WorldWideScience

Sample records for plasmids contained homologs

  1. Effect of chromosome homology an plasmid transformation and plasmid conjugal transfer in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Balganesh, M.; Setlow, J.K.

    1984-05-14

    The pairing between plasmid and the homologous part of the chromosome associated with plasmid establishment may differ from the pairing which results from integration of a homologous region of the plasmid into the chromosome. Thus the rate of novobiocin transformation decreases with duplication of the chromosomal portion in pMB2, but the rate of establishment of the plasmid increases with this duplication. A model to explain these data is given. 17 references, 5 figures, 4 tables.

  2. Homology and repair of UV-irradiated plasmid DNA in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Cabrea-Juarez, E.; Setlow, J.K.

    1983-02-01

    UV-irradiated plasmid pNov1 containing a cloned fragment of chromosomal DNA could be repaired by excision, but plasmid p2265 without homology to the chromosome could not. Establishment of pNov1 was more UV resistant in Rec/sup -/ than in Rec/sup +/ cells. 19 references, 2 figures.

  3. Homology of plasmids in strains of unicellular cyanobacteria

    NARCIS (Netherlands)

    Hondel, C.A.M.J.J. van den; Keegstra, W.; Borrias, W.E.; Arkel, G.A. van

    1979-01-01

    Six strains of unicellular cyanobacteria were examined for the presence of plasmids. Analysis of lysates of these strains by CsCl-ethidium bromide density centrifugation yielded a major chromosomal DNA band and a minor band containing covalently closed circular plasmid DNA, as shown by electron micr

  4. Construction of a novel kind of expression plasmid by homologous recombination in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    CHEN Xiangling; YUAN Hanying; HE Wei; HU Xianghua; LU Hong; LI Yuyang

    2005-01-01

    Based on a previously used plasmid pHC11, a new plasmid pHC11R was constructed. Cutting plasmid pHC11R with proper restriction enzymes, the resulting larger DNA fragment pHC11R' was co-transformed with a PCR amplified expression cassette of human IFNα2b into yeast. By means of the homologous sequences at both ends of two DNA fragments, a novel expression plasmid pHC11R-IFNα2b was formed via homologous recombination in the yeast. Compared with pHC11-IFNα2b, the expression plasmid pHC11R-IFNα2b was smaller in size and in absence of antibiotic resistant gene. The stability and copy number of pHC11R- IFNα2b were greatly increased and the expression level of heterologous protein was improved. As the derivatives of pHC11R, a series of recombination expression vectors pHRs containing different combination of expression elements were developed. This led to a rapid and powerful method for cloning and expressing of different genes in yeast.

  5. Mitochondrial pAL2-1 plasmid homologs are senescence factors in Podospora anserina independent of intrinsic senescence

    NARCIS (Netherlands)

    Diepeningen, van A.D.; Debets, A.J.M.; Slakhorst-Wandel, S.M.; Hoekstra, R.F.

    2008-01-01

    Since the first description of a linear mitochondrial plasmid in Podospora anserina, pAL2-1, and homologous plasmids have gone from being considered beneficial longevity plasmids, via neutral genetic elements, toward mutator plasmids causing senescence. The plasmid has an invertron structure, with t

  6. Mitochondrial pAL2-1 plasmid homologs are senescence factors in Podospora anserina independent of intrinsic senescence

    NARCIS (Netherlands)

    Diepeningen, van A.D.; Debets, A.J.M.; Slakhorst-Wandel, S.M.; Hoekstra, R.F.

    2008-01-01

    Since the first description of a linear mitochondrial plasmid in Podospora anserina, pAL2-1, and homologous plasmids have gone from being considered beneficial longevity plasmids, via neutral genetic elements, toward mutator plasmids causing senescence. The plasmid has an invertron structure, with

  7. Mitochondrial pAL2-1 plasmid homologs are senescence factors in Podospora anserina independent of intrinsic senescence

    NARCIS (Netherlands)

    van Diepeningen, Anne D; Debets, Alfons J M; Slakhorst, S Marijke; Hoekstra, Rolf F

    Since the first description of a linear mitochondrial plasmid in Podospora anserina, pAL2-1, and homologous plasmids have gone from being considered beneficial longevity plasmids, via neutral genetic elements, toward mutator plasmids causing senescence. The plasmid has an invertron structure, with

  8. Transformation of Azotobacter vinelandii OP with a broad host range plasmid containing a cloned chromosomal nif-DNA marker.

    Science.gov (United States)

    Bingle, W H

    1988-05-01

    The non-nitrogen-fixing (Nif-) strain UW10 of Azotobacter vinelandii OP (UW) was naturally induced to competence and transformed with broad host range plasmid pKT210 containing the cloned wild-type nif-10 locus from A. vinelandii UW (Nif+); this marker was unable to complement the nif-10 mutation in trans, but could through recombination with the chromosome. The most frequent type of transformation event observed was recombination between the homologous regions of the plasmid and chromosome (producing Nif+ transformants) with loss of the plasmid vector. At a substantially lower frequency, transformants expressing the plasmid-encoded antibiotic resistance determinants were isolated which were phenotypically Nif-. Agarose gel electrophoresis showed that these transformants contained a plasmid migrating with the same mobility as the original donor plasmid. During culture these transformants acquired a Nif+ phenotype without the loss of the plasmid, as judged by the use of a hybridization probe specific for the cloned nif-DNA fragment. These data indicate that plasmids carrying sequences homologous to chromosomal sequences could be maintained in recombination-proficient A. vinelandii UW. The introduction of plasmids containing sequences homologous to chromosomal sequences was facilitated by prelinearization of the plasmid using a restriction endonuclease generating cohesive ends. Because the site of linearization could be chosen outside the region of shared homology, it was unlikely that the route of plasmid establishment occurred via a homology-facilitated transformation mechanism. The data also indicated that A. vinelandii UW could harbor broad host range cloning vectors based on plasmid RSF1010 without significant impairment of its nitrogen-fixation ability.

  9. Analysis of plasmid genes by phylogenetic profiling and visualization of homology relationships using Blast2Network

    Directory of Open Access Journals (Sweden)

    Bazzicalupo Marco

    2008-12-01

    Full Text Available Abstract Background Phylogenetic methods are well-established bioinformatic tools for sequence analysis, allowing to describe the non-independencies of sequences because of their common ancestor. However, the evolutionary profiles of bacterial genes are often complicated by hidden paralogy and extensive and/or (multiple horizontal gene transfer (HGT events which make bifurcating trees often inappropriate. In this context, plasmid sequences are paradigms of network-like relationships characterizing the evolution of prokaryotes. Actually, they can be transferred among different organisms allowing the dissemination of novel functions, thus playing a pivotal role in prokaryotic evolution. However, the study of their evolutionary dynamics is complicated by the absence of universally shared genes, a prerequisite for phylogenetic analyses. Results To overcome such limitations we developed a bioinformatic package, named Blast2Network (B2N, allowing the automatic phylogenetic profiling and the visualization of homology relationships in a large number of plasmid sequences. The software was applied to the study of 47 completely sequenced plasmids coming from Escherichia, Salmonella and Shigella spps. Conclusion The tools implemented by B2N allow to describe and visualize in a new way some of the evolutionary features of plasmid molecules of Enterobacteriaceae; in particular it helped to shed some light on the complex history of Escherichia, Salmonella and Shigella plasmids and to focus on possible roles of unannotated proteins. The proposed methodology is general enough to be used for comparative genomic analyses of bacteria.

  10. Integration of a pAL2-1 homologous mitochondrial plasmid associated with life span extension in Podospora anserina

    NARCIS (Netherlands)

    Maas, M.F.P.M.; Sellem, C.H.; Rincheval, V.; Hoekstra, R.F.; Debets, A.J.M.; Sainsard-Chanet, A.

    2007-01-01

    We isolated and characterized a novel spontaneous longevity mutant of Podospora anserina strain Wa32 carrying one of the pAL2-1 homologous mitochondrial plasmids. This mutant is at least ten fold longer-lived than the wild type, and is hence a formal suppressor of both the regular and the `plasmid-b

  11. Integration of a pAL2-1 homologous mitochondrial plasmid associated with life span extension in Podospora anserina

    NARCIS (Netherlands)

    Maas, M.F.P.M.; Sellem, C.H.; Rincheval, V.; Hoekstra, R.F.; Debets, A.J.M.; Sainsard-Chanet, A.

    2007-01-01

    We isolated and characterized a novel spontaneous longevity mutant of Podospora anserina strain Wa32 carrying one of the pAL2-1 homologous mitochondrial plasmids. This mutant is at least ten fold longer-lived than the wild type, and is hence a formal suppressor of both the regular and the `plasmid-b

  12. Differential behavior of plasmids containing chromosomal DNA insertions of various sizes during transformation and conjugation in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Balganesh, M.; Setlow, J.K.

    1985-01-01

    Plasmids with chromosomal insertions were constructed by removal of a 1.1-kilobase-pair piece from the 9.8-kilobase-pair vector plasmid pDM2 by EcoRI digestion and inserting in its place various lengths of chromosomal DNA (1.7, 3.4, and 9.0 kilobase pairs) coding for resistance to novobiocin. A fourth plasmid was constructed by insertion of the largest piece of chromosomal DNA into the SmaI site of pDM2. The plasmids without inserts were taken up poorly by competent cells and thus were considered not to contain specific DNA uptake sites. The presence of even the smallest insert of chromosomal DNA caused a large increase in transformation of Rec/sup +/ and Rec/sup -/ strains. The frequency of plasmid establishment in Rec/sup +/ cells by transformation increased exponentially with increasing insert size, but in Rec/sup -/ cells there was less transformation by the larger plasmids. Conjugal transfer of these plasmids was carried out with the 35-kilobase-pair mobilizing plasmid pHD147. The frequency of establishment of plasmids by this method not only was not markedly affected by the presence of the insertions, but also decreased somewhat with increase in insert size and was independent of rec-1 and rec-2 genes. Recombination between plasmid and chromosome was readily detected after transformation, but could not be detected after transconjugation even when the recipient cells were Rec/sup +/ and made competent. These data suggested that there is a special processing of plasmid DNA that enters the competent cells in transformation that makes possible recombination of homologous regions of the plasmid with the chromosome and pairing with the chromosome that aids plasmid establishment.

  13. Construction of a novel kind of expression plasmid by homologous recombination in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    CHEN; Xiangling

    2005-01-01

    [1]Brunelli, J. P., Pall, M. L., A series of yeast vectors for expression of cDNAs and other DNA sequences, Yeast, 1993, 9: 1299―1308.[2]Sikorski, R. S., Hieter, P., A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae, Genetics, 1989, 122: 19―27.[3]Bonneaud, N., Ozier-Kalogerogoulos, O., Li, G. et al., A family of low and high copy replicative, integrative and single-stranded S. cerevisiae /E. coli shuttle vector, Yeast, 1991, 7: 609―615.[4]Huo, K. K., Yu, L. L., Chen, X. J., Li, Y. Y., A stable vector for high-level expression and secretion of human interferon alpha A in yeast, Science in China, Ser. B, 1993, 36(5): 557―567.[5]Zhou, Z. X., Yuan, H. Y., He, W. et al., Expression of the modified HBsAg gene SA-28 directed by a constitutive promoter, Journal of Fudan university (Natural Science), 2000, 39(3): 264―268.[6]Paques, F., Haber, J. E., Multiple pathways of recombination induces by double-strand breaks in Saccharomyces cerevisiae, Microbiology and Molecular Biology Reviews, 1999, 63(2): 349―404.[7]Martin, K., Damage-induced recombination in the yeast Saccharomyces cerevisiae, Mutation Research, 2000, 451: 91―105.[8]Alira, S., Tomoko, O., Homologous recombination and the roles of double-strand breaks, TIBS, 1995, 20: 387―391.[9]Patrick, S., Kelly, M. T., Stephen, V. K., Recombination factor of Saccharomyces cerevisiae, Mutation Research, 2000, 451: 257―275.[10]Manivasakam, P., Weber, S. C., McElver, J., Schiestl, R. H., Micro-homology mediated PCR targeting in Saccharomyces cerevisiae, Nucleic Acids Res., 1995, 23(14): 2799―2800.[11]Baudin, A., Lacroute, F., Cullin, C., A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae, Nucleic Acids Res., 1993, 21(14): 3329―3330.[12]Hua, S. B., Qiu, M., Chan, E., Zhu, L., Luo, Y., Minimum length of sequence homology required for in vivo cloning by homolo-gous recombination in yeast, Plasmid, 1997, 38

  14. Construction of Recombinant Plasmid Containing S. Mutans F-ATPase β Subunit Gene

    Institute of Scientific and Technical Information of China (English)

    YU Dan-ni; JIANG Li

    2005-01-01

    objective: construct a homologous recombinant plasmid which was expected to be transformed into S. mutans Methods: a region at the 5' terminus of the S. mutans F-ATPase β subunit gene was amplified by PCR, the PCR product was inserted into vector pVA891, yielding recombinant plasmid. Results: the DNA sequence of the recombinant plasmid was identified correct in whole by restriction endonuclease and DNA sequence techniques. Conclusion: the recombinant plasmid of S. mutans DNA was cloned in effect ,it may assist in construction of homologues recombinant mutant.

  15. Bacillus stearothermophilus contains a plasmid-borne gene for alpha-amylase.

    Science.gov (United States)

    Mielenz, J R

    1983-01-01

    The gene for thermostable alpha-amylase from the thermophilic bacterium Bacillus stearothermophilus has been cloned and expressed in Escherichia coli. Each alpha-amylase-producing colony contained at least a 9.7-kilobase-pair (kb) chimeric plasmid composed of the vector pBR322 and a common 5.4-kb HindIII fragment of DNA. B. stearothermophilus contains four plasmids with sizes from 12 kb to over 108 kb. Restriction endonuclease analysis of these naturally occurring plasmids showed they also contain a 5.4-kb HindIII fragment of DNA. Cloning experiments with the four plasmids yielded alpha-amylase-producing E. coli that contained the same 9.7-kb chimeric plasmid. Restriction endonuclease analysis and further recombinant DNA experiments identified a 26-kb plasmid that contains the gene for alpha-amylase. A spontaneous mutant of B. stearothermophilus unable to produce alpha-amylase was missing the 26-kb plasmid but contained a 20-kb plasmid. A 6-kb deletion within the region of the 5.4-kb HindIII fragment yielded the 20-kb plasmid unable to code for alpha-amylase. A nick-translated probe for the alpha-amylase coding region did not hybridize to either plasmid or total cellular DNA from this mutant strain of B. stearothermophilus. These results demonstrate the gene for alpha-amylase is located exclusively on a 26-kb plasmid in B. stearothermophilus with no genetic counterpart present on the chromosome. Images PMID:6193526

  16. [Effect of endonuclease G depletion on plasmid DNA uptake and levels of homologous recombination in hela cells].

    Science.gov (United States)

    Misic, V; El-Mogy, M; Geng, S; Haj-Ahmad, Y

    2016-01-01

    Endonuclease G (EndoG) is a mitochondrial apoptosis regulator that also has roles outside of programmed cell death. It has been implicated as a defence DNase involved in the degradation of exogenous DNA after transfection of mammalian cells and in homologous recombination of viral and endogenous DNA. In this study, we looked at the effect of EndoG depletion on plasmid DNA uptake and the levels of homologous recombination in HeLa cells. We show that the proposed defence role of EndoG against uptake of non-viral DNA vectors does not extend to the cervical carcinoma HeLa cells, as targeting of EndoG expression by RNA interference failed to increase intracellular plasmid DNA levels. However, reducing EndoG levels in HeLa cells resulted in a statistically significant reduction of homologous recombination between two plasmid DNA substrates. These findings suggest that non-viral DNA vectors are also substrates for EndoG in its role in homologous recombination.

  17. THE ENDOGENOUS BACILLUS-SUBTILIS (NATTO) PLASMIDS PTA1015 AND PTA1040 CONTAIN SIGNAL PEPTIDASE-ENCODING GENES - IDENTIFICATION OF A NEW STRUCTURAL MODULE ON CRYPTIC PLASMIDS

    NARCIS (Netherlands)

    MEIJER, WJJ; DEJONG, A; BEA, G; WISMAN, A; TJALSMA, H; VENEMA, G; BRON, S; MAARTEN, J; VANDIJL, JM

    Various strains of Bacillus subtilis (natto) contain small cryptic plasmids that replicate via the rolling-circle mechanism. Like plasmids from other Gram-positive bacteria, these plasmids are composed of several distinct structural modules. A new structural module was identified on the B. subtilis

  18. Plasmid containing a DNA ligase gene from Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.; Griffin, K.; Setlow, J.K.

    1984-05-01

    A ligase gene from Haemophilus influenzae was cloned into the shuttle vector pDM2. Although the plasmid did not affect X-ray sensitivity, it caused an increase in UV sensitivity of the wild-type but not excision-defective H. influenzae and a decrease in UV sensitivity of the rec-1 mutant. 14 references, 2 figures.

  19. Streptomyces linear plasmids that contain a phage-like, centrally located, replication origin.

    Science.gov (United States)

    Chang, P C; Kim, E S; Cohen, S N

    1996-12-01

    Unlike previously studied linear replicons containing 5' DNA termini covalently bound to protein, pSLA2, a 17 kb linear plasmid of Streptomyces rochei, initiates replication internally rather than at the telomeres (Chang and Cohen, 1994). Here we identify and characterize the replication origin of pSLA2, showing that it contains a series of direct repeats (iterons) within a centrally located gene encoding an essential DNA-binding protein (Rep1); a second essential protein (Rep2), which resembles prokaryotic DNA helicases and has ATPase activity stimulated by single-stranded DNA, is expressed from the same transcript. A 430 bp locus separated by almost 2 kb from the iterons of the origin specifies an as yet undefined additional function required in cis for plasmid replication. pSCL, a 12 kb linear plasmid of Streptomyces clavuligerus, contains, near the centre of the plasmid, a region configured like the pSLA2 origin. The replication regions of pSLA2 and pSCL, which are capable of propagating plasmid DNA in either a circular or linear form (Shiffman and Cohen, 1992; Chang and Cohen, 1994) resemble those of temperate bacteriophages of the Enterobacteriacae and Bacillus. Our observations suggest that Streptomyces linear plasmids may occupy an evolutionarily intermediate position between circular plasmids and linear phage replicons.

  20. Transformation of indica rice with plasmid pBGll21 containing a tobacco endo-chitinase gene I

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    @@ Several plasmids, which were suitable for cereals transformation, have been reported. In the study, rice was transformed by a new plasmid pBGll21 containing a tobacco endo-chitinase gene ( TchiB ).

  1. High-throughput plasmid construction using homologous recombination in yeast: its mechanisms and application to protein production for X-ray crystallography.

    Science.gov (United States)

    Mizutani, Kimihiko

    2015-01-01

    Homologous recombination is a system for repairing the broken genomes of living organisms by connecting two DNA strands at their homologous sequences. Today, homologous recombination in yeast is used for plasmid construction as a substitute for traditional methods using restriction enzymes and ligases. This method has various advantages over the traditional method, including flexibility in the position of DNA insertion and ease of manipulation. Recently, the author of this review reported the construction of plasmids by homologous recombination in the methanol-utilizing yeast Pichia pastoris, which is known to be an excellent expression host for secretory proteins and membrane proteins. The method enabled high-throughput construction of expression systems of proteins using P. pastoris; the constructed expression systems were used to investigate the expression conditions of membrane proteins and to perform X-ray crystallography of secretory proteins. This review discusses the mechanisms and applications of homologous recombination, including the production of proteins for X-ray crystallography.

  2. Localization and physical mapping of a plasmid-borne 23-kb nif gene cluster from Enterobacter agglomerans showing homology to the entire nif gene cluster of Klebsiella pneumoniae M5a1.

    Science.gov (United States)

    Singh, M; Kreutzer, R; Acker, G; Klingmüller, W

    1988-01-01

    A physical and genetical map of the plasmid pEA3 indigenous to Enterobacter agglomerans is presented. pEA3 is a 111-kb large plasmid containing a 23-kb large cluster of nif genes which shows extensive homology (Southern hybridization and heteroduplex analysis) to the entire nif gene cluster of Klebsiella pneumoniae (Kp) M5a1. All the nif genes on pEA3 are organized in the same manner as in K. pneumoniae, except nifJ, which is located on the left end of pEA3 nif gene cluster (near nifQB). A BamHI restriction map of pEA3 and a detailed restriction map of the 23-kb nif region on pEA3 is also presented. The nif genes of pEA3 showed a low level of acetylene reduction in Escherichia coli, demonstrating that these genes are functional and contain the whole genetic information required to fix nitrogen. The origin of vegetative replication (OriV) of pEA3 was localized about 5.5 kb from the right end of the nif gene cluster. In addition to pEA3, large plasmids from four other strains of E. agglomerans showed homology to all the Kp nif genes tested, indicating that in diazotrophic strains of E. agglomerans nif genes are usually located on plasmids. In contrast, in most of the free-living, nitrogen-fixing bacteria the nif genes are on chromosome.

  3. In vivo transmission of a plasmid containing the KPC-2 gene in a single patient.

    Science.gov (United States)

    Galani, Irene; Panagea, Theofano; Chryssouli, Zoi; Giamarellou, Helen; Souli, Maria

    2013-03-01

    Here we describe a case of in vivo horizontal interspecies transmission of a KPC-2-producing plasmid from a Klebsiella pneumoniae to an Enterobacter aerogenes strain in the same patient. The patient's gut flora initially contained a carbapenem-susceptible E. aerogenes strain and 10 days after admission a KPC-2-positive K. pneumoniae. Three months after admission, a KPC-2-positive E. aerogenes was identified in fecal surveillance cultures. This isolate was isogenic with the initial E. aerogenes and contained a KPC-2-coding plasmid identical to that of the K. pneumoniae. The patient developed bacteraemia by the KPC-2-positive K. pneumoniae 17 days after her first colonization. In vivo horizontal transmission of blaKPC-carrying plasmids between bacterial species underscores the importance of antibiotic stewardship along with implementation of infection control measures for the containment of KPC-producers.

  4. Construction of a recombinant bacterial plasmid containing DNA sequences for a mouse embryonic globin chain.

    Science.gov (United States)

    Fantoni, A; Bozzoni, I; Ullu, E; Farace, M G

    1979-08-10

    Messenger RNAs for mouse embryonic globins were purified from yolk sac derived eyrthroid cells in mouse fetuses. Double stranded DNAs complementary to these messengers were synthesized and blunt end ligated to a EcoRI digested and DNA polymerase I repaired pBR322 plasmid. Of the ampicillin resistant transformants, one contained a plasmid with globin-specific cDNA. The inserted sequence is about 350 base pairs long. It contains one restriction site for EcoRI and one restriction site for HinfI about 170 and 80 base pairs from one end. The insert is not cleaved by HindIII, HindII, BamHI, PstI, SalI, AvaI, TaqI, HpaII, BglI. A mixture of purified messengers coding for alpha chains and for x, y and z embryonic chains was incubated with the recombinant plasmid and the hybridized messenger was translated in a mRNA depleted reticulocyte lysate protein synthesizing system. The product of translation was identified as a z chain by carboxymethylcellulose cromatography. The recombinant plasmid is named "pBR322-egz" after embryonic globin z.

  5. Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids

    Directory of Open Access Journals (Sweden)

    Tetsushi Sakuma

    2015-10-01

    Full Text Available Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc gene, in Chinese hamster ovary (CHO cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins.

  6. In vitro replication of plasmids containing human ribosomal gene sequences: origin localization and dependence on an aprotinin-binding cytosolic protein.

    Science.gov (United States)

    Coffman, F D; Georgoff, I; Fresa, K L; Sylvester, J; Gonzalez, I; Cohen, S

    1993-11-01

    We previously investigated the role of an aprotinin-binding protein (ADR) in the initiation of DNA replication in isolated quiescent nuclei. In the present study, we have used a cell-free DNA replication system to test the ability of plasmid vectors which contain sequences from the human ribosomal RNA gene to serve as replicative templates in vitro when exposed to ADR-containing preparations. Significant dTTP incorporation was seen using DNA from either a 7-kb sequence in the 5' spacer region (CHE) or a 7-kb sequence which begins near the end of the 28S coding region and extends into the 3' spacer region (ADBB), while sequences from other regions of the rRNA gene mediated little or no dTTP incorporation. The characteristics of plasmid-directed dTTP incorporation indicate that most incorporation is due to DNA replication and not repair or damage-initiated processes. To conclusively demonstrate origin-dependent replication in the plasmid system and to further map replication origins, an approach was developed using ddGTP to restrict the length of daughter strands followed by hybridization of these replication products to restriction fragments spanning the putative origin region. This approach allowed us to identify replication origin activity apart from parent strand repair or synthesis initiated at random damaged sites. One of the origins was localized to a 1375-bp fragment within the 5' spacer region, and this fragment contains sequences homologous to those found in other replication origins.

