a promising approach to promote regeneration of tissue defects owing to its simplicity and capacity for expression of a variety of genes. Gene transduction by viral vectors is highly efficient; however, there are safety concerns associated with viruses. As a method for nonviral gene transduction, plasmid DNA delivery is safer and simpler, but requires an efficient carrier substance. Here, we aimed to develop a simple, efficient method for bone regeneration by gene transduction and to identify optimal conditions for plasmid DNA delivery at bone defect sites. We focused on carrier substances and compared the efficiencies of two collagen derivatives, atelocollagen, and gelatin hydrogel, as substrates for plasmid DNA delivery in vivo. To assess the efficiencies of these substrates, we examined exogenous expression of green fluorescence protein (GFP) by fluorescence microscopy, polymerase chain reaction, and immunohistochemistry. GFP expression at the bone defect site was higher when gelatin hydrogel was used as a substrate to deliver plasmids than when atelocollagen was used. Moreover, the gelatin hydrogel was almost completely absorbed at the defect site, whereas some atelocollagen remained. When a plasmid harboring bone morphogenic protein 2 was delivered with the substrate to bony defect sites, more new bone formation was observed in the gelatin group than in the atelocollagen group. These results suggested that the gelatin hydrogel was more efficient than atelocollagen as a substrate for local gene delivery and may be a superior material for induction of bone regeneration.

  13. Plasmids encoding therapeutic agents

    DOEpatents

    Keener, William K.

    2007-08-07

    Plasmids encoding anti-HIV and anti-anthrax therapeutic agents are disclosed. Plasmid pWKK-500 encodes a fusion protein containing DP178 as a targeting moiety, the ricin A chain, an HIV protease cleavable linker, and a truncated ricin B chain. N-terminal extensions of the fusion protein include the maltose binding protein and a Factor Xa protease site. C-terminal extensions include a hydrophobic linker, an L domain motif peptide, a KDEL ER retention signal, another Factor Xa protease site, an out-of-frame buforin II coding sequence, the lacZ.alpha. peptide, and a polyhistidine tag. More than twenty derivatives of plasmid pWKK-500 are described. Plasmids pWKK-700 and pWKK-800 are similar to pWKK-500 wherein the DP178-encoding sequence is substituted by RANTES- and SDF-1-encoding sequences, respectively. Plasmid pWKK-900 is similar to pWKK-500 wherein the HIV protease cleavable linker is substituted by a lethal factor (LF) peptide-cleavable linker.

  14. Sequence and Role in Virulence of the Three Plasmid Complement of the Model Tumor-Inducing Bacterium Pseudomonas savastanoi pv. savastanoi NCPPB 3335

    PubMed Central

    Bardaji, Leire; Pérez-Martínez, Isabel; Rodríguez-Moreno, Luis; Rodríguez-Palenzuela, Pablo; Sundin, George W.; Ramos, Cayo; Murillo, Jesús

    2011-01-01

    Pseudomonas savastanoi pv. savastanoi NCPPB 3335 is a model for the study of the molecular basis of disease production and tumor formation in woody hosts, and its draft genome sequence has been recently obtained. Here we closed the sequence of the plasmid complement of this strain, composed of three circular molecules of 78,357 nt (pPsv48A), 45,220 nt (pPsv48B), and 42,103 nt (pPsv48C), all belonging to the pPT23A-like family of plasmids widely distributed in the P. syringae complex. A total of 152 coding sequences were predicted in the plasmid complement, of which 38 are hypothetical proteins and seven correspond to putative virulence genes. Plasmid pPsv48A contains an incomplete Type IVB secretion system, the type III secretion system (T3SS) effector gene hopAF1, gene ptz, involved in cytokinin biosynthesis, and three copies of a gene highly conserved in plant-associated proteobacteria, which is preceded by a hrp box motif. A complete Type IVA secretion system, a well conserved origin of transfer (oriT), and a homolog of the T3SS effector gene hopAO1 are present in pPsv48B, while pPsv48C contains a gene with significant homology to isopentenyl-diphosphate delta-isomerase, type 1. Several potential mobile elements were found on the three plasmids, including three types of MITE, a derivative of IS801, and a new transposon effector, ISPsy30. Although the replication regions of these three plasmids are phylogenetically closely related, their structure is diverse, suggesting that the plasmid architecture results from an active exchange of sequences. Artificial inoculations of olive plants with mutants cured of plasmids pPsv48A and pPsv48B showed that pPsv48A is necessary for full virulence and for the development of mature xylem vessels within the knots; we were unable to obtain mutants cured of pPsv48C, which contains five putative toxin-antitoxin genes. PMID:22022435

  15. Phytoplasma plasmid DNA extraction.

    PubMed

    Andersen, Mark T; Liefting, Lia W

    2013-01-01

    Phytoplasma plasmids have generally been detected from DNA extracted from plants and insects using methods designed for the purification of total phytoplasma DNA. Methods include extraction from tissues that are high in phytoplasma titre, such as the phloem of plants, with the use of CsCl-bisbenzimide gradients that exploit the low G+C content of phytoplasma DNA. Many of the methods employed for phytoplasma purification have been described elsewhere in this book. Here we describe in detail two methods that are specifically aimed at isolating plasmid DNA.

  16. Plasmid Detection, Characterization, and Ecology.

    PubMed

    Smalla, Kornelia; Jechalke, Sven; Top, Eva M

    2015-02-01

    Plasmids are important vehicles for rapid adaptation of bacterial populations to changing environmental conditions. It is thought that to reduce the cost of plasmid carriage, only a fraction of a local population carries plasmids or is permissive to plasmid uptake. Plasmids provide various accessory traits which might be beneficial under particular conditions. The genetic variation generated by plasmid carriage within populations ensures the robustness toward environmental changes. Plasmid-mediated gene transfer plays an important role not only in the mobilization and dissemination of antibiotic resistance genes but also in the spread of degradative pathways and pathogenicity determinants of pathogens. Here we summarize the state-of-the-art methods to study the occurrence, abundance, and diversity of plasmids in environmental bacteria. Increasingly, cultivation-independent total-community DNA-based methods are being used to characterize and quantify the diversity and abundance of plasmids in relation to various biotic and abiotic factors. An improved understanding of the ecology of plasmids and their hosts is crucial in the development of intervention strategies for antibiotic-resistance-gene spread. We discuss the potentials and limitations of methods used to determine the host range of plasmids, as the ecology of plasmids is tightly linked to their hosts. The recent advances in sequencing technologies provide an enormous potential for plasmid classification, diversity, and evolution studies, but numerous challenges still exist.

  17. Plasmid IL-12 electroporation in melanoma

    PubMed Central

    Cha, Edward; Daud, Adil

    2012-01-01

    Intratumoral gene electroporation uses electric charges to facilitate entry of plasmid DNA into cells in a reproducible and highly efficient manner, especially to accessible sites such as cutaneous and subcutaneous melanomas. Effective for locally treated disease, electroporation of plasmid DNA encoding interleukin-12 can also induce responses in untreated distant disease, suggesting that adaptive immune responses are being elicited that can target melanoma-associated antigens. In vivo electroporation with immunomodulatory cytokine DNA is a promising approach that can trigger systemic anti-tumor immune responses without the systemic toxicity associated with intravenous cytokine delivery and potentially offer complete long-term tumor regression. PMID:23151447

  18. Mechanisms of plasmid segregation: have multicopy plasmids been overlooked?

    PubMed

    Million-Weaver, Samuel; Camps, Manel

    2014-09-01

    Plasmids are self-replicating pieces of DNA typically bearing non-essential genes. Given that plasmids represent a metabolic burden to the host, mechanisms ensuring plasmid transmission to daughter cells are critical for their stable maintenance in the population. Here we review these mechanisms, focusing on two active partition strategies common to low-copy plasmids: par systems type I and type II. Both involve three components: an adaptor protein, a motor protein, and a centromere, which is a sequence area in the plasmid that is recognized by the adaptor protein. The centromere-bound adaptor nucleates polymerization of the motor, leading to filament formation, which can pull plasmids apart (par I) or push them towards opposite poles of the cell (par II). No such active partition mechanisms are known to occur in high copy number plasmids. In this case, vertical transmission is generally considered stochastic, due to the random distribution of plasmids in the cytoplasm. We discuss conceptual and experimental lines of evidence questioning the random distribution model and posit the existence of a mechanism for segregation in high copy number plasmids that moves plasmids to cell poles to facilitate transmission to daughter cells. This mechanism would involve chromosomally-encoded proteins and the plasmid origin of replication. Modulation of this proposed mechanism of segregation could provide new ways to enhance plasmid stability in the context of recombinant gene expression, which is limiting for large-scale protein production and for bioremediation.

  19. Plasmid interference for curing antibiotic resistance plasmids in vivo.

    PubMed

    Kamruzzaman, Muhammad; Shoma, Shereen; Thomas, Christopher M; Partridge, Sally R; Iredell, Jonathan R

    2017-01-01

    Antibiotic resistance increases the likelihood of death from infection by common pathogens such as Escherichia coli and Klebsiella pneumoniae in developed and developing countries alike. Most important modern antibiotic resistance genes spread between such species on self-transmissible (conjugative) plasmids. These plasmids are traditionally grouped on the basis of replicon incompatibility (Inc), which prevents coexistence of related plasmids in the same cell. These plasmids also use post-segregational killing ('addiction') systems, which poison any bacterial cells that lose the addictive plasmid, to guarantee their own survival. This study demonstrates that plasmid incompatibilities and addiction systems can be exploited to achieve the safe and complete eradication of antibiotic resistance from bacteria in vitro and in the mouse gut. Conjugative 'interference plasmids' were constructed by specifically deleting toxin and antibiotic resistance genes from target plasmids. These interference plasmids efficiently cured the corresponding antibiotic resistant target plasmid from different Enterobacteriaceae in vitro and restored antibiotic susceptibility in vivo to all bacterial populations into which plasmid-mediated resistance had spread. This approach might allow eradication of emergent or established populations of resistance plasmids in individuals at risk of severe sepsis, enabling subsequent use of less toxic and/or more effective antibiotics than would otherwise be possible, if sepsis develops. The generalisability of this approach and its potential applications in bioremediation of animal and environmental microbiomes should now be systematically explored.

  20. Chemotherapy of Bacterial Plasmids

    DTIC Science & Technology

    1979-01-29

    multiresistance to chemotherapeutic drugs, mediated drug resistance are the emergence of strains determined by R-plasmids, causes treatment failures of Haemophilus ... influenzae , resistant to ampicillin [8] of hospital infections, foremost in patients with a or chloramphenicol [9] and of Neisseria gonorrhoeae

  1. Physical and genetic analysis of the ColD plasmid.

    PubMed

    Frey, J; Ghersa, P; Palacios, P G; Belet, M

    1986-04-01

    The plasmid ColD-CA23, a high-copy-number plasmid of 5.12 kilobases, encodes colicin D, a protein of approximately 87,000 daltons which inhibits bacterial protein synthesis. Colicin D production is under the control of the Escherichia coli SOS regulatory system and is released to the growth medium via the action of the lysis gene product(s). A detailed map of the ColD plasmid was established for 10 restriction enzymes. Using in vitro insertional omega mutagenesis and in vivo insertional Tn5 mutagenesis, we localized the regions of the plasmid responsible for colicin D activity (cda), for mitomycin C-induced lysis (cdl), and for colicin D immunity (cdi). These genes were all located contiguously on a 2,400-base-pair fragment similar to a large number of other Col plasmids (A, E1, E2, E3, E8, N, and CloDF). The ColD plasmid was mobilizable by conjugative transfer by helper plasmids of the IncFII incompatibility group, but not by plasmids belonging to the groups IncI-alpha or IncP. The location of the mobilization functions was determined by deletion analysis. The plasmid needs a segment of 400 base pairs, which is located between the mob genes and the gene for autolysis, for its replication.

  2. Physical and genetic analysis of the ColD plasmid.

    PubMed Central

    Frey, J; Ghersa, P; Palacios, P G; Belet, M

    1986-01-01

    The plasmid ColD-CA23, a high-copy-number plasmid of 5.12 kilobases, encodes colicin D, a protein of approximately 87,000 daltons which inhibits bacterial protein synthesis. Colicin D production is under the control of the Escherichia coli SOS regulatory system and is released to the growth medium via the action of the lysis gene product(s). A detailed map of the ColD plasmid was established for 10 restriction enzymes. Using in vitro insertional omega mutagenesis and in vivo insertional Tn5 mutagenesis, we localized the regions of the plasmid responsible for colicin D activity (cda), for mitomycin C-induced lysis (cdl), and for colicin D immunity (cdi). These genes were all located contiguously on a 2,400-base-pair fragment similar to a large number of other Col plasmids (A, E1, E2, E3, E8, N, and CloDF). The ColD plasmid was mobilizable by conjugative transfer by helper plasmids of the IncFII incompatibility group, but not by plasmids belonging to the groups IncI-alpha or IncP. The location of the mobilization functions was determined by deletion analysis. The plasmid needs a segment of 400 base pairs, which is located between the mob genes and the gene for autolysis, for its replication. Images PMID:3007432

  3. Electrotransfer of Plasmid Vector DNA into Muscle

    NASA Astrophysics Data System (ADS)

    Miyazaki, Satsuki; Miyazaki, Jun-Ichi

    Wolff et al. (1990) first reported that plasmid DNA injected into skeletal muscle is taken up by muscle cells and the genes in the plasmid are expressed for more than two months thereafter, although the transfected DNA does not usually undergo chromosomal integration (Wolff et al., 1991, 1992). However, the relatively low expression levels attained by this method have hampered its applications for uses other than as a DNA vaccine (Davis et al., 1995). There are a number of reports analyzing the conditions that affect the efficiency of gene transfer by intramuscular DNA injection and assessing the fine structures of expression plasmid vectors that may affect expression levels (Davis et al., 1993; Liang et al., 1996; Norman et al., 1997). Furthermore, various attempts were done to improve the efficiency of gene transfer by intramus cular DNA injection. Consequently, regenerating muscle was shown to produce 80-fold or more protein than did normal muscle, following injection of an expression plas-mid. Muscle regeneration was induced by treatment with cardiotoxin or bupivacaine (Wells, 1993; Vitadello et al., 1994). We previously demonstrated that by combining a strong promoter and bupivacaine pretreatment intramuscular injection of an IL-5 expression plasmid results in IL-5 production in muscle at a level sufficient to induce marked proliferation of eosinophils in the bone marrow and eosinophil infiltration of various organs (Tokui et al., 1997). It was also reported that a single intramuscular injection of an erythropoietin expression plasmid produced physiologically significant elevations in serum erythropoietin levels and increased hematocrits in adult mice (Tripathy et al., 1996). Hematocrits in these animals remained elevated at >60% for at least 90 days after a single injection. However, improvements to this method have not been sufficient to extend its applications including clinical use.

  4. Plasmid interference for curing antibiotic resistance plasmids in vivo

    PubMed Central

    Kamruzzaman, Muhammad; Shoma, Shereen; Thomas, Christopher M.; Partridge, Sally R.

    2017-01-01

    Antibiotic resistance increases the likelihood of death from infection by common pathogens such as Escherichia coli and Klebsiella pneumoniae in developed and developing countries alike. Most important modern antibiotic resistance genes spread between such species on self-transmissible (conjugative) plasmids. These plasmids are traditionally grouped on the basis of replicon incompatibility (Inc), which prevents coexistence of related plasmids in the same cell. These plasmids also use post-segregational killing (‘addiction’) systems, which poison any bacterial cells that lose the addictive plasmid, to guarantee their own survival. This study demonstrates that plasmid incompatibilities and addiction systems can be exploited to achieve the safe and complete eradication of antibiotic resistance from bacteria in vitro and in the mouse gut. Conjugative ‘interference plasmids’ were constructed by specifically deleting toxin and antibiotic resistance genes from target plasmids. These interference plasmids efficiently cured the corresponding antibiotic resistant target plasmid from different Enterobacteriaceae in vitro and restored antibiotic susceptibility in vivo to all bacterial populations into which plasmid-mediated resistance had spread. This approach might allow eradication of emergent or established populations of resistance plasmids in individuals at risk of severe sepsis, enabling subsequent use of less toxic and/or more effective antibiotics than would otherwise be possible, if sepsis develops. The generalisability of this approach and its potential applications in bioremediation of animal and environmental microbiomes should now be systematically explored. PMID:28245276

  5. Cutting edge: retrobulbar inflammation, adipogenesis, and acute orbital congestion in a preclinical female mouse model of Graves' orbitopathy induced by thyrotropin receptor plasmid-in vivo electroporation.

    PubMed

    Moshkelgosha, Sajad; So, Po-Wah; Deasy, Neil; Diaz-Cano, Salvador; Banga, J Paul

    2013-09-01

    Graves' orbitopathy (GO) is a complication in Graves' disease (GD) but mechanistic insights into pathogenesis remain unresolved, hampered by lack of animal model. The TSH receptor (TSHR) and perhaps IGF-1 receptor (IGF-1R) are considered relevant antigens. We show that genetic immunization of human TSHR (hTSHR) A-subunit plasmid leads to extensive remodeling of orbital tissue, recapitulating GO. Female BALB/c mice immunized with hTSHR A-subunit or control plasmids by in vivo muscle electroporation were evaluated for orbital remodeling by histopathology and magnetic resonance imaging (MRI). Antibodies to TSHR and IGF-1R were present in animals challenged with hTSHR A-subunit plasmid, with predominantly TSH blocking antibodies and were profoundly hypothyroid. Orbital pathology was characterized by interstitial inflammation of extraocular muscles with CD3+ T cells, F4/80+ macrophages, and mast cells, accompanied by glycosaminoglycan deposition with resultant separation of individual muscle fibers. Some animals showed heterogeneity in orbital pathology with 1) large infiltrate surrounding the optic nerve or 2) extensive adipogenesis with expansion of retrobulbar adipose tissue. A striking finding that underpins the new model were the in vivo MRI scans of mouse orbital region that provided clear and quantifiable evidence of orbital muscle hypertrophy with protrusion (proptosis) of the eye. Additionally, eyelid manifestations of chemosis, including dilated and congested orbital blood vessels, were visually apparent. Immunization with control plasmids failed to show any orbital pathology. Overall, these findings support TSHR as the pathogenic antigen in GO. Development of a new preclinical model will facilitate molecular investigations on GO and evaluation of new therapeutic interventions.

  6. Differential salt-induced dissociation of the p53 protein complexes with circular and linear plasmid DNA substrates suggest involvement of a sliding mechanism.

    PubMed

    Šebest, Peter; Brázdová, Marie; Fojta, Miroslav; Pivoňková, Hana

    2015-01-30

    A study of the effects of salt conditions on the association and dissociation of wild type p53 with different ~3 kbp long plasmid DNA substrates (supercoiled, relaxed circular and linear, containing or lacking a specific p53 binding site, p53CON) using immunoprecipitation at magnetic beads is presented. Salt concentrations above 200 mM strongly affected association of the p53 protein to any plasmid DNA substrate. Strikingly different behavior was observed when dissociation of pre-formed p53-DNA complexes in increased salt concentrations was studied. While contribution from the p53CON to the stability of the p53-DNA complexes was detected between 100 and 170 mM KCl, p53 complexes with circular DNAs (but not linear) exhibited considerable resistance towards salt treatment for KCl concentrations as high as 2 M provided that the p53 basic C-terminal DNA binding site (CTDBS) was available for DNA binding. On the contrary, when the CTDBS was blocked by antibody used for immunoprecipitation, all p53-DNA complexes were completely dissociated from the p53 protein in KCl concentrations≥200 mM under the same conditions. These observations suggest: (a) different ways for association and dissociation of the p53-DNA complexes in the presence of the CTDBS; and (b) a critical role for a sliding mechanism, mediated by the C-terminal domain, in the dissociation process.

  7. A Recombinant DNA Plasmid Encoding the sIL-4R-NAP Fusion Protein Suppress Airway Inflammation in an OVA-Induced Mouse Model of Asthma.

    PubMed

    Liu, Xin; Fu, Guo; Ji, Zhenyu; Huang, Xiabing; Ding, Cong; Jiang, Hui; Wang, Xiaolong; Du, Mingxuan; Wang, Ting; Kang, Qiaozhen

    2016-08-01

    Asthma is a chronic inflammatory airway disease. It was prevalently perceived that Th2 cells played the crucial role in asthma pathogenesis, which has been identified as the important target for anti-asthma therapy. The soluble IL-4 receptor (sIL-4R), which is the decoy receptor for Th2 cytokine IL-4, has been reported to be effective in treating asthma in phase I/II clinical trail. To develop more efficacious anti-asthma agent, we attempt to test whether the Helicobacter pylori neutrophil-activating protein (HP-NAP), a novel TLR2 agonist, would enhance the efficacy of sIL-4R in anti-asthma therapy. In our work, we constructed a pcDNA3.1-sIL-4R-NAP plasmid, named PSN, encoding fusion protein of murine sIL-4R and HP-NAP. PSN significantly inhibited airway inflammation, decreased the serum OVA-specific IgE levels and remodeled the Th1/Th2 balance. Notably, PSN is more effective on anti-asthma therapy comparing with plasmid only expressing sIL-4R.

  8. Toxin Plasmids of Clostridium perfringens

    PubMed Central

    Li, Jihong; Adams, Vicki; Bannam, Trudi L.; Miyamoto, Kazuaki; Garcia, Jorge P.; Uzal, Francisco A.; Rood, Julian I.

    2013-01-01

    SUMMARY In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch's postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract. PMID:23699255

  9. Conjugative Plasmids of Neisseria gonorrhoeae

    PubMed Central

    Pachulec, Emilia; van der Does, Chris

    2010-01-01

    Many clinical isolates of the human pathogen Neisseria gonorrhoeae contain conjugative plasmids. The host range of these plasmids is limited to Neisseria species, but presence of a tetracycline (tetM) determinant inserted in several of these plasmids is an important cause of the rapid spread of tetracycline resistance. Previously plasmids with different backbones (Dutch and American type backbones) and with and without different tetM determinants (Dutch and American type tetM determinants) have been identified. Within the isolates tested, all plasmids with American or Dutch type tetM determinants contained a Dutch type plasmid backbone. This demonstrated that tetM determinants should not be used to differentiate between conjugal plasmid backbones. The nucleotide sequences of conjugative plasmids with Dutch type plasmid backbones either not containing the tetM determinant (pEP5233) or containing Dutch (pEP5289) or American (pEP5050) type tetM determinants were determined. Analysis of the backbone sequences showed that they belong to a novel IncP1 subfamily divergent from the IncP1α, β, γ, δ and ε subfamilies. The tetM determinants were inserted in a genetic load region found in all these plasmids. Insertion was accompanied by the insertion of a gene with an unknown function, and rearrangement of a toxin/antitoxin gene cluster. The genetic load region contains two toxin/antitoxins of the Zeta/Epsilon toxin/antitoxin family previously only found in Gram positive organisms and the virulence associated protein D of the VapD/VapX toxin/antitoxin family. Remarkably, presence of VapX of pJD1, a small cryptic neisserial plasmid, in the acceptor strain strongly increased the conjugation efficiency, suggesting that it functions as an antitoxin for the conjugative plasmid. The presence of the toxin and antitoxin on different plasmids might explain why the host range of this IncP1 plasmid is limited to Neisseria species. The isolated plasmids conjugated efficiently between

  10. Modulation of in utero total body irradiation induced newborn mouse growth retardation by maternal manganese superoxide dismutase-plasmid liposome (MnSOD-PL) gene therapy.

    PubMed

    Epperly, M W; Smith, T; Zhang, X; Goff, J P; Franicola, D; Greenberger, B; Komanduri, P; Wang, H; Greenberger, J S

    2011-06-01

    To determine the effects of manganese superoxide dismutase (MnSOD) plasmid liposome (PL) maternal radioprotection on fetal mice, timed pregnant female mice (E14 gestation) were irradiated to 3.0 Gy total body irradiation (TBI) dose, and the number, weight and growth and development over 6 months after birth of newborn mice was quantitated compared with irradiated controls. Maternal MnSOD-PL treatment at E13 improved pup survival at birth (5.4±0.9 per litter) compared with non-irradiated 3.0 Gy controls 4.9±1.1. There was no statistically significant difference in newborn abnormalities, male to female ratio in newborn litters, or other evidence of teratogenesis in surviving newborn mice from MnSOD-PL treated compared with irradiated controls. However, E14 3 Gy irradiated pups from gene therapy-treated mothers showed a significant increase in both growth and overall survival over 6 months after birth (P=0.0022). To determine if transgene product crossed the placenta pregnant E13 mice were injected intravenously with hemagglutinin-epitope-tagged MnSOD (100 μg plasmid in 100 μl liposomes), then after 24 h, fetal mice, placentas and maternal tissues were removed and tested by both immunohistochemistry and reverse transcriptase-PCR for transgene and product. There was no evidence of transgene or product in placenta or any fetal tissue while maternal liver was positive by both assays. The data provide evidence for fetal radioprotection by maternal MnSOD-PL gene therapy before irradiation, which is mediated by an indirect bystander effect and is associated with a significant improvement in both survival at birth and growth and development of newborn mice.

  11. Plasmid Rolling-Circle Replication.

    PubMed

    Ruiz-Masó, J A; MachóN, C; Bordanaba-Ruiseco, L; Espinosa, M; Coll, M; Del Solar, G

    2015-02-01

    Plasmids are DNA entities that undergo controlled replication independent of the chromosomal DNA, a crucial step that guarantees the prevalence of the plasmid in its host. DNA replication has to cope with the incapacity of the DNA polymerases to start de novo DNA synthesis, and different replication mechanisms offer diverse solutions to this problem. Rolling-circle replication (RCR) is a mechanism adopted by certain plasmids, among other genetic elements, that represents one of the simplest initiation strategies, that is, the nicking by a replication initiator protein on one parental strand to generate the primer for leading-strand initiation and a single priming site for lagging-strand synthesis. All RCR plasmid genomes consist of a number of basic elements: leading strand initiation and control, lagging strand origin, phenotypic determinants, and mobilization, generally in that order of frequency. RCR has been mainly characterized in Gram-positive bacterial plasmids, although it has also been described in Gram-negative bacterial or archaeal plasmids. Here we aim to provide an overview of the RCR plasmids' lifestyle, with emphasis on their characteristic traits, promiscuity, stability, utility as vectors, etc. While RCR is one of the best-characterized plasmid replication mechanisms, there are still many questions left unanswered, which will be pointed out along the way in this review.

  12. Mechanisms of Theta Plasmid Replication.

    PubMed

    Lilly, Joshua; Camps, Manel

    2015-02-01

    Plasmids are autonomously replicating pieces of DNA. This article discusses theta plasmid replication, which is a class of circular plasmid replication that includes ColE1-like origins of replication popular with expression vectors. All modalities of theta plasmid replication initiate synthesis with the leading strand at a predetermined site and complete replication through recruitment of the host's replisome, which extends the leading strand continuously while synthesizing the lagging strand discontinuously. There are clear differences between different modalities of theta plasmid replication in mechanisms of DNA duplex melting and in priming of leading- and lagging-strand synthesis. In some replicons duplex melting depends on transcription, while other replicons rely on plasmid-encoded trans-acting proteins (Reps); primers for leading-strand synthesis can be generated through processing of a transcript or in other replicons by the action of host- or plasmid-encoded primases. None of these processes require DNA breaks. The frequency of replication initiation is tightly regulated to facilitate establishment in permissive hosts and to achieve a steady state. The last section of the article reviews how plasmid copy number is sensed and how this feedback modulates the frequency of replication.

  13. Phenotypic plasticity in bacterial plasmids.

    PubMed Central

    Turner, Paul E

    2004-01-01

    Plasmid pB15 was previously shown to evolve increased horizontal (infectious) transfer at the expense of reduced vertical (intergenerational) transfer and vice versa, a key trade-off assumed in theories of parasite virulence. Whereas the models predict that susceptible host abundance should determine which mode of transfer is selectively favored, host density failed to mediate the trade-off in pB15. One possibility is that the plasmid's transfer deviates from the assumption that horizontal spread (conjugation) occurs in direct proportion to cell density. I tested this hypothesis using Escherichia coli/pB15 associations in laboratory serial culture. Contrary to most models of plasmid transfer kinetics, my data show that pB15 invades static (nonshaking) bacterial cultures only at intermediate densities. The results can be explained by phenotypic plasticity in traits governing plasmid transfer. As cells become more numerous, the plasmid's conjugative transfer unexpectedly declines, while the trade-off between transmission routes causes vertical transfer to increase. Thus, at intermediate densities the plasmid's horizontal transfer can offset selection against plasmid-bearing cells, but at high densities pB15 conjugates so poorly that it cannot invade. I discuss adaptive vs. nonadaptive causes for the phenotypic plasticity, as well as potential mechanisms that may lead to complex transfer dynamics of plasmids in liquid environments. PMID:15166133

  14. Plasmid acquisition in microgravity

    NASA Technical Reports Server (NTRS)

    Juergensmeyer, Margaret A.; Juergensmeyer, Elizabeth A.; Guikema, James A.

    1995-01-01

    In microgravity, bacteria often show an increased resistance to antibiotics. Bacteria can develop resistance to an antibiotic after transformation, the acquisition of DNA, usually in the form of a plasmid containing a gene for resistance to one or more antibiotics. In order to study the capacity of bacteria to become resistant to antibiotics in microgravity, we have modified the standard protocol for transformation of Escherichia coli for use in the NASA-flight-certified hardware package, The Fluid Processing Apparatus (FPA). Here we report on the ability of E. coli to remain competent for long periods of time at temperatures that are readily available on the Space Shuttle, and present some preliminary flight results.

  15. Microneedle-mediated transcutaneous immunization with plasmid DNA coated on cationic PLGA nanoparticles

    PubMed Central

    Kumar, Amit; Wonganan, Piyanuch; Sandoval, Michael A.; Li, Xinran; Zhu, Saijie; Cui, Zhengrong

    2012-01-01

    Previously, it was shown that microneedle-mediated transcutaneous immunization with plasmid DNA can potentially induce a stronger immune response than intramuscular injection of the same plasmid DNA. In the present study, we showed that the immune responses induced by transcutaneous immunization by applying plasmid DNA onto a skin area pretreated with solid microneedles were significantly enhanced by coating the plasmid DNA on the surface of cationic nanoparticles. In addition, the net surface charge of the DNA-coated nanoparticles significantly affected their in vitro skin permeation and their ability to induce immune responses in vivo. Transcutaneous immunization with plasmid DNA-coated net positively charged anoparticles elicited a stronger immune response than with plasmid DNA-coated net negatively charged nanoparticles or by intramuscular immunization with plasmid DNA alone. Transcutaneous immunization with plasmid DNA-coated net positively charged nanoparticles induced comparable immune responses as intramuscular injection of them, but transcutaneous immunization was able to induce specific mucosal immunity and a more balanced T helper type 1 and type 2 response. The ability of the net positively charged DNA-coated nanoparticles to induce a strong immune response through microneedle-mediated transcutaneous immunization may be attributed to their ability to increase the expression of the antigen gene encoded by the plasmid and to more effectively stimulate the maturation of antigen-presenting cells. PMID:22921518

  16. High-cell Density Shake-flask Expression and Rapid Purification of the Large Fragment of Thermus aquaticus DNA Polymerase I Using a New Chemically and Temperature Inducible Expression Plasmid in Escherichia coli*

    PubMed Central

    Brandis, John W.; Johnson, Kenneth A.

    2008-01-01

    We have developed a new expression vector, pcIts ind+, based upon the powerful rightward promoter of bacteriophage lambda, which is controlled by a temperature-sensitive and chemically-inducible version of the lambda repressor on the same plasmid. Locating the repressor gene on the plasmid makes this vector “portable” in that it can be used to transform any strain of E. coli. Hence, control over strains, induction conditions, and harvest times can be used to optimize yields of heterologous proteins. To provide a proof of concept, we show that Escherichia coli recA+ and recA− host cells transformed with pcIts ind+ modKlenTaq1 (a modified version of the large fragment of Thermus aquaticus DNA polymerase I) could be grown to high cell densities in multiple shake-flasks. A mutant version of modKlenTaq1 (V649C) could be induced by simply raising the thermostat setting from 30 to 37 °C and (in the case of recA+ cells) adding nalidixic acid to achieve full induction (12 to 13% of the total cellular protein). Using a rapid, two-step purification process, it was possible to purify nearly 300 mg of modKlenTaq1 V649C from six 2.8-liter baffle-bottomed shake-flasks each holding 1.5 liters of culture for a final yield of approximately 33 mg per liter or 3 mg of purified enzyme per gram of cells wet weight. PMID:18952180

  17. 5-Aminolevulinic acid induces single-strand breaks in plasmid pBR322 DNA in the presence of Fe2+ ions.

    PubMed

    Onuki, J; Medeiros, M H; Bechara, E J; Di Mascio, P

    1994-02-22

    5-Aminolevulinic acid (ALA), a heme precursor accumulated in chemical and inborn porphyrias, has been demonstrated to produce reactive oxygen species upon metal-catalyzed aerobic oxidation and to cause oxidative damage to proteins, liposomes and subcellular structures. Exposure of plasmid pBR322 DNA to ALA (0.01-3 mM) in the presence of 10 microM Fe2+ ions causes DNA single-strand breaks (ssb), revealed by agarose gel electrophoresis as an increase in the proportion of the open circular form (75 +/- 7.5% at 3 mM ALA) at the expense of the supercoiled form. Addition of either anti-oxidant enzymes such as superoxide dismutase (10 micrograms/ml) and catalase (20 micrograms/ml), or a metal chelator (DTPA, 2.5 mM), or a HO. scavenger (mannitol, 100 mM) inhibited the damage (by 30, 45, 55, and 81%, respectively), evidencing the involvement of O2-., H2O2 and HO. (by the Haber-Weiss reaction) in this process. Hydrogen peroxide (100 microM) or Fe2+ (10 microM) alone were of little effect on the extent of DNA ssb. The present data may shed light on the correlation reported between primary liver-cell carcinoma and intermittent acute porphyria.

  18. Plasmid-Chromosome Recombination of Irradiated Shuttle Vector DNA in African Green Monkey Kidney Cells.

    NASA Astrophysics Data System (ADS)

    Mudgett, John Stuart

    1987-09-01

    An autonomously replicating shuttle vector was used to investigate the enhancement of plasmid-chromosome recombination in mammalian host cells by ultraviolet light and gamma radiation. Sequences homologous to the shuttle vector were stably inserted into the genome of African Green Monkey kidney cells to act as the target substrate for these recombination events. The SV40- and pBR322-derived plasmid DNA was irradiated with various doses of radiation before transfection into the transformed mammalian host cells. The successful homologous transfer of the bacterial ampicillin resistance (amp^{rm r}) gene from the inserted sequences to replace a mutant amp^->=ne on the shuttle vector was identified by plasmid extraction and transformation into E. coli host cells. Ultraviolet light (UV) was found not to induce homologous plasmid-chromosome recombination, while gamma radiation increased the frequency of recombinant plasmids detected. The introduction of specific double -strand breaks in the plasmid or prolonging the time of plasmid residence in the mammalian host cells also enhanced plasmid-chromosome recombination. In contrast, plasmid mutagenesis was found to be increased by plasmid UV irradiation, but not to change with time. Plasmid survival, recombination, and mutagenesis were not affected by treating the mammalian host cells with UV light prior to plasmid transfection. The amp^{rm r} recombinant plasmid molecules analyzed were found to be mostly the result of nonconservative exchanges which appeared to involve both homologous and possibly nonhomologous interactions with the host chromosome. The observation that these recombinant structures were obtained from all of the plasmid alterations investigated suggests a common mechanistic origin for plasmid -chromosome recombination in these mammalian cells.

  19. Methylglyoxal induces G:C to C:G and G:C to T:A transversions in the supF gene on a shuttle vector plasmid replicated in mammalian cells.

    PubMed

    Murata-Kamiya, N; Kamiya, H; Kaji, H; Kasai, H

    2000-07-10

    We previously reported that the majority of base-pair substitutions induced by an endogenous mutagen, methylglyoxal, were G:C-->T:A transversions and G:C-->A:T transitions in wild-type and nucleotide excision repair (NER)-deficient (uvrA or uvrC) Escherichia coli strains. To investigate the mutation spectrum of methylglyoxal in mammalian cells and to compare the spectrum with those detected in other experimental systems, we analyzed mutations in a bacterial suppressor tRNA (supF) gene in the shuttle vector plasmid pMY189. We treated pMY189 with methylglyoxal and immediately transfected it into simian COS-7 cells. The cytotoxicity and the mutation frequency (MF) increased according to the dose of methylglyoxal. In the mutants induced by methylglyoxal, multi-base deletions were predominant (50%), followed by base-pair substitutions (35%), in which 89% of the substitutions occurred at G:C sites. Among them, G:C-->C:G and G:C-->T:A transversions were predominant. The overall distribution of methylglyoxal-induced mutations detected in the supF gene was different from that for the spontaneous mutations. These results suggest that methylglyoxal may take part in causing G:C-->C:G and G:C-->T:A transversions in vivo.

  20. Coelectrotransfer to skeletal muscle of three plasmids coding for antiangiogenic factors and regulatory factors of the tetracycline-inducible system: tightly regulated expression, inhibition of transplanted tumor growth, and antimetastatic effect.

    PubMed

    Martel-Renoir, Dominique; Trochon-Joseph, Véronique; Galaup, Ariane; Bouquet, Céline; Griscelli, Franck; Opolon, Paule; Opolon, David; Connault, Elisabeth; Mir, Lluis; Perricaudet, Michel

    2003-09-01

    We describe an approach employing intramuscular plasmid electrotransfer to deliver secretable forms of K1-5 and K1-3-HSA (a fusion of K1-3 with human serum albumin), which span, respectively, five and three of the five kringle domains of plasminogen. A tetracycline-inducible system (Tet-On) composed of three plasmids coding, respectively, for the transgene, the tetracycline transcriptional activator rtTA, and the silencer tTS was employed. K1-3-HSA and K1-5, produced from C2C12 muscle cells, were found to inhibit endothelial cell (HMEC-1) proliferation by 30 and 51%, respectively. In vivo, the expression of the transgene upon doxycycline stimulation was rapid, stable, and tightly regulated (no background expression) and could be maintained for at least 3 months. Blood half-lives of 2.1 and 3.7 days were found for K1-5 and K1-3-HSA, respectively. The K1-5 protein was secreted from muscle into blood at a level of 45 ng/ml, which was sufficient to inhibit MDA-MB-231 tumor growth by 81% in nude mice and B16-F10 melanoma cell lung invasion in C57BL/6 mice by 73%. PECAM-1 immunostaining studies revealed modest tumor vasculature in mice expressing K1-5. In contrast, K1-3-HSA, although secreted into blood at much higher level (250 ng/ml) than K1-5, had no effect on tumor growth.

  1. Plasmid-mediated quinolone resistance.

    PubMed

    Jacoby, George A; Strahilevitz, Jacob; Hooper, David C

    2014-10-01

    Three mechanisms for plasmid-mediated quinolone resistance (PMQR) have been discovered since 1998. Plasmid genes qnrA, qnrB, qnrC, qnrD, qnrS, and qnrVC code for proteins of the pentapeptide repeat family that protects DNA gyrase and topoisomerase IV from quinolone inhibition. The qnr genes appear to have been acquired from chromosomal genes in aquatic bacteria, are usually associated with mobilizing or transposable elements on plasmids, and are often incorporated into sul1-type integrons. The second plasmid-mediated mechanism involves acetylation of quinolones with an appropriate amino nitrogen target by a variant of the common aminoglycoside acetyltransferase AAC(6')-Ib. The third mechanism is enhanced efflux produced by plasmid genes for pumps QepAB and OqxAB. PMQR has been found in clinical and environmental isolates around the world and appears to be spreading. The plasmid-mediated mechanisms provide only low-level resistance that by itself does not exceed the clinical breakpoint for susceptibility but nonetheless facilitates selection of higher-level resistance and makes infection by pathogens containing PMQR harder to treat.

  2. Plasmid-mediated quinolone resistance

    PubMed Central

    Jacoby, George A.; Strahilevitz, Jacob; Hooper, David C.

    2014-01-01

    Summary Three mechanisms for plasmid-mediated quinolone resistance (PMQR) have been discovered since 1998. Plasmid genes qnrA, qnrB, qnrC, qnrD, qnrS, and qnrVC code for proteins of the pentapeptide repeat family that protects DNA gyrase and topoisomerase IV from quinolone inhibition. The qnr genes appear to have been acquired from chromosomal genes in aquatic bacteria, are usually associated with mobilizing or transposable elements on plasmids, and are often incorporated into sul1-type integrons. The second plasmid-mediated mechanism involves acetylation of quinolones with an appropriate amino nitrogen target by a variant of the common aminoglycoside acetyltransferase AAC(6′)-Ib. The third mechanism is enhanced efflux produced by plasmid genes for pumps QepAB and OqxAB. PMQR has been found in clinical and environmental isolates around the world and appears to be spreading. The plasmid-mediated mechanisms provide only low-level resistance that by itself does not exceed the clinical breakpoint for susceptibility but nonetheless facilitates selection of higher-level resistance and makes infection by pathogens containing PMQR harder to treat. PMID:25584197

  3. Direct and Auger Electron-Induced, Single- and Double-Strand Breaks on Plasmid DNA Caused by 99mTc-Labeled Pyrene Derivatives and the Effect of Bonding Distance.

    PubMed

    Reissig, Falco; Mamat, Constantin; Steinbach, Joerg; Pietzsch, Hans-Juergen; Freudenberg, Robert; Navarro-Retamal, Carlos; Caballero, Julio; Kotzerke, Joerg; Wunderlich, Gerd

    2016-01-01

    It is evident that 99mTc causes radical-mediated DNA damage due to Auger electrons, which were emitted simultaneously with the known γ-emission of 99mTc. We have synthesized a series of new 99mTc-labeled pyrene derivatives with varied distances between the pyrene moiety and the radionuclide. The pyrene motif is a common DNA intercalator and allowed us to test the influence of the radionuclide distance on damages of the DNA helix. In general, pUC 19 plasmid DNA enables the investigation of the unprotected interactions between the radiotracers and DNA that results in single-strand breaks (SSB) or double-strand breaks (DSB). The resulting DNA fragments were separated by gel electrophoresis and quantified by fluorescent staining. Direct DNA damage and radical-induced indirect DNA damage by radiolysis products of water were evaluated in the presence or absence of the radical scavenger DMSO. We demonstrated that Auger electrons directly induced both SSB and DSB in high efficiency when 99mTc was tightly bound to the plasmid DNA and this damage could not be completely prevented by DMSO, a free radical scavenger. For the first time, we were able to minimize this effect by increasing the carbon chain lengths between the pyrene moiety and the 99mTc nuclide. However, a critical distance between the 99mTc atom and the DNA helix could not be determined due to the significantly lowered DSB generation resulting from the interaction which is dependent on the type of the 99mTc binding motif. The effect of variable DNA damage caused by the different chain length between the pyrene residue and the Tc-core as well as the possible conformations of the applied Tc-complexes was supplemented with molecular dynamics (MD) calculations. The effectiveness of the DNA-binding 99mTc-labeled pyrene derivatives was demonstrated by comparison to non-DNA-binding 99mTcO4-, since nearly all DNA damage caused by 99mTcO4- was prevented by incubating with DMSO.

  4. Direct and Auger Electron-Induced, Single- and Double-Strand Breaks on Plasmid DNA Caused by 99mTc-Labeled Pyrene Derivatives and the Effect of Bonding Distance

    PubMed Central

    Reissig, Falco; Mamat, Constantin; Steinbach, Joerg; Pietzsch, Hans-Juergen; Freudenberg, Robert; Navarro-Retamal, Carlos; Caballero, Julio; Kotzerke, Joerg; Wunderlich, Gerd

    2016-01-01

    It is evident that 99mTc causes radical-mediated DNA damage due to Auger electrons, which were emitted simultaneously with the known γ-emission of 99mTc. We have synthesized a series of new 99mTc-labeled pyrene derivatives with varied distances between the pyrene moiety and the radionuclide. The pyrene motif is a common DNA intercalator and allowed us to test the influence of the radionuclide distance on damages of the DNA helix. In general, pUC 19 plasmid DNA enables the investigation of the unprotected interactions between the radiotracers and DNA that results in single-strand breaks (SSB) or double-strand breaks (DSB). The resulting DNA fragments were separated by gel electrophoresis and quantified by fluorescent staining. Direct DNA damage and radical-induced indirect DNA damage by radiolysis products of water were evaluated in the presence or absence of the radical scavenger DMSO. We demonstrated that Auger electrons directly induced both SSB and DSB in high efficiency when 99mTc was tightly bound to the plasmid DNA and this damage could not be completely prevented by DMSO, a free radical scavenger. For the first time, we were able to minimize this effect by increasing the carbon chain lengths between the pyrene moiety and the 99mTc nuclide. However, a critical distance between the 99mTc atom and the DNA helix could not be determined due to the significantly lowered DSB generation resulting from the interaction which is dependent on the type of the 99mTc binding motif. The effect of variable DNA damage caused by the different chain length between the pyrene residue and the Tc-core as well as the possible conformations of the applied Tc-complexes was supplemented with molecular dynamics (MD) calculations. The effectiveness of the DNA-binding 99mTc-labeled pyrene derivatives was demonstrated by comparison to non-DNA-binding 99mTcO4–, since nearly all DNA damage caused by 99mTcO4– was prevented by incubating with DMSO. PMID:27583677

  5. Transcription-replication collision increases recombination efficiency between plasmids.

    PubMed

    Jialiang, Li; Feng, Chen; Zhen, Xu; Jibing, Chen; Xiang, Lv; Lingling, Zhang; Depei, Liu

    2013-11-01

    It has been proposed that the stalling of the replication forks can induce homologous recombination in several organisms, and that arrested replication forks may offer nuclease targets, thereby providing a substrate for proteins involved in double-strand repair. In this article, we constructed a plasmid with the potential for transcription-replication collision (TRC), in which DNA replication and RNA transcription occur on the same DNA template simultaneously. Theoretically, transcription will impede DNA replication and increase homologous recombination. To validate this hypothesis, another plasmid was constructed that contained a homologous sequence with the exception of some mutated sites. Co-transfection of these two plasmids into 293T cells resulted in increased recombination frequency. The ratio of these two plasmids also affected the recombination frequency. Moreover, we found high expression levels of RAD51, which indicated that the increase in the recombination rate was probably via the homologous recombination pathway. These results indicate that mutant genes in plasmids can be repaired by TRC-induced recombination.

  6. Activation-induced CD154 expression abrogates tolerance induced by apoptotic cells*

    PubMed Central

    Gurung, Prajwal; Kucaba, Tamara A.; Ferguson, Thomas A.; Griffith, Thomas S.

    2009-01-01

    The decision to generate a productive immune response or tolerance often depends on the context in which T cells first see Ag. Using a classical system of tolerance induction, we examined the immunological consequence of Ag encountered in the presence of naïve or activated apoptotic cells. Naïve apoptotic cells induced tolerance when injected i.v.; however, previously activated apoptotic cells induced immunity. Further analysis revealed a key role for CD154, as tolerance resulted after i.v. injection of either naïve or activated apoptotic CD154−/− T cells, while co-injection of an agonistic anti-CD40 mAb with naïve apoptotic T cells induced robust immunity. DC fed activated apoptotic T cells in vitro produced IL-12p40 in a CD154-dependent manner, and the use of IL-12p40−/− mice or mAb-mediated neutralization of IL-12 revealed a link between CD154, IL-12, and the ability of activated apoptotic T cells to induce immunity rather than tolerance. Collectively these results show that CD154 expression on apoptotic T cells can determine the outcome of an immune response to Ag recognized within the context of the apoptotic cells, and suggest the balance between naïve and activated apoptotic T cells may dictate whether a productive immune response is encouraged. PMID:19841180

  7. Homology of cryptic plasmid of Neisseria gonorrhoeae with plasmids from Neisseria meningitidis and Neisseria lactamica.

    PubMed

    Ison, C A; Bellinger, C M; Walker, J

    1986-10-01

    DNA probe hybridisation was used to examine the relation between the cryptic plasmid from Neisseria gonorrhoeae and plasmids carried by pharyngeal isolates of Neisseria meningitidis and Neisseria lactamica. The complete gonococcal cryptic plasmid and HinfI derived digestion fragments subcloned into Escherichia coli were used to probe Southern blots of plasmid extracts. Homology was found to a plasmid of approximate molecular weight 4.5 kilobase pairs (Kb) but not to plasmids of less than 3.2 Kb or 6.5 Kb. Eleven of 16 strains of N meningitidis and two of six strains of N lactamica carried plasmids that showed strong hybridisation with the 4.2 Kb gonococcal plasmid. Hybridisation of plasmids from non-gonococcal species of neisseria with the gonococcal cryptic plasmid indicates that caution should be taken when using the cryptic plasmid as a diagnostic probe for gonorrhoea.

  8. Plasmid-mediated degradation of dibenzothiophene by Pseudomonas species

    SciTech Connect

    Monticello, D.J.; Bakker, D.; Finnerty, W.R.

    1985-04-01

    The microbial transformation of dibenzothiophene (DBT) is of interest in the potential desulfurization of oil. The authors isolated three soil Pseuodomonas species which oxidized DBT to characteristic water-soluble, sulfur containing products. Two of the isolates harbored a 55-megadalton plasmid; growth in the presence of novobiocin resulted in both loss of the plasmid and loss of the ability to oxidize DBT. Reintroduction of the plasmid restored the ability to oxidize DBT to water-soluble products. The products resulting from the oxidation of DBT were characterized and included 3-hydroxy-2-formyl benzothiophene, 3-oxo-(3'-hydroxy-thionaphthenyl-(2)-methylene)-dihydrothionaphthene, and the hemiacetal and trans forms of 4-(2-(3-hydroxy)-thianaphthenyl)-2-oxo-3-butenoic acid. The products of DBT oxidation were inhibitory to cell growth and further DBT oxidation. DBT oxidation in the soil isolates was induced by naphthalene or salicylate and to a much lesser extent by DBT and was repressed by succinate.

  9. Production of Plasmid DNA as Pharmaceutical.

    PubMed

    Schmeer, Marco; Schleef, Martin

    2015-01-01

    Pharmaceutical applications of plasmid DNA require certain quality standards, depending on the intended use of the plasmids. That is, for direct gene transfer into human, GMP Grade is mandatory, however, for GMP production of for example viral vectors (AAV or mRNA etc.), the plasmid DNA used has not to be produced under GMP necessarily. Here we summarize important features of producing plasmid DNA, ensuring the required quality for the intended (pharmaceutical) application.

  10. Prevalence and significance of plasmid maintenance functions in the virulence plasmids of pathogenic bacteria.

    PubMed

    Sengupta, Manjistha; Austin, Stuart

    2011-07-01

    Virulence functions of pathogenic bacteria are often encoded on large extrachromosomal plasmids. These plasmids are maintained at low copy number to reduce the metabolic burden on their host. Low-copy-number plasmids risk loss during cell division. This is countered by plasmid-encoded systems that ensure that each cell receives at least one plasmid copy. Plasmid replication and recombination can produce plasmid multimers that hinder plasmid segregation. These are removed by multimer resolution systems. Equitable distribution of the resulting monomers to daughter cells is ensured by plasmid partition systems that actively segregate plasmid copies to daughter cells in a process akin to mitosis in higher organisms. Any plasmid-free cells that still arise due to occasional failures of replication, multimer resolution, or partition are eliminated by plasmid-encoded postsegregational killing systems. Here we argue that all of these three systems are essential for the stable maintenance of large low-copy-number plasmids. Thus, they should be found on all large virulence plasmids. Where available, well-annotated sequences of virulence plasmids confirm this. Indeed, virulence plasmids often appear to contain more than one example conforming to each of the three system classes. Since these systems are essential for virulence, they can be regarded as ubiquitous virulence factors. As such, they should be informative in the search for new antibacterial agents and drug targets.

  11. Building mosaics of therapeutic plasmid gene vectors.

    PubMed

    Tolmachov, Oleg E

    2011-12-01

    Plasmids are circular or linear DNA molecules propagated extra-chromosomally in bacteria. Evolution shaped plasmids are inherently mosaic structures with individual functional units represented by distinct segments in the plasmid genome. The patchwork of plasmid genetic modules is a convenient template and a model for the generation of artificial plasmids used as vehicles for gene delivery into human cells. Plasmid gene vectors are an important tool in gene therapy and in basic biomedical research, where these vectors offer efficient transgene expression in many settings in vitro and in vivo. Plasmid vectors can be attached to nuclear directing ligands or transferred by electroporation as naked DNA to deliver the payload genes to the nuclei of the target cells. Transgene expression silencing by plasmid sequences of bacterial origin and immune stimulation by bacterial unmethylated CpG motifs can be avoided by the generation of plasmid-based minimized DNA vectors, such as minicircles. Systems of efficient site-specific integration into human chromosomes and stable episomal maintenance in human cells are being developed for further reduction of the chances for transgene silencing. The successful generation of plasmid vectors is governed by a number of vector design rules, some of which are common to all gene vectors, while others are specific to plasmid vectors. This review is focused both on the guiding principles and on the technical know-how of plasmid gene vector design.

  12. Plasmid diversity in Vibrio vulnificus biotypes.

    PubMed

    Roig, Francisco J; Amaro, Carmen

    2009-02-01

    Vibrio vulnificus is a heterogeneous bacterial species that can be virulent for humans and fish. Virulence in fish seems to rely on a recently described plasmid that can be transmitted between strains, aided by a conjugative plasmid. The main objective of this work was to analyse the plasmid content of a wide collection of strains from the three biotypes of the species, as well as to identify putative conjugative and virulence plasmids by means of Southern hybridization with specific probes and sequence analysis of selected gene markers. We found 28 different plasmid profiles in a total of 112 strains, which were relatively biotype- or serovar-specific. Biotype 1 lacked high-molecular-mass plasmids, with the exception of a putative conjugative plasmid of 48 kb that was present in 42.8% of clinical and environmental strains isolated worldwide. All biotype 2 strains possessed the virulence plasmid, whose molecular mass ranged between 68 and 70 kb, and 89.65% of these strains also had a putative conjugative plasmid with a molecular size of 52-56 kb. Finally, a 48 kb putative conjugative plasmid was present in all biotype 3 strains. Data from partial sequencing of traD, traI and the whole vep07 (a recently described plasmid-borne virulence gene) from a selection of strains suggest that the plasmids of 48-56 kb probably belong to the same family of F-plasmids as pYJ016 and that the gene vep07 is absolutely essential for fish virulence. Additional cryptic plasmids of low molecular mass were present in the three biotypes. In conclusion, plasmids are widespread among V. vulnificus species and could contribute substantially to genetic plasticity of the species.

  13. Origin and Evolution of Rickettsial Plasmids

    PubMed Central

    El Karkouri, Khalid; Pontarotti, Pierre; Raoult, Didier; Fournier, Pierre-Edouard

    2016-01-01

    Background Rickettsia species are strictly intracellular bacteria that have undergone a reductive genomic evolution. Despite their allopatric lifestyle, almost half of the 26 currently validated Rickettsia species have plasmids. In order to study the origin, evolutionary history and putative roles of rickettsial plasmids, we investigated the evolutionary processes that have shaped 20 plasmids belonging to 11 species, using comparative genomics and phylogenetic analysis between rickettsial, microbial and non-microbial genomes. Results Plasmids were differentially present among Rickettsia species. The 11 species had 1 to 4 plasmid (s) with a size ranging from 12 kb to 83 kb. We reconstructed pRICO, the last common ancestor of the current rickettsial plasmids. pRICO was vertically inherited mainly from Rickettsia/Orientia chromosomes and diverged vertically into a single or multiple plasmid(s) in each species. These plasmids also underwent a reductive evolution by progressive gene loss, similar to that observed in rickettsial chromosomes, possibly leading to cryptic plasmids or complete plasmid loss. Moreover, rickettsial plasmids exhibited ORFans, recent gene duplications and evidence of horizontal gene transfer events with rickettsial and non-rickettsial genomes mainly from the α/γ-proteobacteria lineages. Genes related to maintenance and plasticity of plasmids, and to adaptation and resistance to stress mostly evolved under vertical and/or horizontal processes. Those involved in nucleotide/carbohydrate transport and metabolism were under the influence of vertical evolution only, whereas genes involved in cell wall/membrane/envelope biogenesis, cycle control, amino acid/lipid/coenzyme and secondary metabolites biosynthesis, transport and metabolism underwent mainly horizontal transfer events. Conclusion Rickettsial plasmids had a complex evolution, starting with a vertical inheritance followed by a reductive evolution associated with increased complexity via

  14. Plasmid curing of Oenococcus oeni.

    PubMed

    Mesas, Juan M; Rodríguez, M Carmen; Alegre, M Teresa

    2004-01-01

    Two strains of Oenococcus oeni, RS1 (which carries the plasmid pRS1) and RS2 (which carries the plasmids pRS2 and pRS3), were grown in the presence of different curing agents and at different temperatures. Sublethal temperature together with acriflavine generated all possible types of cured strains, i.e., lacking pRS1 (from strain RS1), and lacking pRS2, pRS3, or both (from strain RS2). Sublethal temperature together with acridine orange only generated cured strains lacking pRS3. These results suggest that acriflavine is a better curing agent than acridine orange for O. oeni, and that pRS3 is the most sensitive to these curing agents. We also observed spontaneous loss of pRS2 or both pRS2 and pRS3 by electroporation. The ability to cure O. oeni strains of plasmids provides a critical new tool for the genetic analysis and engineering of this commercially important bacterium.

  15. Reduced Live Organism Recovery and Lack of Hydrosalpinx in Mice Infected with Plasmid-Free Chlamydia muridarum

    PubMed Central

    Lei, Lei; Chen, Jianlin; Hou, Shuping; Ding, Yiling; Yang, Zhangsheng; Zeng, Hao; Baseman, Joel

    2014-01-01

    Plasmid-free Chlamydia trachomatis and Chlamydia muridarum fail to induce severe pathology. To evaluate whether the attenuated pathogenicity is due to insufficient infection or inability of the plasmidless chlamydial organisms to trigger pathological responses, we compared plasmid-competent and plasmid-free C. muridarum infections in 5 different strains of mice. All 5 strains developed hydrosalpinx following intravaginal inoculation with plasmid-competent, but not inoculation with plasmid-free, C. muridarum. The lack of hydrosalpinx induction by plasmid-free C. muridarum correlated with significantly reduced live organism recovery from the lower genital tract and shortened infection in the upper genital tract. The plasmid-free C. muridarum organisms failed to induce hydrosalpinx even when the organisms were directly inoculated into the oviduct via an intrabursal injection, which was accompanied by significantly reduced survival of the plasmidless organisms in the genital tracts. Furthermore, plasmid-competent C. muridarum organisms after UV inactivation were no longer able to induce hydrosalpinx even when directly delivered into the oviduct at a high dose. Together, these observations suggest that decreased survival of and shortened infection with plasmid-free C. muridarum may contribute significantly to its attenuated pathogenicity. We conclude that adequate live chlamydial infection in the oviduct may be necessary to induce hydrosalpinx. PMID:24343644

  16. Plasmid Diversity and Adaptation Analyzed by Massive Sequencing of Escherichia coli Plasmids.

    PubMed

    de Toro, María; Garcilláon-Barcia, M Pilar; De La Cruz, Fernando

    2014-12-01

    Whole-genome sequencing is revolutionizing the analysis of bacterial genomes. It leads to a massive increase in the amount of available data to be analyzed. Bacterial genomes are usually composed of one main chromosome and a number of accessory chromosomes, called plasmids. A recently developed methodology called PLACNET (for plasmid constellation networks) allows the reconstruction of the plasmids of a given genome. Thus, it opens an avenue for plasmidome analysis on a global scale. This work reviews our knowledge of the genetic determinants for plasmid propagation (conjugation and related functions), their diversity, and their prevalence in the variety of plasmids found by whole-genome sequencing. It focuses on the results obtained from a collection of 255 Escherichia coli plasmids reconstructed by PLACNET. The plasmids found in E. coli represent a nonaleatory subset of the plasmids found in proteobacteria. Potential reasons for the prevalence of some specific plasmid groups will be discussed and, more importantly, additional questions will be posed.

  17. The 2 micron plasmid of Saccharomyces cerevisiae: a miniaturized selfish genome with optimized functional competence.

    PubMed

    Chan, Keng-Ming; Liu, Yen-Ting; Ma, Chien-Hui; Jayaram, Makkuni; Sau, Soumitra

    2013-07-01

    The 2 micron plasmid of Saccharomyces cerevisiae is a relatively small multi-copy selfish DNA element that resides in the yeast nucleus at a copy number of 40-60 per haploid cell. The plasmid is able to persist in host populations with almost chromosome-like stability with the help of a partitioning system and a copy number control system. The first part of this article describes the properties of the partitioning system comprising two plasmid coded proteins, Rep1 and Rep2, and a partitioning locus STB. Current evidence supports a model in which the Rep-STB system couples plasmid segregation to chromosome segregation by promoting the physical association of plasmid molecules with chromosomes. In the second part, the focus is on the Flp site-specific recombination system housed by the plasmid, which plays a critical role in maintaining steady state plasmid copy number. The Flp system corrects any decrease in plasmid population by promoting plasmid amplification via a recombination induced rolling circle replication mechanism. Appropriate plasmid amplification, without runaway increase in copy number, is ensured by positive and negative regulation of FLP gene expression by plasmid coded proteins and by the control of Flp level/activity through post-translational modification of Flp by the cellular sumoylation system. The Flp system has been successfully utilized to understand mechanisms of site-specific recombination and to bring about directed genetic alterations for addressing fundamental problems in biology and for accomplishing bio-engineering objectives. A particularly interesting, and perhaps less well known and underappreciated, application of Flp in revealing unique DNA topologies required to confer functional competence to DNA-protein machines is discussed.

  18. Plasmid-Encoded Iron Uptake Systems.

    PubMed

    Di Lorenzo, Manuela; Stork, Michiel

    2014-12-01

    Plasmids confer genetic information that benefits the bacterial cells containing them. In pathogenic bacteria, plasmids often harbor virulence determinants that enhance the pathogenicity of the bacterium. The ability to acquire iron in environments where it is limited, for instance the eukaryotic host, is a critical factor for bacterial growth. To acquire iron, bacteria have evolved specific iron uptake mechanisms. These systems are often chromosomally encoded, while those that are plasmid-encoded are rare. Two main plasmid types, ColV and pJM1, have been shown to harbor determinants that increase virulence by providing the cell with essential iron for growth. It is clear that these two plasmid groups evolved independently from each other since they do not share similarities either in the plasmid backbones or in the iron uptake systems they harbor. The siderophores aerobactin and salmochelin that are found on ColV plasmids fall in the hydroxamate and catechol group, respectively, whereas both functional groups are present in the anguibactin siderophore, the only iron uptake system found on pJM1-type plasmids. Besides siderophore-mediated iron uptake, ColV plasmids carry additional genes involved in iron metabolism. These systems include ABC transporters, hemolysins, and a hemoglobin protease. ColV- and pJM1-like plasmids have been shown to confer virulence to their bacterial host, and this trait can be completely ascribed to their encoded iron uptake systems.

  19. Virulence Plasmids of Spore-Forming Bacteria.

    PubMed

    Adams, Vicki; Li, Jihong; Wisniewski, Jessica A; Uzal, Francisco A; Moore, Robert J; McClane, Bruce A; Rood, Julian I

    2014-12-01

    Plasmid-encoded virulence factors are important in the pathogenesis of diseases caused by spore-forming bacteria. Unlike many other bacteria, the most common virulence factors encoded by plasmids in Clostridium and Bacillus species are protein toxins. Clostridium perfringens causes several histotoxic and enterotoxin diseases in both humans and animals and produces a broad range of toxins, including many pore-forming toxins such as C. perfringens enterotoxin, epsilon-toxin, beta-toxin, and NetB. Genetic studies have led to the determination of the role of these toxins in disease pathogenesis. The genes for these toxins are generally carried on large conjugative plasmids that have common core replication, maintenance, and conjugation regions. There is considerable functional information available about the unique tcp conjugation locus carried by these plasmids, but less is known about plasmid maintenance. The latter is intriguing because many C. perfringens isolates stably maintain up to four different, but closely related, toxin plasmids. Toxin genes may also be plasmid-encoded in the neurotoxic clostridia. The tetanus toxin gene is located on a plasmid in Clostridium tetani, but the botulinum toxin genes may be chromosomal, plasmid-determined, or located on bacteriophages in Clostridium botulinum. In Bacillus anthracis it is well established that virulence is plasmid determined, with anthrax toxin genes located on pXO1 and capsule genes on a separate plasmid, pXO2. Orthologs of these plasmids are also found in other members of the Bacillus cereus group such as B. cereus and Bacillus thuringiensis. In B. thuringiensis these plasmids may carry genes encoding one or more insecticidal toxins.

  20. Inefficient replication reduces RecA-mediated repair of UV-damaged plasmids introduced into competent Escherichia coli.

    PubMed

    Jeiranian, H A; Courcelle, C T; Courcelle, J

    2012-09-01

    Transformation of Escherichia coli with purified plasmids containing DNA damage is frequently used as a tool to characterize repair pathways that operate on chromosomes. In this study, we used an assay that allowed us to quantify plasmid survival and to compare how efficiently various repair pathways operate on plasmid DNA introduced into cells relative to their efficiency on chromosomal DNA. We observed distinct differences between the mechanisms operating on the transforming plasmid DNA and the chromosome. An average of one UV-induced lesion was sufficient to inactivate ColE1-based plasmids introduced into nucleotide excision repair mutants, suggesting an essential role for repair on newly introduced plasmid DNA. By contrast, the absence of RecA, RecF, RecBC, RecG, or RuvAB had a minimal effect on the survival of the transforming plasmid DNA containing UV-induced damage. Neither the presence of an endogenous homologous plasmid nor the induction of the SOS response enhanced the survival of transforming plasmids. Using two-dimensional agarose-gel analysis, both replication- and RecA-dependent structures that were observed on established, endogenous plasmids following UV-irradiation, failed to form on UV-irradiated plasmids introduced into E. coli. We interpret these observations to suggest that the lack of RecA-mediated survival is likely to be due to inefficient replication that occurs when plasmids are initially introduced into cells, rather than to the plasmid's size, the absence of homologous sequences, or levels of recA expression.