  7. Intergenic regions of Borrelia plasmids contain phylogenetically conserved RNA secondary structure motifs

    Directory of Open Access Journals (Sweden)

    Delihas Nicholas

    2009-03-01

    Full Text Available Abstract Background Borrelia species are unusual in that they contain a large number of linear and circular plasmids. Many of these plasmids have long intergenic regions. These regions have many fragmented genes, repeated sequences and appear to be in a state of flux, but they may serve as reservoirs for evolutionary change and/or maintain stable motifs such as small RNA genes. Results In an in silico study, intergenic regions of Borrelia plasmids were scanned for phylogenetically conserved stem loop structures that may represent functional units at the RNA level. Five repeat sequences were found that could fold into stable RNA-type stem loop structures, three of which are closely linked to protein genes, one of which is a member of the Borrelia lipoprotein_1 super family genes and another is the complement regulator-acquiring surface protein_1 (CRASP-1 family. Modeled secondary structures of repeat sequences display numerous base-pair compensatory changes in stem regions, including C-G→A-U transversions when orthologous sequences are compared. Base-pair compensatory changes constitute strong evidence for phylogenetic conservation of secondary structure. Conclusion Intergenic regions of Borrelia species carry evolutionarily stable RNA secondary structure motifs. Of major interest is that some motifs are associated with protein genes that show large sequence variability. The cell may conserve these RNA motifs whereas allow a large flux in amino acid sequence, possibly to create new virulence factors but with associated RNA motifs intact.

  8. The Completely Sequenced Plasmid pEST4011 Contains a Novel IncP1 Backbone and a Catabolic Transposon Harboring tfd Genes for 2,4-Dichlorophenoxyacetic Acid Degradation

    Science.gov (United States)

    Vedler, Eve; Vahter, Merle; Heinaru, Ain

    2004-01-01

    The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium Achromobacter xylosoxidans subsp. denitrificans strain EST4002 contains plasmid pEST4011. This plasmid ensures its host a stable 2,4-D+ phenotype. We determined the complete 76,958-bp nucleotide sequence of pEST4011. This plasmid is a deletion and duplication derivative of pD2M4, the 95-kb highly unstable laboratory ancestor of pEST4011, and was self-generated during different laboratory manipulations performed to increase the stability of the 2,4-D+ phenotype of the original strain, strain D2M4(pD2M4). The 47,935-bp catabolic region of pEST4011 forms a transposon-like structure with identical copies of the hybrid insertion element IS1071::IS1471 at the two ends. The catabolic regions of pEST4011 and pJP4, the best-studied 2,4-D-degradative plasmid, both contain homologous, tfd-like genes for complete 2,4-D degradation, but they have little sequence similarity other than that. The backbone genes of pEST4011 are most similar to the corresponding genes of broad-host-range self-transmissible IncP1 plasmids. The backbones of the other three IncP1 catabolic plasmids that have been sequenced (the 2,4-D-degradative plasmid pJP4, the haloacetate-catabolic plasmid pUO1, and the atrazine-catabolic plasmid pADP-1) are nearly identical to the backbone of R751, the archetype plasmid of the IncP1 β subgroup. We show that despite the overall similarity in plasmid organization, the pEST4011 backbone is sufficiently different (51 to 86% amino acid sequence identity between individual backbone genes) from the backbones of members of the three IncP1 subgroups (the α, β, and γ subgroups) that it belongs to a new IncP1subgroup, the δ subgroup. This conclusion was also supported by a phylogenetic analysis of the trfA2, korA, and traG gene products of different IncP1 plasmids. PMID:15489427

  9. Homology between nucleotide sequences of promoter regions of nah and sal operons of NAH7 plasmid of Pseudomonas putida.

    OpenAIRE

    1986-01-01

    The in vivo transcription start sites of the nah and sal operons of the NAH7 plasmid were determined by S1 nuclease mapping and the nucleotide sequence surrounding these transcription start sites was determined. Since expression of both of these operons is coordinately controlled by the product of the transcriptional activator gene nahR, the sequences were compared to locate potential sites involved in common regulation. In the 100-base-pair region preceding transcription start sites of both ...

  10. Dothistroma pini, a Forest Pathogen, Contains Homologs of Aflatoxin Biosynthetic Pathway Genes

    OpenAIRE

    2002-01-01

    Homologs of aflatoxin biosynthetic genes have been identified in the pine needle pathogen Dothistroma pini. D. pini produces dothistromin, a difuranoanthraquinone toxin with structural similarity to the aflatoxin precursor versicolorin B. Previous studies with purified dothistromin suggest a possible role for this toxin in pathogenicity. By using an aflatoxin gene as a hybridization probe, a genomic D. pini clone was identified that contained four dot genes with similarity to genes in aflatox...

  11. Homologous recombination in Leishmania enriettii.

    Science.gov (United States)

    Tobin, J F; Laban, A; Wirth, D F

    1991-02-01

    We have used derivatives of the recently developed stable transfection vector pALT-Neo to formally demonstrate that Leishmania enriettii contains the enzymatic machinery necessary for homologous recombination. This observation has implications for gene regulation, gene amplification, genetic diversity, and the maintenance of tandemly repeated gene families in the Leishmania genome as well as in closely related organisms, including Trypanosoma brucei. Two plasmids containing nonoverlapping deletions of the chloramphenicol acetyltransferase (CAT) gene, as well as the neomycin-resistance gene, were cotransfected into L. enriettii. Analysis of the DNA from these cells by Southern blotting and plasmid rescue revealed that a full-length or doubly deleted CAT gene could be reconstructed by homologous crossing-over and/or gene conversion between the two deletion plasmids. Additionally, parasites cotransfected with pALT-Neo and pALT-CAT-S, a plasmid containing two copies of the chimeric alpha-tubulin-CAT gene, resulted in G418-resistant parasites expressing high levels of CAT activity. The structure of the DNA within these cells, as shown by Southern blot analysis and the polymerase chain reaction, is that which would be expected from a homologous exchange event occurring between the two plasmids.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  13. Recombination frequency in plasmid DNA containing direct repeats--predictive correlation with repeat and intervening sequence length.

    Science.gov (United States)

    Oliveira, Pedro H; Lemos, Francisco; Monteiro, Gabriel A; Prazeres, Duarte M F

    2008-09-01

    In this study, a simple non-linear mathematical function is proposed to accurately predict recombination frequencies in bacterial plasmid DNA harbouring directly repeated sequences. The mathematical function, which was developed on the basis of published data on deletion-formation in multicopy plasmids containing direct-repeats (14-856 bp) and intervening sequences (0-3872 bp), also accounts for the strain genotype in terms of its recA function. A bootstrap resampling technique was used to estimate confidence intervals for the correlation parameters. More than 92% of the predicted values were found to be within a pre-established +/-5-fold interval of deviation from experimental data. The correlation does not only provide a way to predict, with good accuracy, the recombination frequency, but also opens the way to improve insight into these processes.

  14. Homologous recombination in Leishmania enriettii.

    OpenAIRE

    1991-01-01

    We have used derivatives of the recently developed stable transfection vector pALT-Neo to formally demonstrate that Leishmania enriettii contains the enzymatic machinery necessary for homologous recombination. This observation has implications for gene regulation, gene amplification, genetic diversity, and the maintenance of tandemly repeated gene families in the Leishmania genome as well as in closely related organisms, including Trypanosoma brucei. Two plasmids containing nonoverlapping del...

  15. Improving the safety of viral DNA vaccines: development of vectors containing both 5' and 3' homologous regulatory sequences from non-viral origin.

    Science.gov (United States)

    Martinez-Lopez, A; Encinas, P; García-Valtanen, P; Gomez-Casado, E; Coll, J M; Estepa, A

    2013-04-01

    Although some DNA vaccines have proved to be very efficient in field trials, their authorisation still remains limited to a few countries. This is in part due to safety issues because most of them contain viral regulatory sequences to driving the expression of the encoded antigen. This is the case of the only DNA vaccine against a fish rhabdovirus (a negative ssRNA virus), authorised in Canada, despite the important economic losses that these viruses cause to aquaculture all over the world. In an attempt to solve this problem and using as a model a non-authorised, but efficient DNA vaccine against the fish rhabdovirus, viral haemorrhagic septicaemia virus (VHSV), we developed a plasmid construction containing regulatory sequences exclusively from fish origin. The result was an "all-fish vector", named pJAC-G, containing 5' and 3' regulatory sequences of β-acting genes from carp and zebrafish, respectively. In vitro and in vivo, pJAC-G drove a successful expression of the VHSV glycoprotein G (G), the only antigen of the virus conferring in vivo protection. Furthermore, and by means of in vitro fusion assays, it was confirmed that G protein expressed from pJAC-G was fully functional. Altogether, these results suggest that DNA vaccines containing host-homologous gene regulatory sequences might be useful for developing safer DNA vaccines, while they also might be useful for basic studies.

  16. Nap1 stimulates homologous recombination by RAD51 and RAD54 in higher-ordered chromatin containing histone H1.

    Science.gov (United States)

    Machida, Shinichi; Takaku, Motoki; Ikura, Masae; Sun, Jiying; Suzuki, Hidekazu; Kobayashi, Wataru; Kinomura, Aiko; Osakabe, Akihisa; Tachiwana, Hiroaki; Horikoshi, Yasunori; Fukuto, Atsuhiko; Matsuda, Ryo; Ura, Kiyoe; Tashiro, Satoshi; Ikura, Tsuyoshi; Kurumizaka, Hitoshi

    2014-05-06

    Homologous recombination plays essential roles in mitotic DNA double strand break (DSB) repair and meiotic genetic recombination. In eukaryotes, RAD51 promotes the central homologous-pairing step during homologous recombination, but is not sufficient to overcome the reaction barrier imposed by nucleosomes. RAD54, a member of the ATP-dependent nucleosome remodeling factor family, is required to promote the RAD51-mediated homologous pairing in nucleosomal DNA. In higher eukaryotes, most nucleosomes form higher-ordered chromatin containing the linker histone H1. However, the mechanism by which RAD51/RAD54-mediated homologous pairing occurs in higher-ordered chromatin has not been elucidated. In this study, we found that a histone chaperone, Nap1, accumulates on DSB sites in human cells, and DSB repair is substantially decreased in Nap1-knockdown cells. We determined that Nap1 binds to RAD54, enhances the RAD54-mediated nucleosome remodeling by evicting histone H1, and eventually stimulates the RAD51-mediated homologous pairing in higher-ordered chromatin containing histone H1.

  17. Characterization of the replication and stability regions of Agrobacterium tumefaciens plasmid pTAR.

    Science.gov (United States)

    Gallie, D R; Zaitlin, D; Perry, K L; Kado, C I

    1984-03-01

    A 5.4-kilobase region containing the origin of replication and stability maintenance of the 44-kilobase Agrobacterium tumefaciens plasmid pTAR has been mapped and characterized. Within this region is a 1.3-kilobase segment that is capable of directing autonomous replication. The remaining segment contains the stability locus for maintenance of pTAR during nonselective growth. Approximately 35% of pTAR shares sequence homology with pAg119, a 44-kilobase cryptic plasmid in grapevine strain 1D1119. However, no homology was detected between pTAR DNA and several Ti plasmids or several other small cryptic plasmids in many A. tumefaciens strains. A recombinant plasmid containing the origin of replication and stability maintenance region of pTAR was compatible with pTiC58, pTi15955, and pTi119 and incompatible with pAg119. A new compatibility group, Inc Ag-1, is discussed.

  18. Preparation and characterization of chitosan/β-cyclodextrin nanoparticles containing plasmid DNA encoding interleukin-12.

    Science.gov (United States)

    Nahaei, M; Valizadeh, H; Baradaran, B; Nahaei, M R; Asgari, D; Hallaj-Nezhadi, S; Dastmalchi, S; Lotfipour, F

    2013-01-01

    Interleukin-12 (IL-12) as a cytokine has been proved to possess antitumor effects via stimulating the immune system. Non-viral gene delivery systems offer several advantages, including easiness in production, low cost, safety; low immunogenicity and can carry higher amounts of genetic material without limitation on their sizes.pUMVC3-hIL12 loaded Low Molecular Weight chitosan/β-cyclodextrin (LMW CS/CD) nanoparticles were prepared using ionotropic gelation method and characterized in terms of size, zeta potential, polydispersity index, morphology, loading efficiency and cytotoxicity against the CT-26 colon carcinoma cell line.All prepared particles were spherical in shape and nano-sized (171.3±2.165 nm, PdI: 0.231±0.014) and exhibited a positive zeta potential (34.3±1.55). The nanoparticles demonstrated good DNA encapsulation efficiencies (83.315%±2.067). Prepared pUMVC3-hIL12 loaded LMW CS/CD nanoparticles showed no cell toxicity in murine CT-26 colon carcinoma cells. At the concentration of 0.1 µg/ml of nanoparticles, the transfection ability was obviously higher than that of the naked DNA.LMW CS/CD-plasmid DNA nanoparticles encoding IL-12 prepared using ionotropic gelation method with no toxic effect on the tested cells can be considered as a basis for further gene delivery studies both in vitro and in vivo to enhance the expression of IL-12.

  19. Recombinogenic engineering of conjugative plasmids with fluorescent marker cassettes

    DEFF Research Database (Denmark)

    Reisner, A.; Molin, Søren; Zechner, E.L.

    2002-01-01

    functions using very short regions of homology. Initial manipulation of the IncFII target plasmids R1 and R1drd19 indicated that the linear targeting DNA should be devoid of all extraneous homologies to. the target molecule for optimal insertion specificity. Indeed, a simple recombination assay proved...... that in the presence of additional homologous regions in the targeting DNA, strand exchanges occurred exclusively within the longest regions of homology. A versatile panel of vectors was created to facilitate convenient PCR amplification of targeting DNAs containing various combinations of different antibiotic...

  20. Enhanced cardioprotective effects mediated by plasmid containing the short-hairpin RNA of angiotensin converting enzyme with a biodegradable hydrogel after myocardial infarction.

    Science.gov (United States)

    Wan, Wei-Guo; Jiang, Xue-Jun; Li, Xiao-Yan; Zhang, Cui; Yi, Xin; Ren, Shan; Zhang, Xian-Zheng

    2014-10-01

    The expression of foreign gene was enhanced and prolonged by sustained releasing a target gene to cells from biodegradable dextran-poly(e-caprolactone)-2-hydroxylethylmethacrylate-poly(N-isopropylacrylamide) (Dex-PCL-HEMA/PNIPAAm) hydrogel in vitro. Moreover, we have demonstrated that injection of the same hydrogel improved post-infarct ventricular remodeling. Therefore, we hypothesized that intramyocardial injection of plasmid containing the short-hairpin RNA (shRNA) of angiotensin converting enzyme (ACE) with the same hydrogel enhances the cardioprotective effects superior to either alone or after rat myocardial infarction (MI). In this study, equal volume of phosphate-buffered solution (PBS), 10 μg ACE-shRNA plasmids, hydrogel containing 10 μg negative control ACE-shRNA plasmids and hydrogel containing 10 μg ACE-shRNA plasmids were shortly injected into the infarct area of rats after MI, respectively. We found that ACE-shRNA plasmid-loaded hydrogel extended the duration of gene expression in vivo. Moreover, it was shown that direct intramyocardial injection of ACE-shRNA plasmid-loaded hydrogel significantly decreased the expression of local ACE expression, inhibited cell apoptosis, reduced infarct size, and improved cardiac function compared with the injection of either alone 30 days after MI in rats. These results suggest that injection of ACE-shRNA plasmid-loaded hydrogel into impaired myocardium obtains more cardioprotective effects than either alone in rat with MI by prolonging the gene silencing of ACE. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 3452-3458, 2014.

  1. Plasmids of the pRM/pRF family occur in diverse Rickettsia species.

    Science.gov (United States)

    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.

  2. Characterization of two novel plasmids from Geobacillus sp. 610 and 1121 strains.

    Science.gov (United States)

    Kananavičiūtė, Rūta; Butaitė, Elena; Citavičius, Donaldas

    2014-01-01

    We describe two cryptic low molecular weight plasmids, pGTD7 (3279bp) and pGTG5 (1540bp), isolated from Geobacillus sp. 610 and 1121 strains, respectively. Homology analysis of the replication protein (Rep) sequences and detection of ssDNA indicate that both of them replicate via rolling circle mechanism. As revealed by sequence similarities of dso region and Rep protein, plasmid pGTD7 belongs to pC194/pUB110 plasmid family. The replicon of pGTD7 was proved to be functional in another Geobacillus host. For this purpose, a construct pUCK7, containing a replicon of the analyzed plasmid, was created and transferred to G. stearothermophilus NUB3621R strain by electroporation. Plasmid pGTG5, based on Rep protein sequence similarity, was found to be related mostly to some poorly characterized bacterial plasmids. Rep proteins encoded by these plasmids contain conservative motifs that are most similar to those of Microviridae phages. This feature suggests that pGTG5, together with other plasmids containing the same motifs, could constitute a new family of bacterial plasmids. To date, pGTG5 is the smallest plasmid identified in bacteria belonging to the genus Geobacillus. The two plasmids described in this study can be used for the construction of new vectors suitable for biotechnologically important bacteria of the genus Geobacillus.

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

    Science.gov (United States)

    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.

  4. CONSTRUCTION AND EXPRESSION OF ADENOASSOCIATED VIRUS- BASED PLASMID EXPRESSING VECTORS CONTAINING hIL- 2 GENE OR mIFN-γ GENE

    Institute of Scientific and Technical Information of China (English)

    张景迎; 梁宏立; 陈诗书

    2000-01-01

    Objective To improve the plasmid vectors in gene therapy, adeno - associated virus (AA V) based plasmid expressing vectors containing hIL-2 gene or mIFN-γ gene were constructed and its expression in transfected cells was studied. Methods By means of step to step cloning, promoter CMVp was placed at the downstream of 5' inverted terminal repeat from AA V (AA V- ITR) of pAP, hIL- 2 gene or mIFN- γ gene inserted into pAC between CMVp and poly A. Then intron A was inserted into pAC- hIL - 2 or pAC- mIFN- γ between CMVp and IL - 2 gene or IFNγ gene to construct pAI- hIL - 2 or pAI- mIFN - γ. Liposome -plasmid complexes were formed by mixing Dosper with these AAV-based plasmids containing hIL-2 gene or mIFN-γgene. Results High biological activities of IL - 2 or IFN- γ could be detected in the supernatants of NIH3T3 and MM45T. Li cells after transfection. Insertion of intron A into pAC-hIL-2 or pAC-mIFN-γ improved the expression of IL- 2 or IFN- γ. Conclusion These data demonstrated that the constructed AA V- based plasmid expressing vectors could efficiently express therapeutic genes in cultured cells and could be used as a nonviral gene transfer system in human gene therapy.

  5. Responses of plasmid-mediated quinolone resistance genes and bacterial taxa to (fluoro)quinolones-containing manure in arable soil.

    Science.gov (United States)

    Xiong, Wenguang; Sun, Yongxue; Ding, Xueyao; Zhang, Yiming; Zhong, Xiaoxia; Liang, Wenfei; Zeng, Zhenling

    2015-01-01

    The aim of the present study was to investigate the fate of plasmid-mediated quinolone resistance (PMQR) genes and the disturbance of soil bacterial communities posed by (fluoro)quinolones (FQNs)-containing manure in arable soil. Representative FQNs (enrofloxacin (ENR), ciprofloxacin (CIP) and norfloxacin (NOR)), PMQR genes (qepA, oqxA, oqxB, aac(6')-Ib-cr and qnrS) and bacterial communities in untreated soil, +manure and +manure+FQNs groups were analyzed using culture independent methods. The significantly higher abundance of oqxA, oqxB and aac(6')-Ib-cr, and significantly higher abundance of qnrS in +manure group than those in untreated soil disappeared at day 30 and day 60, respectively. All PMQR genes (oqxA, oqxB, aac(6')-Ib-cr and qnrS) dissipated 1.5-1.7 times faster in +manure group than those in +manure+FQNs group. The disturbance of soil bacterial communities posed by FQNs-containing manure was also found. The results indicated that significant effects of PMQR genes (oqxA, oqxB, aac(6')-Ib and qnrS) on arable soils introduced by manure disappeared 2 month after manure application. FQNs introduced by manure slowed down the dissipation of PMQR genes. The presence of high FQNs provided a selective advantage for species affiliated to the phylum including Acidobacteria, Verrucomicrobia and Planctomycetes while suppressing Proteobacteria and Actinobacteria.

  6. [A novel Salmonella Typhimurium plasmid, pAnkS: an example for plasmid evolution in antibiotic resistance].

    Science.gov (United States)

    Sahin, Fikret; Karasartova, Djursun; Gerçeker, Devran; Aysev, A Derya; Erdem, Birsel

    2008-07-01

    In this study, a plasmid, carrying ampicillin resistance (ampR) gene, isolated from a clinical isolate of Salmonella enterica serotype Typhimurium presenting ACSSuT (ampicilin, chloramphenicol, streptomycin, sulphonamide, tetracycline) resistance phenotype, was defined. The length of complete sequence of this plasmid was 8271 base pairs (bp), and it was named as pAnkS owing to its isolation place (plasmid-Ankara- Salmonella). The plasmid was analyzed for potential reading frames and structural features indicative of transposons and transposon relics. The Xmnl enzyme restriction fragments of pAnkS were cloned into E. coli plasmid vectors (pBSK), sequenced and analyzed with the BLAST programs. Plasmid pAnkS has contained a previously defined enterohemorrhagic E. coli (EHEC) plasmid p4821 as a core region and also contained a complete Tn3-like transposon of 4950 bp consisting of the left terminal repeat, Tn3-related tnpR and tnpA genes for transposition functions, ampicillin resistance gene bla(TEM), and the right terminal repeats, pAnkS showed strong homology with another Salmonella plasmid, pNTP16, for sequences that belong to p4821 and partial Tn3 segments. It was found that pNTP16 also carries kanamycin resistance gene (kanR) in addition to ampR gene. Plasmid pAnkS is one of the few completely sequenced plasmids from Salmonella Typhimurium and is in the middle of the pathway of evolution of plasmid from p4821 to pNTP16. The identification of pAnkS might help better understanding of plasmid evolution.

  7. The C. elegans Crumbs family contains a CRB3 homolog and is not essential for viability.

    NARCIS (Netherlands)

    Waaijers, S.; Ramalho, J.J.; Koorman, T.; Kruse, E.; Boxem, M.

    2015-01-01

    Crumbs proteins are important regulators of epithelial polarity. In C. elegans, no essential role for the two described Crumbs homologs has been uncovered. Here, we identify and characterize an additional Crumbs family member in C. elegans, which we termed CRB-3 based on its similarity in size and s

  8. The C. elegans Crumbs family contains a CRB3 homolog and is not essential for viability.

    NARCIS (Netherlands)

    Waaijers, S.; Ramalho, J.J.; Koorman, T.; Kruse, E.; Boxem, M.

    2015-01-01

    Crumbs proteins are important regulators of epithelial polarity. In C. elegans, no essential role for the two described Crumbs homologs has been uncovered. Here, we identify and characterize an additional Crumbs family member in C. elegans, which we termed CRB-3 based on its similarity in size and s

  9. Plasmid pP62BP1 isolated from an Arctic Psychrobacter sp. strain carries two highly homologous type II restriction-modification systems and a putative organic sulfate metabolism operon.

    Science.gov (United States)

    Lasek, Robert; Dziewit, Lukasz; Bartosik, Dariusz

    2012-05-01

    The complete nucleotide sequence of plasmid pP62BP1 (34,467 bp), isolated from Arctic Psychrobacter sp. DAB_AL62B, was determined and annotated. The conserved plasmid backbone is composed of several genetic modules, including a replication system (REP) with similarities to the REP region of the iteron-containing plasmid pPS10 of Pseudomonas syringae. The additional genetic load of pP62BP1 includes two highly related type II restriction-modification systems and a set of genes (slfRCHSL) encoding enzymes engaged in the metabolism of organic sulfates, plus a putative transcriptional regulator (SlfR) of the AraC family. The pP62BP1 slf locus has a compact and unique structure. It is predicted that the enzymes SlfC, SlfH, SlfS and SlfL carry out a chain of reactions leading to the transformation of alkyl sulfates into acyl-CoA, with dodecyl sulfate (SDS) as a possible starting substrate. Comparative analysis of the nucleotide sequences of pP62BP1 and other Psychrobacter spp. plasmids revealed their structural diversity. However, the presence of a few highly conserved DNA segments in pP62BP1, plasmid 1 of P. cryohalolentis K5 and pRWF-101 of Psychrobacter sp. PRwf-1 is indicative of recombinational shuffling of genetic information, and is evidence of lateral gene transfer in the Arctic environment.