  1. A Plasmid in Legionella pneumophila

    DTIC Science & Technology

    1980-09-01

    13). which they were isolated and the number of the isolate The Legionnaires disease bacterium, L. pneu. from that city. The following 16... Legionnaires disease bacterium. .1. (un. Micro. biol. 8:320-:t25. appears reasonable that this organism could sup- 1:l. Fraser, D5. W., and J. F. McI~ade. 1979...INFECTION AND IMMUNITY, Sept. 1980, p. 1(92-1095 Vol. 29, No. :1 0I 9-9567/A)/- 1092/14$02.00/0. A Plasmid in Legionella pneumophila_ ( (/’ )GREGORY

  2. Survey of plasmids in various mycoplasmas.

    PubMed Central

    Harasawa, R.; Barile, M. F.

    1983-01-01

    Thirty-three strains representing 15 distinct Mycoplasma, Acholeplasma, and Spiroplasma species were examined for the presence of plasmid DNA by agarose gel electrophoresis. The electrophoretic patterns of the DNAs of three strains, Mycoplasma sp. strain 747, Spiroplasma mirum strain SMCA, and M. hominis strain 1257, suggested the presence of a plasmid with molecular weights of approximately 70, 10, and 9 megadaltons, respectively. The functions of these plasmids are currently unknown. Images FIG. 1 PMID:6679154

  3. Biofilms and the plasmid maintenance question.

    PubMed

    Imran, Mudassar; Jones, Don; Smith, Hal

    2005-02-01

    Can a conjugative plasmid encoding enhanced biofilm forming abilities for its bacterial host facilitate the persistence of the plasmid in a bacterial population despite conferring diminished growth rate and segregative plasmid loss on its bearers? We construct a mathematical model in a chemostat and in a plug flow environment to answer this question. Explicit conditions for an affirmative answer are derived. Numerical simulations support the conclusion.

  4. Plasmid-encoded trimethoprim resistance in staphylococci.

    PubMed Central

    Archer, G L; Coughter, J P; Johnston, J L

    1986-01-01

    High-level (greater than 1,000 micrograms/ml) resistance to the antimicrobial agent trimethoprim was found in 17 of 101 (17%) coagulase-negative staphylococci and 5 of 51 (10%) Staphylococcus aureus from a number of different hospitals in the United States. Resistance was plasmid encoded and could be transferred by conjugation in 4 of the 17 (24%) Tpr coagulase-negative staphylococci and 3 of the 5 (60%) Tpr S. aureus. A 1.2-kilobase segment of plasmid DNA from one of the plasmids (pG01) was cloned on a high-copy-number vector in Escherichia coli and expressed high-level Tpr (MIC, 1,025 micrograms/ml) in the gram-negative host. In situ filter hybridization demonstrated homology between the cloned Tpr gene probe and plasmid DNA from each conjugative Tpr plasmid, a single nonconjugative plasmid from a United States Staphylococcus epidermidis isolate, a nonconjugative plasmid from an Australian methicillin-resistant S. aureus isolate, and chromosomal DNA from three Tpr S. epidermidis isolates that did not contain any plasmid DNA that was homologous with the probe. No homology was seen between the probe and staphylococcal plasmids not mediating Tpr, plasmid DNA from 12 Tpr S. epidermidis isolates not transferring Tpr by conjugation, or plasmid-encoded Tpr genes derived from gram-negative bacteria. Plasmid-encoded Tpr appears to be a relatively new gene in staphylococci and, because it can be transferred by conjugation, could become more prevalent in nonsocomial isolates. Images PMID:3729338

  5. pLS101 plasmid vector

    DOEpatents

    Lacks, S.A.; Balganesh, T.S.

    1985-02-19

    Disclosed is recombinant plasmid pLS101, consisting essentially of a 2.0 Kb ma1M gene fragment ligated to a 4.4 Kb Tcr DNA fragment, which is particularly useful for transforming Gram-positive bacteria. This plasmid contains at least four restriction sites suitable for inserting exogeneous gene sequences. Also disclosed is a method for plasmid isolation by penicillin selection, as well as processes for enrichment of recombinant plasmids in Gram-positive bacterial systems. 5 figs., 2 tabs.

  6. pLS010 plasmid vector

    SciTech Connect

    Lacks, Sanford A.; Balganesh, Tanjore S.

    1988-01-01

    Disclosed is recombinant plasmid pLS101, consisting essentially of a 2.0 Kb malM gene fragment ligated to a 4.4 Kb T.sub.c r DNA fragment, which is particularly useful for transforming Gram-positive bacteria. This plasmid contains at least four restriction sites suitable for inserting exogeneous gene sequences. Also disclosed is a method for plasmid isolation by penicillin selection, as well as processes for enrichment of recombinant plasmids in Gram-positive bacterial systems.

  7. Persistence of Antibiotic Resistance Plasmids in Biofilms

    DTIC Science & Technology

    2013-10-01

    model  broad-­‐host-­‐range  MDR  plasmids  pRGM1  and... model   plasmids,   the   IncU   plasmid   pRGM1   and   the   IncP-­‐1   plasmid   pB10,   with   mini-­‐Tn5-­‐PA1-­‐ 04/03...we  have  focused  on  this   strain   as   our   main   model   host   (from   here   on   often  

  8. Plasmid transfer systems in the rhizobia.

    PubMed

    Ding, Hao; Hynes, Michael F

    2009-08-01

    Rhizobia are agriculturally important bacteria that can form nitrogen-fixing nodules on the roots of leguminous plants. Agricultural application of rhizobial inoculants can play an important role in increasing leguminous crop yields. In temperate rhizobia, genes involved in nodulation and nitrogen fixation are usually located on one or more large plasmids (pSyms) or on symbiotic islands. In addition, other large plasmids of rhizobia carry genes that are beneficial for survival and competition of rhizobia in the rhizosphere. Conjugative transfer of these large plasmids thus plays an important role in the evolution of rhizobia. Therefore, understanding the mechanism of conjugative transfer of large rhizobial plasmids provides foundations for maintaining, monitoring, and predicting the behaviour of these plasmids during field release events. In this minireview, we summarize two types of known rhizobial conjugative plasmids, including quorum sensing regulated plasmids and RctA-repressed plasmids. We provide evidence for the existence of a third type of conjugative plasmid, including pRleVF39c in Rhizobium leguminosarum bv. viciae strain VF39SM, and we provide a comparison of the different types of conjugation genes found in members of the rhizobia that have had their genomes sequenced so far.

  9. Stress responses in pathogenic Yersinia enterocolitica with reference to the stability of the virulence plasmid in food

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Yersinia enterocolitica has been associated with food-borne illness, most often due the ingestion of pork products. The pathogenic effects induced by a Y. enterocolitica infection are caused by the interplay of chromosomal genes and a virulence plasmid, pYV. Generally, the plasmid is lost during g...

  10. Vibrio cholerae conjugative plasmid pSJ15 contains transposable prophage dVcA1.

    PubMed Central

    Johnson, S R; Romig, W R

    1981-01-01

    Evidence is presented that defective prophage dVcA1 in Vibrio cholerae strain 162 was transposed to the hybrid P::Tn1 plasmid pSJ5. Properties of the resulting conjugative plasmid, pSJ15, indicated that bacteriophage VcA1, like coliphage Mu, can insert at many sites. By analogy with other Hfr-like donors, the high-frequency, polarized chromosomal transfer mediated by plasmid pSJ15 in strain 162 appeared to depend on plasmid integration through the homologous dVcA1 sequences in both replicons. When strain 162(pSJ15) donors were mated to the nonlysogenic El Tor strain RJ1, many potential ampicillin-resistant transconjugants were zygotically induced. However, surviving transconjugants (i) were immune to phage VcA1, (ii) cotransferred immunity and ampicillin resistance to nonlysogenic recipients, and (iii) did not preferentially transfer any chromosomal markers. Recombinant plasmids that transferred wild-type VcA1 prophages were readily isolated from strain RJ1 (VcA1+) lysogens that contained plasmid pSJ15. Physical measurements revealed that plasmid pSJ15 and the recombinant plasmids were about one VcA1 genome (22 to 24 megadaltons) larger than the 51-megadalton pSJ5 plasmid. Similar Hfr-like donors were constructed by introducing plasmid pSJ15 into different strain RJ1 (VcA1+) lysogens. Transfer properties of these donors indicated that the VcA1 prophage was integrated at several sites in the strain RJ1 chromosome. Images PMID:6260754

  11. Autotransmissible resident plasmid of Rhizobium meliloti.

    PubMed

    Bedmar, E J; Olivares, J

    1980-01-01

    A resident plasmid of wild-type strains of Rhizobium meliloti of 59.6 megadaltons has been shown to be transferred at a high frequency to "cured" strains of this bacterial species. This plasmid, named pEZ1, that confers phage-sensitivity to cells carrying it is also transmissible to Escherichia coli and from it to "cured" R. meliloti strains.

  12. RepA and RepB exert plasmid incompatibility repressing the transcription of the repABC operon.

    PubMed

    Pérez-Oseguera, Angeles; Cevallos, Miguel A

    2013-11-01

    Rhizobium etli CFN42 has a multipartite genome composed of one chromosome and six large plasmids with low copy numbers, all belonging to the repABC plasmid family. All elements essential for replication and segregation of these plasmids are encoded within the repABC operon. RepA and RepB direct plasmid segregation and are involved in the transcriptional regulation of the operon, and RepC is the initiator protein of the plasmid. Here we show that in addition to RepA (repressor) and RepB (corepressor), full transcriptional repression of the operon located in the symbiotic plasmid (pRetCFN42d) of this strain requires parS, the centromere-like sequence, and the operator sequence. However, the co-expression of RepA and RepB is sufficient to induce the displacement of the parental plasmid. RepA is a Walker-type ATPase that self associates in vivo and in vitro and binds specifically to the operator region in its RepA-ADP form. In contrast, RepA-ATP is capable of binding to non-specific DNA. RepA and RepB form high molecular weight DNA-protein complexes in the presence of ATP and ADP. RepA carrying ATP-pocket motif mutations induce full repression of the repABC operon without the participation of RepB and parS. These mutants specifically bind the operator sequence in their ATP or ADP bound forms. In addition, their expression in trans exerts plasmid incompatibility against the parental plasmid. RepA and RepB expressed in trans induce plasmid incompatibility because of their ability to repress the repABC operon and not only by their capacity to distort the plasmid segregation process.

  13. Large-scale preparation of plasmid DNA.

    PubMed

    Heilig, J S; Elbing, K L; Brent, R

    2001-05-01

    Although the need for large quantities of plasmid DNA has diminished as techniques for manipulating small quantities of DNA have improved, occasionally large amounts of high-quality plasmid DNA are desired. This unit describes the preparation of milligram quantities of highly purified plasmid DNA. The first part of the unit describes three methods for preparing crude lysates enriched in plasmid DNA from bacterial cells grown in liquid culture: alkaline lysis, boiling, and Triton lysis. The second part describes four methods for purifying plasmid DNA in such lysates away from contaminating RNA and protein: CsCl/ethidium bromide density gradient centrifugation, polyethylene glycol (PEG) precipitation, anion-exchange chromatography, and size-exclusion chromatography.

  14. The ABCs of plasmid replication and segregation.

    PubMed

    Pinto, Uelinton M; Pappas, Katherine M; Winans, Stephen C

    2012-11-01

    To ensure faithful transmission of low-copy plasmids to daughter cells, these plasmids must replicate once per cell cycle and distribute the replicated DNA to the nascent daughter cells. RepABC family plasmids are found exclusively in alphaproteobacteria and carry a combined replication and partitioning locus, the repABC cassette, which is also found on secondary chromosomes in this group. RepC and a replication origin are essential for plasmid replication, and RepA, RepB and the partitioning sites distribute the replicons to predivisional cells. Here, we review our current understanding of the transcriptional and post-transcriptional regulation of the Rep proteins and of their functions in plasmid replication and partitioning.

  15. Yeast telomere repeat sequence (TRS) improves circular plasmid segregation, and TRS plasmid segregation involves the RAP1 gene product.

    PubMed Central

    Longtine, M S; Enomoto, S; Finstad, S L; Berman, J

    1992-01-01

    Telomere repeat sequences (TRSs) can dramatically improve the segregation of unstable circular autonomously replicating sequence (ARS) plasmids in Saccharomyces cerevisiae. Deletion analysis demonstrated that yeast TRSs, which conform to the general sequence (C(1-3)A)n, are able to stabilize circular ARS plasmids. A number of TRS clones of different primary sequence and C(1-3)A tract length confer the plasmid stabilization phenotype. TRS sequences do not appear to improve plasmid replication efficiency, as determined by plasmid copy number analysis and functional assays for ARS activity. Pedigree analysis confirms that TRS-containing plasmids are missegregated at low frequency and that missegregated TRS-containing plasmids, like ARS plasmids, are preferentially retained by the mother cell. Plasmids stabilized by TRSs have properties that distinguish them from centromere-containing plasmids and 2 microns-based recombinant plasmids. Linear ARS plasmids, which include two TRS tracts at their termini, segregate inefficiently, while circular plasmids with one or two TRS tracts segregate efficiently, suggesting that plasmid topology or TRS accessibility interferes with TRS segregation function on linear plasmids. In strains carrying the temperature-sensitive mutant alleles rap1grc4 and rap1-5, TRS plasmids are not stable at the semipermissive temperature, suggesting that RAP1 protein is involved in TRS plasmid stability. In Schizosaccharomyces pombe, an ARS plasmid was stabilized by the addition of S. pombe telomere sequence, suggesting that the ability to improve the segregation of ARS plasmids is a general property of telomere repeats. PMID:1569937

  16. Ultrasound enhancement of in vitro transfection of plasmid DNA by a cationized gelatin.

    PubMed

    Hosseinkhani, Hossein; Aoyama, Teruyoshi; Ogawa, Osamu; Tabata, Yasuhiko

    2002-05-01

    In vitro transfection efficiency of a plasmid DNA for rat gastric mucosal (RGM)-1 cells was enhanced by ultrasound (US) irradiation. Ethylenediamine was introduced to the carboxyl groups of gelatin to prepare a cationized gelatin as the vector of plasmid DNA encoding luciferase. An electrophoresis experiment revealed that the cationized gelatin was mixed with plasmid DNA at the weight ratio of 5.0 to form a cationized gelatin-plasmid DNA complex. The complex obtained was about 200nm in diameter with a positive charge. When incubated with the cationized gelatin-plasmid DNA complex and subsequently exposed to US, RGM-1 cells exhibited a significantly enhanced luciferase activity although the extent increased with an increase in the DNA concentration, in contrast to the cationized gelatin alone with or without US irradiation and US irradiation alone. US irradiation was also effective in enhancing the activity by free plasmid DNA although the extent was less than that of the complex. The US-induced enhancement of luciferase activity was influenced by the exposure time period, frequency, and intensity of US. The activity enhancement became higher to be significant at the irradiation time period of 60 s and thereafter decreased. A series of cytotoxicity experiments revealed that an increase in the irradiation time period and intensity of US decreased the viability of cells themselves. It is possible that US irradiation under an appropriate condition enables cells to accelerate the permeation of the cationized gelatin-plasmid DNA complex through the cell membrane, resulted in enhanced transfection efficiency of plasmid DNA. These findings clearly indicate that US exposure is a simple and promising method to enhance the gene expression of plasmid DNA.

  17. Rolling-circle replication of bacterial plasmids.

    PubMed Central

    Khan, S A

    1997-01-01

    Many bacterial plasmids replicate by a rolling-circle (RC) mechanism. Their replication properties have many similarities to as well as significant differences from those of single-stranded DNA (ssDNA) coliphages, which also replicate by an RC mechanism. Studies on a large number of RC plasmids have revealed that they fall into several families based on homology in their initiator proteins and leading-strand origins. The leading-strand origins contain distinct sequences that are required for binding and nicking by the Rep proteins. Leading-strand origins also contain domains that are required for the initiation and termination of replication. RC plasmids generate ssDNA intermediates during replication, since their lagging-strand synthesis does not usually initiate until the leading strand has been almost fully synthesized. The leading- and lagging-strand origins are distinct, and the displaced leading-strand DNA is converted to the double-stranded form by using solely the host proteins. The Rep proteins encoded by RC plasmids contain specific domains that are involved in their origin binding and nicking activities. The replication and copy number of RC plasmids, in general, are regulated at the level of synthesis of their Rep proteins, which are usually rate limiting for replication. Some RC Rep proteins are known to be inactivated after supporting one round of replication. A number of in vitro replication systems have been developed for RC plasmids and have provided insight into the mechanism of plasmid RC replication. PMID:9409148

  18. Evaluating metabolic stress and plasmid stability in plasmid DNA production by Escherichia coli.

    PubMed

    Silva, Filomena; Queiroz, João A; Domingues, Fernanda C

    2012-01-01

    In the context of recombinant DNA technology, the development of feasible and high-yielding plasmid DNA production processes has regained attention as more evidence for its efficacy as vectors for gene therapy and DNA vaccination arise. When producing plasmid DNA in Escherichia coli, a number of biological restraints, triggered by plasmid maintenance and replication as well as culture conditions are responsible for limiting final biomass and product yields. This termed "metabolic burden" can also cause detrimental effects on plasmid stability and quality, since the cell machinery is no longer capable of maintaining an active metabolism towards plasmid synthesis and the stress responses elicited by plasmid maintenance can also cause increased plasmid instability. The optimization of plasmid DNA production bioprocesses is still hindered by the lack of information on the host metabolic responses as well as information on plasmid instability. Therefore, systematic and on-line approaches are required not only to characterise this "metabolic burden" and plasmid stability but also for the design of appropriate metabolic engineering and culture strategies. The monitoring tools described to date rapidly evolve from laborious, off-line and at-line monitoring to online monitoring, at a time-scale that enables researchers to solve these bioprocessing problems as they occur. This review highlights major E. coli biological alterations caused by plasmid maintenance and replication, possible causes for plasmid instability and discusses the ability of currently employed bioprocess monitoring techniques to provide information in order to circumvent metabolic burden and plasmid instability, pointing out the possible evolution of these methods towards online bioprocess monitoring.

  19. Influenza Plasmid DNA Vaccines: Progress and Prospects.

    PubMed

    Bicho, Diana; Queiroz, João António; Tomaz, Cândida Teixeira

    2015-01-01

    Current influenza vaccines have long been used to fight flu infectious; however, recent advances highlight the importance of produce new alternatives. Even though traditional influenza vaccines are safe and usually effective, they need to be uploaded every year to anticipate circulating flu viruses. This limitation together with the use of embryonated chicken eggs as the substrate for vaccine production, is time-consuming and could involve potential biohazards in growth of new virus strains. Plasmid DNA produced by prokaryote microorganisms and encoding foreign proteins had emerged as a promising therapeutic tool. This technology allows the expression of a gene of interest by eukaryotic cells in order to induce protective immune responses against the pathogen of interest. In this review, we discuss the strategies to choose the best DNA vaccine to be applied in the treatment and prevention of influenza. Specifically, we give an update of influenza DNA vaccines developments, all involved techniques, their main characteristics, applicability and technical features to obtain the best option against influenza infections.

  20. Topological Behavior of Plasmid DNA.

    PubMed

    Higgins, N Patrick; Vologodskii, Alexander V

    2015-04-01

    The discovery of the B-form structure of DNA by Watson and Crick led to an explosion of research on nucleic acids in the fields of biochemistry, biophysics, and genetics. Powerful techniques were developed to reveal a myriad of different structural conformations that change B-DNA as it is transcribed, replicated, and recombined and as sister chromosomes are moved into new daughter cell compartments during cell division. This article links the original discoveries of superhelical structure and molecular topology to non-B form DNA structure and contemporary biochemical and biophysical techniques. The emphasis is on the power of plasmids for studying DNA structure and function. The conditions that trigger the formation of alternative DNA structures such as left-handed Z-DNA, inter- and intra-molecular triplexes, triple-stranded DNA, and linked catenanes and hemicatenanes are explained. The DNA dynamics and topological issues are detailed for stalled replication forks and for torsional and structural changes on DNA in front of and behind a transcription complex and a replisome. The complex and interconnected roles of topoisomerases and abundant small nucleoid association proteins are explained. And methods are described for comparing in vivo and in vitro reactions to probe and understand the temporal pathways of DNA and chromosome chemistry that occur inside living cells.

  1. Plasmids as Tools for Containment.

    PubMed

    García, José L; Díaz, Eduardo

    2014-10-01

    Active containment systems are a major tool for reducing the uncertainty associated with the introduction of monocultures, genetically engineered or not, into target habitats for a large number of biotechnological applications (e.g., bioremediation, bioleaching, biopesticides, biofuels, biotransformations, live vaccines, etc.). While biological containment reduces the survival of the introduced organism outside the target habitat and/or upon completion of the projected task, gene containment strategies reduce the lateral spread of the key genetic determinants to indigenous microorganisms. In fundamental research, suicide circuits become relevant tools to address the role of gene transfer, mainly plasmid transfer, in evolution and how this transfer contributes to genome plasticity and to the rapid adaptation of microbial communities to environmental changes. Many lethal functions and regulatory circuits have been used and combined to design efficient containment systems. As many new genomes are being sequenced, novel lethal genes and regulatory elements are available, e.g., new toxin-antitoxin modules, and they could be used to increase further the current containment efficiencies and to expand containment to other organisms. Although the current containment systems can increase the predictability of genetically modified organisms in the environment, containment will never be absolute, due to the existence of mutations that lead to the appearance of surviving subpopulations. In this sense, orthogonal systems (xenobiology) appear to be the solution for setting a functional genetic firewall that will allow absolute containment of recombinant organisms.

  2. Plasmid-mediated degradation of dibenzothiophene by Pseudomonas species.

    PubMed

    Monticello, D J; Bakker, D; Finnerty, W R

    1985-04-01

    The microbial transformation of dibenzothiophene (DBT) is of interest in the potential desulfurization of oil. We isolated three soil Pseudomonas species which oxidized DBT to characteristic water-soluble, sulfur-containing products. Two of our isolates harbored a 55-megadalton plasmid; growth in the presence of novobiocin resulted in both loss of the plasmid and loss of the ability to oxidize DBT. Reintroduction of the plasmid restored the ability to oxidize DBT to water-soluble products. The products resulting from the oxidation of DBT were characterized and included 3-hydroxy-2-formyl benzothiophene, 3-oxo-[3'-hydroxy-thionaphthenyl-(2)-methylene]-dihydrothionaph thene, and the hemiacetal and trans forms of 4-[2-(3-hydroxy)-thianaphthenyl]-2-oxo-3-butenoic acid. The products of DBT oxidation were inhibitory to cell growth and further DBT oxidation. DBT oxidation in our soil isolates was induced by naphthalene or salicylate and to a much lesser extent by DBT and was repressed by succinate.

  3. Isolation and physical characterization of streptomycete plasmids.

    PubMed

    Pernodet, J L; Guerineau, M

    1981-01-01

    Covalently closed circular DNA was isolated from a strain of Streptomyces coelicolor ATCC 10147 and from a strain of Streptomyces coelicolor subspecies flavus ATCC 19894, using two different methods. The two plasmids were of uniform monomer size: 8.9 kb for pS 10147, the plasmid from S. coelicolor ATCC 10147, and around 125 kb for the plasmid from S. coelicolor ATCC 19894. A restriction enzyme map was constructed for pS 10147, using seven enzymes. Four of the enzymes, (BamHI, Bgl,II, PvuII, and XhoI) cut pS 10147 once while PstI made two cuts. The GC content of this plasmid was calculated to be 72%. The possible utilisation of pS 10147 as a cloning vector in Streptomyces is discussed.

  4. Marine Diatom Plasmids and their Biotechnological Applications

    DTIC Science & Technology

    1992-02-27

    plasmid is homologous to the Tn21-type transposable elements. The element carries an open reading frame encoding a DNA invertase gene. Sequence comparisons...of regions upstream and downstream of the invertase gene indicate that the diatom plasmid is most similar to the Staphylococcus aureus transposon...the highly prokaryotic nature (i.e., codon usage bias, promoter sequences, etc.) of the invertase gene we have sequenced, we have tentatively

  5. Plasmid Replication Control by Antisense RNAs.

    PubMed

    Brantl, Sabine

    2014-08-01

    Plasmids are selfish genetic elements that normally constitute a burden for the bacterial host cell. This burden is expected to favor plasmid loss. Therefore, plasmids have evolved mechanisms to control their replication and ensure their stable maintenance. Replication control can be either mediated by iterons or by antisense RNAs. Antisense RNAs work through a negative control circuit. They are constitutively synthesized and metabolically unstable. They act both as a measuring device and a regulator, and regulation occurs by inhibition. Increased plasmid copy numbers lead to increasing antisense-RNA concentrations, which, in turn, result in the inhibition of a function essential for replication. On the other hand, decreased plasmid copy numbers entail decreasing concentrations of the inhibiting antisense RNA, thereby increasing the replication frequency. Inhibition is achieved by a variety of mechanisms, which are discussed in detail. The most trivial case is the inhibition of translation of an essential replication initiator protein (Rep) by blockage of the rep-ribosome binding site. Alternatively, ribosome binding to a leader peptide mRNA whose translation is required for efficient Rep translation can be prevented by antisense-RNA binding. In 2004, translational attenuation was discovered. Antisense-RNA-mediated transcriptional attenuation is another mechanism that has, so far, only been detected in plasmids of Gram-positive bacteria. ColE1, a plasmid that does not need a plasmid-encoded replication initiator protein, uses the inhibition of primer formation. In other cases, antisense RNAs inhibit the formation of an activator pseudoknot that is required for efficient Rep translation.

  6. Protein Diversity Confers Specificity in Plasmid Segregation

    PubMed Central

    Fothergill, Timothy J. G.; Barillà, Daniela; Hayes, Finbarr

    2005-01-01

    The ParG segregation protein (8.6 kDa) of multidrug resistance plasmid TP228 is a homodimeric DNA-binding factor. The ParG dimer consists of intertwined C-terminal domains that adopt a ribbon-helix-helix architecture and a pair of flexible, unstructured N-terminal tails. A variety of plasmids possess partition loci with similar organizations to that of TP228, but instead of ParG homologs, these plasmids specify a diversity of unrelated, but similarly sized, partition proteins. These include the proteobacterial pTAR, pVT745, and pB171 plasmids. The ParG analogs of these plasmids were characterized in parallel with the ParG homolog encoded by the pseudomonal plasmid pVS1. Like ParG, the four proteins are dimeric. No heterodimerization was detectable in vivo among the proteins nor with the prototypical ParG protein, suggesting that monomer-monomer interactions are specific among the five proteins. Nevertheless, as with ParG, the ParG analogs all possess significant amounts of unordered amino acid residues, potentially highlighting a common structural link among the proteins. Furthermore, the ParG analogs bind specifically to the DNA regions located upstream of their homologous parF-like genes. These nucleoprotein interactions are largely restricted to cognate protein-DNA pairs. The results reveal that the partition complexes of these and related plasmids have recruited disparate DNA-binding factors that provide a layer of specificity to the macromolecular interactions that mediate plasmid segregation. PMID:15805511

  7. Protein diversity confers specificity in plasmid segregation.

    PubMed

    Fothergill, Timothy J G; Barillà, Daniela; Hayes, Finbarr

    2005-04-01

    The ParG segregation protein (8.6 kDa) of multidrug resistance plasmid TP228 is a homodimeric DNA-binding factor. The ParG dimer consists of intertwined C-terminal domains that adopt a ribbon-helix-helix architecture and a pair of flexible, unstructured N-terminal tails. A variety of plasmids possess partition loci with similar organizations to that of TP228, but instead of ParG homologs, these plasmids specify a diversity of unrelated, but similarly sized, partition proteins. These include the proteobacterial pTAR, pVT745, and pB171 plasmids. The ParG analogs of these plasmids were characterized in parallel with the ParG homolog encoded by the pseudomonal plasmid pVS1. Like ParG, the four proteins are dimeric. No heterodimerization was detectable in vivo among the proteins nor with the prototypical ParG protein, suggesting that monomer-monomer interactions are specific among the five proteins. Nevertheless, as with ParG, the ParG analogs all possess significant amounts of unordered amino acid residues, potentially highlighting a common structural link among the proteins. Furthermore, the ParG analogs bind specifically to the DNA regions located upstream of their homologous parF-like genes. These nucleoprotein interactions are largely restricted to cognate protein-DNA pairs. The results reveal that the partition complexes of these and related plasmids have recruited disparate DNA-binding factors that provide a layer of specificity to the macromolecular interactions that mediate plasmid segregation.

  8. Polynucleotide sequence relationships among Ent plasmids and the relationship between Ent and other plasmids.

    PubMed Central

    So, M; Crosa, J H; Falkow, S

    1975-01-01

    Deoxyribonucleic acid-deoxyribonucleic acid hybridization studies reveal that the plasmids coding for the production of heat stable and heat labile enteroxtoxins of Escherichia coli, regardless of their origin, have a majority of their polynucleotide sequences in common, but are not related in any significant way to those plasmids coding for the synthesis of only ST toxin. The heat stable and heat labile plasmids also share a significant degree of their polynucleotide sequences with plasmids of the FI and FII incompatibility groups, but not with R factors belonging to the I, N, W, P, or X incompatibility groups. PMID:1090570

  9. Recombination-dependent concatemeric plasmid replication.

    PubMed Central

    Viret, J F; Bravo, A; Alonso, J C

    1991-01-01

    The replication of covalently closed circular supercoiled (form I) DNA in prokaryotes is generally controlled at the initiation level by a rate-limiting effector. Once initiated, replication proceeds via one of two possible modes (theta or sigma replication) which do not rely on functions involved in DNA repair and general recombination. Recently, a novel plasmid replication mode, leading to the accumulation of linear multigenome-length plasmid concatemers in both gram-positive and gram-negative bacteria, has been described. Unlike form I DNA replication, an intermediate recombination step is most probably involved in the initiation of concatemeric plasmid DNA replication. On the basis of structural and functional studies, we infer that recombination-dependent plasmid replication shares important features with phage late replication modes and, in several aspects, parallels the synthesis of plasmid concatemers in phage-infected cells. The characterization of the concatemeric plasmid replication mode has allowed new insights into the mechanisms of DNA replication and recombination in prokaryotes. PMID:1779931

  10. Plasmid-mediated mineralization of 4-chlorobiphenyl.

    PubMed Central

    Shields, M S; Hooper, S W; Sayler, G S

    1985-01-01

    Strains of Alcaligenes and Acinetobacter spp. were isolated from a mixed culture already proven to be proficient at complete mineralization of monohalogenated biphenyls. These strains were shown to harbor a 35 X 10(6)-dalton plasmid mediating a complete pathway for 4-chlorobiphenyl (4CB) oxidation. Subsequent plasmid curing of these bacteria resulted in the abolishment of the 4CB mineralization phenotype and loss of even early 4CB metabolism by Acinetobacter spp. Reestablishment of the Alcaligenes plasmid, denoted pSS50, in the cured Acinetobacter spp. via filter surface mating resulted in the restoration of 4CB mineralization abilities. 4CB mineralization, however, proved to be an unstable characteristic in some subcultured strains. Such loss was not found to coincide with any detectable alteration in plasmid size. Cultures capable of complete mineralization, as well as those limited to partial metabolism of 4CB, produced 4-chlorobenzoate as a metabolite. Demonstration of mineralization of a purified 14C-labeled chlorobenzoate showed it to be a true intermediate in 4CB mineralization. Unlike the mineralization capability, the ability to produce a metabolite has proven to be stable on subculture. These results indicate the occurrence of a novel plasmid, or evolved catabolic plasmid, that mediates the complete mineralization of 4CB. Images PMID:2993249

  11. [Progress in endogenous plasmid curing of bacteria--a review].

    PubMed

    Feng, Jun; Zhang, Wei; Song, Cunjiang

    2013-11-04

    To investigate the functions of the bacteria endogenous plasmid, which include bacterial drug resistance, symbiosis, capsular formation and heavy metal resistance, the endogenous plasmid needs to be cured first. We reviewed physical, chemical and molecular biological methods of endogenous plasmid curing, clarified the curing principles. The prospective of research on plasmid curing was also discussed, based on our own studies.

  12. Clostridium perfringens type A-E toxin plasmids.

    PubMed

    Freedman, John C; Theoret, James R; Wisniewski, Jessica A; Uzal, Francisco A; Rood, Julian I; McClane, Bruce A

    2015-05-01

    Clostridium perfringens relies upon plasmid-encoded toxin genes to cause intestinal infections. These toxin genes are associated with insertion sequences that may facilitate their mobilization and transfer, giving rise to new toxin plasmids with common backbones. Most toxin plasmids carry a transfer of clostridial plasmids locus mediating conjugation, which likely explains the presence of similar toxin plasmids in otherwise unrelated C. perfringens strains. The association of many toxin genes with insertion sequences and conjugative plasmids provides virulence flexibility when causing intestinal infections. However, incompatibility issues apparently limit the number of toxin plasmids maintained by a single cell.

  13. Clostridium perfringens type A–E toxin plasmids

    PubMed Central

    Freedman, John C.; Theoret, James R.; Wisniewski, Jessica A.; Uzal, Francisco A.; Rood, Julian I.; McClane, Bruce A.

    2014-01-01

    Clostridium perfringens relies upon plasmid-encoded toxin genes to cause intestinal infections. These toxin genes are associated with insertion sequences that may facilitate their mobilization and transfer, giving rise to new toxin plasmids with common backbones. Most toxin plasmids carry a transfer of clostridial plasmids locus mediating conjugation, which likely explains the presence of similar toxin plasmids in otherwise unrelated C. perfringens strains. The association of many toxin genes with insertion sequences and conjugative plasmids provides virulence flexibility when causing intestinal infections. However, incompatibility issues apparently limit the number of toxin plasmids maintained by a single cell. PMID:25283728

  14. Historical Events That Spawned the Field of Plasmid Biology.

    PubMed

    Kado, Clarence I

    2014-10-01

    This chapter revisits the historical development and outcome of studies focused on the transmissible, extrachromosomal genetic elements called plasmids. Early work on plasmids involved structural and genetic mapping of these molecules, followed by the development of an understanding of how plasmids replicate and segregate during cell division. The intriguing property of plasmid transmission between bacteria and between bacteria and higher cells has received considerable attention. The utilitarian aspects of plasmids are described, including examples of various plasmid vector systems. This chapter also discusses the functional attributes of plasmids needed for their persistence and survival in nature and in man-made environments. The term plasmid biology was first conceived at the Fallen Leaf Lake Conference on Promiscuous Plasmids, 1990, Lake Tahoe, California. The International Society for Plasmid Biology was established in 2004 (www.ISPB.org).

  15. Sequencing of IncX-plasmids suggests ubiquity of mobile forms of a biofilm-promoting gene cassette recruited from Klebsiella pneumoniae.

    PubMed

    Burmølle, Mette; Norman, Anders; Sørensen, Søren J; Hansen, Lars Hestbjerg

    2012-01-01

    Plasmids are a highly effective means with which genetic traits that influence human health, such as virulence and antibiotic resistance, are disseminated through bacterial populations. The IncX-family is a hitherto sparsely populated group of plasmids that are able to thrive within Enterobacteriaceae. In this study, a replicon-centric screening method was used to locate strains from wastewater sludge containing plasmids belonging to the IncX-family. A transposon aided plasmid capture method was then employed to transport IncX-plasmids from their original hosts (and co-hosted plasmids) into a laboratory strain (Escherichia coli Genehogs®) for further study. The nucleotide sequences of the three newly isolated IncX-plasmids (pLN126_33, pMO17_54, pMO440_54) and the hitherto un-sequenced type-plasmid R485 revealed a remarkable occurrence of whole or partial gene cassettes that promote biofilm-formation in Klebsiella pneumonia or E. coli, in all four instances. Two of the plasmids (R485 and pLN126_33) were shown to directly induce biofilm formation in a crystal violet retention assay in E. coli. Sequence comparison revealed that all plasmid-borne forms of the type 3 fimbriae encoding gene cassette mrkABCDF were variations of a composite transposon Tn6011 first described in the E. coli IncX plasmid pOLA52. In conclusion, IncX-plasmids isolated from Enterobacteriaceae over almost 40 years and on three different continents have all been shown to carry a type 3 fimbriae gene cassette mrkABCDF stemming from pathogenic K. pneumoniae. Apart from contributing general knowledge about IncX-plasmids, this study also suggests an apparent ubiquity of a mobile form of an important virulence factor and is an illuminating example of the recruitment, evolution and dissemination of genetic traits through plasmid-mediated horizontal gene transfer.

  16. Construction of pTM series plasmids for gene expression in Brucella species.

    PubMed

    Tian, Mingxing; Qu, Jing; Bao, Yanqing; Gao, Jianpeng; Liu, Jiameng; Wang, Shaohui; Sun, Yingjie; Ding, Chan; Yu, Shengqing

    2016-04-01

    Brucellosis, the most common widespread zoonotic disease, is caused by Brucella spp., which are facultative, intracellular, Gram-negative bacteria. With the development of molecular biology techniques, more and more virulence-associated factors have been identified in Brucella spp. A suitable plasmid system is an important tool to study virulence genes in Brucella. In this study, we constructed three constitutive replication plasmids (pTM1-Cm, pTM2-Amp, and pTM3-Km) using the replication origin (rep) region derived from the pBBR1-MCS vector. Also, a DNA fragment containing multiple cloning sites (MCSs) and a terminator sequence derived from the pCold vector were produced for complementation of the deleted genes. Besides pGH-6×His, a plasmid containing the groE promoter of Brucella spp. was constructed to express exogenous proteins in Brucella with high efficiency. Furthermore, we constructed the inducible expression plasmid pZT-6×His, containing the tetracycline-inducible promoter pzt1, which can induce expression by the addition of tetracycline in the Brucella culture medium. The constructed pTM series plasmids will play an important role in the functional investigation of Brucella spp.

  17. Loss of plasmids containing cloned inserts coding for novobiocin resistance or novobiocin sensitivity in Haemophilus influenzae

    SciTech Connect

    Setlow, J.K.; Spikes, D.; Ledbetter, M.

    1984-06-01

    Plasmids pNov1 and pNov1s, coding for resistance and sensitivity to novobiocin, respectively, were readily lost from wild-type Haemophilus influenzae but retained in a strain lacking an inducible defective prophage. The plasmid loss could be partly or wholly eliminated by a low-copy-number mutation in the plasmid or by the presence of certain antibiotic resistance markers in the host chromosome. Release of both phage HP1c1, measured by plaque assay, and defective phage, measured by electron microscopy, was increased when the plasmids were present. The frequency of recombination between pNov1 and the chromosome, causing the plasmid to be converted to pNov1s, could under some circumstances be decreased from the normal 60 to 70% to below 10% by the presence of a kanamycin resistance marker in the chromosome. This suggested that a gene product coded for by the plasmid, the expression of which was affected by the kanamycin resistance marker, was responsible for the high recombination frequency. Evidence was obtained from in vitro experiments that the gene product was a gyrase.

  18. Enhanced recognition of hydroxyl radical modified plasmid DNA by circulating cancer antibodies.

    PubMed

    Khan, F; Ali, A; Ali, R

    2005-06-01

    Reactive oxygen species have been implicated in various human diseases which are also responsible for the elimination of invading pathogens. In disease state and inflammatory responses, the excess of these radicals damage cellular macromolecules. DNA is susceptible to attacks by OH-induced damage. Oxidative DNA damage is an important factor in mutagenesis and carcinogenesis. In the present study, purified plasmid Bluescript DNA was modified by hydroxyl radical. Modifications incurred in DNA were characterized by physico-chemical techniques. Sera from patients of cancer were studied for their binding to native and hydroxyl radical modified plasmid DNA. Direct binding ELISA and competition binding results indicated that autoantibodies in cancer showed higher recognition to ROS-plasmid DNA as compared to the native form. Retarded mobility of the immune complex formation between IgG isolated from cancer sera using native and ROS-plasmid DNA as antigens reiterated preferential recognition of modified plasmid DNA by cancer autoantibodies. Therefore, it can be concluded that circulating autoantibodies in cancer sera bind preferentially to ROS-plasmid DNA as compared to native polymer. The data presented in the present communication suggest a role of ROS in the etiology of cancer.

  19. Identification of bacterial plasmids based on mobility and plasmid population biology.

    PubMed

    Garcillán-Barcia, Maria Pilar; Alvarado, Andrés; de la Cruz, Fernando

    2011-09-01

    Plasmids contain a backbone of core genes that remains relatively stable for long evolutionary periods, making sense to speak about plasmid species. The identification and characterization of the core genes of a plasmid species has a special relevance in the study of its epidemiology and modes of transmission. Besides, this knowledge will help to unveil the main routes that genes, for example antibiotic resistance (AbR) genes, use to travel from environmental reservoirs to human pathogens. Global dissemination of multiple antibiotic resistances and virulence traits by plasmids is an increasing threat for the treatment of many bacterial infectious diseases. To follow the dissemination of virulence and AbR genes, we need to identify the causative plasmids and follow their path from reservoirs to pathogens. In this review, we discuss how the existing diversity in plasmid genetic structures gives rise to a large diversity in propagation strategies. We would like to propose that, using an identification methodology based on plasmid mobility types, we can follow the propagation routes of most plasmids in Gammaproteobacteria, as well as their cargo genes, in complex ecosystems. Once the dissemination routes are known, designing antidissemination drugs and testing their efficacy will become feasible. We discuss in this review how the existing diversity in plasmid genetic structures gives rise to a large diversity in propagation strategies. We would like to propose that, by using an identification methodology based on plasmid mobility types, we can follow the propagation routes of most plasmids in ?-proteobacteria, as well as their cargo genes, in complex ecosystems.

  20. BIP induces mice CD19(hi) regulatory B cells producing IL-10 and highly expressing PD-L1, FasL.

    PubMed

    Tang, Youfa; Jiang, Qing; Ou, Yanghui; Zhang, Fan; Qing, Kai; Sun, Yuanli; Lu, Wenjie; Zhu, Huifen; Gong, Feili; Lei, Ping; Shen, Guanxin

    2016-01-01

    Many studies have shown that B cells possess a regulatory function in mouse models of autoimmune diseases. Regulatory B cells can modulate immune response through many types of molecular mechanisms, including the production of IL-10 and the expression of PD-1 Ligand and Fas Ligand, but the microenvironmental factors and mechanisms that induce regulatory B cells have not been fully identified. BIP (binding immunoglobulin protein), a member of the heat shock protein 70 family, is a type of evolutionarily highly conserved protein. In this article, we have found that IL-10(+), PD-L1(hi) and FasL(hi) B cells are discrete cell populations, but enriched in CD19(hi) cells. BIP can induce IL-10-producing splenic B cells, IL-10 secretion and B cells highly expressing PD-L1 and FasL. CD40 signaling acts in synergy with BIP to induce regulatory B cells. BIP increased surface CD19 molecule expression intensity and IL-10(+), PD-L1(hi) and FasL(hi) B cells induced by BIP share the CD19(hi) phenotype. Furthermore, B cells treated with BIP and anti-CD40 can lead to suppression of T cell proliferation and the effect is partially IL-10-dependent and mainly BIP-induced. Taken together, our findings identify a novel function of BIP in the induction of regulatory B cells and add a new reason for the therapy of autoimmune disorders or other inflammatory conditions.

  1. Cold atmospheric pressure plasma jet interactions with plasmid DNA

    SciTech Connect

    O'Connell, D.; Cox, L. J.; Hyland, W. B.; McMahon, S. J.; Reuter, S.; Graham, W. G.; Gans, T.; Currell, F. J.

    2011-01-24

    The effect of a cold (<40 deg. C) radio frequency-driven atmospheric pressure plasma jet on plasmid DNA has been investigated. Gel electrophoresis was used to analyze the DNA forms post-treatment. The experimental data are fitted to a rate equation model that allows for quantitative determination of the rates of single and double strand break formation. The formation of double strand breaks correlates well with the atomic oxygen density. Taken with other measurements, this indicates that neutral components in the jet are effective in inducing double strand breaks.

  2. N15: the linear phage-plasmid.

    PubMed

    Ravin, Nikolai V

    2011-03-01

    The lambdoid phage N15 of Escherichia coli is very unusual among temperate phages in that its prophage is not integrated into chromosome but is a linear plasmid molecule with covalently closed ends. Upon infection the phage DNA circularises via cohesive ends, then phage-encoded enzyme, protelomerase, cuts at an inverted repeat site and forms hairpin ends (telomeres) of the linear plasmid prophage. Replication of the N15 prophage is initiated at an internally located ori site and proceeds bidirectionally resulting in formation of duplicated telomeres. Then the N15 protelomerase cuts duplicated telomeres generating two linear plasmid molecules with hairpin telomeres. Stable inheritance of the plasmid prophage is ensured by partitioning operon similar to the F factor sop operon. Unlike F sop, the N15 centromere consists of four inverted repeats dispersed in the genome. The multiplicity and dispersion of centromeres are required for efficient partitioning of a linear plasmid. The centromeres are located in N15 genome regions involved in phage replication and control of lysogeny, and binding of partition proteins at these sites regulates these processes. Two N15-related lambdoid Siphoviridae phages, φKO2 in Klebsiella oxytoca and pY54 in Yersinia enterocolitica, also lysogenize their hosts as linear plasmids, as well as Myoviridae marine phages VP882 and VP58.5 in Vibrio parahaemolyticus and ΦHAP-1 in Halomonas aquamarina. The genomes of all these phages contain similar protelomerase genes, lysogeny modules and replication genes, as well as plasmid-partitioning genes, suggesting that these phages may belong to a group diverged from a common ancestor.

  3. Using Plasmids as DNA Vaccines for Infectious Diseases.

    PubMed

    Tregoning, John S; Kinnear, Ekaterina

    2014-12-01

    DNA plasmids can be used to induce a protective (or therapeutic) immune response by delivering genes encoding vaccine antigens. That naked DNA (without the refinement of coat proteins or host evasion systems) can cross from outside the cell into the nucleus and be expressed is particularly remarkable given the sophistication of the immune system in preventing infection by pathogens. As a result of the ease, low cost, and speed of custom gene synthesis, DNA vaccines dangle a tantalizing prospect of the next wave of vaccine technology, promising individual designer vaccines for cancer or mass vaccines with a rapid response time to emerging pandemics. There is considerable enthusiasm for the use of DNA vaccination as an approach, but this enthusiasm should be tempered by the successive failures in clinical trials to induce a potent immune response. The technology is evolving with the development of improved delivery systems that increase expression levels, particularly electroporation and the incorporation of genetically encoded adjuvants. This review will introduce some key concepts in the use of DNA plasmids as vaccines, including how the DNA enters the cell and is expressed, how it induces an immune response, and a summary of clinical trials with DNA vaccines. The review also explores the advances being made in vector design, delivery, formulation, and adjuvants to try to realize the promise of this technology for new vaccines. If the immunogenicity and expression barriers can be cracked, then DNA vaccines may offer a step change in mass vaccination.

  4. Electrotransformation of Yersinia ruckeri by plasmid DNA.

    PubMed

    Cutrín, J M; Conchas, R F; Barja, J L; Toranzo, A E

    1994-01-01

    Yersinia ruckeri, a fish pathogenic bacterium in aquaculture, was used to evaluate the electroporation as a new transformation method for this species. DNA used for the electrotransformation were plasmids of molecular mass ranging from 2.3 kb to 33 kb, and diverse replicons. To optimize this method we used Y. ruckeri 11.29 strain (from serotype 02) and pSU2718 DNA. The best transformation efficiency (6.0 x 10(5) transformants/micrograms DNA) was obtained with 12.5 kV/cm, 25 microF, 400 omega and 2 hours of incubation after pulse. When these conditions were applied to other strains belonging to different serotypes and other plasmids, we obtained transformants in all strains assayed, but only when using low molecular weight plasmids. Plasmid vectors and resident plasmid were not modified in host strains after electrotransformation. In studies of conformation we confirmed that only circular DNA was able for transformation. The utilization of this technique for direct cloning in Y. ruckeri makes possible further studies on recombinant DNA.

  5. Complete nucleotide sequence of plasmid pNA6 reveals the high plasticity of IncU family plasmids.

    PubMed

    Dang, Bingjun; Xu, Yan; Mao, Daqing; Luo, Yi

    2016-10-10

    Antibiotic resistance is a serious problem in health care and is of widespread public concern. Conjugative plasmids are the most important vectors in the dissemination of antibiotic resistance genes. In this study, we determined the complete sequence of plasmid pNA6, a plasmid which was isolated from the sediments of Haihe River. This plasmid confers reduced susceptibility to ampicillin, erythromycin and sulfamethoxazole. The complete sequence of plasmid pNA6 was 52,210bp in length with an average G+C content of 52.70%. Plasmid pNA6 belongs to the IncU group by sequence queries against the GenBank database. This plasmid has a typical IncU backbone and shows the highest similarities with plasmid RA3 and plasmid pFBAOT6. Plasmid pNA6 carries a class 1 integron consisting of aacA4, ereA and dfrA1 genes. Moreover, plasmid pNA6 also harbors a blaTEM-1-containing complex structure which inserted into the replication region and maintenance region. This insertion site has never been found on other IncU plasmids. The sequencing of plasmid pNA6 will add new sequence information to IncU family plasmids and enhance our understanding of the plasticity of IncU family plasmids.

  6. Targeting of plasmid DNA to renal interstitial fibroblasts by cationized gelatin.

    PubMed

    Kushibiki, Toshihiro; Nagata-Nakajima, Natsuki; Sugai, Manabu; Shimizu, Akira; Tabata, Yasuhiko

    2005-10-01

    Renal interstitial fibrosis is the common pathway of chronic renal disease, while it causes end-stage renal failure. A lot of cytokines and biologically active substances are well recognized to be the candidates of primary mediators to induce accumulation of extracelluar matrix (ECM) in the interstitial fibrotic area. Interstitial fibroblasts are played a crucial role in the accumulation of excess ECM during renal interstitial fibrogenesis. Therefore, the targeting of therapeutic drugs and genes to interstitial renal fibroblasts is effective in suppressing the progress of interstitial renal failure. However, despite various approaches and techniques, few successful results have been reported on the in vivo targeting for interstitial fibroblasts. The objective of this study is to deliver an enhanced green fluorescent protein (EGFP) plasmid DNA, as a model plasmid DNA, into renal interstitial space by a cationized gelatin. After the plasmid DNA with or without complexation of the cationized gelatin was injected to the left kidney of mice via the ureter, unilateral ureteral obstruction (UUO) was performed for the mice injected to induce the renal interstitial fibrosis. When the EGFP plasmid DNA complexed with the cationized gelatin was injected, EGFP expression was observed in the fibroblasts in the interstitial area of renal cortex. It is concluded that the retrograde injection of EGFP plasmid DNA complexed with the cationized gelatin is available to target the interstitial renal fibroblasts which are currently considered as the cell source responsible for excessive ECM synthesis.

  7. 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.

  8. Proteolysis in plasmid DNA stable maintenance in bacterial cells.

    PubMed

    Karlowicz, Anna; Wegrzyn, Katarzyna; Dubiel, Andrzej; Ropelewska, Malgorzata; Konieczny, Igor

    2016-07-01

    Plasmids, as extrachromosomal genetic elements, need to work out strategies that promote independent replication and stable maintenance in host bacterial cells. Their maintenance depends on constant formation and dissociation of nucleoprotein complexes formed on plasmid DNA. Plasmid replication initiation proteins (Rep) form specific complexes on direct repeats (iterons) localized within the plasmid replication origin. Formation of these complexes along with a strict control of Rep protein cellular concentration, quaternary structure, and activity, is essential for plasmid maintenance. Another important mechanism for maintenance of low-copy-number plasmids are the toxin-antitoxin (TA) post-segregational killing (psk) systems, which prevent plasmid loss from the bacterial cell population. In this mini review we discuss the importance of nucleoprotein complex processing by energy-dependent host proteases in plasmid DNA replication and plasmid type II toxin-antitoxin psk systems, and draw attention to the elusive role of DNA in this process.

  9. The partitioning and copy number control systems of the selfish yeast plasmid: an optimized molecular design for stable persistence in host cells.

    PubMed

    Yen-Ting-Liu; Sau, Saumitra; Ma, Chien-Hui; Kachroo, Aashiq H; Rowley, Paul A; Chang, Keng-Ming; Fan, Hsiu-Fang; Jayaram, Makkuni

    2014-10-01

    The multi-copy 2 micron plasmid of Saccharomyces cerevisiae, a resident of the nucleus, is remarkable for its high chromosome-like stability. The plasmid does not appear to contribute to the fitness of the host, nor does it impose a significant metabolic burden on the host at its steady state copy number. The plasmid may be viewed as a highly optimized selfish DNA element whose genome design is devoted entirely towards efficient replication, equal segregation and copy number maintenance. A partitioning system comprised of two plasmid coded proteins, Rep1 and Rep2, and a partitioning locus STB is responsible for equal or nearly equal segregation of plasmid molecules to mother and daughter cells. Current evidence supports a model in which the Rep-STB system promotes the physical association of the plasmid with chromosomes and thus plasmid segregation by a hitchhiking mechanism. The Flp site-specific recombination system housed by the plasmid plays a critical role in maintaining steady state plasmid copy number. A decrease in plasmid population due to rare missegregation events is rectified by plasmid amplification via a recombination induced rolling circle replication mechanism. Appropriate plasmid amplification, without runaway increase in copy number, is ensured by positive and negative regulation of FLP gene expression by plasmid coded proteins and by the control of Flp level/activity through host mediated post-translational modification(s) of Flp. The Flp system has been successfully utilized to understand mechanisms of site-specific recombination, to bring about directed genetic alterations for addressing fundamental problems in biology, and as a tool in biotechnological applications.

  10. Mutation detection in plasmid-based biopharmaceuticals.

    PubMed

    Oliveira, Pedro H; Prather, Kristala L J; Prazeres, Duarte M F; Monteiro, Gabriel A

    2011-04-01

    As the number of applications involving therapeutic plasmid DNA (pDNA) increases worldwide, there is a growing concern over maintaining rigorous quality control through a panel of high-quality assays. For this reason, efficient, cost-effective and sensitive technologies enabling the identification of genetic variants and unwanted side products are needed to successfully establish the identity and stability of a plasmid-based biopharmaceutical. This review highlights several bioinformatic tools for ab initio detection of potentially unstable DNA regions, as well as techniques used for mutation detection in nucleic acids, with particular emphasis on pDNA.

  11. Small-plasmid-mediated antibiotic resistance is enhanced by increases in plasmid copy number and bacterial fitness.

    PubMed

    San Millan, Alvaro; Santos-Lopez, Alfonso; Ortega-Huedo, Rafael; Bernabe-Balas, Cristina; Kennedy, Sean P; Gonzalez-Zorn, Bruno

    2015-01-01

    Plasmids play a key role in the horizontal spread of antibiotic resistance determinants among bacterial pathogens. When an antibiotic resistance plasmid arrives in a new bacterial host, it produces a fitness cost, causing a competitive disadvantage for the plasmid-bearing bacterium in the absence of antibiotics. On the other hand, in the presence of antibiotics, the plasmid promotes the survival of the clone. The adaptations experienced by plasmid and bacterium in the presence of antibiotics during the first generations of coexistence will be crucial for the progress of the infection and the maintenance of plasmid-mediated resistance once the treatment is over. Here we developed a model system using the human pathogen Haemophilus influenzae carrying the small plasmid pB1000 conferring resistance to β-lactam antibiotics to investigate host and plasmid adaptations in the course of a simulated ampicillin therapy. Our results proved that plasmid-bearing clones compensated for the fitness disadvantage during the first 100 generations of plasmid-host adaptation. In addition, ampicillin treatment was associated with an increase in pB1000 copy number. The augmentation in both bacterial fitness and plasmid copy number gave rise to H. influenzae populations with higher ampicillin resistance levels. In conclusion, we show here that the modulations in bacterial fitness and plasmid copy number help a plasmid-bearing bacterium to adapt during antibiotic therapy, promoting both the survival of the host and the spread of the plasmid.

  12. Plasmid Introduction in Metal-Stressed, Subsurface-Derived Microcosms: Plasmid Fate and Community Response

    PubMed Central

    Smets, Barth F.; Morrow, Jayne B.; Arango Pinedo, Catalina

    2003-01-01

    The nonconjugal IncQ plasmids pMOL187 and pMOL222, which contain the metal resistance-encoding genes czc and ncc, were introduced by using Escherichia coli as a transitory delivery strain into microcosms containing subsurface-derived parent materials. The microcosms were semicontinuously dosed with an artificial groundwater to set a low-carbon flux and a target metal stress (0, 10, 100, and 1,000 μM CdCl2), permitting long-term community monitoring. The broad-host-range IncPα plasmid RP4 was also transitorily introduced into a subset of microcosms. No novel community phenotype was detected after plasmid delivery, due to the high background resistances to Cd and Ni. At fixed Cd doses, however, small but consistent increases in Cdr or Nir density were measured due to the introduction of a single pMOL plasmid, and this effect was enhanced by the joint introduction of RP4; the effects were most significant at the highest Cd doses. The pMOL plasmids introduced could, however, be monitored via czc- and ncc-targeted infinite-dilution PCR (ID-PCR) methods, because these genes were absent from the indigenous community: long-term presence of czc (after 14 or 27 weeks) was contingent on the joint introduction of RP4, although RP4 cointroduction was not yet required to ensure retention of ncc after 8 weeks. Plasmids isolated from Nir transconjugants further confirmed the presence and retention of a pMOL222-sized plasmid. ID-PCR targeting the RP4-specific trafA gene revealed retention of RP4 for at least 8 weeks. Our findings confirm plasmid transfer and long-term retention in low-carbon-flux, metal-stressed subsurface communities but indicate that the subsurface community examined has limited mobilization potential for the IncQ plasmids employed. PMID:12839785

  13. Maintenance of a Pseudomonas fluorescens plasmid in heterologous hosts: metabolic burden as a more reliable variable to predict plasmid instability.

    PubMed

    Chandrasekaran, S; Lalithakumari, D

    1998-07-01

    The stability of a large, multiresistance plasmid, pSCL of P. fluorescens CAS102 was studied in Pseudomonas putida and E. coli under various non-stress conditions. Both the strains lost the plasmid within 25 days when repeatedly subcultured in LB broth without any antibiotic. The transformants survived in sterile soil and water without any marked reduction in the viability. In sterile soil, P. putida lost 93% and E. coli, 98% of their plasmid containing population in 30 days, while in sterile water the plasmid loss was 92.5% and 97% respectively. The two variables, viz. the efficiency of plasmid-partitioning during cell division and measurement of relative specific growth rates of plasmid-plus and plasmid-minus cells which are used to predict plasmid instability cannot be used to predict plasmid loss during starvation. The utility of a third variable, viz. the metabolic burden due to plasmid maintenance in predicting plasmid instability in different hosts is discussed. The rate of plasmid loss was found to be comparatively faster in E. coli than in P. putida. The biosynthetic burden due to plasmid maintenance was also more in E. coli than in P. putida when compared to the plasmid-plus and plasmid-minus cells of the two strains which was evident from the increased nutrient uptake rates (glucose, O2, and amino acid) and increased protein content of the plasmid-plus cells of E. coli. From the results, a correlation could be found between the degree of metabolic burden and the rate of plasmid loss. The reliability of metabolic burden, to predict plasmid instability versus the relative specific growth rates is discussed.

  14. Construction of disarmed Ti plasmids transferable between Escherichia coli and Agrobacterium species.

    PubMed

    Kiyokawa, Kazuya; Yamamoto, Shinji; Sakuma, Kei; Tanaka, Katsuyuki; Moriguchi, Kazuki; Suzuki, Katsunori

    2009-04-01

    Agrobacterium-mediated plant transformation has been used widely, but there are plants that are recalcitrant to this type of transformation. This transformation method uses bacterial strains harboring a modified tumor-inducing (Ti) plasmid that lacks the transfer DNA (T-DNA) region (disarmed Ti plasmid). It is desirable to develop strains that can broaden the host range. A large number of Agrobacterium strains have not been tested yet to determine whether they can be used in transformation. In order to improve the disarming method and to obtain strains disarmed and ready for the plant transformation test, we developed a simple scheme to make certain Ti plasmids disarmed and simultaneously maintainable in Escherichia coli and mobilizable between E. coli and Agrobacterium. To establish the scheme in nopaline-type Ti plasmids, a neighboring segment to the left of the left border sequence, a neighboring segment to the right of the right border sequence of pTi-SAKURA, a cassette harboring the pSC101 replication gene between these two segments, the broad-host-range IncP-type oriT, and the gentamicin resistance gene were inserted into a suicide-type sacB-containing vector. Replacement of T-DNA with the cassette in pTiC58 and pTi-SAKURA occurred at a high frequency and with high accuracy when the tool plasmid was used. We confirmed that there was stable maintenance of the modified Ti plasmids in E. coli strain S17-1lambdapir and conjugal transfer from E. coli to Ti-less Agrobacterium strains and that the reconstituted Agrobacterium strains were competent to transfer DNA into plant cells. As the modified plasmid delivery system was simple and efficient, conversion of strains to the disarmed type was easy and should be applicable in studies to screen for useful strains.

  15. R plasmid in Escherichia coli O103 coding for colonization of the rabbit intestinal tract.

    PubMed Central

    Reynaud, A; Federighi, M; Licois, D; Guillot, J F; Joly, B

    1991-01-01

    One rabbit pathogenic Escherichia coli strain, belonging to serogroup O103, harbors a self-transferable 117-kb plasmid (pREC-1) encoding resistance to several antibiotics. The role of this R plasmid in the colonization of the digestive tract in specific-pathogen-free (E. coli O103-free) rabbits was studied. Five-week-old rabbits were inoculated with the wild-type strain, with its variant cured of the plasmid, with an E. coli K-12 strain, or with an untypeable E. coli strain from a healthy rabbit. No symptoms and no mortality were observed in animals inoculated with strains without the plasmid pREC-1, but 87.5% of the rabbits infected by the wild strain died, generally with bloody diarrhea, between days 5 and 15 postinfection. The weight gain of animals was strongly reduced. Transfer of the plasmid to the cured strain or to nonvirulent strains led these strains to induce the same pathology but with a lower mortality. Colonization of the gut by the O103 strain and symptoms of bloody diarrhea are thus related to the presence of the pREC-1 plasmid. The GV strain, which does not produce classical heat-labile enterotoxin or heat-stable enterotoxin and is not invasive, could be considered an enteropathogenic E. coli-like strain. The presence of a conjugative plasmid such as pREC-1 encoding both antibiotic resistance and virulence determinants in O103 E. coli from rabbits could represent a prominent epidemiological hazard under selective pressure by antibiotic therapy. Images PMID:2037350

  16. The immunogenicity of viral haemorragic septicaemia rhabdovirus (VHSV) DNA vaccines can depend on plasmid regulatory sequences.

    PubMed

    Chico, V; Ortega-Villaizan, M; Falco, A; Tafalla, C; Perez, L; Coll, J M; Estepa, A

    2009-03-18

    A plasmid DNA encoding the viral hemorrhagic septicaemia virus (VHSV)-G glycoprotein under the control of 5' sequences (enhancer/promoter sequence plus both non-coding 1st exon and 1st intron sequences) from carp beta-actin gene (pAE6-G(VHSV)) was compared to the vaccine plasmid usually described the gene expression is regulated by the human cytomegalovirus (CMV) immediate-early promoter (pMCV1.4-G(VHSV)). We observed that these two plasmids produced a markedly different profile in the level and time of expression of the encoded-antigen, and this may have a direct effect upon the intensity and suitability of the in vivo immune response. Thus, fish genetic immunisation assays were carried out to study the immune response of both plasmids. A significantly enhanced specific-antibody response against the viral glycoprotein was found in the fish immunised with pAE6-G(VHSV). However, the protective efficacy against VHSV challenge conferred by both plasmids was similar. Later analysis of the transcription profile of a set of representative immune-related genes in the DNA immunized fish suggested that depending on the plasmid-related regulatory sequences controlling its expression, the plasmid might activate distinct patterns of the immune system. All together, the results from this study mainly point out that the selection of a determinate encoded-antigen/vector combination for genetic immunisation is of extraordinary importance in designing optimised DNA vaccines that, when required for inducing protective immune response, could elicit responses biased to antigen-specific antibodies or cytotoxic T cells generation.

  17. Molecular delivery of plasmids for genetic vaccination.

    PubMed

    Mazid, Romiza; Tan, Melvin X; Danquah, Michael K

    2013-01-01

    Plasmid vaccination is a smart gene delivery application mostly achieved through the utilisation of viral or copolymeric systems as surrogated carriers in micro or nano formulations. A common polymeric protocol for plasmid vaccine formulation, which as somewhat been successful, is via the complexation of the DNA molecules with a cationic polymer, and encapsulating in a vehicular carrier polymer. Even though plasmid vaccination research has not witnessed the much anticipated success, due a number of cellular and physicochemical reasons, application of copolymeric carriers with tight functionalities is a promising strategy to optimally deliver the DNA molecules; in view of the available chemistries and physical properties that could be tuned to enable enhanced targeted delivery, uptake and specific transfection. This also enables the targeting of specific epitopes and antigen presenting cells for the treatment of many pathogenic infections and cancer. This paper provides a brief critical review of the current state of plasmid vaccines formulation and molecular delivery with analysis of performance data obtained from clinical trials.

  18. The Virulence Plasmid of Yersinia, an Antihost Genome

    PubMed Central

    Cornelis, Guy R.; Boland, Anne; Boyd, Aoife P.; Geuijen, Cecile; Iriarte, Maite; Neyt, Cécile; Sory, Marie-Paule; Stainier, Isabelle

    1998-01-01

    The 70-kb virulence plasmid enables Yersinia spp. (Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica) to survive and multiply in the lymphoid tissues of their host. It encodes the Yop virulon, an integrated system allowing extracellular bacteria to disarm the cells involved in the immune response, to disrupt their communications, or even to induce their apoptosis by the injection of bacterial effector proteins. This system consists of the Yop proteins and their dedicated type III secretion apparatus, called Ysc. The Ysc apparatus is composed of some 25 proteins including a secretin. Most of the Yops fall into two groups. Some of them are the intracellular effectors (YopE, YopH, YpkA/YopO, YopP/YopJ, YopM, and YopT), while the others (YopB, YopD, and LcrV) form the translocation apparatus that is deployed at the bacterial surface to deliver the effectors into the eukaryotic cells, across their plasma membrane. Yop secretion is triggered by contact with eukaryotic cells and controlled by proteins of the virulon including YopN, TyeA, and LcrG, which are thought to form a plug complex closing the bacterial secretion channel. The proper operation of the system also requires small individual chaperones, called the Syc proteins, in the bacterial cytosol. Transcription of the genes is controlled both by temperature and by the activity of the secretion apparatus. The virulence plasmid of Y. enterocolitica and Y. pseudotuberculosis also encodes the adhesin YadA. The virulence plasmid contains some evolutionary remnants including, in Y. enterocolitica, an operon encoding resistance to arsenic compounds. PMID:9841674

  19. Diversity, biology and evolution of IncQ-family plasmids.

    PubMed

    Loftie-Eaton, Wesley; Rawlings, Douglas E

    2012-01-01

    Plasmids of IncQ-family are distinguished by having a unique strand-displacement mechanism of replication that is capable of functioning in a wide variety of bacterial hosts. In addition, these plasmids are highly mobilizable and therefore very promiscuous. Common features of the replicons have been used to identify IncQ-family plasmids in DNA sequence databases and in this way several unstudied plasmids have been compared to more well-studied IncQ plasmids. We propose that IncQ plasmids can be divided into four subgroups based on a number of mutually supportive criteria. The most important of these are the amino acid sequences of their three essential replication proteins and the observation that the replicon of each subgroup has become fused to four different lineages of mobilization genes. This review of IncQ-family plasmid diversity has highlighted several events in the evolution of these plasmids and raised several questions for further research.