  10. Mobilization of Bacillus thuringiensis plasmid pTX14-3.

    Science.gov (United States)

    Andrup, L; Bendixen, H H; Jensen, G B

    1995-05-01

    The Bacillus thuringiensis subsp. israelensis (Bti) plasmid pTX14-3 has been reported to contain a gene, mob14-3, with considerable homology to genes encoding mobilization proteins from other gram-positive bacteria. We have used the aggregation-mediated conjugation system recently discovered in Bti to compare the mobilization kinetics of different derivatives of plasmid pTX14-3. Plasmid pTX14-3 has been found to replicate by the rolling-circle mechanism and to contain a locus suppressing the formation of high-molecular-weight DNA. We found that deleting a DNA fragment containing this locus increased the transfer frequency about twofold. The mobilization frequency of the plasmid containing the intact mob14-3 gene did not indicate a mobilization-enhancing activity of the encoded polypeptide. However, the presence of the mob14-3 gene seemed to increase the stability of the plasmid in exponential growth.

  11. Transmissible Plasmid Containing Salmonella enterica Heidelberg Isolates Modulate Cytokine Production During Early Stage of Interaction with Intestinal Epithelial Cells.

    Science.gov (United States)

    Gokulan, Kuppan; Khare, Sangeeta; Williams, Katherine; Foley, Steven L

    2016-08-01

    The variation in cytokine production during bacterial invasion of human intestinal epithelial cells (IECs) is a contributing factor for progression of the infection. A few Salmonella enterica Heidelberg strains isolated from poultry products harbor transmissible plasmids (TPs), including those that encode a type-IV secretion system. Earlier, we showed that these TPs are responsible for increased virulence during infection. This study examines the potential role of these TPs in cytokine production in IECs. This study showed that S. Heidelberg strains containing TPs (we refer as virulent strains) caused decreased interleukin (IL)-10 production in IECs after 1 h infection. The virulent strains induced a high level of tumor necrosis factor-α production under identical conditions. The virulent strains of S. Heidelberg also altered the production of IL-2, IL-17, and granulocyte macrophage colony-stimulating factor compared to an avirulent strain. As a part of infection, bacteria cross the epithelial barrier and encounter intestinal macrophages. Hence, we examined the cytotoxic mechanism of strains of S. Heidelberg in macrophages. Scanning electron microscopy showed cell necrosis occurs during the early stage of infection. In conclusion, virulent S. Heidelberg strains were able to modify the host cytokine profile during the early stages of infection and also caused necrosis in macrophages.

  12. The C. elegans Crumbs family contains a CRB3 homolog and is not essential for viability

    Directory of Open Access Journals (Sweden)

    Selma Waaijers

    2015-02-01

    Full Text Available Crumbs proteins are important regulators of epithelial polarity. In C. elegans, no essential role for the two described Crumbs homologs has been uncovered. Here, we identify and characterize an additional Crumbs family member in C. elegans, which we termed CRB-3 based on its similarity in size and sequence to mammalian CRB3. We visualized CRB-3 subcellular localization by expressing a translational GFP fusion. CRB-3::GFP was expressed in several polarized tissues in the embryo and larval stages, and showed apical localization in the intestine and pharynx. To identify the function of the Crumbs family in C. elegans development, we generated a triple Crumbs deletion mutant by sequentially removing the entire coding sequence for each crumbs homolog using a CRISPR/Cas9-based approach. Remarkably, animals lacking all three Crumbs homologs are viable and show normal epithelial polarity. Thus, the three C. elegans Crumbs family members do not appear to play an essential role in epithelial polarity establishment.

  13. Characterization of a collection of plasmid-containing bacteria isolated from an on-farm biopurification system used for pesticide removal.

    Science.gov (United States)

    Martini, María Carla; Albicoro, Francisco Javier; Nour, Eman; Schlüter, Andreas; van Elsas, Jan Dirk; Springael, Dirk; Smalla, Kornelia; Pistorio, Mariano; Lagares, Antonio; Del Papa, María Florencia

    2015-07-01

    Biopurification systems (BPS) are complex soil-related and artificially-generated environments usually designed for the removal of toxic compounds from contaminated wastewaters. The present study has been conducted to isolate and characterize a collection of cultivable plasmid-carrying bacterial isolates recovered from a BPS established for the decontamination of wastewater generated in a farmyard. Out of 1400 isolates, a collection of 75 plasmid-containing bacteria was obtained, of which 35 representative isolates comprising in total at least 50 plasmids were chosen for further characterization. Bacterial hosts were taxonomically assigned by 16S ribosomal RNA gene sequencing and phenotypically characterized according to their ability to grow in presence of different antibiotics and heavy metals. The study demonstrated that a high proportion of the isolates was tolerant to antibiotics and/or heavy metals, highlighting the on-farm BPS enrichment in such genetic traits. Several plasmids conferring such resistances in the bacterial collection were detected to be either mobilizable or selftransmissible. Occurrence of broad host range plasmids of the incompatibility groups IncP, IncQ, IncN and IncW was examined with positive results only for the first group. Presence of the IS1071 insertion sequence, frequently associated with xenobiotics degradation genes, was detected in DNA obtained from 24 of these isolates, strongly suggesting the presence of yet-hidden catabolic activities in the collection of isolates. The results showed a remarkable diversity in the plasmid mobilome of cultivable bacteria in the BPS with the presence of abundant resistance markers of different types, thus providing a suitable environment to investigate the genetic structure of the mobile genetic pool in a model on-farm biofilter for wastewater decontamination in intensive agricultural production.

  14. A plasmid containing the human metallothionein II gene can function as an antibody-assisted electrophoretic biosensor for heavy metals.

    Science.gov (United States)

    Wooten, Dennis C; Starr, Clarise R; Lyon, Wanda J

    2016-01-01

    Different forms of heavy metals affect biochemical systems in characteristic ways that cannot be detected with typical metal analysis methods like atomic absorption spectrometry. Further, using living systems to analyze interaction of heavy metals with biochemical systems can be laborious and unreliable. To generate a reliable easy-to-use biologically-based biosensor system, the entire human metallothionein-II (MT-II) gene was incorporated into a plasmid (pUC57-MT) easily replicated in Escherichia coli. In this system, a commercial polyclonal antibody raised against human metal-responsive transcription factor-1 protein (MTF-1 protein) could modify the electrophoretic migration patterns (i.e. cause specific decreases in agarose gel electrophoretic mobility) of the plasmid in the presence or absence of heavy metals other than zinc (Zn). In the study here, heavy metals, MTF-1 protein, and polyclonal anti-MTF-1 antibody were used to assess pUC57-MT plasmid antibody-assisted electrophoretic mobility. Anti-MTF-1 antibody bound both MTF-1 protein and pUC57-MT plasmid in a non-competitive fashion such that it could be used to differentiate specific heavy metal binding. The results showed that antibody-inhibited plasmid migration was heavy metal level-dependent. Zinc caused a unique mobility shift pattern opposite to that of other metals tested, i.e. Zn blocked the antibody ability to inhibit plasmid migration, despite a greatly increased affinity for DNA by the antibody when Zn was present. The Zn effect was reversed/modified by adding MTF-1 protein. Additionally, antibody inhibition of plasmid mobility was resistant to heat pre-treatment and trypsinization, indicating absence of residual DNA extraction-resistant bacterial DNA binding proteins. DNA binding by anti-DNA antibodies may be commonly enhanced by xenobiotic heavy metals and elevated levels of Zn, thus making them potentially effective tools for assessment of heavy metal bioavailability in aqueous solutions and

  15. Remarkable stability of an instability-prone lentiviral vector plasmid in Escherichia coli Stbl3.

    Science.gov (United States)

    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.

  16. Conjugation efficiency depends on intra and intercellular interactions between distinct plasmids: Plasmids promote the immigration of other plasmids but repress co-colonizing plasmids.

    Science.gov (United States)

    Gama, João Alves; Zilhão, Rita; Dionisio, Francisco

    2017-08-24

    Conjugative plasmids encode the genes responsible for the synthesis of conjugative pili and plasmid transfer. Expression of the conjugative machinery (including conjugative pili) may be costly to bacteria, not only due to the energetic/metabolic cost associated with their expression but also because they serve as receptors for certain viruses. Consequently, the presence of two plasmids in the same cell may be disadvantageous to each plasmid, because they may impose a higher fitness cost on the host. Therefore, plasmids may encode mechanisms to cope with co-resident plasmids. Moreover, it is possible that the transfer rate of a plasmid is affected by the presence of a distinct plasmid in the recipient cell. In this work, we measured transfer rates of twelve natural plasmids belonging to seven incompatibility groups in three situations, namely when: (i) donor cells contain a plasmid and recipient cells are plasmid-free; (ii) donor cells contain two unrelated plasmids and recipient cells are plasmid-free; and (iii) half of the cells contain a given plasmid and the other half contain another, unrelated, plasmid. In the third situation, recipient cells of a plasmid are the donor cells of the other plasmid. We show that there are more negative interactions (reduction of a plasmid's conjugative efficiency) between plasmids if they reside in the same cell than if they reside in different cells. However, if plasmids interacted intercellularly, the transfer rate of one of the plasmids was often higher (when the unrelated conjugative plasmid was present in the recipient cell) than if the recipient cell was plasmid-free - a positive effect. Experimental data retrieved from the study of mutant plasmids not expressing conjugative pili on the cell surface suggest that positive effects result from a higher efficiency of mating pair formation. Overall, our results suggest that negative interactions are significantly more frequent when plasmids occupy the same cell. Such

  17. Draft Genome Sequences of Salmonella enterica Isolates Containing Incompatibility Group I1 Plasmids from Swine, Poultry, and Human Sources.

    Science.gov (United States)

    Kaldhone, Pravin R; Khajanchi, Bijay K; Han, Jing; Nayak, Rajesh; Ricke, Steven C; Foley, Steven L

    2017-09-28

    The draft genome sequences of eight Salmonella enterica isolates from various sources were evaluated for the influence of incompatibility group I1 (IncI1) plasmids on virulence. Strains SE142, SE143, SE144, and SE146 originated from swine, SE36N and SE89N from poultry-related sources, and SE991 and SE1148 from human patients.

  18. Pleckstrin homology domain-containing protein PHLDB3 supports cancer growth via a negative feedback loop involving p53

    Science.gov (United States)

    Chao, Tengfei; Zhou, Xiang; Cao, Bo; Liao, Peng; Liu, Hongbing; Chen, Yun; Park, Hee-Won; Zeng, Shelya X.; Lu, Hua

    2016-01-01

    The tumour suppressor p53 transactivates the expression of its target genes to exert its functions. Here, we identify a pleckstrin homology domain-containing protein (PHLDB3)-encoding gene as a p53 target. PHLDB3 overexpression increases proliferation and restrains apoptosis of wild-type p53-harboring cancer cells by reducing p53 protein levels. PHLDB3 binds to MDM2 (mouse double minute 2 homolog) and facilitates MDM2-mediated ubiquitination and degradation of p53. Knockdown of PHLDB3 more efficiently inhibits the growth of mouse xenograft tumours derived from human colon cancer HCT116 cells that contain wild type p53 compared with p53-deficient HCT116 cells, and also sensitizes tumour cells to doxorubicin and 5-Fluorouracil. Analysis of cancer genomic databases reveals that PHLDB3 is amplified and/or highly expressed in numerous human cancers. Altogether, these results demonstrate that PHLDB3 promotes tumour growth by inactivating p53 in a negative feedback fashion and suggest PHLDB3 as a potential therapeutic target in various human cancers. PMID:28008906

  19. INVESTIGATION OF POLYDL-LACTIDE-b-POLY(ETHYLENE GLYCOL)-b-POLYDL-LACTIDE MICROSPHERES CONTAINING PLASMID DNA

    Institute of Scientific and Technical Information of China (English)

    Yu Liu; Long Huang; Xiao-rong Qiao; Xian-mo Deng; Wen-xiang Jia; Xiao-hong Li

    2004-01-01

    PolyDL-lactide (PDLLA) and the block copolymer, polyDL-lactide-b-poly(ethylene glycol)-b-polyDL-lactide (PELA) were used as the microsphere matrix to encapsulate plasmid DNA. The PDLLA, PELA, pBR322-1oaded PDLLA and pBR322-1oaded PELA microspheres were prepared by solvent extraction method based on the formation of multiple w1/o/w2 emulsion. The microspheres were characterized by surface morphology, mean particle size, particle size distribution and loading efficiency. The integrity of DNA molecules after being extracted from microspheres was determined by agarose gel electrophoresis. The result suggested that plasmid DNA molecules could retain their integrity after being encapsulated by PELA. The PELA microspheres could prevent plasmid DNA from being digested by DNase. The in vitro degradation and release profiles of plasmid DNA-loaded microspheres were measured in pH = 7.4 buffer solution at 37℃. The in vitro degradation profiles of the microspheres were evaluated by the deterioration in microspheres surface morphology, the molecular weight reduction of polymer, the mass loss of microspheres, the changes of pH values of degradation medium, and the changes of particle size. The in vitro release profiles of the microspheres were assessed by measurement of the amount of DNA presented in the release medium at determined intervals. The release profiles were correlation with the degradation profiles. The release of plasmid DNA from PELA microspheres showed a similar biphasic trend, that is, an initial burst release was followed by a slow, but sustained release.

  20. Construction and Expression of Periplanete fuliginosa densovirus Recombinant Plasmid Which Contains Luciferase Gene%含荧光素酶基因的黑胸大蠊浓核病毒重组质粒的构建与表达

    Institute of Scientific and Technical Information of China (English)

    杨娟; 张珈敏; 蒋洪; 邓晓军; 胡建芳; 胡远扬

    2003-01-01

    A luciferase gene has been inserted into the recombinant plasmid PfDNV-pUC119 which contained partly deletion of genome of Periplanete fuliginosa densovirus(PfDNV.)The recombinant plasmid with luciferase gene was co-transfrected with PfDNV-pUC 119 into Periplanele fuliginosa larvae and had a high luciferase gene expression in enteron of the transfected larvae.

  1. First environmental sample containing plasmid-mediated colistin-resistant ESBL-producing Escherichia coli detected in Norway.

    Science.gov (United States)

    Jørgensen, Silje Bakken; Søraas, Arne; Arnesen, Lotte Stenfors; Leegaard, Truls; Sundsfjord, Arnfinn; Jenum, Pål A

    2017-09-01

    We hereby report the detection of the plasmid borne mcr-1 gene conferring colistin resistance in an extended-spectrum β-lactamase (ESBL) producing Escherichia coli ST10 strain retrieved from seawater at a public beach in Norway. The sample was collected in September 2010 and was investigated by whole-genome sequencing in 2016. This report illustrates that E. coli strains carrying plasmid-mediated colistin resistance genes have also reached areas where this drug is hardly used at all. Surveillance of colistin resistance in environmental, veterinary, and human strains is warranted also in countries where colistin resistance is rare in clinical settings. © 2017 APMIS. Published by John Wiley & Sons Ltd.

  2. Pleckstrin homology domain containing 6 protein (PLEKHA6) polymorphisms are associated with psychopathology and response to treatment in schizophrenic patients.

    Science.gov (United States)

    Spellmann, Ilja; Rujescu, Dan; Musil, Richard; Meyerwas, Sebastian; Giegling, Ina; Genius, Just; Zill, Peter; Dehning, Sandra; Cerovecki, Anja; Seemüller, Florian; Schennach, Rebecca; Hartmann, Annette M; Schäfer, Martin; Müller, Norbert; Möller, Hans-Jürgen; Riedel, Michael

    2014-06-03

    Pleckstrin homology domain (PH domain) comprises approximately 120 amino acids and is integrated in a wide range of proteins involved in intracellular signaling or as constituents of the cytoskeleton. This domain can bind phosphatidylinositol (3,4,5)-triphosphate and phosphatidylinositol (4,5)-biphosphate and proteins such as the βγ-subunits of heterotrimeric G proteins and protein kinase C. Associations with psychiatric diseases have not been investigated yet. To identify genes involved in response to antipsychotics, mice were treated with haloperidol (1mg/kg, n = 11) or saline (n = 12) for one week. By analyzing microarray data, we observed an increase of pleckstrin homology domain containing 6 (PLEKHA6) gene expression. Furthermore, we genotyped 263 schizophrenic patients, who were treated monotherapeutically with different antipsychotics within randomized-controlled trials. Psychopathology was measured weekly using the PANSS for a minimum of four and a maximum of twelve weeks. Correlations between PANSS subscale scores at baseline and PANSS improvement scores after four weeks of treatment and genotypes were calculated by using a linear model for all investigated SNPs. We found associations between four PLEKHA6 polymorphisms (rs17333933 (T/G), rs3126209 (C/T), rs4951338 (A/G) and rs100900571 (T/C)) and different PANSS subscales at baseline. Furthermore two different polymorphisms (rs7513240 (T/C), rs4951353 (A/G)) were found to be associated with therapy response in terms of a significant correlation with different PANSS improvement subscores after four weeks of antipsychotic treatment. Our observation of an association between genetic polymorphisms of a protein of the PH domain and psychopathology data in schizophrenic patients might be indicative for an involvement of PLEKHA6 in the pathophysiology of schizophrenia and the therapy response towards antipsychotics.

  3. 携带鸡贫血病毒凋亡素基因重组腺病毒质粒的构建及鉴定%Construction and Identification of Recombinant Adenovirus Plasmid Containing Apoptin VP3 Gene

    Institute of Scientific and Technical Information of China (English)

    邓守明; 蔡召忠

    2015-01-01

    目的:构建携带鸡贫血病毒凋亡素 VP3基因的重组腺病毒质粒。方法设计 VP3 cDNA 扩增引物,从PET15b-VP3质粒中扩增 VP3的 DNA 序列,与线性 pShuttle-IRES-hrGFP-2连接,PCR 和 EcoR Ⅴ酶切电泳鉴定;穿梭质粒pShuttle-VP3-EGFP 经 Pmel 酶切线性化后,转化含 pAdeasy-1的超感受态 BJ5183大肠杆菌,细菌内同源重组法构建重组腺病毒质粒 pAd-VP3-EGFP,质粒经 PCR、PacI 酶切电泳及测序鉴定。结果线性化的 pShuttle-VP3-EGFP 转化含pAdeasy-1的超感受态 BJ5183大肠杆菌,重组质粒经酶切获得一大于23 kb 的大片段和4.5 kb 的片段,PCR 反应扩增出了402 bp 的片段,重组质粒测序证实 VP3-EGFP 编码区成功克隆入了腺病毒 pAd 中,且其序列与 GeneBank 中 VP3 CD-NA 序列完全一致。结论用细菌内同源重组法可快速、高效地制备携带凋亡素 VP3基因的重组腺病毒质粒,为深入研究 VP3的抗肿瘤效应及机制奠定了基础。%Objective To construct recombinant adenovirus plasmid containing apoptin VP 3 gene.Methods The VP3 cDNA primers were designed,the DNA sequence of VP3 was amplified from recombinant vector PET15b-VP3 and ligated into pShuttle-IRES-hrGFP-2,the recombinant plasmid was named after pShuttle -VP3-EGFP and was identified with PCR and EcoR Ⅴdigestion;pShuttle-VP3-EGFP was linealized with PmeI and transformed into ultracompletent BJ 5183 containing pAdeasy-1,then recombinant adenovirus pAd-vp3-EGFP was constructed by homologous recombination in bacteria .The recombinant adenoviral plasmid pAd-VP3-EGFP was identified by PCR,PacI digestion and DNA sequencing .Results There were 2 bands 4.5 kb and larger than 23 kb when pAd-VP3-EGFP was digested with PacI.A 402 bp VP3 cDNA fragment was amplified by PCR .The target gene VP3-EGFP was successfully cloned into adenovirus ,The sequence of VP3 segment was identical with that published in Gen -Bank

  4. Plasmid Biopharmaceuticals.

    Science.gov (United States)

    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.

  5. Plasmid recombination in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.

    1982-01-01

    DNA recombination in exponential phase and competent Haemophilus influenzae was measured by an electron microscopic assay that relies on the conversion of plasmid RSF0885 monomers into multimeric forms. Dimer circles were present at a frequency of 2% in plasmid preparations from competent Rd (wild-type) cells; multimers were present at a frequency of 0.2% in preparations from exponential phase cells. Thus, plasmid recombination was stimulated in competent cells. Multimer formation occurred efficiently in cells of the transformation defective mutant rec2, implying that the rec2 gene product is not required for plasmid recombination. However, the absence of multimer plasmids in preparations from competent cells of the transformation defective mutant rec1 suggests that the rec1 gene product is required. Digestion of purified plasmids with restriction endonuclease PvuII, which makes a single cut in the monomer, revealed the presence of recombination intermediates composed of two linear plasmids joined to form two pairs of arms resembling the Greek letter chi. Length measurements of these arms taken from a population of recombination intermediates gave evidence that the plasmids were joined at sites of homology. The distributions of individual DNA strands, at the intersections of the four arms, could be resolved in some recombination intermediates and were of two types. The first type of junction appeared as a single-stranded arm appended to each corner. The second type of junction consisted of a single strand of DNA linking the two linear plasmids at a site of homology. The single-stranded linker was frequently situated at the edge of a short gap on one of the plasmids in the pair. The fine structures of the recombinational joints have been interpreted in terms of previously proposed models of recombination.

  6. Transformation-associated recombination between diverged and homologous DNA repeats is induced by strand breaks

    Energy Technology Data Exchange (ETDEWEB)

    Larionov, V.; Kouprina, N. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States)]|[Institute of Cytology, St. Petersburg, (Russian Federation); Edlarov, M. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States)]|[Center of Bioengineering, Moscow, (Russian Federation); Perkins, E.; Porter, G.; Resnick, M.A. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States)

    1993-12-31

    Rearrangement and deletion within plasmid DNA is commonly observed during transformation. We have examined the mechanisms of transformation-associated recombination in the yeast Saccharomyces cerevisiae using a plasmid system which allowed the effects of physical state and/or extent of homology on recombination to be studied. The plasmid contains homologous or diverged (19%) DNA repeats separated by a genetically detectable color marker. Recombination during transformation for covalently closed circular plasmids was over 100-fold more frequent than during mitotic growth. The frequency of recombination is partly dependent on the method of transformation in that procedures involving lithium acetate or spheroplasting yield higher frequencies than electroporation. When present in the repeats, unique single-strand breaks that are ligatable, as well as double-strand breaks, lead to high levels of recombination between diverged and identical repeats. The transformation-associated recombination between repeat DNA`s is under the influence of the RADS2, RADI and the RNCI genes,

  7. Frequency and diversity of small cryptic plasmids in the genus Rahnella

    Directory of Open Access Journals (Sweden)

    Summers David K

    2010-02-01

    Full Text Available Abstract Background Rahnella is a widely distributed genus belonging to the Enterobacteriaceae and frequently present on vegetables. Although Rahnella has interesting agro-economical and industrial properties and several strains possess antibiotic resistances and toxin genes which might spread within microbial communities, little is known about plasmids of this genus. Thus, we isolated a number of Rahnella strains and investigated their complements of small plasmids. Results In total 53 strains were investigated and 11 plasmids observed. Seven belonged to the ColE1 family; one was ColE2-like and three shared homology to rolling circle plasmids. One of them belonged to the pC194/pUB110 family and two showed similarity to poorly characterised plasmid groups. The G+C content of two rolling circle plasmids deviated considerably from that of Rahnella, indicating that their usual hosts might belong to other genera. Most ColE1-like plasmids formed a subgroup within the ColE1 family that seems to be fairly specific for Rahnella. Intriguingly, the multimer resolution sites of all ColE1-like plasmids had the same orientation with respect to the origin of replication. This arrangement might be necessary to prevent inappropriate synthesis of a small regulatory RNA that regulates cell division. Although the ColE1-like plasmids did not possess any mobilisation system, they shared large parts with high sequence identity in coding and non-coding regions. In addition, highly homologous regions of plasmids isolated from Rahnella and the chromosomes of Erwinia tasmaniensis and Photorhabdus luminescens could be identified. Conclusions For the genus Rahnella we observed plasmid-containing isolates at a frequency of 19%, which is in the average range for Enterobacteriaceae. These plasmids belonged to diffent groups with members of the ColE1-family most frequently found. Regions of striking sequence homology of plasmids and bacterial chromosomes highlight the

  8. Plasmids of Carotenoid-Producing Paracoccus spp. (Alphaproteobacteria) - Structure, Diversity and Evolution

    Science.gov (United States)

    Maj, Anna; Dziewit, Lukasz; Czarnecki, Jakub; Wlodarczyk, Miroslawa; Baj, Jadwiga; Skrzypczyk, Grazyna; Giersz, Dorota; Bartosik, Dariusz

    2013-01-01

    Plasmids are components of many bacterial genomes. They enable the spread of a large pool of genetic information via lateral gene transfer. Many bacterial strains contain mega-sized replicons and these are particularly common in Alphaproteobacteria. Considerably less is known about smaller alphaproteobacterial plasmids. We analyzed the genomes of 14 such plasmids residing in 4 multireplicon carotenoid-producing strains of the genus Paracoccus (Alphaproteobacteria): P. aestuarii DSM 19484, P. haeundaensis LG P-21903, P. marcusii DSM 11574 and P. marcusii OS22. Comparative analyses revealed mosaic structures of the plasmids and recombinational shuffling of diverse genetic modules involved in (i) plasmid replication, (ii) stabilization (including toxin-antitoxin systems of the relBE/parDE, tad-ata, higBA, mazEF and toxBA families) and (iii) mobilization for conjugal transfer (encoding relaxases of the MobQ, MobP or MobV families). A common feature of the majority of the plasmids is the presence of AT-rich sequence islets (located downstream of exc1-like genes) containing genes, whose homologs are conserved in the chromosomes of many bacteria (encoding e.g. RelA/SpoT, SMC-like proteins and a retron-type reverse transcriptase). The results of this study have provided insight into the diversity and plasticity of plasmids of Paracoccus spp., and of the entire Alphaproteobacteria. Some of the identified plasmids contain replication systems not described previously in this class of bacteria. The composition of the plasmid genomes revealed frequent transfer of chromosomal genes into plasmids, which significantly enriches the pool of mobile DNA that can participate in lateral transfer. Many strains of Paracoccus spp. have great biotechnological potential, and the plasmid vectors constructed in this study will facilitate genetic studies of these bacteria. PMID:24260361

  9. Reduction of Intimal Hyperplasia in Injured Rat Arteries Promoted by Catheter Balloons Coated with Polyelectrolyte Multilayers that Contain Plasmid DNA Encoding PKCδ

    Science.gov (United States)

    Bechler, Shane L.; Si, Yi; Yu, Yan; Ren, Jun; Liu, Bo; Lynn, David M.