  20. A novel method of plasmid isolation using laundry detergent.

    PubMed

    Yadav, P; Yadav, A; Garg, V; Datta, T K; Goswami, S L; De, S

    2011-07-01

    Since the discovery of plasmid, various methods have been developed to isolate plasmid DNA. All the methods have one common and important target of isolating plasmid DNA of high quality and quantity in less time. These methods are not completely safe because of use of toxic chemicals compounds. The developed protocol for plasmid extraction is based on the alkaline lysis method of plasmid preparation (extraction atpH 8.0) with slight modifications. Cell lysis reagent sodium dodecyl sulfate is replaced by lipase enzyme present in laundry detergent. A good plasmid preparation can be made, which is well suited for subsequent molecular biology applications. By taking safety measures on count, contaminants like, RNA and protein can be completely avoided with maximized plasmid yield. The resultant plasmid quality and quantity can be well comparable to other prevalent methods.

  1. Antibiotic resistance and R-plasmids in food chain Salmonella: evidence of plasmid relatedness.

    PubMed Central

    Bezanson, G S; Pauzé, M; Lior, H

    1981-01-01

    A large number of strains (1,783) belonging to 15 Salmonella serovars isolated, in Canada, from the three major links of the human food chain were screened for multiple antibiotic resistance and the presence of R-plasmids. Multiresistant strains occurred among animal feed, livestock, and human isolates at frequencies of 4, 22, and 14%, respectively. Conjugation analysis revealed that 58% of the isolates from feeds, 87% of those from livestock, and 89% of the human strains carried all or part of their resistance determinants extrachromosomally on R-plasmids. Conjugative plasmids representing nine different incompatibility groups were detected, with the Inc I alpha group being predominant. Within the limits of the parameters measured, certain of these plasmids show a degree of relatedness suggestive of a common ancestry. PMID:7013704

  2. Endogenous mutagenesis in recombinant sulfolobus plasmids.

    PubMed

    Sakofsky, Cynthia J; Grogan, Dennis W

    2013-06-01

    Low rates of replication errors in chromosomal genes of Sulfolobus spp. demonstrate that these extreme thermoacidophiles can maintain genome integrity in environments with high temperature and low pH. In contrast to this genetic stability, we observed unusually frequent mutation of the β-D-glycosidase gene (lacS) of a shuttle plasmid (pJlacS) propagated in Sulfolobus acidocaldarius. The resulting Lac(-) mutants also grew faster than the Lac(+) parent, thereby amplifying the impact of the frequent lacS mutations on the population. We developed a mutant accumulation assay and corrections for the effects of copy number and differential growth for this system; the resulting measurements and calculations yielded a corrected rate of 5.1 × 10(-4) mutational events at the lacS gene per plasmid replication. Analysis of independent lacS mutants revealed three types of mutations: (i) G · C-to-A · T transitions, (ii) slipped-strand events, and (iii) deletions. These mutations were frequent in plasmid-borne lacS expressed at a high level but not in single-copy lacS in the chromosome or at lower levels of expression in a plasmid. Substitution mutations arose at only two of 12 potential priming sites of the DNA primase of the pRN1 replicon, but nearly all these mutations created nonsense (chain termination) codons. The spontaneous mutation rate of plasmid-borne lacS was 175-fold higher under high-expression than under low-expression conditions. The results suggest that important DNA repair or replication fidelity functions are impaired or overwhelmed in pJlacS, with results analogous to those of the "transcription-associated mutagenesis" seen in bacteria and eukaryotes.

  3. Plasmid regulation and temperature-sensitive behavior of the Yersinia pestis penicillin-binding proteins.

    PubMed Central

    Ferreira, R C; Park, J T; Ferreira, L C

    1994-01-01

    Six major bands corresponding to penicillin-binding proteins (PBPs) with molecular weights ranging from 43,000 to 97,000 were detected in cell envelopes of Yersinia pestis EV76 grown at 28 degrees C. When cells were transferred to 37 degrees C and incubated for extended periods of time, the amounts of all PBPs, except for PBP2, were gradually reduced in cell envelopes of a strain carrying a 75-kb virulence-associated plasmid (as measured by penicillin-binding capacity), whereas in a strain cured of the plasmid, all PBPs were stable. The results indicated that the stability and/or the expression of Y. pestis PBPs is affected by a temperature-inducible pathway associated with the virulence-associated plasmid. Images PMID:8188365

  4. Generation of Small Colony Variants in Biofilms by Escherichia coli Harboring a Conjugative F Plasmid

    PubMed Central

    Tashiro, Yosuke; Eida, Hiroaki; Ishii, Satoshi; Futamata, Hiroyuki; Okabe, Satoshi

    2017-01-01

    A conjugative F plasmid induces mature biofilm formation by Escherichia coli by promoting F-pili-mediated cell-cell interactions and increasing the expression of biofilm-related genes. We herein demonstrated another function for the F plasmid in E. coli biofilms; it contributes to the emergence of genetic and phenotypic variations by spontaneous mutations. Small colony variants (SCVs) were more frequently generated in a continuous flow-cell biofilm than in the planktonic state of E. coli harboring the F plasmid. E. coli SCVs represented typical phenotypic changes such as slower growth, less biofilm formation, and greater resistance to aminoglycoside antibiotics than the parent strain. Genomic and complementation analyses indicated that the small colony phenotype was caused by the insertion of Tn1000, which was originally localized in the F plasmid, into the hemB gene. Furthermore, the Tn1000 insertion was removed from hemB in the revertant, which showed a normal colony phenotype. This study revealed that the F plasmid has the potential to increase genetic variations not only by horizontal gene transfer via F pili, but also by site-specific recombination within a single cell. PMID:28302951

  5. Elimination of plasmids by SILA compounds that inhibit efflux pumps of bacteria and cancer cells.

    PubMed

    Schelz, Zsuzsanna; Martins, Marta; Martins, Ana; Viveiros, Miguel; Molnar, Joseph; Amaral, Leonard

    2007-01-01

    Patented SILA compounds 409 and 421, previously shown to inhibit the efflux pumps of bacteria and cancer cells, have been studied for their ability to reduce or eliminate the presence of plasmids from Escherichia coli strains that have been induced to high level resistance to tetracycline by gradual exposure to increasing concentrations of the antibiotic. The results demonstrate that SILA compound 421, which has greater efflux pump inhibitory activity than its parent SILA compound 409, can reduce plasmid loads by 5 logs, over that present in the absence of the drug. The ability of the SILA compound to eliminate much larger plasmids is substantially lower. Because in vivo studies have shown that these compounds are not toxic to the mouse, the results obtained in our study suggest a potential role for SILA compound 421 as an adjunct for the therapy of antibiotic-resistant E. coli infections whose resistance is plasmid-mediated. In addition, because plasmid-mediated resistance is often found in tetracycline-treated cattle, SILA compound 421 may have potential as an adjunct during the time that the cattle are maintained on tetracycline prior to slaughter.

  6. Photoreactivation of Ultraviolet-Irradiated, Plasmid-Bearing and Plasmid-Free Strains of Bacillus anthracis

    DTIC Science & Technology

    1985-12-19

    NUMBER __ vation Bacillus anthracis) ś 7. AUTHOR(’a) B.KusnShD . CONTRACT OR GRANT NUMBER(a) PERFORMING ORGANIZATION NAME AND ADDRESS 10. PROGRAM ELEMENT... Bacillus anthracis, anthrax, photoreactivation, DNA repair, plasmid A6SSTACT (Cinvt ass,.yme eEb ir "mease wy f dentif by block nlmbaw) Iee. he...effects of toxin- a’nd capsule-encoding plasmids on the kinetics of UIV inactivation of various strains of Bacillus anthracis were investigated. :Z

  7. Isolation and screening of plasmids from the epilithon which mobilize recombinant plasmid pD10

    SciTech Connect

    Hill, K.E.; Weightman, A.J.; Fry, J.C. )

    1992-04-01

    This study examined the potential of bacteria from river epilithon to mobilize a recombinant catabolic plasmid, pD10, encoding 3-chlorobenzoate degradation and kanamycin resistance. Fifty-four mobilizing plasmids were exogenously isolated by triparental matings between strains of Pseudomonas putida and epilithic bacteria from the River Taff (South Wales, United Kingdom). Frequencies for mobilization ranged from 1.7 {times} 10{sup {minus}8} to 4.5 {times} 10{sup {minus}3} per recipient at 20C. The sizes of the mobilizing plasmids isolated ranged from 40 kb to over 200 kb, and 19 of 54 were found to encode mercury resistance. Plasmid-encoded resistance from 40 kb to over 200 kb, and 19 of 54 were found to encode mercury resistance. Plasmid-encoded resistance to tetracycline and streptomycin was also found but not resistance to UV light or various heavy metals. Eight plasmids of epilithic bacteria, analyzed by comparing restriction fragmentation patterns, showed significant differences between those isolated from different independent matings. Optimal temperatures for mobilization of pD10 were between 15 and 25C. Four mercury resistance plasmids were found to be broad host range, transferring mercury resistance and mobilizing pD10 readily to representative species of {beta}- and {gamma}-purple bacteria. In general, frequencies of pD10 mobilization by plasmids of epilithic bacteria were 2 to 3 orders of magnitude lower than conjugal transfer frequencies. Thus, there is a high potential for exchange of recombinant genes introduced into the epilithon by mobilization between a variety of bacterial species.

  8. Murine Langerin+ dermal dendritic cells prime CD8+ T cells while Langerhans cells induce cross-tolerance

    PubMed Central

    Flacher, Vincent; Tripp, Christoph H; Mairhofer, David G; Steinman, Ralph M; Stoitzner, Patrizia; Idoyaga, Juliana; Romani, Nikolaus

    2014-01-01

    Skin dendritic cells (DCs) control the immunogenicity of cutaneously administered vaccines. Antigens targeted to DCs via the C-type lectin Langerin/CD207 are cross-presented to CD8+ T cells in vivo. We investigated the relative roles of Langerhans cells (LCs) and Langerin+ dermal DCs (dDCs) in different vaccination settings. Poly(I:C) and anti-CD40 agonist antibody promoted cytotoxic responses upon intradermal immunization with ovalbumin (OVA)-coupled anti-Langerin antibodies (Langerin/OVA). This correlated with CD70 upregulation in Langerin+ dDCs, but not LCs. In chimeric mice where Langerin targeting was restricted to dDCs, CD8+ T-cell memory was enhanced. Conversely, providing Langerin/OVA exclusively to LCs failed to prime cytotoxicity, despite initial antigen cross-presentation to CD8+ T cells. Langerin/OVA combined with imiquimod could not prime CD8+ T cells and resulted in poor cytotoxicity in subsequent responses. This tolerance induction required targeting and maturation of LCs. Altogether, Langerin+ dDCs prime long-lasting cytotoxic responses, while cross-presentation by LCs negatively influences CD8+ T-cell priming. Moreover, this highlights that DCs exposed to TLR agonists can still induce tolerance and supports the existence of qualitatively different DC maturation programs. PMID:25085878

  9. Characterization of mal recombination plasmids cloned in Streptococcus pneumoniae

    SciTech Connect

    Stassi, D.L.; Lopez, P.; Espinosa, M.; Lacks, S.A.

    1981-01-01

    The malM locus of Streptococcus pneumoniae was cloned into one of the two PstI sites of the multicopy S. pneumoniae plasmid pMV158. To eliminate chromosomal transformants in the simultaneous selection for tetracycline resistance (coded by pMV158) and maltose utilization, the host cells contained a chromosomal deletion of the mal gene cluster. Two clones were isolated; one with a 3.3 kb insert (pLS70) which behaved like wild type with respect to maltose utilization, and another with a 2.9 kb insert (pLS69) which behaved as though it contained a down promoter mutation. Preliminary mapping of these clones by restriction analysis placed the 0.4kb deletion on a HindIII fragment in the interior of the chromosomal insert. The recombinant plasmids were able to transform over 50% of a recipient population to Mal/sup +/. Enzyme measurements of the clones indicated an overproduction of amylomaltase, constituting up to 10% of the total cellular protein, and supported the theory that the deletion in the pLS69 is in the promoter region. Protein analysis by polyacrylamide gel electrophoresis confirmed that the amylomaltase polypeptide was produced in large amounts in induced cells containing the pLS70. Another polypeptide, possibly a fragment of the phosphorylase or X protein of the mal gene cluster, was also produced to a similar extent.

  10. IL-27 induces the production of IgG1 by human B cells.

    PubMed

    Boumendjel, Amel; Tawk, Lina; Malefijt, René de Waal; Boulay, Vera; Yssel, Hans; Pène, Jérôme

    2006-12-01

    It has been reported that IL-27 specifically induces the production of IgG2a by mouse B cells and inhibits IL-4-induced IgG1 synthesis. Here, we show that human naïve cord blood expresses a functional IL-27 receptor, consisting of the TCCR and gp130 subunits, although at lower levels as compared to naïve and memory splenic B cells. IL-27 does not induce proliferative responses and does not increase IgG1 production by CD19(+)CD27(+) memory B cells. However, it induces a low, but significant production of IgG1 by naïve CD19(+)CD27(-)IgD(+)IgG(-) spleen and cord blood B cells, activated via CD40, whereas it has no effect on the production of the other IgG subclasses. In addition, IL-27 induces the differentiation of a population of B cells that express high levels of CD38, in association with a down-regulation of surface IgD expression, and that are surface IgG(+/int), CD20(low), CD27(high), indicating that IL-27 promotes isotype switching and plasma cell differentiation of naive B cells. However, as compared to the effects of IL-21 and IL-10, both switch factors for human IgG1 and IgG3, those of IL-27 are modest and regulate exclusively the production of IgG1. Finally, although IL-27 has no effect on IL-4 and anti-CD40-induced Cepsilon germline promoter activity, it up-regulates IL-4-induced IgE production by naive B cells. These results point to a partial redundancy of switch factors regulating the production of IgG1 in humans, and furthermore indicate the existence of a common regulation of the human IgG1and murine IgG2a isotypes by IL-27.

  11. Bacteriophage selection against a plasmid-encoded sex apparatus leads to the loss of antibiotic-resistance plasmids.

    PubMed

    Jalasvuori, Matti; Friman, Ville-Petri; Nieminen, Anne; Bamford, Jaana K H; Buckling, Angus

    2011-12-23

    Antibiotic-resistance genes are often carried by conjugative plasmids, which spread within and between bacterial species. It has long been recognized that some viruses of bacteria (bacteriophage; phage) have evolved to infect and kill plasmid-harbouring cells. This raises a question: can phages cause the loss of plasmid-associated antibiotic resistance by selecting for plasmid-free bacteria, or can bacteria or plasmids evolve resistance to phages in other ways? Here, we show that multiple antibiotic-resistance genes containing plasmids are stably maintained in both Escherichia coli and Salmonella enterica in the absence of phages, while plasmid-dependent phage PRD1 causes a dramatic reduction in the frequency of antibiotic-resistant bacteria. The loss of antibiotic resistance in cells initially harbouring RP4 plasmid was shown to result from evolution of phage resistance where bacterial cells expelled their plasmid (and hence the suitable receptor for phages). Phages also selected for a low frequency of plasmid-containing, phage-resistant bacteria, presumably as a result of modification of the plasmid-encoded receptor. However, these double-resistant mutants had a growth cost compared with phage-resistant but antibiotic-susceptible mutants and were unable to conjugate. These results suggest that bacteriophages could play a significant role in restricting the spread of plasmid-encoded antibiotic resistance.

  12. Plasmids of Distinct IncK Lineages Show Compatible Phenotypes

    PubMed Central

    Rozwandowicz, Marta; Brouwer, Michael S. M.; Zomer, Aldert L.; Bossers, Alex; Harders, Frank; Mevius, Dik J.; Wagenaar, Jaap A.

    2017-01-01

    ABSTRACT IncK plasmids are some of the main carriers of blaCTX-M-14 and blaCMY-2 genes and show high similarity to other plasmids belonging to the I complex, including IncB/O plasmids. Here, we studied the phylogenetic relationship of 37 newly sequenced IncK and IncB/O plasmids. We show that IncK plasmids can be divided into two compatible lineages named IncK1 and IncK2. PMID:28052854

  13. Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation network (PLACNET), a new method for plasmid reconstruction from whole genome sequences.

    PubMed

    Lanza, Val F; de Toro, María; Garcillán-Barcia, M Pilar; Mora, Azucena; Blanco, Jorge; Coque, Teresa M; de la Cruz, Fernando

    2014-12-01

    Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ-proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages.

  14. Plasmid Flux in Escherichia coli ST131 Sublineages, Analyzed by Plasmid Constellation Network (PLACNET), a New Method for Plasmid Reconstruction from Whole Genome Sequences

    PubMed Central

    Garcillán-Barcia, M. Pilar; Mora, Azucena; Blanco, Jorge; Coque, Teresa M.; de la Cruz, Fernando

    2014-01-01

    Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ–proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages. PMID:25522143

  15. Comparative genetic organization of incompatibility group P degradative plasmids.

    PubMed Central

    Burlage, R S; Bemis, L A; Layton, A C; Sayler, G S; Larimer, F

    1990-01-01

    Plasmids that encode genes for the degradation of recalcitrant compounds are often examined only for characteristics of the degradative pathways and ignore regions that are necessary for plasmid replication, incompatibility, and conjugation. If these characteristics were known, then the mobility of the catabolic genes between species could be predicted and different catabolic pathways might be combined to alter substrate range. Two catabolic plasmids, pSS50 and pSS60, isolated from chlorobiphenyl-degrading strains and a 3-chlorobenzoate-degrading plasmid, pBR60, were compared with the previously described IncP group (Pseudomonas group P-1) plasmids pJP4 and R751. All three of the former plasmids were also members of the IncP group, although pBR60 is apparently more distantly related. DNA probes specific for known genetic loci were used to determine the order of homologous loci on the plasmids. In all of these plasmids the order is invariant, demonstrating the conservation of this "backbone" region. In addition, all five plasmids display at least some homology with the mercury resistance transposon, Tn501, which has been suggested to be characteristic of the beta subgroup of the IncP plasmids. Plasmids pSS50 and pSS60 have been mapped in detail, and repeat sequences that surround the suspected degradation genes are described. Images PMID:2254257

  16. Plasmids of ’Legionella’ Species

    DTIC Science & Technology

    1982-03-09

    reports of cytotoxin and 8-lactamase production and the virulent to avirulent conversion of Legionella pneumophila through serial passage on...strains of L. pneumophila and 12 strains representing four other species of Legionella were screened for the presence of plasmid DNA’ by a variety of lysing...several well-established methods. Table 1. Legionella -like Strains Legionella pneumophila Legionella bozemanii OLDA WIGA, MI-15 Legionella micdadei

  17. Plasmid Stabilization to Insure Gene Expression

    DTIC Science & Technology

    1992-10-10

    suspended colony was used to initiate growth), independent growth rate measurements and a simple mathematical model, the kinetics of the loss of the LacZ...thermophilic anaerobe, C. thermocellum, an organism which degrades cellulose and hemicellulose at high temperature and carries out a direct fermentation... Kinetics of loss of a recombinant plasmid in Bacillus subtilis. Biotechnol. Bioeng. 37: 927-935. Shoham, Y., E. Israeli, A. L. Sonenshein and A. L

  18. The Influence of Biofilms in the Biology of Plasmids.

    PubMed

    Cook, Laura C C; Dunny, Gary M

    2014-10-01

    The field of plasmid biology has historically focused on bacteria growing in liquid culture. Surface-attached communities of bacterial biofilms have recently been understood to be the normal environment of bacteria in the natural world. Thus, studies examining plasmid replication, maintenance, and transfer in biofilms are essential for a true understanding of bacterial plasmid biology. This article reviews the current knowledge of the interplay between bacterial biofilms and plasmids, focusing on the role of plasmids in biofilm development and the role of biofilms in plasmid maintenance, copy-number control, and transfer. The studies examined herein highlight the importance of biofilms as an important ecological niche in which bacterial plasmids play an essential role.

  19. Modeling sRNA-Regulated Plasmid Maintenance

    PubMed Central

    Klumpp, Stefan

    2017-01-01

    We study a theoretical model for the toxin-antitoxin (hok/sok) mechanism for plasmid maintenance in bacteria. Toxin-antitoxin systems enforce the maintenance of a plasmid through post-segregational killing of cells that have lost the plasmid. Key to their function is the tight regulation of expression of a protein toxin by an sRNA antitoxin. Here, we focus on the nonlinear nature of the regulatory circuit dynamics of the toxin-antitoxin mechanism. The mechanism relies on a transient increase in protein concentration rather than on the steady state of the genetic circuit. Through a systematic analysis of the parameter dependence of this transient increase, we confirm some known design features of this system and identify new ones: for an efficient toxin-antitoxin mechanism, the synthesis rate of the toxin’s mRNA template should be lower that of the sRNA antitoxin, the mRNA template should be more stable than the sRNA antitoxin, and the mRNA-sRNA complex should be more stable than the sRNA antitoxin. Moreover, a short half-life of the protein toxin is also beneficial to the function of the toxin-antitoxin system. In addition, we study a therapeutic scenario in which a competitor mRNA is introduced to sequester the sRNA antitoxin, causing the toxic protein to be expressed. PMID:28085919

  20. Plasmid R6K replication control.

    PubMed

    Rakowski, Sheryl A; Filutowicz, Marcin

    2013-05-01

    The focus of this minireview is the replication control of the 39.9-kb plasmid R6K and its derivatives. Historically, this plasmid was thought to have a narrow host range but more recent findings indicate that its derivatives can replicate in a variety of enteric and non-enteric bacterial species (Wild et al., 2004). In the four-plus decades since it was first described, R6K has proven to be an excellent model for studies of plasmid DNA replication. In part this is because of its similarities to other systems in which replication is activated and regulated by Rep protein and iteron-containing DNA. However its apparent idiosynchracies have also added to its significance (e.g., independent and co-dependent replication origins, and Rep dimers that stably bind iterons). Here, we survey the current state of knowledge regarding R6K replication and place individual regulatory elements into a proposed homeostatic model with implications for the biological significance of R6K and its multiple origins of replication.

  1. Plasmid-cured Chlamydia caviae activates TLR2-dependent signaling and retains virulence in the guinea pig model of genital tract infection.

    PubMed

    Frazer, Lauren C; Darville, Toni; Chandra-Kuntal, Kumar; Andrews, Charles W; Zurenski, Matthew; Mintus, Margaret; AbdelRahman, Yasser M; Belland, Robert J; Ingalls, Robin R; O'Connell, Catherine M

    2012-01-01

    Loss of the conserved "cryptic" plasmid from C. trachomatis and C. muridarum is pleiotropic, resulting in reduced innate inflammatory activation via TLR2, glycogen accumulation and infectivity. The more genetically distant C. caviae GPIC is a natural pathogen of guinea pigs and induces upper genital tract pathology when inoculated intravaginally, modeling human disease. To examine the contribution of pCpGP1 to C. caviae pathogenesis, a cured derivative of GPIC, strain CC13, was derived and evaluated in vitro and in vivo. Transcriptional profiling of CC13 revealed only partial conservation of previously identified plasmid-responsive chromosomal loci (PRCL) in C. caviae. However, 2-deoxyglucose (2DG) treatment of GPIC and CC13 resulted in reduced transcription of all identified PRCL, including glgA, indicating the presence of a plasmid-independent glucose response in this species. In contrast to plasmid-cured C. muridarum and C. trachomatis, plasmid-cured C. caviae strain CC13 signaled via TLR2 in vitro and elicited cytokine production in vivo similar to wild-type C. caviae. Furthermore, inflammatory pathology induced by infection of guinea pigs with CC13 was similar to that induced by GPIC, although we observed more rapid resolution of CC13 infection in estrogen-treated guinea pigs. These data indicate that either the plasmid is not involved in expression or regulation of virulence in C. caviae or that redundant effectors prevent these phenotypic changes from being observed in C. caviae plasmid-cured strains.

  2. Plasmid-Cured Chlamydia caviae Activates TLR2-Dependent Signaling and Retains Virulence in the Guinea Pig Model of Genital Tract Infection

    PubMed Central

    Frazer, Lauren C.; Darville, Toni; Chandra-Kuntal, Kumar; Andrews, Charles W.; Zurenski, Matthew; Mintus, Margaret; AbdelRahman, Yasser M.; Belland, Robert J.; Ingalls, Robin R.; O'Connell, Catherine M.

    2012-01-01

    Loss of the conserved “cryptic” plasmid from C. trachomatis and C. muridarum is pleiotropic, resulting in reduced innate inflammatory activation via TLR2, glycogen accumulation and infectivity. The more genetically distant C. caviae GPIC is a natural pathogen of guinea pigs and induces upper genital tract pathology when inoculated intravaginally, modeling human disease. To examine the contribution of pCpGP1 to C. caviae pathogenesis, a cured derivative of GPIC, strain CC13, was derived and evaluated in vitro and in vivo. Transcriptional profiling of CC13 revealed only partial conservation of previously identified plasmid-responsive chromosomal loci (PRCL) in C. caviae. However, 2-deoxyglucose (2DG) treatment of GPIC and CC13 resulted in reduced transcription of all identified PRCL, including glgA, indicating the presence of a plasmid-independent glucose response in this species. In contrast to plasmid-cured C. muridarum and C. trachomatis, plasmid-cured C. caviae strain CC13 signaled via TLR2 in vitro and elicited cytokine production in vivo similar to wild-type C. caviae. Furthermore, inflammatory pathology induced by infection of guinea pigs with CC13 was similar to that induced by GPIC, although we observed more rapid resolution of CC13 infection in estrogen-treated guinea pigs. These data indicate that either the plasmid is not involved in expression or regulation of virulence in C. caviae or that redundant effectors prevent these phenotypic changes from being observed in C. caviae plasmid-cured strains. PMID:22292031

  3. Plasmid and clonal interference during post horizontal gene transfer evolution.

    PubMed

    Bedhomme, S; Perez Pantoja, D; Bravo, I G

    2017-02-16

    Plasmids are nucleic acid molecules that can drive their own replication in a living cell. They can be transmitted horizontally and can thrive in the host cell to high-copy numbers. Plasmid replication and gene expression consume cellular resources and cells carrying plasmids incur fitness costs. But many plasmids carry genes that can be beneficial under certain conditions, allowing the cell to endure in the presence of antibiotics, toxins, competitors or parasites. Horizontal transfer of plasmid-encoded genes can thus instantaneously confer differential adaptation to local or transient selection conditions. This conflict between cellular fitness and plasmid spread sets the scene for multilevel selection processes. We have engineered a system to study the short-term evolutionary impact of different synonymous versions of a plasmid-encoded antibiotic resistance gene. Applying experimental evolution under different selection conditions and deep sequencing allowed us to show rapid local adaptation to the presence of antibiotic and to the specific version of the resistance gene transferred. We describe the presence of clonal interference at two different levels: at the within-cell level, because a single cell can carry several plasmids, and at the between-cell level, because a bacterial population may contain several clones carrying different plasmids and displaying different fitness in the presence/absence of antibiotic. Understanding the within-cell and between-cell dynamics of plasmids after horizontal gene transfer is essential to unravel the dense network of mobile elements underlying the worldwide threat to public health of antibiotic resistance.

  4. Plasmid accumulation reduces life span in Saccharomyces cerevisiae.

    PubMed

    Falcón, Alaric A; Aris, John P

    2003-10-24

    Aging in the yeast Saccharomyces cerevisiae is under the control of multiple pathways. The production and accumulation of extrachromosomal rDNA circles (ERCs) is one pathway that has been proposed to bring about aging in yeast. To test this proposal, we have developed a plasmid-based model system to study the role of DNA episomes in reduction of yeast life span. Recombinant plasmids containing different replication origins, cis-acting partitioning elements, and selectable marker genes were constructed and analyzed for their effects on yeast replicative life span. Plasmids containing the ARS1 replication origin reduce life span to the greatest extent of the plasmids analyzed. This reduction in life span is partially suppressed by a CEN4 centromeric element on ARS1 plasmids. Plasmids containing a replication origin from the endogenous yeast 2 mu circle also reduce life span, but to a lesser extent than ARS1 plasmids. Consistent with this, ARS1 and 2 mu origin plasmids accumulate in approximately 7-generation-old cells, but ARS1/CEN4 plasmids do not. Importantly, ARS1 plasmids accumulate to higher levels in old cells than 2 mu origin plasmids, suggesting a correlation between plasmid accumulation and life span reduction. Reduction in life span is neither an indirect effect of increased ERC levels nor the result of stochastic cessation of growth. The presence of a fully functional 9.1-kb rDNA repeat on plasmids is not required for, and does not augment, reduction in life span. These findings support the view that accumulation of DNA episomes, including episomes such as ERCs, cause cell senescence in yeast.

  5. Effect of cumin (Cuminum cyminum) seed essential oil on biofilm formation and plasmid Integrity of Klebsiella pneumoniae.

    PubMed

    Derakhshan, Safoura; Sattari, Morteza; Bigdeli, Mohsen

    2010-01-01

    Seeds of the cumin plant (Cuminum cyminum L.) have been used since many years in Iranian traditional medicine. We assessed the effect of cumin seed essential oil on the biofilm-forming ability of Klebsiella pneumoniae strains and on the integrity of a native resistance plasmid DNA from K. pneumoniae isolates, treated with essential oil. Antibacterial coaction between the essential oil and selected antibiotic disks were determined for inhibiting K. pneumoniae. The essential oil of the cumin seeds was obtained by hydrodistillation in a Clavenger system. A simple method for the formation of biofilms on semiglass lamellas was established. The biofilms formed were observed by scanning electron microscopy (SEM). The effect of essential oil on plasmid integrity was studied through the induction of R-plasmid DNA degradation. The plasmid was incubated with essential oil, and agarose gel electrophoresis was performed. Disk diffusion assay was employed to determine the coaction. The essential oil decreased biofilm formation and enhanced the activity of the ciprofloxacin disk. The incubation of the R-plasmid DNA with essential oil could not induce plasmid DNA degradation. The results of this study suggest the potential use of cumin seed essential oil against K. pneumoniae in vitro, may contribute to the in vivo efficacy of this essential oil.

  6. [Stability of the NPL-1 and NPL-41 plasmids of naphthalene biodegradation in Pseudomonas putida populations in continuous culture].

    PubMed

    Boronin, A M; Filonov, A E; Balakshina, V V; Kulakova, A N

    1985-01-01

    The stability of biodegradation plasmids NPL-1 and NPL-41, which control the synthesis of enzymes for naphthalene oxidation to salicylate, was studied in Pseudomonas putida BSA under the conditions of its continuous cultivation with limitation in glucose or salicylate in the chemostat regime and without limitation in the pH-stat regime. Plasmid NPL-1, which controls the inducible synthesis of naphthalene oxygenase, is stable in the population of P. putida cells under the conditions of continuous cultivation on glucose, but is not stable in the course of cultivation on salicylate, an inductor of the naphthalene oxygenase synthesis. Plasmid NPL-41, which controls the constitutive synthesis of naphthalene oxygenase, is not stable in the population of P. putida cells under the conditions of continuous cultivation on glucose. The operation of genes, which control the oxidation of naphthalene to salicylate (nah), makes plasmids NPL-1 and NPL-41 unstable under the conditions of continuous cultivation in the absence of naphthalene from the medium, i.e. under the conditions when the expression of these genes is not necessary. In that case, cells containing plasmids with a deletion of nah-genes as well as cells without plasmids appear in the population of P. putida, which causes a decline in its futile energy and metabolic processes.

  7. Therapeutic option of plasmid-DNA based gene transfer.

    PubMed

    Taniyama, Yoshiaki; Azuma, Junya; Kunugiza, Yasuo; Iekushi, Kazuma; Rakugi, Hiromi; Morishita, Ryuichi

    2012-01-01

    Gene therapy offers a novel approach for the prevention and treatment of a variety of diseases, but it is not yet a common method in clinical cases because of various problems. Viral vectors show high efficiency of gene transfer, but they have some problems with toxicity and immunity. On the other hand, plasmid deoxyribonucleic acid (DNA)-based gene transfer is very safe, but its efficiency is relatively low. Especially, plasmid DNA gene therapy is used for cardiovascular disease because plasmid DNA transfer is possible for cardiac or skeletal muscle. Clinical angiogenic gene therapy using plasmid DNA gene transfer has been attempted in patients with peripheral artery disease, but a phase III clinical trial did not show sufficient efficiency. In this situation, more efficient plasmid DNA gene transfer is needed all over the world. This review focuses on plasmid DNA gene transfer and its enhancement, including ultrasound with microbubbles, electroporation, hydrodynamic method, gene gun, jet injection, cationic lipids and cationic polymers.

  8. Identification and sequence homology relationships of plasmids from various micrococci

    SciTech Connect

    Mathis, J.N.

    1983-01-01

    Plasmids have been found in strains of the following Micrococcus species M. nishinomiyaensis (9/22), M. luteus (8/47), and M. agilis (1/5). No plasmids were detected in strains of M. lylae (0/16) or M. sedentarius (0/20). Thirty-eight antibiotics and 23 inorganic salts were screened in an attempt to determine plasmid function. None of these antibiotics and inorganic salts were found to be associated with the presence or absence of plasmid DNA within these strains. Minimum inhibitory concentration experiments and curing experiments in which phenotypic change occurred without plasmid loss are the basis for this conclusion. Hydrocarbon biosynthesis parameters in certain Micrococcus strains previously analyzed were also shown not to be clearly associated to the presence or absence of plasmid DNA.

  9. Differential requirement for SUB1 in chromosomal and plasmid double-strand DNA break repair.

    PubMed

    Yu, Lijian; Volkert, Michael R

    2013-01-01

    Non homologous end joining (NHEJ) is an important process that repairs double strand DNA breaks (DSBs) in eukaryotic cells. Cells defective in NHEJ are unable to join chromosomal breaks. Two different NHEJ assays are typically used to determine the efficiency of NHEJ. One requires NHEJ of linearized plasmid DNA transformed into the test organism; the other requires NHEJ of a single chromosomal break induced either by HO endonuclease or the I-SceI restriction enzyme. These two assays are generally considered equivalent and rely on the same set of NHEJ genes. PC4 is an abundant DNA binding protein that has been suggested to stimulate NHEJ. Here we tested the role of PC4's yeast homolog SUB1 in repair of DNA double strand breaks using different assays. We found SUB1 is required for NHEJ repair of DSBs in plasmid DNA, but not in chromosomal DNA. Our results suggest that these two assays, while similar are not equivalent and that repair of plasmid DNA requires additional factor(s) that are not required for NHEJ repair of chromosomal double-strand DNA breaks. Possible roles for Sub1 proteins in NHEJ of plasmid DNA are discussed.

  10. Bovine papillomavirus vector that propagates as a plasmid in both mouse and bacterial cells.

    PubMed

    DiMaio, D; Treisman, R; Maniatis, T

    1982-07-01

    We report the construction of a bovine papillomavirus (BPV)-derived recombinant plasmid that propagates as an extrachromosomal element in both mouse and bacterial cells. Plasmids composed of a subgenomic transforming fragment of BPV DNA, a deletion derivative of pBR322, and a 7.6-kilobase fragment of DNA from the human beta-globin gene cluster efficiently induce focus formation on mouse C127 cells. BPV-beta-globin hybrids are maintained in the transformed cells as plasmids with a copy number of about 10-30 per cell. Plasmids indistinguishable from the input DNA have been recovered by transformation of bacteria with low molecular weight DNA from transformed mouse cells. The human beta-globin gene linked to BPV DNA is transcribed from its own promoter at a high level in these cells. The expression of BPV-linked cellular genes in conjunction with the ability to shuttle DNA between bacteria and mammalian cells may provide a rapid means of analyzing and recovering genes that confer an identifiable phenotype upon mammalian cells.

  11. Proteomic profiling of salivary gland after nonviral gene transfer mediated by conventional plasmids and minicircles

    PubMed Central

    Geguchadze, Ramaz; Wang, Zhimin; Zourelias, Lee; Perez-Riveros, Paola; Edwards, Paul C; Machen, Laurie; Passineau, Michael J

    2014-01-01

    In this study, we compared gene transfer efficiency and host response to ultrasound-assisted, nonviral gene transfer with a conventional plasmid and a minicircle vector in the submandibular salivary glands of mice. Initially, we looked at gene transfer efficiency with equimolar amounts of the plasmid and minicircle vectors, corroborating an earlier report showing that minicircle is more efficient in the context of a physical method of gene transfer. We then sought to characterize the physiological response of the salivary gland to exogenous gene transfer using global proteomic profiling. Somewhat surprisingly, we found that sonoporation alone, without a gene transfer vector present, had virtually no effect on the salivary gland proteome. However, when a plasmid vector was used, we observed profound perturbations of the salivary gland proteome that compared in magnitude to that seen in a previous report after high doses of adeno-associated virus. Finally, we found that gene transfer with a minicircle induces only minor proteomic alterations that were similar to sonoporation alone. Using mass spectrometry, we assigned protein IDs to 218 gel spots that differed between plasmid and minicircle. Bioinformatic analysis of these proteins demonstrated convergence on 68 known protein interaction pathways, most notably those associated with innate immunity, cellular stress, and morphogenesis. PMID:25414909

  12. Community-wide plasmid gene mobilization and selection

    PubMed Central

    Sentchilo, Vladimir; Mayer, Antonia P; Guy, Lionel; Miyazaki, Ryo; Green Tringe, Susannah; Barry, Kerrie; Malfatti, Stephanie; Goessmann, Alexander; Robinson-Rechavi, Marc; van der Meer, Jan R

    2013-01-01

    Plasmids have long been recognized as an important driver of DNA exchange and genetic innovation in prokaryotes. The success of plasmids has been attributed to their independent replication from the host's chromosome and their frequent self-transfer. It is thought that plasmids accumulate, rearrange and distribute nonessential genes, which may provide an advantage for host proliferation under selective conditions. In order to test this hypothesis independently of biases from culture selection, we study the plasmid metagenome from microbial communities in two activated sludge systems, one of which receives mostly household and the other chemical industry wastewater. We find that plasmids from activated sludge microbial communities carry among the largest proportion of unknown gene pools so far detected in metagenomic DNA, confirming their presumed role of DNA innovators. At a system level both plasmid metagenomes were dominated by functions associated with replication and transposition, and contained a wide variety of antibiotic and heavy metal resistances. Plasmid families were very different in the two metagenomes and grouped in deep-branching new families compared with known plasmid replicons. A number of abundant plasmid replicons could be completely assembled directly from the metagenome, providing insight in plasmid composition without culturing bias. Functionally, the two metagenomes strongly differed in several ways, including a greater abundance of genes for carbohydrate metabolism in the industrial and of general defense factors in the household activated sludge plasmid metagenome. This suggests that plasmids not only contribute to the adaptation of single individual prokaryotic species, but of the prokaryotic community as a whole under local selective conditions. PMID:23407308

  13. Drug resistance plasmids in Lactobacillus acidophilus and Lactobacillus reuteri.

    PubMed Central

    Vescovo, M; Morelli, L; Bottazzi, V

    1982-01-01

    Sixteen strains of Lactobacillus reuteri and 20 strains of Lactobacillus acidophilus were tested for resistance to 22 antibiotics by using commercially available sensitivity disks. Evidence suggesting linkage of these resistances to plasmids was obtained by "curing" experiments with acridine dyes and high growth temperatures. Examination of plasmid patterns of agarose gel electrophoresis provided further evidence of loss in plasmid DNA under curing conditions in some of the strains examined. Images PMID:6798933

  14. Large Linear Plasmids of Borrelia Species That Cause Relapsing Fever

    PubMed Central

    Porcella, Stephen F.; Raffel, Sandra J.; Schwan, Tom G.; Barbour, Alan G.

    2013-01-01

    Borrelia species of relapsing fever (RF) and Lyme disease (LD) lineages have linear chromosomes and both linear and circular plasmids. Unique to RF species, and little characterized to date, are large linear plasmids of ∼160 kb, or ∼10% of the genome. By a combination of Sanger and next-generation methods, we determined the sequences of large linear plasmids of two New World species: Borrelia hermsii, to completion of its 174-kb length, and B. turicatae, partially to 114 kb of its 150 kb. These sequences were then compared to corresponding sequences of the Old World species B. duttonii and B. recurrentis and to plasmid sequences of LD Borrelia species. The large plasmids were largely colinear, except for their left ends, about 27 kb of which was inverted in New World species. Approximately 60% of the B. hermsii lp174 plasmid sequence was repetitive for 6 types of sequence, and half of its open reading frames encoded hypothetical proteins not discernibly similar to proteins in the database. The central ∼25 kb of all 4 linear plasmids was syntenic for orthologous genes for plasmid maintenance or partitioning in Borrelia species. Of all the sequenced linear and circular plasmids in Borrelia species, the large plasmid's putative partition/replication genes were most similar to those of the 54-kb linear plasmids of LD species. Further evidence for shared ancestry was the observation that two of the hypothetical proteins were predicted to be structurally similar to the LD species' CspA proteins, which are encoded on the 54-kb plasmids. PMID:23749977

  15. Large linear plasmids of Borrelia species that cause relapsing fever.

    PubMed

    Miller, Shelley Campeau; Porcella, Stephen F; Raffel, Sandra J; Schwan, Tom G; Barbour, Alan G

    2013-08-01

    Borrelia species of relapsing fever (RF) and Lyme disease (LD) lineages have linear chromosomes and both linear and circular plasmids. Unique to RF species, and little characterized to date, are large linear plasmids of ∼160 kb, or ∼10% of the genome. By a combination of Sanger and next-generation methods, we determined the sequences of large linear plasmids of two New World species: Borrelia hermsii, to completion of its 174-kb length, and B. turicatae, partially to 114 kb of its 150 kb. These sequences were then compared to corresponding sequences of the Old World species B. duttonii and B. recurrentis and to plasmid sequences of LD Borrelia species. The large plasmids were largely colinear, except for their left ends, about 27 kb of which was inverted in New World species. Approximately 60% of the B. hermsii lp174 plasmid sequence was repetitive for 6 types of sequence, and half of its open reading frames encoded hypothetical proteins not discernibly similar to proteins in the database. The central ∼25 kb of all 4 linear plasmids was syntenic for orthologous genes for plasmid maintenance or partitioning in Borrelia species. Of all the sequenced linear and circular plasmids in Borrelia species, the large plasmid's putative partition/replication genes were most similar to those of the 54-kb linear plasmids of LD species. Further evidence for shared ancestry was the observation that two of the hypothetical proteins were predicted to be structurally similar to the LD species' CspA proteins, which are encoded on the 54-kb plasmids.

  16. Photonic plasmid stability of transformed Salmonella typhimurium: A comparison of three unique plasmids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Acquiring a highly stable photonic plasmid in transformed Salmonella typhimurium for use in biophotonic studies of bacterial tracking in vivo is critical to experimental paradigm development. The objective of this study was to determine stability of transformed Salmonella typhimurium (S. typh-lux) u...

  17. Photonic Plasmid Stability of Transformed Salmonella Typhimurium: A Comparison of Three Unique Plasmids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Acquiring a highly stable photonic plasmid in transformed Salmonella Typhimurium for use in biophotonic studies of bacterial tracking in vivo is critical to experimental paradigm development. The objective of this study was to determine stability of transformed Salmonella Typhimurium (S....

  18. Ornamental fish as a source of plasmid-mediated quinolone resistance genes and antibiotic resistance plasmids.

    PubMed

    Dobiasova, Hana; Kutilova, Iva; Piackova, Veronika; Vesely, Tomas; Cizek, Alois; Dolejska, Monika

    2014-07-16

    Growing ornamental fish industry is associated with public health concerns including extensive antibiotic use accompanied by increasing antibiotic resistance. The aim of this study was to analyze Aeromonas isolates from imported tropical ornamental fish and coldwater koi carps bred in the Czech Republic to assess the potential risk of ornamental fish as a source of plasmid-mediated quinolone resistance genes (PMQR) and antibiotic resistance plasmids. A collection of Aeromonas spp. with reduced susceptibility to ciprofloxacin (MIC ≥ 0.05 mg/L) was selected for the detection of PMQR genes. Isolates harbouring PMQR genes were further analyzed for the additional antibiotic resistance, integron content, clonality, biofilm production and transferability of PMQR genes by conjugation and transformation. Comparative analysis of plasmids carrying PMQR genes was performed. Fifteen (19%, n=80) isolates from koi carps and 18 (24%, n=76) isolates from imported ornamental fish were positive for qnrS2, aac(6')-Ib-cr or qnrB17 genes. PMQR-positive isolates from imported ornamental fish showed higher MIC levels to quinolones, multiresistance and diverse content of antibiotic resistance genes and integrons compared to the isolates from the carps. Related IncU plasmids harbouring qnrS2 and aac(6')-Ib-cr genes were found in Aeromonas spp. from imported ornamental fish and koi carps from various geographical areas. Ornamental fish may represent a potential source of multiresistant bacteria and mobile genetic elements for the environment and for humans.

  19. Why is entry exclusion an essential feature of conjugative plasmids?

    PubMed

    Garcillán-Barcia, M Pilar; de la Cruz, Fernando

    2008-07-01

    Entry exclusion is a property of plasmids by which the cells that contain them become bad recipients in additional conjugation rounds. This work reviews entry exclusion essential features and analyzes the mechanisms of action of the best studied systems. We searched for homologs of the proteins responsible for experimentally known exclusion systems. Results were used to classify exclusion systems in families of related elements. We arrive to the conclusion that all conjugative plasmids contain at least one entry exclusion gene. Although entry exclusion genes seem to be part of the plasmid conjugative machinery, they are systematically absent in phylogenetically related type IV protein exporting machines involved in virulence for plants and animals. We infer from this fact that entry exclusion is an essential feature of conjugative plasmid biology. Mathematical models suggest that plasmids expressing entry exclusion selectively eliminate plasmids lacking it, reinforcing its essential character and suggesting that entry exclusion plays a direct role in plasmid survival. Other experimental results confirm that entry exclusion is essential for the stability of a conjugative plasmid. We suggest that entry exclusion limits the damage of lethal zygosis (bacterial death produced by excessive rounds of conjugation). Additionally, it avoids competition in a host among identical plasmid backbones. Conversely, the lack of entry exclusion in conjugative transposons can be understood as a means of generating rapid evolutionary change.

  20. [Isolation of the R'his plasmids of Vibrio cholerae].

    PubMed

    Rusina, O Iu; Tiganova, I G; Aleshkin, G I; Andreeva, I V; Skavronskaia, A G

    1987-06-01

    V. cholerae strain VT5104 capable of donor activity in conjugation has been constructed by the genetic technique based on plasmid RP4::Mucts62 integration into V. cholerae chromosome due to plasmid homology with Mucts62 inserted into the chromosome. The gene for histidine synthesis has been mobilized and transferred into the recipient cells from VT5104 donor. The conjugants obtained are able to efficiently transfer his+ gene included into the plasmid structure in conjugation with eltor recipient. Thus, the constructed strain VT5104 generates R' plasmids carrying V. cholerae chromosomal genes.

  1. Bacteriophages Limit the Existence Conditions for Conjugative Plasmids

    PubMed Central

    Wood, A. Jamie; Dytham, Calvin; Pitchford, Jonathan W.; Truman, Julie; Spiers, Andrew; Paterson, Steve; Brockhurst, Michael A.

    2015-01-01

    ABSTRACT Bacteriophages are a major cause of bacterial mortality and impose strong selection on natural bacterial populations, yet their effects on the dynamics of conjugative plasmids have rarely been tested. We combined experimental evolution, mathematical modeling, and individual-based simulations to explain how the ecological and population genetics effects of bacteriophages upon bacteria interact to determine the dynamics of conjugative plasmids and their persistence. The ecological effects of bacteriophages on bacteria are predicted to limit the existence conditions for conjugative plasmids, preventing persistence under weak selection for plasmid accessory traits. Experiments showed that phages drove faster extinction of plasmids in environments where the plasmid conferred no benefit, but they also revealed more complex effects of phages on plasmid dynamics under these conditions, specifically, the temporary maintenance of plasmids at fixation followed by rapid loss. We hypothesized that the population genetic effects of bacteriophages, specifically, selection for phage resistance mutations, may have caused this. Further mathematical modeling and individual-based simulations supported our hypothesis, showing that conjugative plasmids may hitchhike with phage resistance mutations in the bacterial chromosome. PMID:26037122

  2. Plasmid genes required for microcin B17 production.

    PubMed Central

    San Millán, J L; Kolter, R; Moreno, F

    1985-01-01

    The production of the antibiotic substance microcin B17 (Mcc) is determined by a 3.5-kilobase DNA fragment from plasmid pMccB17. Several Mcc- mutations on plasmid pMccB17 were obtained by both transposon insertion and nitrosoguanidine mutagenesis. Plasmids carrying these mutations were tested for their ability to complement Mcc- insertion or deletion mutations on pMM102 (pMM102 is a pBR322 derivative carrying the region encoding microcin B17). Results from these experiments indicate that at least four plasmid genes are required for microcin production. PMID:2993228

  3. A comparison of the kinetics of plasmid transfer in the conjugation systems encoded by the F plasmid from Escherichia coli and plasmid pCF10 from Enterococcus faecalis.

    PubMed

    Andrup, L; Andersen, K

    1999-08-01

    Quantitative measurements of horizontal DNA transfer are critical if one wishes to address questions relating to ecology, evolution and the safe use of recombinant bacteria. Traditionally, the efficiency of a conjugation system has been described by its transfer frequency. However, transfer frequencies can be determined in many ways and may be sensitive to physical, chemical and biological conditions. In this study the authors have used the mechanistic similarity between bacterial conjugation and simple enzyme catalysis in order to calculate the maximal conjugation rate (Vmax) and the recipient concentration (K(m)) at which the conjugation rate is half its maximal value, for two different conjugation systems: the F plasmid from Escherichia coli and plasmid pCF10 from Enterococcus faecalis. The results are compared with the data obtained from the aggregation-mediated conjugation system encoded on pXO16 from Bacillus thuringiensis. The conjugation systems analysed are fundamentally different; however, they have some characteristics in common: they are able to sustain conjugative transfer in liquid medium and the transfer efficiencies are very high. Conjugation encoded by the F plasmid in E. coli involves the formation of small aggregates (2-20 cells), established by sex pili, and the plasmid's maximal conjugation rate was estimated to be approximately 0.15 transconjugants per donor per minute. Pheromone-induced conjugation in Ent. faecalis, which involves the formation of large aggregates, was found to proceed at a maximal conjugation rate of 0.29 transconjugants per donor per minute. Also, the K(m) value differed significantly between these conjugation systems; this may reflect the inherent differences in mating pair formation and transfer mechanisms. In these conjugation systems, the donors underwent a 'recovery period' between rounds of conjugative transfer and newly formed transconjugants required a period of about 40-80 min to mature into proficient donors.

  4. Plasmid DNA hydrogels for biomedical applications.

    PubMed

    Costa, Diana; Valente, Artur J M; Miguel, M Graça; Queiroz, João

    2014-03-01

    In the last few years, our research group has focused on the design and development of plasmid DNA (pDNA) based systems as devices to be used therapeutically in the biomedical field. Biocompatible macro and micro plasmid DNA gels were prepared by a cross-linking reaction. For the first time, the pDNA gels have been investigated with respect to their swelling in aqueous solution containing different additives. Furthermore, we clarified the fundamental and basic aspects of the solute release mechanism from pDNA hydrogels and the significance of this information is enormous as a basic tool for the formulation of pDNA carriers for drug/gene delivery applications. The co-delivery of a specific gene and anticancer drugs, combining chemical and gene therapies in the treatment of cancer was the main challenge of our research. Significant progresses have been made with a new p53 encoding pDNA microgel that is suitable for the loading and release of pDNA and doxorubicin. This represents a strong valuable finding in the strategic development of systems to improve cancer cure through the synergetic effect of chemical and gene therapy.

  5. Development of plasmid cloning vectors for Thermus thermophilus HB8: Expression of a heterologous, plasmid-borne kanamycin nucleotidyltransferase gene

    SciTech Connect

    Mather, M.W.; Fee, J.A. )

    1992-01-01

    While several thermus genes have been cloned and T. thermophilus has been shown to be transformable, molecular genetic studies of these thermophiles have been hampered by the absence of selectable cloning vectors. The authors have constructed a selectable plasmid by random insertion of a heterologous gene encoding a thermostable kanamycin nucleotidyltransferase activity into a cryptic, multicopy plasmid from T. thermophilus HB8. This plasmid should serve as a suitable starting point for the development of a gene expression system for T. thermophilus.

  6. Plasmids in different strains of Streptomyces ambofaciens: free and integrated form of plasmid pSAM2.

    PubMed

    Pernodet, J L; Simonet, J M; Guérineau, M

    1984-01-01

    Five strains of Streptomyces ambofaciens were examined for their plasmid content. Among these strains, four belong to the same lineage (strains B) and the other was isolated independently (strain A). A large plasmid (ca. 80 kb), called pSAM1 in this paper and already described, was present in all B strains, and absent in strain A. A second plasmid, not described before, was found as covalently closed circular DNA in two of the four B strains. This plasmid with a size of 11.1 kb was called pSAM2. A restriction map for 14 enzymes was established. Hybridization experiments showed that a unique sequence homologous to this plasmid is integrated in a larger replicon, which is not pSAM1 and is probably the chromosome, in all B strains and not in strain A. It seems probable that the integrated sequence is the origin of the free plasmid found in two strains of the B family. It is noteworthy that the integrated form and the free plasmid may be found together. Transformation experiments proved that pSAM2 may be maintained autonomously in S. ambofaciens strain A and in S. lividans. pSAM2 is a self-transmissible plasmid, able to elicit the lethal zygosis reaction. pSAM2 was compared to the plasmids SLP1, pIJ110 and pIJ408, which all come from integrated sequences in three Streptomyces species and are found as autonomous plasmids after transfer to S. lividans. If pSAM2 resembles these plasmids in its origin, it does not appear to be related directly to them. Concerning their plasmid content, the two isolates of S. ambofaciens are very different.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Restriction endonuclease analysis of the lactose plasmid in Streptococcus lactis ML3 and two recombinant lactose plasmids.

    PubMed

    Walsh, P M; McKay, L L

    1982-05-01

    We investigated the molecular relationship between the 60-megadalton (Mdal) recombinant lactose plasmids in ML 3 x LM2301 lactose-positive (Lac+) transconjugants and the genetic material of Streptococcus lactis ML3. Lactose metabolism is linked to the 33-Mdal plasmid pSK08 in ML3, and the recipient LM2301 is cured of plasmid DNA. The plasmids were analyzed with a series of restriction enzymes. We found that the 60-Mdal plasmids of Lac+ transconjugants contained pSK08 DNA, but were not simply dimers of pSK08. The 60-Mdal plasmids contained a segment of DNA not apparent in pSK08. The restriction patterns of the 60-Mdal plasmid in a Lac+ nonclumping transconjugant and that in a Lac+ clumping transconjugant were different. This suggested that there was a molecular differences between these two recombinant plasmids. We conclude that the segment of DNA in the 60-Mdal plasmids that was not present in pSK08 was the proposed transfer factor responsible for cell aggregation and high-frequency conjugation.

  8. Plasmid pGA1 from Corynebacterium glutamicum codes for a gene product that positively influences plasmid copy number.

    PubMed Central

    Nesvera, J; Pátek, M; Hochmannová, J; Abrhámová, Z; Becvárová, V; Jelínkova, M; Vohradský, J

    1997-01-01

    The complete nucleotide sequence (4,826 bp) of the cryptic plasmid pGA1 from Corynebacterium glutamicum was determined. DNA sequence analysis revealed four putative coding regions (open reading frame A [ORFA], ORFA2, ORFB, and ORFC). ORFC was identified as a rep gene coding for an initiator of plasmid replication (Rep) according to the high level of homology of its deduced amino acid sequence with the Rep proteins of plasmids pSR1 (from C. glutamicum) and pNG2 (from Corynebacterium diphtheriae). This function was confirmed by deletion mapping of the minimal replicon of pGA1 (1.7 kb) which contains only ORFC. Deletion derivatives of pGA1 devoid of ORFA exhibited significant decreases in the copy number in C. glutamicum cells and displayed segregational instability. Introduction of ORFA in trans into the cells harboring these deletion plasmids dramatically increased their copy number and segregational stability. The ORFA gene product thus positively influences plasmid copy number. This is the first report on such activity associated with a nonintegrating bacterial plasmid. The related plasmids pGA1, pSR1, and pNG2 lacking significant homology with any other plasmid seem to be representatives of a new group of plasmids replicating in the rolling-circle mode. PMID:9045809

  9. Restriction-Stimulated Homologous Recombination of Plasmids by the Rece Pathway of Escherichia Coli

    PubMed Central

    Nussbaum, A.; Shalit, M.; Cohen, A.

    1992-01-01

    To test the double-strand break (DSB) repair model in recombination by the RecE pathway of Escherichia coli, we constructed chimeric phages that allow restriction-mediated release of linear plasmid substrates of the bioluminescence recombination assay in infected EcoRI(+) cells. Kinetics of DSB repair and expression of recombination products were followed by Southern hybridization and by the bioluminescence recombination assay, respectively. Plasmid recombinants were analyzed with restriction endonucleases. Our results indicate that a DSB can induce more than one type of RecE-mediated recombination. A DSB within the homology induced intermolecular recombination that followed the rules of the DSB repair model: (1) Recombination was enhanced by in vivo restriction. (2) Repair of the break depended on homologous sequences on the resident plasmid. (3) Break-repair was frequently associated with conversion of alleles that were cis to the break. (4) Conversion frequency decreased as the distance from the break increased. (5) Some clones contained a mixture of plasmid recombinants as expected by replication of a heteroduplex in the primary recombinant. The rules of the DSB repair model were not followed when recombination was induced by a DSB outside the homology. Both the cut and the uncut substrates were recipients in conversion events. Recombination events were associated with deletions that spanned the break site, but these deletions did not reach the homology. We propose that a break outside the homology may stimulate a RecE-mediated recombination pathway that does not involve direct participation of DNA ends in the homologous pairing reaction. PMID:1732167

  10. An oligonucleotide microarray to characterize multidrug resistant plasmids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteria plasmids are fragments of extra-chromosomal double stranded deoxyribonucleic acid (DNA) that can contain a variety of genes beneficial to the host organism like antibiotic drug resistance. Many of the Enterobacteriaceae carry multiple drug resistance (MDR) genes on large plasmids of replic...

  11. Plasmid-Mediated Antimicrobial Resistance in Staphylococci and Other Firmicutes.

    PubMed

    Schwarz, Stefan; Shen, Jianzhong; Wendlandt, Sarah; Fessler, Andrea T; Wang, Yang; Kadlec, Kristina; Wu, Cong-Ming

    2014-12-01

    In staphylococci and other Firmicutes, resistance to numerous classes of antimicrobial agents, which are commonly used in human and veterinary medicine, is mediated by genes that are associated with mobile genetic elements. The gene products of some of these antimicrobial resistance genes confer resistance to only specific members of a certain class of antimicrobial agents, whereas others confer resistance to the entire class or even to members of different classes of antimicrobial agents. The resistance mechanisms specified by the resistance genes fall into any of three major categories: active efflux, enzymatic inactivation, and modification/replacement/protection of the target sites of the antimicrobial agents. Among the mobile genetic elements that carry such resistance genes, plasmids play an important role as carriers of primarily plasmid-borne resistance genes, but also as vectors for nonconjugative and conjugative transposons that harbor resistance genes. Plasmids can be exchanged by horizontal gene transfer between members of the same species but also between bacteria belonging to different species and genera. Plasmids are highly flexible elements, and various mechanisms exist by which plasmids can recombine, form cointegrates, or become integrated in part or in toto into the chromosomal DNA or into other plasmids. As such, plasmids play a key role in the dissemination of antimicrobial resistance genes within the gene pool to which staphylococci and other Firmicutes have access. This chapter is intended to provide an overview of the current knowledge of plasmid-mediated antimicrobial resistance in staphylococci and other Firmicutes.

  12. Plasmid-encoded copper resistance and precipitation by Mycobacterium scrofulaceum.

    PubMed Central

    Erardi, F X; Failla, M L; Falkinham, J O

    1987-01-01

    A copper-tolerant Mycobacterium scrofulaceum strain was able to remove copper from culture medium by sulfate-dependent precipitation as copper sulfide. Such precipitation of copper sulfide was not observed in a derivative that lacks a 173-kilobase plasmid. In addition, the plasmid-carrying strain has a sulfate-independent copper resistance mechanism. PMID:3662522

  13. Identification of IncA/C Plasmid Replication and Maintenance Genes and Development of a Plasmid Multilocus Sequence Typing Scheme.

    PubMed

    Hancock, Steven J; Phan, Minh-Duy; Peters, Kate M; Forde, Brian M; Chong, Teik Min; Yin, Wai-Fong; Chan, Kok-Gan; Paterson, David L; Walsh, Timothy R; Beatson, Scott A; Schembri, Mark A

    2017-02-01

    Plasmids of incompatibility group A/C (IncA/C) are becoming increasingly prevalent within pathogenic Enterobacteriaceae They are associated with the dissemination of multiple clinically relevant resistance genes, including blaCMY and blaNDM Current typing methods for IncA/C plasmids offer limited resolution. In this study, we present the complete sequence of a blaNDM-1-positive IncA/C plasmid, pMS6198A, isolated from a multidrug-resistant uropathogenic Escherichia coli strain. Hypersaturated transposon mutagenesis, coupled with transposon-directed insertion site sequencing (TraDIS), was employed to identify conserved genetic elements required for replication and maintenance of pMS6198A. Our analysis of TraDIS data identified roles for the replicon, including repA, a toxin-antitoxin system; two putative partitioning genes, parAB; and a putative gene, 053 Construction of mini-IncA/C plasmids and examination of their stability within E. coli confirmed that the region encompassing 053 contributes to the stable maintenance of IncA/C plasmids. Subsequently, the four major maintenance genes (repA, parAB, and 053) were used to construct a new plasmid multilocus sequence typing (PMLST) scheme for IncA/C plasmids. Application of this scheme to a database of 82 IncA/C plasmids identified 11 unique sequence types (STs), with two dominant STs. The majority of blaNDM-positive plasmids examined (15/17; 88%) fall into ST1, suggesting acquisition and subsequent expansion of this blaNDM-containing plasmid lineage. The IncA/C PMLST scheme represents a standardized tool to identify, track, and analyze the dissemination of important IncA/C plasmid lineages, particularly in the context of epidemiological studies.

  14. Manipulating yeast genome using plasmid vectors.

    PubMed

    Stearns, T; Ma, H; Botstein, D

    1990-01-01

    The vectors and techniques described here enable one to manipulate the yeast genome to meet specific needs. Genes can be cloned, and the clone used to delete the wild-type gene from the chromosome, or replace it with mutant versions. Mutants derived by classical methods, such as mutagenesis of whole cells, or by reversion of a phenotype, can be cloned and analyzed in vitro. Yeast genes and foreign genes can either be inserted into autonomously replicating plasmid vectors that are reasonably stable or integrated into a yeast chromosome where they are maintained at one copy per genome. The combination of these techniques with the characterized promoter systems available in yeast make it possible to express almost any gene in yeast. Once this is achieved, the entire repertoire of yeast genetics is available to probe the function of the gene, or to engineer the expression in useful ways.

  15. Plasmid addiction systems: perspectives and applications in biotechnology.

    PubMed

    Kroll, Jens; Klinter, Stefan; Schneider, Cornelia; Voss, Isabella; Steinbüchel, Alexander

    2010-11-01

    Biotechnical production processes often operate with plasmid-based expression systems in well-established prokaryotic and eukaryotic hosts such as Escherichia coli or Saccharomyces cerevisiae, respectively. Genetically engineered organisms produce important chemicals, biopolymers, biofuels and high-value proteins like insulin. In those bioprocesses plasmids in recombinant hosts have an essential impact on productivity. Plasmid-free cells lead to losses in the entire product recovery and decrease the profitability of the whole process. Use of antibiotics in industrial fermentations is not an applicable option to maintain plasmid stability. Especially in pharmaceutical or GMP-based fermentation processes, deployed antibiotics must be inactivated and removed. Several plasmid addiction systems (PAS) were described in the literature. However, not every system has reached a full applicable state. This review compares most known addiction systems and is focusing on biotechnical applications.

  16. Intradermal naked plasmid DNA immunization: mechanisms of action.

    PubMed

    Elnekave, Mazal; Furmanov, Karina; Hovav, Avi-Hai

    2011-08-01

    Plasmid DNA is a promising vaccine modality that is regularly examined in prime-boost immunization regimens. Recent advances in skin immunity increased our understanding of the sophisticated cutaneous immune network, which revived scientific interest in delivering vaccines to the skin. Intradermal administration of plasmid DNA via needle injection is a simple and inexpensive procedure that exposes the plasmid and its encoded antigen to the dermal immune surveillance system. This triggers unique mechanisms for eliciting local and systemic immunity that can confer protection against pathogens and tumors. Understanding the mechanisms of intradermal plasmid DNA immunization is essential for enhancing and modulating its immunogenicity. With regard to vaccination, this is of greater importance as this routine injection technique is highly desirable for worldwide immunization. This article will focus on the current understanding of the mechanisms involved in antigen expression and presentation during primary and secondary syringe and needle intradermal plasmid DNA immunization.