    2012-01-01

    New therapeutic approaches that eliminate or reduce the occurrence of intimal hyperplasia following balloon angioplasty could improve the efficacy of vascular interventions and improve the quality of life of patients suffering from vascular diseases. Here, we report that treatment of arteries using catheter balloons coated with thin polyelectrolyte-based films (‘polyelectrolyte multilayers’, PEMs) can substantially reduce intimal hyperplasia in an in vivo rat model of vascular injury. We used a layer-by-layer (LbL) process to coat the surfaces of inflatable catheter balloons with PEMs composed of nanolayers of a cationic poly(β-amino ester) (polymer 1) and plasmid DNA (pPKCδ) encoding the δ isoform of protein kinase C (PKCδ), a regulator of apoptosis and other cell processes that has been demonstrated to reduce intimal hyperplasia in injured arterial tissue when administered via perfusion using viral vectors. Insertion of balloons coated with polymer 1/pPKCδ multilayers into injured arteries for 20 min resulted in local transfer of DNA and elevated levels of PKCδ expression in the media of treated tissue 3 days after delivery. IFC and IHC analysis revealed these levels of expression to promote downstream cellular processes associated with up-regulation of apoptosis. Analysis of arterial tissue 14 days after treatment revealed polymer 1/pPKCδ-coated balloons to reduce the occurrence of intimal hyperplasia by ~60% compared to balloons coated with films containing empty plasmid vectors. Our results demonstrate the potential therapeutic value of this nanotechnology-based approach to local gene delivery in the clinically important context of balloon-mediated vascular interventions. These PEM-based methods could also prove useful for other in vivo applications that require short-term, surface-mediated transfer of plasmid DNA. PMID:23069712

  10. Sprouty-Related Ena/Vasodilator-Stimulated Phosphoprotein Homology 1-Domain-Containing Protein-2 Critically Regulates Influenza A Virus-Induced Pneumonia.

    Science.gov (United States)

    Ito, Toshihiro; Itakura, Junya; Takahashi, Sakuma; Sato, Miwa; Mino, Megumi; Fushimi, Soichiro; Yamada, Masao; Morishima, Tuneo; Kunkel, Steven L; Matsukawa, Akihiro

    2016-07-01

    Influenza A virus causes acute respiratory infections that induce annual epidemics and occasional pandemics. Although a number of studies indicated that the virus-induced intracellular signaling events are important in combating influenza virus infection, the mechanism how specific molecule plays a critical role among various intracellular signaling events remains unknown. Raf/MEK/extracellular signal-regulated kinase cascade is one of the key signaling pathways during influenza virus infection, and the Sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein has recently been identified as a negative regulator of Raf-dependent extracellular signal-regulated kinase activation. Here, we examined the role of Raf/MEK/extracellular signal-regulated kinase cascade through sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein in influenza A viral infection because the expression of sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein was significantly enhanced in human influenza viral-induced pneumonia autopsy samples. Prospective animal trial. Research laboratory. Wild-type and sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein-2 knockout mice inoculated with influenza A. Wild-type or sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein-2 knockout mice were infected by intranasal inoculation of influenza A (A/PR/8). An equal volume of phosphate-buffered saline was inoculated intranasally into mock-infected mice. Influenza A infection of sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1-domain-containing protein-2 knockout mice led to higher mortality with greater viral load, excessive inflammation, and enhanced cytokine production than wild-type mice. Administration of MEK inhibitor, U0126, improved mortality and reduced both viral load and

  11. A 2,4-dichlorophenoxyacetic acid degradation plasmid pM7012 discloses distribution of an unclassified megaplasmid group across bacterial species.

    Science.gov (United States)

    Sakai, Yoriko; Ogawa, Naoto; Shimomura, Yumi; Fujii, Takeshi

    2014-03-01

    Analysis of the complete nucleotide sequence of plasmid pM7012 from 2,4-dichlorophenoxyacetic-acid (2,4-D)-degrading bacterium Burkholderia sp. M701 revealed that the plasmid had 582 142 bp, with 541 putative protein-coding sequences and 39 putative tRNA genes for the transport of the standard 20 aa. pM7012 contains sequences homologous to the regions involved in conjugal transfer and plasmid maintenance found in plasmids byi_2p from Burkholderia sp. YI23 and pBVIE01 from Burkholderia sp. G4. No relaxase gene was found in any of these plasmids, although genes for a type IV secretion system and type IV coupling proteins were identified. Plasmids with no relaxase gene have been classified as non-mobile plasmids. However, nucleotide sequences with a high level of similarity to the genes for plasmid transfer, plasmid maintenance, 2,4-D degradation and arsenic resistance contained on pM7012 were also detected in eight other megaplasmids (~600 or 900 kb) found in seven Burkholderia strains and a strain of Cupriavidus, which were isolated as 2,4-D-degrading bacteria in Japan and the United States. These results suggested that the 2,4-D degradation megaplasmids related to pM7012 are mobile and distributed across various bacterial species worldwide, and that the plasmid group could be distinguished from known mobile plasmid groups.

  12. Fermentation of starch by Klebsiella oxytoca P2, containing plasmids with {alpha}-amylase and pullulanase genes

    Energy Technology Data Exchange (ETDEWEB)

    Santos, V.L. dos; Araujo, E.F.; Barros, E.G. de; Guimaraes, W.V.

    1999-12-20

    Klebsiella oxytoca P2(pC46), an ethanol-producing recombinant, has been evaluated in fermentation of maltose and starch. The maximum ethanol produced by P2(pC46) was 0.34 g ethanol/g maltose and 0.38, 0.40, or 0.36 g ethanol/g starch in fermentation of 1, 2, or 4% starch, representing 68, 71, and 64% the theoretical yield. The pC46 plasmid transformed to cells of K. oxytoca P2 reduced the ethanol production from maltose and starch. In fermentation of starch after its digestion at 60 C for 24 h, in two-step fermentation, the time for maximum ethanol production was reduced to 12--24 h and the theoretical yield was around 90%. The increase in starch concentration resulted in lower {alpha}-amylase activity but in higher pullulanase activity. The high activity and thermostability of the amylolytic enzymes from this transformant suggest that it has a potential for amylolytic enzymes source.

  13. The minimum amount of homology required for homologous recombination in mammalian cells.

    OpenAIRE

    Rubnitz, J; Subramani, S

    1984-01-01

    Although DNA sequence homology is believed to be a prerequisite for homologous recombination events in procaryotes and eucaryotes, no systematic study has been done on the minimum amount of homology required for homologous recombination in mammalian cells. We have used simian virus 40-pBR322 hybrid plasmids constructed in vitro as substrates to quantitate intramolecular homologous recombination in cultured monkey cells. Excision of wild-type simian virus 40 DNA by homologous recombination was...

  14. Chlamydial plasmids and bacteriophages.

    Science.gov (United States)

    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.

  15. Merkel cell carcinoma expresses K homology domain-containing protein overexpressed in cancer similar to other high-grade neuroendocrine carcinomas.

    Science.gov (United States)

    Pryor, Jennifer G; Simon, Rochelle A; Bourne, Patricia A; Spaulding, Betsy O; Scott, Glynis A; Xu, Haodong

    2009-02-01

    Merkel cell carcinoma is an uncommon and aggressive primary cutaneous neuroendocrine carcinoma with a high rate of recurrence and metastasis. Optimal management is controversial; consequently, it is imperative to identify the signaling pathways involved in the pathogenesis of Merkel cell carcinoma so that effective therapeutic targeting agents can be developed. We previously reported that K homology domain-containing protein overexpressed in cancer is expressed in high-grade neuroendocrine carcinomas of the lung and extrapulmonary small cell carcinomas. The K homology domain-containing protein overexpressed in cancer (KOC), also known as L523S and IMP-3, is an insulin-like growth factor II messenger RNA-binding protein that promotes tumor cell proliferation by enhancing insulin-like growth factor II protein expression. Expression of KOC in Merkel cell carcinoma has not been investigated. We studied 20 Merkel cell carcinomas by immunohistochemistry using a monoclonal antibody against L523S/KOC. Of 20 Merkel cell carcinomas, 18 (90%) overexpressed KOC, with 11 (55%) overexpressing KOC in greater than 90% of tumor cells, 3 (15%) overexpressing KOC in 50% to 90% of tumor cells, 3 (15%) overexpressing KOC in 10% to 50% of tumor cells, and 1 (5%) overexpressing KOC in less than 10% of tumor cells. The immunostaining intensity was variable, with moderate to strong staining in 14 cases and weak staining in the remaining 4. Extent of expression of K homology domain-containing protein overexpressed in cancer predicted metastasis (P = .04) and was weakly correlated with increased tumor size (P = .08). In conclusion, Merkel cell carcinoma expresses K homology domain-containing protein overexpressed in cancer with an expression pattern similar to high-grade neuroendocrine carcinomas of the lung and extrapulmonary small cell carcinomas. We propose K homology domain-containing protein overexpressed in cancer as a potential target molecule for the treatment of high

  16. Characterization of the replication and stability regions of Agrobacterium tumefaciens plasmid pTAR.

    OpenAIRE

    Gallie, D R; Zaitlin, D; Perry, K L; Kado, C I

    1984-01-01

    A 5.4-kilobase region containing the origin of replication and stability maintenance of the 44-kilobase Agrobacterium tumefaciens plasmid pTAR has been mapped and characterized. Within this region is a 1.3-kilobase segment that is capable of directing autonomous replication. The remaining segment contains the stability locus for maintenance of pTAR during nonselective growth. Approximately 35% of pTAR shares sequence homology with pAg119, a 44-kilobase cryptic plasmid in grapevine strain 1D11...

  17. Pheromone-responsive conjugative vancomycin resistance plasmids in Enterococcus faecalis isolates from humans and chicken feces.

    Science.gov (United States)

    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

  18. Crystal structure of an archaeal actin homolog.

    Science.gov (United States)

    Roeben, Annette; Kofler, Christine; Nagy, István; Nickell, Stephan; Hartl, F Ulrich; Bracher, Andreas

    2006-04-21

    Prokaryotic homologs of the eukaryotic structural protein actin, such as MreB and ParM, have been implicated in determination of bacterial cell shape, and in the segregation of genomic and plasmid DNA. In contrast to these bacterial actin homologs, little is known about the archaeal counterparts. As a first step, we expressed a predicted actin homolog of the thermophilic archaeon Thermoplasma acidophilum, Ta0583, and determined its crystal structure at 2.1A resolution. Ta0583 is expressed as a soluble protein in T.acidophilum and is an active ATPase at physiological temperature. In vitro, Ta0583 forms sheets with spacings resembling the crystal lattice, indicating an inherent propensity to form filamentous structures. The fold of Ta0583 contains the core structure of actin and clearly belongs to the actin/Hsp70 superfamily of ATPases. Ta0583 is approximately equidistant from actin and MreB on the structural level, and combines features from both eubacterial actin homologs, MreB and ParM. The structure of Ta0583 co-crystallized with ADP indicates that the nucleotide binds at the interface between the subdomains of Ta0583 in a manner similar to that of actin. However, the conformation of the nucleotide observed in complex with Ta0583 clearly differs from that in complex with actin, but closely resembles the conformation of ParM-bound nucleotide. On the basis of sequence and structural homology, we suggest that Ta0583 derives from a ParM-like actin homolog that was once encoded by a plasmid and was transferred into a common ancestor of Thermoplasma and Ferroplasma. Intriguingly, both genera are characterized by the lack of a cell wall, and therefore Ta0583 could have a function in cellular organization.

  19. Kalilo plasmids are a family of four distinct members with individual global distributions across species.

    Science.gov (United States)

    He, C; Nastasja de Groot; Bok, J W; Griffiths, A J

    2000-01-01

    Kalilo is a linear 9-kb plasmid, isolated originally from Hawaiian strains of the heterothallic fungus Neurospora intermedia. Its properties include terminal inverted repeats, two ORFs coding for a presumptive DNA and an RNA polymerase, and the ability to cause senescence in its original host and in the closely related species Neurospora crassa. We have examined natural isolates alleged to contain plasmids homologous to kalilo. Most of these isolates do in fact contain plasmids with so close an identity to kalilo as to be certain relatives. We found a new case of kalilo in Neurospora tetrasperma from Moorea-Tahiti, and a new case of LA-kalilo (previously found only in N. tetrasperma) in N. crassa from Haiti. A previously unreported, substantially shorter, kalilo variant has been found in three geographically separate isolates of the heterothallic species Neurospora discreta. Therefore, if the previously reported kalilo variant from the genus Gelasinospora is included, in all there are four members of the kalilo plasmid family. The main differences between these plasmids are in the terminal inverted repeats (TIRs). The phylogeny of the TIR sequences is largely congruent with that of nuclear DNA in the species in which they are found, suggesting that the plasmids are related by vertical descent throughout the evolution of these species. However, there are two cases of a plasmid found in a heterothallic and a pseudohomothallic species in the same global area; these cases might have arisen from more recent horizontal transmission or introgression.

  20. Conjugative plasmids of Neisseria gonorrhoeae.

    Directory of Open Access Journals (Sweden)

    Emilia Pachulec

    Full Text Available 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 IncP1alpha, beta, gamma, delta and epsilon 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

  1. Plasmid construction using recombination activity in the fission yeast Schizosaccharomyces pombe.

    Directory of Open Access Journals (Sweden)

    Ayako Chino

    Full Text Available BACKGROUND: Construction of plasmids is crucial in modern genetic manipulation. As of now, the common method for constructing plasmids is to digest specific DNA sequences with restriction enzymes and to ligate the resulting DNA fragments with DNA ligase. Another potent method to construct plasmids, known as gap-repair cloning (GRC, is commonly used in the budding yeast Saccharomyces cerevisiae. GRC makes use of the homologous recombination activity that occurs within the yeast cells. Due to its flexible design and efficiency, GRC has been frequently used for constructing plasmids with complex structures as well as genome-wide plasmid collections. Although there have been reports indicating GRC feasibility in the fission yeast Schizosaccharomyces pombe, this species is not commonly used for GRC as systematic studies of reporting GRC efficiency in S. pombe have not been performed till date. METHODOLOGY/PRINCIPAL FINDINGS: We investigated GRC efficiency in S. pombe in this study. We first showed that GRC was feasible in S. pombe by constructing a plasmid that contained the LEU2 auxotrophic marker gene in vivo and showed sufficient efficiency with short homology sequences (>25 bp. No preference was shown for the sequence length from the cut site in the vector plasmid. We next showed that plasmids could be constructed in a proper way using 3 DNA fragments with 70% efficiency without any specific selections being made. The GRC efficiency with 3 DNA fragments was dramatically increased >95% in lig4Delta mutant cell, where non-homologous end joining is deficient. Following this approach, we successfully constructed plasmid vectors with leu1+, ade6+, his5+, and lys1+ markers with the low-copy stable plasmid pDblet as a backbone by applying GRC in S. pombe. CONCLUSIONS/SIGNIFICANCE: We concluded that GRC was sufficiently feasible in S. pombe for genome-wide gene functional analysis as well as for regular plasmid construction. Plasmids with different

  2. Distinct regions of Galpha13 participate in its regulatory interactions with RGS homology domain-containing RhoGEFs.

    Science.gov (United States)

    Kreutz, Barry; Hajicek, Nicole; Yau, Douglas M; Nakamura, Susumu; Kozasa, Tohru

    2007-08-01

    Galpha12 and Galpha13 transduce signals from G protein-coupled receptors to RhoA through RhoGEFs containing an RGS homology (RH) domain, such as p115 RhoGEF or leukemia-associated RhoGEF (LARG). The RH domain of p115 RhoGEF or LARG binds with high affinity to active forms of Galpha12 and Galpha13 and confers specific GTPase-activating protein (GAP) activity, with faster GAP responses detected in Galpha13 than in Galpha12. At the same time, Galpha13, but not Galpha12, directly stimulates the RhoGEF activity of p115 RhoGEF or nonphosphorylated LARG in reconstitution assays. In order to better understand the molecular mechanism by which Galpha13 regulates RhoGEF activity through interaction with RH-RhoGEFs, we sought to identify the region(s) of Galpha13 involved in either the GAP response or RhoGEF activation. For this purpose, we generated chimeras between Galpha12 and Galpha13 subunits and characterized their biochemical activities. In both cell-based and reconstitution assays of RhoA activation, we found that replacing the carboxyl-terminal region of Galpha12 (residues 267-379) with that of Galpha13 (residues 264-377) conferred gain-of-function to the resulting chimeric subunit, Galpha12C13. The inverse chimera, Galpha13C12, exhibited basal RhoA activation which was similar to Galpha12. In contrast to GEF assays, GAP assays showed that Galpha12C13 or Galpha13C12 chimeras responded to the GAP activity of p115 RhoGEF or LARG in a manner similar to Galpha12 or Galpha13, respectively. We conclude from these results that the carboxyl-terminal region of Galpha13 (residues 264-377) is essential for its RhoGEF stimulating activity, whereas the amino-terminal alpha helical and switch regions of Galpha12 and Galpha13 are responsible for their differential GAP responses to the RH domain.

  3. Development and host compatibility of plasmids for two important ruminant pathogens, Mycoplasma bovis and Mycoplasma agalactiae.

    Directory of Open Access Journals (Sweden)

    Shukriti Sharma

    Full Text Available Mycoplasma bovis is a cause of pneumonia, mastitis, arthritis and otitis media in cattle throughout the world. However, despite its clinical significance, there is a paucity of tools to genetically manipulate it, impeding our capacity to further explore the molecular basis of its virulence. To address this limitation, we developed a series of homologous and heterologous replicable plasmids from M. bovis and M. agalactiae. The shortest replicable oriC plasmid based on the region downstream of dnaA in M. bovis was 247 bp and contained two DnaA boxes, while oriC plasmids based on the region downstream of dnaA in M. agalactiae strains 5632 and PG2 were 219 bp and 217 bp in length, respectively, and contained only a single DnaA box. The efficiency of transformation in M. bovis and M. agalactiae was inversely correlated with the size of the oriC region in the construct, and, in general, homologous oriC plasmids had a higher transformation efficiency than heterologous oriC plasmids. The larger pWholeoriC45 and pMM21-7 plasmids integrated into the genomic oriC region of M. bovis, while the smaller oriC plasmids remained extrachromosomal for up to 20 serial passages in selective media. Although specific gene disruptions were not be achieved in M. bovis in this study, the oriC plasmids developed here could still be useful as tools in complementation studies and for expression of exogenous genes in both M. bovis and M. agalactiae.

  4. Toxin Plasmids of Clostridium perfringens

    Science.gov (United States)

    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

  5. Complete nucleotide sequence of pGA45, a 140,698-bp incFIIY plasmid encoding blaIMI-3-mediated carbapenem resistance, from river sediment

    Directory of Open Access Journals (Sweden)

    Bingjun eDang

    2016-02-01

    Full Text Available Plasmid pGA45 was isolated from the sediment of Haihe River using E. coli CV601 (gfp-tagged as recipients and indigenous bacteria from sediment as donors. This plasmid confers reduced susceptibility to imipenem which belongs to carbapenem group. Plasmid pGA45 was fully sequenced on an Illumina HiSeq 2000 sequencing system. The complete sequence of plasmid pGA45 was 140,698 bp in length with an average G+C content of 52.03%. Sequence analysis shows that pGA45 belongs to incFIIY group and harbors a backbone region shares high homology and gene synteny to several other incF plasmids including pNDM1_EC14653, pYDC644, pNDM-Ec1GN574, pRJF866, pKOX_NDM1 and pP10164-NDM. In addition to the backbone region, plasmid pGA45 harbors two notable features including one blaIMI-3-containing region and one type VI secretion system region. The blaIMI-3-containing region is responsible for bacteria carbapenem resistance and the type VI secretion system region is probably involved in bacteria virulence, respectively. Plasmid pGA45 represents the first complete nucleotide sequence of the blaIMI-harboring plasmid from environment sample and the sequencing of this plasmid provided insight into the architecture used for the dissemination of blaIMI carbapenemase genes.

  6. [Labelling of nif-plasmid pEA9 from Enterobacter agglomerans 339].

    Science.gov (United States)

    Liu, Cheng-jun; Klingmüller, Walter

    2002-07-01

    The authors describe the in vivo labelling of the plasmid pEA9 in Enterobacter agglomerans 339 with a kanamycin resistance gene. For labelling purposes the donor plasmid pST5 was constructed. This plasmid contains the nif ENX region from pEA9,in which a kanamycin resistance gene is cloned.pST5 was transformed into E.a.339 and subsequently cured from the host. Curing was achieved with AP medium. Fourty strains that had lost pST5,but retained the kanamycin resistance, could be isolated. It showed that none of these clones contained co-integrates of pST5 and pEA9. This is evident that in all clones the kanamycin resistance gene was integrated into pEA9 by homologous recombination.

  7. Characterization of a cryptic plasmid from an alpha-proteobacterial endosymbiont of Amoeba proteus.

    Science.gov (United States)

    Park, Miey; Kim, Min-Soo; Lee, Kyung-Min; Hwang, Sue-Yun; Ahn, Tae In

    2009-01-01

    A new cryptic plasmid pAP3.9 was discovered in symbiotic alpha-proteobacteria present in the cytoplasm of Amoeba proteus. The plasmid is 3869bp with a GC content of 34.66% and contains replication origins for both double-strand (dso) and single-strand (sso). It has three putative ORFs encoding Mob, Rep and phosphoglycolate phosphatase (PGPase). The pAP3.9 plasmid appears to propagate by the conjugative rolling-circle replication (RCR), since it contains all required factors such as Rep, sso and dso. Mob and Rep showed highest similarities to those of the cryptic plasmid pBMYdx in Bacillus mycoides. The PGPase was homologous to that of Bacillus cereus and formed a clade with those of Bacillus sp. in molecular phylogeny. These results imply that the pAP3.9 plasmid evolved by the passage through Bacillus species. We hypothesize that the plasmid-encoded PGPase may have contributed to the establishment of bacterial symbiosis within the hostile environment of amoeba cytoplasm.

  8. Replication regions of two pairs of incompatible lactococcal theta-replicating plasmids.

    Science.gov (United States)

    Gravesen, A; von Wright, A; Josephsen, J; Vogensen, F K

    1997-01-01

    Incompatibility tests were performed employing 12 replicons belonging to a family of homologous lactococcal theta-replicating plasmids. Two pairs of incompatible plasmids were found, namely, pFV1001 and pFV1201, and pJW565 and pFW094. The replicons of plasmids pFV1001, pFV1201, pJW565, pJW566, and pFW094 were sequenced. Alignments were made of the replicational origins (repA) and putative replication proteins (RepB) of these and 11 related plasmid sequences. Comparison of the alignments with the incompatibility data indicated that the incompatibility determinant could be contained within the 22-bp tandem repeats DRII and/or the inverted repeat IR1 in repA. In support, the incompatibility determinant of pJW563 was localized to a 743-bp fragment encompassing repA. A stretch of 13 amino acids of RepB was proposed to be responsible for the plasmid-specific initiation of replication. This stretch is part of a domain containing features that are highly conserved within the proposed DNA binding regions of the initiation proteins from several well-characterized plasmids from Gram-negative bacteria, including pSC101, R6K, and mini-F.