  17. Marker-free plasmids for biotechnological applications - implications and perspectives.

    PubMed

    Oliveira, Pedro H; Mairhofer, Juergen

    2013-09-01

    Nonviral gene therapy and DNA vaccines have become the first promising approaches to treat, cure, or ultimately prevent disease by providing genetic information encoded on a plasmid. Since 1989, more than 1800 clinical trials have been approved worldwide, and approximately 20% of them are using plasmid DNA (pDNA) as a vector system. Although much safer than viral approaches, DNA vectors generally do encode antibiotic resistance genes in the plasmid backbone. These antibiotic resistance markers constitute a possible safety risk, and they are associated with structural plasmid instabilities and decreased gene delivery efficiency. These drawbacks have initiated the development of various antibiotic marker-free selection approaches. We provide an overview on the potential implications of marker-free plasmids and perspectives for their successful biotechnological use in the future.

  18. Characterization of chimeric plasmid cloning vehicles in Bacillus subtilis.

    PubMed

    Gryczan, T; Shivakumar, A G; Dubnau, D

    1980-01-01

    Restriction endonuclease cleavage maps of seven chimeric plasmids that may be used for molecular cloning in Bacillus subtilis are presented. These plasmids all carry multiple antibiotic resistance markers and were constructed by in vitro molecular cloning techniques. Several of the antibiotic resistance markers were shown to undergo insertional inactivation at specific restriction endonuclease sites. Kanamycin inactivation occurred at the BglII site of pUB110 derivatives, erythromycin inactivation occurred at the HpaI and BclI sites of pE194 derivatives, and streptomycin inactivation occurred at the HindIII site of pSA0501 derivatives. A stable mini-derivative of pBD12 was isolated and characterized. By using these plasmids, we identified proteins involved in plasmid-coded kanamycin and erythromycin resistance. The properties and uses of these chimeric plasmids in the further development of recombinant deoxyribonucleic acid technology in B. subtilis are discussed.

  19. Antibiotic resistance of vibrio cholerae: special considerations of R-plasmids.

    PubMed

    Kuwahara, S

    1978-09-01

    Studies on the transmission of R plasmid by conjugation between enterobacteria and vibrio or related bacteria were reviewed. The majority of the reports confirmed successful transmission from enterobacteria to Vibrio cholerae and related species, although the transmission frequencies were extremely low and the transmitted R plasmid was very unstable except for thermosensitive kanamycin plasmid and usual R plasmid coexisting with P plasmid. Strains of V. cholerae and Aeromonas liquefaciens as well as A. salmonicida bearing R plasmid were detected in nature. R plasmid was relatively unstable in V. cholerae strains with which transmission of R plasmid to enterobacteria was confirmed. At present, only 3 R plasmids have been obtained from naturally occurring strains of V. cholerae. Although the 2 European plasmids belong to the C incompatibility group with 98 megadalton closed covalent circular DNA molecule, one plasmid belongs to the J group with more than 25 megadalton molecular weight, and no CCC of satelite DNA was detected in bacteria harboring this plasmid.

  20. Complex nature of enterococcal pheromone-responsive plasmids.

    PubMed

    Wardal, Ewa; Sadowy, Ewa; Hryniewicz, Waleria

    2010-01-01

    Pheromone-responsive plasmids constitute a unique group of approximately 20 plasmids identified, as yet, only among enterococcal species. Several of their representatives, e.g. pAD1, pCF10, pPD1 and pAM373 have been extensively studied. These plasmids possess a sophisticated conjugation mechanism based on response to sex pheromones--small peptides produced by plasmid-free recipient cells. Detailed analysis of regulation and function of the pheromone response process revealed its great complexity and dual role--in plasmid conjugation and modulation of enterococcal virulence. Among other functional modules identified in pheromone plasmids, the stabilization/partition systems play a crucial role in stable maintenance of the plasmid molecule in host bacteria. Among them, the par locus of pAD1 is one of the exceptional RNA addiction systems. Pheromone-responsive plasmids contribute also to enterococcal phenotype being an important vehicle of antibiotic resistance in this genus. Both types of acquired vancomycin resistance determinants, vanA and vanB, as well many other resistant phenotypes, were found to be located on these plasmids. They also encode two basic agents of enterococcal virulence, i.e. aggregation substance (AS) and cytolysin. AS participates in mating-pair formation during conjugation but can also facilitate the adherence ofenterococci to human tissues during infection. The second protein, cytolysin, displays hemolytic activity and helps to invade eukaryotic cells. There are still many aspects of the nature of pheromone plasmids that remain unclear and more detailed studies are needed to understand their uniqueness and complexity.

  1. Plasmid pORF-hTRAIL targeting to glioma using transferrin-modified polyamidoamine dendrimer

    PubMed Central

    Gao, Song; Li, Jianfeng; Jiang, Chen; Hong, Bo; Hao, Bing

    2016-01-01

    A gene drug delivery system for glioma therapy based on transferrin (Tf)-modified polyamidoamine dendrimer (PAMAM) was prepared. Gene drug, tumor necrosis factor-related apoptosis-inducing ligand (hTRAIL)-encoding plasmid open reading frame (pORF-hTRAIL, Trail), was condensed by Tf-modified PAMAM to form nanoparticles (NPs). PAMAM-PEG-Tf/DNA NPs showed higher cellular uptake, in vitro gene expression, and cytotoxicity than PAMAM-PEG/DNA NPs in C6 cells. The in vivo targeting efficacy of NPs was visualized by ex vivo fluorescence imaging. Tf-modified NPs showed obvious glioma-targeting trend. Plasmid encoding green fluorescence protein (GFP) was also condensed by modified or unmodified PAMAM to evaluate the in vivo gene expression level. The PAMAM-PEG-Tf/plasmid encoding enhanced green fluorescence protein (pEGFP) NPs exhibited higher GFP expression level than PAMAM-PEG/pEGFP NPs. TUNEL assay revealed that Tf-modified NPs could induce much more tumor apoptosis. The median survival time of PAMAM-PEG-Tf/Trail-treated rats (28.5 days) was longer than that of rats treated with PAMAM-PEG/Trail (25.5 days), temozolomide (24.5 days), PAMAM-PEG-Tf/pEGFP (19 days), or saline (17 days). The therapeutic effect was further confirmed by magnetic resonance imaging. This study demonstrated that targeting gene delivery system had potential application for the treatment of glioma. PMID:26719669

  2. DNA repair in cells sensitive and resistant to cis-diamminedichloroplatinum(II): Host cell reactivation of damaged plasmid DNA

    SciTech Connect

    Sheibani, N.; Jennerwein, M.M.; Eastman, A. )

    1989-04-04

    cis-Diamminedichloroplatinum(II) (cis-DDP) has a broad clinical application as an effective anticancer drug. However, development of resistance to the cytotoxic effects is a limiting factor. In an attempt to understand the mechanism of resistance, the authors have employed a host cell reactivation assay of DNA repair using a cis-DDP-damaged plasmid vector. The efficiency of DNA repair was assayed by measuring the activity of an enzyme coded for by the plasmid vector. The plasmid expression vector pRSV cat contains the bacterial gene coding for chloramphenicol acetyltransferase (CAT) in a configuration which permits expression in mammalian cells. The plasmid was transfected into repair-proficient and -deficient Chinese hamster ovary cells, and CAT activity was subsequently measured in cell lysates. In the repair-deficient cells, one cis-DDP adduct per cat gene was sufficient to eliminate expression. An equivalent inhibition of CAT expression in the repair-proficient cells did not occur until about 8 times the amount of damage was introduced into the plasmid. These results implicate DNA intrastrand cross-links as the lesions responsible for the inhibition of CAT expression. This assay was used to investigate the potential role of DNA repair in mediating cis-DDP resistance in murine leukemia L1210 cells. The assay readily detects the presence or absence of repair and confirms that these resistant L1210 cells have an enhanced capacity for repair of cis-DDP-induced intrastrand cross-links.

  3. Plasmid DNA is internalized from the apical plasma membrane of the salivary gland epithelium in live animals.

    PubMed

    Sramkova, Monika; Masedunskas, Andrius; Weigert, Roberto

    2012-08-01

    Non-viral-mediated gene delivery represents an alternative way to express the gene of interest without inducing immune responses or other adverse effects. Understanding the mechanisms by which plasmid DNAs are delivered to the proper target in vivo is a fundamental issue that needs to be addressed in order to design more effective strategies for gene therapy. As a model system, we have used the submandibular salivary glands in live rats and we have recently shown that reporter transgenes can be expressed in different cell populations of the glandular epithelium, depending on the modality of administration of plasmid DNA. Here, by using a combination of immunofluorescence and intravital microscopy, we have explored the relationship between the pattern of transgenes expression and the internalization of plasmid DNA. We found that plasmid DNA is internalized: (1) by all the cells in the salivary gland epithelium, when administered alone, (2) by large ducts, when mixed with empty adenoviral particles, and (3) by acinar cells upon stimulation of compensatory endocytosis. Moreover, we showed that plasmid DNA utilizes different routes of internalization, and evades both the lysosomal degradative pathway and the retrograde pathway towards the Golgi apparatus. This study clearly shows that in vivo approaches have the potential to address fundamental questions on the cellular mechanisms regulating gene delivery.

  4. The mosaic structure of the symbiotic plasmid of Rhizobium etli CFN42 and its relation to other symbiotic genome compartments

    PubMed Central

    González, Víctor; Bustos, Patricia; Ramírez-Romero, Miguel A; Medrano-Soto, Arturo; Salgado, Heladia; Hernández-González, Ismael; Hernández-Celis, Juan Carlos; Quintero, Verónica; Moreno-Hagelsieb, Gabriel; Girard, Lourdes; Rodríguez, Oscar; Flores, Margarita; Cevallos, Miguel A; Collado-Vides, Julio; Romero, David; Dávila, Guillermo

    2003-01-01

    Background Symbiotic bacteria known as rhizobia interact with the roots of legumes and induce the formation of nitrogen-fixing nodules. In rhizobia, essential genes for symbiosis are compartmentalized either in symbiotic plasmids or in chromosomal symbiotic islands. To understand the structure and evolution of the symbiotic genome compartments (SGCs), it is necessary to analyze their common genetic content and organization as well as to study their differences. To date, five SGCs belonging to distinct species of rhizobia have been entirely sequenced. We report the complete sequence of the symbiotic plasmid of Rhizobium etli CFN42, a microsymbiont of beans, and a comparison with other SGC sequences available. Results The symbiotic plasmid is a circular molecule of 371,255 base-pairs containing 359 coding sequences. Nodulation and nitrogen-fixation genes common to other rhizobia are clustered in a region of 125 kilobases. Numerous sequences related to mobile elements are scattered throughout. In some cases the mobile elements flank blocks of functionally related sequences, thereby suggesting a role in transposition. The plasmid contains 12 reiterated DNA families that are likely to participate in genomic rearrangements. Comparisons between this plasmid and complete rhizobial genomes and symbiotic compartments already sequenced show a general lack of synteny and colinearity, with the exception of some transcriptional units. There are only 20 symbiotic genes that are shared by all SGCs. Conclusions Our data support the notion that the symbiotic compartments of rhizobia genomes are mosaic structures that have been frequently tailored by recombination, horizontal transfer and transposition. PMID:12801410

  5. Examination of a plasmid-based reverse genetics system for human astrovirus.

    PubMed

    Chapellier, Benoit; Tange, Shoichiro; Tasaki, Hidetaka; Yoshida, Kazuhiro; Zhou, Yan; Sakon, Naomi; Katayama, Kazuhiko; Nakanishi, Akira

    2015-10-01

    A plasmid-based reverse genetics system for human astrovirus type 1 (HAstV1) is examined. Upon transfection into 293T cells, the plasmid vector, which harbors a HAstV1 expression cassette, expressed astroviral RNA that appeared to be capable of viral RNA replication, as indicated by the production of subgenomic RNA and capsid protein expression irrespective of the heterologous 5' ends of the transcribed RNA. Particles infectious to Caco-2 cells were made in this system; however, their infectivity was much lower than would be expected from the amount of particles apparently produced. Using Huh-7 cells as the transfection host with the aim of improving viral capsid processing for virion maturation partially restored the efficiency of infectious particle formation. Our results support the possibility that the DNA transfection process induces a cellular response that targets late, but not early, stages of HAstV1 infection.

  6. Impact of carbondiimide crosslinker used for magnetic carbon nanotube mediated GFP plasmid delivery

    NASA Astrophysics Data System (ADS)

    Hao, Yuzhi; Xu, Peng; He, Chuan; Yang, Xiaoyan; Huang, Min; Xing, James; Chen, Jie

    2011-07-01

    1-ethyl-3-(3-dimethylaminopropyl) carbondiimide hydrochloride (EDC) is commonly used as a crosslinker to help bind biomolecules, such as DNA plasmids, with nanostructures. However, EDC often remains, after a crosslink reaction, in the micro-aperture of the nanostructure, e.g., carbon nanotube. The remaining EDC shows positive green fluorescent signals and makes a nanostructure with a strong cytotoxicity which induces cell death. The toxicity of EDC was confirmed on a breast cancer cell line (MCF-7) and two leukemic cell lines (THP-1 and KG-1). The MCF-7 cells mainly underwent necrosis after treatment with EDC, which was verified by fluorescein isothiocyanate (FITC) annexin V staining, video microscopy and scanning electronic microscopy (SEM). If the EDC was not removed completely, the nanostructures with remaining EDC produced a green fluorescent background that could interfere with flow cytometry (FACS) measurement and result in false information about GFP plasmid delivery. Effective methods to remove residual EDC on macromolecules were also developed.

  7. [Effect of He-Ne-laser irradiation on plasmid transformation of Escherichia coli bacteria].

    PubMed

    Tiflova, O A; Leonov, P G; Karbysheva, E A; Shakhnabatian, L G

    1997-01-01

    The influence of the of radiation a He-Ne laser (632.8 nm, 30 W/m2, 5-20 J/m2) on the transformation of Escherichia coli cells with plasmid DNA was studied. The irradiation of a mixture of bacterial cells and plasmid DNA increased the transformation efficiency 2.5-3 times, thus offering an alternative to the heat treatment commonly used. In contrast to the standard techniques, the laser-induced increase in the transformation efficiency was accompanied by a 1.7- to 2-fold increase in cell survival. The effect of the 632.8-nm light, know to be absorbed by membrane porphyrin components, is supposed to be mediated via a modification in the replication and transformation DNA-membrane complexes in E. coli cells.

  8. Parameters controlling interbacterial plasmid spreading in a gnotoxenic chicken gut system: influence of plasmid and bacterial mutations.

    PubMed Central

    Sansonetti, P; Lafont, J P; Jaffé-Brachet, A; Guillot, J F; Chaslus-Dancla, E

    1980-01-01

    Conjugative transfer of R plasmids R64 and R64drd-11 has been compared in vitro and in vivo without selective pressure by antibiotics in a simplified experimental system; the ecosystem was the bowel of germfree chickens, with the host bacteria almost isogenic, and the plasmids differing only in their conjugative transfer frequency. The spread of repressed and derepressed (drd) R plasmids in recipient bacterial populations was very extensive. The repressed phenotype had only a transient effect during the first 4 h. The level of implantation of the donor bacterial population seems to be of minor importance. Only with a poor recipient (con strain) could the spread of R plasmids be reduced and a steady state with a predominantly sensitive bacterial population be established. It is suggested that this steady state results from an equilibrium between the frequencies of R plasmid transfer and loss. PMID:6999980

  9. PCR-based typing of IncC plasmids.

    PubMed

    Harmer, Christopher J; Hall, Ruth M

    IncC (A/C2) plasmids are known to play an important role in the spread of multiple antibiotic resistance determinants, including extended-spectrum β-lactamases and carbapenamases, amongst Gram negative bacterial populations. The ability to identify and track these plasmids is valuable in epidemiological and clinical studies. A recent comparative analysis of the backbones of sequenced IncC plasmids identified two distinct lineages, type 1 and type 2, with different evolutionary histories. Here, a simple PCR method to rapidly assign plasmids to one of these lineages by detecting variable regions in the backbone was developed. This PCR scheme uses two primer pairs to assign the plasmid to a lineage, and an additional two PCRs can be used to detect the i1 and i2 insertions, which are only found in type 2. PCRs were also developed to detect the presence or absence of the sul2-containing ARI-B island, which is found in some plasmids belonging to both type 1 and type 2, and the ARI-A island found in most type 1 plasmids. The PCR strategy was validated using sequenced type 1 plasmids pRMH760 and pDGO100, and the type 2 plasmid pSRC119-A/C, and a collection of non-IncC plasmids in Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae backgrounds. An IncC plasmid detected in an antibiotic susceptible commensal E. coli isolate was examined and found to be a type 1, lacking any antibiotic resistance islands and missing a large backbone segment. Examination of pIP40a, an IncC plasmid isolated in Paris in 1969, by PCR revealed that it belongs to type 1 but lacks ARI-A. However, it includes both ends of the integrative element GIsul2, whereas only remnants of one end of this element are found in more recently isolated IncC plasmids. The sequence of pIP40a was determined and confirmed the assignment to type 1 and revealed the presence of a complete copy of GIsul2.

  10. Tom, a new aromatic degradative plasmid from Burkholderia (Pseudomonas) cepacia G4

    SciTech Connect

    Shields, M.S.; Reagin, J.J.; Campbell, R.

    1995-04-01

    Burkholderia (Pseudomonas) cepacia PR1{sub 23} has been shown to constitutively express a toluene catabolic pathway distinguished by a unique toluene ortho-monooxygenase (Tom). This strain has also been shown to contain two extrachromosomal elements of <70 and> 100 kb. A derivative strain cured of the largest plasmid, PR1{sub 23} Cure, was unable to grow on phenol or toluene as the sole source of carbon and energy, which requires expression of the Tom pathway. Transfer of the larger plasmid from strain G4 J(the parent strain inducible for Tom) enabled PR1{sub 23} Cure to grow on toluene or phenol via inducible Tom pathway expression. Conjugal transfer of TOM{sub 23c} from PR1{sub 23} to an antibiotic-resistant derivative of PR1{sub 23} Cure enabled the transconjugant to grow with either phenol or toluene as the sole source of carbon and energy through constitutive expression of the Tom pathway. A cloned 11.2-kb EcoRI restriction fragment of Tom{sub 23c} resulted in the expression of both Tom and catechol 2,3-dioxygenase in Escherichia coli, as evidenced by its ability to oxidize trichloroethylene, toluene, m-cresol, o-cresol, phenol, and catechol. The largest resident plasmid of PR1 was identified as the source of these genes by DNA hybridization. These results indicate that the genes which encode Tom and catechol 2,3-dioxygenase are located on TOM, an approximately 108-kb degradative plasmid of B. cepacia G4. 35 refs., 3 figs., 3 tabs.

  11. Vaccine-induced but not tumor-derived Interleukin-10 dictates the efficacy of Interleukin-10 blockade in therapeutic vaccination.

    PubMed

    Llopiz, Diana; Aranda, Fernando; Díaz-Valdés, Nancy; Ruiz, Marta; Infante, Stefany; Belsúe, Virginia; Lasarte, Juan José; Sarobe, Pablo

    2016-02-01

    Blocking antibodies against immunosuppressive molecules have shown promising results in cancer patients. However, there are not enough data to define those conditions dictating treatment efficacy. In this scenario, IL-10 is a cytokine with controversial effects on tumor growth. Thus, our aim was to characterize in which setting IL-10 blockade may potentiate the beneficial effects of a therapeutic vaccine In the IL-10-expressing B16-OVA and TC-1 P3 (A15) tumor models, therapeutic vaccination with tumor antigens plus the TLR7 ligand Imiquimod increased IL-10 production. Although blockade of IL-10 signal with anti-IL-10R antibodies did not inhibit tumor growth, when combined with vaccination it enhanced tumor rejection, associated with stronger innate and adaptive immune responses. Interestingly, a similar enhancement on immune responses was observed after simultaneous vaccination and IL-10 blockade in naive mice. However, when using vaccines containing as adjuvants the TLR3 ligand poly(I:C) or anti-CD40 agonistic antibodies, despite tumor IL-10 expression, anti-IL-10R antibodies did not provide any beneficial effect on tumor growth and antitumor immune responses. Of note, as opposed to Imiquimod, vaccination with this type of adjuvants did not induce IL-10 and correlated with a lack of in vitro IL-10 production by dendritic cells (DC). Finally, in B16-OVA-bearing mice, blockade of IL-10 during therapeutic vaccination with a multiple adjuvant combination (MAC) with potent immunostimulatory properties but still inducing IL-10 led to superior antitumor immunity and complete tumor rejection. These results suggest that for therapeutic antitumor vaccination, blockade of vaccine-induced IL-10 is more relevant than tumor-associated IL-10.

  12. Plasmid incidence in bacteria from deep subsurface sediments

    SciTech Connect

    Fredrickson, J.K.; Hicks, R.J.; Li, S.W.; Brockman, F.J. )

    1988-12-01

    Bacteria were isolated from deep terrestrial subsurface sediments underlying the coastal plain of South Carolina. A total of 163 isolates from deep sediments, surface soil, and return drill muds were examined for plasmid DNA content and resistance to the antibiotics penicillin, ampicillin, carbenicillin, streptomycin, kanamycin, and tetracycline. MICs of Cu{sup 2+}, Cr{sup 3+}, and Hg{sup 2+} for each isolate were also determined. The overall frequency of plasmid occurrence in the subsurface bacteria was 33%. Resistance was most frequent to penicillin (70% of all isolates), ampicillin (49%), and carbenicillin (32%) and was concluded to be related to the concentrations of the individual antibiotics in the disks used for assaying resistance and to the production of low levels of {beta}-lactamase. The frequencies of resistance to penicillin and ampicillin were significantly greater for isolates bearing plasmids than for plasmidless isolates; however, resistance was not transferable to penicillin-sensitive Escherichia coli. Hybridization of subsurface bacterial plasmids and chromosomal DNA with a whole-TOL-plasmid (pWWO) probe revealed some homology of subsurface bacterial plasmid and chromosomal DNAs, indicating a potential for those bacterial to harbor catabolic genes on plasmids or chromosomes. The incidences of antibiotic resistance and MICs of metals for subsurface bacteria were significantly different from those drill mud bacteria, ruling out the possibility that bacteria from sediments were derived from drill muds.

  13. Identifying stabilizers of plasmid DNA for pharmaceutical use.

    PubMed

    Zeng, Yuhong; Ramsey, Joshua D; King, Robert; Leviten, Michael; Mcguire, Ruth; Volkin, David B; Joshi, Sangeeta B; Middaugh, C Russell

    2011-03-01

    To better address the need for developing stable formulations of plasmid DNA-based biopharmaceuticals, 37 compounds from a generally regarded as safe library were examined for their potential use as stabilizers. A plasmid DNA-based therapeutic vaccine, BHT-DNA, was used as a model system. Initial studies were performed to compare the biophysical properties of BHT-DNA plasmid from bulk drug substance and finished drug product. An agarose gel electrophoresis-based assay was then employed in excipient compatibility studies for the drug product by monitoring supercoiled plasmid DNA content in various formulations. After incubation at 40 °C for 30 days, eight out of the 37 excipients tested were able to better retain the supercoil content compared to the control. Sodium citrate appeared to be the most effective stabilizer and its protective capability plateaued at an ionic strength of about 0.4. Several other excipients including malic acid, ethanol, and Pluronic F-68 were also identified as promising stabilizers for BHT-DNA plasmid DNA. Additionally, compounds, including ferrous chloride, ascorbic acid, human serum albumin, and PEG 1000, which significantly destabilized the supercoiled plasmid DNA were identified. These data may also be applicable to other plasmid DNA-based pharmaceuticals for storage stability improvement, due to chemical and structural similarities of these macromolecules.

  14. Tubular cationized pullulan hydrogels as local reservoirs for plasmid DNA.

    PubMed

    San Juan, Aurélie; Ducrocq, Grégory; Hlawaty, Hanna; Bataille, Isabelle; Guénin, Erwann; Letourneur, Didier; Feldman, Laurent J

    2007-12-01

    In the present study, we measured the ability of various cationized pullulan tubular hydrogels to retain plasmid DNA, and tested the ability of retained plasmid DNA to transfect vascular smooth muscle cells (VSMCs). Cationized pullulans were obtained by grafting at different charge densities ethylamine (EA) or diethylaminoethylamine (DEAE) on the pullulan backbone. Polymers were characterized by elemental analysis, acid-base titration, size exclusion chromatography, Fourier-transform infrared spectroscopy, and proton nuclear magnetic resonance. The complexation of cationized pullulans in solution with plasmid DNA was evidenced by fluorescence quenching with PicoGreen. Cationized pullulans were then chemically crosslinked with phosphorus oxychloride to obtain tubular cationized pullulan hydrogels. Native pullulan tubes did not retain loaded plasmid DNA. In contrast, the ability of cationized pullulan tubes to retain plasmid DNA was dependent on both the amine content and the type of amine. The functional integrity of plasmid DNA in cationized pullulan tubes was demonstrated by in vitro transfection of VSMCs. Hence, cationized pullulan hydrogels can be designed as tubular structures with high affinity for plasmid DNA, which may provide new biomaterials to enhance the efficiency of local arterial gene transfer strategies.

  15. Plasmid carriage can limit bacteria-phage coevolution.

    PubMed

    Harrison, Ellie; Truman, Julie; Wright, Rosanna; Spiers, Andrew J; Paterson, Steve; Brockhurst, Michael A

    2015-08-01

    Coevolution with bacteriophages is a major selective force shaping bacterial populations and communities. A variety of both environmental and genetic factors has been shown to influence the mode and tempo of bacteria-phage coevolution. Here, we test the effects that carriage of a large conjugative plasmid, pQBR103, had on antagonistic coevolution between the bacterium Pseudomonas fluorescens and its phage, SBW25ϕ2. Plasmid carriage limited bacteria-phage coevolution; bacteria evolved lower phage-resistance and phages evolved lower infectivity in plasmid-carrying compared with plasmid-free populations. These differences were not explained by effects of plasmid carriage on the costs of phage resistance mutations. Surprisingly, in the presence of phages, plasmid carriage resulted in the evolution of high frequencies of mucoid bacterial colonies. Mucoidy can provide weak partial resistance against SBW25ϕ2, which may have limited selection for qualitative resistance mutations in our experiments. Taken together, our results suggest that plasmids can have evolutionary consequences for bacteria that go beyond the direct phenotypic effects of their accessory gene cargo.

  16. Plasmid-associated sensitivity of Bacillus thuringiensis to UV light

    SciTech Connect

    Benoit, T.G.; Wilson, G.R.; Bull, D.L.; Aronson, A.I. )

    1990-08-01

    Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores.

  17. Plasmid-mediated mineralization of naphthalene, phenanthrene, and anthracene.

    PubMed Central

    Sanseverino, J; Applegate, B M; King, J M; Sayler, G S

    1993-01-01

    The well-characterized plasmid-encoded naphthalene degradation pathway in Pseudomonas putida PpG7(NAH7) was used to investigate the role of the NAH plasmid-encoded pathway in mineralizing phenanthrene and anthracene. Three Pseudomonas strains, designated 5R, DFC49, and DFC50, were recovered from a polynuclear aromatic hydrocarbon-degrading inoculum developed from a manufactured gas plant soil slurry reactor. Plasmids pKA1, pKA2, and pKA3, approximately 100 kb in size, were isolated from these strains and characterized. These plasmids have homologous regions of upper and lower NAH7 plasmid catabolic genes. By conjugation experiments, these plasmids, including NAH7, have been shown to encode the genotype for mineralization of [9-14C]phenanthrene and [U-14C]anthracene, as well as [1-14C]naphthalene. One strain, Pseudomonas fluorescens 5RL, which has the complete lower pathway inactivated by transposon insertion in nahG, accumulated a metabolite from phenanthrene and anthracene degradation. This is the first direct evidence to indicate that the NAH plasmid-encoded catabolic genes are involved in degradation of polynuclear aromatic hydrocarbons other than naphthalene. Images PMID:8328809

  18. General method for plasmid construction using homologous recombination.

    PubMed

    Raymond, C K; Pownder, T A; Sexson, S L

    1999-01-01

    We describe a general method for plasmid assembly that uses yeast and extends beyond yeast-specific research applications. This technology exploits the homologous recombination, double-stranded break repair pathway in Saccharomyces cerevisiae to join DNA fragments. Synthetic, double-stranded "recombination linkers" were used to "subclone" a DNA fragment into a plasmid with > 80% efficiency. Quantitative data on the influence of DNA concentration and overlap length on the efficiency of recombination are presented. Using a simple procedure, plasmids were shuttled from yeast into E. coli for subsequent screening and large-scale plasmid preps. This simple method for plasmid construction has several advantages. (i) It bypasses the need for extensive PCR amplification and for purification, modification and/or ligation techniques routinely used for plasmid constructions. (ii) The method does not rely on available restriction sites, thus fragment and vector DNA can be joined within any DNA sequence. This enables the use of multifunctional cloning vectors for protein expression in mammalian cells, other yeast species, E. coli and other expression systems as discussed. (iii) Finally, the technology exploits yeast strains, plasmids and microbial techniques that are inexpensive and readily available.

  19. Analysis of chromosomal integration and deletions of yeast plasmids.

    PubMed Central

    Cameron, J R; Philippsen, P; Davis, R W

    1977-01-01

    Plasmid DNAs from six strains of Saccharomyces cerevisiae were compared. Three different plasmids were found, designated Scp 1, Scp 2 and Scp 3, with monomer lengths of 6.19, 6.06 and 5.97 kilobases as referenced to sequenced phiX174 DNA. DNA from each of the plasmids was inserted into a lambda vector DNA. Hybrid phage containing inserted DNA of the desired size were enriched by genetic selection and their DNAs analysed by rapid techniques. All three plasmids share the same organization, two unique sequences separated by two inverted repeats, and share basically the same DNA sequences. Scp 2 and Scp 3 differ from Scp 1 by missing a unique HpaI site and by having small overlapping deletions in the same region. The HpaI site in Scp 1 is, therefore, in a nonessential region and suitable for insertion of foreign DNA in the potential use of the yeast plasmid as a vector. Hybridization of labelled cloned plasmid DNA to restriction fragments of linear yeast DNA separated on agarose gels showed that the plasmid DNA was not stably integrated into the yeast chromosomal DNA. Images PMID:331256

  20. Development of a microfluidic chip-based plasmid miniprep.

    PubMed

    Northrup, Victoria A; Backhouse, Christopher J; Glerum, D Moira

    2010-07-15

    Plasmids are the workhorse of contemporary molecular biology, serving as vectors in the multitude of molecular cloning approaches now available. Plasmid minipreps are a routine and essential means of extracting plasmid DNA from bacteria, such as Escherichia coli, for identification, characterization, and further manipulation. Although there have been many approaches described and miniprep kits are commercially available, traditional minipreps typically require more than 16h, including the time needed for bacterial cell culture. Here we describe the development of a microfluidic chip (MFC)-based miniprep that uses on-chip lysis and trapping of large DNA in agarose to differentially separate plasmid DNA from the bacterial chromosome. Our approach greatly decreases both the time required for the miniprep itself and the time required for growth of the bacterial cultures because our on-chip miniprep uses 10(5) times fewer E. coli cells. Because the quality of the isolated plasmid is comparable to that obtained using conventional miniprep protocols, this approach allows growth of E. coli and isolation of plasmid within hours, thereby making it ideal for rapid screening approaches. This MFC-based miniprep, coupled with recently demonstrated on-chip transfection capabilities, lays the groundwork for seamless manipulation of plasmids on MFC platforms.

  1. An updated view of plasmid conjugation and mobilization in Staphylococcus

    PubMed Central

    Ramsay, Joshua P.; Kwong, Stephen M.; Murphy, Riley J. T.; Yui Eto, Karina; Price, Karina J.; Nguyen, Quang T.; O'Brien, Frances G.; Grubb, Warren B.; Coombs, Geoffrey W.; Firth, Neville

    2016-01-01

    ABSTRACT The horizontal gene transfer facilitated by mobile genetic elements impacts almost all areas of bacterial evolution, including the accretion and dissemination of antimicrobial-resistance genes in the human and animal pathogen Staphylococcus aureus. Genome surveys of staphylococcal plasmids have revealed an unexpected paucity of conjugation and mobilization loci, perhaps suggesting that conjugation plays only a minor role in the evolution of this genus. In this letter we present the DNA sequences of historically documented staphylococcal conjugative plasmids and highlight that at least 3 distinct and widely distributed families of conjugative plasmids currently contribute to the dissemination of antimicrobial resistance in Staphylococcus. We also review the recently documented “relaxase-in trans” mechanism of conjugative mobilization facilitated by conjugative plasmids pWBG749 and pSK41, and discuss how this may facilitate the horizontal transmission of around 90% of plasmids that were previously considered non-mobilizable. Finally, we enumerate unique sequenced S. aureus plasmids with a potential mechanism of mobilization and predict that at least 80% of all non-conjugative S. aureus plasmids are mobilizable by at least one mechanism. We suggest that a greater research focus on the molecular biology of conjugation is essential if we are to recognize gene-transfer mechanisms from our increasingly in silico analyses. PMID:27583185

  2. Characterization of the temperate bacteriophage phi adh and plasmid transduction in Lactobacillus acidophilus ADH.

    PubMed

    Raya, R R; Kleeman, E G; Luchansky, J B; Klaenhammer, T R

    1989-09-01

    Lactobacillus acidophilus ADH is lysogenic and harbors an inducible prophage, phi adh. Bacteriophage were detected in cell lysates induced by treatment with mitomycin C or UV light. Electron microscopy of lysates revealed phage particles with a hexagonal head (62 nm) and a long, noncontractile, flexible tail (398 nm) ending in at last five short fibers. Phage phi adh was classified within Bradley's B1 phage group and the Siphoviridae family. The phi adh genome is a linear double-stranded DNA molecule of 41.7 kilobase pairs with cohesive ends: a physical map of the phi adh genome was constructed. A prophage-cured derivative of strain ADH, designated NCK102, was isolated from cells that survived UV exposure. NCK102 did not exhibit mitomycin C-induced lysis, but broth cultures lysed upon addition of phage. Phage phi adh produced clear plaques on NCK102 in media containing 10 mM CaCl2 at pH values between 5.2 and 5.5. A relysogenized derivative (NCK103) of NCK102 was isolated that exhibited mitomycin C-induced lysis and superinfection immunity to phage phi adh. Hybridization experiments showed that the phi adh genome was present in the ADH and NCK103 chromosomes, but absent in NCK102. These results demonstrated classic lytic and lysogenic cycles of replication for the temperate phage phi adh induced from L. acidophilus ADH. Phage phi adh also mediates transduction of plasmid DNA. Transductants of strain ADH containing pC194, pGK12, pGB354, and pVA797 were detected at frequencies in the range of 3.6 x 10(-8) to 8.3 x 10(-10) per PFU. Rearrangements or deletions were not detected in these plasmids as a consequence of transduction. This is the first description of plasmid transduction in the genus Lactobacillus.

  3. F'-plasmid transfer from Escherichia coli to Pseudomonas fluorescens.

    PubMed Central

    Mergeay, M; Gerits, J

    1978-01-01

    Various F' plasmids of Escherichia coli K-12 could be transferred into mutants of the soil strain 6.2, classified herein as a Pseudomonas fluorescens biotype IV. This strain was previously found to receive Flac plasmid (N. Datta and R.W. Hedges, J. Gen Microbiol. 70:453-460, 1972). ilv, leu, met, arg, and his auxotrophs were complemented by plasmids carrying isofunctional genes; trp mutants were not complemented or were very poorly complemented. The frequency of transfer was 10(-5). Subsequent transfer into other P. fluorescens recipients was of the same order of magnitude. Some transconjugants were unable to act as donors, and these did not lose the received information if subcultured on nonselective media. Use of F' plasmids helped to discriminate metabolic blocks in P. fluorescens. In particular, metA, metB, and argH mutants were so distinguished. In addition, F131 plasmid carrying the his operon and a supD mutation could partially relieve the auxotrophy of thr, ilv, and metA13 mutants, suggesting functional expression of E. coli tRNA in P. fluorescens. In P. fluorescens metA Rifr mutants carrying the F110 plasmid, which carried the E. coli metA gene and the E. coli rifs allele, sensitivity to rifampin was found to be dominant at least temporarily over resistance. This suggests interaction of E. coli and P. fluorescens subunits of RNA polymerase. his mutations were also complemented by composite P plasmids containing the his-nif region of Klebsiella pneumoniae (plasmids FN68 and RP41). nif expression could be detected by acetylene reduction in some his+ transconjugants. The frequency of transfer of these P plasmids was 5 X 10(-4). PMID:97267

  4. Comparative study of the lethal effects of near-UV light (360 nm) and 8-methoxypsoralen plus near-UV on plasmid DNA

    SciTech Connect

    Paramio, J.M.; Bauluz, C.; de Vidania, R. )

    1991-01-01

    The authors have studied the lethality produced on pBR322 by near-UV radiation and by 8-Methoxypsoralen plus near-UV (PUV treatment). Samples of pBR322 DNA were irradiated with increasing fluences of 360 nm-light either in the absence or presence of 400 molecules of 8-Methoxypsoralen (8-MOP) per plasmid molecule. They have estimated to what extent the global lethality of PUVA treatment is due to the presence of psoralen adducts in DNA or to radiation itself. In order to analyse the involvement of DNA repair mechanisms in the removal of plasmid lesions, several strains of E. coli (differing in their repair capacities) they are used as recipients of the treated plasmids. Results showed that excision and recombination participate in the repair of near-UV-induced plasmid lesions. Repair of PUV-induced lesions showed an even greater requirement of the excision pathway. Besides, a slight increase on plasmid mutation frequencies was observed after near-UV or PUV treatment in wild type and uvrA cells. Estimation of the contribution of 8-MOP to the global lethality of PUV treatment showed that only the excision pathway was involved in removing psoralen adducts from plasmid DNA, suggesting the involvement of the recombinational pathway in the repair of near-UV-derived lesions.

  5. Compatibility of plasmids encoding bovine viral diarrhea virus type 1 and type 2 E2 in a single DNA vaccine formulation.

    PubMed

    Liang, Rong; Babiuk, Lorne A; van Drunen Littel-van den Hurk, Sylvia

    2007-08-10

    Type 2 bovine viral diarrhea virus (BVDV) has become increasingly prevalent worldwide, and currently the ratio of type 2 to type 1 strains in the USA approaches 50%. Although there is cross-reactivity between BVDV type 1 and type 2 strains, BVDV1 vaccine strains poorly protect from type 2 infection, so vaccines against BVDV should contain antigens from both BVDV types. Previously we demonstrated efficacy of a BVDV1 E2 DNA vaccine, and in this study we optimized a BVDV2 E2 DNA vaccine. Furthermore, as an approach to vaccinate with a DNA vaccine against both BVDV types, we compared two strategies, mixing of plasmids encoding type 1 and type 2 E2, and co-expression of type 1 and type 2 E2 from one plasmid with an internal ribosomal entry site (IRES). An evaluation of the IRES-containing plasmids demonstrated that the C-terminally expressed protein is produced at lower levels and induces weaker immune responses than the N-terminally expressed protein, regardless of the position of the type 1 and type 2 E2 genes. In contrast, when both plasmids encoding type 1 and type 2 E2 were administered to mice, the immune responses were similar to those induced by the individual plasmids. Thus, a mixture of plasmids encoding type 1 and type 2 E2 could be a potential DNA vaccine candidate against both BVDV1 and BVDV2.

  6. [Epidemiologic study of 2 S. typhimurium outbreaks using plasmid fingerprints].

    PubMed

    Baumgartner, A; Breer, C; Schopfer, K

    1989-04-05

    An outbreak of salmonellosis in an old people's home is reported. The infectious agent, S. typhi-murium, was isolated not only from several inmates but also from sick cows of the farm belonging to the home, in animal feed, from employees of the local butcher's shop, and finally in sludge from the local sewage plant. Plasmid analysis provided evidence of a common origin for the isolated S. typhi-murium strains. The incriminated strains harboured, together with two low-molecular-weight plasmids, a plasmid of approximately 50 Mdal, which was also demonstrated in some other S. typhi-murium strains isolated from clinical cases in the area around St. Gallen.

  7. Plasmid-determined resistance to fosfomycin in Serratia marcescens.

    PubMed Central

    Mendoza, C; Garcia, J M; Llaneza, J; Mendez, F J; Hardisson, C; Ortiz, J M

    1980-01-01

    Multiple-antibiotic-resistant strains of Serratia marcescens isolated from hospitalized patients were examined for their ability to transfer antibiotic resistance to Escherichia coli by conjugation. Two different patterns of linked transferable resistance were found among the transconjugants. The first comprised resistance to carbenicillin, streptomycin, and fosfomycin; the second, and more common, pattern included resistance to carbenicillin, streptomycin, kanamycin, gentamicin, tetracycline, chloramphenicol, sulfonamide, and fosfomycin. The two types of transconjugant strains carried a single plasmid of either 57 or 97 megadaltons in size. Both of these plasmids are present in parental S. marcescens strains resistant to fosfomycin. The 57-megadalton plasmid was transformed into E. coli. Images PMID:7004337

  8. A nonalkaline method for isolating sequencing-ready plasmids.

    PubMed

    Paul, Bonnie; Cloninger, Cheri; Felton, Marilyn; Khachatoorian, Ronik; Metzenberg, Stan

    2008-06-15

    We describe a simple method of isolating plasmid DNA directly from Escherichia coli culture medium by addition of lithium acetate and Sodium dodecyl sulphate, followed by centrifugation and alcohol precipitation. The plasmid is sufficiently pure that it can be used in many enzyme-based reactions, including DNA sequencing and restriction analysis. Chromosomal DNA contamination is significantly reduced by pretreatment of the culture with DNase I, suggesting that much of the contaminant is associated with permeable dead cells. Chromosomal DNA contaminant can also be selectively denatured without damage to the supercoiled plasmid by alkaline denaturation in an arginine buffer or heat treatment in the presence of urea or N,N-dimethylformamide.

  9. Fasciation induction by the phytopathogen Rhodococcus fascians depends upon a linear plasmid encoding a cytokinin synthase gene.

    PubMed

    Crespi, M; Messens, E; Caplan, A B; van Montagu, M; Desomer, J

    1992-03-01

    Rhodococcus fascians is a nocardiform bacteria that induces leafy galls (fasciation) on dicotyledonous and several monocotyledonous plants. The wild-type strain D188 contained a conjugative, 200 kb linear extrachromosomal element, pFiD188. Linear plasmid-cured strains were avirulent and reintroduction of this linear element restored virulence. Pulsed field electrophoresis indicated that the chromosome might also be a linear molecule of 4 megabases. Three loci involved in phytopathogenicity have been identified by insertion mutagenesis of this Fi plasmid. Inactivation of the fas locus resulted in avirulent strains, whereas insertions in the two other loci affected the degree of virulence, yielding attenuated (att) and hypervirulent (hyp) bacteria. One of the genes within the fas locus encoded an isopentenyltranferase (IPT) with low homology to analogous proteins from Gram-negative phytopathogenic bacteria. IPT activity was detected after expression of this protein in Escherichia coli cells. In R.fascians, ipt expression could only be detected in bacteria induced with extracts from fasciated tissue. R.fascians strains without the linear plasmid but containing this fas locus alone could not provoke any phenotype on plants, indicating additional genes from the linear plasmid were also essential for virulence. These studies, the first genetic analysis of the interaction of a Gram-positive bacterium with plants, suggest that a novel mechanism for plant tumour induction has evolved in R.fascians independently from the other branches of the eubacteria.

  10. Infrared laser effects at fluences used for treatment of dentin hypersensitivity on DNA repair in Escherichia coli and plasmids

    NASA Astrophysics Data System (ADS)

    Rocha Teixeira, Gleica; da Silva Marciano, Roberta; da Silva Sergio, Luiz Philippe; Castanheira Polignano, Giovanni Augusto; Roberto Guimarães, Oscar; Geller, Mauro; de Paoli, Flavia; de Souza da Fonseca, Adenilson

    2014-12-01

    Low-intensity infrared lasers are proposed in clinical protocols based on biostimulative effects, yet dosimetry is inaccurate and their effects on DNA at therapeutic doses are controversial. The aim of this work was to evaluate the effects of low-intensity infrared laser on survival and induction of filamentation of Escherichia coli cells, and induction of DNA lesions in bacterial plasmids. E. coli cultures were exposed to laser (808 nm, 100 mW, 40 and 60 J/cm2) to study bacterial survival and filamentation. Also, bacterial plasmids were exposed to laser to study DNA lesions by electrophoretic profile and action of DNA repair enzymes. Data indicate low-intensity infrared laser has no effect on survival of E. coli wild type and exonuclease III, but decreases the survival of formamidopyrimidine DNA glycosylase/MutM protein and endonuclease III deficient cells in stationary growth phase, induces bacterial filamentation, does not alter the electrophoretic profile of plasmids in agarose gels and does not alter the electrophoretic profile of plasmids incubated with endonuclease III, formamidopyrimidine DNA glycosylase/MutM protein and exonuclease III. Our findings show that low-intensity laser exposure causes DNA lesions at sub-lethal level and induces cellular mechanisms involved in repair of oxidative lesions in DNA. Studies about laser dosimetry and safety strategies are necessary for professionals and patients exposed to low-intensity lasers at therapeutic doses.

  11. Asbestos fibers mediate transformation of monkey cells by exogenous plasmid DNA

    SciTech Connect

    Appel, J.D.; Fasy, T.M.; Kohtz, D.S.; Kohtz, J.D.; Johnson, E.M. )

    1988-10-01

    The authors have tested the ability of chrysotile asbestos fibers to introduce plasmid DNA into monkey COS-7 cells and the ability of this DNA to function in both replication and gene expression. Chrysotile fibers are at least as effective as calcium phosphate in standard transfection assays at optimal ratios of asbestos to DNA. After transfection with chrysotile, a minor percentage of introduced plasmid DNA bearing a simian virus 40 origin of replication replicates after 24 hr. Fragmentation of entering DNA is more prominent with asbestos than with calcium phosphate, and after 72 hr most DNA introduced by asbestos is associated with chromosomal DNA. Cells transfected with plasmid p11-4, bearing the p53 protooncogene, express this gene. Cells transfected with pSV2-neo express a gene conferring resistance of antibiotic G418, allowing isolation of colonies of transformed cells after 18 days. The introduction of exogenous DNA into eukaryotic cells could cause mutations in several ways and thus contribute to asbestos-induced oncogenesis.

  12. Bacterial expression system with tightly regulated gene expression and plasmid copy number.

    PubMed

    Bowers, Lisa M; Lapoint, Kathleen; Anthony, Larry; Pluciennik, Anna; Filutowicz, Marcin

    2004-09-29

    A new Escherichia coli host/vector system has been engineered to allow tight and uniform modulation of gene expression and gamma origin (ori) plasmid copy number. Regulation of gamma ori plasmid copy number is achieved through arabinose-inducible expression of the necessary Rep protein, pi, whose gene was integrated into the chromosome of the host strain under control of the P(BAD) promoter. gamma ori replication can be uniformly modulated over 100-fold by changing the concentration of l-arabinose in the growth medium. This strain avoids the problem of all-or-nothing induction of P(BAD) because it is deficient in both arabinose uptake and degradation genes. Arabinose enters the cell by a mutant LacY transporter, LacYA177C, which is expressed from the host chromosome. Although this strain could be compatible with any gamma ori plasmid, we describe the utility of a gamma ori expression vector that allows especially tight regulation of gene expression. With this host/vector system, it is possible to independently modulate gene expression and gene dosage, facilitating the cloning and overproduction of toxic gene products. We describe the successful use of this system for cloning a highly potent toxin, Colicin E3, in the absence of its cognate immunity protein. This system could be useful for cloning genes encoding other potent toxins, screening libraries for potential toxins, and maintaining any gamma ori vector at precise copy levels in a cell.

  13. Construction and characterization of gelonin and saporin plasmids for toxic gene-based cancer therapy.

    PubMed

    Min, Kyoung Ah; He, Huining; Yang, Victor C; Shin, Meong Cheol

    2016-05-01

    Toxic gene therapy (or suicidal gene therapy) is gaining enormous interest, specifically for the treatment of cancer. The success of this therapy lies in several crucial factors, including the potency of gene products to kill the transfected tumor cells and the transfection ability of the transfection vehicles. To address the potency problem, in the present study, we engineered two separate mammalian transfection plasmids (pSAP and pGEL) containing genes encoding ribosome inactivating proteins (RIPs), gelonin and saporin. After the successful preparation and amplification of the plasmids, they were tested on various cancer cell lines (HeLa, U87, 9L, and MDA-MB-435) and a noncancerous cell line (293 HEK) using polyethyleneimine (PEI) as the transfection agent. Transfection studies performed under varying gene concentration, incubation time, and gene-to-PEI ratios revealed that, compared to the treatment of pGFP (GFP expression plasmid)/PEI, both pGEL/PEI and pSAP/PEI complexes could induce significantly augmented cytotoxic effects at only 2 μg/mL gene concentration. Importantly, these cytotoxic effects were observed universally in all tested cancer cell lines. Overall, this study demonstrated the potential of pGEL and pSAP as effective gene candidates for the toxic gene-based cancer therapy.

  14. Cloning and expression of plasmid genes encoding resistances to chromate and cobalt in Alcaligenes eutrophus.

    PubMed Central

    Nies, A; Nies, D H; Silver, S

    1989-01-01

    Resistances to chromate and cobalt were cloned on a 30-kilobase-pair (kb) DNA region from the large Alcaligenes eutrophus plasmid pMOL28 into the broad-host-range mobilizable cosmid vector pVK102. A restriction nuclease map of the 30-kb region was generated. The resistances expressed from the hybrid plasmids after transfer back into A. eutrophus were inducible and conferred the same degree of resistance as the parent plasmid pMOL28. Resistances were expressed in metal-sensitive Alcaligenes strains and related bacteria but not in Escherichia coli. Resistance to chromate was further localized on a 2.6-kb EcoRI fragment, and resistance to cobalt was localized on an adjoining 8.5-kb PstI-EcoRI fragment. When the 2.6-kb EcoRI fragment was expressed in E. coli under the control of a bacteriophage T7 promoter, three polypeptides with molecular masses of 31,500, 21,000, and 14,500 daltons were visible on autoradiograms. The 31,500- and 21,000-dalton polypeptides were membrane bound; the 14,500-dalton polypeptide was soluble. Images PMID:2549011

  15. DKK1 eukaryotic expression plasmid and expression product identification.

    PubMed

    Bao, G Y; Lu, K Y; Cui, S F; Xu, L

    2015-06-11

    We constructed the human dickkopf 1 (DKK1) eukaryotic expression plasmid and expressed, purified, and identified its expression product. We extracted cancer cells from cervical cancer tissue, followed by extraction of mRNA. Reverse transcription-polymerase chain reaction was conducted to obtain DKK1 gene fragments. Using these fragments, we prepared the recombinant plasmid pCMV-HA2/DKK1. The recombinant plasmid was restriction enzyme-digested and sequenced, and using liposome vectors, was transiently transfected into Free-Style 293-F cells (serum-free medium). DKK1 protein was detected by western blotting. The amplification product showed the expected size. Restriction enzyme digestion and sequence analysis showed that the recombinant plasmid was PCMV-HA2/DKK1. The expression product was verified properly by western blotting using an anti-DKKI antibody. The successful cloning of the DKKI gene and expression of DKKI protein will be useful for studying the biological activity of tumorigenesis.

  16. Mechanisms of Evolution in High-Consequence Drug Resistance Plasmids

    PubMed Central

    He, Susu; Chandler, Michael; Varani, Alessandro M.; Hickman, Alison B.; Dekker, John P.

    2016-01-01

    ABSTRACT The dissemination of resistance among bacteria has been facilitated by the fact that resistance genes are usually located on a diverse and evolving set of transmissible plasmids. However, the mechanisms generating diversity and enabling adaptation within highly successful resistance plasmids have remained obscure, despite their profound clinical significance. To understand these mechanisms, we have performed a detailed analysis of the mobilome (the entire mobile genetic element content) of a set of previously sequenced carbapenemase-producing Enterobacteriaceae (CPE) from the National Institutes of Health Clinical Center. This analysis revealed that plasmid reorganizations occurring in the natural context of colonization of human hosts were overwhelmingly driven by genetic rearrangements carried out by replicative transposons working in concert with the process of homologous recombination. A more complete understanding of the molecular mechanisms and evolutionary forces driving rearrangements in resistance plasmids may lead to fundamentally new strategies to address the problem of antibiotic resistance. PMID:27923922

  17. Complete nucleotide sequence of a native plasmid from Brevibacterium linens.

    PubMed

    Moore, Mathew; Svenson, Charles; Bowling, David; Glenn, Dianne

    2003-03-01

    Brevibacterium linens has commercial significance in the dairy industry and potential application in the production of bacteriocins and carotenoids. Strain development of these industrially significant organisms would be facilitated by the use of vectors, yet few are available. In this study we report the isolation of four novel plasmids from the Gram-positive coryneform B. linens, and determine the first complete nucleotide sequence of a native plasmid of B. linens. The cryptic plasmid pLIM is 7610 bp in length, and belongs to a subfamily of theta replicating ColE2-related plasmids. Initial investigation suggests that replication in pLIM requires two replicases, a primase (RepA) and a DNA binding protein (RepB), encoded by a single operon repAB. The origin of replication is located upstream of repAB transcription.

  18. The A to Z of A/C plasmids.

    PubMed

    Harmer, Christopher J; Hall, Ruth M

    2015-07-01

    Plasmids belonging to incompatibility groups A and C (now A/C) were among the earliest to be associated with antibiotic resistance in Gram-negative bacteria. A/C plasmids are large, conjugative plasmids with a broad host range. The prevalence of A/C plasmids in collections of clinical isolates has revealed their importance in the dissemination of extended-spectrum β-lactamases and carbapenemases. They also mobilize SGI1-type resistance islands. Revived interest in the family has yielded many complete A/C plasmid sequences, revealing that RA1, designated A/C1, is different from the remainder, designated A/C2. There are two distinct A/C2 lineages. Backbones of 128-130 kb include over 120 genes or ORFs encoding proteins of at least 100 amino acids, but very few have been characterized. Genes potentially required for replication, stability and transfer have been identified, but only the replication system of RA1 and the regulation of transfer have been studied. There is enormous variety in the antibiotic resistance genes carried by A/C2 plasmids but they are usually clustered in larger regions at various locations in the backbone. The ARI-A and ARI-B resistance islands are always at a specific location but have variable content. ARI-A is only found in type 1 A/C2 plasmids, which disseminate blaCMY-2 and blaNDM-1 genes, whereas ARI-B, carrying the sul2 gene, is found in both type 1 and type 2. This review summarizes current knowledge of A/C plasmids, and highlights areas of research to be considered in the future.

  19. Modelling the spatial dynamics of plasmid transfer and persistence.

    PubMed

    Krone, Stephen M; Lu, Ruinan; Fox, Randal; Suzuki, Haruo; Top, Eva M

    2007-08-01

    Bacterial plasmids are extra-chromosomal genetic elements that code for a wide variety of phenotypes in their bacterial hosts and are maintained in bacterial communities through both vertical and horizontal transfer. Current mathematical models of plasmid-bacteria dynamics, based almost exclusively on mass-action differential equations that describe these interactions in completely mixed environments, fail to adequately explain phenomena such as the long-term persistence of plasmids in natural and clinical bacterial communities. This failure is, at least in part, due to the absence of any spatial structure in these models, whereas most bacterial populations are spatially structured in microcolonies and biofilms. To help bridge the gap between theoretical predictions and observed patterns of plasmid spread and persistence, an individual-based lattice model (interacting particle system) that provides a predictive framework for understanding the dynamics of plasmid-bacteria interactions in spatially structured populations is presented here. To assess the accuracy and flexibility of the model, a series of experiments that monitored plasmid loss and horizontal transfer of the IncP-1beta plasmid pB10 : : rfp in Escherichia coli K12 and other bacterial populations grown on agar surfaces were performed. The model-based visual patterns of plasmid loss and spread, as well as quantitative predictions of the effects of different initial parental strain densities and incubation time on densities of transconjugants formed on a 2D grid, were in agreement with this and previously published empirical data. These results include features of spatially structured populations that are not predicted by mass-action differential equation models.

  20. The Native Plasmid pML21 Plays a Role in Stress Tolerance in Enterococcus faecalis ML21, as Analyzed by Plasmid Curing Using Plasmid Incompatibility.

    PubMed

    Zuo, Fang-Lei; Chen, Li-Li; Zeng, Zhu; Feng, Xiu-Juan; Yu, Rui; Lu, Xiao-Ming; Ma, Hui-Qin; Chen, Shang-Wu

    2016-02-01

    To investigate the role of the native plasmid pML21 in Enterococcus faecalis ML21's response to abiotic stresses, the plasmid pML21 was cured based on the principle of plasmid incompatibility and segregational instability, generating E. faecalis mutant strain ML0. The mutant and the wild strains were exposed to abiotic stresses: bile salts, low pH, H2O2, ethanol, heat, and NaCl, and their survival rate was measured. We found that curing of pML21 lead to reduced tolerance to stress in E. faecalis ML0, especially oxidative and osmotic stress. Complementation analysis suggested that the genes from pML21 played different role in stress tolerance. The result indicated that pML21 plays a role in E. faecalis ML21's response to abiotic stresses.

  1. Biomedical application of plasmid DNA in gene therapy: a new challenge for chromatography.

    PubMed

    Sousa, F; Passarinha, L; Queiroz, J A

    2010-01-01

    Gene therapy and DNA vaccination are clinical fields gradually emerging in the last few decades, in particular after the discovery of some gene-related diseases. The increased relevance of biomedical applications of plasmid DNA (pDNA) to induce therapeutic effects has had a great impact on biopharmaceutical research and industry. Although there are several steps involved in the pDNA manufacturing process, the several unit operations must be designed and integrated into a global process. After the plasmid has been designed according to the requirements for clinical administeration to humans, it is biosynthesised mainly by an E. coli host. The overriding priority of the production process is to improve plasmid quantity - the production conditions need to be optimised to guarantee pDNA stability and biological activity. The complexity and diversity of biomolecules present on the pDNA-containing extracts represent the main concern and limitation to achieve pure and biologically active pDNA. There has been a recent intenstification of the improvement of existing purification procedures or the establishment of novel schemes for plasmid purification. This review focuses on the progress and relevance of chromatographic methodologies in the purification of pDNA-based therapeutic products. The review will attempt to assemble their different contributions of the different chromatographic procedures that are being used in the pDNA purification area. The advantages and disadvantages of the different chromatographic techniques, as well as the most significant improvements in response to the challenge of purifying pDNA will be discussed, emphasizing the future directions in this field.

  2. Cloning and sequencing of a plasmid-borne gene (opd) encoding a phosphotriesterase.

    PubMed Central

    McDaniel, C S; Harper, L L; Wild, J R

    1988-01-01

    Plasmid pCMS1 was isolated from Pseudomonas diminuta MG, a strain which constitutively hydrolyzes a broad spectrum of organophosphorus compounds. The native plasmid was restricted with PstI, and individual DNA fragments were subcloned into pBR322. A recombinant plasmid transformed into Escherichia coli possessed weak hydrolytic activity, and Southern blotting with the native plasmid DNA verified that the DNA sequence originated from pCMS1. When the cloned 1.3-kilobase fragment was placed behind the lacZ' promoter of M13mp10 and retransformed into E. coli, clear-plaque isolates with correctly sized inserts exhibited isopropyl-beta-D-thiogalactopyranoside-inducible whole-cell activity. Sequence determination of the M13 constructions identified an open reading frame of 975 bases preceded by a putative ribosome-binding site appropriately positioned upstream of the first ATG codon in the open reading frame. An intragenic fusion of the opd gene with the lacZ gene produced a hybrid polypeptide which was purified by beta-galactosidase immunoaffinity chromatography and used to confirm the open reading frame of opd. The gene product, an organophosphorus phosphotriesterase, would have a molecular weight of 35,418 if the presumed start site is correct. Eighty to ninety percent of the enzymatic activity was associated with the pseudomonad membrane fractions. When dissociated by treatment with 0.1% Triton and 1 M NaCl, the enzymatic activity was associated with a molecular weight of approximately 65,000, suggesting that the active enzyme was dimeric. Images PMID:2834339

  3. Transformation of Actinobacillus actinomycetemcomitans by electroporation, utilizing constructed shuttle plasmids.

    PubMed Central

    Sreenivasan, P K; LeBlanc, D J; Lee, L N; Fives-Taylor, P

    1991-01-01

    Actinobacillus actinomycetemcomitans, a periodontal pathogen, has been strongly implicated in human periodontal disease. Advances in the molecular analysis of A. actinomycetemcomitans virulence factors have been limited due to the unavailability of systems for genetic transfer, transposon mutagenesis, and gene complementation. Slow progress can be traced almost exclusively to the lack of gene vector systems and methods for the introduction of DNA into A. actinomycetemcomitans. An electrotransformation system that allowed at least five strains of A. actinomycetemcomitans to be transformed with stable shuttle plasmids which efficiently replicated in both Escherichia coli and A. actinomycetemcomitans was developed. One plasmid, a potential shuttle vector designated pDL282, is 5.7 kb in size, has several unique restriction enzyme sites, and codes for resistance to spectinomycin and ampicillin. E. coli and A. actinomycetemcomitans were transformed with equal efficiencies of approximately 10(5) transformants per micrograms of DNA. Similar transformation efficiencies were obtained whether the plasmid DNA was isolated from A. actinomycetemcomitans or E. coli. In addition, frozen competent cells of A. actinomycetemcomitans yielded comparable efficiencies of transformation. Restriction enzyme analysis of pDL282 isolated after transformation confirmed the presence of intact donor plasmids. A plasmid isolated from A. pleuropneumoniae was also capable of transforming some isolates of A. actinomycetemcomitans, although generally at a lower frequency. The availability of these shuttle plasmids and an efficient transformation procedure should significantly facilitate the molecular analysis of virulence factors of A. actinomycetemcomitans. PMID:1937823

  4. The Addgene repository: an international nonprofit plasmid and data resource

    PubMed Central

    Kamens, Joanne

    2015-01-01

    The Addgene Repository (http://www.addgene.org) was founded to accelerate research and discovery by improving access to useful, high-quality research materials and information. The repository archives plasmids generated by scientists, conducts quality control, annotates the associated data and makes the plasmids and their data available to the scientific community. Plasmid associated data undergoes ongoing curation by members of the scientific community and by Addgene scientists. The growing database contains information on >31 000 unique plasmids spanning most experimental biological systems and organisms. The library includes a large number of plasmid tools for use in a wide variety of research areas, such as empty backbones, lentiviral resources, fluorescent protein vectors and genome engineering tools. The Addgene Repository database is always evolving with new plasmid deposits so it contains currently pertinent resources while ensuring the information on earlier deposits is still available. Custom search and browse features are available to access information on the diverse collection. Extensive educational materials and information are provided by the database curators to support the scientists that are accessing the repository's materials and data. PMID:25392412

  5. Plasmid copy number noise in monoclonal populations of bacteria

    NASA Astrophysics Data System (ADS)

    Wong Ng, Jérôme; Chatenay, Didier; Robert, Jérôme; Poirier, Michael Guy

    2010-01-01

    Plasmids are extra chromosomal DNA that can confer to their hosts’ supplementary characteristics such as antibiotic resistance. Plasmids code for their copy number through their own replication frequency. Even though the biochemical networks underlying the plasmid copy number (PCN) regulation processes have been studied and modeled, no measurement of the heterogeneity in PCN within a whole population has been done. We have developed a fluorescent-based measurement system, which enables determination of the mean and noise in PCN within a monoclonal population of bacteria. Two different fluorescent protein reporters were inserted: one on the chromosome and the other on the plasmid. The fluorescence of these bacteria was measured with a microfluidic flow cytometry device. We show that our measurements are consistent with known plasmid characteristics. We find that the partitioning system lowers the PCN mean and standard deviation. Finally, bacterial populations were allowed to grow without selective pressure. In this case, we were able to determine the plasmid loss rate and growth inhibition effect.

  6. Homologous Recombination between Autonomously Replicating Plasmids in Mammalian Cells

    PubMed Central

    Ayares, David; Spencer, James; Schwartz, Faina; Morse, Brian; Kucherlapati, Raju

    1985-01-01

    The ability of autonomously replicating plasmids to recombine in mammalian cells was investigated. Two deletion plasmids of the eukaryotic-prokaryotic shuttle vector pSV2neo were cotransfected into transformed monkey COS cells. Examination of the low molecular weight DNA isolated after 48 hr of incubation revealed that recombination between the plasmids had occurred. The DNA was also used to transform recA- E. coli. Yield of neo R colonies signified homologous recombination. Examination of the plasmid DNA from these colonies confirmed this view. Double-strand breaks in one or both of the input plasmids at the sites of deletion resulted in an enhancement of recombination frequency. The recombination process yielded monomeric and dimeric molecules. Examination of these molecules revealed that reciprocal recombination as well as gene conversion events were involved in the generation of plasmids bearing an intact neo gene. The COS cell system we describe is analogous to study of bacteriophage recombination and yeast random-spore analysis. PMID:2996980

  7. 99mTc-labeled HYNIC-DAPI causes plasmid DNA damage with high efficiency.

    PubMed

    Kotzerke, Joerg; Punzet, Robert; Runge, Roswitha; Ferl, Sandra; Oehme, Liane; Wunderlich, Gerd; Freudenberg, Robert

    2014-01-01

    (99m)Tc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, (99m)Tc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs) or double-strand breaks (DSBs); the resulting DNA fragments can be separated by gel electrophoresis and quantified by fluorescent staining. This study aimed to compare the plasmid DNA damage potential of a (99m)Tc-labeled HYNIC-DAPI compound with that of (99m)Tc pertechnetate ((99m)TcO4(-)). pUC19 plasmid DNA was irradiated for 2 or 24 hours. Direct and radical-induced DNA damage were evaluated in the presence or absence of the radical scavenger DMSO. For both compounds, an increase in applied activity enhanced plasmid DNA damage, which was evidenced by an increase in the open circular and linear DNA fractions and a reduction in the supercoiled DNA fraction. The number of SSBs elicited by 99mTc-HYNIC-DAPI (1.03) was twice that caused by (99m)TcO4(-) (0.51), and the number of DSBs increased fivefold in the (99m)Tc-HYNIC-DAPI-treated sample compared with the (99m)TcO4(-) treated sample (0.02 to 0.10). In the presence of DMSO, the numbers of SSBs and DSBs decreased to 0.03 and 0.00, respectively, in the (99m)TcO4(-) treated samples, whereas the numbers of SSBs and DSBs were slightly reduced to 0.95 and 0.06, respectively, in the (99m)Tc-HYNIC-DAPI-treated samples. These results indicated that (99m)Tc-HYNIC-DAPI induced SSBs and DSBs via a direct interaction of the (99m)Tc-labeled compound with DNA. In contrast to these results, (99m)TcO4(-) induced SSBs via radical formation, and DSBs were formed by two nearby SSBs. The biological effectiveness of (99m)Tc-HYNIC-DAPI increased by approximately 4-fold in terms of inducing SSBs and by

  8. 99mTc-Labeled HYNIC-DAPI Causes Plasmid DNA Damage with High Efficiency

    PubMed Central

    Kotzerke, Joerg; Punzet, Robert; Runge, Roswitha; Ferl, Sandra; Oehme, Liane; Wunderlich, Gerd; Freudenberg, Robert

    2014-01-01

    99mTc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, 99mTc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs) or double-strand breaks (DSBs); the resulting DNA fragments can be separated by gel electrophoresis and quantified by fluorescent staining. This study aimed to compare the plasmid DNA damage potential of a 99mTc-labeled HYNIC-DAPI compound with that of 99mTc pertechnetate (99mTcO4−). pUC19 plasmid DNA was irradiated for 2 or 24 hours. Direct and radical-induced DNA damage were evaluated in the presence or absence of the radical scavenger DMSO. For both compounds, an increase in applied activity enhanced plasmid DNA damage, which was evidenced by an increase in the open circular and linear DNA fractions and a reduction in the supercoiled DNA fraction. The number of SSBs elicited by 99mTc-HYNIC-DAPI (1.03) was twice that caused by 99mTcO4− (0.51), and the number of DSBs increased fivefold in the 99mTc-HYNIC-DAPI-treated sample compared with the 99mTcO4− treated sample (0.02 to 0.10). In the presence of DMSO, the numbers of SSBs and DSBs decreased to 0.03 and 0.00, respectively, in the 99mTcO4– treated samples, whereas the numbers of SSBs and DSBs were slightly reduced to 0.95 and 0.06, respectively, in the 99mTc-HYNIC-DAPI-treated samples. These results indicated that 99mTc-HYNIC-DAPI induced SSBs and DSBs via a direct interaction of the 99mTc-labeled compound with DNA. In contrast to these results, 99mTcO4− induced SSBs via radical formation, and DSBs were formed by two nearby SSBs. The biological effectiveness of 99mTc-HYNIC-DAPI increased by approximately 4-fold in terms of inducing SSBs and by approximately 10-fold in terms of

  9. Plasmid content of isolates of Erwinia amylovora from orchards in Washington and Oregon in the USA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nearly all strains of Erwinia amylovora carry plasmid pEA29, which has not been found in other species of bacteria. Additional plasmids have been reported in the pathogen isolates from Western states, such as a plasmid in strain CA11 that carries streptomycin-resistance genes and the plasmid pEU30,...

  10. Allelopathy of plasmid-bearing and plasmid-free organisms competing for two complementary resources in a chemostat.

    PubMed

    Bhattacharyya, Joydeb; Smith, Hal L; Pal, Samares

    2012-01-01

    We consider a model of competition between plasmid-bearing and plasmid-free organisms for two complementary nutrients in a chemostat. We assume that the plasmid-bearing organism produces an allelopathic agent at the cost of its reproductive abilities which is lethal to plasmid-free organism. Our analysis leads to different thresholds in terms of the model parameters acting as conditions under which the organisms associated with the system cannot thrive even in the absence of competition. Local stability of the system is obtained in the absence of one or both the organisms. Also, global stability of the system is obtained in the presence of both the organisms. Computer simulations have been carried out to illustrate various analytical results.

  11. Biology of the staphylococcal conjugative multiresistance plasmid pSK41.

    PubMed

    Liu, Michael A; Kwong, Stephen M; Jensen, Slade O; Brzoska, Anthony J; Firth, Neville

    2013-07-01

    Plasmid pSK41 is a large, low-copy-number, conjugative plasmid from Staphylococcus aureus that is representative of a family of staphylococcal plasmids that confer multiple resistances to a wide range of antimicrobial agents. The plasmid consists of a conserved plasmid backbone containing the genes for plasmid housekeeping functions, which is punctuated by copies of IS257 that flank a Tn4001-hybrid structure and cointegrated plasmids that harbour resistance genes. This review summarises the current understanding of the biology of pSK41, focussing on the systems responsible for its replication, maintenance and transmission, and their regulation.

  12. Exposing Plasmids as the Achilles’ Heel of Drug-Resistant Bacteria

    PubMed Central

    Williams, Julia J.; Hergenrother, Paul J.

    2008-01-01

    Many multi-drug resistant bacterial pathogens harbor large plasmids that encode proteins conferring resistance to antibiotics. While the acquisition of these plasmids often enables bacteria to survive in the presence of antibiotics, it is possible that plasmids also represent a vulnerability that can be exploited in tailored antibacterial therapy. This review highlights three recently described strategies designed to specifically combat bacteria harboring such plasmids: Inhibition of plasmid conjugation, inhibition of plasmid replication, and exploitation of plasmid-encoded toxin-antitoxin systems. PMID:18625335

  13. A segregated model for plasmid content and product synthesis in unstable binary fission recombinant organisms.

    PubMed

    Seo, J H; Bailey, J E

    1985-02-01

    Plasmid propagation in populations of unstable, binary fission recombinant organisms has been studied using a segregated, population balance mathematical model. Segregated models have the advantage of direct incorporation of basic information on mechanisms and kinetics of plasmid replication and segregation at the single-cell level. The distribution of cellular plasmid content and specific rates of plasmid gene expression have been obtained for several single-cell models of plasmid replication, partition, and gene expression. Plasmid replication kinetics during cell growth significantly influence the plasmid content distribution. In the case of transient growth of plasmid-containing and plasmid-free cells in partially selective medium, the degree of selection required for stable maintenance of plasmid-containing cells has been determined. Guidelines are presented for applicability of simpler, nonsegregated models and for evaluation of the parameters in these models based on single-cell mechanisms and associated parameters.

  14. [SOS-induction in the presence of the plasmid pKM101 in the bacterial cells Escherichia coli K12].

    PubMed

    Tiganova, I G; Rusina, O Iu; Andreeva, I V

    2006-01-01

    Induction of transcription by the plasmid pKM101 (mutability mediating derivate of the plasmid R46) of the sfiA gene controlling cell division and of the fruA gene encoding the fructose specific enzyme II of the phosphoenolpyruvate-phosphotransferase system in intact cultures of Escherichia coli was studied. The genes under study were fused to the bacteriophage Mu dl (Ap lac). Activation of the sfiA gene, a typical member of the SOS-regulon, was demonstrated to depend on the key genes of the SOS-system-recA and lexA. In contrast, the fruA gene that is non-inducible by the UV-light, a classical SOS-inducing agent, is not activated by the presence of the plasmid pKMIO1 in the bacterial cells. The data obtained suggest that the presence of pKMIO1 plasmid in the Escherichia coli cells induces a SOS-signal as a consequence of the plasmid DNA replication or its conjugative transfer.

  15. Plasmid Stability in Dried Cells of the Desert Cyanobacterium Chroococcidiopsis and its Potential for GFP Imaging of Survivors on Earth and in Space

    NASA Astrophysics Data System (ADS)

    Billi, Daniela

    2012-06-01

    Two GFP-based plasmids, namely pTTQ18-GFP-pDU1mini and pDUCA7-GFP, of about 7 kbp and 15 kbp respectively, able to replicate in Chroococcidiopsis sp. CCMEE 029 and CCMEE 123, were developed. Both plasmids were maintained in Chroococcidiopsis cells after 18 months of dry storage as demonstrated by colony PCR, plasmid restriction analysis, GFP imaging and colony-forming ability under selection of dried transformants; thus suggesting that strategies employed by this cyanobacterium to stabilize dried chromosomal DNA, must have protected plasmid DNA. The suitability of pDU1mini-plasmid for GFP tagging in Chroococcidiopsis was investigated by using the RecA homolog of Synechocystis sp. PCC 6803. After 2 months of dry storage, the presence of dried cells with a GFP-RecASyn distribution resembling that of hydrated cells, supported its capability of preventing desiccation-induced genome damage, whereas the rewetted cells with filamentous GFP-RecASyn structures revealed sub-lethal DNA damage. The long-term stability of plasmid DNA in dried Chroococcidiopsis has implication for space research, for example when investigating the recovery of dried cells after Martian and space simulations or when developing life support systems based on phototrophs with genetically enhanced stress tolerance and stored in the dry state for prolonged periods.

  16. Plasmid stability in dried cells of the desert cyanobacterium Chroococcidiopsis and its potential for GFP imaging of survivors on Earth and in space.

    PubMed

    Billi, Daniela

    2012-06-01

    Two GFP-based plasmids, namely pTTQ18-GFP-pDU1(mini) and pDUCA7-GFP, of about 7 kbp and 15 kbp respectively, able to replicate in Chroococcidiopsis sp. CCMEE 029 and CCMEE 123, were developed. Both plasmids were maintained in Chroococcidiopsis cells after 18 months of dry storage as demonstrated by colony PCR, plasmid restriction analysis, GFP imaging and colony-forming ability under selection of dried transformants; thus suggesting that strategies employed by this cyanobacterium to stabilize dried chromosomal DNA, must have protected plasmid DNA. The suitability of pDU1(mini)-plasmid for GFP tagging in Chroococcidiopsis was investigated by using the RecA homolog of Synechocystis sp. PCC 6803. After 2 months of dry storage, the presence of dried cells with a GFP-RecA(Syn) distribution resembling that of hydrated cells, supported its capability of preventing desiccation-induced genome damage, whereas the rewetted cells with filamentous GFP-RecA(Syn) structures revealed sub-lethal DNA damage. The long-term stability of plasmid DNA in dried Chroococcidiopsis has implication for space research, for example when investigating the recovery of dried cells after Martian and space simulations or when developing life support systems based on phototrophs with genetically enhanced stress tolerance and stored in the dry state for prolonged periods.

  17. Radiosensitivity of plasmid DNA: role of topology and concentration

    NASA Astrophysics Data System (ADS)

    Giustranti, C.; Pérez, C.; Rousset, S.; Balanzat, E.; Sage, E.

    1999-01-01

    Using the plasmid relaxation assay, the induction of single strand breaks (SSB) by ionizing radiation was investigated in two plasmids of different length, pBS and pSP189. The dose-response was linear for both plasmids but pSP189 exhibited a three times higher sensitivity than pBS. This disparity may be explained by a reduced accessibility to hydroxyl radicals due to a different topology of each plasmid, i.e. degree of compaction, as observed with electron microscopy. pBS plasmid was also exposed at various DNA concentrations to rays. The yield of SSB decreased with increasing concentration, suggesting a diminution in the amount of hydroxyl radicals efficient for radiolytic attack. This effect of concentration was also observed with densely ionizing radiation. In conclusion, the accessibility of DNA is a key-parameter in the formation of damage in vitro and in vivo as well. En utilisant la technique de relaxation de plasmide, l'induction de cassures simple brin (SSB) par les radiations ? a été comparée dans deux plasmides de taille différente, pSP189 et pBS. La relation dose-effet est linéaire pour les deux plasmides, mais il se forme trois fois plus de SSB dans pSP189 que dans pBS. Cette disparité semble pouvoir être reliée au degré de compaction différent des plasmides, observé en microscopie électronique. Elle s'expliquerait en terme d'accessibilité aux espèces radicalaires formées lors de la radiolyse de l'eau. Le plasmide pBS, à différentes concentrations, a été ensuite exposé aux radiations γ. Le taux de cassures décroit lorsque la concentration en ADN croit, suggérant une diminution du nombre de radicaux pouvant efficacement réagir avec l'ADN. Cet effet a également été mis en évidence lors d'une irradiation avec des particules de TEL élevé. En conclusion, l'accessibilité de l'ADN est un paramètre- clé dans la formation des dommages, tant in vitro que in vivo.

  18. Integration of pT181-like tetracycline resistance plasmids into large staphylococcal plasmids involves IS257.

    PubMed Central

    Werckenthin, C; Schwarz, S; Roberts, M C

    1996-01-01

    Four large staphylococcal plasmids ranging in size from 31 to 82 kbp have been shown to mediate tetracycline resistance via an integrated copy of the tet(K)-encoding plasmid pT181 which was flanked by copies of the insertion element IS257. In two cases, IS257 elements interrupted the repC reading frame of pT181 and an 8-bp sequence from within the repC gene was duplicated at the interrupted site. In the third plasmid, the IS257 elements interrupted the pT181 DNA immediately upstream of the repC coding sequence with an 8-bp duplication. In the fourth case, the IS257 elements flanked a pT181-like plasmid with one IS257 in the repC coding sequence and the other within the recombinase (pre) coding sequence, so that a section of the pT181 sequence was deleted. All four integration sites detected in this study differ from those previously described for the IS257-mediated integration of pT181-like plasmids into large plasmids or into the chromosomal DNA. PMID:8913460

  19. Serum resistance encoded by colicin V plasmids in Escherichia coli and its relationship to the plasmid transfer system.

    PubMed Central

    Nilius, A M; Savage, D C

    1984-01-01

    Eight colicin V plasmids were conjugated into a plasmidless Escherichia coli (K-12) strain that was susceptible to the bactericidal effects of normal rabbit serum. The resulting colicin V-positive strains were examined for their capacity to resist the lethal effects of serum. Serum resistance was assessed as growth of the bacterial strain in medium containing 5% normal rabbit serum inoculated from a culture in the early exponential phase. Only three of the eight colicin V plasmids were found to confer the serum resistance phenotype on the host strain. Derepression of the transfer system was associated with serum resistance in two of the plasmids. Two other derepressed plasmids did not confer serum resistance on the host bacterium. Therefore, such derepression alone was insufficient to produce serum resistance. The factor(s) encoded by colicin V plasmids and responsible for the serum resistance of the bacterial strains bearing the plasmids was shown to be a property associated with the cell and not an extracellular factor excreted into the growth medium. PMID:6365788

  20. Plasmids and Rickettsial Evolution: Insight from Rickettsia felis

    PubMed Central

    Gillespie, Joseph J.; Beier, Magda S.; Rahman, M. Sayeedur; Ammerman, Nicole C.; Shallom, Joshua M.; Purkayastha, Anjan; Sobral, Bruno S.; Azad, Abdu F.

    2007-01-01

    Background The genome sequence of Rickettsia felis revealed a number of rickettsial genetic anomalies that likely contribute not only to a large genome size relative to other rickettsiae, but also to phenotypic oddities that have confounded the categorization of R. felis as either typhus group (TG) or spotted fever group (SFG) rickettsiae. Most intriguing was the first report from rickettsiae of a conjugative plasmid (pRF) that contains 68 putative open reading frames, several of which are predicted to encode proteins with high similarity to conjugative machinery in other plasmid-containing bacteria. Methodology/Principal Findings Using phylogeny estimation, we determined the mode of inheritance of pRF genes relative to conserved rickettsial chromosomal genes. Phylogenies of chromosomal genes were in agreement with other published rickettsial trees. However, phylogenies including pRF genes yielded different topologies and suggest a close relationship between pRF and ancestral group (AG) rickettsiae, including the recently completed genome of R. bellii str. RML369-C. This relatedness is further supported by the distribution of pRF genes across other rickettsiae, as 10 pRF genes (or inactive derivatives) also occur in AG (but not SFG) rickettsiae, with five of these genes characteristic of typical plasmids. Detailed characterization of pRF genes resulted in two novel findings: the identification of oriV and replication termination regions, and the likelihood that a second proposed plasmid, pRFδ, is an artifact of the original genome assembly. Conclusion/Significance Altogether, we propose a new rickettsial classification scheme with the addition of a fourth lineage, transitional group (TRG) rickettsiae, that is unique from TG and SFG rickettsiae and harbors genes from possible exchanges with AG rickettsiae via conjugation. We offer insight into the evolution of a plastic plasmid system in rickettsiae, including the role plasmids may have played in the acquirement of

  1. Thioredoxin-like proteins in F and other plasmid systems.

    PubMed

    Hemmis, Casey W; Schildbach, Joel F

    2013-09-01

    Bacterial conjugation is the process by which a conjugative plasmid transfers from donor to recipient bacterium. During this process, single-stranded plasmid DNA is actively and specifically transported from the cytoplasm of the donor, through a large membrane-spanning assembly known as the pore complex, and into the cytoplasm of the recipient. In Gram negative bacteria, construction of the pore requires localization of a subset of structural and catalytically active proteins to the bacterial periplasm. Unlike the cytoplasm, the periplasm contains proteins that promote disulfide bond formation within or between cysteine-containing proteins. To ensure proper protein folding and assembly, bacteria employ periplasmic redox systems for thiol oxidation, disulfide bond/sulfenic acid reduction, and disulfide bond isomerization. Recent data suggest that plasmid-based proteins belonging to the disulfide bond formation family play an integral role in the conjugative process by serving as mediators in folding and/or assembly of pore complex proteins. Here we report the identification of 165 thioredoxin-like family members across 89 different plasmid systems. Using phylogenetic analysis, all but nine family members were categorized into thioredoxin-like subfamilies. In addition, we discuss the diversity, conservation, and putative roles of thioredoxin-like proteins in plasmid systems, which include homologs of DsbA, DsbB, DsbC, DsbD, DsbG, and CcmG from Escherichia coli, TlpA from Bradyrhizobium japonicum, Com1 from Coxiella burnetii, as well as TrbB and TraF from plasmid F, and the absolute conservation of a disulfide isomerase in plasmids containing homologs of the transfer proteins TraH, TraN, and TraU.

  2. Dichromatic laser radiation effects on DNA of Escherichia coli and plasmids

    NASA Astrophysics Data System (ADS)

    Martins, W. A.; Polignano, G. A. C.; Guimarães, O. R.; Geller, M.; Paoli, F.; Fonseca, A. S.

    2015-04-01

    Dichromatic and consecutive laser radiations have attracted increased attention for clinical applications as offering new tools for the treatment of dysfunctional tissues in situations where monochromatic radiation is not effective. This work evaluated the survival, filamentation and morphology of Escherichia coli cells, and the induction of DNA lesions, in plasmid DNA exposed to low-intensity consecutive dichromatic laser radiation. Exponential and stationary wild type and formamidopyrimidine DNA glycosylase/MutM protein deficient E. coli cultures were exposed to consecutive low-intensity dichromatic laser radiation (infrared laser immediately after red laser) to study the survival, filamentation and morphology of bacterial cells. Plasmid DNA samples were exposed to dichromatic radiation to study DNA lesions by electrophoretic profile. Dichromatic laser radiation affects the survival, filamentation and morphology of E. coli cultures depending on the growth phase and the functional repair mechanism of oxidizing lesions in DNA, but does not induce single/double strands breaks or alkali-labile DNA lesions. Results show that low-intensity consecutive dichromatic laser radiation induces biological effects that differ from those induced by monochromatic laser radiation, suggesting that other therapeutic effects could be obtained using dichromatic radiation.

  3. Pheromone-inducible conjugation in Enterococcus faecalis

    PubMed Central

    Kozlowicz, Briana K.; Dworkin, Martin; Dunny, Gary M.

    2009-01-01

    Pheromone-inducible transfer of the plasmid pCF10 in Enterococcus faecalis is regulated using a complicated network of proteins and RNAs. The plasmid itself has been assembled from parts garnered from a variety of sources, and many aspects of the system resemble a biological kluge. Recently several new functions of various pCF10 gene products that participate in regulation of plasmid transfer have been identified. The results indicate that selective pressures controlling the evolution of the plasmid have produced a highly complex regulatory network with multiple biological functions that may serve well as a model for the evolution of biological complexity. PMID:16503196

  4. Skin Electroporation of a Plasmid Encoding hCAP-18/LL-37 Host Defense Peptide Promotes Wound Healing

    PubMed Central

    Steinstraesser, Lars; Lam, Martin C; Jacobsen, Frank; Porporato, Paolo E; Chereddy, Kiran Kumar; Becerikli, Mustafa; Stricker, Ingo; Hancock, Robert EW; Lehnhardt, Marcus; Sonveaux, Pierre; Préat, Véronique; Vandermeulen, Gaëlle

    2014-01-01

    Host defense peptides, in particular LL-37, are emerging as potential therapeutics for promoting wound healing and inhibiting bacterial growth. However, effective delivery of the LL-37 peptide remains limiting. We hypothesized that skin-targeted electroporation of a plasmid encoding hCAP-18/LL-37 would promote the healing of wounds. The plasmid was efficiently delivered to full-thickness skin wounds by electroporation and it induced expression of LL-37 in the epithelium. It significantly accelerated reepithelialization of nondiabetic and diabetic wounds and caused a significant VEGFa and interleukin (IL)-6 induction. IL-6 was involved in LL-37–mediated keratinocyte migration in vitro and IL-6 neutralizing antibodies delivered to mice were able to suppress the wound healing activity of the hCAP-18/LL-37 plasmid. In a hindlimb ischemia model, electroporation of the hCAP-18/LL-37 plasmid increased blood perfusion, reduced muscular atrophy, and upregulated the angiogenic chemokines VEGFa and SDF-1a, and their receptors VEGF-R and CXCR-4. These findings demonstrate that a localized gene therapy with LL-37 is a promising approach for the treatment of wounds. PMID:24394186

  5. Protein H encoded by plasmid CloDF13 is involved in excretion of cloacin DF13.

    PubMed Central

    Oudega, B; Stegehuis, F; van Tiel-Menkveld, G J; de Graaf, F K

    1982-01-01

    Excretion of cloacin DF13 was studied in Escherichia coli cells harboring different CloDF13 insertion and deletion mutant plasmids. Insertions of a transposon at position 9.8 or 11.5% of the CloDF13 plasmid blocked the expression of gene H and strongly reduced the specific excretion of cloacin DF13 into the culture medium, but had no effect on the production of cloacin DF13. Insertions in or deletions of regions of the CloDF13 DNA upstream the cloacin operon did not affect the excretion or production of the bacteriocin. Introduction of a CloDF13 plasmid that encodes for the gene H product in cells harboring a CloDF13 plasmid with an insertion in gene H stimulated the excretion of cloacin DF13 significantly in mitomycin C-induced and in noninduced cultures. Cloacin DF13 in cloacinogenic cells that did not produce the gene H protein was found to be about 90% located in the cytoplasm. In cells that did produce the gene H product, about 30% of the cloacin DF13 molecules were found in the cytoplasm, about 18% were found in the periplasm, about 2% were in the membranes, and about 50% were located in the culture supernatant. Cyclic AMP stimulated the production but not the excretion of cloacin DF13 in cells cultivated in the presence of glucose. PMID:6281236

  6. Bacterial plasmid transfer under space flight conditions: The Mobilisatsia experience

    NASA Astrophysics Data System (ADS)

    de Boever, P.; Ilyin, V.; Mahillon, J.; Mergeay, M.

    Background Microorganisms are subject to a genetic evolution which may lead to the capacity to colonize new environments and to cause infections Central players in this evolutionary process are mobile genetic elements phages plasmids and transposons The latter help to mobilize and reorganize genes be it within a given genome intragenomic mobility or between bacterial cells intercellular mobility Confined environment and space flight related factors such as microgravity and cosmic radiation may influence the frequency with which mobile genetic elements are exchanged between microorganisms Aim Within the frame of the Mobilisatsia experiment a triparental microbial plasmid transfer was promoted aboard the International Space Station ISS The efficiency of the plasmid exchange process was compared with a synchronously performed ground control experiment An experiment was carried out with well-characterized Gram-negative test strains and one experiment was done with Gram-positive test strains Results The experiment took place during the Soyouz Mission 8 to the ISS from April 19th until April 30th 2004 Liquid cultures of the bacterial strains Cupriavidus metallidurans AE815 final recipient Escherichia coli CM1962 carrying a mobilisable vector with a nickel-resistance marker and E coli CM140 carrying the Broad Host Range plasmid RP4 for the Gram-negative experiment and Bacillus thuringiensis Bti AND931 carrying the conjugative plasmid pXO16 Bti 4Q7 with mobilisable vector pC194 carrying a resistance to chloramphenicol and Bti GBJ002

  7. Plasmid DNA-based gene transfer with ultrasound and microbubbles.

    PubMed

    Taniyama, Yoshiaki; Azuma, Junya; Rakugi, Hiromi; Morishita, Ryuichi

    2011-12-01

    Gene therapy offers a novel approach for the prevention and treatment of a variety of diseases, but it is not yet a common option in the real world because of various problems. Viral vectors show high efficiency of gene transfer, but they have some problems with toxicity and immunity. On the other hand, plasmid DNA-based gene transfer is very safe, but its efficiency is relatively low. Especially, plasmid DNA gene therapy is used for cardiovascular disease because plasmid DNA transfer is possible for cardiac or skeletal muscle. Clinical angiogenic gene therapy using plasmid DNA gene transfer has been attempted in patients with peripheral artery disease, but a Phase III clinical trial did not show sufficient efficiency. Recently, a Phase III clinical trial of hepatocyte growth factor gene therapy in peripheral artery disease (PAD) showed improvement of ischemic ulcers, but it could not salvage limbs from amputation. In addition, a Phase I/II clinical study of fibroblast growth factor gene therapy in PAD extended amputation-free survival, but it seemed to fail in Phase III. In this situation, we and others have developed plasmid DNA-based gene transfer using ultrasound with microbubbles to enhance its efficiency while maintaining safety. Ultrasound-mediated gene transfer has been reported to augment the gene transfer efficiency and select the target organ using cationic microbubble phospholipids which bind negatively charged DNA. Ultrasound with microbubblesis likely to create new therapeutic options inavariety of diseases.

  8. Functional amyloids as inhibitors of plasmid DNA replication

    PubMed Central

    Molina-García, Laura; Gasset-Rosa, Fátima; Moreno-del Álamo, María; Fernández-Tresguerres, M. Elena; Moreno-Díaz de la Espina, Susana; Lurz, Rudi; Giraldo, Rafael

    2016-01-01

    DNA replication is tightly regulated to constrain the genetic material within strict spatiotemporal boundaries and copy numbers. Bacterial plasmids are autonomously replicating DNA molecules of much clinical, environmental and biotechnological interest. A mechanism used by plasmids to prevent over-replication is ‘handcuffing’, i.e. inactivating the replication origins in two DNA molecules by holding them together through a bridge built by a plasmid-encoded initiator protein (Rep). Besides being involved in handcuffing, the WH1 domain in the RepA protein assembles as amyloid fibres upon binding to DNA in vitro. The amyloid state in proteins is linked to specific human diseases, but determines selectable and epigenetically transmissible phenotypes in microorganisms. Here we have explored the connection between handcuffing and amyloidogenesis of full-length RepA. Using a monoclonal antibody specific for an amyloidogenic conformation of RepA-WH1, we have found that the handcuffed RepA assemblies, either reconstructed in vitro or in plasmids clustering at the bacterial nucleoid, are amyloidogenic. The replication-inhibitory RepA handcuff assembly is, to our knowledge, the first protein amyloid directly dealing with DNA. Built on an amyloid scaffold, bacterial plasmid handcuffs can bring a novel molecular solution to the universal problem of keeping control on DNA replication initiation. PMID:27147472

  9. Dcm methylation is detrimental to plasmid transformation in Clostridium thermocellum

    SciTech Connect

    Guss, Adam M; Olson, Daniel G.; Caiazza, Nicky; Lynd, Lee R

    2012-01-01

    BACKGROUND: Industrial production of biofuels and other products by cellulolytic microorganisms is of interest but hindered by the nascent state of genetic tools. Although a genetic system for Clostridium thermocellum DSM1313 has recently been developed, available methods achieve relatively low efficiency and similar plasmids can transform C. thermocellum at dramatically different efficiencies. RESULTS: We report an increase in transformation efficiency of C. thermocellum for a variety of plasmids by using DNA that has been methylated by Escherichia coli Dam but not Dcm methylases. When isolated from a dam+ dcm+ E. coli strain, pAMG206 transforms C. thermocellum 100-fold better than the similar plasmid pAMG205, which contains an additional Dcm methylation site in the pyrF gene. Upon removal of Dcm methylation, transformation with pAMG206 showed a four- to seven-fold increase in efficiency; however, transformation efficiency of pAMG205 increased 500-fold. Removal of the Dcm methylation site from the pAM205 pyrF gene via silent mutation resulted in increased transformation efficiencies equivalent to that of pAMG206. Upon proper methylation, transformation efficiency of plasmids bearing the pMK3 and pB6A origins of replication increased ca. three orders of magnitude. CONCLUSION: E. coli Dcm methylation decreases transformation efficiency in C. thermocellum DSM1313. The use of properly methylated plasmid DNA should facilitate genetic manipulation of this industrially relevant bacterium.

  10. Plasma-activated air mediates plasmid DNA delivery in vivo

    PubMed Central

    Edelblute, Chelsea M; Heller, Loree C; Malik, Muhammad A; Bulysheva, Anna; Heller, Richard

    2016-01-01

    Plasma-activated air (PAA) provides a noncontact DNA transfer platform. In the current study, PAA was used for the delivery of plasmid DNA in a 3D human skin model, as well as in vivo. Delivery of plasmid DNA encoding luciferase to recellularized dermal constructs was enhanced, resulting in a fourfold increase in luciferase expression over 120 hours compared to injection only (P < 0.05). Delivery of plasmid DNA encoding green fluorescent protein (GFP) was confirmed in the epidermal layers of the construct. In vivo experiments were performed in BALB/c mice, with skin as the delivery target. PAA exposure significantly enhanced luciferase expression levels 460-fold in exposed sites compared to levels obtained from the injection of plasmid DNA alone (P < 0.001). Expression levels were enhanced when the plasma reactor was positioned more distant from the injection site. Delivery of plasmid DNA encoding GFP to mouse skin was confirmed by immunostaining, where a 3-minute exposure at a 10 mm distance displayed delivery distribution deep within the dermal layers compared to an exposure at 3 mm where GFP expression was localized within the epidermis. Our findings suggest PAA-mediated delivery warrants further exploration as an alternative approach for DNA transfer for skin targets. PMID:27110584

  11. Dendritic cell secretion of IL-15 is induced by recombinant huCD40LT and augments the stimulation of antigen-specific cytolytic T cells.

    PubMed

    Kuniyoshi, J S; Kuniyoshi, C J; Lim, A M; Wang, F Y; Bade, E R; Lau, R; Thomas, E K; Weber, J S

    1999-04-10

    Dendritic cells (DC) are professional antigen-presenting cells which stimulate strong proliferative and cytolytic T cell responses. Stimulation of CD40 on dendritic cells by its ligands and anti-CD40 antibodies induces maturation and enhances DC stimulatory ability. In order to understand the mechanism by which ligand:CD40 interactions augment DC function, we assessed the role of T cell stimulatory cytokines IL-12 and IL-15 in the function of DC stimulated with soluble trimeric CD40L, a recombinant fusion protein incorporating three covalently linked extracellular CD40L domains (huCD40LT). Peripheral blood derived DC treated with huCD40LT and/or IFN-gamma were used to stimulate T cell responses in vitro to specific antigens. DC treated with huCD40LT or IFN-gamma/huCD40LT stimulated enhanced T cell proliferation to CASTA, a soluble protein from C. albicans, induced T cells with augmented antigen-specific lysis, and increased the yield of antigen-specific IFN-gamma-producing T cells. IL-15 production by DC was enhanced in cultures treated with huCD40LT and correlated with expansion of antigen-specific cytolytic T cells. Addition of a neutralizing anti-IL-15 monoclonal antibody inhibited the expansion of viral and tumor antigen-specific T cells stimulated by IFN-gamma and huCD40LT-treated DC. In contrast, this enhanced stimulatory ability of DC did not appear to depend on synthesis of IL-12 since huCD40LT treatment stimulated the generation of antigen-specific cytokine producing and cytolytic T cells without increased IL-12 production. Addition of anti-IL-12 monoclonal antibody did not inhibit expansion of these cells. These data suggest that production of IL-15 but not IL-12 is an important factor in the enhanced immunostimulatory ability of huCD40LT-treated DC.

  12. Plasmid Vector-Linked Maturation of Natural Killer (NK) Cells Is Coupled to Antigen-Dependent NK Cell Activation during DNA-Based Immunization in Mice ▿

    PubMed Central

    Zhu, Ren; Mancini-Bourgine, Maryline; Zhang, Xiao Ming; Bayard, Florence; Deng, Qiang; Michel, Marie-Louise

    2011-01-01

    Plasmid DNA vaccines serve in a wide array of applications ranging from prophylactic vaccines to potential therapeutic tools against infectious diseases and cancer. In this study, we analyzed the mechanisms underlying the activation of natural killer (NK) cells and their potential role in adaptive immunity during DNA-based immunization against hepatitis B virus surface antigen in mice. We observed that the mature Mac-1+ CD27− NK cell subset increased in the liver of mice early after DNA injection, whereas the number of the less mature Mac-1+ CD27+ NK cells in the liver and spleen was significantly reduced. This effect was attributed to bacterial sequences present in the plasmid backbone rather than to the encoded antigen and was not observed in immunized MyD88-deficient mice. The activation of NK cells by plasmid-DNA injection was associated with an increase in their effector functions that depended on the expressed antigen. Maturation of NK cells was abrogated in the absence of T cells, suggesting that cross talk exists between NK cells and antigen-specific T cells. Taken together, our data unravel the mechanics of plasmid vector-induced maturation of NK cells and plasmid-encoded antigen-dependent activation of NK cells required for a crucial role of NK cells in DNA vaccine-induced immunogenicity. PMID:21775455

  13. [Plasmid P85 from Azospirillum brasilense SP245: study of the circle of possible hosts and incompatibility with plasmids from Azospirillum brasilense SP7].

    PubMed

    Katsy, E I

    1992-01-01

    The possibility of the stable inheritance of the plasmid p85 mobilized derivatives from Azospirillum brasilense Sp245 in the cells of the bacterial genera Rizobiaceae (Agrobacterium tumfaciens) and Pseudomonadaceae (Pseudomonas putida) has been shown. The plasmid p85 participates in coding for the physiologically active products (the plant hormones). It is not inherited by the Escherichia coli strains. For the first time the incompatibility of azospirillium plasmids has been demonstrated on the example of the plasmid p85 from Azospirillum brasilense Sp245 and the plasmid p115 from Azospirillum brasilense Sp7.

  14. Bacterial plasmid partition machinery: a minimalist approach to survival.

    PubMed

    Schumacher, Maria A

    2012-02-01

    The accurate segregation or partition of replicated DNA is essential for ensuring stable genome transmission. Partition of bacterial plasmids requires only three elements: a centromere-like DNA site and two proteins, a partition NTPase, and a centromere-binding protein (CBP). Because of this simplicity, partition systems have served as tractable model systems to study the fundamental molecular mechanisms required for DNA segregation at an atomic level. In the last few years, great progress has been made in this endeavor. Surprisingly, these studies have revealed that although the basic partition components are functionally conserved between three types of plasmid partition systems, these systems employ distinct mechanisms of DNA segregation. This review summarizes the molecular insights into plasmid segregation that have been achieved through these recent structural studies.

  15. Plasmid-mediated mineralization of naphthalene, phenanthrene, and anthracene

    SciTech Connect

    Sanseverino, J. IT Corp., Knoxville, TN ); Applegate, B.M.; King, J.M.H.; Sayler, G.S. )

    1993-06-01

    The biochemistry and genetics of the naphthalene degradation pathway contained on plasmid NAH7 have been well characterized. However, not much is known about the substrate specificity of the enzymes of nah operons and whether the nah-encoded enzymes are capable of metabolizing higher polyaromatic hydrocarbons. This paper shows that NAH7 and NAH7-like plasmids can mediate metabolism of phenanthrene and anthracene as well as naphthalene. In addition, a mutant blocked in the nahG (salicylate hydroxylase) gene produced unidentified metabolites when it is grown in the presence of phenanthrene and anthracene. This implies that phenanthrene and anthracene are degraded through the nah plasmid-encoded system. 29 refs., 3 figs., 2 tabs.

  16. Fingerprinting of Flavobacterium psychrophilum isolates by ribotyping and plasmid profiling.

    PubMed

    Chakroun, C; Grimont, F; Urdaci, M C; Bernardet, J F

    1998-07-30

    Flavobacterium psychrophilum is the agent of cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide. Ribosomal RNA gene restriction patterns (ribotypes) and plasmid profiles were determined on a collection of 85 strains isolated from different countries and fish species. Several ribotypes were obtained by using the restriction endonucleases Hinc II and Pvu II. Computer analysis of the ribotypes revealed that some of them were clearly associated with the fish species from which the strains were isolated, whereas no correlation with the geographical origin was found. Most of the strains harboured at least one plasmid and several different plasmid profiles were observed, even among strains sharing the same ribotype. These methods, used alone or in combination with other typing techniques, can be considered powerful tools for the epidemiological tracing of F. psychrophilum infections.

  17. Resolution of Multimeric Forms of Circular Plasmids and Chromosomes.

    PubMed

    Crozat, Estelle; Fournes, Florian; Cornet, François; Hallet, Bernard; Rousseau, Philippe

    2014-10-01

    One of the disadvantages of circular plasmids and chromosomes is their high sensitivity to rearrangements caused by homologous recombination. Odd numbers of crossing-over occurring during or after replication of a circular replicon result in the formation of a dimeric molecule in which the two copies of the replicon are fused. If they are not converted back to monomers, the dimers of replicons may fail to correctly segregate at the time of cell division. Resolution of multimeric forms of circular plasmids and chromosomes is mediated by site-specific recombination, and the enzymes that catalyze this type of reaction fall into two families of proteins: the serine and tyrosine recombinase families. Here we give an overview of the variety of site-specific resolution systems found on circular plasmids and chromosomes.

  18. A novel plasmid pEA68 of Erwinia amylovora and the description of a new family of plasmids.

    PubMed

    Ismail, Emadeldeen; Blom, Jochen; Bultreys, Alain; Ivanović, Milan; Obradović, Aleksa; van Doorn, Joop; Bergsma-Vlami, Maria; Maes, Martine; Willems, Anne; Duffy, Brion; Stockwell, Virginia O; Smits, Theo H M; Puławska, Joanna

    2014-12-01

    Recent genome analysis of Erwinia amylovora, the causal agent of fire blight disease on Rosaceae, has shown that the chromosome is highly conserved among strains and that plasmids are the principal source of genomic diversity. A new circular plasmid, pEA68, was found in E. amylovora strain 692 (LMG 28361), isolated in Poland from Sorbus (mountain ash) with fire blight symptoms. Annotation of the 68,763-bp IncFIIa-type plasmid revealed that it contains 79 predicted CDS, among which two operons (tra, pil) are associated with mobility. The plasmid is maintained stably in E. amylovora and does not possess genes associated with antibiotic resistance or known virulence genes. Curing E. amylovora strain 692 of pEA68 did not influence its virulence in apple shoots nor amylovoran synthesis. Of 488 strains of E. amylovora from seventeen countries, pEA68 was only found in two additional strains from Belgium. Although the spread of pEA68 is currently limited to Europe, pEA68 comprises, together with pEA72 and pEA78 both found in North America, a new plasmid family that spans two continents.

  19. Conjugation of plasmids of Neisseria gonorrhoeae to other Neisseria species: potential reservoirs for the beta-lactamase plasmid.

    PubMed

    Genco, C A; Knapp, J S; Clark, V L

    1984-09-01

    The discovery that penicillinase production in Neisseria gonorrhoeae was plasmid mediated and the spread of the beta-lactamase encoding plasmids in gonococcal isolates since 1976, raise the possibility that a nonpathogenic indigenous bacterium could serve as a reservoir for these plasmids. We initiated studies to define the ability of commensal Neisseria species and Branhamella catarrhalis strains, as well as strains of the pathogen Neisseria meningitidis, to serve as recipients in conjugation with Neisseria gonorrhoeae. We found that with N. gonorrhoeae as the donor, 3 of 5 Neisseria cinerea, 2 of 5 Neisseria flava, 0 of 1 Neisseria flavescens, 1 of 3 Neisseria subflava, 0 of 6 B. catarrhalis, 0 of 7 Neisseria lactamica, 1 of 5 Neisseria mucosa, 1 of 7 Neisseria perflava/sicca, and 0 of 13 N. meningitidis strains gave detectable conjugation frequencies (greater than 10(-8). N. cinerea was the only species found to maintain the gonococcal conjugal plasmid (pLE2451). A N. cinerea transconjugant containing pLE2451 was observed to transfer both the beta-lactamase plasmid and pLE2451 to N. gonorrhoeae at high frequency.

  20. Replication and Maintenance of Linear Phage-Plasmid N15.

    PubMed

    Ravin, Nikolai V

    2015-02-01

    The lambdoid phage N15 of Escherichia coli is very unusual among temperate phages in that its prophage is not integrated into the chromosome but is a linear plasmid molecule with covalently closed ends (telomeres). Upon infection, the phage DNA circularizes via cohesive ends, and then a special phage enzyme of the tyrosine recombinase family, protelomerase, cuts at another site and joins the ends, forming hairpin telomeres of the linear plasmid prophage. Replication of the N15 prophage is initiated at an internally located ori site and proceeds bidirectionally, resulting in the formation of duplicated telomeres. The N15 protelomerase cuts them, generating two linear plasmid molecules with hairpin telomeres. Stable inheritance of the plasmid prophage is ensured by a partitioning operon similar to the F factor sop operon. Unlike the F centromere, the N15 centromere consists of four inverted repeats dispersed in the genome. The multiplicity and dispersion of centromeres are required for efficient partitioning of a linear plasmid. The centromeres are located in the N15 genome regions involved in phage replication and control of lytic development, and binding of partition proteins at these sites regulates these processes. The family of N15-like linear phage-plasmids includes lambdoid phages ɸKO2 and pY54, as well as Myoviridae phages ΦHAP-1, VHML, VP882, Vp58.5, and vB_VpaM_MAR of marine gamma-proteobacteria. The genomes of these phages contain similar protelomerase genes, lysogeny control modules, and replication genes, suggesting that these phages may belong to a group diverged from a common ancestor.

  1. Conjugative Plasmid Transfer in Gram-Positive Bacteria

    PubMed Central

    Grohmann, Elisabeth; Muth, Günther; Espinosa, Manuel

    2003-01-01

    Conjugative transfer of bacterial plasmids is the most efficient way of horizontal gene spread, and it is therefore considered one of the major reasons for the increase in the number of bacteria exhibiting multiple-antibiotic resistance. Thus, conjugation and spread of antibiotic resistance represents a severe problem in antibiotic treatment, especially of immunosuppressed patients and in intensive care units. While conjugation in gram-negative bacteria has been studied in great detail over the last decades, the transfer mechanisms of antibiotic resistance plasmids in gram-positive bacteria remained obscure. In the last few years, the entire nucleotide sequences of several large conjugative plasmids from gram-positive bacteria have been determined. Sequence analyses and data bank comparisons of their putative transfer (tra) regions have revealed significant similarities to tra regions of plasmids from gram-negative bacteria with regard to the respective DNA relaxases and their targets, the origins of transfer (oriT), and putative nucleoside triphosphatases NTP-ases with homologies to type IV secretion systems. In contrast, a single gene encoding a septal DNA translocator protein is involved in plasmid transfer between micelle-forming streptomycetes. Based on these clues, we propose the existence of two fundamentally different plasmid-mediated conjugative mechanisms in gram-positive microorganisms, namely, the mechanism taking place in unicellular gram-positive bacteria, which is functionally similar to that in gram-negative bacteria, and a second type that occurs in multicellular gram-positive bacteria, which seems to be characterized by double-stranded DNA transfer. PMID:12794193

  2. blaCMY-2-positive IncA/C plasmids from Escherichia coli and Salmonella enterica are a distinct component of a larger lineage of plasmids.

    PubMed

    Call, Douglas R; Singer, Randall S; Meng, Da; Broschat, Shira L; Orfe, Lisa H; Anderson, Janet M; Herndon, David R; Kappmeyer, Lowell S; Daniels, Joshua B; Besser, Thomas E

    2010-02-01

    Large multidrug resistance plasmids of the A/C incompatibility complex (IncA/C) have been found in a diverse group of Gram-negative commensal and pathogenic bacteria. We present three completed sequences from IncA/C plasmids that originated from Escherichia coli (cattle) and Salmonella enterica serovar Newport (human) and that carry the cephamycinase gene blaCMY-2. These large plasmids (148 to 166 kbp) share extensive sequence identity and synteny. The most divergent plasmid, peH4H, has lost several conjugation-related genes and has gained a kanamycin resistance region. Two of the plasmids (pAM04528 and peH4H) harbor two copies of blaCMY-2, while the third plasmid (pAR060302) harbors a single copy of the gene. The majority of single-nucleotide polymorphisms comprise nonsynonymous mutations in floR. A comparative analysis of these plasmids with five other published IncA/C plasmids showed that the blaCMY-2 plasmids from E. coli and S. enterica are genetically distinct from those originating from Yersinia pestis and Photobacterium damselae and distal to one originating from Yersinia ruckeri. While the overall similarity of these plasmids supports the likelihood of recent movements among E. coli and S. enterica hosts, their greater divergence from Y. pestis or Y. ruckeri suggests less recent plasmid transfer among these pathogen groups.

  3. Ribonucleases, antisense RNAs and the control of bacterial plasmids.

    PubMed

    Saramago, Margarida; Bárria, Cátia; Arraiano, Cecília M; Domingues, Susana

    2015-03-01

    In the last decade regulatory RNAs have emerged as powerful tools to regulate the expression of genes both in prokaryotes and in eukaryotes. RNases, by degrading these RNA molecules, control the right amount of regulatory RNAs, which is fundamental for an accurate regulation of gene expression in the cell. Remarkably the first antisense RNAs identified were plasmid-encoded and their detailed study was crucial for the understanding of prokaryotic antisense RNAs. In this review we highlight the role of RNases in the precise modulation of antisense RNAs that control plasmid replication, maintenance and transfer.

  4. DNA Assembly Tools and Strategies for the Generation of Plasmids.

    PubMed

    Baek, Chang-Ho; Liss, Michael; Clancy, Kevin; Chesnut, Jonathan; Katzen, Federico

    2014-10-01

    Since the discovery of restriction enzymes and the generation of the first recombinant DNA molecule over 40 years ago, molecular biology has evolved into a multidisciplinary field that has democratized the conversion of a digitized DNA sequence stored in a computer into its biological counterpart, usually as a plasmid, stored in a living cell. In this article, we summarize the most relevant tools that allow the swift assembly of DNA sequences into useful plasmids for biotechnological purposes. We cover the main components and stages in a typical DNA assembly workflow, namely in silico design, de novo gene synthesis, and in vitro and in vivo sequence assembly methodologies.

  5. Conjugative transfer of cadmium resistance plasmids in Rhodococcus fascians strains.

    PubMed

    Desomer, J; Dhaese, P; Van Montagu, M

    1988-05-01

    The presence of a 138-kilobase plasmid (pD188) correlated with increased resistance to cadmium in Rhodococcus fascians D188. This plasmid could be transferred by a conjugation-like system in matings between R. fascians strains. Transconjugants expressed the cadmium resistance and could be used as donors in subsequent matings. Four other R. fascians strains (NCPPB 1488, NCPPB 1675, NCPPB 2551, and ATCC 12974) could also be used as donors for cadmium resistance in matings. Strain NCPPB 1675 showed a 100% cotransfer of cadmium and chloramphenicol resistance markers.

  6. Replication of staphylococcal plasmid pT48.

    PubMed

    Catchpole, I R; Dyke, K G

    1992-02-01

    Insertion of a synthetic DNA linker into the repL gene of staphylococcal plasmid pT48 inactivates the replication system. This defect can be complemented in trans by the presence of a pT48 repL gene, but not by the rep genes of the related Staphylococcus areus plasmids pSN2 and pOX1000. Comparison of the sequences of the three replication proteins indicates that specificity may be determined by a putative helix-turn-helix region.

  7. Mutagenesis of dimeric plasmids by the transposon. gamma. delta. (Tn1000)

    SciTech Connect

    Liu, L.; Berg, C.M. )

    1990-05-01

    The Escherichia coli F factor mediates conjugal transfer of a plasmid such as pBR322 primarily by replicative transposition of transposon {gamma}{delta} (Tn1000) from F to that plasmid to form a cointegrate intermediate. Although resolution of this cointegrate always yields a plasmid containing a single {gamma}{delta} insertion, the occasional recovery of transposon-free plasmids after connuugal transfer has led to alternative hypotheses for F mobilization. The authors show here that {gamma}{delta}-free plasmids are found after F-mediated conjugal transfer only when the donor plasmid is a dimer and the recipient is Rec{sup +}.

  8. Plasmid DNA damage by heavy ions at spread-out Bragg peak energies

    NASA Astrophysics Data System (ADS)

    Dang, H. M.; van Goethem, M. J.; van der Graaf, E. R.; Brandenburg, S.; Hoekstra, R.; Schlathölter, T.

    2010-10-01

    Interaction of ionizing radiation with plasmid DNA can lead to formation of single strand breaks, double strand breaks and clustered lesions. We have investigated the response of the synthetic plasmid pBR322 in aqueous solution upon irradiation with 12C ions under spread-out Bragg peak conditions (densely ionizing) and with 137Cs γ-photons (sparsely ionizing) as a function of dose. To evaluate the relevance of indirect effects, i.e. influences of diffusion limited radical induced DNA damage triggered by water radiolysis, the experiments were performed at various concentrations of the radical scavenger mannitol. Agarose gel electrophoresis was employed to quantify the DNA damage. At low scavenger concentration for a given dose DNA damage is higher for γ-photons than for 12C. For the latter, the microscopic dose distribution is inhomogeneous, with very high dose deposited along the few tracks through the solution. This is in agreement with the concept that scavengers efficiently reduce damage for γ-photons, implying that the underlying damage mechanism is single strand break induction by OH radicals. For 12C induced damage, the fraction of SSB and DSB that is unaffected by radical scavengers and thus due to direct effect is quantified.

  9. Effect of naked eukaryotic expression plasmid encoding rat augmenter of liver regeneration on acute hepatic injury and hepatic failure in rats

    PubMed Central

    Zhang, Li-Mei; Liu, Dian-Wu; Liu, Jian-Bo; Zhang, Xiao-Lin; Wang, Xiao-Bo; Tang, Long-Mei; Wang, Li-Qin

    2005-01-01

    AIM: To study the protective effect of eukaryotic expression plasmid encoding augmenter of liver regeneration (ALR) on acute hepatic injury and hepatic failure in rats. METHODS: The PCR-amplified ALR gene was recombined with pcDNA3 plasmid, and used to treat rats with acute hepatic injury. The rats with acute hepatic injury induced by intraperitoneal injection of 2 mL/kg 50% carbon tetrachloride (CCl4) were randomly divided into saline control group and recombinant pcDNA3-ALR plasmid treatment groups. Recombinant pcDNA3-ALR plasmid DNA (50 or 200 μg/kg) was injected into the rats with acute hepatic injury intraven-ously, intraperitoneally, or intravenously and intraperitoneally in combination 4 h after CCl4 administration, respectively. The recombinant plasmid was injected once per 12 h into all treatment groups four times, and the rats were decapitated 12 h after the last injection. Hepatic histopathological alterations were observed after HE staining, the expression of proliferating cell nuclear antigen (PCNA) in liver tissue was detected by immunohistochemical staining, and the level of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was determined by biochemical method. The recombinant plasmid DNA (200 μg/kg) and saline were intraperitoneally injected into the rats with acute hepatic failure induced by intraperitoneal injection of 4 mL/kg 50% CCl4 after 4 h of CCl4 administration, respectively. Rats living over 96 h were considered as survivals. RESULTS: The sequence of ALR cDNA of recombinant pcDNA3-ALR plasmid was accordant with the reported sequence of rat ALR cDNA. After the rats with acute hepatic injury were treated with recombinant pcDNA3-ALR plasmid, the degree of liver histopathological injury markedly decreased. The pathologic liver tissues, in which hepatic degeneration and necrosis of a small amount of hepatocytes and a large amount of infiltrating inflammatory cells were observed, and they became basically normal in the

  10. Transcriptional Profiling of Human Epithelial Cells Infected with Plasmid-Bearing and Plasmid-Deficient Chlamydia trachomatis

    PubMed Central

    Carlson, John H.; Sturdevant, Daniel E.; Sturdevant, Gail L.; Kanakabandi, Kishore; Virtaneva, Kimmo; Wilder, Hannah; Whitmire, William M.; Caldwell, Harlan D.

    2014-01-01

    Chlamydia trachomatis is an obligate intracellular epitheliotropic bacterial pathogen of humans. Infection of the eye can result in trachoma, the leading cause of preventable blindness in the world. The pathophysiology of blinding trachoma is driven by multiple episodes of reinfection of conjunctival epithelial cells, producing an intense chronic inflammatory response resulting in submucosal tissue remodeling and scarring. Recent reports have shown that infection with trachoma organisms lacking the cryptic chlamydial plasmid is highly attenuated in macaque eyes, a relevant experimental model of human trachoma infection. To better understand the molecular basis of plasmid-mediated infection attenuation and the potential modulation of host immunity, we conducted transcriptional profiling of human epithelial cells infected with C. trachomatis plasmid-bearing (A2497) and plasmid-deficient (A2497P−) organisms. Infection of human epithelial cells with either strain increased the expression of host genes coding for proinflammatory (granulocyte-macrophage colony-stimulating factor [GM-CSF], macrophage colony-stimulating factor [MCSF], interleukin-6 [IL-6], IL-8, IL-1α, CXCL1, CXCL2, CXCL3, intercellular adhesion molecule 1 [ICAM1]), chemoattraction (CCL20, CCL5, CXCL10), immune suppression (PD-L1, NFKB1B, TNFAIP3, CGB), apoptosis (CASP9, FAS, IL-24), and cell growth and fibrosis (EGR1 and IL-20) proteins. Statistically significant increases in the levels of expression of many of these genes were found in A2497-infected cells compared to the levels of expression in A2497P−-infected cells. Our findings suggest that the chlamydial plasmid plays a focal role in the host cell inflammatory response to infection and immune avoidance. These results provide new insights into the role of the chlamydial plasmid as a chlamydial virulence factor and its contributions to trachoma pathogenesis. PMID:25404022

  11. Plasmid pEC156, a Naturally Occurring Escherichia coli Genetic Element That Carries Genes of the EcoVIII Restriction-Modification System, Is Mobilizable among Enterobacteria

    PubMed Central

    Werbowy, Olesia; Kaczorowski, Tadeusz

    2016-01-01

    Type II restriction-modification systems are ubiquitous in prokaryotes. Some of them are present in naturally occurring plasmids, which may facilitate the spread of these systems in bacterial populations by horizontal gene transfer. However, little is known about the routes of their dissemination. As a model to study this, we have chosen an Escherichia coli natural plasmid pEC156 that carries the EcoVIII restriction modification system. The presence of this system as well as the cis-acting cer site involved in resolution of plasmid multimers determines the stable maintenance of pEC156 not only in Escherichia coli but also in other enterobacteria. We have shown that due to the presence of oriT-type F and oriT-type R64 loci it is possible to mobilize pEC156 by conjugative plasmids (F and R64, respectively). The highest mobilization frequency was observed when pEC156-derivatives were transferred between Escherichia coli strains, Enterobacter cloacae and Citrobacter freundii representing coliform bacteria. We found that a pEC156-derivative with a functional EcoVIII restriction-modification system was mobilized in enterobacteria at a frequency lower than a plasmid lacking this system. In addition, we found that bacteria that possess the EcoVIII restriction-modification system can efficiently release plasmid content to the environment. We have shown that E. coli cells can be naturally transformed with pEC156-derivatives, however, with low efficiency. The transformation protocol employed neither involved chemical agents (e.g. CaCl2) nor temperature shift which could induce plasmid DNA uptake. PMID:26848973

  12. Sequence-based analysis of pQBR103; a representative of a unique, transfer-proficient mega plasmid resident in the microbial community of sugar beet.

    PubMed

    Tett, Adrian; Spiers, Andrew J; Crossman, Lisa C; Ager, Duane; Ciric, Lena; Dow, J Maxwell; Fry, John C; Harris, David; Lilley, Andrew; Oliver, Anna; Parkhill, Julian; Quail, Michael A; Rainey, Paul B; Saunders, Nigel J; Seeger, Kathy; Snyder, Lori A S; Squares, Rob; Thomas, Christopher M; Turner, Sarah L; Zhang, Xue-Xian; Field, Dawn; Bailey, Mark J

    2007-08-01

    The plasmid pQBR103 was found within Pseudomonas populations colonizing the leaf and root surfaces of sugar beet plants growing at Wytham, Oxfordshire, UK. At 425 kb it is the largest self-transmissible plasmid yet sequenced from the phytosphere. It is known to enhance the competitive fitness of its host, and parts of the plasmid are known to be actively transcribed in the plant environment. Analysis of the complete sequence of this plasmid predicts a coding sequence (CDS)-rich genome containing 478 CDSs and an exceptional degree of genetic novelty; 80% of predicted coding sequences cannot be ascribed a function and 60% are orphans. Of those to which function could be assigned, 40% bore greatest similarity to sequences from Pseudomonas spp, and the majority of the remainder showed similarity to other gamma-proteobacterial genera and plasmids. pQBR103 has identifiable regions presumed responsible for replication and partitioning, but despite being tra+ lacks the full complement of any previously described conjugal transfer functions. The DNA sequence provided few insights into the functional significance of plant-induced transcriptional regions, but suggests that 14% of CDSs may be expressed (11 CDSs with functional annotation and 54 without), further highlighting the ecological importance of these novel CDSs. Comparative analysis indicates that pQBR103 shares significant regions of sequence with other plasmids isolated from sugar beet plants grown at the same geographic location. These plasmid sequences indicate there is more novelty in the mobile DNA pool accessible to phytosphere pseudomonas than is currently appreciated or understood.

  13. Quinolone resistance in bacteria: emphasis on plasmid-mediated mechanisms.

    PubMed

    Li, Xian-Zhi

    2005-06-01

    Bacterial resistance to quinolones/fluoroquinolones has emerged rapidly and such resistance has traditionally been attributed to the chromosomally mediated mechanisms that alter the quinolone targets (i.e. DNA gyrase and topoisomerase IV) and/or overproduce multidrug resistance efflux pumps. However, the discovery of the plasmid-borne quinolone resistance determinant, named qnr, has substantially broadened our horizon on the molecular mechanisms of quinolone resistance. Several recent reports of Qnr or its homologues encoded by transferable plasmids in Gram-negative bacteria isolated worldwide highlight the significance of the emerging plasmid-mediated mechanism(s). This also alerts us to the potential rapid dissemination of quinolone resistance determinants. Qnr belongs to the pentapeptide repeat family and protects DNA gyrase from the action of quinolone agents including the newer fluoroquinolones. This protection interplays with chromosomal mechanisms to raise significantly the resistance levels. The qnr-bearing strains generate quinolone-resistant mutants at a much higher frequency than those qnr-free strains. Furthermore, the qnr-plasmids are integron-associated and carry multiple resistance determinants providing resistance to several classes of antimicrobials including beta-lactams and aminoglycosides. The high quinolone resistance rates in Escherichia coli are used to address issues of quinolone resistance, and possible strategies for minimising quinolone resistance are discussed.

  14. Plasmid-determined copper resistance in Pseudomonas syringae from impatiens

    SciTech Connect

    Cooksey, D.A. )

    1990-01-01

    A strain of Pseudomonas syringae was recently identified as the cause of a new foliar blight of impatiens. The bacterium was resistant to copper compounds, which are used on a variety of crops for bacterial and fungal disease control. The bacterium contained a single 47-kilobase plasmid (pPSI1) that showed homology to a copper resistance operon previously cloned and characterized from P. syringae pv. tomato plasmid pPT23D (D. Cooksey, Appl. Environ. Microbiol. 53:454-456, 1987). pPSI1 was transformed by electroporation into a copper-sensitive P. syringae strain, and the resulting transformants were copper resistant. A physical map of pPSI1 was constructed, and the extent of homology to pPT23D outside the copper resistance operon was determined in Southern hybridizations. The two plasmids shared approximately 20 kilobases of homologous DNA, with the remainder of each plasmid showing no detectable homology. The homologous regions hybridized strongly, but there was little or no conservation of restriction enzyme recognition sites.

  15. Synthetic Fatty Acids Prevent Plasmid-Mediated Horizontal Gene Transfer

    PubMed Central

    Getino, María; Sanabria-Ríos, David J.; Fernández-López, Raúl; Campos-Gómez, Javier; Sánchez-López, José M.; Fernández, Antonio; Carballeira, Néstor M.

    2015-01-01

    ABSTRACT Bacterial conjugation constitutes a major horizontal gene transfer mechanism for the dissemination of antibiotic resistance genes among human pathogens. Antibiotic resistance spread could be halted or diminished by molecules that interfere with the conjugation process. In this work, synthetic 2-alkynoic fatty acids were identified as a novel class of conjugation inhibitors. Their chemical properties were investigated by using the prototype 2-hexadecynoic acid and its derivatives. Essential features of effective inhibitors were the carboxylic group, an optimal long aliphatic chain of 16 carbon atoms, and one unsaturation. Chemical modification of these groups led to inactive or less-active derivatives. Conjugation inhibitors were found to act on the donor cell, affecting a wide number of pathogenic bacterial hosts, including Escherichia, Salmonella, Pseudomonas, and Acinetobacter spp. Conjugation inhibitors were active in inhibiting transfer of IncF, IncW, and IncH plasmids, moderately active against IncI, IncL/M, and IncX plasmids, and inactive against IncP and IncN plasmids. Importantly, the use of 2-hexadecynoic acid avoided the spread of a derepressed IncF plasmid into a recipient population, demonstrating the feasibility of abolishing the dissemination of antimicrobial resistances by blocking bacterial conjugation. PMID:26330514

  16. Effects of maternal plasmid GHRH treatment on offspring growth

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To differentiate prenatal effects of plasmid growth hormone-releasing hormone (GHRH) treatment from maternal effects mediated by lactation on long-term growth of offspring, a cross-fostering study was designed. Pregnant sows (n = 12) were untreated (n = 6), or received either a Wt-GHRH (n = 2), or H...

  17. Plasmid transfer and genetic recombination by protoplast fusion in staphylococci.

    PubMed

    Götz, F; Ahrné, S; Lindberg, M

    1981-01-01

    The experimental conditions for plasmid transfer and genetic recombination in Staphylococcus aureus and some coagulase-negative staphylococci by protoplast fusion are described. Protoplasts were prepared by treatment with lysostaphin and lysozyme in a buffered medium with 0.7 to 0.8 M sucrose. Regeneration of cell walls was accomplished on a hypertonic agar medium containing succinate and bovine serum albumin. Transfer of plasmids occurred after treatment of the protoplast mixtures with polyethylene glycol (molecular weight, 6,000) not only between strains of the same species but also between parents of different species, although at approximately 100 times lower frequency in the latter case. Recombination of the chromosomal genes in fused protoplasts required simultaneous treatment of the mixed protoplasts with polyethylene glycol and CaCl2. A method was developed for isolation of recombinants after fusion between mutants of S. areus carrying unselectable markers. Antibiotic resistance plasmids were introduced into the parental strains and used as primary markers to detect protoplast fusion. Chromosomal recombinants were found among the clones with both parental plasmids at a high frequency. The method appears to have simple applications in the construction of strains with multiple mutant characters.

  18. Plasmid transfer and genetic recombination by protoplast fusion in staphylococci.

    PubMed Central

    Götz, F; Ahrné, S; Lindberg, M

    1981-01-01

    The experimental conditions for plasmid transfer and genetic recombination in Staphylococcus aureus and some coagulase-negative staphylococci by protoplast fusion are described. Protoplasts were prepared by treatment with lysostaphin and lysozyme in a buffered medium with 0.7 to 0.8 M sucrose. Regeneration of cell walls was accomplished on a hypertonic agar medium containing succinate and bovine serum albumin. Transfer of plasmids occurred after treatment of the protoplast mixtures with polyethylene glycol (molecular weight, 6,000) not only between strains of the same species but also between parents of different species, although at approximately 100 times lower frequency in the latter case. Recombination of the chromosomal genes in fused protoplasts required simultaneous treatment of the mixed protoplasts with polyethylene glycol and CaCl2. A method was developed for isolation of recombinants after fusion between mutants of S. areus carrying unselectable markers. Antibiotic resistance plasmids were introduced into the parental strains and used as primary markers to detect protoplast fusion. Chromosomal recombinants were found among the clones with both parental plasmids at a high frequency. The method appears to have simple applications in the construction of strains with multiple mutant characters. PMID:7007333

  19. Immune Response to Plasmid- and Chromosome-Encoded Yersinia Antigens,

    DTIC Science & Technology

    The immune response of humans and mice to temperature-specific, plasmid- or chromosome-encoded proteins of Yersinia pestis and Yersinia ... enterocolitica was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Extracts from Y. pestis and Y. enterocolitica

  20. From plasmids to protection: a review of DNA vaccines against infectious diseases.

    PubMed

    Laddy, Dominick J; Weiner, David B

    2006-01-01

    The field of DNA vaccine development began over 16 years ago with the observation that plasmid DNA could be injected into and expressed in vivo and drive adaptive immune responses. Since then, there has been great interest in developing this technology to create a new generation of vaccines with the ability to elicit both humoral and cellular immune responses from an inherently innocuous injection. However, DNA vaccines have yet to proceed past phase I/II clinical trials in humans--primarily due to a desire to induce more potent immune responses. This review will examine how DNA vaccines function to induce an immune response and how this information might be useful in future vaccine design.

  1. Transformation of heat-treated Clostridium acetobutylicum protoplasts with pUB110 plasmid DNA

    SciTech Connect

    Lin, Y.L.; Blaschek, H.P.

    1984-10-01

    Heat treatment of Clostridium acetobutylicum SA-1 protoplasts at 55/sup 0/C for 15 min before transformation resulted in expression in this microorganism of the kanamycin resistance determinant associated with plasmid pUB110. No heat treatment, or heat treatment at 65 or 44/sup 0/C for various time intervals, resulted in no kanamycin resistance transformants being recovered on selective kanamycin-containing regeneration medium. DNase plate assay indicated that treatment at 55/sup 0/C for 15 min completely inactivated the DNase activity associated with SA-1 protoplasts. Treatment of protoplasts at 65 or 55/sup 0/C for various periods under simulated transformation conditions had an inhibitory effect, although prolonged treatment at 55 or 44/sup 0/C appeared to stimulate DNase activity. Inactivation of protoplast-associated DNase activity by heat treatment at 55/sup 0/C for 15 min correlated with successful expression of kanamycin resistance and suggests that an extremely active, heat-sensitive, protoplast-associated DNase may be a factor in the polyethylene glycol-induced transformation of C. acetobutylicum SA-1 protoplasts. Plasmid pUB110 DNA was isolated from C. acetobutylicum SA-1 kanamycin-resistant (Km/sup r/) transformant cultures by a modification of the procedure used for C. perfringens plasmids. Detection of pUB110 DNA was possible only when diethyl pyrocarbonate was incorporated into isolation protocols to inactivate DNase activity. Restriction studies further verified the presence of pUB110 DNA in C. acetobutylicum SA-1 Km/sup r/ transformants. 36 references, 4 figures, 1 table.