  9. Mechanism of acquisition of chromosomal markers by plasmids in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Setlow, J.K.; Cabrera-Juarez, E.; Griffin, K.

    1984-11-01

    The hybrid plasmid pNov1 readily acquired genetic information from the chromosome of wild-type, but not rec-2, cells. Most of the recombination had taken place 1 h after entrance of the plasmid into the cell, as judged by transformation of rec-2 by lysates made from wild-type cells exposed to pNova. Measurement of physical transfer from radioactively labeled cellular DNA to plasmids recombining in wild-type cells failed, since there was little more radioactivity in plasmids from such cells than from labeled rec-2 recipients, in which no recombination took place. EcoRI digestion of pNov1 divided the DNA into a 1.7-kilobase-pair fragment containing the novobiocin resistance marker and a 13-kilobase-pair fragment containing all of the original vector and considerable portions homologous to the chromosome. Transformation by the large fragment alone resulted in a plasmid the size of the original pNov1. The hypothesis to explain the data is that genetic transfer from chromosome to plasmid took place by a copy choice mechanism.

  10. Self-transmissible nif plasmid (pEA9) of Enterobacter agglomerans 339: molecular cloning and evidence for the existence of similar nif clusters on dissimilar plasmids in Enterobacter strains.

    Science.gov (United States)

    Steibl, H D; Siddavattam, D; Klingmüller, W

    1995-11-01

    A cosmid library was generated to the 200-kb self-transmissible nif plasmid pEA9 isolated from Enterobacter agglomerans 339. The cosmid clone identified to contain the complete nif cluster was used to determine the nif gene organization and the physical map. The restriction pattern and nif gene organization of this nif cluster showed remarkable similarities to the nif cluster identified on the 110-kb plasmid pEA3 of Enterobacter agglomerans 333. Nucleotide sequence of several randomly selected regions of the nif cluster of pEA9 showed 96% similarity when compared to the known sequences of the nif cluster of pEA3. However, the homology ended abruptly at the flanking regions of the nif clusters and no similarity could be detected with the rest of the DNA of these plasmids. This reveals the existence of similar nif clusters on dissimilar plasmids, implying the horizontal transfer of the entire nif gene cluster.

  11. Dendritic cells primed with a chimeric plasmid containing HIV-1-gag associated with lysosomal-associated protein-1 (LAMP/gag) is a potential therapeutic vaccine against HIV.

    Science.gov (United States)

    Lucas, Carolina G D O; Matassoli, Flavio L; Peçanha, Ligia M T; Santillo, Bruna Tereso; Oliveira, Luanda Mara da Silva; Oshiro, Telma Miyuki; Marques, Ernesto T D A; Oxenius, Annette; de Arruda, Luciana B

    2016-08-01

    The decline in number and function of T cells is a hallmark of HIV infection, and preservation or restoration of HIV-specific cellular immune response is a major goal of AIDS treatment. Dendritic cells (DCs) play a key role in the initiation and maintenance of the immune response, and their use as a vaccine vehicle is a promising strategy for enhancing vaccine efficacy. We evaluated the potential of DC-mediated immunization with a DNA vaccine consisting of HIV-1-p55gag (gag, group-specific antigen) associated to lysosomal associated protein (LAMP) sequence (LAMP/gag vaccine). Immunization of mice with mouse DCs transfected with LAMP/gag (Lg-mDCs) stimulated more potent B- and T-cell responses than naked DNA or DCs pulsed with inactivated HIV. Anti-Gag antibody levels were sustained for at least 3 mo after immunization, and recall T-cell responses were also strongly detected at this time point. Human DCs transfected with LAMP/gag (Lg-hDCs) were also activated and able to stimulate greater T-cell response than native gag-transfected DCs. Coculture between Lg-hDCs and T lymphocytes obtained from patients with HIV resulted in upregulation of CD38, CD69, HLA-DR, and granzyme B by CD4(+) and CD8(+) T cells, and increased IFN-γ and TNF-α production. These results indicate that the use of LAMP/gag-DC may be an efficient strategy for enhancing immune function in patients with HIV.-Lucas, C. G. D. O., Matassoli, F. L., Peçanha, L. M. T., Santillo, B. T., Oliveira, L. M. D. S., Oshiro, T. M., Marques, E. T. D. A., Jr., Oxenius, A., de Arruda, L. B. Dendritic cells primed with a chimeric plasmid containing HIV-1-gag associated with lysosomal-associated protein-1 (LAMP/gag) is a potential therapeutic vaccine against HIV.

  12. The expression of a plasmid-specified exported protein causes structural plasmid instability in Bacillus subtilis

    NARCIS (Netherlands)

    Cordes, C.; Meima, R; Twiest, B; Kazemier, B; Venema, G; vanDijl, JM; Bron, S

    The rolling-circle plasmid pGP1 was used to study the effects of the expression of a plasmid-specified exported protein on structural plasmid stability in Bacillus subtilis. pGP1 contains a fusion between the Bacillus licheniformis penP gene, encoding a C-terminally truncated penicillinase, and the

  13. Recombinational instability of F' plasmids in Escherichia coli K-12: localization of fre-sites.

    Science.gov (United States)

    Bresler, S E; Krivonogov, S V; Lanzov, V A

    1981-01-01

    The F' plasmids ORF-1 (purE+ tsxs proC+ lac+) and F'14 (argE+ metB+ ilv+) contain active regions of recombination, fre I and fre II correspondingly. The plasmid ORF-1 is stable in recF- cells (i.e., with the RecBC pathway of recombination) and decays in rec+ cells (RecBCF pathway) giving two types of product: F+ and plasmid pCK-1 (tsxs proC+ lac+) containing part of the initial DNA. They are extremely instable in the presence of the RecF pathway, (recBC- sbcB-), yielding F+ and plasmid pCK-2 (proC+ lac+). The instability of plasmids depends on a region of homology between the chromosome and the episome. The instability of ORF-1 shows the participation of IS3 elements (alpha 1 beta 3 and alpha 3 beta 1) in the recA, recF-dependent recombinational decay and allows localization of two active sites on the chromosome: fre I1 between purE and tsx markers and fre I2 between tsx and proC. The plasmid F'14, in accordance with published data, is able to yield F+ cells by recA-independent recombination. But eventually this plasmid may undergo a recA, recF-dependent decay. Genetic analysis of these events allows localization of an active point of recombination, freII1, between argE and metB. Another active point is localized inside the F factor. The recA-dependent decay of plasmid F-14 is also excluded on the RecBC pathway (recF- strains).

  14. Characterization of the replicon from the lactococcal theta-replicating plasmid pJW563.

    Science.gov (United States)

    Gravesen, A; Josephsen, J; von Wright, A; Vogensen, F K

    1995-09-01

    The replication region of the lactococcal plasmid pJW563 was localized to a 2.3-kb EcoRI fragment. This DNA fragment was sequenced ans a 1155-bp open reading frame, repB563, encoding a putative protein RepB563 of 385 amino acids was found. An AT-rich noncoding region, repA563, was found upstream of repB563. This segment included several direct and inverted repeats. A downstream 591-bp open reading frame, ORF X, which was not necessary for replication, was putatively translationally coupled to repB563, RepB563 supplied in trans could support replication of a plasmid containing repA563 and a truncated repB563. This observation suggests that RepB563 is a trans-acting replication protein, and repA563 the cis-acting origin of replication, repA563, repB563, and the beginning of ORF X showed high homology to similar regions in a family of lactococcal theta-replicating plasmids. The repA DNA sequences and the RepB amino acid sequences of the plasmids were aligned and the consensus sequences generated. The comparison revealed highly conserved areas among this family of plasmids. In addition, variable domains emerged, presumably having a plasmid specific function, pVS40 and pC1305 were plasmids with replication proteins showing high homology to RepB563. Despite this homology, replication from repA563 could not be supported by the pVS40 or pC1305 replication protein supplied in trans. Likewise the pJW563 protein could not support replication from the pVS40 origin. pJW563 was found to be compatible with the pVS40 and pC1305 replicons. The results indicate that pJW563 belongs to the widespread family of lactococcal theta-replicating pladmids. Despite the high homology between their replicons, the interaction between the replication origin and the protein is highly specific in many cases rendering the plasmids compatible.

  15. Complete Sequences of Four Plasmids of Lactococcus lactis subsp. cremoris SK11 Reveal Extensive Adaptation to the Dairy Environment†

    Science.gov (United States)

    Siezen, Roland J.; Renckens, Bernadet; van Swam, Iris; Peters, Sander; van Kranenburg, Richard; Kleerebezem, Michiel; de Vos, Willem M.

    2005-01-01

    Lactococcus lactis strains are known to carry plasmids encoding industrially important traits. L. lactis subsp. cremoris SK11 is widely used by the dairy industry in cheese making. Its complete plasmid complement was sequenced and found to contain the plasmids pSK11A (10,372 bp), pSK11B (13,332 bp), pSK11L (47,165 bp), and pSK11P (75,814 bp). Six highly homologous repB-containing replicons were found, all belonging to the family of lactococcal theta-type replicons. Twenty-three complete insertion sequence elements segment the plasmids into numerous modules, many of which can be identified as functional units or containing functionally related genes. Plasmid-encoded functions previously known to reside on L. lactis SK11 plasmids were now mapped in detail, e.g., lactose utilization (lacR-lacABCDFEGX), the proteolytic system (prtM-prtP, pepO, pepF), and the oligopeptide permease system (oppDFBCA). Newly identified plasmid-encoded functions could facilitate the uptake of various cations, while the pabA and pabB genes could be essential for folate biosynthesis. A competitive advantage could be obtained by using the putative flavin adenine dinucleotide-dependent d-lactate dehydrogenase and oxalate:formate antiporter for enhanced ATP synthesis, while the activity of the predicted α-acetolactate decarboxylase may contribute to the formation of an additional electron sink. Various stress response proteins are plasmid encoded, which could enhance strain robustness. A substantial number of these “adaptation” genes have not been described before on L. lactis plasmids. Moreover, several genes were identified for the first time in L. lactis, possibly reflecting horizontal gene transfer. PMID:16332824

  16. Characterization of a collection of plasmid-containing bacteria isolated from an on-farm biopurification system used for pesticide removal

    NARCIS (Netherlands)

    Carla Martini, Maria; Javier Albicoro, Francisco; Nour, Eman; Schlueter, Andreas; van Elsas, Jan Dirk; Springael, Dirk; Smalla, Kornelia; Pistorio, Mariano; Lagares, Antonio; Florencia Del Papa, Maria

    2015-01-01

    Biopurification systems (BPS) are complex soil-related and artificially-generated environments usually designed for the removal of toxic compounds from contaminated wastewaters. The present study has been conducted to isolate and characterize a collection of cultivable plasmid-carrying bacterial iso

  17. Complex integrons containing qnrB4-ampC (bla(DHA-1)) in plasmids of multidrug-resistant Citrobacter freundii from wastewater.

    Science.gov (United States)

    Yim, Grace; Kwong, Waldan; Davies, Julian; Miao, Vivian

    2013-02-01

    Microbial populations in wastewater treatment plants (WWTPs) are increasingly being recognized as environmental reservoirs of antibiotic resistance genes. PCR amplicons for plasmid-mediated quinolone resistance determinants qnrA, qnrB, and qnrS were recorded in samples from a WWTP in Vancouver, British Columbia. Six strains of ciprofloxacin-resistant Citrobacter freundii were isolated and found to carry mutations in gyrA and parC, as well as multiple plasmid-borne resistance genes, collectively including qnrB; aac(6')-Ib-cr; β-lactamase-encoding genes from molecular classes A (blaTEM-1), C (ampC), D (blaOXA-1, blaOXA-10); and genes for resistance to 5 other types of antibiotics. In 3 strains, large (>60 kb) plasmids carried qnrB4 and ampC as part of a complex integron in a 14 kb arrangement that has been reported worldwide but, until recently, only among pathogenic strains of Klebsiella. Analysis of single-nucleotide polymorphisms in the qnrB4-ampC regions infers 2 introductions into the WWTP environment. These results suggest recent passage of plasmid-borne fluoroquinolone and β-lactam resistance genes from pathogens to bacteria that may be indigenous inhabitants of WWTPs, thus contributing to an environmental pool of antibiotic resistance.

  18. Plasmid and Host Strain Characteristics of Escherichia coli Resistant to Extended-Spectrum Cephalosporins in the Norwegian Broiler Production.

    Science.gov (United States)

    Mo, Solveig Sølverød; Slettemeås, Jannice Schau; Berg, Einar Sverre; Norström, Madelaine; Sunde, Marianne

    2016-01-01

    Escherichia coli resistant to extended-spectrum cephalosporins have been detected in the Norwegian broiler production, despite the fact that antimicrobial agents are rarely used. The genetic mechanism responsible for cephalosporin resistance is mainly attributed to the presence of the blaCMY-2 gene encoding a plasmid-mediated AmpC-beta-lactamase (pAmpC). The aim of this study was to characterize and compare blaCMY-2 containing Escherichia coli isolated from the intestinal flora of broilers and retail chicken meat (fillets) to identify possible successful clones and/or resistance plasmids widespread in the Norwegian broiler production. Methods used included PCR based phylotyping, conjugation experiments, plasmid replicon typing, pulsed-field gel electrophoresis, multiple locus variable-number tandem-repeats analysis and whole genome sequencing. The nucleotide sequence of an IncK plasmid carrying blaCMY-2 was determined. Intestinal isolates displayed a higher degree of genetic diversity than meat isolates. A cluster of genetically related isolates belonging to ST38, phylogroup D, carrying blaCMY-2 containing IncK plasmids was identified. Furthermore, genes encoding plasmid stability systems (relBE/stbDE and pndAC) were identified on the IncK plasmid. Single nucleotide polymorphism (SNP) analysis of a subset of isolates confirmed a close genetic relationship within the two most prevalent STs. The IncK plasmids within these two STs also shared a high degree of similarity. Cephalosporin-resistant E. coli with the same genetic characteristics have been identified in the broiler production in other European countries, and the IncK plasmid characterized in this study showed close homology to a plasmid isolated from retail chicken meat in the Netherlands. The results indicate that both clonal expansion and horizontal transfer of blaCMY-2 containing plasmids contribute to dissemination of cephalosporin resistant E. coli in the broiler production. The presence of plasmid

  19. Protein diversity confers specificity in plasmid segregation.

    Science.gov (United States)

    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.

  20. The mammalian hypusine-containing protein, eukaryotic initiation factor 4D. Structural homology of this protein from several species.

    Science.gov (United States)

    Park, M H; Chung, S I; Cooper, H L; Folk, J E

    1984-04-10

    A single cellular protein of Mr approximately 18,000 and pI near 5.1, recently identified as eukaryotic translation initiation factor eIF-4D, contains the unusual amino acid hypusine [N epsilon-(4-amino--2-hydroxybutyl)lysine] formed post-translationally from lysine with a structural contribution from the polyamine spermidine. When the 3H-labeled hypusine-containing protein isolated from Chinese hamster ovary (CHO) cells that were grown with radioactive polyamine is digested with trypsin and the digest is subjected to two-dimensional separation, a single radioactive peptide is seen. A labeled peptide that occupies this same position is found in a digest of the [3H]hypusine protein from human lymphocytes and the single hypusine-containing tryptic peptide from purified rabbit reticulocyte eIF-4D also moves to this identical position. Stepwise Edman degradation of the tryptic digest of CHO cell hypusine-protein releases the radioactivity as a single peak in accordance with our earlier evidence for a single hypusine residue per molecule of eIF-4D. The similar patterns of radioactive peptides obtained from tryptic digests of radioiodinated eIF-4D from CHO cells, human lymphocytes, and rabbit reticulocytes suggest a highly conserved primary structure for this protein.

  1. Mutants of Streptomyces roseosporus that express enhanced recombination within partially homologous genes.

    Science.gov (United States)

    Hosted, T J; Baltz, R H

    1996-10-01

    Streptomyces roseosporus mutants that express enhanced recombination between partially homologous (homeologous) sequences were isolated by selection for recombination between the bacteriophage phi C31 derivative KC570 containing the Streptomyces coelicolor glucose kinase (glk) gene and the S. roseosporus chromosome. The frequencies of homeologous recombination in the ehr mutants were determined by measuring the chromosomal insertion frequencies of plasmids containing S. coelicolor glnA or whiG genes. S. roseosporus ehr mutants showed 10(2)- to 10(4)-fold increases in homeologous recombination relative to Ehr+ strains, but no increase in homologous recombination. Southern hybridization analysis revealed single unique sites for the insertion of each of the plasmids, and the crossovers occurred in frame and in proper translational register, yielding functional chimeric glnA and whiG genes.

  2. Apple contains receptor-like genes homologous to the Cladosporium fulvum resistance gene family of tomato with a cluster of genes cosegregating with Vf apple scab resistance.

    Science.gov (United States)

    Vinatzer, B A; Patocchi, A; Gianfranceschi, L; Tartarini, S; Zhang, H B; Gessler, C; Sansavini, S

    2001-04-01

    Scab caused by the fungal pathogen Venturia inaequalis is the most common disease of cultivated apple (Malus x domestica Borkh.). Monogenic resistance against scab is found in some small-fruited wild Malus species and has been used in apple breeding for scab resistance. Vf resistance of Malus floribunda 821 is the most widely used scab resistance source. Because breeding a high-quality cultivar in perennial fruit trees takes dozens of years, cloning disease resistance genes and using them in the transformation of high-quality apple varieties would be advantageous. We report the identification of a cluster of receptor-like genes with homology to the Cladosporium fulvum (Cf) resistance gene family of tomato on bacterial artificial chromosome clones derived from the Vf scab resistance locus. Three members of the cluster were sequenced completely. Similar to the Cf gene family of tomato, the deduced amino acid sequences coded by these genes contain an extracellular leucine-rich repeat domain and a transmembrane domain. The transcription of three members of the cluster was determined by reverse transcriptionpolymerase chain reaction to be constitutive, and the transcription and translation start of one member was verified by 5' rapid amplification of cDNA ends. We discuss the parallels between Cf resistance of tomato and Vf resistance of apple and the possibility that one of the members of the gene cluster is the Vf gene. Cf homologs from other regions of the apple genome also were identified and are likely to present other scab resistance genes.

  3. Properties and nucleotide se- quence of linear plasmid-like DNA pC4 from mitochondria of Cucumis sativus

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Four kinds of mitochondrial plasmid-like DNAs, designated pC1, pC2, pC3 and pC4, were detected in Cucumis sativus Jinyan No. 4. The electron microscopy ob- servation showed that pC4 was linear conformation. Complete sequence of pC4 was cloned into pUC19 with E. coli JM109 as host. Sequence analysis revealed that pC4 was 370 bp long, the shortest one among all the reported mitochondrial plasmid-like DNAs. pC4 was AT rich. It contained terminal direct repeat sequence (35 bp in length) as well as many short direct and inverted repeats. ORFs in pC4 were short. pC4 was found to be homologous to nuclear DNAs, but lack homology with main mitochondrial and chloroplast DNAs. pC4-homologous sequence also occurred in nuclear genome of Jinyan No. 7 which contained no mito- chondrial plasmid-like DNAs. The hybridization pattern of Jinyan No. 7 was slightly different from that of Jinyan No. 4. This suggested that pC4 occurred at the forepart of Cucumis sativus species divergence and integrated into the nuclear genome, and the pC4-homologous sequence in nucleus varied during species diverging.

  4. Double-stranded DNA homology produces a physical signature

    Science.gov (United States)

    Wang, Xing; Zhang, Xiaoping; Mao, Chengde; Seeman, Nadrian C.

    2010-01-01

    DNA is found in the cell largely as a negatively supercoiled molecule. This high-energy form of the genetic material can engender sequence-dependent structures, such as cruciforms, Z-DNA, or H-DNA, even though they are not favored by conventional conditions in relaxed DNA. A key feature of DNA in living systems is the presence of homology. We have sought homology-dependent structural phenomena based on topological relaxation. Using two-dimensional electrophoresis, we demonstrate a structural transition in supercoiled plasmid molecules containing homologous segments. Atomic force microscopy (AFM) reveals a dumbbell structure in molecules whose linking difference is beyond the transition point. The position of the dumbbell shaft is a function of the site of homology, and its extent is proportional to the linking difference. Second-site-reversion electrophoresis data support the notion that the shaft contains PX-DNA. Predicted cross-linking patterns generated in vivo suggest that homology-dependent structures can occur within the cell. PMID:20616051

  5. Conditional Knockout of Src Homology 2 Domain-containing Protein Tyrosine Phosphatase-2 in Myeloid Cells Attenuates Renal Fibrosis after Unilateral Ureter Obstruction

    Institute of Scientific and Technical Information of China (English)

    Jing-Fei Teng; Kai Wang; Yao Li; Fa-Jun Qu; Qing Yuan; Xin-Gang Cui; Quan-Xing Wang

    2015-01-01

    Background:Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) is a kind of intracellular protein tyrosine phosphatase.Studies have revealed its roles in various disease,however,whether SHP-2 involves in renal fibrosis remains unclear.The aim of this study was to explore the roles of myeloid cells SHP-2 in renal interstitial fibrosis.Methods:Myeloid cells SHP-2 gene was conditionally knocked-out (CKO) in mice using loxP-Cre system,and renal interstitial fibrosis was induced by unilateral ureter obstruction (UUO).The total collagen deposition in the renal interstitium was assessed using picrosirius red stain.F4/80 immunostaing was used to evaluate macrophage infiltration in renal tubular interstitium.Quantitative real-time polymerase chain reaction and enzyme linked immunosorbent assay were used to analyze the production of cytokines in the kidney.Transferase-mediated dUTP nick-end labeling stain was used to assess the apoptotic renal tubular epithelial cells.Results:Src homology 2 domain-containing protein tyrosine phosphatase-2 gene CKO in myeloid cells significantly reduced collagen deposition in the renal interstitium after UUO.Macrophage infiltration was evidently decreased in renal tubular interstitium of SHP-2 CKO mice.Meanwhile,the production of pro-inflammatory cytokines was significantly suppressed in SHP-2 CKO mice.However,no significant difference was observed in the number of apoptotic renal tubular epithelial cells between wild-type and SHP-2 CKO mice.Conclusions:Our observations suggested that SHP-2 in myeloid cells plays a pivotal role in the pathogenesis of renal fibrosis,and that silencing of SHP-2 gene in myeloid cells may protect renal from inflammatory damage and prevent renal fibrosis after renal injury.

  6. Eps15 homology domain containing protein of Plasmodium falciparum (PfEHD) associates with endocytosis and vesicular trafficking towards neutral lipid storage site.

    Science.gov (United States)

    Thakur, Vandana; Asad, Mohd; Jain, Shaifali; Hossain, Mohammad E; Gupta, Akanksha; Kaur, Inderjeet; Rathore, Sumit; Ali, Shakir; Khan, Nida J; Mohmmed, Asif

    2015-11-01

    The human malaria parasite, Plasmodium falciparum, takes up numerous host cytosolic components and exogenous nutrients through endocytosis during the intra-erythrocytic stages. Eps15 homology domain-containing proteins (EHDs) are conserved NTPases, which are implicated in membrane remodeling and regulation of specific endocytic transport steps in eukaryotic cells. In the present study, we have characterized the dynamin-like C-terminal Eps15 homology domain containing protein of P. falciparum (PfEHD). Using a GFP-targeting approach, we studied localization and trafficking of PfEHD in the parasite. The PfEHD-GFP fusion protein was found to be a membrane bound protein that associates with vesicular network in the parasite. Time-lapse microscopy studies showed that these vesicles originate at parasite plasma membrane, migrate through the parasite cytosol and culminate into a large multi-vesicular like structure near the food-vacuole. Co-staining of food vacuole membrane showed that the multi-vesicular structure is juxtaposed but outside the food vacuole. Labeling of parasites with neutral lipid specific dye, Nile Red, showed that this large structure is neutral lipid storage site in the parasites. Proteomic analysis identified endocytosis modulators as PfEHD associated proteins in the parasites. Treatment of parasites with endocytosis inhibitors obstructed the development of PfEHD-labeled vesicles and blocked their targeting to the lipid storage site. Overall, our data suggests that the PfEHD is involved in endocytosis and plays a role in the generation of endocytic vesicles at the parasite plasma membrane, that are subsequently targeted to the neutral lipid generation/storage site localized near the food vacuole.