  2. Ca2+ Promoted the Low Transformation Efficiency of Plasmid DNA Exposed to PAH Contaminants

    PubMed Central

    Gao, Yanzheng; Long, Jian; Wang, Qian

    2013-01-01

    The effects of interactions between genetic materials and polycyclic aromatic hydrocarbons (PAHs) on gene expression in the extracellular environment remain to be elucidated and little information is currently available on the effect of ionic strength on the transformation of plasmid DNA exposed to PAHs. Phenanthrene and pyrene were used as representative PAHs to evaluate the transformation of plasmid DNA after PAH exposure and to determine the role of Ca2+ during the transformation. Plasmid DNA exposed to the test PAHs demonstrated low transformation efficiency. In the absence of PAHs, the transformation efficiency was 4.7 log units; however, the efficiency decreased to 3.72–3.14 log units with phenanthrene/pyrene exposures of 50 µg·L–1. The addition of Ca2+ enhanced the low transformation efficiency of DNA exposed to PAHs. Based on the co-sorption of Ca2+ and phenanthrene/pyrene by DNA, we employed Fourier-transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and mass spectrometry (MS) to determine the mechanisms involved in PAH-induced DNA transformation. The observed low transformation efficiency of DNA exposed to either phenanthrene or pyrene can be attributed to a broken hydrogen bond in the double helix caused by planar PAHs. Added Ca2+ formed strong electrovalent bonds with “–POO––” groups in the DNA, weakening the interaction between PAHs and DNA based on weak molecular forces. This decreased the damage of PAHs to hydrogen bonds in double-stranded DNA by isolating DNA molecules from PAHs and consequently enhanced the transformation efficiency of DNA exposed to PAH contaminants. The findings provide insight into the effects of anthropogenic trace PAHs on DNA transfer in natural environments. PMID:23484001

  3. Campylobacter fetus Subspecies Contain Conserved Type IV Secretion Systems on Multiple Genomic Islands and Plasmids

    PubMed Central

    van der Graaf–van Bloois, Linda; Miller, William G.; Yee, Emma; Gorkiewicz, Gregor; Forbes, Ken J.; Zomer, Aldert L.; Wagenaar, Jaap A.; Duim, Birgitta

    2016-01-01

    The features contributing to differences in pathogenicity of the Campylobacter fetus subspecies are unknown. Putative factors involved in pathogenesis are located in genomic islands that encode a type IV secretion system (T4SS) and fic domain (filamentation induced by cyclic AMP) proteins, which may disrupt host cell processes. In the genomes of 27 C. fetus strains, three phylogenetically-different T4SS-encoding regions (T4SSs) were identified: one was located in both the chromosome and in extra-chromosomal plasmids; one was located exclusively in the chromosome; and one exclusively in extra-chromosomal plasmids. We observed that C. fetus strains can contain multiple T4SSs and that homologous T4SSs can be present both in chromosomal genomic islands (GI) and on plasmids in the C. fetus strains. The GIs of the chromosomally located T4SS differed mainly by the presence of fic genes, insertion sequence elements and phage-related or hypothetical proteins. Comparative analysis showed that T4SS sequences, inserted in the same locations, were conserved in the studied C. fetus genomes. Using phylogenetic analysis of the T4SSs, it was shown that C. fetus may have acquired the T4SS regions from other Campylobacter species by horizontal gene transfer. The identified T4SSs and fic genes were found in Cff and Cfv strains, although the presence of T4SSs and fic genes were significantly associated with Cfv strains. The T4SSs and fic genes could not be associated with S-layer serotypes or geographical origin of the strains. PMID:27049518

  4. Characterization of the Lactobacillus plantarum plasmid pCD033 and generation of the plasmid free strain L. plantarum 3NSH.

    PubMed

    Heiss, Silvia; Grabherr, Reingard; Heinl, Stefan

    2015-09-01

    Lactobacillus plantarum CD033, a strain isolated from grass silage in Austria, harbors a 7.9 kb plasmid designated pCD033. Sequence analysis identified 14 open reading frames and 8 of these were supposed to be putative coding sequences. Gene annotation revealed no putative essential genes being plasmid encoded, but a plasmid addiction system based on a PemI/PemK-like toxin-antitoxin system, able to stabilize plasmid maintenance. Absence of a replication initiation protein, a double strand origin as well as a single strand origin on plasmid pCD033 suggests replication via a new type of theta mechanism, whereby plasmid replication is potentially initiated and regulated by non-coding RNA. Detailed examination of segregational stability of plasmid vectors consisting of pCD033-fragments, combined with a selection marker, resulted in definition of a stably maintained minimal replicon. A gene encoding a RepB/OrfX-like protein was found to be not essential for plasmid replication. Alignment of the amino acid sequence of this protein with related proteins unveiled a highly conserved amino acid motif (LLDQQQ). L. plantarum CD033 was cured of pCD033 resulting in the novel plasmid free strain L. plantarum 3NSH. Plasmid curing demonstrated that no essential features are provided by pCD033 under laboratory conditions.

  5. [Integration of plasmid pPL 7065 into chromosome of Bac. pumilis].

    PubMed

    Lysenko, A M; Koz'mipa, L M; Abromova, M A; Lukin, A A

    1980-01-01

    Hybridization of tritium-labelled plasmid 7065 with total DNA of several Bac. pumilis strains differing in the degree of spore-formation showed that strain 7065-k contains the plasmid in an integral state.

  6. A Time-Efficient and User-Friendly Method for Plasmid DNA Restriction Analysis.

    ERIC Educational Resources Information Center

    LaBanca, Frank; Berg, Claire M.

    1998-01-01

    Describes an experiment in which plasmid DNA is digested with restriction enzymes that cleave the plasmid either once or twice. The DNA is stained, loaded on a gel, electrophoresed, and viewed under normal laboratory conditions during electrophoresis. (DDR)

  7. Transfer of plasmid-mediated ampicillin resistance from Haemophilus to Neisseria gonorrhoeae requires an intervening organism.

    PubMed

    McNicol, P J; Albritton, W L; Ronald, A R

    1986-01-01

    Haemophilus species have been implicated as the source of plasmid-mediated ampicillin resistance in Neisseria gonorrhoeae. Previous attempts to transfer conjugally the resistance plasmids from Haemophilus species to N. gonorrhoeae have met with limited success. Using both biparental and triparental mating systems, it was found that transfer will occur if the commensal Neisseria species, Neisseria cinerea, is used as a transfer intermediate. This organism stably maintains resistance plasmids of Haemophilus and facilitates transfer of these plasmids to N. gonorrhoeae, in a triparental mating system, at a transfer frequency of 10(-8). Both Haemophilus ducreyi and N. gonorrhoeae carry mobilizing plasmids capable of mediating conjugal transfer of the same resistance plasmids. However, restriction endonuclease mapping and DNA hybridization studies indicate that the mobilizing plasmids are distinctly different molecules. Limited homology is present within the transfer region of these plasmids.

  8. Plasmid profiling of bacterial isolates from confined environments

    NASA Astrophysics Data System (ADS)

    van Houdt, Rob; Provoost, Ann; Coninx, Ilse; Leys, Natalie; Mergeay, Max

    Plasmid profiling of bacterial isolates from confined environments R. Van Houdt, I. Coninx, A. Provoost, N. Leys, and M. Mergeay Expertise group for Molecular and Cellular Biology, Institute for Environment, Health and Safety, Belgian Nuclear Research Centre (SCK•CEN), Boeretang 200, B-2400 Mol, Belgium. Human exploration of extreme and isolated hostile environments such as space requires special confined small volume habitats to protect and house the crew. However, human confinement in such small volume habitats has restrictions on waste disposal and personal hygiene and inevitably generates a particular community of microorganisms within the habitat. These microorganisms are mainly originating from the crew (skin, mucous membranes, upper respiratory tract, mouth, and gastrointestinal tract) but also include the residing environmental microorganisms. Earth-based confined habitats such as the Antarctic Research Station Concordia are used as test beds for long-duration spaceflights to study the physiologic and psychological adaptation to isolated environments. The dynamics of the environmental microbial population in such a test bed could render additional insights in assessing the potential health risks in long-duration space missions. Not only total bacterial contamination levels are important, but it is essential to identify also the predominant microbial taxa and their mobile genetic elements (MGE). These MGEs could be exchanged between bacteria by horizontal gene transfer and may alter the pathogenic potential since they often carry antibiotic resistance or more in general adaptation-enhancing traits. In this study several bacterial strains isolated in the Concordia research station were examined for their plasmid content. An optimized protocol for extraction of large plasmids showed the present of at least one plasmid in 50% of the strains. For all strains the minimal inhibitory concentration of a range of antibiotics was determined indicating resistance to

  9. Effect of plasmid pKM101 in ultraviolet irradiated uvr+ and uvr- Escherichia coli.

    PubMed

    Slezáriková, V; Sedliaková, M; Andreeva, I V; Rusina OYu; Skavronskaya, A G

    1992-11-16

    The effect of plasmid pKM101 on UV irradiated excision proficient and excision deficient cells was investigated. The plasmid increased the survival of excision proficient cells while partially inhibiting thymine dimer excision. The frequency of mutations was almost unchanged. In excision deficient cells the effect of the plasmid on survival was less pronounced while cell mutability was increased. Our data indicate that the mucAB genes (carried by the plasmid) influence the two types of cells in a different way.

  10. Plasmid- and chromosome-coded aerobactin synthesis in enteric bacteria: insertion sequences flank operon in plasmid-mediated systems.

    PubMed Central

    McDougall, S; Neilands, J B

    1984-01-01

    Large plasmids were detected in two aerobactin-producing enteric bacterial species (Aerobacter aerogenes 62-I, Salmonella arizona SA1, and S. arizona SL5301) and designated pSMN1, pSMN2, and pSMN3, respectively. Other Salmonella spp., namely, S. arizona SL5302, S. arizona SLS, Salmonella austin, and Salmonella memphis, formed aerobactin but contained no detectable large plasmids. S. arizona SL5283 made no aerobactin. A probe consisting of the aerobactin biosynthetic genes cloned on plasmid pABN5 hybridized to a HindIII digest of pSMN1 but not to digests of pSMN2 or pSMN3. A larger probe, the insert of pABN1 containing the complete aerobactin operon, hybridized to four fragments in HindIII digests of the parent plasmid, pColV-K30. A 2.0-kilobase PvuII fragment responsible for this multiple-hybridization pattern was cloned into vector pUC9 to form pSMN30. The latter was mapped and shown to correspond to either IS1 or to a closely related insertion sequence. Images PMID:6330037

  11. Plasmid vectors for Xylella fastidiosa utilizing a toxin-antitoxin system for plasmid stability in the absence of antibiotic selection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacte...

  12. Novel plasmid conferring kanamycin and tetracycline resistance in turkey-derived Campylobacter jejuni strain 11601MD

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In Campylobacter spp., resistance to the antibiotics kanamycin and tetracycline is frequently associated with plasmid-borne genes. However, relatively few plasmids of Campylobacter jejuni have been fully characterized to date. A novel plasmid (p11601MD; 44,095 bp.) harboring tet(O) was identified in...

  13. Characterisation of a mobilisable plasmid conferring florfenicol and chloramphenicol resistance in Actinobacillus pleuropneumoniae.

    PubMed

    Bossé, Janine T; Li, Yanwen; Atherton, Tom G; Walker, Stephanie; Williamson, Susanna M; Rogers, Jon; Chaudhuri, Roy R; Weinert, Lucy A; Holden, Matthew T G; Maskell, Duncan J; Tucker, Alexander W; Wren, Brendan W; Rycroft, Andrew N; Langford, Paul R

    2015-08-05

    The complete nucleotide sequence of a 7.7kb mobilisable plasmid (pM3446F), isolated from a florfenicol resistant isolate of Actinobacillus pleuropneumoniae, showed extended similarity to plasmids found in other members of the Pasteurellaceae containing the floR gene as well as replication and mobilisation genes. Mobilisation into other Pasteurellaceae species confirmed that this plasmid can be transferred horizontally.

  14. Plasmid-determined silver resistance in Pseudomonas stutzeri isolated from a silver mine.

    PubMed Central

    Haefeli, C; Franklin, C; Hardy, K

    1984-01-01

    A silver-resistant strain of Pseudomonas stutzeri was isolated from a silver mine. It harbored three plasmids, the largest of which (pKK1; molecular weight, 49.4 X 10(6)) specified silver resistance. Plasmid pKK1 was apparently nonconjugative but could be transferred to Pseudomonas putida by mobilization with plasmid R68.45. Images PMID:6715284

  15. Prevalence of ColE1-like plasmids and kanamycinr resistance genes in Salmonella enterica serotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Multi-antibiotic resistant Salmonella enterica serotypes are increasing in prevalence and concern in human and animal health. Many strains carry resistance determinants on plasmids; current practices focus heavily on large plasmids, and the role that small plasmids play in resistance gene transfer ...

  16. Occurrence and persistence of indigenous transconjugants carrying conjugative plasmids in soil.

    PubMed

    Inoue, Daisuke; Soda, Satoshi; Tsutsui, Hirofumi; Yamazaki, Yuji; Murashige, Katsushi; Sei, Kazunari; Fujita, Masanori; Ike, Michihiko

    2009-09-01

    The transfer of the self-transmissible plasmids, RP4 and pJP4, from introduced bacteria to indigenous bacteria was examined in soil and slurry microcosms. The introduced plasmids persisted in indigenous transconjugants despite the low survival of introduced donors. The potential of the transconjugants for growth and conjugation affects the persistence of introduced plasmids in soil.

  17. The Sphingomonas Plasmid pCAR3 Is Involved in Complete Mineralization of Carbazole▿ †

    PubMed Central

    Shintani, Masaki; Urata, Masaaki; Inoue, Kengo; Eto, Kaori; Habe, Hiroshi; Omori, Toshio; Yamane, Hisakazu; Nojiri, Hideaki

    2007-01-01

    We determined the complete 254,797-bp nucleotide sequence of the plasmid pCAR3, a carbazole-degradative plasmid from Sphingomonas sp. strain KA1. A region of about 65 kb involved in replication and conjugative transfer showed similarity to a region of plasmid pNL1 isolated from the aromatic-degrading Novosphingobium aromaticivorans strain F199. The presence of many insertion sequences, transposons, repeat sequences, and their remnants suggest plasticity of this plasmid in genetic structure. Although pCAR3 is thought to carry clustered genes for conjugative transfer, a filter-mating assay between KA1 and a pCAR3-cured strain (KA1W) was unsuccessful, indicating that pCAR3 might be deficient in conjugative transfer. Several degradative genes were found on pCAR3, including two kinds of carbazole-degradative gene clusters (car-I and car-II), and genes for electron transfer components of initial oxygenase for carbazole (fdxI, fdrI, and fdrII). Putative genes were identified for the degradation of anthranilate (and), catechol (cat), 2-hydroxypenta-2,4-dienoate (carDFE), dibenzofuran/fluorene (dbf/fln), protocatechuate (lig), and phthalate (oph). It appears that pCAR3 may carry clustered genes (car-I, car-II, fdxI, fdrI, fdrII, and, and cat) for the degradation of carbazole into tricarboxylic acid cycle intermediates; KA1W completely lost the ability to grow on carbazole, and the carbazole-degradative genes listed above were all expressed when KA1 was grown on carbazole. Reverse transcription-PCR analysis also revealed that the transcription of car-I, car-II, and cat genes was induced by carbazole or its metabolic intermediate. Southern hybridization analyses with probes prepared from car-I, car-II, repA, parA, traI, and traD genes indicated that several Sphingomonas carbazole degraders have DNA regions similar to parts of pCAR3. PMID:17172338

  18. Integrable alpha-amylase plasmid for generating random transcriptional fusions in Bacillus subtilis.

    PubMed Central

    O'Kane, C; Stephens, M A; McConnell, D

    1986-01-01

    An integrable plasmid, pOK4, which replicated independently in Escherichia coli was constructed for generating transcriptional fusions in vivo in Bacillus DNA. It did not replicate independently in Bacillus subtilis, but it could be made to integrate into the chromosome of B. subtilis if sequences homologous to chromosomal sequences were inserted into it. It had a selectable marker for chloramphenicol resistance and carried unique sites for EcoRI and SmaI just to the 5' side of a promoterless alpha-amylase gene from Bacillus licheniformis. When B. subtilis DNA fragments were ligated into one of these sites and the ligation mixture was used to transform an alpha-amylase-negative B. subtilis strain, chloramphenicol-resistant transformants could be isolated conveniently. Many of these were alpha-amylase positive, owing to the fusion of the plasmid amylase gene to chromosomal operons. In principle, because integration need not be mutagenic, it is possible to obtain fusions to any chromosomal operon. The site of each integration can be mapped, and the flanking sequences can be cloned into E. coli. The alpha-amylase gene can be used to detect regulated genes. We used it as an indicator to detect operons which are DNA-damage-inducible (din), and we identified insertions in both SP beta and PBSX prophages. Images PMID:3096966

  19. High-Voltage Electroporation of Bacteria: Genetic Transformation of Campylobacter jejuni with Plasmid DNA

    NASA Astrophysics Data System (ADS)

    Miller, Jeff F.; Dower, William J.; Tompkins, Lucy S.

    1988-02-01

    Electroporation permits the uptake of DNA by mammalian cells and plant protoplasts because it induces transient permeability of the cell membrane. We investigated the utility of high-voltage electroporation as a method for genetic transformation of intact bacterial cells by using the enteric pathogen Campylobacter jejuni as a model system. This report demonstrates that the application of high-voltage discharges to bacterial cells permits genetic transformation. Our method involves exposure of a Campylobacter cell suspension to a high-voltage exponential decay discharge (5-13 kV/cm) for a brief period of time (resistance-capacitance time constant = 2.4-26 msec) in the presence of plasmid DNA. Electrical transformation of C. jejuni results in frequencies as high as 1.2 × 106 transformants per μ g of DNA. We have investigated the effects of pulse amplitude and duration, cell growth conditions, divalent cations, and DNA concentration on the efficiency of transformation. Transformants of C. jejuni obtained by electroporation contained structurally intact plasmid molecules. In addition, evidence is presented that indicates that C. jejuni possesses DNA restriction and modification systems. The use of electroporation as a method for transforming other bacterial species and guidelines for its implementation are also discussed.

  20. Insertion element analysis and mapping of the Pseudomonas plasmid alk regulon.

    PubMed Central

    Fennewald, M; Benson, S; Oppici, M; Shapiro, J

    1979-01-01

    We characterized and mapped new mutations of the alk (alkane utilization) genes found on Pseudomonas plasmids of the Inc P-2 group. These mutations were isolated after (i) nitrosoguanidine mutagenesis, (ii) transposition of the Tn7 trimethoprim and streptomycin resistance determinant, and (iii) reversion of polarity effects of alk::Tn7 insertion mutations. Our results indicate the existence of two alk loci not previously described--alkD, whose product is required for synthesis of membrane alkane-oxidizing activities, and alkE, whose product is required for synthesis of inducible membrane alcohol dehydrogenase activity. Polarity of alk::Tn7 insertion mutations indicates the existence of an alkBAE operon. Mapping of alk loci by transduction in P. aeruginosa shows that there are at least three alk clusters in the CAM-OCT plasmid--alkRD, containing regulatory genes; alkBAE, containing genes for specific biochemical activities; and alkC, containing one or more genes needed for normal synthesis of membrane alcohol dehydrogenase. The alkRD and alkBAE clusters are linked but separated by about 42 kilobases. The alkC cluster is not linked to either of the other two alk regions. Altogether, these results indicate a complex genetic control of the alkane utilization phenotype in P. putida and P. aeruginosa involving at least six separate genes. Images PMID:479111

  1. Expansion of a plasmid classification system for Gram-positive bacteria and determination of the diversity of plasmids in Staphylococcus aureus strains of human, animal, and food origins.

    PubMed

    Lozano, Carmen; García-Migura, Lourdes; Aspiroz, Carmen; Zarazaga, Myriam; Torres, Carmen; Aarestrup, Frank Møller

    2012-08-01

    An expansion of a previously described plasmid classification was performed and used to reveal the plasmid content of a collection of 92 Staphylococcus aureus strains of different origins. rep genes of other genera were detected in Staphylococcus. S1 pulsed-field gel electrophoresis (PFGE) hybridizations were performed with 18 representative S. aureus strains, and a high number of plasmids of different sizes and organizations were detected.

  2. Remarkable stability of an instability-prone lentiviral vector plasmid in Escherichia coli Stbl3.

    PubMed

    Al-Allaf, Faisal A; Tolmachov, Oleg E; Zambetti, Lia Paola; Tchetchelnitski, Viktoria; Mehmet, Huseyin

    2013-02-01

    Large-scale production of plasmid DNA to prepare therapeutic gene vectors or DNA-based vaccines requires a suitable bacterial host, which can stably maintain the plasmid DNA during industrial cultivation. Plasmid loss during bacterial cell divisions and structural changes in the plasmid DNA can dramatically reduce the yield of the desired recombinant plasmid DNA. While generating an HIV-based gene vector containing a bicistronic expression cassette 5'-Olig2cDNA-IRES-dsRed2-3', we encountered plasmid DNA instability, which occurred in homologous recombination deficient recA1 Escherichia coli strain Stbl2 specifically during large-scale bacterial cultivation. Unexpectedly, the new recombinant plasmid was structurally changed or completely lost in 0.5 L liquid cultures but not in the preceding 5 mL cultures. Neither the employment of an array of alternative recA1 E. coli plasmid hosts, nor the lowering of the culture incubation temperature prevented the instability. However, after the introduction of this instability-prone plasmid into the recA13E. coli strain Stbl3, the transformed bacteria grew without being overrun by plasmid-free cells, reduction in the plasmid DNA yield or structural changes in plasmid DNA. Thus, E. coli strain Stbl3 conferred structural and maintenance stability to the otherwise instability-prone lentivirus-based recombinant plasmid, suggesting that this strain can be used for the faithful maintenance of similar stability-compromised plasmids in large-scale bacterial cultivations. In contrast to Stbl2, which is derived wholly from the wild type isolate E. coli K12, E. coli Stbl3 is a hybrid strain of mixed E. coli K12 and E. coli B parentage. Therefore, we speculate that genetic determinants for the benevolent properties of E. coli Stbl3 for safe plasmid propagation originate from its E. coli B ancestor.

  3. Regulation of the degradative pathway enzymes coded for by the TOL plasmid (pWWO) from Pseudomonas putida mt-2.

    PubMed Central

    Worsey, M J; Franklin, F C; Williams, P A

    1978-01-01

    Pseudomonas putida mt-2 carries a plasmid (TOL, pWWO) which codes for a single set of enzymes responsible for the catabolism of toluene and m- and p-xylene to central metabolites by way of benzoate and m- and p-toluate, respectively, and subsequently by a meta cleavage pathway. Characterization of strains with mutations in structural genes of this pathway demonstrates that the inducers of the enzymes responsible for further degradation of m-toluate include m-xylene, m-methylbenzyl alcohol, and m-toluate, whereas the inducers of the enzymes responsible for oxidation of m-xylene to m-toluate include m-xylene and m-methylbenzyl alcohol but not m-toluate. A regulatory mutant is described in which m-xylene and m-methylbenzyl alcohol no longer induce any of the pathway enzymes, but m-toluate is still able to induce the enzymes responsible for its own degradation. Among revertants of this mutant are some strains in which all the enzymes are expressed constitutively and are not further induced by m-xylene. A model is proposed for the regulation of the pathway in which the enzymes are in two regulatory blocks, which are under the control of two regulator gene products. The model is essentially the same as proposed earlier for the regulation of the isofunctional pathway on the TOL20 plasmid from P. putida MT20. PMID:659369

  4. Complete sequence of three plasmids from Bacillus thuringiensis INTA-FR7-4 environmental isolate and comparison with related plasmids from the Bacillus cereus group.

    PubMed

    Amadio, Ariel F; Benintende, Graciela B; Zandomeni, Rubén O

    2009-11-01

    Bacillus thuringiensis is an insect pathogen used worldwide as a bioinsecticide. It belongs to the Bacillus cereus sensu lato group as well as Bacillus anthracis and B. cereus. Plasmids from this group of organisms have been implicated in pathogenicity as they carry the genes responsible for different types of diseases that affect mammals and insects. Some plasmids, like pAW63 and pBT9727, encode a functional conjugation machinery allowing them to be transferred to a recipient cell. They also share extensive homology with the non-functional conjugation apparatus of pXO2 from B. anthracis. In this study we report the complete sequence of three plasmids from an environmental B. thuringiensis isolate from Argentina, obtained by a shotgun sequencing method. We obtained the complete nucleotide sequence of plasmids pFR12 (12,095bp), pFR12.5 (12,459bp) and pFR55 (55,712bp) from B. thuringiensis INTA-FR7-4. pFR12 and pFR12.5 were classified as cryptic as they do not code for any obvious functions besides replication and mobilization. Both small plasmids were classified as RCR plasmids due to similarities with the replicases they encode. Plasmid pFR55 showed a structural organization similar to that observed for plasmids pAW63, pBT9727 and pXO2. pFR55 also shares a tra region with these plasmids, containing genes related to T4SS and conjugation. A comparison between pFR55 and conjugative plasmids led to the postulation that pFR55 is a conjugative plasmid. Genes related to replication functions in pFR55 are different to those described for plasmids with known complete sequences. pFR55 is the first completely sequenced plasmid with a replication machinery related to that of ori44. The analysis of the complete sequence of plasmids from an environmental isolate of B. thuringiensis permitted the identification of a near complete conjugation apparatus in pFR55, resembling those of plasmids pAW63, pBT9727 and pXO2. The availability of this sequence is a step forward in the study

  5. Coupling between the basic replicon and the Kis-Kid maintenance system of plasmid R1: modulation by Kis antitoxin levels and involvement in control of plasmid replication.

    PubMed

    López-Villarejo, Juan; Lobato-Márquez, Damián; Díaz-Orejas, Ramón

    2015-02-05

    kis-kid, the auxiliary maintenance system of plasmid R1 and copB, the auxiliary copy number control gene of this plasmid, contribute to increase plasmid replication efficiency in cells with lower than average copy number. It is thought that Kis antitoxin levels decrease in these cells and that this acts as the switch that activates the Kid toxin; activated Kid toxin reduces copB-mRNA levels and this increases RepA levels that increases plasmid copy number. In support of this model we now report that: (i) the Kis antitoxin levels do decrease in cells containing a mini-R1 plasmid carrying a repA mutation that reduces plasmid copy number; (ii) kid-dependent replication rescue is abolished in cells in which the Kis antitoxin levels or the CopB levels are increased. Unexpectedly we found that this coordination significantly increases both the copy number of the repA mutant and of the wt mini-R1 plasmid. This indicates that the coordination between plasmid replication functions and kis-kid system contributes significantly to control plasmid R1 replication.

  6. Conservation of Plasmid-Encoded Traits among Bean-Nodulating Rhizobium Species

    PubMed Central

    Brom, Susana; Girard, Lourdes; García-de los Santos, Alejandro; Sanjuan-Pinilla, Julio M.; Olivares, José; Sanjuan, Juan

    2002-01-01

    Rhizobium etli type strain CFN42 contains six plasmids. We analyzed the distribution of genetic markers from some of these plasmids in bean-nodulating strains belonging to different species (Rhizobium etli, Rhizobium gallicum, Rhizobium giardinii, Rhizobium leguminosarum, and Sinorhizobium fredii). Our results indicate that independent of geographic origin, R. etli strains usually share not only the pSym plasmid but also other plasmids containing symbiosis-related genes, with a similar organization. In contrast, strains belonging to other bean-nodulating species seem to have acquired only the pSym plasmid from R. etli. PMID:11976134

  7. Rearrangement and mutagenesis of a shuttle vector plasmid after passage in mammalian cells.

    PubMed Central

    Razzaque, A; Mizusawa, H; Seidman, M M

    1983-01-01

    A shuttle vector plasmid that contains sequences from simian virus 40, pBR322, and a bacterial marker gene, galactokinase, has been constructed. After replication in cells permissive for virus progeny, plasmid DNA was introduced into a galactokinase-deficient bacterial strain and the relative frequency of colonies with plasmids but without galactokinase activity was determined. This assay showed that 1% of the plasmids were defective after passage in the mammalian cells. Individual mutant plasmids were examined and found to contain deletions, duplications, point mutations, and insertions of cell DNA. Images PMID:6304690

  8. Large plasmids of Escherichia coli and Salmonella encode highly diverse arrays of accessory genes on common replicon families.

    PubMed

    Williams, Laura E; Wireman, Joy; Hilliard, Valda C; Summers, Anne O

    2013-01-01

    Plasmids are important in evolution and adaptation of host bacteria, yet we lack a comprehensive picture of their own natural variation. We used replicon typing and RFLP analysis to assess diversity and distribution of plasmids in the ECOR, SARA, SARB and SARC reference collections of Escherichia coli and Salmonella. Plasmids, especially large (≥30 kb) plasmids, are abundant in these collections. Host species and genotype clearly impact plasmid prevalence; plasmids are more abundant in ECOR than SAR, but, within ECOR, subgroup B2 strains have the fewest large plasmids. The majority of large plasmids have unique RFLP patterns, suggesting high variation, even within dominant replicon families IncF and IncI1. We found only four conserved plasmid types within ECOR, none of which are widely distributed. Within SAR, conserved plasmid types are primarily serovar-specific, including a pSLT-like plasmid in 13 Typhimurium strains. Conservation of pSLT contrasts with variability of other plasmids, suggesting evolution of serovar-specific virulence plasmids is distinct from that of most enterobacterial plasmids. We sequenced a conserved serovar Heidelberg plasmid but did not detect virulence or antibiotic resistance genes. Our data illustrate the high degree of natural variation in large plasmids of E. coli and Salmonella, even among plasmids sharing backbone genes.

  9. Plasmids and packaging cell lines for use in phage display

    DOEpatents

    Bradbury, Andrew M.

    2012-07-24

    The invention relates to a novel phagemid display system for packaging phagemid DNA into phagemid particles which completely avoids the use of helper phage. The system of the invention incorporates the use of bacterial packaging cell lines which have been transformed with helper plasmids containing all required phage proteins but not the packaging signals. The absence of packaging signals in these helper plasmids prevents their DNA from being packaged in the bacterial cell, which provides a number of significant advantages over the use of both standard and modified helper phage. Packaged phagemids expressing a protein or peptide of interest, in fusion with a phage coat protein such as g3p, are generated simply by transfecting phagemid into the packaging cell line.

  10. Current trends in separation of plasmid DNA vaccines: a review.

    PubMed

    Ghanem, Ashraf; Healey, Robert; Adly, Frady G

    2013-01-14

    Plasmid DNA (pDNA)-based vaccines offer more rapid avenues for development and production if compared to those of conventional virus-based vaccines. They do not rely on time- or labour-intensive cell culture processes and allow greater flexibility in shipping and storage. Stimulating antibodies and cell-mediated components of the immune system are considered as some of the major advantages associated with the use of pDNA vaccines. This review summarizes the current trends in the purification of pDNA vaccines for practical and analytical applications. Special attention is paid to chromatographic techniques aimed at reducing the steps of final purification, post primary isolation and intermediate recovery, in order to reduce the number of steps necessary to reach a purified end product from the crude plasmid.

  11. Conjugative transfer of cadmium resistance plasmids in Rhodococcus fascians strains

    SciTech Connect

    Desomer, J.; Dhaese, P.; Montagu, M.V.

    1988-05-01

    The presence of a 138-kilobase plasmid (pD188) correlated with increased resistance to cadmium in Rhodococcus fascians D188. This plasmid could be transferred by a conjugation-like system in matings between R. fascians strains. To examine this correlation we used large /sup 32/P-labeled pD188 subclones as probes in hybridization analyses with Southern blots of restricted total DNAs of D188 and its derivative mutants. Transconjugants expressed the cadmium resistance and could be used as donors in subsequent matings. Four other R. fascians strains (NCPPB 1488, NCPPB 1675, NCPPB 2551, and ATCC 12974) could also be used as donors for cadmium resistance in matings. Strain NCPPB 1675 showed a 100% cotransfer of cadmium and chloramphenicol resistance markers.

  12. Replisome Assembly at Bacterial Chromosomes and Iteron Plasmids

    PubMed Central

    Wegrzyn, Katarzyna E.; Gross, Marta; Uciechowska, Urszula; Konieczny, Igor

    2016-01-01

    The proper initiation and occurrence of DNA synthesis depends on the formation and rearrangements of nucleoprotein complexes within the origin of DNA replication. In this review article, we present the current knowledge on the molecular mechanism of replication complex assembly at the origin of bacterial chromosome and plasmid replicon containing direct repeats (iterons) within the origin sequence. We describe recent findings on chromosomal and plasmid replication initiators, DnaA and Rep proteins, respectively, and their sequence-specific interactions with double- and single-stranded DNA. Also, we discuss the current understanding of the activities of DnaA and Rep proteins required for replisome assembly that is fundamental to the duplication and stability of genetic information in bacterial cells. PMID:27563644

  13. Quantification of plasmid DNA copies in the nucleus after lipoplex and polyplex transfection.

    PubMed

    Cohen, Richard N; van der Aa, Marieke A E M; Macaraeg, Nichole; Lee, Ai Ping; Szoka, Francis C

    2009-04-17

    Nuclear uptake of plasmid DNA is one of the many cellular barriers that limit the efficiency of non-viral gene delivery systems. We have determined the number of plasmids that reach the nucleus of a transfected cell using an internally standardized quantitative PCR (qPCR) assay. We isolated nuclei using two different protocols: a density gradient technique and a detergent-based method. The density gradient procedure yielded nuclei with substantially less adhering plasmids on the outside of the nuclei. Using the density gradient protocol we determined that cells transfected with Lipofectamine lipoplexes or polyethylenimine polyplexes contained between 75 and 50,000 plasmids/nucleus, depending on the applied plasmid dose. Any increase above 3000 plasmids/nucleus resulted in only marginal increases in transgene expression. Furthermore, lipoplex-delivered plasmids were more efficiently expressed, on the basis of protein expression per plasmid number in the nucleus, than polyplex-delivered plasmids. This indicates that polymer may remain bound to some plasmids in the nucleus. Lastly, by sorting transfected cells into high- and low-expressing sub-populations, we observe that a sub-population of cells contain 3x greater plasmids/nucleus but express nearly 100x more transgene than other cells within a single transfection reaction. Taken together these results suggest the importance of considering the processes downstream from nuclear entry for strategies to improve the efficiency of gene transfer reagents.

  14. Dynamics of the IncW genetic backbone imply general trends in conjugative plasmid evolution.

    PubMed

    Fernández-López, Raúl; Garcillán-Barcia, M Pilar; Revilla, Carlos; Lázaro, Miguel; Vielva, Luis; de la Cruz, Fernando

    2006-11-01

    Plasmids cannot be understood as mere tools for genetic exchange: they are themselves subject to the forces of evolution. Their genomic and phylogenetic features have been less studied in this respect. Focusing on the IncW incompatibility group, which includes the smallest known conjugative plasmids, we attempt to unveil some common trends in plasmid evolution. The functional modules of IncW genetic backbone are described, with emphasis on their architecture and relationships to other plasmid groups. Some plasmid regions exhibit strong phylogenetic mosaicism, in striking contrast to others of unusual synteny conservation. The presence of genes of unknown function that are widely distributed in plasmid genomes is also emphasized, exposing the existence of ill-defined yet conserved plasmid functions. Conjugation is an essential hallmark of IncW plasmid biology and special attention is given to the organization and evolution of its transfer modules. Genetic exchange between plasmids and their hosts is analysed by following the evolution of the type IV secretion system. Adaptation of the trw conjugative machinery to pathogenicity functions in Bartonella is discussed as an example of how plasmids can change their host modus vivendi. Starting from the phage paradigm, our analysis articulates novel concepts that apply to plasmid evolution.

  15. Plasmid Isolation in Legionella pneumophila and Legionella-like Organisms.

    DTIC Science & Technology

    1980-08-22

    AD-A090 844 ARMY MEDICAL RESEARCH INST OF INFECTIOUS DISEASES FR-ETC FIG 6/5 PLASMID ISOLATI ON IN LEGIONELLA PNEUMOPHILA AND LEGIONELLA -LIKE--ETC(U... Legionella -like Organisms PERRY MIKESELL, J. W. EZZELL AND GREGORY B. KNUDSON United States Army Medical Research Institute of Infectious Diseases Fort... Hospital , Pittsburgh, Pa.). Growth conditions. Legionella -like bacteria were cultured on charcoal yeast extract agar (5), yeast extract broth (20) or

  16. Controlled release of plasmid DNA from hyaluronan nanoparticles.

    PubMed

    Mahor, Sunil; Collin, Estelle; Dash, Biraja C; Pandit, Abhay

    2011-07-01

    Encapsulation of plasmid DNA (pDNA) in nanoparticulate gene delivery systems offers the possibility of control in dosing, enhanced pDNA uptake, increased resistance to nuclease degradation and sustained release of functionally active pDNA over time. Extracellular matrix based biomaterial i.e. hyaluronan (HA) was used to encapsulate pDNA (pCMV-GLuc, Gaussia Luciferase reporter plasmid DNA having CMV promoter) in submicron size particulate system. Nano size range (~400-600 nm) pDNA loaded hyaluronan nanoparticles were formulated by ionic gelation followed by the cross-linking method with high encapsulation efficiency (~75-85%). The particle preparation process was further optimized for molecular weight, cross-linking method, cross-linking time and plasmid/polymer ratio. The entrapped plasmid maintained its structural and functional integrity as revealed by agarose gel electrophoresis. The pDNA was released from the hyaluronan nanoparticles in a controlled manner over a period of one month. In vitro transfection by one-week released pDNA from nanoparticles with transfecting agent branched polyethyleneimine (bPEI) resulted in significantly higher expression levels than those in pDNA alone which demonstrated the functional bioactivity of released pDNA. For cellular localization studies, the hyaluronan nanoparticles encapsulated with FITC-dextran were incubated with adipose derived stem cells (ADSCs) and localization in the cellular environment were investigated. The results of this study illustrate that hyaluronan nanoparticles were rapidly internalized by the cells through nonspecific endocytosis and remained intact in the cytosol for up to 24 h.

  17. Biological behavior of plasmid in Rhizobium sp. strain S25 from Tephrosia candida.

    PubMed

    Zou, X; Feng, X L; Chen, W X; Li, F D

    1998-09-01

    Rhizobium sp. strain S25 was isolated from the nodule on Tephrosia candida in Hainan Province, China. The strain showed high stress tolerance. The plasmid profile of strain S25, examined by the Eckhardt procedure, indicated that the strain harbors only one plasmid with an estimated size of 150 kb. The plasmid was shown to carry nod and nif genes by hybridization with probes of nodABC and nifHDK genes. Plasmid curing was carried out using the Bacillus subtilis sacB to generate derivatives of strain S25. In comparison with the parent strain S25, the cured derivative lost its ability to nodulate the host plant. Loss of the plasmid reduced significantly the strain's tolerance to acid, nitrous, and multiple antibiotics. The properties of the cured strain also indicated that the plasmid was involved in carbon and nitrogen metabolism. Reintroduction of the plasmid from S25 in the cured derivative restored its original biological phenotypes.

  18. Physical comparison of parathion hydrolase plasmids from Pseudomonas diminuta and Flavobacterium sp.

    PubMed

    Mulbry, W W; Kearney, P C; Nelson, J O; Karns, J S

    1987-09-01

    Restriction maps of two plasmids encoding parathion hydrolase have been determined. pPDL2 is a 39-kb plasmid harbored by Flavobacterium sp. (ATCC 27551), while pCMS1 is a 70-kb plasmid found in Pseudomonas diminuta (strain MG). Both plasmids previously have been shown to share homologous parathion hydrolase genes (termed opd for organophosphate degradation) as judged by DNA-DNA hybridization and restriction mapping. In the present study, we conducted DNA hybridization experiments using each of nine PstI restriction fragments from pCMS1 as probes against Flavobacterium plasmid DNA. The opd genes of both plasmids are located within a highly conserved region of approximately 5.1 kb. This region of homology extends approximately 2.6 kb upstream and 1.7 kb downstream from the opd genes. No homology between the two plasmids is evident outside of this region.

  19. Origin, duplication and reshuffling of plasmid genes: Insights from Burkholderia vietnamiensis G4 genome.

    PubMed

    Maida, Isabel; Fondi, Marco; Orlandini, Valerio; Emiliani, Giovanni; Papaleo, Maria Cristiana; Perrin, Elena; Fani, Renato

    2014-01-01

    Using a computational pipeline based on similarity networks reconstruction we analysed the 1133 genes of the Burkholderia vietnamiensis (Bv) G4 five plasmids, showing that gene and operon duplication played an important role in shaping the plasmid architecture. Several single/multiple duplications occurring at intra- and/or interplasmids level involving 253 paralogous genes (stand-alone, clustered or operons) were detected. An extensive gene/operon exchange between plasmids and chromosomes was also disclosed. The larger the plasmid, the higher the number and size of paralogous fragments. Many paralogs encoded mobile genetic elements and duplicated very recently, suggesting that the rearrangement of the Bv plastic genome is ongoing. Concerning the "molecular habitat" and the "taxonomical status" (the Preferential Organismal Sharing) of Bv plasmid genes, most of them have been exchanged with other plasmids of bacteria belonging (or phylogenetically very close) to Burkholderia, suggesting that taxonomical proximity of bacterial strains is a crucial issue in plasmid-mediated gene exchange.

  20. Comparison between plasmids of Salmonella and other enterobacteria isolated from the same patients.

    PubMed

    Palomares, J C; Perea, E J

    1982-01-01

    The ecology of R plasmids was studied in the intestinal flora of 19 patients with salmonellosis without antibiotic treatment. The plasmids found in the Salmonella strains and the accompanying non-pathogenic Enterobacteriaceae were characterized in each patient. We determined the transferability by conjugation, the fi character and the incompatibility group and did enzyme restriction analysis of these plasmids. The results obtained showed that S. typhimurium is the species of this genus with the highest incidence of R plasmids, and Escherichia coli among the non-pathogenic Enterobacteriaceae. The plasmids found in Salmonella are different from the plasmids found in the other Enterobacteriaceae in fi character (50% fi+ in Salmonella and 5% in the other Enterobacteriaceae) and incompatibility group (33% belong to the FII group in Salmonella plasmids and none on the other Enterobacteriaceae), thereby expressing a different origin.

  1. Production of toxin by Clostridium botulinum type A strains cured by plasmids.

    PubMed Central

    Weickert, M J; Chambliss, G H; Sugiyama, H

    1986-01-01

    Twelve strains of Clostridium botulinum type A and seven strains of Clostridium sporogenes were screened for plasmids by agarose gel electrophoresis of cleared lysates of cells from 5 ml of mid-log-phase culture. Nine type A strains had one or more plasmids of 4.3, 6.8, or 36 megadaltons (MDa); several strains showed a large plasmid of 61 MDa, but it was not consistently recovered. Four C. sporogenes strains had one or more plasmids of 4.3, 5.6 or 36 MDa. Isolates obtained from cultures of plasmid-containing C. botulinum type A strains grown in ionic detergent broth and from spontaneously arising variants were screened both for toxin production and for plasmid content. Toxigenicity of C. botulinum could not be correlated with the presence of any one plasmid. Images PMID:3082278

  2. Scaling-up recombinant plasmid DNA for clinical trial: current concern, solution and status.

    PubMed

    Ismail, Ruzila; Allaudin, Zeenathul Nazariah; Lila, Mohd-Azmi Mohd

    2012-09-07

    Gene therapy and vaccines are rapidly developing field in which recombinant nucleic acids are introduced in mammalian cells for enhancement, restoration, initiation or silencing biochemical function. Beside simplicity in manipulation and rapid manufacture process, plasmid DNA-based vaccines have inherent features that make them promising vaccine candidates in a variety of diseases. This present review focuses on the safety concern of the genetic elements of plasmid such as propagation and expression units as well as their host genome for the production of recombinant plasmid DNA. The highlighted issues will be beneficial in characterizing and manufacturing plasmid DNA for save clinical use. Manipulation of regulatory units of plasmid will have impact towards addressing the safety concerns raised in human vaccine applications. The gene revolution with plasmid DNA by alteration of their plasmid and production host genetics will be promising for safe delivery and obtaining efficient outcomes.

  3. Analysis of plasmids in nosocomial strains of multiple-antibiotic-resistant Staphylococcus aureus.

    PubMed Central

    Lyon, B R; May, J W; Skurray, R A

    1983-01-01

    Nosocomial infections caused by Staphylococcus aureus strains resistant to methicillin and multiple antibiotics have reached epidemic proportions in Melbourne, Australia, over the past 5 years. Plasmid analysis of representative clinical isolates demonstrated the presence of three classes of plasmid DNA in most strains. Resistance to gentamicin, kanamycin, and tobramycin was usually mediated by an 18-megadalton plasmid but could also be encoded by a related 22-megadalton plasmid. Two distinguishable plasmids of 3 megadaltons each endowed resistance to chloramphenicol, and the third class consisted of small plasmids, each approximately 1 megadalton in size, with no attributable function. An extensive array of resistance determinants, including some which have usually been associated with a plasmid locus, were found to exist on the chromosome. Evidence that resistance to gentamicin, kanamycin, and tobramycin is chromosomally encoded in some clinical isolates suggests that this determinant may have undergone genetic translocation onto the staphylococcal chromosome. Images PMID:6311086

  4. Immunization of chickens with an agonistic monoclonal anti-chicken CD40 antibody-hapten complex: rapid and robust IgG response induced by a single subcutaneous injection.

    PubMed

    Chen, Chang-Hsin; Abi-Ghanem, Daad; Waghela, Suryakant D; Chou, Wen-Ko; Farnell, Morgan B; Mwangi, Waithaka; Berghman, Luc R

    2012-04-30

    Producing diagnostic antibodies in chicken egg yolk represents an alternate animal system that offers many advantages including high productivity at low cost. Despite being an excellent counterpart to mammalian antibodies, chicken IgG from yolk still represents an underused resource. The potential of agonistic monoclonal anti-CD40 antibodies (mAb) as a powerful immunological adjuvant has been demonstrated in mammals, but not in chickens. We recently reported an agonistic anti-chicken CD40 mAb (designated mAb 2C5) and showed that it may have potential as an immunological adjuvant. In this study, we examined the efficacy of targeting a short peptide to chicken CD40 [expressed by the antigen-presenting cells (APCs)] in enhancing an effective IgG response in chickens. For this purpose, an immune complex consisting of one streptavidin molecule, two directionally biotinylated mAb 2C5 molecules, and two biotinylated peptide molecules was produced. Chickens were immunized subcutaneously with doses of this complex ranging from 10 to 90 μg per injection once, and relative quantification of the peptide-specific IgG response showed that the mAb 2C5-based complex was able to elicit a strong IgG response as early as four days post-immunization. This demonstrates that CD40-targeting antigen to chicken APCs can significantly enhance antibody responses and induce immunoglobulin isotype-switching. This immunization strategy holds promise for rapid production of hapten-specific IgG in chickens.

  5. Virulence Plasmids of Nonsporulating Gram-Positive Pathogens

    PubMed Central

    Van Tyne, Daria; Gilmore, Michael S.

    2014-01-01

    SUMMARY Gram-positive bacteria are leading causes of many types of human infection, including pneumonia, skin and nasopharyngeal infections, as well as urinary tract and surgical wound infections among hospitalized patients. These infections have become particularly problematic because many of the species causing them have become highly resistant to antibiotics. The role of mobile genetic elements, such as plasmids, in the dissemination of antibiotic resistance among Gram-positive bacteria has been well studied; less well understood is the role of mobile elements in the evolution and spread of virulence traits among these pathogens. While these organisms are leading agents of infection, they are also prominent members of the human commensal ecology. It appears that these bacteria are able to take advantage of the intimate association between host and commensal, via virulence traits that exacerbate infection and cause disease. However, evolution into an obligate pathogen has not occurred, presumably because it would lead to rejection of pathogenic organisms from the host ecology. Instead, in organisms that exist as both commensal and pathogen, selection has favored the development of mechanisms for variability. As a result, many virulence traits are localized on mobile genetic elements, such as virulence plasmids and pathogenicity islands. Virulence traits may occur within a minority of isolates of a given species, but these minority populations have nonetheless emerged as a leading problem in infectious disease. This chapter reviews virulence plasmids in nonsporulating Gram-positive bacteria, and examines their contribution to disease pathogenesis. PMID:25544937

  6. Genetic immunization with plasmid DNA mediated by electrotransfer.

    PubMed

    Rochard, Alice; Scherman, Daniel; Bigey, Pascal

    2011-07-01

    The concept of DNA immunization was first advanced in the early 1990s, but was not developed because of an initial lack of efficiency. Recent technical advances in plasmid design and gene delivery techniques have allowed renewed interest in the idea. Particularly, a better understanding of genetic immunization has led to construction of optimized plasmids and the use of efficient molecular adjuvants. The field also took great advantage of new delivery techniques such as electrotransfer. This is a simple physical technique consisting of injecting plasmid DNA into a target tissue and applying an electric field, allowing up to a thousandfold more expression of the transgene than naked DNA. DNA immunization mediated by electrotransfer is now effective in a variety of preclinical models against infectious or acquired diseases such as cancer or autoimmune diseases, and is making its way through the clinics in several ongoing phase I human clinical trials. This review will briefly describe genetic immunization mediated by electrotransfer and the main fields of application.

  7. An insight of traditional plasmid curing in Vibrio species.

    PubMed

    Letchumanan, Vengadesh; Chan, Kok-Gan; Lee, Learn-Han

    2015-01-01

    As the causative agent of foodborne related illness, Vibrio species causes a huge impact on the public health and management. Vibrio species is often associated with seafood as the latter plays a role as a vehicle to transmit bacterial infections. Hence, antibiotics are used not to promote growth but rather to prevent and treat bacterial infections. The extensive use of antibiotics in the aquaculture industry and environment has led to the emerging of antibiotic resistant strains. This phenomenon has triggered an alarming public health concern due to the increase number of pathogenic Vibrio strains that are resistant to clinically used antibiotics and is found in the environment. Antibiotic resistance and the genes location in the strains can be detected through plasmid curing assay. The results derived from plasmid curing assay is fast, cost effective, sufficient in providing insights, and influence the antibiotic management policies in the aquaculture industry. This presentation aims in discussing and providing insights on various curing agents in Vibrio species. To our best of knowledge, this is a first review written discussing on plasmid curing in Vibrio species.

  8. Cloning-independent plasmid construction for genetic studies in streptococci

    PubMed Central

    Xie, Zhoujie; Qi, Fengxia; Merritt, Justin

    2013-01-01

    Shuttle plasmids are among the few routinely utilized tools in the Streptococcus mutans genetic system that still require the use of classical cloning methodologies and intermediate hosts for genetic manipulation. Accordingly, it typically requires considerably less time and effort to introduce mutations onto the S. mutans chromosome than it does to construct shuttle vectors for expressing genes in trans. Occasionally, shuttle vector constructs also exhibit toxicity in E. coli, which prevents their proper assembly. To circumvent these limitations, we modified a prolonged overlap extension PCR (POE-PCR) protocol to facilitate direct plasmid assembly in S. mutans. Using solely PCR, we created the reporter vector pZX7, which contains a single minimal streptococcal replication origin and harbors a spectinomycin resistance cassette and the gusA gene encoding β-glucuronidase. We compared the efficiency of pZX7 assembly using multiple strains of S. mutans and were able to obtain from 5×103 – 2×105 CFU/μg PCR product. Likewise, we used pZX7 to further demonstrate that Streptococcus sanguinis and Streptococcus gordonii are also excellent hosts for cloning-independent plasmid assembly, which suggests that this system is likely to function in numerous other streptococci. Consequently, it should be possible to completely forgo the use of E. coli – Streptococcus shuttle vectors in many streptococcal species, thereby decreasing the time and effort required to assemble constructs and eliminating any toxicity issues associated with intermediate hosts. PMID:23673081

  9. Identification of two replicons in phage-plasmid P4.

    PubMed

    Tocchetti, A; Serina, S; Terzano, S; Dehò, G; Ghisotti, D

    1998-06-05

    DNA replication of phage-plasmid P4 proceeds bidirectionally from the ori1 site (previously named ori), but requires a second cis-acting region, crr. Replication depends on the product of the P4 alpha gene, a protein with primase and helicase activity, that binds both ori1 and crr. A negative regulator of P4 DNA replication, the Cnr protein, is required for copy number control of plasmid P4. Using a plasmid complementation test for replication, we found that two replicons, both dependent on the alpha gene product, coexist in P4. The first replicon is made by the cnr and alpha genes and the ori1 and crr sites. The second is limited to the alpha and crr region. Thus, in the absence of the ori1 region, replication can initiate at a different site. By deletion mapping, a cis-acting region, ori2, essential for replication of the alpha-crr replicon was mapped within a 270-bp fragment in the first half of the alpha gene. The ori2 site was found to be dispensable in a replicon that contains ori1. A construct that besides crr and alpha carries also the cnr gene was unable to replicate, suggesting that Cnr not only controls replication from ori1, but also silences ori2.

  10. An insight of traditional plasmid curing in Vibrio species

    PubMed Central

    Letchumanan, Vengadesh; Chan, Kok-Gan; Lee, Learn-Han

    2015-01-01

    As the causative agent of foodborne related illness, Vibrio species causes a huge impact on the public health and management. Vibrio species is often associated with seafood as the latter plays a role as a vehicle to transmit bacterial infections. Hence, antibiotics are used not to promote growth but rather to prevent and treat bacterial infections. The extensive use of antibiotics in the aquaculture industry and environment has led to the emerging of antibiotic resistant strains. This phenomenon has triggered an alarming public health concern due to the increase number of pathogenic Vibrio strains that are resistant to clinically used antibiotics and is found in the environment. Antibiotic resistance and the genes location in the strains can be detected through plasmid curing assay. The results derived from plasmid curing assay is fast, cost effective, sufficient in providing insights, and influence the antibiotic management policies in the aquaculture industry. This presentation aims in discussing and providing insights on various curing agents in Vibrio species. To our best of knowledge, this is a first review written discussing on plasmid curing in Vibrio species. PMID:26347714

  11. Antibiotic resistance free plasmid DNA expressing LACK protein leads towards a protective Th1 response against Leishmania infantum infection.

    PubMed

    Ramos, I; Alonso, A; Peris, A; Marcen, J M; Abengozar, M A; Alcolea, P J; Castillo, J A; Larraga, V

    2009-11-12

    Canine visceral leishmaniasis is a serious public health concern in the Mediterranean basin since dogs are the main Leishmania infantum reservoir. However, there is not a vaccination method in veterinary use in this area, and therefore the development of a vaccine against this parasite is essential for the possible control of the disease. Previous reports have shown the efficacy of heterologous prime-boost vaccination with the pCIneo plasmid and the poxvirus VV (both Western Reserve and MVA strains) expressing L. infantum LACK antigen against canine leishmaniasis. As pCIneo-LACK plasmid contains antibiotic resistance genes, its use as a profilactic method is not recommended. Hence, the antibiotic resistance gene free pORT-LACK plasmid is a more suitable tool for its use as a vaccine. Here we report the protective and immunostimulatory effect of the prime-boost pORT-LACK/MVA-LACK vaccination tested in a canine experimental model. Vaccination induced a reduction in clinical signs and in parasite burden in the liver, an induction of the Leishmania-specific T cell activation, as well as an increase of the expression of Th1 type cytokines in PBMC and target organs.

  12. The broad-host-range plasmid pSFA231 isolated from petroleum-contaminated sediment represents a new member of the PromA plasmid family

    PubMed Central

    Li, Xiaobin; Top, Eva M.; Wang, Yafei; Brown, Celeste J.; Yao, Fei; Yang, Shan; Jiang, Yong; Li, Hui

    2015-01-01

    A self-transmissible broad-host-range (BHR) plasmid pSFA231 was isolated from petroleum-contaminated sediment in Shen-fu wastewater irrigation zone, China, using the triparental mating exogenous plasmid capture method. Based on its complete sequence the plasmid has a size of 41.5 kb and codes for 50 putative open reading frames (orfs), 29 of which represent genes involved in replication, partitioning and transfer functions of the plasmid. Phylogenetic analysis grouped pSFA231 into the newly defined PromA plasmid family, which currently includes five members. Further comparative genomic analysis shows that pSFA231 shares the common backbone regions with the other PromA plasmids, i.e., genes involved in replication, maintenance and control, and conjugative transfer. Nevertheless, phylogenetic divergence was found in specific gene products. We propose to divide the PromA group into two subgroups, PromA-α (pMRAD02, pSB102) and PromA-β (pMOL98, pIPO2T, pSFA231, pTer331), based on the splits network analysis of the RepA protein. Interestingly, a cluster of hypothetical orfs located between parA and traA of pSFA231 shows high similarity with the corresponding regions on pMOL98, pIPO2T, and pTer331, suggesting these hypothetical orfs may represent “essential” plasmid backbone genes for the PromA-β subgroup. Alternatively, they may also be accessory genes that were first acquired and then stayed as the plasmid diverged. Our study increases the available collection of complete genome sequences of BHR plasmids, and since pSFA231 is the only characterized PromA plasmid from China, our findings also enhance our understanding of the genetic diversity of this plasmid group in different parts of the world. PMID:25628616

  13. DETECTION OF LOW DOSE RADIATION INDUCED DNA DAMAGE USING TEMPERATURE DIFFERENTIAL FLUORESCENCE ASSAY

    EPA Science Inventory

    A rapid and sensitive fluorescence assay for radiation-induced DNA damage is reported. Changes in temperature-induced strand separation in both calf thymus DNA and plasmid DNA (puc 19 plasmid from Escherichia coli) were measured after exposure to low doses of radiation. Exposur...

  14. DETECTION OF LOW DOSE RADIATION INDUCED DNA DAMAGE USING TEMPERATURE DIFFERENNTIAL FLUORESENCE ASSAY

    EPA Science Inventory

    A rapid and sensitive fluorescence assay for radiation-induced DNA damage is reported. Changes in temperature-induced strand separation in both calf thymus DNA and plasmid DNA (puc 19 plasmid from Escherichia coli) were measured after exposure to low doses of radiation. Exposures...

  15. Bacillus anthracis Virulent Plasmid pX02 Genes Found in Large Plasmids of Two Other Bacillus Species

    PubMed Central

    Luna, Vicki A.; King, Debra S.; Peak, K. Kealy; Reeves, Frank; Heberlein-Larson, Lea; Veguilla, William; Heller, L.; Duncan, Kathleen E.; Cannons, Andrew C.; Amuso, Philip; Cattani, Jacqueline

    2006-01-01

    In order to cause the disease anthrax, Bacillus anthracis requires two plasmids, pX01 and pX02, which carry toxin and capsule genes, respectively, that are used as genetic targets in the laboratory detection of the bacterium. Clinical, forensic, and environmental samples that test positive by PCR protocols established by the Centers for Disease Control and Prevention for B. anthracis are considered to be potentially B. anthracis until confirmed by culture and a secondary battery of tests. We report the presence of 10 genes (acpA, capA, capB, capC, capR, capD, IS1627, ORF 48, ORF 61, and repA) and the sequence for the capsule promoter normally found on pX02 in Bacillus circulans and a Bacillus species closely related to Bacillus luciferensis. Tests revealed these sequences to be present on a large plasmid in each isolate. The 11 sequences consistently matched to B. anthracis plasmid pX02, GenBank accession numbers AF188935.1, AE011191.1, and AE017335.3. The percent nucleotide identities for capD and the capsule promoter were 99.9% and 99.7%, respectively, and for the remaining nine genes, the nucleotide identity was 100% for both isolates. The presence of these genes, which are usually associated with the pX02 plasmid, in two soil Bacillus species unrelated to B. anthracis alerts us to the necessity of identifying additional sequences that will signal the presence of B. anthracis in clinical, forensic, and environmental samples. PMID:16825351

  16. Mechanistic basis of plasmid-specific DNA binding of the F plasmid regulatory protein, TraM.

    PubMed

    Peng, Yun; Lu, Jun; Wong, Joyce J W; Edwards, Ross A; Frost, Laura S; Mark Glover, J N

    2014-11-11

    The conjugative transfer of bacterial F plasmids relies on TraM, a plasmid-encoded protein that recognizes multiple DNA sites to recruit the plasmid to the conjugative pore. In spite of the high degree of amino acid sequence conservation between TraM proteins, many of these proteins have markedly different DNA binding specificities that ensure the selective recruitment of a plasmid to its cognate pore. Here we present the structure of F TraM RHH (ribbon-helix-helix) domain bound to its sbmA site. The structure indicates that a pair of TraM tetramers cooperatively binds an underwound sbmA site containing 12 base pairs per turn. The sbmA is composed of 4 copies of a 5-base-pair motif, each of which is recognized by an RHH domain. The structure reveals that a single conservative amino acid difference in the RHH β-ribbon between F and pED208 TraM changes its specificity for its cognate 5-base-pair sequence motif. Specificity is also dictated by the positioning of 2-base-pair spacer elements within sbmA; in F sbmA, the spacers are positioned between motifs 1 and 2 and between motifs 3 and 4, whereas in pED208 sbmA, there is a single spacer between motifs 2 and 3. We also demonstrate that a pair of F TraM tetramers can cooperatively bind its sbmC site with an affinity similar to that of sbmA in spite of a lack of sequence similarity between these DNA elements. These results provide a basis for the prediction of the DNA binding properties of the family of TraM proteins.

  17. Structure and regulation of gene expression of a Clo DF13 plasmid DNA region involved in plasmid segregation and incompatibility.

    PubMed Central

    van den Elzen, P J; Hakkaart, M J; van Putten, A J; Walters, H H; Veltkamp, E; Nijkamp, H J

    1983-01-01

    The bacteriocinogenic plasmid Clo DF13 contains genetic information involved in the accurate partitioning of the plasmid (parA and parB) as well as in incompatibility phenomena (incA, B, C and D). In this paper we report on the primary structure and regulation of gene expression of the 29% - 50% part of Clo DF13, containing the DNA regions incA, incB and parB as well as genes K and L. According to the results of our DNA sequence analysis, mapping of transposon insertions, RNA blotting and S1 mapping experiments, we conclude that: a) genes K and L are transcribed as one operon; transcription of this operon is initiated at a promoter (P2) located at 32.5% and proceeds in a clockwise direction. b) treatment of cells with mitomycin-C, significantly enhances transcription from P2, although this promoter is probably not directly repressed by lexA protein. c) Termination of transcription of this operon occurs between genes K and L, as well as distal to gene L. The possible role of gene products and/or sites, located within the 29-50% DNA region, in plasmid incompatibility and segregation is discussed. Images PMID:6324101

  18. Vaccination with Human Papillomavirus Pseudovirus-Encapsidated Plasmids Targeted to Skin Using Microneedles

    PubMed Central

    Kines, Rhonda C.; Zarnitsyn, Vladimir; Johnson, Teresa R.; Pang, Yuk-Ying S.; Corbett, Kizzmekia S.; Nicewonger, John D.; Gangopadhyay, Anu; Chen, Man; Liu, Jie; Prausnitz, Mark R.; Schiller, John T.; Graham, Barney S.

    2015-01-01

    Human papilloma virus-like particles (HPV VLP) serve as the basis of the current licensed vaccines for HPV. We have previously shown that encapsidation of DNA expressing the model antigen M/M2 from respiratory syncytial virus (RSV) in HPV pseudovirions (PsV) is immunogenic when delivered intravaginally. Because the HPV capsids confer tropism for basal epithelium, they represent attractive carriers for vaccination targeted to the skin using microneedles. In this study we asked: 1) whether HPV16 VLP administered by microneedles could induce protective immune responses to HPV16 and 2) whether HPV16 PsV-encapsidated plasmids delivered by microneedles could elicit immune responses to both HPV and the antigen delivered by the transgene. Mice immunized with HPV16 VLP coated microneedles generated robust neutralizing antibody responses and were protected from HPV16 challenge. Microneedle arrays coated with HPV16-M/M2 or HPV16-F protein (genes of RSV) were then tested and dose-dependent HPV and F-specific antibody responses were detected post-immunization, and M/M2-specific T-cell responses were detected post RSV challenge, respectively. HPV16 PsV-F immunized mice were fully protected from challenge with HPV16 PsV and had reduced RSV viral load in lung and nose upon intranasal RSV challenge. In summary, HPV16 PsV-encapsidated DNA delivered by microneedles induced neutralizing antibody responses against HPV and primed for antibody and T-cell responses to RSV antigens encoded by the encapsidated plasmids. Although the immunogenicity of the DNA component was just above the dose response threshold, the HPV-specific immunity was robust. Taken together, these data suggest microneedle delivery of lyophilized HPV PsV could provide a practical, thermostable combined vaccine approach that could be developed for clinical evaluation. PMID:25785935

  19. Conserved Plasmid Hydrogen-Uptake (hup)-Specific Sequences within Hup+Rhizobium leguminosarum Strains

    PubMed Central

    Leyva, Antonio; Palacios, José M.; Ruiz-Argüeso, Tomás

    1987-01-01

    Thirteen Rhizobium leguminosarum strains previously reported as H2-uptake hydrogenase positive (Hup+) or negative (Hup−) were analyzed for the presence and conservation of DNA sequences homologous to cloned Bradyrhizobium japonicum hup-specific DNA from cosmid pHU1 (M. A. Cantrell, R. A. Haugland, and H. J. Evans, Proc. Natl. Acad. Sci. USA 80:181-185, 1983). The Hup phenotype of these strains was reexamined by determining hydrogenase activity induced in bacteroids from pea nodules. Five strains, including H2 oxidation-ATP synthesis-coupled and -uncoupled strains, induced significant rates of H2-uptake hydrogenase activity and contained DNA sequences homologous to three probe DNA fragments (5.9-kilobase [kb] HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) from pHU1. The pattern of genomic DNA HindIII and EcoRI fragments with significant homology to each of the three probes was identical in all five strains regardless of the H2-dependent ATP generation trait. The restriction fragments containing the homology totalled about 22 kb of DNA common to the five strains. In all instances the putative hup sequences were located on a plasmid that also contained nif genes. The molecular sizes of the identified hup-sym plasmids ranged between 184 and 212 megadaltons. No common DNA sequences homologous to B. japonicum hup DNA were found in genomic DNA from any of the eight remaining strains showing no significant hydrogenase activity in pea bacteroids. These results suggest that the identified DNA region contains genes essential for hydrogenase activity in R. leguminosarum and that its organization is highly conserved within Hup+ strains in this symbiotic species. Images PMID:16347471

  20. Mulberry strains of Xylella fastidiosa contain a 25 kilobase pair plasmid with extensive sequence identity to a plasmid from Verminephrobacter eiseniae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A 25 kbp plasmid was present in each of four Californian strains of Xylella fastidiosa from mulberry affected with leaf scorch disease. Fragments of each plasmid were cloned into E. coli, sequenced, and assembled into circular contigs of 25,105 bp (pXF-RIV11 and pXF-RIV16) or 24,372 bp (pXF-RIV19 an...

  1. Xylella fastidiosa isolates from mulberry harbor a 25 kilobase pair plasmid with extensive sequence identity to a plasmid from Verminephrobacter eiseniae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A 25 kbp plasmid was present in each of four Californian isolates of Xylella fastidiosa from mulberry affected with leaf scorch disease. Fragments of each plasmid were cloned into E. coli, sequenced, and assembled into circular contigs of 25,105 bp (pXF-RIV11 and pXF-RIV16) or 24,372 bp (pXF-RIV19 a...

  2. blaCMY-2-positive IncA/C plasmids from escherichia coli and salmonella enterica are a distinct component of a larger lineage of plasmids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Large multidrug resistance plasmids of the A/C incompatibility complex (IncA/C) have been found in a diverse group of Gram-negative commensal and pathogenic bacteria. We present three completed sequences from IncA/C plasmids that originated from Escherichia coli (cattle) and Salmonella enterica sero...

  3. Conjugative transferability of the A/C plasmids from Salmonella enterica isolates that possess or lack blaCMY in the A/C plasmid backbone

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to gain a better understanding of the conjugative transfer of antimicrobial resistance plasmids from 205 Salmonella enterica strains, isolated from cattle to E. coli or Salmonella recipients. PCR-based replicon typing (PBRT) was used to type incompatibility plasmid r...

  4. Complementation of Conjugation Functions of Streptomyces lividans Plasmid pIJ101 by the Related Streptomyces Plasmid pSB24.2

    PubMed Central

    Pettis, Gregg S.; Prakash, Shubha

    1999-01-01

    A database search revealed extensive sequence similarity between Streptomyces lividans plasmid pIJ101 and Streptomyces plasmid pSB24.2, which is a deletion derivative of Streptomyces cyanogenus plasmid pSB24.1. The high degree of relatedness between the two plasmids allowed the construction of a genetic map of pSB24.2, consisting of putative transfer and replication loci. Two pSB24.2 loci, namely, the cis-acting locus for transfer (clt) and the transfer-associated korB gene, were shown to be capable of complementing the pIJ101 clt and korB functions, respectively, a result that is consistent with the notion that pIJ101 and the parental plasmid pSB24.1 encode highly similar, if not identical, conjugation systems. PMID:10419972

  5. Insights into dynamics of mobile genetic elements in hyperthermophilic environments from five new Thermococcus plasmids.

    PubMed

    Krupovic, Mart; Gonnet, Mathieu; Hania, Wajdi Ben; Forterre, Patrick; Erauso, Gaël

    2013-01-01

    Mobilome of hyperthermophilic archaea dwelling in deep-sea hydrothermal vents is poorly characterized. To gain insight into genetic diversity and dynamics of mobile genetic elements in these environments we have sequenced five new plasmids from different Thermococcus strains that have been isolated from geographically remote hydrothermal vents. The plasmids were ascribed to two subfamilies, pTN2-like and pEXT9a-like. Gene content and phylogenetic analyses illuminated a robust connection between pTN2-like plasmids and Pyrococcus abyssi virus 1 (PAV1), with roughly half of the viral genome being composed of genes that have homologues in plasmids. Unexpectedly, pEXT9a-like plasmids were found to be closely related to the previously sequenced plasmid pMETVU01 from Methanocaldococcus vulcanius M7. Our data suggests that the latter observation is most compatible with an unprecedented horizontal transfer of a pEXT9a-like plasmid from Thermococcales to Methanococcales. Gene content analysis revealed that thermococcal plasmids encode Hfq-like proteins and toxin-antitoxin (TA) systems of two different families, VapBC and RelBE. Notably, although abundant in archaeal genomes, to our knowledge, TA and hfq-like genes have not been previously found in archaeal plasmids or viruses. Finally, the plasmids described here might prove to be useful in developing new genetic tools for hyperthermophiles.

  6. Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity.

    PubMed

    Münch, Karin M; Müller, Johannes; Wienecke, Sarah; Bergmann, Simone; Heyber, Steffi; Biedendieck, Rebekka; Münch, Richard; Jahn, Dieter

    2015-09-01

    During the past 2 decades, Bacillus megaterium has been systematically developed for the gram-per-liter scale production of recombinant proteins. The plasmid-based expression systems employed use a xylose-controlled promoter. Protein production analyses at the single-cell level using green fluorescent protein as a model product revealed cell culture heterogeneity characterized by a significant proportion of less productive bacteria. Due to the enormous size of B. megaterium, such bistable behavior seen in subpopulations was readily analyzed by time lapse microscopy and flow cytometry. Cell culture heterogeneity was not caused simply by plasmid loss: instead, an asymmetric distribution of plasmids during cell division was detected during the exponential-growth phase. Multicopy plasmids are generally randomly distributed between daughter cells. However, in vivo and in vitro experiments demonstrated that under conditions of strong protein production, plasmids are retained at one of the cell poles. Furthermore, it was found that cells with accumulated plasmids and high protein production ceased cell division. As a consequence, the overall protein production of the culture was achieved mainly by the subpopulation with a sufficient plasmid copy number. Based on our experimental data, we propose a model whereby the distribution of multicopy plasmids is controlled by polar fixation under protein production conditions. Thereby, cell lines with fluctuating plasmid abundance arise, which results in population heterogeneity. Our results provide initial insights into the mechanism of cellular heterogeneity during plasmid-based recombinant protein production in a Bacillus species.

  7. Peaceful coexistence amongst Borrelia plasmids: getting by with a little help from their friends?

    PubMed

    Chaconas, George; Norris, Steven J

    2013-09-01

    Borrelia species comprise a unique genus of bacterial pathogens. These organisms contain a segmented genome with up to two dozen plasmids ranging in size from 5 kb up to about 200 kb. The plasmids have also been referred to as mini-chromosomes or essential genetic elements, as some of them carry information important for infection of vertebrates or for survival in the tick vector. Most of the plasmids are linear with covalently closed hairpin telomeres and these linear plasmids are in a constant state of genetic rearrangement. The mechanisms of plasmid replication, maintenance and partitioning remain largely obscure and are complicated by a long doubling time, the requirement for expensive media and inefficient genetic manipulation. A set of five parologous protein families (PFs) are believed to confer the ability for autonomous replication and plasmid maintenance. The number of plasmids also complicates analyses because of the possibility that PFs from one plasmid may sometimes function in trans on other plasmids. Two papers in the last year have moved the field forward and their combined data suggest that trans complementation amongst Borrelia plasmids may sometimes occur.

  8. Conservation of plasmids among Escherichia coli K1 isolates of diverse origins.

    PubMed

    Mercer, A A; Morelli, G; Heuzenroeder, M; Kamke, M; Achtman, M

    1984-12-01

    Escherichia coli K1 isolates of various O types were previously assigned to different clonal groups. Members of the two clones defined by membrane pattern 9 (MP9) and serotypes O18:K1 and O1:K1 had been found to be very similar to each other. The plasmid contents of these bacteria confirmed this conclusion. Both groups carried a self-transmissible plasmid of the FI incompatibility group that coded for colicin production and a major outer membrane protein called the plasmid-coded protein (PCP). The size of this plasmid varied from 76 to 96 megadaltons, but restriction endonuclease digestion and DNA heteroduplex analysis revealed that these plasmids were highly related. O18:K1 bacteria of MP6 had previously been determined to represent a subclone, related to but different from O18:K1 MP9 bacteria. These MP6 bacteria carried a different, smaller IncFI plasmid which did not code for colicin production or the PCP protein. This smaller plasmid was primarily related to the larger plasmid within the regions of DNA encoding incompatibility, replication, and conjugation. O1:K1 bacteria of MP5 contained other unrelated plasmids in agreement with the previous conclusion that they are unrelated to O1:K1 bacteria of MP9. The bacteria examined had been isolated from two continents over a time span of 38 years, and the results attest to conservative inheritance of plasmids within bacteria of common descent.

  9. Insights into Dynamics of Mobile Genetic Elements in Hyperthermophilic Environments from Five New Thermococcus Plasmids

    PubMed Central

    Krupovic, Mart; Gonnet, Mathieu; Hania, Wajdi Ben; Forterre, Patrick; Erauso, Gaël

    2013-01-01

    Mobilome of hyperthermophilic archaea dwelling in deep-sea hydrothermal vents is poorly characterized. To gain insight into genetic diversity and dynamics of mobile genetic elements in these environments we have sequenced five new plasmids from different Thermococcus strains that have been isolated from geographically remote hydrothermal vents. The plasmids were ascribed to two subfamilies, pTN2-like and pEXT9a-like. Gene content and phylogenetic analyses illuminated a robust connection between pTN2-like plasmids and Pyrococcus abyssi virus 1 (PAV1), with roughly half of the viral genome being composed of genes that have homologues in plasmids. Unexpectedly, pEXT9a-like plasmids were found to be closely related to the previously sequenced plasmid pMETVU01 from Methanocaldococcus vulcanius M7. Our data suggests that the latter observation is most compatible with an unprecedented horizontal transfer of a pEXT9a-like plasmid from Thermococcales to Methanococcales. Gene content analysis revealed that thermococcal plasmids encode Hfq-like proteins and toxin-antitoxin (TA) systems of two different families, VapBC and RelBE. Notably, although abundant in archaeal genomes, to our knowledge, TA and hfq-like genes have not been previously found in archaeal plasmids or viruses. Finally, the plasmids described here might prove to be useful in developing new genetic tools for hyperthermophiles. PMID:23326305

  10. CRISPR-Cas systems preferentially target the leading regions of MOBF conjugative plasmids.

    PubMed

    Westra, Edze R; Staals, Raymond H J; Gort, Gerrit; Høgh, Søren; Neumann, Sarah; de la Cruz, Fernando; Fineran, Peter C; Brouns, Stan J J

    2013-05-01

    Most prokaryotes contain CRISPR-Cas immune systems that provide protection against mobile genetic elements. We have focused on the ability of CRISPR-Cas to block plasmid conjugation, and analyzed the position of target sequences (protospacers) on conjugative plasmids. The analysis reveals that protospacers are non-uniformly distributed over plasmid regions in a pattern that is determined by the plasmid's mobilization type (MOB). While MOBP plasmids are most frequently targeted in the region entering the recipient cell last (lagging region), MOBF plasmids are mostly targeted in the region entering the recipient cell first (leading region). To explain this protospacer distribution bias, we propose two mutually non-exclusive hypotheses: (1) spacers are acquired more frequently from either the leading or lagging region depending on the MOB type (2) CRISPR-interference is more efficient when spacers target these preferred regions. To test the latter hypothesis, we analyzed Type I-E CRISPR-interference against MOBF prototype plasmid F in Escherichia coli. Our results show that plasmid conjugation is effectively inhibited, but the level of immunity is not affected by targeting the plasmid in the leading or lagging region. Moreover, CRISPR-immunity levels do not depend on whether the incoming single-stranded plasmid DNA, or the DNA strand synthesized in the recipient is targeted. Our findings indicate that single-stranded DNA may not be a target for Type I-E CRISPR-Cas systems, and suggest that the protospacer distribution bias might be due to spacer acquisition preferences.

  11. RK2 plasmid dynamics in Caulobacter crescentus cells--two modes of DNA replication initiation.

    PubMed

    Wegrzyn, Katarzyna; Witosinska, Monika; Schweiger, Pawel; Bury, Katarzyna; Jenal, Urs; Konieczny, Igor

    2013-06-01

    Undisturbed plasmid dynamics is required for the stable maintenance of plasmid DNA in bacterial cells. In this work, we analysed subcellular localization, DNA synthesis and nucleoprotein complex formation of plasmid RK2 during the cell cycle of Caulobacter crescentus. Our microscopic observations showed asymmetrical distribution of plasmid RK2 foci between the two compartments of Caulobacter predivisional cells, resulting in asymmetrical allocation of plasmids to progeny cells. Moreover, using a quantitative PCR (qPCR) method, we estimated that multiple plasmid particles form a single fluorescent focus and that the number of plasmids per focus is approximately equal in both swarmer and predivisional Caulobacter cells. Analysis of the dynamics of TrfA-oriV complex formation during the Caulobacter cell cycle revealed that TrfA binds oriV primarily during the G1 phase, however, plasmid DNA synthesis occurs during the S and G2 phases of the Caulobacter cell cycle. Both in vitro and in vivo analysis of RK2 replication initiation in C. crescentus cells demonstrated that it is independent of the Caulobacter DnaA protein in the presence of the longer version of TrfA protein, TrfA-44. However, in vivo stability tests of plasmid RK2 derivatives suggested that a DnaA-dependent mode of plasmid replication initiation is also possible.

  12. Prevalence of ColE1-like plasmids and kanamycin resistance genes in Salmonella enterica serovars.

    PubMed

    Chen, Chin-Yi; Lindsey, Rebecca L; Strobaugh, Terence P; Frye, Jonathan G; Meinersmann, Richard J

    2010-10-01

    Multi-antimicrobial-resistant Salmonella enterica strains frequently carry resistance genes on plasmids. Recent studies focus heavily on large conjugative plasmids, and the role that small plasmids play in resistance gene transfer is largely unknown. To expand our previous studies in assessing the prevalence of the isolates harboring ColE1-like plasmids carrying the aph gene responsible for kanamycin resistance (Kan(r)) phenotypes, 102 Kan(r) Salmonella isolates collected through the National Antimicrobial Resistance Monitoring System (NARMS) in 2005 were screened by PCR using ColE1 primer sets. Thirty isolates were found to be positive for ColE1-like replicon. Plasmids from 23 isolates were able to propagate in Escherichia coli and were subjected to further characterization. Restriction mapping revealed three major plasmid groups found in three or more isolates, with each group consisting of two to three subtypes. The aph genes from the Kan(r) Salmonella isolates were amplified by PCR, sequenced, and showed four different aph(3')-I genes. The distribution of the ColE1 plasmid groups in association with the aph gene, Salmonella serovar, and isolate source demonstrated a strong linkage of the plasmid with S. enterica serovar Typhimurium DT104. Due to their high copy number and mobility, the ColE1-like plasmids may play a critical role in transmission of antibiotic resistance genes among enteric pathogens, and these findings warrant a close monitoring of this plasmid incompatibility group.

  13. Comparative genomics of the IncA/C multidrug resistance plasmid family.

    PubMed

    Fricke, W Florian; Welch, Timothy J; McDermott, Patrick F; Mammel, Mark K; LeClerc, J Eugene; White, David G; Cebula, Thomas A; Ravel, Jacques

    2009-08-01

    Multidrug resistance (MDR) plasmids belonging to the IncA/C plasmid family are widely distributed among Salmonella and other enterobacterial isolates from agricultural sources and have, at least once, also been identified in a drug-resistant Yersinia pestis isolate (IP275) from Madagascar. Here, we present the complete plasmid sequences of the IncA/C reference plasmid pRA1 (143,963 bp), isolated in 1971 from the fish pathogen Aeromonas hydrophila, and of the cryptic IncA/C plasmid pRAx (49,763 bp), isolated from Escherichia coli transconjugant D7-3, which was obtained through pRA1 transfer in 1980. Using comparative sequence analysis of pRA1 and pRAx with recent members of the IncA/C plasmid family, we show that both plasmids provide novel insights into the evolution of the IncA/C MDR plasmid family and the minimal machinery necessary for stable IncA/C plasmid maintenance. Our results indicate that recent members of the IncA/C plasmid family evolved from a common ancestor, similar in composition to pRA1, through stepwise integration of horizontally acquired resistance gene arrays into a conserved plasmid backbone. Phylogenetic comparisons predict type IV secretion-like conjugative transfer operons encoded on the shared plasmid backbones to be closely related to a group of integrating conjugative elements, which use conjugative transfer for horizontal propagation but stably integrate into the host chromosome during vegetative growth. A hipAB toxin-antitoxin gene cluster found on pRA1, which in Escherichia coli is involved in the formation of persister cell subpopulations, suggests persistence as an early broad-spectrum antimicrobial resistance mechanism in the evolution of IncA/C resistance plasmids.

  14. Testing plasmid stability of Escherichia coli using the Continuously Operated Shaken BIOreactor System.

    PubMed

    Sieben, Michaela; Steinhorn, Gregor; Müller, Carsten; Fuchs, Simone; Ann Chin, Laura; Regestein, Lars; Büchs, Jochen

    2016-11-01

    Plasmids are common vectors to genetically manipulate Escherichia coli or other microorganisms. They are easy to use and considerable experience has accumulated on their application in heterologous protein production. However, plasmids can be lost during cell growth, if no selection pressure like, e.g., antibiotics is used, hampering the production of the desired protein and endangering the economic success of a biotechnological production process. Thus, in this study the Continuously Operated Shaken BIOreactor System (COSBIOS) is applied as a tool for fast parallel testing of strain stability and operation conditions and to evaluate measures to counter such plasmid loss. In specific, by applying various ampicillin concentrations, the lowest effective ampicillin dosage is investigated to secure plasmid stability while lowering adverse ecological effects. A significant difference was found in the growth rates of plasmid-bearing and plasmid-free cells. The undesired plasmid-free cells grew 30% faster than the desired plasmid-bearing cells. During the testing of plasmid stability without antibiotics, the population fraction of plasmid-bearing cells rapidly decreased in continuous culture to zero within the first 48 h. An initial single dosage of ampicillin did not prevent plasmid loss. By contrast, a continuous application of a low dosage of 10 µg/mL ampicillin in the feed medium maintained plasmid stability in the culture. Consequently, the COSBIOS is an apt reactor system for measuring plasmid stability and evaluating methods to enhance this stability. Hence, decreased production of heterologous protein can be prevented. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1418-1425, 2016.

  15. Plasmids of the pRM/pRF family occur in diverse Rickettsia species.

    PubMed

    Baldridge, Gerald D; Burkhardt, Nicole Y; Felsheim, Roderick F; Kurtti, Timothy J; Munderloh, Ulrike G

    2008-02-01

    The recent discoveries of the pRF and pRM plasmids of Rickettsia felis and R. monacensis have contravened the long-held dogma that plasmids are not present in the bacterial genus Rickettsia (Rickettsiales; Rickettsiaceae). We report the existence of plasmids in R. helvetica, R. peacockii, R. amblyommii, and R. massiliae isolates from ixodid ticks and in an R. hoogstraalii isolate from an argasid tick. R. peacockii and four isolates of R. amblyommii from widely separated geographic locations contained plasmids that comigrated with pRM during pulsed-field gel electrophoresis and larger plasmids with mobilities similar to that of pRF. The R. peacockii plasmids were lost during long-term serial passage in cultured cells. R. montanensis did not contain a plasmid. Southern blots showed that sequences similar to those of a DnaA-like replication initiator protein, a small heat shock protein 2, and the Sca12 cell surface antigen genes on pRM and pRF were present on all of the plasmids except for that of R. massiliae, which lacked the heat shock gene and was the smallest of the plasmids. The R. hoogstraalii plasmid was most similar to pRM and contained apparent homologs of proline/betaine transporter and SpoT stringent response genes on pRM and pRF that were absent from the other plasmids. The R. hoogstraalii, R. helvetica, and R. amblyommii plasmids contained homologs of a pRM-carried gene similar to a Nitrobacter sp. helicase RecD/TraA gene, but none of the plasmids hybridized with a probe derived from a pRM-encoded gene similar to a Burkholderia sp. transposon resolvase gene.

  16. Detection and Characterization of Conjugative Degradative Plasmids in Xenobiotic-Degrading Sphingomonas Strains

    PubMed Central

    Basta, Tamara; Keck, Andreas; Klein, Joachim; Stolz, Andreas

    2004-01-01

    A systematic survey for the presence of plasmids in 17 different xenobiotic-degrading Sphingomonas strains was performed. In almost all analyzed strains, two to five plasmids with sizes of about 50 to 500 kb were detected by using pulsed-field gel electrophoresis. A comparison of plasmid preparations untreated or treated with S1 nuclease suggested that, in general, Sphingomonas plasmids are circular. Hybridization experiments with labeled gene probes suggested that large plasmids are involved in the degradation of dibenzo-p-dioxin, dibenzofuran, and naphthalenesulfonates in S. wittichii RW1, Sphingomonas sp. HH69, and S. xenophaga BN6, respectively. The plasmids which are responsible for the degradation of naphthalene, biphenyl, and toluene by S. aromaticivorans F199 (pNL1) and of naphthalenesulfonates by S. xenophaga BN6 (pBN6) were site-specifically labeled with a kanamycin resistance cassette. The conjugative transfer of these labeled plasmids was attempted with various bacterial strains as putative recipient strains. Thus, a conjugative transfer of plasmid pBN6 from S. xenophaga BN6 to a cured mutant of strain BN6 and to Sphingomonas sp. SS3 was observed. The conjugation experiments with plasmid pNL1 suggested a broader host range of this plasmid, because it was transferred without any obvious structural changes to S. yanoikuyae B1, Sphingomonas sp. SS3, and S. herbicidovorans. In contrast, major plasmid rearrangements were observed in the transconjugants after the transfer of plasmid pNL1 to Sphingomonas sp. HH69 and of pBN6 to Sphingomonas sp. SS3. No indications for the transfer of a Sphingomonas plasmid to bacteria outside of the Sphingomonadaceae were obtained. PMID:15175300

  17. Detection and characterization of conjugative degradative plasmids in xenobiotic-degrading Sphingomonas strains.

    PubMed

    Basta, Tamara; Keck, Andreas; Klein, Joachim; Stolz, Andreas

    2004-06-01

    A systematic survey for the presence of plasmids in 17 different xenobiotic-degrading Sphingomonas strains was performed. In almost all analyzed strains, two to five plasmids with sizes of about 50 to 500 kb were detected by using pulsed-field gel electrophoresis. A comparison of plasmid preparations untreated or treated with S1 nuclease suggested that, in general, Sphingomonas plasmids are circular. Hybridization experiments with labeled gene probes suggested that large plasmids are involved in the degradation of dibenzo-p-dioxin, dibenzofuran, and naphthalenesulfonates in S. wittichii RW1, Sphingomonas sp. HH69, and S. xenophaga BN6, respectively. The plasmids which are responsible for the degradation of naphthalene, biphenyl, and toluene by S. aromaticivorans F199 (pNL1) and of naphthalenesulfonates by S. xenophaga BN6 (pBN6) were site-specifically labeled with a kanamycin resistance cassette. The conjugative transfer of these labeled plasmids was attempted with various bacterial strains as putative recipient strains. Thus, a conjugative transfer of plasmid pBN6 from S. xenophaga BN6 to a cured mutant of strain BN6 and to Sphingomonas sp. SS3 was observed. The conjugation experiments with plasmid pNL1 suggested a broader host range of this plasmid, because it was transferred without any obvious structural changes to S. yanoikuyae B1, Sphingomonas sp. SS3, and S. herbicidovorans. In contrast, major plasmid rearrangements were observed in the transconjugants after the transfer of plasmid pNL1 to Sphingomonas sp. HH69 and of pBN6 to Sphingomonas sp. SS3. No indications for the transfer of a Sphingomonas plasmid to bacteria outside of the Sphingomonadaceae were obtained.

  18. Novel Plasmids and Resistance Phenotypes in Yersinia pestis: Unique Plasmid Inventory of Strain Java 9 Mediates High Levels of Arsenic Resistance

    PubMed Central

    Eppinger, Mark; Radnedge, Lyndsay; Andersen, Gary; Vietri, Nicholas; Severson, Grant; Mou, Sherry; Ravel, Jacques; Worsham, Patricia L.

    2012-01-01

    Growing evidence suggests that the plasmid repertoire of Yersinia pestis is not restricted to the three classical virulence plasmids. The Java 9 strain of Y. pestis is a biovar Orientalis isolate obtained from a rat in Indonesia. Although it lacks the Y. pestis-specific plasmid pMT, which encodes the F1 capsule, it retains virulence in mouse and non-human primate animal models. While comparing diverse Y. pestis strains using subtractive hybridization, we identified sequences in Java 9 that were homologous to a Y. enterocolitica strain carrying the transposon Tn2502, which is known to encode arsenic resistance. Here we demonstrate that Java 9 exhibits high levels of arsenic and arsenite resistance mediated by a novel promiscuous class II transposon, named Tn2503. Arsenic resistance was self-transmissible from Java 9 to other Y. pestis strains via conjugation. Genomic analysis of the atypical plasmid inventory of Java 9 identified pCD and pPCP plasmids of atypical size and two previously uncharacterized cryptic plasmids. Unlike the Tn2502-mediated arsenic resistance encoded on the Y. enterocolitica virulence plasmid; the resistance loci in Java 9 are found on all four indigenous plasmids, including the two novel cryptic plasmids. This unique mobilome introduces more than 105 genes into the species gene pool. The majority of these are encoded by the two entirely novel self-transmissible plasmids, which show partial homology and synteny to other enterics. In contrast to the reductive evolution in Y. pestis, this study underlines the major impact of a dynamic mobilome and lateral acquisition in the genome evolution of the plague bacterium. PMID:22479347

  19. Wound-released chemical signals may elicit multiple responses from an Agrobacterium tumefaciens strain containing an octopine-type Ti plasmid.

    PubMed

    Kalogeraki, V S; Winans, S C

    1998-11-01

    The vir regions of octopine-type and nopaline-type Ti plasmids direct the transfer of oncogenic T-DNA from Agrobacterium tumefaciens to the nuclei of host plant cells. Previous studies indicate that at least two genetic loci at the left ends of these two vir regions are sufficiently conserved to form heteroduplexes visible in the electron microscope. To initiate an investigation of these genetic loci, we determined the DNA sequences of these regions of both Ti plasmids and identified both conserved loci. One of these is the 2.5-kb virH locus, which was previously identified on the octopine-type Ti plasmid but thought to be absent from the nopaline-type Ti plasmid. The virH operon contains two genes that resemble P-450-type monooxygenases. The other locus encodes a 0.5-kb gene designated virK. In addition, we identified other potential genes in this region that are not conserved between these two plasmids. To determine (i) whether these genes are members of the vir regulon and, (ii) whether they are required for tumorigenesis, we used a genetic technique to disrupt each gene and simultaneously fuse its promoter to lacZ. Expression of these genes was also measured by nuclease S1 protection assays. virK and two nonconserved genes, designated virL and virM, were strongly induced by the vir gene inducer acetosyringone. Disruptions of virH, virK, virL, or virM did not affect tumorigenesis of Kalanchöe diagramontiana leaves or carrot disks, suggesting that they may play an entirely different role during pathogenesis.

  20. Novel synthetic (S,S) and (R,R)-secoisolariciresinol diglucosides (SDGs) protect naked plasmid and genomic DNA From gamma radiation damage.

    PubMed

    Mishra, Om P; Pietrofesa, Ralph; Christofidou-Solomidou, Melpo

    2014-07-01

    Secoisolariciresinol diglucoside (SDG) is the major lignan in wholegrain flaxseed. However, extraction methods are complex and are associated with low yield and high costs. Using a novel synthetic pathway, our group succeeded in chemically synthesizing SDG (S,S and R,R enantiomers), which faithfully recapitulates the properties of their natural counterparts, possessing strong antioxidant and free radical scavenging properties. This study further extends initial findings by now investigating the DNA-radioprotective properties of the synthetic SDG enantiomers compared to the commercial SDG. DNA radioprotection was assessed by cell-free systems such as: (a) plasmid relaxation assay to determine the extent of the supercoiled (SC) converted to open-circular (OC) plasmid DNA (pBR322) after exposure of the plasmid to gamma radiation; and (b) determining the extent of genomic DNA fragmentation. Exposure of plasmid DNA to 25 Gy of γ radiation resulted in decreased supercoiled form and increased open-circular form, indicating radiation-induced DNA damage. Synthetic SDG (S,S) and SDG (R,R), and commercial SDG at concentrations of 25-250 μM significantly and equipotently reduced the radiation-induced supercoiled to open-circular plasmid DNA in a dose-dependent conversion. In addition, exposure of calf thymus DNA to 50 Gy of gamma radiation resulted in DNA fragments of low-molecular weight (<6,000 bps), which was prevented in a dose-dependence manner by all synthetic and natural SDG enantomers, at concentrations as low as 0.5 μM. These novel results demonstrated that synthetic SDG (S,S) and SDG (R,R) isomers and commercial SDG possess DNA-radioprotective properties. Such properties along with their antioxidant and free radical scavenging activity, reported earlier, suggest that SDGs are promising candidates for radioprotection for normal tissue damage as a result of accidental exposure during radiation therapy for cancer treatment.

  1. Metabolism of toluene and xylenes by Pseudomonas (putida (arvilla) mt-2: evidence for a new function of the TOL plasmid.

    PubMed Central

    Worsey, M J; Williams, P A

    1975-01-01

    Pseudomonas putida (arvilla) mt-2 carries genes for the catabolism of toluene, m-xylene, and p-xylene on a transmissible plasmid, TOL. These compounds are degraded by oxidation of one of the methyl substituents via the corresponding alcohols and aldehydes to benzoate and m- and p-toluates, respectively, which are then further metabolised by the meta pathway, also coded for by the TOL plasmid. The specificities of the benzyl alcohol dehydrogenase and the benzaldehyde dehydrogenase for their three respective substrates are independent of the carbon source used for growth, suggesting that a single set of nonspecific enzymes is responsible for the dissimilation of the breakdown products of toluene and m- and p-xylene. Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase are coincidently and possible coordinately induced by toluene and the xylenes, and by the corresponding alcohols and aldehydes. They are not induced in cells grown on m-toluate but catechol 2,3-oxygenase can be induced by m-xylene. PMID:1176436

  2. Strategies and approaches in plasmidome studies—uncovering plasmid diversity disregarding of linear elements?

    PubMed Central

    Dib, Julián R.; Wagenknecht, Martin; Farías, María E.; Meinhardt, Friedhelm

    2015-01-01

    The term plasmid was originally coined for circular, extrachromosomal genetic elements. Today, plasmids are widely recognized not only as important factors facilitating genome restructuring but also as vehicles for the dissemination of beneficial characters within bacterial communities. Plasmid diversity has been uncovered by means of culture-dependent or -independent approaches, such as endogenous or exogenous plasmid isolation as well as PCR-based detection or transposon-aided capture, respectively. High-throughput-sequencing made possible to cover total plasmid populations in a given environment, i.e., the plasmidome, and allowed to address the quality and significance of self-replicating genetic elements. Since such efforts were and still are rather restricted to circular molecules, here we put equal emphasis on the linear plasmids which—despite their frequent occurrence in a large number of bacteria—are largely neglected in prevalent plasmidome conceptions. PMID:26074886

  3. Strategies and approaches in plasmidome studies-uncovering plasmid diversity disregarding of linear elements?

    PubMed

    Dib, Julián R; Wagenknecht, Martin; Farías, María E; Meinhardt, Friedhelm

    2015-01-01

    The term plasmid was originally coined for circular, extrachromosomal genetic elements. Today, plasmids are widely recognized not only as important factors facilitating genome restructuring but also as vehicles for the dissemination of beneficial characters within bacterial communities. Plasmid diversity has been uncovered by means of culture-dependent or -independent approaches, such as endogenous or exogenous plasmid isolation as well as PCR-based detection or transposon-aided capture, respectively. High-throughput-sequencing made possible to cover total plasmid populations in a given environment, i.e., the plasmidome, and allowed to address the quality and significance of self-replicating genetic elements. Since such efforts were and still are rather restricted to circular molecules, here we put equal emphasis on the linear plasmids which-despite their frequent occurrence in a large number of bacteria-are largely neglected in prevalent plasmidome conceptions.

  4. Processing of plasmid DNA with ColE1-like replication origin.

    PubMed

    Wang, Zhijun; Yuan, Zhenghong; Hengge, Ulrich R

    2004-05-01

    With the increasing utilization of plasmid DNA as a biopharmaceutical drug, there is a rapidly growing need for high quality plasmid DNA for drug applications. Although there are several different kinds of replication origins, ColE1-like replication origin is the most extensively used origin in biotechnology. This review addresses problems in upstream and downstream processing of plasmid DNA with ColE1-like origin as drug applications. In upstream processing of plasmid DNA, regulation of replication of ColE1-like origin was discussed. In downstream processing of plasmid DNA, we analyzed simple, robust, and scalable methods, which can be used in the efficient production of pharmaceutical-grade plasmid DNA.

  5. IncA/C plasmids: An emerging threat to human and animal health?

    PubMed

    Johnson, Timothy J; Lang, Kevin S

    2012-01-01

    Incompatibility group IncA/C plasmids are large, low copy, theta-replicating plasmids that have been described in the literature for over 40 years. However, they have only recently been intensively studied on the genomic level because of their associations with the emergence of multidrug resistance in enteric pathogens of humans and animals. These plasmids are unique among other enterobacterial plasmids in many aspects, including their modular structure and gene content. While the IncA/C plasmid genome structure has now been well defined, many questions remain pertaining to their basic biological mechanisms of dissemination and regulation. Here, we discuss the history of IncA/C plasmids in light of our recent understanding of their population distribution, genomics, and effects on host bacteria.

  6. Polintons: a hotbed of eukaryotic virus, transposon and plasmid evolution.

    PubMed

    Krupovic, Mart; Koonin, Eugene V

    2015-02-01

    Polintons (also known as Mavericks) are large DNA transposons that are widespread in the genomes of eukaryotes. We have recently shown that Polintons encode virus capsid proteins, which suggests that these transposons might form virions, at least under some conditions. In this Opinion article, we delineate the evolutionary relationships among bacterial tectiviruses, Polintons, adenoviruses, virophages, large and giant DNA viruses of eukaryotes of the proposed order 'Megavirales', and linear mitochondrial and cytoplasmic plasmids. We hypothesize that Polintons were the first group of eukaryotic double-stranded DNA viruses to evolve from bacteriophages and that they gave rise to most large DNA viruses of eukaryotes and various other selfish genetic elements.

  7. Gene regulation of plasmid- and chromosome-determined inorganic ion transport in bacteria.

    PubMed Central

    Silver, S; Walderhaug, M

    1992-01-01

    Regulation of chromosomally determined nutrient cation and anion uptake systems shows important similarities to regulation of plasmid-determined toxic ion resistance systems that mediate the outward transport of deleterious ions. Chromosomally determined transport systems result in accumulation of K+, Mg2+, Fe3+, Mn2+, PO4(3-), SO4(2-), and additional trace nutrients, while bacterial plasmids harbor highly specific resistance systems for AsO2-, AsO4(3-), CrO4(2-), Cd2+, Co2+, Cu2+, Hg2+, Ni2+, SbO2-, TeO3(2-), Zn2+, and other toxic ions. To study the regulation of these systems, we need to define both the trans-acting regulatory proteins and the cis-acting target operator DNA regions for the proteins. The regulation of gene expression for K+ and PO4(3-) transport systems involves two-component sensor-effector pairs of proteins. The first protein responds to an extracellular ionic (or related) signal and then transmits the signal to an intracellular DNA-binding protein. Regulation of Fe3+ transport utilizes the single iron-binding and DNA-binding protein Fur. The MerR regulatory protein for mercury resistance both represses and activates transcription. The ArsR regulatory protein functions as a repressor for the arsenic and antimony(III) efflux system. Although the predicted cadR regulatory gene has not been identified, cadmium, lead, bismuth, zinc, and cobalt induce this system in a carefully regulated manner from a single mRNA start site. The cadA Cd2+ resistance determinant encodes an E1(1)-1E2-class efflux ATPase (consisting of two polypeptides, rather than the one earlier identified). Cadmium resistance is also conferred by the czc system (which confers resistances to zinc and cobalt in Alcaligenes species) via a complex efflux pump consisting of four polypeptides. These two cadmium efflux systems are not otherwise related. For chromate resistance, reduced cellular accumulation is again the resistance mechanism, but the regulatory components are not identified

  8. [Construction and preliminary applications of a Saccharomyces cerevisiae detection plasmid using for screening promoter elements].

    PubMed

    Wang, Zhi-Fang; Wang, Zhi-Biao; Li, Li-Na; Jian-Mei, A N; Wang-Wei; Cheng, Ke-Di; Kong, Jian-Qiang

    2013-02-01

    Synthetic biology of natural products is the design and construction of new biological systems by transferring a metabolic pathway of interest products into a chassis. Large-scale production of natural products is achieved by coordinate expression of multiple genes involved in genetic pathway of desired products. Promoters are cis-elements and play important roles in the balance of the metabolic pathways controlled by multiple genes by regulating gene expression. A detection plasmid of Saccharomyces cerevisiae was constructed based on DsRed-Monomer gene encoding for a red fluorescent protein. This plasmid was used for screening the efficient promoters applying for multiple gene-controlled pathways. First of all, eight pairs of primers specific to DsRed-Monomer gene were synthesized. The rapid cloning of DsRed-Monomer gene was performed based on step-by-step extension of a short region of the gene through a series of PCR reactions. All cloned sequences were confirmed by DNA sequencing. A vector named pEASYDs-M containing full-length DsRed-Monomer gene was constructed and was used as the template for the construction of S. cerevisiae expression vector named for pYeDP60-Ds-M. pYeDP60-Ds-M was then transformed into S. cerevisiae for heterologous expression of DsRed-Monomer gene. SDS-PAGE, Western blot and fluorescence microscopy results showed that the recombinant DsRed-Monomer protein was expressed successfully in S. cerevisiae. The well-characterized DsRed-Monomer gene was then cloned into a yeast expression vector pGBT9 to obtain a promoter detection plasmid pGBT9Red. For determination efficacy of pGBT9Red, six promoters (including four inducible promoters and two constitutive promoters) were cloned by PCR from the S. cerevisiae genome, and cloned into pGBT9Red by placing upstream of DsRed-Monomer gene, separately. The fluorescence microscopy results indicated that the six promoters (GAL1, GAL2, GAL7, GAL10, TEF2 and PGK1) can regulate the expression of Ds

  9. Abnormal ultraviolet mutagenic spectrum in plasmid DNA replicated in cultured fibroblasts from a patient with the skin cancer-prone disease, xeroderma pigmentosum

    SciTech Connect

    Seetharam, S.; Protic-Sabljic, M.; Seidman, M.M.; Kraemer, K.H.

    1987-12-01

    A shuttle vector plasmid, pZ189, was utilized to assess the types of mutations that cells from a patient with xeroderma pigmentosum, complementation group D, introduce into ultraviolet (UV) damaged, replicating DNA. Patients with xeroderma pigmentosum have clinical and cellular UV hypersensitivity, increased frequency of sun-induced skin cancer, and deficient DNA repair. In comparison to UV-treated pZ189 replicated in DNA repair-proficient cells, there were fewer surviving plasmids, a higher frequency of plasmids with mutations, fewer plasmids with two or more mutations in the marker gene, and a new mutagenic hotspot. The major type of base substitution mutation was the G:C to A:T transition with both cell lines. These results, together with similar findings published earlier with cells from a xeroderma pigmentosum patient in complementation group A, suggest that isolated G:C to A:T somatic mutations may be particularly important in generation of human skin cancer by UV radiation.

  10. Assessment of aryl hydrocarbon receptor complex interactions using pBEVY plasmids: expressionvectors with bi-directional promoters for use in Saccharomyces cerevisiae.

    PubMed

    Miller, C A; Martinat, M A; Hyman, L E

    1998-08-01

    The pBEVY (bi-directional expression vectors for yeast) plasmids were designed with constitutive and galactose-induced bi-directional promoters to direct the expression of multiple proteins in Saccharomyces cerevisiae . Using human estrogen receptor as a test gene, relatively balanced expression levels from each side of a bi-directional promoter were observed. Expression of a functional heterodimeric transcription factor composed of human aryl hydrocarbon receptor (Ahr) and aryl hydrocarbon receptor nuclear translocator (Arnt) proteins was accomplished using a single pBEVY plasmid. Previous studies suggest that inhibitory cross-talk between the estrogen receptor and the Ahr/Arnt complex may occur and that Hsp90-Ahr complex formation is important for Ahr-mediated signal transduction. Evidence for functional interaction among these proteins was investigated using pBEVY plasmids in a yeast system. No inhibitory cross-talk was observed in signaling assays performed with yeast that co-expressed Ahr, Arnt and estrogen receptor. In contrast, Ahr/Arnt-mediated signal transduction was reduced by 80% in a temperature-sensitive Hsp90 strain grown under non-permissive conditions. We conclude that pBEVY plasmids facilitate the examination of multiple protein interactions in yeast model systems.

  11. Preparation of Plasmid DNA by Alkaline Lysis with Sodium Dodecyl Sulfate: Minipreps.

    PubMed

    Green, Michael R; Sambrook, Joseph

    2016-10-03

    In this protocol, plasmid DNA is isolated from small-scale (1-2 mL) bacterial cultures. Yields vary between 100 and 5 µg of DNA, depending on the copy number of the plasmid. Miniprep DNA is sufficiently pure for use as a substrate or template in many in vitro enzymatic reactions. However, further purification is required if the plasmid DNA is used as the substrate in sequencing reactions.

  12. Linear Plasmids and the Rate of Sequence Evolution in Plant Mitochondrial Genomes

    PubMed Central

    Warren, Jessica M.; Simmons, Mark P.; Wu, Zhiqiang; Sloan, Daniel B.

    2016-01-01

    The mitochondrial genomes of flowering plants experience frequent insertions of foreign sequences, including linear plasmids that also exist in standalone forms within mitochondria, but the history and phylogenetic distribution of plasmid insertions is not well known. Taking advantage of the increased availability of plant mitochondrial genome sequences, we performed phylogenetic analyses to reconstruct the evolutionary history of these plasmids and plasmid-derived insertions. Mitochondrial genomes from multiple land plant lineages (including liverworts, lycophytes, ferns, and gymnosperms) include fragmented remnants from ancient plasmid insertions. Such insertions are much more recent and widespread in angiosperms, in which approximately 75% of sequenced mitochondrial genomes contain identifiable plasmid insertions. Although conflicts between plasmid and angiosperm phylogenies provide clear evidence of repeated horizontal transfers, we were still able to detect significant phylogenetic concordance, indicating that mitochondrial plasmids have also experienced sustained periods of (effectively) vertical transmission in angiosperms. The observed levels of sequence divergence in plasmid-derived genes suggest that nucleotide substitution rates in these plasmids, which often encode their own viral-like DNA polymerases, are orders of magnitude higher than in mitochondrial chromosomes. Based on these results, we hypothesize that the periodic incorporation of mitochondrial genes into plasmids contributes to the remarkable heterogeneity in substitution rates among genes that has recently been discovered in some angiosperm mitochondrial genomes. In support of this hypothesis, we show that the recently acquired ψtrnP-trnW gene region in a maize linear plasmid is evolving significantly faster than homologous sequences that have been retained in the mitochondrial chromosome in closely related grasses. PMID:26759362

  13. Role of Plasmids in Lactobacillus brevis BSO 464 Hop Tolerance and Beer Spoilage

    PubMed Central

    Bergsveinson, Jordyn; Baecker, Nina; Pittet, Vanessa

    2014-01-01

    Specific isolates of lactic acid bacteria (LAB) can grow in the harsh beer environment, thus posing a threat to brew quality and the economic success of breweries worldwide. Plasmid-localized genes, such as horA, horC, and hitA, have been suggested to confer hop tolerance, a trait required for LAB survival in beer. The presence and expression of these genes among LAB, however, do not universally correlate with the ability to grow in beer. Genome sequencing of the virulent beer spoilage organism Lactobacillus brevis BSO 464 revealed the presence of eight plasmids, with plasmids 1, 2, and 3 containing horA, horC, and hitA, respectively. To investigate the roles that these and the other five plasmids play in L. brevis BSO 464 growth in beer, plasmid curing with novobiocin was used to derive 10 plasmid variants. Multiplex PCRs were utilized to determine the presence or absence of each plasmid, and how plasmid loss affected hop tolerance and growth in degassed (noncarbonated) beer was assessed. Loss of three of the eight plasmids was found to affect hop tolerance and growth in beer. Loss of plasmid 2 (horC and 28 other genes) had the most dramatic effect, with loss of plasmid 4 (120 genes) and plasmid 8 (47 genes) having significant, but smaller, impacts. These results support the contention that genes on mobile genetic elements are essential for bacterial growth in beer and that beer spoilage ability is not dependent solely on the three previously described hop tolerance genes or on the chromosome of a beer spoilage LAB isolate. PMID:25501474

  14. Role of plasmids in Lactobacillus brevis BSO 464 hop tolerance and beer spoilage.

    PubMed

    Bergsveinson, Jordyn; Baecker, Nina; Pittet, Vanessa; Ziola, Barry

    2015-02-01

    Specific isolates of lactic acid bacteria (LAB) can grow in the harsh beer environment, thus posing a threat to brew quality and the economic success of breweries worldwide. Plasmid-localized genes, such as horA, horC, and hitA, have been suggested to confer hop tolerance, a trait required for LAB survival in beer. The presence and expression of these genes among LAB, however, do not universally correlate with the ability to grow in beer. Genome sequencing of the virulent beer spoilage organism Lactobacillus brevis BSO 464 revealed the presence of eight plasmids, with plasmids 1, 2, and 3 containing horA, horC, and hitA, respectively. To investigate the roles that these and the other five plasmids play in L. brevis BSO 464 growth in beer, plasmid curing with novobiocin was used to derive 10 plasmid variants. Multiplex PCRs were utilized to determine the presence or absence of each plasmid, and how plasmid loss affected hop tolerance and growth in degassed (noncarbonated) beer was assessed. Loss of three of the eight plasmids was found to affect hop tolerance and growth in beer. Loss of plasmid 2 (horC and 28 other genes) had the most dramatic effect, with loss of plasmid 4 (120 genes) and plasmid 8 (47 genes) having significant, but smaller, impacts. These results support the contention that genes on mobile genetic elements are essential for bacterial growth in beer and that beer spoilage ability is not dependent solely on the three previously described hop tolerance genes or on the chromosome of a beer spoilage LAB isolate.

  15. Broad host range plasmids can invade an unexpectedly diverse fraction of a soil bacterial community

    PubMed Central

    Klümper, Uli; Riber, Leise; Dechesne, Arnaud; Sannazzarro, Analia; Hansen, Lars H; Sørensen, Søren J; Smets, Barth F

    2015-01-01

    Conjugal plasmids can provide microbes with full complements of new genes and constitute potent vehicles for horizontal gene transfer. Conjugal plasmid transfer is deemed responsible for the rapid spread of antibiotic resistance among microbes. While broad host range plasmids are known to transfer to diverse hosts in pure culture, the extent of their ability to transfer in the complex bacterial communities present in most habitats has not been comprehensively studied. Here, we isolated and characterized transconjugants with a degree of sensitivity not previously realized to investigate the transfer range of IncP- and IncPromA-type broad host range plasmids from three proteobacterial donors to a soil bacterial community. We identified transfer to many different recipients belonging to 11 different bacterial phyla. The prevalence of transconjugants belonging to diverse Gram-positive Firmicutes and Actinobacteria suggests that inter-Gram plasmid transfer of IncP-1 and IncPromA-type plasmids is a frequent phenomenon. While the plasmid receiving fractions of the community were both plasmid- and donor- dependent, we identified a core super-permissive fraction that could take up different plasmids from diverse donor strains. This fraction, comprising 80% of the identified transconjugants, thus has the potential to dominate IncP- and IncPromA-type plasmid transfer in soil. Our results demonstrate that these broad host range plasmids have a hitherto unrecognized potential to transfer readily to very diverse bacteria and can, therefore, directly connect large proportions of the soil bacterial gene pool. This finding reinforces the evolutionary and medical significances of these plasmids. PMID:25333461

  16. Plasmid Profiles of Virulent Rhodococcus equi Strains Isolated from Infected Foals in Poland

    PubMed Central

    Kalinowski, Marcin; Grądzki, Zbigniew; Jarosz, Łukasz; Kato, Kiyoko; Hieda, Yu; Kakuda, Tsutomu; Takai, Shinji

    2016-01-01

    Rhodococcus equi is an important bacterial pathogen in foals up to 6 months old, widespread in horse farms all over the world. It was found that only virulent R. equi strains expressing 15–17 kDa virulence-associated protein (VapA) and having large virulence plasmid of 85–90 kb containing vapA gene are pathogenic for horses. To date, 12 plasmid types have been reported in VapA positive strains from horses. There are no data concerning plasmid types of Polish field R. equi strains isolated from horses and horse farm environment. The aim of the study is to determine plasmid profiles of virulent R. equi strains isolated in Poland from dead foals as well as from soil samples taken from horse breeding farms. Plasmid profiles of 10 clinical strains derived from 8 farms and 11 environmental strains from 3 farms, confirmed as virulent by PCR, were compared with 12 reference strains containing the known plasmid size and type. Plasmid DNAs were analysed by digestion with the restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII for detailed comparison and estimation of plasmid sizes. The results of RFLP analysis revealed that all except one isolates used in the study are classified as VapA 85 kb type I plasmid. One strain harboured VapA 87 kb type I plasmid. This is the first report of plasmid types of Polish field R. equi strains. The results of our preliminary investigations on horse farms located in central and eastern Poland indicate that the virulent R. equi strains thus far isolated from diseased foals and horse farms environment represent a highly uniform plasmid pattern. PMID:27074033

  17. Transfer of Plasmids to an Antibiotic-Sensitive Mutant of Zymomonas mobilis†

    PubMed Central

    Buchholz, Steven E.; Eveleigh, Douglas E.

    1986-01-01

    Wild-type strains of Zymomonas mobilis exhibit multiple antibiotic resistance and thus restrict the use of many broad-host-range plasmids in them as cloning vehicles. Antibiotic-sensitive mutants of Z. mobilis were isolated and used as hosts for the conjugal transfer of broad-host-range plasmids from Escherichia coli. Such antibiotic-sensitive strains can facilitate the application of broad-host-range plasmids to the study of Z. mobilis. Images PMID:16347136

  18. Cloning in Streptococcus lactis of plasmid-mediated UV resistance and effect on prophage stability

    SciTech Connect

    Chopin, M.C.; Chopin, A.; Rouault, A.; Simon, D.

    1986-02-01

    Plasmid pIL7 (33 kilobases) from Streptococcus lactis enhances UV resistance and prophage stability. A 5.4-kilobase pIL7 fragment carrying genes coding for both characters was cloned into S. lactis, using plasmid pHV1301 as the cloning vector. The recombinant plasmid was subsequently transferred to three other S. lactis strains by transformation or protoplast fusion. Cloned genes were expressed in all tested strains.

  19. 8-Methoxypsoralen photoinduced plasmid-chromosome recombination in Saccharomyces cerevisiae using a centromeric vector.

    PubMed Central

    Meira, L B; Henriques, J A; Magaña-Schwencke, N

    1995-01-01

    The characterization of a new system to study the induction of plasmid-chromosome recombination is described. Single-stranded and double-stranded centromeric vectors bearing 8-methoxypsoralen photoinduced lesions were used to transform a wild-type yeast strain bearing the leu2-3,112 marker. Using the SSCP methodology and DNA sequencing, it was demonstrated that repair of the lesions in plasmid DNA was mainly due to conversion of the chromosomal allele to the plasmid DNA. Images PMID:7784218

  20. Molecular cloning of complementary DNA: preparation of a plasmid vector with low transformation background.

    PubMed

    Leriche, A; Christophe, D; Brocas, H; Vassart, G

    1983-02-15

    A simple method that allows the rapid preparation of oligo dG-tailed plasmid vectors is presented. The procedure involves purification of the tailed molecules by hybridization to oligo dC-cellulose followed by a stepwise thermal elution. The resulting plasmid is virtually devoid of transformation activity in the absence of oligo dC-tailed DNA fragments. It allows construction of cDNA libraries with as low as 1% of colonies harboring wild-type plasmids.

  1. Evaluation of pKD1-based plasmid systems for heterologous protein production in Kluyveromyces lactis.

    PubMed

    Panuwatsuk, W; Da Silva, N A

    2002-02-01

    The stability of pKD1-based vectors was evaluated during the synthesis of intracellular and extracellular gene products in the yeast Kluyveromyces lactis. The Escherichia coli lacZ and MFalpha1 leader-BPTI (bovine pancreatic trypsin inhibitor) cassettes were placed under the control of the inducible K. lactis LAC4 promoter and inserted into the pKD1-based plasmids. To induce gene expression while maintaining inducer level, a gratuitous gal1-209 K. lactis strain was employed. Selective medium containing 5 g glucose/l and 0.5 g galactose (inducer)/l allowed optimum expression and secretion of heterologous products without a significant effect on the growth of the recombinant cells. During long-term sequential batch cultures (60 generations), plasmid instability was mainly the result of structural instability. The expression and secretion of BPTI resulted in greater structural instability relative to the intracellular beta-galactosidase. For both products, vectors carrying the pKD1 replication origin and the cis-acting stability locus (partial-pKD1 vectors) were more stable than vectors carrying the full pKD1 sequence (full-pKD1 vectors). However, after 55 generations, the beta-galactosidase and BPTI activities were still higher with the full-pKD1 vectors. This was due to the significantly higher initial beta-galactosidase and BPTI activities for the full-pKD1 vectors (approximately 85% and 47% higher, respectively) relative to the partial-pKDI vectors. Southern blots confirmed that these increases were due to the higher copy number of the vectors carrying the full pKD1 sequence. In contrast to our previously reported results for the secretion of invertase, full-pKD1 vectors were preferred for the expression/secretion of beta-galactosidase and BPTI for at least 55 generations. Due to their structural stability, partial-pKD1 vectors will be advantageous for very long cultivation times.

  2. Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft.

    PubMed

    Mohiuddin, Muhammad M; Singh, Avneesh K; Corcoran, Philip C; Thomas, Marvin L; Clark, Tannia; Lewis, Billeta G; Hoyt, Robert F; Eckhaus, Michael; Pierson, Richard N; Belli, Aaron J; Wolf, Eckhard; Klymiuk, Nikolai; Phelps, Carol; Reimann, Keith A; Ayares, David; Horvath, Keith A

    2016-04-05

    Preventing xenograft rejection is one of the greatest challenges of transplantation medicine. Here, we describe a reproducible, long-term survival of cardiac xenografts from alpha 1-3 galactosyltransferase gene knockout pigs, which express human complement regulatory protein CD46 and human thrombomodulin (GTKO.hCD46.hTBM), that were transplanted into baboons. Our immunomodulatory drug regimen includes induction with anti-thymocyte globulin and αCD20 antibody, followed by maintenance with mycophenolate mofetil and an intensively dosed αCD40 (2C10R4) antibody. Median (298 days) and longest (945 days) graft survival in five consecutive recipients using this regimen is significantly prolonged over our recently established survival benchmarks (180 and 500 days, respectively). Remarkably, the reduction of αCD40 antibody dose on day 100 or after 1 year resulted in recrudescence of anti-pig antibody and graft failure. In conclusion, genetic modifications (GTKO.hCD46.hTBM) combined with the treatment regimen tested here consistently prevent humoral rejection and systemic coagulation pathway dysregulation, sustaining long-term cardiac xenograft survival beyond 900 days.

  3. Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft

    PubMed Central

    Mohiuddin, Muhammad M.; Singh, Avneesh K.; Corcoran, Philip C.; Thomas III, Marvin L.; Clark, Tannia; Lewis, Billeta G.; Hoyt, Robert F.; Eckhaus, Michael; Pierson III, Richard N.; Belli, Aaron J.; Wolf, Eckhard; Klymiuk, Nikolai; Phelps, Carol; Reimann, Keith A.; Ayares, David; Horvath, Keith A.

    2016-01-01

    Preventing xenograft rejection is one of the greatest challenges of transplantation medicine. Here, we describe a reproducible, long-term survival of cardiac xenografts from alpha 1-3 galactosyltransferase gene knockout pigs, which express human complement regulatory protein CD46 and human thrombomodulin (GTKO.hCD46.hTBM), that were transplanted into baboons. Our immunomodulatory drug regimen includes induction with anti-thymocyte globulin and αCD20 antibody, followed by maintenance with mycophenolate mofetil and an intensively dosed αCD40 (2C10R4) antibody. Median (298 days) and longest (945 days) graft survival in five consecutive recipients using this regimen is significantly prolonged over our recently established survival benchmarks (180 and 500 days, respectively). Remarkably, the reduction of αCD40 antibody dose on day 100 or after 1 year resulted in recrudescence of anti-pig antibody and graft failure. In conclusion, genetic modifications (GTKO.hCD46.hTBM) combined with the treatment regimen tested here consistently prevent humoral rejection and systemic coagulation pathway dysregulation, sustaining long-term cardiac xenograft survival beyond 900 days. PMID:27045379

  4. High instability of a nematicidal Cry toxin plasmid in Bacillus thuringiensis.

    PubMed

    Sheppard, Anna E; Nakad, Rania; Saebelfeld, Manja; Masche, Anna C; Dierking, Katja; Schulenburg, Hinrich

    2016-01-01

    In bacterial pathogens, virulence factors are often carried on plasmids and other mobile genetic elements, and as such, plasmid evolution is central in understanding pathogenicity. Bacillus thuringiensis is an invertebrate pathogen that uses plasmid-encoded crystal (Cry) toxins to establish infections inside the host. Our study aimed to quantify stability of two Cry toxin-encoding plasmids, BTI_23p and BTI_16p, under standard laboratory culturing conditions. These two plasmids are part of the genome of the B. thuringiensis strain MYBT18679, which is of particular interest because of its high pathogenicity towards nematodes. One of the plasmids, BTI_23p, was found to be highly unstable, with substantial loss occurring within a single growth cycle. Nevertheless, longer term experimental evolution in the absence of a host revealed maintenance of the plasmid at low levels in the bacterial populations. BTI_23p encodes two nematicidal Cry toxins, Cry21Aa2 and Cry14Aa1. Consistent with previous findings, loss of the plasmid abolished pathogenicity towards the nematode Caenorhabditis elegans, which could be rescued by addition of Cry21Aa2-expressing Escherichia coli. These results implicate BTI_23p as a plasmid that is required for successful infection, yet unstable when present at high frequency in the population, consistent with the role of Cry toxins as public goods.

  5. Construction of pBR322-ara hybrid plasmids by in vivo recombination.

    PubMed

    Horwitz, A H; Heffernan, L; Cass, L; Miyada, C G; Wilcox, G

    1980-01-01

    In vivo recombination was used to clone deletions of the araBAD-araC genes of Escherichia coli onto a hybrid pBR322-ara plasmid. Genetic and physical analyses demonstrated that the desired deletions had been recombined onto the plasmid. In addition to permitting a detailed physical analysis of various ara deletions, this procedure has generated a series of plasmid cloning vehicles that can be used to clone, by in vivo recombination, any ara point mutation located within the region covered by the deletions. Hybrid plasmids containing the cloned point mutation can be distinguished from the original cloning vehicle by genetic complementation. The desired recombinant plasmid can be easily obtained because the frequency of recombination between the plasmid ara region and the chromosomal ara region is 0.025%--3%. A plasmid containing a deletion which removes the ara controlling site region and the araC gene was used to clone two types of araBAD promoter mutations and an araC mutation by in vivo recombination. Genetic and physical analysis of these plasmids established that the mutations in question had been recombined on to the ara deletion plasmid. The application of this procedure to the ara genes and to other genetic systems is discussed.

  6. Plasmid rolling-circle replication: highlights of two decades of research.

    PubMed

    Khan, Saleem A

    2005-03-01

    This review provides a historical perspective of the major findings that contributed to our current understanding of plasmid rolling-circle (RC) replication. Rolling-circle-replicating (RCR) plasmids were discovered approximately 20 years ago. The first of the RCR plasmids to be identified were native to Gram-positive bacteria, but later such plasmids were also identified in Gram-negative bacteria and in archaea. Further studies revealed mechanistic similarities in the replication of RCR plasmids and the single-stranded DNA bacteriophages of Escherichia coli, although there were important differences as well. Three important elements, a gene encoding the initiator protein, the double strand origin, and the single strand origin, are contained in all RCR plasmids. The initiator proteins typically contain a domain involved in their sequence-specific binding to the double strand origin and a domain that nicks within the double strand origin and generates the primer for DNA replication. The double strand origins include the start-site of leading strand synthesis and contain sequences that are bound and nicked by the initiator proteins. The single strand origins are required for synthesis of the lagging strand of RCR plasmids. The single strand origins are non-coding regions that are strand-specific, and contain extensive secondary structures. This minireview will highlight the major findings in the study of plasmid RC replication over the past twenty years. Regulation of replication of RCR plasmids will not be included since it is the subject of another review.

  7. Postsymbiotic plasmid acquisition and evolution of the repA1-replicon in Buchnera aphidicola

    PubMed Central

    Van Ham, Roeland C. H. J.; González-Candelas, Fernando; Silva, Francisco J.; Sabater, Beatriz; Moya, Andrés; Latorre, Amparo

    2000-01-01

    Buchnera aphidicola is an obligate, strictly vertically transmitted, bacterial symbiont of aphids. It supplies its host with essential amino acids, nutrients required by aphids but deficient in their diet of plant phloem sap. Several lineages of Buchnera show adaptation to their nutritional role in the form of plasmid-mediated amplification of key-genes involved in the biosynthesis of tryptophan (trpEG) and leucine (leuABCD). Phylogenetic analyses of these plasmid-encoded functions have thus far suggested the absence of horizontal plasmid exchange among lineages of Buchnera. Here, we describe three new Buchnera plasmids, obtained from species of the aphid host families Lachnidae and Pemphigidae. All three plasmids belong to the repA1 family of Buchnera plasmids, which is characterized by the presence of a repA1-replicon responsible for replication initiation. A comprehensive analysis of this family of plasmids unexpectedly revealed significantly incongruent phylogenies for different plasmid and chromosomally encoded loci. We infer from these incongruencies a case of horizontal plasmid transfer in Buchnera. This process may have been mediated by secondary endosymbionts, which occasionally undergo horizontal transmission in aphids. PMID:10984505

  8. Genomics of microbial plasmids: classification and identification based on replication and transfer systems and host taxonomy

    PubMed Central

    Shintani, Masaki; Sanchez, Zoe K.; Kimbara, Kazuhide

    2015-01-01

    Plasmids are important “vehicles” for the communication of genetic information between bacteria. The exchange of plasmids transmits pathogenically and environmentally relevant traits to the host bacteria, promoting their rapid evolution and adaptation to various environments. Over the past six decades, a large number of plasmids have been identified and isolated from different microbes. With the revolution of sequencing technology, more than 4600 complete sequences of plasmids found in bacteria, archaea, and eukaryotes have been determined. The classification of a wide variety of plasmids is not only important to understand their features, host ranges, and microbial evolution but is also necessary to effectively use them as genetic tools for microbial engineering. This review summarizes the current situation of the classification of fully sequenced plasmids based on their host taxonomy and their features of replication and conjugative transfer. The majority of the fully sequenced plasmids are found in bacteria in the Proteobacteria, Firmicutes, Spirochaetes, Actinobacteria, Cyanobacteria and Euryarcheota phyla, and key features of each phylum are included. Recent advances in the identification of novel types of plasmids and plasmid transfer by culture-independent methods using samples from natural environments are also discussed. PMID:25873913

  9. Enhancing yields of low and single copy number plasmid DNAs from Escherichia coli cells.

    PubMed

    Wood, Whitney N; Smith, Kyle D; Ream, Jennifer A; Kevin Lewis, L

    2017-02-01

    Many plasmids used for gene cloning and heterologous protein expression in Escherichia coli cells are low copy number or single copy number plasmids. The extraction of these types of plasmids from small bacterial cell cultures produces low DNA yields. In this study, we have quantitated yields of low copy and single copy number plasmid DNAs after growth of cells in four widely used broths (SB, SOC, TB, and 2xYT) and compared results to those obtained with LB, the most common E. coli cell growth medium. TB (terrific broth) consistently generated the greatest amount of plasmid DNA, in agreement with its ability to produce higher cell titers. The superiority of TB was primarily due to its high levels of yeast extract (24g/L) and was independent of glycerol, a unique component of this broth. Interestingly, simply preparing LB with similarly high levels of yeast extract (LB24 broth) resulted in plasmid yields that were equivalent to those of TB. By contrast, increasing ampicillin concentration to enhance plasmid retention did not improve plasmid DNA recovery. These experiments demonstrate that yields of low and single copy number plasmid DNAs from minipreps can be strongly enhanced using simple and inexpensive media.

  10. Structural plasmid evolution as a result of coupled recombinations at bom and cer sites.

    PubMed

    Zakharova, M V; Beletskaya, I V; Bolovin, D V; Yurkova, T V; Semenova, L M; Solonin, A S

    2003-12-01

    We have studied the recombination of plasmids bearing bom and cer sites. The bom ( basis of mobilization) site is required for conjugative transfer, while the cer ( Col E1 resolution) site is involved in the resolution of plasmid multimers, which increases plasmid stability. We constructed a pair of parent plasmids in such a way as to allow us select clones containing recombinant plasmids directly. Clone selection was based on the McrA sensitivity of recipient host DNA modified by M. Ecl18kI, which is encoded by one of the parent plasmids. The recombinant plasmid contains segments originating from both parental DNAs, which are bounded by bom and cer sites. Its structure is in accordance with our previously proposed model for recombination mediated by bom and cer sequences. The frequency of recombinant plasmid formation coincided with the frequency of recombination at the bom site. We also show that bom-mediated recombination in trans, unlike in cis, is independent of other genetic determinants on the conjugative plasmids.

  11. Pheromone-responsive conjugative vancomycin resistance plasmids in Enterococcus faecalis isolates from humans and chicken feces.