  7. Nucleotide Sequence and Analysis of an orotate transporter-containing plasmid isolated from the Lactococcus lactis ssp. lactis biovar diacetylactis strain DB0410

    DEFF Research Database (Denmark)

    Defoor, Els Marie Celine; Martinussen, Jan

    determined. Fifteen open reading frames (ORFs) were encountered of which three insertion-sequence (IS) elements identified as two IS946 and one IS982. Two ORFs are incomplete due to the insertion of an IS element in their carboxy terminal end. Homologs for four ORFs were found on the IL1403 sequence: the cop...... and molecular cloning, we identified the open reading frames on pDBORO necessary for the utilization of orotate as the sole pyrimidine source. Surprisingly, homologs are found on the Lactococcus lactis IL1403 and MG1363 chromosomes despite the fact that they are resistant towards fluoroorotate....

  8. The structure of the transposable genetic element ISBsu2 from the cryptic plasmid p1516 of a soil Bacillus subtilis strain and the presence of homologues of this element in the chromosomes of various Bacillus subtilis strains

    NARCIS (Netherlands)

    Holsappel, S; Gagarina, EY; Poluektova, EU; Nezametdinova, VZ; Gel'fand, MS; Prozorov, AA; Bron, S

    2003-01-01

    A cryptic plasmid from a soil strain of Bacillus subtilis was found to contain a sequence having features of an IS element. Homologous sequences were also found in the chromosome of this strain and in the chromosomes of some other B. subtilis strains.

  9. Plasmid profiles of Moraxella bovis isolates.

    Science.gov (United States)

    McDonald, T J; Pugh, G W

    1986-04-01

    Two-hundred isolates of Moraxella bovis were selected at random and examined for the presence of plasmid DNA by a rapid alkaline-detergent lysis method. All isolates contained from 1 to 6 plasmids, with varying agarose-gel electrophoretic migration patterns. Most (80%) isolates carried 2 to 4 plasmids, which ranged in molecular weight from 2.6 to 80 megadaltons. Seemingly, plasmid profiles can be used as a simple, reliable epizootiologic tool to establish a strain identification scheme for M bovis.

  10. International Spread and Persistence of TEM-24 Is Caused by the Confluence of Highly Penetrating Enterobacteriaceae Clones and an IncA/C2 Plasmid Containing Tn1696::Tn1 and IS5075-Tn21▿

    Science.gov (United States)

    Novais, Ângela; Baquero, Fernando; Machado, Elisabete; Cantón, Rafael; Peixe, Luísa; Coque, Teresa M.

    2010-01-01

    TEM-24 remains one of the most widespread TEM-type extended-spectrum β-lactamases (ESBLs) among Enterobacteriaceae. To analyze the reasons influencing its spread and persistence, a multilevel population genetics study was carried out on 28 representative TEM-24 producers from Belgium, France, Portugal, and Spain (13 Enterobacter aerogenes isolates, 6 Escherichia coli isolates, 6 Klebsiella pneumoniae isolates, 2 Proteus mirabilis isolates, and 1 Klebsiella oxytoca isolate, from 1998 to 2004). Clonal relatedness (XbaI pulsed-field gel electrophoresis [PFGE] and E. coli phylogroups) and antibiotic susceptibility were determined by standard procedures. Plasmid analysis included determination of the incompatibility group (by PCR, hybridization, and/or sequencing) and comparison of restriction fragment length polymorphism (RFLP) patterns. Characterization of genetic elements conferring antibiotic resistance included integrons (classes 1, 2, and 3) and transposons (Tn3, Tn21, and Tn402). Similar PFGE patterns were identified among E. aerogenes, K. pneumoniae, and P. mirabilis isolates, while E. coli strains were diverse (phylogenetic groups A, B2, and D). Highly related 180-kb IncA/C2 plasmids conferring resistance to kanamycin, tobramycin, chloramphenicol, trimethoprim, and sulfonamides were identified. Each plasmid contained defective In0-Tn402 (dfrA1-aadA1, aacA4, or aacA4-aacC1-orfE-aadA2-cmlA1) and In4-Tn402 (aacA4 or dfrA1-aadA1) variants. These integrons were located within Tn21, Tn1696, or hybrids of these transposons, with IS5075 interrupting their IRtnp and IRmer. In all cases, blaTEM-24 was part of an IS5075-ΔTn1 transposon within tnp1696, mimicking other genetic elements containing blaTEM-2 and blaTEM-3 variants. The international dissemination of TEM-24 is fuelled by an IncA/C2 plasmid acquired by different enterobacterial clones which seem to evolve by gaining diverse genetic elements. This work highlights the risks of a confluence between highly

  11. HCV NS5A protein containing potential ligands for both Src homology 2 and 3 domains enhances autophosphorylation of Src family kinase Fyn in B cells.

    Directory of Open Access Journals (Sweden)

    Kenji Nakashima

    Full Text Available Hepatitis C virus (HCV infects B lymphocytes and induces mixed cryoglobulinemia and B cell non-Hodgkin's lymphoma. The molecular mechanism for the pathogenesis of HCV infection-mediated B cell disorders remains obscure. To identify the possible role for HCV nonstructural 5A (NS5A protein in B cells, we generated the stable B cell lines expressing Myc-His tagged NS5A. Immunoprecipitation study in the presence or absence of pervanadate (PV implied that NS5A was tyrosine phosphorylated by pervanadate (PV treatment of the cells. Therefore we examined pull-down assay by using glutathione S-transferase (GST-fusion proteins of various Src homology 2 (SH2 domains, which associates with phosphotyrosine within a specific amino acid sequence. The results showed that NS5A specifically bound to SH2 domain of Fyn from PV-treated B cells in addition to Src homology 3 (SH3 domain. Substitution of Arg(176 to Lys in the SH2 domain of Fyn abrogated this interaction. Deletion mutational analysis demonstrated that N-terminal region of NS5A was not required for the interaction with the SH2 domain of Fyn. Tyr(334 was identified as a tyrosine phosphorylation site in NS5A. Far-western analysis revealed that SH2 domain of Fyn directly bound to NS5A. Fyn and NS5A were colocalized in the lipid raft. These results suggest that NS5A directly binds to the SH2 domain of Fyn in a tyrosine phosphorylation-dependent manner. Lastly, we showed that the expression of NS5A in B cells increased phosphorylation of activation loop tyrosine in the kinase domain of Fyn. NS5A containing ligand for both SH2 and SH3 domains enhances an aberrant autophosphorylation and kinase activity of Fyn in B cells.

  12. Induction of Th1-Type Immune Response by Chitosan Nanoparticles Containing Plasmid DNA Encoding House Dust Mite Allergen Der p 2 for Oral Vaccination in Mice

    Institute of Scientific and Technical Information of China (English)

    Guoping Li; Zhigang Liu; Bin Liao; Nanshan Zhong

    2009-01-01

    This study was to prepare the chitosan-pDer p 2 nanoparticles and to investigate the effect of chitosan-DNA nanoparticles on immune response in mice by oral delivery of chitosan-DNA nanoparticles. The nanoparticles were synthesized by complexing chitosan with plasmid DNA. The DNA was fully complexed into chitosan-DNA nanoparticles, suggesting a 100% encapsulation efficiency. Chitosan-DNA complex renders a significant protection of the plasmid. No effect on cell viability was observed in both cell types and average cell viability over 100% was obtained. Oral gene delivery with chitosan-DNA nanoparticles can generate a higher level expression of gene in vivo. Oral chitosan-pDer p 2 nanoparticles in BALB/c mice can induce IFN-γ in serum and prevent subsequent sensitization of Th2 cell-regulated specific IgE responses. The data indicate that the oral administration of chitosan-pDer p 2 nanoparticles results in the expression of Der p 2 in the epithelial cells of both stomach and small intestine and the induction of Th1-type immune response.

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

    Science.gov (United States)

    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.

  14. A mutational analysis of the ColE1-encoded cell cycle regulator Rcd confirms its role in plasmid stability.

    Science.gov (United States)

    Balding, Claire; Blaby, Ian; Summers, David

    2006-07-01

    Multimers of multicopy plasmids cause instability. They arise by homologous recombination and accumulate by over-replication in a process known as the dimer catastrophe. Dimers are resolved to monomers by site-specific recombination systems such as Xer-cer of plasmid ColE1. In addition, the Rcd checkpoint hypothesis proposes that a short transcript (Rcd) coded within ColE1 cer delays the division of multimer-containing cells. The crucial observation underpinning the checkpoint hypothesis is that when the Rcd promoter (P(cer)) is inactivated by mutation of its invariant T, the plasmid becomes unstable. Recently, we discovered that this mutation also alters a potential Fis binding site in cer. ColE1-like plasmids are less stable in fis mutant hosts and it is conceivable that instability caused by the mutation is due to altered Fis binding, rather than the loss of Rcd expression per se. We have therefore undertaken an independent test of the role of P(cer)-Rcd in multicopy plasmid stability. We have generated a series of loss-of-function mutants of Rcd and detailed analysis of two of these shows that they cause a level of instability indistinguishable from P(cer) inactivation. This result is consistent with the predictions of the checkpoint hypothesis and confirms the role of Rcd in plasmid stability.

  15. Directed homology

    DEFF Research Database (Denmark)

    Fahrenberg, Uli

    2004-01-01

    We introduce a new notion of directed homology for semicubical sets. We show that it respects directed homotopy and is functorial, and that it appears to enjoy some good algebraic properties. Our work has applications to higher-dimensional automata.......We introduce a new notion of directed homology for semicubical sets. We show that it respects directed homotopy and is functorial, and that it appears to enjoy some good algebraic properties. Our work has applications to higher-dimensional automata....

  16. Chemotherapy of Bacterial Plasmids

    Science.gov (United States)

    1979-01-29

    render them non-susceptible to K: z plasmid-encoded enzymes. (3) Development of drugs which are selective inhibitor! 1 4, of plasmid DNA replication. (4... Development of drugs which inhibit phenotypic as expression of plasmid genes, and (5) Development of drugs which are inhibitors o, drug-inactivating...Barnes [2] them non-susceptible to plasmid-encoded enzymes, tabulated data on the incidence of Gram-negative 3) development of drugs which are

  17. Plasmid segregation mechanisms

    DEFF Research Database (Denmark)

    Ebersbach, Gitte; Gerdes, Kenn; Charbon, Gitte Ebersbach

    2005-01-01

    Bacterial plasmids encode partitioning (par) loci that ensure ordered plasmid segregation prior to cell division. par loci come in two types: those that encode actin-like ATPases and those that encode deviant Walker-type ATPases. ParM, the actin-like ATPase of plasmid R1, forms dynamic filaments ...

  18. Identification of Toxoplasma TgPH1, a pleckstrin homology domain-containing protein that binds to the phosphoinositide PI(3,5)P2.

    Science.gov (United States)

    Daher, Wassim; Morlon-Guyot, Juliette; Alayi, Tchilabalo Dilezitoko; Tomavo, Stan; Wengelnik, Kai; Lebrun, Maryse

    2016-05-01

    The phosphoinositide phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2) plays crucial roles in the maintenance of lysosome/vacuole morphology, membrane trafficking and regulation of endolysosome-localized membrane channel activity. In Toxoplasma gondii, we previously reported that PI(3,5)P2 is essential for parasite survival by controlling homeostasis of the apicoplast, a particular organelle of algal origin. Here, by using a phosphoinositide pull-down assay, we identified TgPH1 in Toxoplasma a protein conserved in many apicomplexan parasites. TgPH1 binds specifically to PI(3,5)P2, shows punctate intracellular localization, but plays no vital role for tachyzoite growth in vitro. TgPH1 is a protein predominantly formed by a pleckstrin homology (PH) domain. So far, PH domains have been described to bind preferentially to bis- or trisphosphate phosphoinositides containing two adjacent phosphates (i.e. PI(3,4)P2, PI(4,5)P2, PI(3,4,5)P3). Therefore, our study reveals an unusual feature of TgPH1 which binds preferentially to PI(3,5)P2.

  19. The S-layer homology domain-containing protein SlhA from Paenibacillus alvei CCM 2051(T is important for swarming and biofilm formation.

    Directory of Open Access Journals (Sweden)

    Bettina Janesch

    Full Text Available Swarming and biofilm formation have been studied for a variety of bacteria. While this is well investigated for Gram-negative bacteria, less is known about Gram-positive bacteria, including Paenibacillus alvei, a secondary invader of diseased honeybee colonies infected with Melissococcus pluton, the causative agent of European foulbrood (EFB.Paenibacillus alvei CCM 2051(T is a Gram-positive bacterium which was recently shown to employ S-layer homology (SLH domains as cell wall targeting modules to display proteins on its cell surface. This study deals with the newly identified 1335-amino acid protein SlhA from P. alvei which carries at the C‑terminus three consecutive SLH-motifs containing the predicted binding sequences SRGE, VRQD, and LRGD instead of the common TRAE motif. Based on the proof of cell surface location of SlhA by fluorescence microscopy using a SlhA-GFP chimera, the binding mechanism was investigated in an in vitro assay. To unravel a putative function of the SlhA protein, a knockout mutant was constructed. Experimental data indicated that one SLH domain is sufficient for anchoring of SlhA to the cell surface, and the SLH domains of SlhA recognize both the peptidoglycan and the secondary cell wall polymer in vitro. This is in agreement with previous data from the S-layer protein SpaA, pinpointing a wider utilization of that mechanism for cell surface display of proteins in P. alvei. Compared to the wild-type bacterium ΔslhA revealed changed colony morphology, loss of swarming motility and impaired biofilm formation. The phenotype was similar to that of the flagella knockout Δhag, possibly due to reduced EPS production influencing the functionality of the flagella of ΔslhA.This study demonstrates the involvement of the SLH domain-containing protein SlhA in swarming and biofilm formation of P. alvei CCM 2051(T.

  20. Regulation of abiotic stress signalling by Arabidopsis C-terminal domain phosphatase-like 1 requires interaction with a k-homology domain-containing protein.

    Directory of Open Access Journals (Sweden)

    In Sil Jeong

    Full Text Available Arabidopsis thaliana CARBOXYL-TERMINAL DOMAIN (CTD PHOSPHATASE-LIKE 1 (CPL1 regulates plant transcriptional responses to diverse stress signals. Unlike typical CTD phosphatases, CPL1 contains two double-stranded (ds RNA binding motifs (dsRBMs at its C-terminus. Some dsRBMs can bind to dsRNA and/or other proteins, but the function of the CPL1 dsRBMs has remained obscure. Here, we report identification of REGULATOR OF CBF GENE EXPRESSION 3 (RCF3 as a CPL1-interacting protein. RCF3 co-purified with tandem-affinity-tagged CPL1 from cultured Arabidopsis cells and contains multiple K-homology (KH domains, which were predicted to be important for binding to single-stranded DNA/RNA. Yeast two-hybrid, luciferase complementation imaging, and bimolecular fluorescence complementation analyses established that CPL1 and RCF3 strongly associate in vivo, an interaction mediated by the dsRBM1 of CPL1 and the KH3/KH4 domains of RCF3. Mapping of functional regions of CPL1 indicated that CPL1 in vivo function requires the dsRBM1, catalytic activity, and nuclear targeting of CPL1. Gene expression profiles of rcf3 and cpl1 mutants were similar during iron deficiency, but were distinct during the cold response. These results suggest that tethering CPL1 to RCF3 via dsRBM1 is part of the mechanism that confers specificity to CPL1-mediated transcriptional regulation.

  1. antimicrobial susceptibility and plasmids from escherichia coli ...

    African Journals Online (AJOL)

    2001-10-10

    Oct 10, 2001 ... transmission to humans of E. coli containing antibiotic resistance plasmids ... resistant micro-organisms, which may in turn transfer resistance to .... cells were washed with sterile normal saline to remove leached. Я-lactamase ...

  2. Homological descent for motivic homology theories

    OpenAIRE

    Geisser, Thomas

    2014-01-01

    The purpose of this paper is to give homological descent theorems for motivic homology theories (for example, Suslin homology) and motivic Borel-Moore homology theories (for example, higher Chow groups) for certain hypercoverings.

  3. Transformation-associated recombination between diverged and homologous DNA repeats is induced by strand breaks

    Energy Technology Data Exchange (ETDEWEB)

    Larionov, V.; Kouprina, N. [National Institute of Environmental Health Sciences (NIH), Research Triangle Park, NC (United States)]|[Institute of Cytology, St. Petersburg (Russian Federation); Eldarov, M. [National Institute of Environmental Health Sciences (NIH), Research Triangle Park, NC (United States)]|[Center for Bioengineering, Moscow (Russian Federation); Perkins, E.; Porter, G.; Resnick, M.A. [National Institute of Environmental Health Sciences (NIH), Research Triangle Park, NC (United States)

    1994-10-01

    Rearrangement and deletion within plasmid DNA is commonly observed during transformation. We have examined the mechanisms of transformation-associated recombination in the yeast Saccharomyces cerevisiae using a plasmid system which allowed the effects of physical state and/or extent of homology on recombination to be studied. The plasmid contains homologous or diverged (19%) DNA repeats separated by a genetically detectable color marker. Recombination during transformation for covalently closed circular plasmids was over 100-fold more frequent than during mitotic-growth. The frequency of recombination is partly dependent on the method of transformation In that procedures involving lithium acetate or spheroplasting yield higher frequencies than electroporation. When present in the repeats, unique single-strand breaks that are ligatable, as well as double-strand breaks, lead to high levels of recombination between diverged and identical repeats. The transformation-associated recombination between repeat DNA`s is under the influence of the RAD52, RAD1 and the RNC1 genes.

  4. Kloning Gen Melanoma Antigen 1 (Mage-1 dari Jaringan Testis untuk mendapatkan Plasmid Rekombinan Mage-1

    Directory of Open Access Journals (Sweden)

    Gondo Mastutik

    2015-12-01

    Full Text Available Melanoma antigen-1 (Mage-1 is expressed by spermatogonia cells of normal testicular tissue and 60−80% is expressed by the liver of hepatocellular carcinoma (HC patients. Mage-1 expression is a marker for diagnosing HC and predicting gastric and colorectal cancers. Isolation of messenger ribonucleid acid (mRNA Mage-1 from the liver tissue of HC patients is difficult; therefore, Mage-1 mRNA isolates can be obtained from tissues that express Mage-1 such as normal testicular tissues . This is an explorative research that was conducted at the Institute of Tropical Diseases of Airlangga University during August 2006–August 2008. The aim was to clone the coding sequence of Mage-1 gene from testicular tissues into a vector and to get recombinant plasmid Mage-1. Isolation of the full-length Mage-1 was performed using semi-nested polymerase chain reaction (PCR which was then cloned into plasmid pET101/D-TOPO and transformed into Escherichia coli (E. coli Top10 to get recombinant plasmid Mage-1. The length of pET101/D-TOPO was 5,753 bp and Mage-1 was 927 bp. The length of recombinant plasmid was 6,680 bp (5,753+927. Restriction analysis using EcoRV showed 4,230 and 2,450 bp bands (4,230+2,450=6,680. Sequence analyses showed that Mage-1 was 100% homologous with M77481 and NM_004988, 99% homologous with BC01755. In conclusion, according to the results of the restriction and sequences analysis, the recombinant plasmid pETGM/MAGE1-Testis contains the full length coding region of Mage-1 and is useful for developing the hepatocellular carcinoma diagnostic kits.

  5. Eps15 Homology Domain-containing Protein 3 Regulates Cardiac T-type Ca2+ Channel Targeting and Function in the Atria*

    Science.gov (United States)

    Curran, Jerry; Musa, Hassan; Kline, Crystal F.; Makara, Michael A.; Little, Sean C.; Higgins, John D.; Hund, Thomas J.; Band, Hamid; Mohler, Peter J.

    2015-01-01

    Proper trafficking of membrane-bound ion channels and transporters is requisite for normal cardiac function. Endosome-based protein trafficking of membrane-bound ion channels and transporters in the heart is poorly understood, particularly in vivo. In fact, for select cardiac cell types such as atrial myocytes, virtually nothing is known regarding endosomal transport. We previously linked the C-terminal Eps15 homology domain-containing protein 3 (EHD3) with endosome-based protein trafficking in ventricular cardiomyocytes. Here we sought to define the roles and membrane protein targets for EHD3 in atria. We identify the voltage-gated T-type Ca2+ channels (CaV3.1, CaV3.2) as substrates for EHD3-dependent trafficking in atria. Mice selectively lacking EHD3 in heart display reduced expression and targeting of both Cav3.1 and CaV3.2 in the atria. Furthermore, functional experiments identify a significant loss of T-type-mediated Ca2+ current in EHD3-deficient atrial myocytes. Moreover, EHD3 associates with both CaV3.1 and CaV3.2 in co-immunoprecipitation experiments. T-type Ca2+ channel function is critical for proper electrical conduction through the atria. Consistent with these roles, EHD3-deficient mice demonstrate heart rate variability, sinus pause, and atrioventricular conduction block. In summary, our findings identify CaV3.1 and CaV3.2 as substrates for EHD3-dependent protein trafficking in heart, provide in vivo data on endosome-based trafficking pathways in atria, and implicate EHD3 as a key player in the regulation of atrial myocyte excitability and cardiac conduction. PMID:25825486

  6. Homology among extra-cryptic DNA bands and the typical plasmids in Brazilian Yersinia pestis strains Homologia entre bandas extras de DNA críptico e os plasmídios típicos em cepas brasileiras de Yersinia pestis

    Directory of Open Access Journals (Sweden)

    Nilma Cintra Leal

    2000-03-01

    Full Text Available Yersinia pestis, the etiologic agent of plague, harbors three well-characterized plasmids: pFra (90-110kb, pYV (70 kb and pPst (9.5 kb. Furthermore, some extra-cryptic DNA bands have been observed in a number of wild strains from several foci of the world. Additional bands have also been reported in Brazilian strains. Looking for any relationship among these cryptic DNA bands and the three-prototypical plasmids, we analyzed twelve strains displaying different plasmid content. The DNA bands were hybridized by southern blot with probes directed at the genes caf1, lcrV and pla located respectively on the plasmids pFra, pYV and pPst. The probes were constructed by PCR amplification and labeled with digoxigenin. The Pla probe hybridized with its target (pPst and with bands of about 35 kb suggesting some homology among them. The Caf1 probe hybridized with the target (pFra as well as with higher bands. The LcrV also hybridized with the target (pYV and both with the bands higher than pFra and the bands between pFra and pYV. These results suggest that the large-cryptic bands could represent some rearrangement, open circular or linearized forms of the pFra and pYV plasmids.Yersinia pestis, o agente causador da peste, possui três plasmídios bem caracterizados: pFra (90-110 kb, pYV (70 kb e pPst (9.5 kb. Adicionalmente, algumas bandas extras de DNA críptico têm sido observadas em numerosas cepas selvagens em vários focos do mundo. Bandas extras também foram observadas em cepas brasileiras. Para verificar se existe alguma homologia entre as bandas extras de DNA críptico e os três plasmídios típicos, foram analisadas 12 culturas de Y. pestis através de hibridização com sondas dirigidas aos genes caf1, lcrV e pla localizados respectivamente nos plasmídios pFra, pYV e pPst. As sondas foram construídas através de amplificação por PCR e marcadas com digoxigenina. A sonda Pla reconheceu seu alvo (pPst e bandas de cerca de 35 kb sugerindo que

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

    Science.gov (United States)

    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.

  8. Helper plasmid cloning in Streptococcus sanguis: cloning of a tetracycline resistance determinant from the Streptococcus mutans chromosome.

    Science.gov (United States)

    Tobian, J A; Macrina, F L

    1982-10-01

    A model system for testing the helper plasmid cloning system of Gryczan et al. (Mol. Gen. Genet. 177:459-467, 1980) was devised for the Streptococcus sanguis (Challis) host-vector system. In this system, linearized pVA736 plasmid efficiently transformed an S. sanguis (Challis) host containing a homologous plasmid, pVA380-1, but did not transform a plasmidless host or a host containing a nonhomologous plasmid, pVA380. In addition, whereas monomeric circular pVA736 transformed a plasmidless host with two-hit kinetics, it transformed a pVA380-1-containing host with one-hit kinetics. This helper plasmid cloning system was used to isolate two HindIII fragments (5.0 megadaltons [Mdal] and 1.9 Mdal in size) from the chromosome of Streptococcus mutans V825 which conferred high-level tetracycline resistance. One tetracycline-resistant clone was examined and found to contain three plasmids which were sized and designated pVA868 (9.0 Mdal), pVA869 (9.5 Mdal), and pVA870 (9.8 Mdal). Results of Southern blot hybridization and restriction endonuclease digestion confirmed that all three chimeras were composed of two HindIII fragments of the S. mutans V825 chromosome, as well as a large portion, varying in size for each chimera, of the 2.8 Mdal cloning vector, pVA380-1. Incompatibility observed between pVA380-1 and each of the chimeras indicated that replication of the chimeras was governed by the pVA380-1 replicative origin. Southern blotting experiments revealed that the chimeras hybridized to Tn916, providing the first evidence that transposon-related genes of enteric streptococcal origin are disseminated among oral streptococci.

  9. HBD3基因真核表达质粒载体的构建及其体外表达和活性鉴定%Construction and in expression of eukaryotic expression plasmid containing HBD3 gene

    Institute of Scientific and Technical Information of China (English)

    夏章权; 张从纪; 王涛

    2012-01-01

    Objective To further determine the possible effect on antimicrobial activity, plasmid vector containing recombinant human beta defensins 3(HBD3)gene was constructed and the expression of exogenous gene in transformed bone marrow-derived mesenchymal stem cells (BMSCs)was observed. Methods By using RT-PCR obtained HBD3 from gingiva tissue,then recombinat- ed the gene to plasmid Pvivo1-mcs. The recombinated plasmid vector was named as pVIVOl-HBD3 and identified by restriction enzyme analysis and DNA sequencin. After the recombinated plasmid transformed BMSCs, expression of HBD3 in BMSCs was detected by RT-PCR,immunofluorescence and Western blot,and the function was determined by Kleihauer-Betke (K-B) test. Results We successfully constructed recombinant plasmid vector that expressed HBD3. The expression of HBD3 were confirmed by RT-PCR, immunofluorescence and Western blot on transformed BMSCs. The function of HBD3 were confirmed by K-B test. Conclusion The established BMSCs that overexpressed HBD3 provide a new strategy of gene therapy to promote wound healing, especially the infected one.%目的 构建人β防御素3(HBD3)基因真核表达质粒载体,观察其在大鼠骨髓间充质干细胞(BMSCs)中的表达,并检测其活性.方法 利用RT-PCR方法从牙龈组织中扩增出HBD3基因片段,通过DNA重组技术将基因片段重组于pVIVO1-mcs真核表达质粒载体上,构建成pVIVO1-HBD3重组质粒载体,分别用酶切电泳分析及DNA测序的方法对重组质粒载体进行鉴定.运用jetPEI转染试剂将重组质粒转染BMSCs后,RT-PCR、免疫荧光和蛋白质印迹法检测目的 基因的表达.取转染重组质粒的BMSCs无血清上清的浓缩液,采用K-B纸片扩散法进行抗菌活性实验.结果 (1)酶切电泳分析得到相应的目的 片段,大小与理论计算值一致,DNA测序证实目的 基因序列正确无突变,成功构建真核表达质粒载体pVIVO1-HBD3.(2)RT-PCR和免疫荧光、蛋白质印

  10. Kaposi's sarcoma-associated herpesvirus contains G protein-coupled receptor and cyclin D homologs which are expressed in Kaposi's sarcoma and malignant lymphoma.

    OpenAIRE

    1996-01-01

    A new human herpesvirus was recently identified in all forms of Kaposi's sarcoma (Kaposi's sarcoma-associated herpesvirus [KSHV] or human herpesvirus 8), as well as in primary effusion (body cavity-based) lymphomas (PELs). A 12.3-kb-long KSHV clone was obtained from a PEL genomic library. Sequencing of this clone revealed extensive homology and colinearity with the right end of the herpesvirus saimiri (HVS) genome and more limited homology to the left end of the Epstein-Barr virus genome. Fou...

  11. 长片断引物反向PCR方法构建重复序列的重组质粒%Construction of Recombinant Plasmid Containing Repeated Sequence by Megaprimer Reverse PCR

    Institute of Scientific and Technical Information of China (English)

    刘猛; 刘亚娟; 徐文选; 贺添艳; 刘亮伟

    2016-01-01

    构建重复序列的重组质粒比较困难,因为扩增重复序列会产生多聚物.利用长片断引物反向PCR方法构建重组质粒pET20b-C1-Xyn-C1,将碳水化合物结合模块C1连接在木聚糖酶Xyn两端,为类似质粒的构建提供新方法.第一步,以C1特异性正向引物LF、载体特异性反向引物VRP从pET20b-Xyn-C1模板上扩增C1-pET20b长片断DNA,作为第二步的正向引物;第二步,以pET20b-C1-Xyn为模板,Xyn特异性反向引物RX,正向引物与模板上pET20b同源区域匹配,阻止了模板上C1同源区域的匹配,从而抑制多聚物产生;反向PCR扩增得到线性重组质粒C1-pET20b-C1-Xyn,将此线性产物用T4 DNA连接酶连接后转化DH5α感受态细胞,得到含重复序列的pET20b-C1-Xyn-C1转化子.%It is difficult to construct recombinant plasmid containing repeated DNA sequences,because multimer would be produced in PCR amplification. Megaprimer reverse PCR was used to construct recombinant plasmid pET20b-C1-Xyn-C1,which contained a xylanase(Xyn)DNA flanked by repeated DNA sequence,a Carbohydrate binding module C1. Firstly,megaprimer C1-pET20b was amplified using forward primer LF for C1,reverse primer VRP for pET20b,and pET20b-Xyn-C1 template. Secondly,the linear recombinant plasmid C1-pET20b-C1-Xyn DNA was amplified by using reverse primer RX for Xyn,pET20b-C1-Xyn template,and the amplified C1-pET20b as forward megaprimer. The forward megaprimer annealed to pET20b instead of region of the template. The linear recombinant plasmid was ligated with T4 DNA ligase and transformed to DH5α competent cell. The recombinant pET20b-C1-Xyn-C1 containing repeated sequences C1 was constructed.

  12. KOC (K homology domain containing protein overexpressed in cancer): a novel molecular marker that distinguishes between benign and malignant lesions of the pancreas.

    Science.gov (United States)

    Yantiss, Rhonda K; Woda, Bruce A; Fanger, Gary R; Kalos, M; Whalen, Giles F; Tada, Hiroomi; Andersen, Dana K; Rock, Kenneth L; Dresser, Karen

    2005-02-01

    KOC (K homology domain containing protein overexpressed in cancer) is a novel oncofetal RNA-binding protein highly expressed in pancreatic carcinomas. Recently, Corixa Corporation developed a monoclonal antibody specific for KOC that can be used with standard immunohistochemical techniques. The purposes of this study were 1) to assess KOC mRNA expression in pancreatic carcinoma, 2) to determine the pattern of KOC immunoexpression among benign, borderline, and malignant pancreatic epithelial lesions, and 3) to evaluate the utility of the KOC antibody in distinguishing between these entities. mRNA was isolated from fresh pancreatic tissues (19 carcinomas, 2 normal pancreas, 1 chronic pancreatitis) and amplified using standard RT-PCR techniques. Fifteen of 19 (79%) carcinomas overexpressed KOC mRNA relative to non-neoplastic tissue samples and expression increased progressively with tumor stage: the mean copy number of KOC mRNA transcripts was 1.5, 11.1, 31, and 28 for stage I, II, III, and IV carcinomas, respectively, compared with 0.9 and 1 for normal pancreatic tissue and chronic pancreatitis, respectively. Immunostains using the KOC antibody were performed on 50 surgical resection specimens (38 invasive adenocarcinomas, 3 intraductal papillary-mucinous neoplasms, 2 mucinous cystic neoplasms, 7 chronic pancreatitis). KOC staining was present in 37 of 38 (97%) carcinomas: the staining reaction was moderate or strong in 36 of 38 (94%) and present in >50% of the tumor cells in 35 of 38 (92%) cases. Severe dysplasia of the ductal epithelium, present in 19 foci of intraductal papillary mucinous carcinoma, mucinous cystadenocarcinoma, and grade 3 pancreatic intraepithelial neoplasia (PanIN3) showed strong or moderate staining in 15 (79%) cases, whereas foci of mild and moderate dysplasia (intraductal papillary-mucinous neoplasms and mucinous cystic neoplasms with adenoma and/or moderate dysplasia, PanIN1, and PanIN2) were uniformly negative for this marker in 25 and 22

  13. Complete nucleotide sequence of CTX-M-15-plasmids from clinical Escherichia coli isolates: insertional events of transposons and insertion sequences.

    Directory of Open Access Journals (Sweden)

    Annemieke Smet

    Full Text Available BACKGROUND: CTX-M-producing Escherichia coli strains are regarded as major global pathogens. METHODOLOGY/PRINCIPAL FINDINGS: The nucleotide sequence of three plasmids (pEC_B24: 73801-bp; pEC_L8: 118525-bp and pEC_L46: 144871-bp from Escherichia coli isolates obtained from patients with urinary tract infections and one plasmid (pEC_Bactec: 92970-bp from an Escherichia coli strain isolated from the joint of a horse with arthritis were determined. Plasmid pEC_Bactec belongs to the IncI1 group and carries two resistance genes: bla(TEM-1 and bla(CTX-M-15. It shares more than 90% homology with a previously published bla(CTX-M-plasmid from E. coli of human origin. Plasmid pEC_B24 belongs to the IncFII group whereas plasmids pEC_L8 and pEC_L46 represent a fusion of two replicons of type FII and FIA. On the pEC_B24 backbone, two resistance genes, bla(TEM-1 and bla(CTX-M-15, were found. Six resistance genes, bla(TEM-1, bla(CTX-M-15, bla(OXA-1, aac6'-lb-cr, tetA and catB4, were detected on the pEC_L8 backbone. The same antimicrobial drug resistance genes, with the exception of tetA, were also identified on the pEC_L46 backbone. Genome analysis of all 4 plasmids studied provides evidence of a seemingly frequent transposition event of the bla(CTX-M-15-ISEcp1 element. This element seems to have a preferred insertion site at the tnpA gene of a bla(TEM-carrying Tn3-like transposon, the latter itself being inserted by a transposition event. The IS26-composite transposon, which contains the bla(OXA-1, aac6'-lb-cr and catB4 genes, was inserted into plasmids pEC_L8 and pEC_L46 by homologous recombination rather than a transposition event. Results obtained for pEC_L46 indicated that IS26 also plays an important role in structural rearrangements of the plasmid backbone and seems to facilitate the mobilisation of fragments from other plasmids. CONCLUSIONS: Collectively, these data suggests that IS26 together with ISEcp1 could play a critical role in the evolution of

  14. Nucleotide sequence of pOLA52: a conjugative IncX1 plasmid from Escherichia coli which enables biofilm formation and multidrug efflux

    DEFF Research Database (Denmark)

    Norman, Anders; Hansen, Lars H.; She, Qunxin

    2008-01-01

    The large conjugative multidrug resistance (MDR) plasmid pOLA52 was sequenced and annotated. The plasmid encodes two phenotypes normally associated with the chromosomes of opportunistic pathogens, namely MDR via a resistance-nodulation-division (RND)-type efflux-pump (oqxAB), and the formation....... The plasmid was also classified as IncX1 with incompatibility testing. The conjugal transfer and plasmid maintenance regions of pOLA52 therefore seem to represent IncX1 orthologues of the well-characterized IncX2 plasmid R6K. Sequence homology searches in GenBank also suggested a considerably higher...... prevalence of IncX1 group plasmids than IncX2. The 21 kb 'genetic load' region of pOLA52 was shown to consist of a mosaic, among other things a fragmented Tn3 transposon encoding ampicillin resistance. Most notably the oqxAB and mrkABCDF cassettes were contained within two composite transposons (Tn6010...

  15. The RepA_N replicons of Gram-positive bacteria: a family of broadly distributed but narrow host range plasmids.

    Science.gov (United States)

    Weaver, Keith E; Kwong, Stephen M; Firth, Neville; Francia, Maria Victoria

    2009-03-01

    The pheromone-responsive conjugative plasmids of Enterococcus faecalis and the multiresistance plasmids pSK1 and pSK41 of Staphylococcus aureus are among the best studied plasmids native to Gram-positive bacteria. Although these plasmids seem largely restricted to their native hosts, protein sequence comparison of their replication initiator proteins indicates that they are clearly related. Homology searches indicate that these replicons are representatives of a large family of plasmids and a few phage that are widespread among the low G+C Gram-positive bacteria. We propose to name this family the RepA_N family of replicons after the annotated conserved domain that the initiator protein contains. Detailed sequence comparisons indicate that the initiator protein phylogeny is largely congruent with that of the host, suggesting that the replicons have evolved along with their current hosts and that intergeneric transfer has been rare. However, related proteins were identified on chromosomal regions bearing characteristics indicative of ICE elements, and the phylogeny of these proteins displayed evidence of more frequent intergeneric transfer. Comparison of stability determinants associated with the RepA_N replicons suggests that they have a modular evolution as has been observed in other plasmid families.

  16. Transfer of plasmid-mediated ampicillin resistance from Haemophilus to Neisseria gonorrhoeae requires an intervening organism.

    Science.gov (United States)

    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.

  17. Effect of plasmid R391 and other IncJ plasmids on the survival of Escherichia coli after UV irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Pembroke, J.T.; Stevens, E. (University Coll., Galway (Ireland))

    1984-07-01

    The presence of the IncJ plasmids R391, R997, R705, R706, R748, and R749 was shown to sensitize Escherichia coli AB1157 and both its uvr A and lexA derivatives to UV irradiation. No alteration in post-irradiation survival was observed in a recA mutant containing these plasmids, compared with the non-plasmid-containing recA strain. Analysis of recombination frequency in Hfr crosses to recA/sup +/ cells containing plasmid R391 indicated a reduction in recombination frequency compared with that obtained in similar crosses to a non-plasmid-containing strain. This effect was not due to plasmid-encoded restriction or entry exclusion systems and therefore must be considered as a real block in recombination. When cells containing plasmid R391 were irradiated and allowed to photoreactivate, an increase in survival was observed which was comparable to that observed in the non-plasmid-containing derivative. This indicated that post-irradiation processing of UV-induced damage, or lack of such processing, by mechanisms other than photoreactivation was responsible for the UV sensitivity associated with plasmid R391.

  18. Role of Plasmid in Production of Acetobacter Xylinum Biofilms

    Directory of Open Access Journals (Sweden)

    Abbas Rezaee

    2005-01-01

    Full Text Available Acetobacter xylinum has the ability to produce cellulotic biofilms. Bacterial cellulose is expected to be used in many industrial or biomedical materials for its unique characteristics. A. xylinum contains a complex system of plasmid DNA molecules. A 44 kilobases (kb plasmid was isolated in wild type of A. xylinum. To improve the cellulose producing ability of A. xylinum, role of the plasmid in production of cellulose was studied. The comparisons between wild type and cured cells of A. xylinum showed that there is considerably difference in cellulose production. In order to study the relationship between plasmid and the rate of cellulose production, bacteria were screened for plasmid profile by a modified method for preparation of plasmid. This method yields high levels of pure plasmid DNA that can be used for common molecular techniques, such as digestion and transformation, with high efficiency.

  19. Alignment of Homologous Chromosomes and Effective Repair of Programmed DNA Double-Strand Breaks during Mouse Meiosis Require the Minichromosome Maintenance Domain Containing 2 (MCMDC2) Protein.

    Science.gov (United States)

    Finsterbusch, Friederike; Ravindranathan, Ramya; Dereli, Ihsan; Stanzione, Marcello; Tränkner, Daniel; Tóth, Attila

    2016-10-01

    Orderly chromosome segregation during the first meiotic division requires meiotic recombination to form crossovers between homologous chromosomes (homologues). Members of the minichromosome maintenance (MCM) helicase family have been implicated in meiotic recombination. In addition, they have roles in initiation of DNA replication, DNA mismatch repair and mitotic DNA double-strand break repair. Here, we addressed the function of MCMDC2, an atypical yet conserved MCM protein, whose function in vertebrates has not been reported. While we did not find an important role for MCMDC2 in mitotically dividing cells, our work revealed that MCMDC2 is essential for fertility in both sexes due to a crucial function in meiotic recombination. Meiotic recombination begins with the introduction of DNA double-strand breaks into the genome. DNA ends at break sites are resected. The resultant 3-prime single-stranded DNA overhangs recruit RAD51 and DMC1 recombinases that promote the invasion of homologous duplex DNAs by the resected DNA ends. Multiple strand invasions on each chromosome promote the alignment of homologous chromosomes, which is a prerequisite for inter-homologue crossover formation during meiosis. We found that although DNA ends at break sites were evidently resected, and they recruited RAD51 and DMC1 recombinases, these recombinases were ineffective in promoting alignment of homologous chromosomes in the absence of MCMDC2. Consequently, RAD51 and DMC1 foci, which are thought to mark early recombination intermediates, were abnormally persistent in Mcmdc2-/- meiocytes. Importantly, the strand invasion stabilizing MSH4 protein, which marks more advanced recombination intermediates, did not efficiently form foci in Mcmdc2-/- meiocytes. Thus, our work suggests that MCMDC2 plays an important role in either the formation, or the stabilization, of DNA strand invasion events that promote homologue alignment and provide the basis for inter-homologue crossover formation during

  20. 重组溶菌酶质粒pcDNAKLYZ治疗泌乳期奶牛乳房炎%The Treatment of Lactating Bovine Mastitis by Using Recombinant Plasmid pcDNAKLYZ Containing Lysozyme Gene

    Institute of Scientific and Technical Information of China (English)

    沈诚; 林源; 叶承荣; 金耀忠; 俞向前; 孙怀昌; 朱建国

    2011-01-01

    通过比较注射人溶菌酶重组质粒pcDNAKLYZ的隐性乳房炎奶牛注射前后的奶样中细菌计数结果,对重组溶菌酶基因工程质粒治疗奶牛乳房炎的效果进行分析.对乳房炎患牛的156个乳区治疗前后312份奶样进行细菌培养,其中在注射前的乳样的培养结果中,阴性率为0(0/156),菌落数在50以内的比率为12.82%(20/156),在51~100的比率为16.67%(26/156),大于等于100的比率为70.51%(110/150);在注射人溶菌酶的重组质粒pcDNAKLYz后的奶牛乳样的培养结果中,阴性率为51.92%(81/156),菌落数在50以内的比率为45.51%(71/156),菌落数在51~100的比率为0%(0/156),大于等于100的比率为2.56%(4/156).因此,从细菌计数结果来看,重组质粒组的阴性及小于50的比率(97.44%)远高于治疗前(12.82%),重组质粒的抑菌效果显著(P<0.05),该重组质粒对奶牛乳房炎具有较好的治疗效果.%To analysis the curative effect to bovine mastitis by using the recombinant plasmid pcDNAKLYZ containing lysozyme gene, We make several bacterial cultures with 312 parts of milk samples collected from 156 mammary area of cattle infected with subclinical or clinical mastitis to compare the bacterial colonies between the pre-and-post-injection of the recombinant plasmid. In the sample of pre-injection, the culture result showing negative result of samples accounts for 0(0/156), the number of samples showing bacterial colonies from 1 to 50 accounts for 12.82%(20/156), the number of samples showing bacterial colonies from 51 to 100 accounts for 16.67% (26/156), the number of samples show bacterial colonies above 100 accounts for 70.51% (110/156); In the post-injection sample, the culture result showing negative result of samples accounts for 51.92%(81/156), the number of samples showing bacterial colonies from 1 to 50 accounts for 45.51%(71/156), the number of samples showing bacterial colonies from 51 to 100 accounts for 0(0/156), the number of samples

  1. The Plasmid of Escherichia coli Strain S88 (O45:K1:H7) That Causes Neonatal Meningitis Is Closely Related to Avian Pathogenic E. coli Plasmids and Is Associated with High-Level Bacteremia in a Neonatal Rat Meningitis Model▿

    Science.gov (United States)

    Peigne, Chantal; Bidet, Philippe; Mahjoub-Messai, Farah; Plainvert, Céline; Barbe, Valérie; Médigue, Claudine; Frapy, Eric; Nassif, Xavier; Denamur, Erick; Bingen, Edouard; Bonacorsi, Stéphane

    2009-01-01

    A new Escherichia coli virulent clonal group, O45:K1, belonging to the highly virulent subgroup B21 was recently identified in France, where it accounts for one-third of E. coli neonatal meningitis cases. Here we describe the sequence, epidemiology and function of the large plasmid harbored by strain S88, which is representative of the O45:K1 clonal group. Plasmid pS88 is 133,853 bp long and contains 144 protein-coding genes. It harbors three different iron uptake systems (aerobactin, salmochelin, and the sitABCD genes) and other putative virulence genes (iss, etsABC, ompTP, and hlyF). The pS88 sequence is composed of several gene blocks homologous to avian pathogenic E. coli plasmids pAPEC-O2-ColV and pAPEC-O1-ColBM. PCR amplification of 11 open reading frames scattered throughout the plasmid was used to investigate the distribution of pS88 and showed that a pS88-like plasmid is present in other meningitis clonal groups such as O18:K1, O1:K1, and O83:K1. A pS88-like plasmid was also found in avian pathogenic strains and human urosepsis strains belonging to subgroup B21. A variant of S88 cured of its plasmid displayed a marked loss of virulence relative to the wild-type strain in a neonatal rat model, with bacteremia more than 2 log CFU/ml lower. The salmochelin siderophore, a known meningovirulence factor, could not alone explain the plasmid's contribution to virulence, as a salmochelin mutant displayed only a minor fall in bacteremia (0.9 log CFU/ml). Thus, pS88 is a major virulence determinant related to avian pathogenic plasmids that has spread not only through meningitis clonal groups but also human urosepsis and avian pathogenic strains. PMID:19307211

  2. Mutations of Conserved Residues in the Major Homology Region Arrest Assembling HIV-1 Gag as a Membrane-Targeted Intermediate Containing Genomic RNA and Cellular Proteins.

    Science.gov (United States)

    Tanaka, Motoko; Robinson, Bridget A; Chutiraka, Kasana; Geary, Clair D; Reed, Jonathan C; Lingappa, Jaisri R

    2015-12-09

    The major homology region (MHR) is a highly conserved motif that is found within the Gag protein of all orthoretroviruses and some retrotransposons. While it is widely accepted that the MHR is critical for assembly of HIV-1 and other retroviruses, how the MHR functions and why it is so highly conserved are not understood. Moreover, consensus is lacking on when HIV-1 MHR residues function during assembly. Here, we first addressed previous conflicting reports by confirming that MHR deletion, like conserved MHR residue substitution, leads to a dramatic reduction in particle production in human and nonhuman primate cells expressing HIV-1 proviruses. Next, we used biochemical analyses and immunoelectron microscopy to demonstrate that conserved residues in the MHR are required after assembling Gag has associated with genomic RNA, recruited critical host factors involved in assembly, and targeted to the plasma membrane. The exact point of inhibition at the plasma membrane differed depending on the specific mutation, with one MHR mutant arrested as a membrane-associated intermediate that is stable upon high-salt treatment and other MHR mutants arrested as labile, membrane-associated intermediates. Finally, we observed the same assembly-defective phenotypes when the MHR deletion or conserved MHR residue substitutions were engineered into Gag from a subtype B, lab-adapted provirus or Gag from a subtype C primary isolate that was codon optimized. Together, our data support a model in which MHR residues act just after membrane targeting, with some MHR residues promoting stability and another promoting multimerization of the membrane-targeted assembling Gag oligomer. The retroviral Gag protein exhibits extensive amino acid sequence variation overall; however, one region of Gag, termed the major homology region, is conserved among all retroviruses and even some yeast retrotransposons, although the reason for this conservation remains poorly understood. Highly conserved residues

  3. Homologous recombination and gene replacement at the dihydrofolate reductase-thymidylate synthase locus in Toxoplasma gondii.

    Science.gov (United States)

    Donald, R G; Roos, D S

    1994-02-01

    To investigate the feasibility of genomic transgene expression and gene targeting in Toxoplasma gondii, parasites have been transfected with constructs differing in the length of contiguous genomic sequence spanning the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene. We have previously reported that vectors derived from a DHFR-TS cDNA 'minigene' containing mutations in the DHFR coding sequence confer pyrimethamine resistance to transfected parasites (Donald and Roos, 1993). Stably resistant parasite clones arise at high frequency, generally by virtue of transgene integration into parasite chromosomes at locations scattered throughout the genome. In contrast, using a vector which contains 8 kb of contiguous genomic sequence (vs. homologous recombination. Homologous recombination appears to occur at even higher frequency when a 16 kb genomic clone is used. Circular plasmids were more efficient than linearized molecules at producing homologous recombination in this system, integrating by reciprocal crossing-over to produce a duplication of the DHFR-TS locus. Double crossing-over (or gene conversion) was also observed at low frequency, resulting in complete allelic replacement in this haploid stage of the parasite. The ability to produce either homologous or non-homologous recombinants, by the selection of appropriate transformation constructs, has considerable genetic potential.

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

    Science.gov (United States)

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

    1992-01-01

    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. Images PMID:1547783

  5. Isolation and characterization of yeast DNA repair genes. II. Isolation of plasmids that complement the mutations rad50-1, rad51-1, rad54-3, and rad55-3

    Energy Technology Data Exchange (ETDEWEB)

    Calderon, I.L.; Contopoulou, C.R.; Mortimer, R.K.