    PubMed

    Lim, Suk-Kyung; Tanimoto, Koichi; Tomita, Haruyoshi; Ike, Yasuyoshi

    2006-10-01

    The drug resistances and plasmid contents of a total of 85 vancomycin-resistant enterococcus (VRE) strains that had been isolated in Korea were examined. Fifty-four of the strains originated from samples of chicken feces, and 31 were isolated from hospital patients in Korea. Enterococcus faecalis KV1 and KV2, which had been isolated from a patient and a sample of chicken feces, respectively, were found to carry the plasmids pSL1 and pSL2, respectively. The plasmids transferred resistances to vancomycin, gentamicin, kanamycin, streptomycin, and erythromycin to E. faecalis strains at a high frequency of about 10(-3) per donor cell during 4 hours of broth mating. E. faecalis strains containing each of the pSL plasmids formed clumps after 2 hours of incubation in broth containing E. faecalis FA2-2 culture filtrate (i.e., the E. faecalis sex pheromone), and the plasmid subsequently transferred to the recipient strain in a 10-min short mating in broth, indicating that the plasmids are responsive to E. faecalis pheromones. The pSL plasmids did not respond to any of synthetic pheromones for the previously characterized plasmids. The pheromone specific for pSL plasmids has been designated cSL1. Southern hybridization analysis showed that specific FspI fragments from each of the pSL plasmids hybridized with the aggregation substance gene (asa1) of the pheromone-responsive plasmid pAD1, indicating that the plasmids had a gene homologous to asa1. The restriction maps of the plasmids were identical, and the size of the plasmids was estimated to be 128.1 kb. The plasmids carried five drug resistance determinants for vanA, ermB, aph(3'), aph(6'), and aac(6')/aph(2'), which encode resistance to vancomycin, erythromycin, kanamycin, streptomycin, and gentamicin/kanamycin, respectively. Nucleotide sequence analyses of the drug resistance determinants and their flanking regions are described in this report. The results described provide evidence for the exchange of genetic information

  12. Single molecule sequencing to track plasmid diversity of hospital-associated carbapenemase-producing Enterobacteriaceae

    PubMed Central

    Conlan, Sean; Thomas, Pamela J.; Deming, Clayton; Park, Morgan; Lau, Anna F.; Dekker, John P.; Snitkin, Evan S.; Clark, Tyson A.; Luong, Khai; Song, Yi; Tsai, Yu-Chih; Boitano, Matthew; Gupta, Jyoti; Brooks, Shelise Y.; Schmidt, Brian; Young, Alice C.; Thomas, James W.; Bouffard, Gerard G.; Blakesley, Robert W.; Mullikin, James C.; Korlach, Jonas; Henderson, David K.; Frank, Karen M.; Palmore, Tara N.; Segre, Julia A.

    2014-01-01

    Public health officials have raised concerns that plasmid transfer between Enterobacteriaceae species may spread resistance to carbapenems, an antibiotic class of last resort, thereby rendering common healthcare-associated infections nearly impossible to treat. We performed comprehensive surveillance and genomic sequencing to identify carbapenem-resistant Enterobacteriaceae in the NIH Clinical Center patient population and hospital environment in order to to articulate the diversity of carbapenemase-encoding plasmids and survey the mobility of and assess the mobility of these plasmids between bacterial species. We isolated a repertoire of carbapenemase-encoding Enterobacteriaceae, including multiple strains of Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, Enterobacter cloacae, Citrobacter freundii, and Pantoea species. Long-read genome sequencing with full end-to-end assembly revealed that these organisms carry the carbapenem-resistance genes on a wide array of plasmids. Klebsiella pneumoniae and Enterobacter cloacae isolated simultaneously from a single patient harbored two different carbapenemase-encoding plasmids, overriding the epidemiological scenario of plasmid transfer between organisms within this patient. We did, however, find evidence supporting horizontal transfer of carbapenemase-encoding plasmids between Klebsiella pneumoniae, Enterobacter cloacae and Citrobacter freundii in the hospital environment. Our comprehensive sequence data, with full plasmid identification, challenges assumptions about horizontal gene transfer events within patients and identified wider possible connections between patients and the hospital environment. In addition, we identified a new carbapenemase-encoding plasmid of potentially high clinical impact carried by Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae and Pantoea species, from unrelated patients and the hospital environment. PMID:25232178

  13. Hofmeister series salts enhance purification of plasmid DNA by non-ionic detergents.

    PubMed

    Lezin, George; Kuehn, Michael R; Brunelli, Luca

    2011-08-01

    Ion-exchange chromatography is the standard technique used for plasmid DNA purification, an essential molecular biology procedure. Non-ionic detergents (NIDs) have been used for plasmid DNA purification, but it is unclear whether Hofmeister series salts (HSS) change the solubility and phase separation properties of specific NIDs, enhancing plasmid DNA purification. After scaling-up NID-mediated plasmid DNA isolation, we established that NIDs in HSS solutions minimize plasmid DNA contamination with protein. In addition, large-scale NID/HSS solutions eliminated lipopolysaccharides (LPS) contamination of plasmid DNA more effectively than Qiagen ion-exchange columns. Large-scale NID isolation/NID purification generated increased yields of high-quality DNA compared to alkali isolation/column purification. This work characterizes how HSS enhance NID-mediated plasmid DNA purification, and demonstrates that NID phase transition is not necessary for LPS removal from plasmid DNA. Specific NIDs such as IGEPAL CA-520 can be utilized for rapid, inexpensive, and efficient laboratory-based large-scale plasmid DNA purification, outperforming Qiagen-based column procedures.

  14. Influence of Plasmid Type on the Replication of Rhodococcus equi in Host Macrophages

    PubMed Central

    Willingham-Lane, Jennifer M.; Berghaus, Londa J.; Giguère, Steeve

    2016-01-01

    ABSTRACT The soil-dwelling, saprophytic actinomycete Rhodococcus equi is a multihost, facultative intracellular pathogen of macrophages. When inhaled by susceptible foals, it causes severe bronchopneumonia. It is also a pathogen of pigs, which may develop submaxillary lymphadenitis upon exposure. R. equi isolates obtained from foals and pigs possess conjugative plasmids housing a pathogenicity island (PAI) containing a novel family of genes of unknown function called the virulence-associated protein or vap family. The PAI regions of the equine and swine plasmids differ in vap gene composition, with equine isolates possessing six vap genes, including the major virulence determinant vapA, while the PAIs of swine isolates house vapB and five other unique vap genes. Possession of the pVAPA-type virulence plasmid by equine isolates bestows the capacity for intramacrophage replication essential for disease development in vivo. Swine isolates of R. equi are largely unstudied. Here, we show that R. equi isolates from pigs, carrying pVAPB-type plasmids, are able to replicate in a plasmid-dependent manner in macrophages obtained from a variety of species (murine, swine, and equine) and anatomical locations. Similarly, equine isolates carrying pVAPA-type plasmids are capable of replication in swine macrophages. Plasmid swapping between equine and swine strains through conjugation did not alter the intracellular replication capacity of the parental strain, indicating that coevolution of the plasmid and chromosome is not crucial for this attribute. These results demonstrate that while distinct plasmid types exist among R. equi isolates obtained from equine and swine sources, this tropism is not determined by host species-specific intramacrophage replication capabilities. IMPORTANCE This work greatly advances our understanding of the opportunistic pathogen Rhodococcus equi, a disease agent of animals and immunocompromised people. Clinical isolates from diseased foals carry a

  15. Plasmid-Borne Antimicrobial Resistance of Staphylococcus aureus Isolated in a Hospital in Lisbon, Portugal.

    PubMed

    Costa, Sofia Santos; Palma, Cláudia; Kadlec, Kristina; Fessler, Andrea T; Viveiros, Miguel; Melo-Cristino, José; Schwarz, Stefan; Couto, Isabel

    2016-12-01

    Plasmids play a key role in the genetic plasticity and survival of Staphylococcus aureus in challenging environments. Although many S. aureus plasmids have been described, still few studies portray the plasmid content of a given S. aureus population. The aim of this work was to characterize the plasmids carried by a collection of 53 S. aureus isolates collected in a large hospital in Lisbon, Portugal, and investigate their role in conferring resistance to several antimicrobial agents. Plasmids were present in 44 out of the 53 isolates and were grouped into eleven AccI restriction profiles. Plasmid curing of representative strains and comparison of antimicrobial susceptibility profiles between pairs of isogenic strains proved to be a valuable guidance tool in the identification of plasmid-located resistance genes. The plasmids harbored several resistance genes, namely blaZ (resistance to β-lactams), erm(C) (resistance to macrolides, lincosamides, and streptogramin B), cadA (resistance to cadmium and zinc), cadD (resistance to cadmium), and qacA and smr (resistance to biocides and dyes). This study demonstrates the impact of plasmids on the resistance properties of S. aureus, highlighting their role in the dissemination of antibiotic, heavy metal, and biocide resistance genes, and survival of this major pathogen in the hospital environment.

  16. Characteristics and restriction analysis of the 4-chlorobiphenyl catabolic plasmid, pSS50.

    PubMed Central

    Hooper, S W; Dockendorff, T C; Sayler, G S

    1989-01-01

    The plasmid pSS50 is a 53-kilobase self-transmissible plasmid of broad host range that has been isolated from several Alcaligenes and Acinetobacter species. This plasmid has previously been shown to mediate the mineralization of 4-chlorobiphenyl to carbon dioxide and water. Physical characterization of this plasmid by restriction analysis indicates that most hexanucleotide cleavage sites are clustered in a 5-kilobase region, leaving large regions without restriction sites. The paucity of restriction sites is not due to DNA methylation. PMID:2757383

  17. Epidemiology of virulence-associated plasmids and outer membrane protein patterns within seven common Salmonella serotypes.

    PubMed

    Helmuth, R; Stephan, R; Bunge, C; Hoog, B; Steinbeck, A; Bulling, E

    1985-04-01

    Antibiotic-sensitive Salmonella isolates belonging to seven common serotypes and originating from 29 different countries from all continents were investigated for their plasmid DNA content (337 isolates) and their outer membrane protein profiles (216 isolates). Of the S. typhimurium, S. enteritidis, S. dublin, and S. choleraesuis isolates, 90% or more carried a serotype-specific plasmid. The molecular sizes of the plasmids were 60 megadaltons (Md) for S. typhimurium, 37 Md for S. enteritidis, 56 Md for S. dublin, and 30 Md for S. choleraesuis. The outer membrane protein profiles were homogeneous within each of the seven serotypes, except that a minority of S. enteritidis and S. dublin strains were lacking one major outer membrane protein. Virulence studies were performed with 39 representative strains by measuring the 50% lethal doses (LD50S) after oral infection of mice. The LD50 values obtained for plasmid-positive strains of S. typhimurium, S. enteritidis, and S. dublin were up to 10(6)-fold lower than the values obtained for the plasmid-free strains of the same serotype. Only the plasmid-positive strains could invade the livers of orally infected mice, and only they were resistant to the bactericidal activity of 90% guinea pig serum. Strains of S. infantis were generally plasmid free, whereas S. panama and S. heidelberg isolates carried heterogeneous plasmid populations. The virulence properties of the latter three serotypes could not be correlated with the predominant plasmids found in these strains.

  18. Hofmeister series salts enhance purification of plasmid DNA by non-ionic detergents

    PubMed Central

    Lezin, George; Kuehn, Michael R.; Brunelli, Luca

    2011-01-01

    Ion-exchange chromatography is the standard technique used for plasmid DNA purification, an essential molecular biology procedure. Non-ionic detergents (NIDs) have been used for plasmid DNA purification, but it is unclear whether Hofmeister series salts (HSS) change the solubility and phase separation properties of specific NIDs, enhancing plasmid DNA purification. After scaling-up NID-mediated plasmid DNA isolation, we established that NIDs in HSS solutions minimize plasmid DNA contamination with protein. In addition, large-scale NID/HSS solutions eliminated LPS contamination of plasmid DNA more effectively than Qiagen ion-exchange columns. Large-scale NID isolation/NID purification generated increased yields of high quality DNA compared to alkali isolation/column purification. This work characterizes how HSS enhance NID-mediated plasmid DNA purification, and demonstrates that NID phase transition is not necessary for LPS removal from plasmid DNA. Specific NIDs such as IGEPAL CA-520 can be utilized for rapid, inexpensive and efficient laboratory-based large-scale plasmid DNA purification, outperforming Qiagen-based column procedures. PMID:21351074

  19. The 2-micron plasmid as a nonselectable, stable, high copy number yeast vector

    NASA Technical Reports Server (NTRS)

    Ludwig, D. L.; Bruschi, C. V.

    1991-01-01

    The endogenous 2-microns plasmid of Saccharomyces cerevisiae has been used extensively for the construction of yeast cloning and expression plasmids because it is a native yeast plasmid that is able to be maintained stably in cells at high copy number. Almost invariably, these plasmid constructs, containing some or all 2-microns sequences, exhibit copy number levels lower than 2-microns and are maintained stably only under selective conditions. We were interested in determining if there was a means by which 2-microns could be utilized for vector construction, without forfeiting either copy number or nonselective stability. We identified sites in the 2-microns plasmid that could be used for the insertion of genetic sequences without disrupting 2-microns coding elements and then assessed subsequent plasmid constructs for stability and copy number in vivo. We demonstrate the utility of a previously described 2-microns recombination chimera, pBH-2L, for the manipulation and transformation of 2-microns as a pure yeast plasmid vector. We show that the HpaI site near the STB element in the 2-microns plasmid can be utilized to clone yeast DNA of at least 3.9 kb with no loss of plasmid stability. Additionally, the copy number of these constructs is as high as levels reported for the endogenous 2-microns.

  20. Rapid plasmid library screening using RecA-coated biotinylated probes.

    PubMed

    Rigas, B; Welcher, A A; Ward, D C; Weissman, S M

    1986-12-01

    A method for the rapid physical isolation of recombinant plasmids of interest from a mixture of plasmids such as a plasmid cDNA library is presented. This method utilizes the ability of RecA protein to form stable complexes between linear single-stranded and circular double-stranded DNA molecules sharing sequence homology, and procedures allowing isolation of biotinylated nucleic acid. Biotinylated linear DNA probes coated with RecA have been used to screen reconstituted plasmid libraries consisting of two plasmid species, one homologous and the other heterologous to the probe. When the link between biotin and the nucleotide base could be cleaved by reducing agents, the complex was purified by streptavidin-agarose chromatography and the recovered plasmid was propagated in Escherichia coli. When the link was not cleavable the complex was bound to avidin in solution and purified by cupric iminodiacetic acid-agarose chromatography. The complex was then dissociated and the plasmids were propagated in E. coli. With either protocol, homologous plasmid recovery was between 10% and 20%, and enrichment was between 10(4)- and 10(5)-fold. Potential applications and extensions of this method, such as plasmid, cosmid, and phage library screening and facilitation of physical mapping of macroregions of mammalian genomes are presented and discussed.

  1. Occurrence of Plasmids in the Aromatic Degrading Bacterioplankton of the Baltic Sea

    PubMed Central

    Jutkina, Jekaterina; Heinaru, Eeva; Vedler, Eve; Juhanson, Jaanis; Heinaru, Ain

    2011-01-01

    Plasmids are mobile genetic elements that provide their hosts with many beneficial traits including in some cases the ability to degrade different aromatic compounds. To fulfill the knowledge gap regarding catabolic plasmids of the Baltic Sea water, a total of 209 biodegrading bacterial strains were isolated and screened for the presence of these mobile genetic elements. We found that both large and small plasmids are common in the cultivable Baltic Sea bacterioplankton and are particularly prevalent among bacterial genera Pseudomonas and Acinetobacter. Out of 61 plasmid-containing strains (29% of all isolates), 34 strains were found to carry large plasmids, which could be associated with the biodegradative capabilities of the host bacterial strains. Focusing on the diversity of IncP-9 plasmids, self-transmissible m-toluate (TOL) and salicylate (SAL) plasmids were detected. Sequencing the repA gene of IncP-9 carrying isolates revealed a high diversity within IncP-9 plasmid family, as well as extended the assumed bacterial host species range of the IncP-9 representatives. This study is the first insight into the genetic pool of the IncP-9 catabolic plasmids in the Baltic Sea bacterioplankton. PMID:24710296

  2. Reduction in horizontal transfer of conjugative plasmid by UV irradiation and low-level chlorination.

    PubMed

    Lin, Wenfang; Li, Shuai; Zhang, Shuting; Yu, Xin

    2016-03-15

    The widespread presence of antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB) in the drinking water system facilitates their horizontal gene transfer among microbiota. In this study, the conjugative gene transfer of RP4 plasmid after disinfection including ultraviolet (UV) irradiation and low-level chlorine treatment was investigated. It was found that both UV irradiation and low-level chlorine treatment reduced the conjugative gene transfer frequency. The transfer frequency gradually decreased from 2.75 × 10(-3) to 2.44 × 10(-5) after exposure to UV doses ranging from 5 to 20 mJ/cm(2). With higher UV dose of 50 and 100 mJ/cm(2), the transfer frequency was reduced to 1.77 × 10(-6) and 2.44 × 10(-8). The RP4 plasmid transfer frequency was not significantly affected by chlorine treatment at dosages ranging from 0.05 to 0.2 mg/l, but treatment with 0.3-0.5 mg/l chlorine induced a decrease in conjugative transfer to 4.40 × 10(-5) or below the detection limit. The mechanisms underlying these phenomena were also explored, and the results demonstrated that UV irradiation and chlorine treatment (0.3 and 0.5 mg/l) significantly reduced the viability of bacteria, thereby lowering the conjugative transfer frequency. Although the lower chlorine concentrations tested (0.05-0.2 mg/l) were not sufficient to damage the cells, exposure to these concentrations may still depress the expression of a flagellar gene (FlgC), an outer membrane porin gene (ompF), and a DNA transport-related gene (TraG). Additionally, fewer pili were scattered on the bacteria after chlorine treatment. These findings are important in assessing and controlling the risk of ARG transfer and dissemination in the drinking water system.

  3. Single-cell Transfection by Electroporation Using an Electrolyte/Plasmid-Filled Capillary

    PubMed Central

    Wang, Manyan; Orwar, Owe; Weber, Stephen G.

    2009-01-01

    Single-cell transfection of adherent cells has been accomplished using single-cell electroporation (SCEP) with a pulled capillary. HEPES-buffered physiological saline solution containing pEGFP plasmid at a low concentration (0.16 ∼ 0.78 µg/µL) filled a 15 cm long capillary with a tip opening of 2 µm. The electric field is applied to individual cells by bringing the tip close to the cell and subsequently applying one or two brief electric pulses. Many individual cells can thus be transfected with a small volume of plasmid-containing solution (∼ 1 µL). The extent of electroporation is determined by measuring the percentage loss of freely diffusing thiols (chiefly reduced glutathione) that have been derivatized with the fluorogenic ThioGlo 1. A mass transport model is used to fit the time-dependent fluorescence intensity decay in the target cells. The fits, which are excellent, yield the electroporation-induced fluorescence loss at steady state and the mass transfer rate through the electroporated cell membrane. Steady-state fluorescence loss ranged approximately from 0 to about 80% (based on the fluorescence intensity before electroporation). For the cells having a loss of thiol-ThioGlo 1 fluorescence intensity greater than 10%, and mass transfer rate greater than 0.03 s−1, EGFP fluorescence is observed after 24 hours. The EGFP fluorescence is increased at 48 hours. With a loss smaller than 10% and a mass transfer rate smaller than 0.03 s−1, no EGFP fluorescence is detected. Thus, transfection success is closely related to the small molecule mass transport dynamics as indicated by the loss of fluorescence from thiol-ThioGlo 1 conjugates. The EGFP expression is weaker than bulk lipid-mediated transfection, as indicated by the EGFP fluorescence intensities. However, the success with the single-cell approach is considerably greater than lipid-mediated transfection. PMID:19351139

  4. Process considerations related to the microencapsulation of plasmid DNA via ultrasonic atomization.

    PubMed

    Ho, Jenny; Wang, Huanting; Forde, Gareth M

    2008-09-01

    An effective means of facilitating DNA vaccine delivery to antigen presenting cells is through biodegradable microspheres. Microspheres offer distinct advantages over other delivery technologies by providing release of DNA vaccine in its bioactive form in a controlled fashion. In this study, biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres containing polyethylenimine (PEI) condensed plasmid DNA (pDNA) were prepared using a 40 kHz ultrasonic atomization system. Process synthesis parameters, which are important to the scale-up of microspheres that are suitable for nasal delivery (i.e., less than 20 microm), were studied. These parameters include polymer concentration; feed flowrate; volumetric ratio of polymer and pDNA-PEI (plasmid DNA-polyethylenimine) complexes; and nitrogen to phosphorous (N/P) ratio. PDNA encapsulation efficiencies were predominantly in the range 82-96%, and the mean sizes of the particle were between 6 and 15 microm. The ultrasonic synthesis method was shown to have excellent reproducibility. PEI affected morphology of the microspheres, as it induced the formation of porous particles that accelerate the release rate of pDNA. The PLGA microspheres displayed an in vitro release of pDNA of 95-99% within 30 days and demonstrated zero order release kinetics without an initial spike of pDNA. Agarose electrophoresis confirmed conservation of the supercoiled form of pDNA throughout the synthesis and in vitro release stages. It was concluded that ultrasonic atomization is an efficient technique to overcome the key obstacles in scaling-up the manufacture of encapsulated vaccine for clinical trials and ultimately, commercial applications.

  5. Delivery of plasmid DNA to vascular tissue in vivo using catheter balloons coated with polyelectrolyte multilayers.

    PubMed

    Saurer, Eric M; Yamanouchi, Dai; Liu, Bo; Lynn, David M

    2011-01-01

    We report an approach for the localized delivery of plasmid DNA to vascular tissue from the surfaces of inflatable embolectomy catheter balloons. Using a layer-by-layer approach, ultrathin multilayered polyelectrolyte films were fabricated on embolectomy catheter balloons by alternately adsorbing layers of a hydrolytically degradable poly(β-amino ester) and plasmid DNA. Fluorescence microscopy revealed that the films coated the surfaces of the balloons uniformly. Coated balloons that were incubated in phosphate-buffered saline at 37 °C released ∼25 μg DNA/cm(2) over 24 h. Analysis of the DNA by gel electrophoresis showed that the DNA was released in open-circular ('nicked') and supercoiled conformations, and in vitro cell transfection assays confirmed that the released DNA was transcriptionally active. Arterial injury was induced in the left common, carotid arteries of Sprague-Dawley rats using uncoated balloons, followed by treatment with film-coated balloons for 20 min. X-gal, immunohistochemical, and immunofluorescence staining of sectioned arteries indicated high levels of β-galactosidase or enhanced green fluorescent protein (EGFP) expression in arteries treated with film-coated balloons. β-galactosidase and EGFP expression were observed throughout the medial layers of arterial tissue, and around approximately two-thirds of the circumference of the treated arteries. The layer-by-layer approach reported here provides a general platform for the balloon-mediated delivery of DNA to vascular tissue. Our results suggest the potential of this approach to deliver therapeutically relevant DNA to prevent complications such as intimal hyperplasia that arise after vascular interventions.

  6. Transposon mutagenesis identified chromosomal and plasmid genes essential for adaptation of the marine bacterium Dinoroseobacter shibae to anaerobic conditions.

    PubMed

    Ebert, Matthias; Laaß, Sebastian; Burghartz, Melanie; Petersen, Jörn; Koßmehl, Sebastian; Wöhlbrand, Lars; Rabus, Ralf; Wittmann, Christoph; Tielen, Petra; Jahn, Dieter

    2013-10-01

    Anaerobic growth and survival are integral parts of the life cycle of many marine bacteria. To identify genes essential for the anoxic life of Dinoroseobacter shibae, a transposon library was screened for strains impaired in anaerobic denitrifying growth. Transposon insertions in 35 chromosomal and 18 plasmid genes were detected. The essential contribution of plasmid genes to anaerobic growth was confirmed with plasmid-cured D. shibae strains. A combined transcriptome and proteome approach identified oxygen tension-regulated genes. Transposon insertion sites of a total of 1,527 mutants without an anaerobic growth phenotype were determined to identify anaerobically induced but not essential genes. A surprisingly small overlap of only three genes (napA, phaA, and the Na(+)/Pi antiporter gene Dshi_0543) between anaerobically essential and induced genes was found. Interestingly, transposon mutations in genes involved in dissimilatory and assimilatory nitrate reduction (napA, nasA) and corresponding cofactor biosynthesis (genomic moaB, moeB, and dsbC and plasmid-carried dsbD and ccmH) were found to cause anaerobic growth defects. In contrast, mutation of anaerobically induced genes encoding proteins required for the later denitrification steps (nirS, nirJ, nosD), dimethyl sulfoxide reduction (dmsA1), and fermentation (pdhB1, arcA, aceE, pta, acs) did not result in decreased anaerobic growth under the conditions tested. Additional essential components (ferredoxin, cccA) of the anaerobic electron transfer chain and central metabolism (pdhB) were identified. Another surprise was the importance of sodium gradient-dependent membrane processes and genomic rearrangements via viruses, transposons, and insertion sequence elements for anaerobic growth. These processes and the observed contributions of cell envelope restructuring (lysM, mipA, fadK), C4-dicarboxylate transport (dctM1, dctM3), and protease functions to anaerobic growth require further investigation to unravel the

  7. A new plasmid vector for DNA delivery using lactococci

    PubMed Central

    Guimarães, Valeria; Innocentin, Sylvia; Chatel, Jean-Marc; Lefèvre, François; Langella, Philippe; Azevedo, Vasco; Miyoshi, Anderson

    2009-01-01

    Background The use of food-grade lactococci as bacterial carriers to DNA delivery into epithelial cells is a new strategy to develop live oral DNA vaccine. Our goal was to develop a new plasmid, named pValac, for antigen delivery for use in lactococci. The pValac plasmid was constructed by the fusion of: i) a eukaryotic region, allowing the cloning of an antigen of interest under the control of the pCMV eukaryotic promoter to be expressed by a host cell and ii) a prokaryotic region allowing replication and selection of bacteria. In order to evaluate pValac functionality, the gfp ORF was cloned into pValac (pValac:gfp) and was analysed by transfection in PK15 cells. The applicability of pValac was demonstrated by invasiveness assays of Lactococcus lactis inlA+ strains harbouring pValac:gfp into Caco-2 cells. Results After transfection with pValac:gfp, we observed GFP expression in PK15 cells. L. lactis inlA+ were able to invade Caco-2 cells and delivered a functional expression cassette (pCMV:gfp) into epithelial cells. Conclusion We showed the potential of an invasive L. lactis harbouring pValac to DNA delivery and subsequent triggering DNA expression by epithelial cells. Further work will be to examine whether these strains are able to deliver DNA in intestinal cells in vivo. PMID:19208231

  8. Transformation of vegetative cells of Bacillus thuringiensis by plasmid DNA.

    PubMed

    Heierson, A; Landén, R; Lövgren, A; Dalhammar, G; Boman, H G

    1987-03-01

    Plasmid DNA-mediated transformation of vegetative cells of Bacillus thuringiensis was studied with the following two plasmids: pBC16 coding for tetracycline resistance and pC194 expressing chloramphenicol resistance. A key step was the induction of competence by treatment of the bacteria with 50 mM Tris hydrochloride buffer (pH 8.9) containing 30% sucrose. Transformation frequency was strongly influenced by culture density during the uptake of DNA and required the presence of polyethylene glycol. Growth in a minimal medium supplemented with Casamino Acids gave 35 times more transformants than growth in a rich medium. The highest frequencies were obtained with covalently closed circular DNA. With all parameters optimized, the frequency was 10(-3) transformants per viable cell or 10(4) transformants per microgram of DNA. Cells previously frozen were also used as recipients in transformation experiments; such cells gave frequencies similar to those obtained with freshly grown cells. The procedure was optimized for B. thuringiensis subsp. gelechiae, but B. thuringiensis subsp. kurstaki, B. thuringiensis subsp. galleriae, B. thuringiensis subsp. thuringiensis, and B. thuringiensis subsp. israelensis were also transformed. Compared with protoplast transformation, our method is much faster and 3 orders of magnitude more efficient per microgram of added DNA.

  9. pTAR-encoded proteins in plasmid partitioning.

    PubMed

    Kalnin, K; Stegalkina, S; Yarmolinsky, M

    2000-04-01

    Partition cassettes, essential for the segregational stability of low-copy-number bacterial plasmids, typically encode two autoregulated proteins and an adjacent cis-acting centromere analog to which one or perhaps both proteins bind. The diminutive partition region of pTAR of Agrobacterium spp. was reported to be exceptional, encoding only a single protein, ParA (D. R. Gallie and C. I. Kado, J. Mol. Biol. 193:465-478, 1987). However, resequencing of the region revealed two small downstream genes, parB and orf-84, of which only parB was found to be essential for partitioning in A. tumefaciens. Purified ParA exhibited a weak ATPase activity that was modestly increased by nonspecific DNA. ParB bound in vitro to repeated sequences present in a region, parS, that possesses centromere and operator functions and within which we identified the primary transcription start site by primer extension. In certain respects the Par proteins behave normally in the foreign host Escherichia coli. In E. coli, as in A. tumefaciens, ParB repressed the partition operon; ParA, inactive alone, augmented this repression. Functional similarities between the partition system of pTAR and those of other plasmids and bacteria are prominent, despite differences in size, organization, and amino acid sequence.

  10. Biodegradable poly(ethylenimine) for plasmid DNA delivery.

    PubMed

    Ahn, Cheol-Hee; Chae, Su Young; Bae, You Han; Kim, Sung Wan

    2002-04-23

    Poly(ethylenimine) (PEI) has been known as an efficient gene carrier with the highest cationic charge potential. High transfection efficiency of PEI, along with its cytotoxicity, strongly depends on the molecular weight. Synthesis of cationic copolymers derived from the low molecular weight of PEI and hydrophilic poly(ethylene glycol) (PEG), which are water soluble and degradable under physiological conditions, was investigated for plasmid delivery. Hydrophilic PEG is expected to reduce the toxicity of the copolymer, improve the poor solubility of the PEI and DNA complexes, and help to introduce degradable bonds by reaction with the primary amines in the PEI. Considering the dependence of transfection efficiency and cytotoxicity on the molecular weight of the PEI, high transfection efficiency is expected from an increased molecular weight of the copolymer and low cytotoxicity from the introduction of PEG and the degradation of the copolymer into low molecular weight PEIs. Reaction conditions were carefully controlled to produce water soluble copolymers. Results from a gel retardation assay and zetapotentiometer indicated that complete neutralization of the complexes was achieved at the charge ratios of copolymer/pSV-beta-gal plasmid from 0.8 to 1.0 with the mean particle size of the polyplexes ranging from 129.8+/-0.9 to 151.8+/-3.4 nm. In vitro transfection efficiency of the synthesized copolymer increased up to three times higher than that of starting low molecular weight PEI, while the cell viability was maintained over 80%.

  11. Use of plasmid profiles in epidemiologic surveillance of disease outbreaks and in tracing the transmission of antibiotic resistance.

    PubMed Central

    Mayer, L W

    1988-01-01

    Plasmids are circular deoxyribonucleic acid molecules that exist in bacteria, usually independent of the chromosome. The study of plasmids is important to medical microbiology because plasmids can encode genes for antibiotic resistance or virulence factors. Plasmids can also serve as markers of various bacterial strains when a typing system referred to as plasmid profiling, or plasmid fingerprinting is used. In these methods partially purified plasma deoxyribonucleic acid species are separated according to molecular size by agarose gel electrophoresis. In a second procedure, plasmid deoxyribonucleic acid which has been cleaved by restriction endonucleases can be separated by agarose gel electrophoresis and the resulting pattern of fragments can be used to verify the identity of bacterial isolates. Because many species of bacteria contain plasmids, plasmid profile typing has been used to investigate outbreaks of many bacterial diseases and to trace inter- and intra-species spread of antibiotic resistance. Images PMID:2852997

  12. Increased STAT3 phosphorylation on CD27(+) B-cells from common variable immunodeficiency disease patients.

    PubMed

    Clemente, Antonio; Pons, Jaume; Lanio, Nallibe; Cunill, Vanesa; Frontera, Guillem; Crespí, Catalina; Matamoros, Núria; Ferrer, Joana M

    2015-12-01

    Maturation and differentiation of B-cells are driven by T-cells' help through IL-21/STAT3 axis in GC centers or through extrafollicular pathways, in a T-independent manner. B-cell differentiation is defective in common variable immunodeficiency disease (CVID) patients. We investigated if IL-21/STAT3 axis alterations could influence B-cell fate. We activated purified CVID B-cells with surrogate T-dependent (anti-CD40), T-independent (TLR-9 ligand) stimuli or through B-cell receptor engagement (anti-IgM) with or without IL-21. IL-21 mediated STAT3 activation was greater on CD27(-) than CD27(+) B-cells depending on the stimulus. IL-21 alone induced STAT3 phosphorylation (pSTAT3) only on CD27(-) B-cells and IL-21 induced higher pSTAT3 levels on CD27(-) than CD27(+) B-cells after anti-IgM or anti-CD40 activation. CVID CD27(+) B-cells showed selective STAT3 hyperphosphorylation after activation with anti-IgM or anti-CD40 alone and anti-IgM, anti-CD40 or ODN combined with IL-21. Increased STAT3 activation during immune responses could result in B-cell differentiation defects in CVID.

  13. Plasmid Classification in an Era of Whole-Genome Sequencing: Application in Studies of Antibiotic Resistance Epidemiology.

    PubMed

    Orlek, Alex; Stoesser, Nicole; Anjum, Muna F; Doumith, Michel; Ellington, Matthew J; Peto, Tim; Crook, Derrick; Woodford, Neil; Walker, A Sarah; Phan, Hang; Sheppard, Anna E

    2017-01-01

    Plasmids are extra-chromosomal genetic elements ubiquitous in bacteria, and commonly transmissible between host cells. Their genomes include variable repertoires of 'accessory genes,' such as antibiotic resistance genes, as well as 'backbone' loci which are largely conserved within plasmid families, and often involved in key plasmid-specific functions (e.g., replication, stable inheritance, mobility). Classifying plasmids into different types according to their phylogenetic relatedness provides insight into the epidemiology of plasmid-mediated antibiotic resistance. Current typing schemes exploit backbone loci associated with replication (replicon typing), or plasmid mobility (MOB typing). Conventional PCR-based methods for plasmid typing remain widely used. With the emergence of whole-genome sequencing (WGS), large datasets can be analyzed using in silico plasmid typing methods. However, short reads from popular high-throughput sequencers can be challenging to assemble, so complete plasmid sequences may not be accurately reconstructed. Therefore, localizing resistance genes to specific plasmids may be difficult, limiting epidemiological insight. Long-read sequencing will become increasingly popular as costs decline, especially when resolving accurate plasmid structures is the primary goal. This review discusses the application of plasmid classification in WGS-based studies of antibiotic resistance epidemiology; novel in silico plasmid analysis tools are highlighted. Due to the diverse and plastic nature of plasmid genomes, current typing schemes do not classify all plasmids, and identifying conserved, phylogenetically concordant genes for subtyping and phylogenetics is challenging. Analyzing plasmids as nodes in a network that represents gene-sharing relationships between plasmids provides a complementary way to assess plasmid diversity, and allows inferences about horizontal gene transfer to be made.

  14. Characterization and Comparative Overview of Complete Sequences of the First Plasmids of Pandoraea across Clinical and Non-clinical Strains

    PubMed Central

    Yong, Delicia; Tee, Kok Keng; Yin, Wai-Fong; Chan, Kok-Gan

    2016-01-01

    To date, information on plasmid analysis in Pandoraea spp. is scarce. To address the gap of knowledge on this, the complete sequences of eight plasmids from Pandoraea spp. namely Pandoraea faecigallinarum DSM 23572T (pPF72-1, pPF72-2), Pandoraea oxalativorans DSM 23570T (pPO70-1, pPO70-2, pPO70-3, pPO70-4), Pandoraea vervacti NS15 (pPV15) and Pandoraea apista DSM 16535T (pPA35) were studied for the first time in this study. The information on plasmid sequences in Pandoraea spp. is useful as the sequences did not match any known plasmid sequence deposited in public databases. Replication genes were not identified in some plasmids, a situation that has led to the possibility of host interaction involvement. Some plasmids were also void of par genes and intriguingly, repA gene was also not discovered in these plasmids. This further leads to the hypothesis of host-plasmid interaction. Plasmid stabilization/stability protein-encoding genes were observed in some plasmids but were not established for participating in plasmid segregation. Toxin-antitoxin systems MazEF, VapBC, RelBE, YgiT-MqsR, HigBA, and ParDE were identified across the plasmids and their presence would improve plasmid maintenance. Conjugation genes were identified portraying the conjugation ability amongst Pandoraea plasmids. Additionally, we found a shared region amongst some of the plasmids that consists of conjugation genes. The identification of genes involved in replication, segregation, toxin-antitoxin systems and conjugation, would aid the design of drugs to prevent the survival or transmission of plasmids carrying pathogenic properties. Additionally, genes conferring virulence and antibiotic resistance were identified amongst the plasmids. The observed features in the plasmids shed light on the Pandoraea spp. as opportunistic pathogens. PMID:27790203

  15. Plasmid Classification in an Era of Whole-Genome Sequencing: Application in Studies of Antibiotic Resistance Epidemiology

    PubMed Central

    Orlek, Alex; Stoesser, Nicole; Anjum, Muna F.; Doumith, Michel; Ellington, Matthew J.; Peto, Tim; Crook, Derrick; Woodford, Neil; Walker, A. Sarah; Phan, Hang; Sheppard, Anna E.

    2017-01-01

    Plasmids are extra-chromosomal genetic elements ubiquitous in bacteria, and commonly transmissible between host cells. Their genomes include variable repertoires of ‘accessory genes,’ such as antibiotic resistance genes, as well as ‘backbone’ loci which are largely conserved within plasmid families, and often involved in key plasmid-specific functions (e.g., replication, stable inheritance, mobility). Classifying plasmids into different types according to their phylogenetic relatedness provides insight into the epidemiology of plasmid-mediated antibiotic resistance. Current typing schemes exploit backbone loci associated with replication (replicon typing), or plasmid mobility (MOB typing). Conventional PCR-based methods for plasmid typing remain widely used. With the emergence of whole-genome sequencing (WGS), large datasets can be analyzed using in silico plasmid typing methods. However, short reads from popular high-throughput sequencers can be challenging to assemble, so complete plasmid sequences may not be accurately reconstructed. Therefore, localizing resistance genes to specific plasmids may be difficult, limiting epidemiological insight. Long-read sequencing will become increasingly popular as costs decline, especially when resolving accurate plasmid structures is the primary goal. This review discusses the application of plasmid classification in WGS-based studies of antibiotic resistance epidemiology; novel in silico plasmid analysis tools are highlighted. Due to the diverse and plastic nature of plasmid genomes, current typing schemes do not classify all plasmids, and identifying conserved, phylogenetically concordant genes for subtyping and phylogenetics is challenging. Analyzing plasmids as nodes in a network that represents gene-sharing relationships between plasmids provides a complementary way to assess plasmid diversity, and allows inferences about horizontal gene transfer to be made. PMID:28232822

  16. Experimental and clinical application of plasmid DNA in the field of central nervous diseases.

    PubMed

    Shimamura, Munehisa; Sato, Naoyuki; Morishita, Ryuichi

    2011-12-01

    Novel therapeutic strategies utilizing plasmid DNA (pDNA) have been sought for non-treatable neurological disorders, such as ischemic stroke, Parkinson disease (PD), Alzheimer disease (AD), and multiple sclerosis (MS). One strategy is to induce overexpression of growth factors, such as vascular endothelial growth factor (VEGF), glial cell-line derived neurotrophic factor (GDNF), and hepatocyte growth factor (HGF), in the brain. Since ischemic stroke, PD, and AD show damage of neurons, the transfer of pDNA encoding these genes has been examined and shown to protect neurons from damage, associated with a better behavioral outcome. These growth factors have also been shown to accelerate angiogenesis, neurite outgrowth, and neurogenesis in the brain, and overexpression of these factors showed therapeutic effects in cerebral ischemia in rodents. Another application of pDNA is as a "DNA vaccine" to induce immunity against amyloid Aβ in AD, which requires a predominantly Th2 response to avoid autoimmune encephalomyelitis evoked by a Th1 response. Since the combination of pDNA and special devices and/or modification of pDNA could induce a predominantly Th2 response to a targeted antigen, a pDNA-based vaccine would be ideal for AD. Interestingly, pDNA could also induce immune tolerance, and pDNA-based vaccines to induce immune tolerance to autoimmune antibodies have been extensively examined in an animal model of MS. Based on the results, a pDNA vaccine has already been tried in MS patients and reported to be safe and partly effective in phase I/II clinical studies. In this review, we discuss the potential and problems of pDNA-mediated medicine in neurological disorders.

  17. IncHI2 Plasmids Are Predominant in Antibiotic-Resistant Salmonella Isolates

    PubMed Central

    Chen, Wenyao; Fang, Tingzi; Zhou, Xiujuan; Zhang, Daofeng; Shi, Xianming; Shi, Chunlei

    2016-01-01

    The wide usage of antibiotics contributes to the increase in the prevalence of antibiotic-resistant Salmonella. Plasmids play a critical role in horizontal transfer of antibiotic resistance markers in Salmonella. This study aimed to screen and characterize plasmid profiles responsible for antibiotic resistance in Salmonella and ultimately to clarify the molecular mechanism of transferable plasmid-mediated antibiotic resistance. A total of 226 Salmonella isolates were examined for antimicrobial susceptibility by a disk diffusion method. Thirty-two isolates (14.2%) were resistant to at least one antibiotic. The presence of plasmid-mediated quinolone resistance (PMQR) genes and β-lactamase genes were established by PCR amplification. PCR-based replicon typing revealed that these 32 isolates represented seven plasmid incompatibility groups (IncP, HI2, A/C, FIIs, FIA, FIB, and I1), and the IncHI2 (59.4%) was predominant. Antibiotic resistance markers located on plasmids were identified through plasmid curing. Fifteen phenotypic variants were obtained with the curing efficiency of 46.9% (15/32). The cured plasmids mainly belong to the HI2 incompatibility group. The elimination of IncHI2 plasmids correlated with the loss of β-lactamase genes (blaOXA-1 and blaTEM-1) and PMQR genes (qnrA and aac(6′)-Ib-cr). Both IncHI2 and IncI1 plasmids in a S. enterica serovar Indiana isolate SJTUF 10584 were lost by curing. The blaCMY -2-carrying plasmid pS10584 from SJTUF 10584 was fully sequenced. Sequence analysis revealed that it possessed a plasmid scaffold typical for IncI1 plasmids with the unique genetic arrangement of IS1294-ΔISEcp1-blaCMY -2-blc-sugE-ΔecnR inserted into the colicin gene cia. These data suggested that IncHI2 was the major plasmid lineage contributing to the dissemination of antibiotic resistance in Salmonella and the activity of multiple mobile genetic elements may contribute to antibiotic resistance evolution and dissemination between different plasmid

  18. Ultrasound enhances in vivo tumor expression of plasmid DNA by PEG-introduced cationized dextran.

    PubMed

    Hosseinkhani, Hossein; Tabata, Yasuhiko

    2005-11-28

    This study is an investigation to experimentally confirm whether or not ultrasound (US) irradiation is effective in enhancing the in vivo gene expression of plasmid DNA in tumor. Dextran was cationized by introducing spermine to the hydroxyl groups to allow to polyionically complex with a plasmid DNA. The cationized dextran prepared was additionally modified with poly(ethylene glycol) (PEG) molecules which have an active ester and methoxy groups at each terminal, to obtain cationized dextran with different percentages of PEG introduced. Various cationized dextrans with or without PEG introduction were mixed with a plasmid DNA of LacZ to form cationized dextran-plasmid DNA complexes. Electrophoretical examination revealed that the plasmid DNA was complexed both with the cationized dextran and PEG-introduced cationized dextran, irrespective of the PEG introduction percentage, although the higher N/P ratio was needed for plasmid DNA complexation with the latter. By complexation with the cationized dextran, the zeta potential of plasmid DNA was changed to be positive. The charge of PEG-introduced cationized dextran-plasmid DNA complexes became close to 0 mV as their percentage of PEG introduced increased, although the molecular size was about 250 nm, irrespective of the PEG introduction. When cationized dextran-plasmid DNA complexes with or without PEG introduction were intravenously injected to mice carrying a subcutaneous Meth-AR-1 fibrosarcoma mass and the subsequent US irradiation to the tumor mass percutaneously, the PEG-introduced cationized dextran-plasmid DNA complex plus US irradiation enhanced the tumor level of gene expression to a significantly high extent compared with the cationized dextran-plasmid DNA complex and free plasmid DNA with or without US irradiation. The enhanced level depended on the time period and timing of US irradiation. Fluorescent microscopic studies revealed that the localization of plasmid DNA and the gene expression were observed in

  19. DNASU plasmid and PSI:Biology-Materials repositories: resources to accelerate biological research.

    PubMed

    Seiler, Catherine Y; Park, Jin G; Sharma, Amit; Hunter, Preston; Surapaneni, Padmini; Sedillo, Casey; Field, James; Algar, Rhys; Price, Andrea; Steel, Jason; Throop, Andrea; Fiacco, Michael; LaBaer, Joshua

    2014-01-01

    The mission of the DNASU Plasmid Repository is to accelerate research by providing high-quality, annotated plasmid samples and online plasmid resources to the research community through the curated DNASU database, website and repository (http://dnasu.asu.edu or http://dnasu.org). The collection includes plasmids from grant-funded, high-throughput cloning projects performed in our laboratory, plasmids from external researchers, and large collections from consortia such as the ORFeome Collaboration and the NIGMS-funded Protein Structure Initiative: Biology (PSI:Biology). Through DNASU, researchers can search for and access detailed information about each plasmid such as the full length gene insert sequence, vector information, associated publications, and links to external resources that provide additional protein annotations and experimental protocols. Plasmids can be requested directly through the DNASU website. DNASU and the PSI:Biology-Materials Repositories were previously described in the 2010 NAR Database Issue (Cormier, C.Y., Mohr, S.E., Zuo, D., Hu, Y., Rolfs, A., Kramer, J., Taycher, E., Kelley, F., Fiacco, M., Turnbull, G. et al. (2010) Protein Structure Initiative Material Repository: an open shared public resource of structural genomics plasmids for the biological community. Nucleic Acids Res., 38, D743-D749.). In this update we will describe the plasmid collection and highlight the new features in the website redesign, including new browse/search options, plasmid annotations and a dynamic vector mapping feature that was developed in collaboration with LabGenius. Overall, these plasmid resources continue to enable research with the goal of elucidating the role of proteins in both normal biological processes and disease.

  20. Molecular and population analyses of a recombination event in the catabolic plasmid pJP4.

    PubMed

    Larraín-Linton, Juanita; De la Iglesia, Rodrigo; Melo, Francisco; González, Bernardo

    2006-10-01

    Cupriavidus necator JMP134(pJP4) harbors a catabolic plasmid, pJP4, which confers the ability to grow on chloroaromatic compounds. Repeated growth on 3-chlorobenzoate (3-CB) results in selection of a recombinant strain, which degrades 3-CB better but no longer grows on 2,4-dichlorophenoxyacetate (2,4-D). We have previously proposed that this phenotype is due to a double homologous recombination event between inverted repeats of the multicopies of this plasmid within the cell. One recombinant form of this plasmid (pJP4-F3) explains this phenotype, since it harbors two copies of the chlorocatechol degradation tfd gene clusters, which are essential to grow on 3-CB, but has lost the tfdA gene, encoding the first step in degradation of 2,4-D. The other recombinant plasmid (pJP4-FM) should harbor two copies of the tfdA gene but no copies of the tfd gene clusters. A molecular analysis using a multiplex PCR approach to distinguish the wild-type plasmid pJP4 from its two recombinant forms, was carried out. Expected PCR products confirming this recombination model were found and sequenced. Few recombinant plasmid forms in cultures grown in several carbon sources were detected. Kinetic studies indicated that cells containing the recombinant plasmid pJP4-FM were not selectable by sole carbon source growth pressure, whereas those cells harboring recombinant plasmid pJP4-F3 were selected upon growth on 3-CB. After 12 days of repeated growth on 3-CB, the complete plasmid population in C. necator JMP134 apparently corresponds to this form. However, wild-type plasmid forms could be recovered after growing this culture on 2,4-D, indicating that different plasmid forms can be found in C. necator JMP134 at the population level.

  1. Characterization and comparative analysis of antibiotic resistance plasmids isolated from a wastewater treatment plant.

    PubMed

    Rahube, Teddie O; Viana, Laia S; Koraimann, Günther; Yost, Christopher K

    2014-01-01

    A wastewater treatment plant (WWTP) is an environment high in nutrient concentration with diverse bacterial populations and can provide an ideal environment for the proliferation of mobile elements such as plasmids. WWTPs have also been identified as reservoirs for antibiotic resistance genes that are associated with human pathogens. The objectives of this study were to isolate and characterize self-transmissible or mobilizable resistance plasmids associated with effluent from WWTP. An enrichment culture approach designed to capture plasmids conferring resistance to high concentrations of erythromycin was used to capture plasmids from an urban WWTP servicing a population of ca. 210,000. DNA sequencing of the plasmids revealed diversity of plasmids represented by incompatibility groups IncU, col-E, IncFII and IncP-1β. Genes coding resistance to clinically relevant antibiotics (macrolide, tetracycline, beta-lactam, trimethoprim, chloramphenicol, sulphonamide), quaternary ammonium compounds and heavy metals were co-located on these plasmids, often within transposable and integrative mobile elements. Several of the plasmids were self-transmissible or mobilizable and could be maintained in the absence of antibiotic selection. The IncFII plasmid pEFC36a showed the highest degree of sequence identity to plasmid R1 which has been isolated in England more than 50 years ago from a patient suffering from a Salmonella infection. Functional conservation of key regulatory features of this F-like conjugation module were demonstrated by the finding that the conjugation frequency of pEFC36a could be stimulated by the positive regulator of plasmid R1 DNA transfer genes, TraJ.

  2. Infectivity acts as in vivo selection for maintenance of the chlamydial cryptic plasmid.

    PubMed

    Russell, Marsha; Darville, Toni; Chandra-Kuntal, Kumar; Smith, Bennett; Andrews, Charles W; O'Connell, Catherine M

    2011-01-01

    Chlamydia trachomatis contains a conserved ∼7.5-kb plasmid. Loss of the plasmid results in reduced glycogen accumulation, failure to activate TLR2, and reduced infectivity. We hypothesized that reduced infectivity functions as a means of selection for plasmid maintenance. We directly examined the biological significance of the reduced infectivity associated with plasmid deficiency by determining the relative fitness of plasmid-deficient CM972 versus that of wild-type C. muridarum Nigg in mixed inocula in vitro and in vivo. C. muridarum Nigg rapidly out-competed its plasmid-cured derivative CM972 in vitro but was not competitive with CM3.1, a derivative of CM972 that has reverted to a normal infectivity phenotype. C. muridarum Nigg also effectively competed with CM972 during lower and upper genital tract infection in the mouse, demonstrating that strong selective pressure for plasmid maintenance occurs during infection. The severity of oviduct inflammation and dilatation resulting from these mixed infections correlated directly with the amount of C. muridarum Nigg in the initial inoculum, confirming the role of the plasmid in virulence. Genetic characterization of CM972 and CM3.1 revealed no additional mutations (other than loss of the plasmid) to account for the reduced infectivity of CM972 and detected a single base substitution in TC_0236 in CM3.1 that may be responsible for its restored infectivity. These data demonstrate that a chlamydial strain that differs genetically from its wild-type parent only with respect to the lack of the chlamydial plasmid is unable to compete in vitro and in vivo, likely explaining the rarity of plasmid-deficient isolates in nature.

  3. Characterization of Plasmids in Extensively Drug-Resistant Acinetobacter Strains Isolated in India and Pakistan

    PubMed Central

    Carvalho, Maria J.; Toleman, Mark A.; White, P. Lewis; Connor, Thomas R.; Mushtaq, Ammara; Weeks, Janis L.; Kumarasamy, Karthikeyan K.; Raven, Katherine E.; Török, M. Estée; Peacock, Sharon J.; Howe, Robin A.; Walsh, Timothy R.

    2014-01-01

    The blaNDM-1 gene is associated with extensive drug resistance in Gram-negative bacteria. This probably spread to Enterobacteriaceae from Acinetobacter spp., and we characterized plasmids associated with blaNDM-1 in Acinetobacter spp. to gain insight into their role in this dissemination. Four clinical NDM-1-producing Acinetobacter species strains from India and Pakistan were investigated. A plasmid harboring blaNDM-1, pNDM-40-1, was characterized by whole-genome sequencing of Acinetobacter bereziniae CHI-40-1 and comparison with related plasmids. The presence of similar plasmids in strains from Pakistan was sought by PCR and sequencing of amplicons. Conjugation frequency was tested and stability of pNDM-40-1 investigated by real-time PCR of isolates passaged with and without antimicrobial selection pressure. A. bereziniae and Acinetobacter haemolyticus strains contained plasmids similar to the pNDM-BJ01-like plasmids identified in Acinetobacter spp. in China. The backbone of pNDM-40-1 was almost identical to that of pNDM-BJ01-like plasmids, but the transposon harboring blaNDM-1, Tn125, contained two short deletions. Escherichia coli and Acinetobacter pittii transconjugants were readily obtained. Transconjugants retained pNDM-40-1 after a 14-day passage experiment, although stability was greater with meropenem selection. Fragments of pNDM-BJ01-like plasmid backbones are found near blaNDM-1 in some genetic contexts from Enterobacteriaceae, suggesting that cross-genus transfer has occurred. pNDM-BJ01-like plasmids have been described in isolates originating from a wide geographical region in southern Asia. In vitro data on plasmid transfer and stability suggest that these plasmids could have contributed to the spread of blaNDM-1 into Enterobacteriaceae. PMID:25421466

  4. Characterization of plasmids in extensively drug-resistant acinetobacter strains isolated in India and Pakistan.

    PubMed

    Jones, Lim S; Carvalho, Maria J; Toleman, Mark A; White, P Lewis; Connor, Thomas R; Mushtaq, Ammara; Weeks, Janis L; Kumarasamy, Karthikeyan K; Raven, Katherine E; Török, M Estée; Peacock, Sharon J; Howe, Robin A; Walsh, Timothy R

    2015-02-01

    The blaNDM-1 gene is associated with extensive drug resistance in Gram-negative bacteria. This probably spread to Enterobacteriaceae from Acinetobacter spp., and we characterized plasmids associated with blaNDM-1 in Acinetobacter spp. to gain insight into their role in this dissemination. Four clinical NDM-1-producing Acinetobacter species strains from India and Pakistan were investigated. A plasmid harboring blaNDM-1, pNDM-40-1, was characterized by whole-genome sequencing of Acinetobacter bereziniae CHI-40-1 and comparison with related plasmids. The presence of similar plasmids in strains from Pakistan was sought by PCR and sequencing of amplicons. Conjugation frequency was tested and stability of pNDM-40-1 investigated by real-time PCR of isolates passaged with and without antimicrobial selection pressure. A. bereziniae and Acinetobacter haemolyticus strains contained plasmids similar to the pNDM-BJ01-like plasmids identified in Acinetobacter spp. in China. The backbone of pNDM-40-1 was almost identical to that of pNDM-BJ01-like plasmids, but the transposon harboring blaNDM-1, Tn125, contained two short deletions. Escherichia coli and Acinetobacter pittii transconjugants were readily obtained. Transconjugants retained pNDM-40-1 after a 14-day passage experiment, although stability was greater with meropenem selection. Fragments of pNDM-BJ01-like plasmid backbones are found near blaNDM-1 in some genetic contexts from Enterobacteriaceae, suggesting that cross-genus transfer has occurred. pNDM-BJ01-like plasmids have been described in isolates originating from a wide geographical region in southern Asia. In vitro data on plasmid transfer and stability suggest that these plasmids could have contributed to the spread of blaNDM-1 into Enterobacteriaceae.

  5. Structural and replicative diversity of large plasmids from sphingomonads that degrade polycyclic aromatic compounds and xenobiotics.

    PubMed

    Basta, Tamara; Buerger, Sibylle; Stolz, Andreas

    2005-06-01

    The plasmids from 16 sphingomonads which degrade various xenobiotics and polycyclic aromatic compounds were compared with the previously sequenced plasmid pNL1 from Sphingomonas aromaticivorans F199. The replicase genes repAaAb from plasmid pNL1 were amplified by PCR and used as a gene probe for the identification of plasmids belonging to the same incompatibility group as plasmid pNL1. Plasmids were prepared from various sphingomonads and hybridized with the repA gene probe. Positive hybridization signals were obtained with plasmids of approximately 160-195 kb from Sphingomonas subterranea and S. aromaticivorans B0695, which had been isolated from the same subsurface location as S. aromaticivorans F199. The repA probe also hybridized with plasmids from Sphingomonas xenophaga BN6, Sphingomonas sp. HH69 and Sphingomonas macrogoltabidus, which had been isolated from different continents and which utilize different organic compounds than S. aromaticivorans F199 and the other subsurface strains. The results of the hybridization experiments were confirmed by PCR experiments using primers deduced from the repAaAb region of plasmid pNL1. Nucleotide sequence comparisons suggested that three gene clusters were conserved between plasmid pNL1 and plasmid pBN6 from the naphthalenesulfonate- degrading strain S. xenophaga BN6. From these sequence comparisons, PCR primers were derived in order to detect the respective gene clusters in the other strains and to deduce their position relative to each other. These experiments demonstrated that all analysed subsurface strains harboured the same three gene clusters, but that the position and distance from each other of the clusters varied considerably among the different strains.

  6. Comparison of nanoparticle-mediated transfection methods for DNA expression plasmids: efficiency and cytotoxicity

    PubMed Central

    2011-01-01

    Background Reproducibly high transfection rates with low methodology-induced cytotoxic side effects are essential to attain the required effect on targeted cells when exogenous DNA is transfected. Different approaches and modifications such as the use of nanoparticles (NPs) are being evaluated to increase transfection efficiencies. Several studies have focused on the attained transfection efficiency after NP-mediated approaches. However, data comparing toxicity of these novel approaches with conventional methods is still rare. Transfection efficiency and methodology-induced cytotoxicity were analysed after transfection with different NP-mediated and conventional approaches. Two eukaryotic DNA-expression-plasmids were used to transfect the mammalian cell line MTH53A applying six different transfection protocols: conventional transfection reagent (FuGENE HD, FHD), FHD in combination with two different sizes of stabilizer-free laser-generated AuNPs (PLAL-AuNPs_S1,_S2), FHD and commercially available AuNPs (Plano-AuNP), and two magnetic transfection protocols. 24 h post transfection efficiency of each protocol was analysed using fluorescence microscopy and GFP-based flow cytometry. Toxicity was assessed measuring cell proliferation and percentage of propidium iodide (PI%) positive cells. Expression of the respective recombinant proteins was evaluated by immunofluorescence. Results The addition of AuNPs to the transfection protocols significantly increased transfection efficiency in the pIRES-hrGFPII-eIL-12 transfections (FHD: 16%; AuNPs mean: 28%), whereas the magnet-assisted protocols did not increase efficiency. Ligand-free PLAL-AuNPs had no significant cytotoxic effect, while the ligand-stabilized Plano-AuNPs induced a significant increase in the PI% and lower cell proliferation. For pIRES-hrGFPII-rHMGB1 transfections significantly higher transfection efficiency was observed with PLAL-AuNPs (FHD: 31%; PLAL-AuNPs_S1: 46%; PLAL-AuNPs_S2: 50%), while the magnet

  7. Induction of mucosal immunity against herpes simplex virus by plasmid DNA immunization.

    PubMed Central

    Kuklin, N; Daheshia, M; Karem, K; Manickan, E; Rouse, B T

    1997-01-01

    The ability of mucosally delivered plasmid DNA encoding glycoprotein B (gB) of herpes simplex virus type 1 (HSV-1) to generate systemic as well as distal mucosal immunity was evaluated. BALB/c mice were immunized intranasally (i.n.) with gB DNA or DNA expressing beta-galactosidase (beta-Gal). Two days following immunization, gB and beta-Gal gene expression was detected by reverse transcription (RT)-PCR in lungs and cervical lymph nodes (CLN). Histological analysis showed that beta-Gal protein was expressed in vivo in the lungs and the CLN of animals immunized with i.n. administered beta-Gal DNA. The immune responses generated by i.n. administration of gB DNA with or without cholera toxin (CT) were compared to those generated by intramuscular (i.m.) gB DNA and i.n. live HSV administration. Three i.n. doses of gB DNA over a 3-week period resulted in a distal mucosal immunoglobulin A (IgA) response. In addition, the mucosal IgA response was enhanced by coadministration of CT with gB DNA. The i.m. route of immunization induced a strong IgG response in the serum and vagina but was inefficient in generating a mucosal IgA response. Antigen-specific cytokine ELISPOT analyses as well as the serum IgG1/IgG2a ratio indicated induction of stronger Th2 responses following the additional i.n. administration of CT compared to i.n. or i.m. gB DNA or i.n. live HSV immunization. In addition, mucosal immunization with gB DNA induced anti-HSV cell-mediated immunity in vivo as measured by delayed-type hypersensitivity. Although i.n. DNA immunization was an effective means of inducing mucosal antibody, it was inferior to i.m. DNA delivery in providing protection against lethal HSV challenge via the vaginal route. In addition, both i.m. and i.n. plasmid immunizations failed to generate an immune barrier to viral invasion of the mucosa. PMID:9060677

  8. Genomic and Functional Characterization of the Unusual pLOCK 0919 Plasmid Harboring the spaCBA Pili Cluster in Lactobacillus casei LOCK 0919

    PubMed Central

    Aleksandrzak-Piekarczyk, Tamara; Koryszewska-Bagińska, Anna; Grynberg, Marcin; Nowak, Adriana; Cukrowska, Bożena; Kozakova, Hana; Bardowski, Jacek

    2016-01-01

    Here, we report the extensive bioinformatic and functional analyses of the unusual pLOCK 0919, a plasmid originating from the probiotic Lactobacillus casei LOCK 0919 strain. This plasmid is atypical because it harbors the spaCBA-srtC gene cluster encoding SpaCBA pili. We show that all other spaCBA-srtC sequences of the Lactobacillus genus that have been previously described and deposited in GenBank are present in the chromosomal DNA. Another important observation for pLOCK 0919 is that the spaCBA-srtC gene cluster and its surrounding genes are highly similar to the respective DNA region that is present in the most well-known and active SpaCBA pili producer, the probiotic Lactobacillus rhamnosus GG strain. Our results demonstrate that the spaCBA-srtC clusters of pLOCK 0919 and L. rhamnosus GG are genealogically similar, located in DNA regions that are rich in transposase genes and are poorly conserved among the publicly available sequences of Lactobacillus sp. In contrast to chromosomally localized pilus gene clusters from L. casei and Lactobacillus paracasei, the plasmidic spaC of L. casei LOCK 0919 is expressed and undergoes a slight glucose-induced repression. Moreover, results of series of in vitro tests demonstrate that L. casei LOCK 0919 has an adhesion potential, which is largely determined by the presence of the pLOCK 0919 plasmid. In particular, the plasmid occurrence positively influenced the hydrophobicity and aggregation abilities of L. casei LOCK 0919. Moreover, in vivo studies indicate that among the three Lactobacillus strains used to colonize the gastrointestinal tract of germ-free mice, already after 2 days of colonization, L. casei LOCK 0919 became the dominant strain and persisted there for at least 48 days. PMID:26637469

  9. Complete nucleotide sequence of the self-transmissible TOL plasmid pD2RT provides new insight into arrangement of toluene catabolic plasmids.

    PubMed

    Jutkina, Jekaterina; Hansen, Lars Hestbjerg; Li, Lili; Heinaru, Eeva; Vedler, Eve; Jõesaar, Merike; Heinaru, Ain

    2013-11-01

    In the present study we report the complete nucleotide sequence of the toluene catabolic plasmid pD2RT of Pseudomonas migulae strain D2RT isolated from Baltic Sea water. The pD2RT is 129,894 base pairs in size with an average G+C content of 53.75%. A total of 135 open reading frames (ORFs) were predicted to encode proteins, among them genes for catabolism of toluene, plasmid replication, maintenance and conjugative transfer. ORFs encoding proteins with putative functions in stress response, transposition and site-specific recombination were also predicted. Analysis of the organization and nucleotide sequence of pD2RT backbone region revealed high degree of similarity to the draft genome sequence data of the plant-pathogenic pseudomonad Pseudomonas syringae pv. glycinea strain B076, exhibiting relatedness to pPT23A plasmid family. The pD2RT backbone is also closely related to that of pGRT1 of Pseudomonas putida strain DOT-T1E and pBVIE04 of Burkholderia vietnamiensis strain G4, both plasmids are associated with resistance to toluene. The ability of pD2RT to self-transfer by conjugation to P. putida recipient strain PaW340 was experimentally determined. Genetic organization of toluene-degrading (xyl) genes and flanking DNA segments resembles the structure of Tn1721-related class II transposon Tn4656 of TOL plasmid pWW53 of P. putida strain MT53. The complete sequence of the plasmid pD2RT extends the known range of xyl genes carriers, being the first completely sequenced TOL plasmid, which is not related to well-studied IncP plasmid groups. We also verified the functionality of the catabolic route encoded by pD2RT by monitoring the expression of the xylE gene in pD2RT bearing hosts along with bacterial strains containing TOL plasmid of IncP-9 group. The growth kinetics of plasmid-bearing strains was found to be affected by particular TOL plasmid.

  10. Identical plasmid AmpC beta-lactamase genes and plasmid types in E. coli isolates from patients and poultry meat in the Netherlands.

    PubMed

    Voets, Guido M; Fluit, Ad C; Scharringa, Jelle; Schapendonk, Claudia; van den Munckhof, Thijs; Leverstein-van Hall, Maurine A; Stuart, James Cohen

    2013-11-01

    The increasing prevalence of third-generation cephalosporin-resistant Enterobacteriaceae is a worldwide problem. Recent studies showed that poultry meat and humans share identical Extended-Spectrum Beta-Lactamase genes, plasmid types, and Escherichia coli strain types, suggesting that transmission from poultry meat to humans may occur. The aim of this study was to compare plasmid-encoded Ambler class C beta-lactamase (pAmpC) genes, their plasmids, and bacterial strain types between E. coli isolates from retail chicken meat and clinical isolates in the Netherlands. In total, 98 Dutch retail chicken meat samples and 479 third-generation cephalosporin non-susceptible human clinical E. coli isolates from the same period were screened for pAmpC production. Plasmid typing was performed using PCR-based replicon typ