    1983-01-01

    Plasmids that complement the yeast mutations rad50-1, rad51-1, rad54-3, and rad55-3 were obtained by transforming strains that carried a leu2 marker and the particular rad mutation, with YEp 13 plasmids containing near random yeast DNA inserts. Integration of these plasmids or of fragments of these plasmids was accomplished. Genetic studies using the integrants established the presence of the genes RAD50, RAD54 and RAD55 in the respective plasmids. However, a BamHI subclone of the rad50-1 complementing plasmid failed to integrate at the RAD50 locus, indicating that no homology exists between this fragment and the RAD50 gene. A BamHI fragment for the RAD54 plasmid was shown to be internal to the RAD54 gene: its integration within a wild type copy of RAD54 causes the cell to become Rad/sup -/; its excision is X-ray inducible and restores the Rad/sup -/ phenotype. Since cells bearing a disrupted copy of RAD54 are able to survive, the author concludes that this is not essential.

  6. Antibiotic multiresistance plasmid pRSB101 isolated from a wastewater treatment plant is related to plasmids residing in phytopathogenic bacteria and carries eight different resistance determinants including a multidrug transport system.

    Science.gov (United States)

    Szczepanowski, Rafael; Krahn, Irene; Linke, Burkhard; Goesmann, Alexander; Pühler, Alfred; Schlüter, Andreas

    2004-11-01

    Ten different antibiotic resistance plasmids conferring high-level erythromycin resistance were isolated from an activated sludge bacterial community of a wastewater treatment plant by applying a transformation-based approach. One of these plasmids, designated pRSB101, mediates resistance to tetracycline, erythromycin, roxythromycin, sulfonamides, cephalosporins, spectinomycin, streptomycin, trimethoprim, nalidixic acid and low concentrations of norfloxacin. Plasmid pRSB101 was completely sequenced and annotated. Its size is 47 829 bp. Conserved synteny exists between the pRSB101 replication/partition (rep/par) module and the pXAC33-replicon from the phytopathogen Xanthomonas axonopodis pv. citri. The second pRSB101 backbone module encodes a three-Mob-protein type mobilization (mob) system with homology to that of IncQ-like plasmids. Plasmid pRSB101 is mobilizable with the help of the IncP-1alpha plasmid RP4 providing transfer functions in trans. A 20 kb resistance region on pRSB101 is located within an integron-containing Tn402-like transposon. The variable region of the class 1 integron carries the genes dhfr1 for a dihydrofolate reductase, aadA2 for a spectinomycin/streptomycin adenylyltransferase and bla(TLA-2) for a so far unknown Ambler class A extended spectrum beta-lactamase. The integron-specific 3'-segment (qacEDelta1-sul1-orf5Delta) is connected to a macrolide resistance operon consisting of the genes mph(A) (macrolide 2'-phosphotransferase I), mrx (hydrophobic protein of unknown function) and mphR(A) (regulatory protein). Finally, a putative mobile element with the tetracycline resistance genes tetA (tetracycline efflux pump) and tetR was identified upstream of the Tn402-specific transposase gene tniA. The second 'genetic load' region on pRSB101 harbours four distinct mobile genetic elements, another integron belonging to a new class and footprints of two more transposable elements. A tripartite multidrug (MDR) transporter consisting of an ATP

  7. Rapid plasmid library screening using RecA-coated biotinylated probes.

    Science.gov (United States)

    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.

  8. Structural similarity and distribution of small cryptic plasmids of Lactobacillus curvatus and L. sake.

    Science.gov (United States)

    Vogel, R F; Lohmann, M; Weller, A N; Hugas, M; Hammes, W P

    1991-11-15

    Plasmid profiles of strains of Lactobacillus curvatus and L. sake isolated from meat or sauerkraut were analysed to investigate plasmid homology and distribution in relation to the ecology of these organisms in fermenting foods. A hybridisation probe was constructed by cloning of pLc2, a cryptic, 2.6-kbp plasmid from L. curvatus LTH683, into the Escherichia coli plasmid pRV50. In Southern hybridisations with the digoxygenine labeled pLc2 probe, pLc2-related small plasmids were frequently detected in meat-borne strains of L. casei subsp. pseudoplantarum, L. curvatus, L. sake, L. alimentarius, L. farciminis and L. halotolerans and in L. curvatus and L. sake isolated from sauerkraut. Among 27 Lactobacillus type strains originally isolated from habitats other than meat this type of homology was detected only with plasmids of L. buchneri and L. mali. Restriction-enzyme mapping of six small cryptic plasmids from L. curvatus and L. sake revealed strong structural homology but no similarity to previously characterized plasmids of lactobacilli. The presence of a variable region in addition to a conserved one and the occurrence of deletions during cloning of pLc2 suggest that vectors derived from these plasmids are likely to be structurally unstable.

  9. Recombinogenic engineering of conjugative plasmids with fluorescent marker cassettes

    DEFF Research Database (Denmark)

    Reisner, A.; Molin, Søren; Zechner, E.L.

    2002-01-01

    An efficient approach for the insertion of fluorescent marker genes with sequence specificity into conjugative plasmids in Escherichia coli is described. For this purpose, homologous recombination of linear double-stranded targeting DNA was mediated by the bacteriophage lambda recombination funct...

  10. Homologous gene replacement in Physarum

    Energy Technology Data Exchange (ETDEWEB)

    Burland, T.G. [Univ. of Wisconsin, Madison, WI (United States); Pallotta, D. [Laval Univ., Quebec (Canada)

    1995-01-01

    The protist Physarum polycephalum is useful for analysis of several aspects of cellular and developmental biology. To expand the opportunities for experimental analysis of this organism, we have developed a method for gene replacement. We transformed Physarum amoebae with plasmid DNA carrying a mutant allele, ardD{Delta}1, of the ardD actin gene; ardD{Delta}1 mutates the critical carboxy-terminal region of the gene product. Because ardD is not expressed in the amoeba, replacement of ardD{sup +} with ardD{Delta}1 should not be lethal for this cell type. Transformants were obtained only when linear plasmid DNA was used. Most transformants carried one copy of ardD{Delta}1 in addition to ardD{sup +}, but in two (5%), ardD{sup +} was replaced by a single copy of ardD{Delta}1. This is the first example of homologous gene replacement in Physarum. ardD{Delta}1 was stably maintained in the genome through growth, development and meiosis. We found no effect of ardD{Delta}l on viability, growth, or development of any of the various cell types of Physarum. Thus, the carboxy-terminal region of the ardD product appears not to perform a unique essential role in growth or development. Nevertheless, this method for homologous gene replacement can be applied to analyze the function of any cloned gene. 38 refs., 6 figs., 1 tab.

  11. A universal cloning method based on yeast homologous recombination that is simple, efficient, and versatile

    Science.gov (United States)

    Joska, Tammy M.; Mashruwala, Ameya; Boyd, Jeffrey M.; Belden, William J.

    2014-01-01

    Cloning by homologous recombination (HR) in Saccharomyces cerevisiae is an extremely efficient and cost-effective alternative to other methods of recombinant DNA technologies. Unfortunately, it is incompatible with all the various specialized plasmids currently used in microbiology and biomedical research laboratories, and is therefore, not widely adopted. In an effort to dramatically improve the versatility of yeast gap-repair cloning and make it compatible with any DNA plasmid, we demonstrate that by simply including a yeast-cloning cassette (YCC) that contains the 2-micron origin of replication (2 μm ori) and the ura3 gene for selection, multiple DNA fragments can be assembled into any DNA vector. We show this has almost unlimited potential by building a variety of plasmid for different uses including: recombinant protein production, epitope tagging, site-directed mutagenesis, and expression of fluorescent fusion proteins. We demonstrate the use in a variety of plasmids for use in microbial systems and even demonstrate it can be used in a vertebrate model. This method is remarkably simple and extremely efficient, plus it provides a significant cost saving over commercially available kits. PMID:24418681

  12. Dissemination and Persistence of blaCTX-M-9 Are Linked to Class 1 Integrons Containing CR1 Associated with Defective Transposon Derivatives from Tn402 Located in Early Antibiotic Resistance Plasmids of IncHI2, IncP1-α, and IncFI Groups

    Science.gov (United States)

    Novais, Ângela; Cantón, Rafael; Valverde, Aránzazu; Machado, Elisabete; Galán, Juan-Carlos; Peixe, Luísa; Carattoli, Alessandra; Baquero, Fernando; Coque, Teresa M.

    2006-01-01

    This study analyzes the diversity of In60, a class 1 integron bearing CR1 and containing blaCTX-M-9, and its association with Tn402, Tn21, and classical conjugative plasmids among 45 CTX-M-9-producing clinical strains (41 Escherichia coli strains, 2 Klebsiella pneumoniae strains, 1 Salmonella enterica strain, and 1 Enterobacter cloacae strain). Forty-five patients in a Spanish tertiary care hospital were studied (1996 to 2003). The diversity of In60 and association of In60 with Tn402 or mercury resistance transposons were investigated by overlapping PCR assays and/or hybridization. Plasmid characterization included comparison of restriction fragment length polymorphism patterns and determination of incompatibility group by PCR-based replicon typing, sequencing, and hybridization. CTX-M-9 plasmids belonged to IncHI2 (n = 26), IncP-1α (n = 10), IncFI (n = 4), and IncI (n = 1) groups. Genetic platforms containing blaCTX-M-9 were classified in six types in relation to the In60 backbone and in eight subtypes in relation to Tn402 derivatives. They were associated with Tn21 sequences when located in IncP-1α or IncHI2 plasmids. Our study identified blaCTX-M-9 in a high diversity of CR1-bearing class 1 integrons linked to different Tn402 derivatives, often to Tn21, highlighting the role of recombination events in the evolution of antibiotic resistance plasmids. The presence of blaCTX-M-9 on broad-host-range IncP-1α plasmids might contribute to its dissemination to hosts that were not members of the family Enterobacteriaceae. PMID:16870767

  13. Algebra V homological algebra

    CERN Document Server

    Shafarevich, I

    1994-01-01

    This book, the first printing of which was published as volume 38 of the Encyclopaedia of Mathematical Sciences, presents a modern approach to homological algebra, based on the systematic use of the terminology and ideas of derived categories and derived functors. The book contains applications of homological algebra to the theory of sheaves on topological spaces, to Hodge theory, and to the theory of modules over rings of algebraic differential operators (algebraic D-modules). The authors Gelfand and Manin explain all the main ideas of the theory of derived categories. Both authors are well-known researchers and the second, Manin, is famous for his work in algebraic geometry and mathematical physics. The book is an excellent reference for graduate students and researchers in mathematics and also for physicists who use methods from algebraic geometry and algebraic topology.

  14. Plasmid diversity and phylogenetic consistency in the Lyme disease agent Borrelia burgdorferi.

    Science.gov (United States)

    Casjens, Sherwood R; Gilcrease, Eddie B; Vujadinovic, Marija; Mongodin, Emmanuel F; Luft, Benjamin J; Schutzer, Steven E; Fraser, Claire M; Qiu, Wei-Gang

    2017-02-15

    Bacteria from the genus Borrelia are known to harbor numerous linear and circular plasmids. We report here a comparative analysis of the nucleotide sequences of 236 plasmids present in fourteen independent isolates of the Lyme disease agent B. burgdorferi. We have sequenced the genomes of 14 B. burgdorferi sensu stricto isolates that carry a total of 236 plasmids. These individual isolates carry between seven and 23 plasmids. Their chromosomes, the cp26 and cp32 circular plasmids, as well as the lp54 linear plasmid, are quite evolutionarily stable; however, the remaining plasmids have undergone numerous non-homologous and often duplicative recombination events. We identify 32 different putative plasmid compatibility types among the 236 plasmids, of which 15 are (usually) circular and 17 are linear. Because of past rearrangements, any given gene, even though it might be universally present in these isolates, is often found on different linear plasmid compatibility types in different isolates. For example, the arp gene and the vls cassette region are present on plasmids of four and five different compatibility types, respectively, in different isolates. A majority of the plasmid types have more than one organizationally different subtype, and the number of such variants ranges from one to eight among the 18 linear plasmid types. In spite of this substantial organizational diversity, the plasmids are not so variable that every isolate has a novel version of every plasmid (i.e., there appears to be a limited number of extant plasmid subtypes). Although there have been many past recombination events, both homologous and nonhomologous, among the plasmids, particular organizational variants of these plasmids correlate with particular chromosomal genotypes, suggesting that there has not been rapid horizontal transfer of whole linear plasmids among B. burgdorferi lineages. We argue that plasmid rearrangements are essentially non-revertable and are present at a frequency of

  15. Conservation of Plasmid-Encoded Traits among Bean-Nodulating Rhizobium Species

    OpenAIRE

    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 organi...

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

    Directory of Open Access Journals (Sweden)

    Susu He

    2016-12-01

    Full Text Available 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.

  17. A simple method for construction of pir+ Enterobacterial hosts for maintenance of R6K replicon plasmids

    Directory of Open Access Journals (Sweden)

    Kvitko Brian H

    2012-03-01

    Full Text Available Abstract Background The R6K replicon is one of the best studied bacterial plasmid replicons. Replication of the R6K plasmid and derivatives harboring its γ origin of replication (oriR6Kγ is dependent on the pir gene-encoded π protein. Originally encoded by R6K, this protein is usually provided in trans in hosts engineered to support replication of plasmids harboring oriR6Kγ. In Escherichia coli this is commonly achieved by chromosomal integration of pir either via lysogenization with a λpir phage or homologous recombination at a pre-determined locus. Findings Current methods for construction of host strains for oriR6Kγ-containing plasmids involve procedures that do not allow selection for presence of the pir gene and require cumbersome and time-consuming screening steps. In this study, we established a mini-Tn7-based method for rapid and reliable construction of pir+ host strains. Using a curable mini-Tn7 delivery plasmid, pir expressing derivatives of several commonly used E. coli cloning and mobilizer strains were isolated using both the wild-type pir+ gene as well as the copy-up pir-116 allele. In addition, we isolated pir+ and pir-116 expressing derivatives of a clinical isolate of Salmonella enterica serovar Typhimurium. In both E. coli and S. enterica serovar Typhimurium, the presence of the pir+ wild-type or pir-116 alleles allowed the replication of oriR6Kγ-containing plasmids. Conclusions A mini-Tn7 system was employed for rapid and reliable engineering of E. coli and S. enterica serovar Typhimurium host strains for plasmids containing oriR6Kγ. Since mini-Tn7 elements transpose in most, if not all, Gram negative bacteria, we anticipate that with relatively minor modifications this newly established method will for the first time allow engineering of other bacterial species to enable replication of plasmids with oriR6Kγ.

  18. Plasmid P1 replication: negative control by repeated DNA sequences.

    OpenAIRE

    Chattoraj, D; Cordes, K.; Abeles, A

    1984-01-01

    The incompatibility locus, incA, of the unit-copy plasmid P1 is contained within a fragment that is essentially a set of nine 19-base-pair repeats. One or more copies of the fragment destabilizes the plasmid when present in trans. Here we show that extra copies of incA interfere with plasmid DNA replication and that a deletion of most of incA increases plasmid copy number. Thus, incA is not essential for replication but is required for its control. When cloned in a high-copy-number vector, pi...

  19. Linear plasmid in the genome of Clavibacter michiganensis subsp. sepedonicus.

    Science.gov (United States)

    Brown, Susan E; Knudson, Dennis L; Ishimaru, Carol A

    2002-05-01

    Contour-clamped homogeneous electric field gel analysis of genomic DNA of the plant pathogen Clavibacter michiganensis subsp. sepedonicus revealed the presence of a previously unreported extrachromosomal element. This new element was demonstrated to be a linear plasmid. Of 11 strains evaluated, all contained either a 90-kb (pCSL1) or a 140-kb (pCSL2) linear plasmid.

  20. Plasmid cloning vehicle for Haemophilus influenzae and Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.; Clayton, N.L.; Setlow, J.K.

    1982-09-01

    A new plasmid cloning vehicle (pDM2) was used to introduce a library of Haemophilus influenzae chromosomal fragments into H. influenzae. Transformants of the higly recombination-defective rec-1 mutant were more likely to contain exclusively recombinant plasmids after exposure to ligated DNA mixtures than was the wild type. pDM2 could replicate in Escherichia coli K-12.

  1. Functional analysis of three plasmids from Lactobacillus plantarum

    NARCIS (Netherlands)

    Kranenburg, R. van; Golic, N.; Bongers, R.; Leer, R.J.; Vos, W.M. de; Siezen, R.J.; Kleerebezem, M.

    2005-01-01

    Lactobacillus plantarum WCFS1 harbors three plasmids, pWCFS101, pWCFS102, and pWCFS103, with sizes of 1,917, 2,365, and 36,069 bp, respectively. The two smaller plasmids are of unknown function and contain replication genes that are likely to function via the rolling-circle replication mechanism. Th

  2. [Localization of denitrification genes in plasmid DNA of bacteria Azospirillum brasilense].

    Science.gov (United States)

    Petrova, L P; Varshalomidze, O É; Shelud'ko, A V; Katsy, E I

    2010-07-01

    In 85-Mda plasmid (p85) of plant-associated bacteria Azospirillum brasilense Sp245 model strain, the genes encoding copper-containing nitrite reductase (nirK); heterodimeric NO-reductase (norCB); NorQ and NorD proteins affecting synthesis and (or) activation of NirK and (or) NO-reductase (norQD); catalytic subunit I ofcytochrom c oxidase (CccoN); presumable NO sensor carrying two hemeerythrine domains (orf181); and an enzyme required for synthesis of presumable NO antagonist, homocystein (metC) were identified. In the same region of p85, orf293 encoding transcriptional regulator of LysR type, orf208 whose protein product carries a formylmethanofuran dehydrogenase subunit E domain, and an orf164-encoding conservative secretory protein with unknown function were also found. Localization of a set of denitrification genes in the plasmid DNA A. brasilense Sp245 adjacent to IS elements ISAzba1 and ISAzba2 indicates potential mobility of these genes and high probability of their horizontal transfer among populations of rhizospheric bacteria. A site homologous to p85 nirK-orf208-orf181 genes was detected in the 115 kb plasmid of A. brasilense Sp7 type strain.

  3. Homology and causes.

    Science.gov (United States)

    Van Valen, L M

    1982-09-01

    Homology is resemblance caused by a continuity of information. In biology it is a unified developmental phenomenon. Homologies among and within individuals intergrade in several ways, so historical homology cannot be separated sharply from repetitive homology. Nevertheless, the consequences of historical and repetitive homologies can be mutually contradictory. A detailed discussion of the rise and fall of the "premolar-analogy" theory of homologies of mammalian molar-tooth cusps exemplifies such a contradiction. All other hypotheses of historical homology which are based on repetitive homology, such as the foliar theory of the flower considered phyletically, are suspect.

  4. Construction of yellow fever virus subgenomic replicons by yeast-based homologous recombination cloning technique

    Directory of Open Access Journals (Sweden)

    Sabrina R.A. Queiroz

    2013-03-01

    Full Text Available RNA replicon derived from Flavivirus genome is a valuable tool for studying viral replication independent of virion assembly and maturation, besides being a great potencial for heterologous gene expression. In this study we described the construction of subgenomic replicons of yellow fever virus by yeast-based homologous recombination technique. The plasmid containing the yellow fever 17D strain replicon (pBSC-repYFV-17D, previously characterized, was handled to heterologous expression of the green fluorescent protein (repYFV-17D-GFP and firefly luciferase (repYFV-17D-Luc reporter genes. Both replicons were constructed by homologous recombination between the linearized vector pBSC-repYFV-17D and the PCR product containing homologous 25 nucleotides ends incorporated into PCR primers. The genomic organization of these constructs is similar to repYFV-17D, but with insertion of the reporter gene between the remaining 63 N-terminal nucleotides of the capsid protein and 72 C-terminal nucleotides of the E protein. The replicons repYFV-17D-GFP and repYFV-17D-Luc showed efficient replication and expression of the reporter genes. The yeast-based homologous recombination technique used in this study proved to be applicable for manipulation of the yellow fever virus genome in order to construct subgenomic replicons.

  5. Definition and identification of homology domains.

    Science.gov (United States)

    Lawrence, C B; Goldman, D A

    1988-03-01

    A method is described for identifying and evaluating regions of significant similarity between two sequences. The notion of a 'homology domain' is employed which defines the boundaries of a region of sequence homology containing no insertions or deletions. The relative significance of different potential homology domains is evaluated using a non-linear similarity score related to the probability of finding the observed level of similarity in the region by chance. The sensitivity of the method is demonstrated by simulating the evolution of homology domains and applying the method to their detection. Several examples of the use of homology domain identification are given.

  6. The fate of linear DNA in Saccharomyces cerevisiae and Candida glabrata: the role of homologous and non-homologous end joining.

    Directory of Open Access Journals (Sweden)

    Mary W Corrigan

    Full Text Available In vivo assembly of plasmids has become an increasingly used process, as high throughput studies in molecular biology seek to examine gene function. In this study, we investigated the plasmid construction technique called gap repair cloning (GRC in two closely related species of yeast - Saccharomyces cerevisiae and Candida glabrata. GRC utilizes homologous recombination (HR activity to join a linear vector and a linear piece of DNA that contains base pair homology. We demonstrate that a minimum of 20 bp of homology on each side of the linear DNA is required for GRC to occur with at least 10% efficiency. Between the two species, we determine that S. cerevisiae is slightly more efficient at performing GRC. GRC is less efficient in rad52 deletion mutants, which are defective in HR in both species. In dnl4 deletion mutants, which perform less non-homologous end joining (NHEJ, the frequency of GRC increases in C. glabrata, whereas GRC frequency only minimally increases in S. cerevisiae, suggesting that NHEJ is more prevalent in C. glabrata. Our studies allow for a model of the fate of linear DNA when transformed into yeast cells. This model is not the same for both species. Most significantly, during GRC, C. glabrata performs NHEJ activity at a detectable rate (>5%, while S. cerevisiae does not. Our model suggests that S. cerevisiae is more efficient at HR because NHEJ is less prevalent than in C. glabrata. This work demonstrates the determinants for GRC and that while C. glabrata has a lower efficiency of GRC, this species still provides a viable option for GRC.

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

    Science.gov (United States)

    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.

  8. Plasmid-to-plasmid recombination in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Balganesh, M.; Setlow, J.K.

    1986-01-01

    No recombination between plasmids was observed after conjugal transfer of a plasmid into a cell carrying another plasmid. Two types of such recombination took place after transformation, one type being Rec/sup +/ dependent and suggesting a preferred site of recombination. The other much rarer type was at least partially Rec/sup +/ independent.

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

    Science.gov (United States)

    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.

  10. Arabidopsis Indole Synthase,a Homolog of Tryptophan Synthase Alpha,is an Enzyme Involved in the Trp-independent Indole-containing Metabolite Biosynthesis

    Institute of Scientific and Technical Information of China (English)

    Rui Zhang; Bing Wang; Jian Ouyang; Jiayang Li; Yonghong Wang

    2008-01-01

    The plant tryptophan (Trp) biosynthetic pathway produces many secondary metabolites with diverse functions.Indole-3-acetic acid (IAA),proposed as a derivative from Trp or its precursors,plays an essential role in plant growth and development.Although the Trp-dependant and Trp-independent IAA biosynthetic pathways have been proposed,the enzymes,reactions and regulatory mechanisms are largely unknown.In Arabidopsis,indole-3-glycerol phosphate (IGP) is suggested to serve as a branchpoint component in the Trp-independent IAA biosynthesis.To address whether other enzymes in addition to Trp synthase α(TSA1) catalyze IGP cleavage,we identified and characterized an indole synthase (INS) gene,a homolog of TSA1 in Arabidopsis.INS exhibits different subcellular localization from TSA1 owing to the lack of chloroplast transit peptide (cTP).In silico data show that the expression levels of INS and TSA1 in all examined organs are quite different.Histochemical staining of INS promoter-GUS transgenic lines indicates that INS is expressed in vascular tissue of cotyledons,hypocotyls,roots and rosette leaves as well as in flowers and siliques.INS is capable of complementing the Trp auxotrophy of Escherichia coil △trpA strain,which is defective in Trp synthesis due to the deletion of TSA.This implies that INS catalyzes the conversion of IGP to indole and may be involved in the biosynthesis of Trp-independent IAA or other secondary metabolites in Arabidopsis.

  11. An Escherichia coli chromosomal ars operon homolog is functional in arsenic detoxification and is conserved in gram-negative bacteria.

    Science.gov (United States)

    Diorio, C; Cai, J; Marmor, J; Shinder, R; DuBow, M S

    1995-04-01

    Arsenic is a known toxic metalloid, whose trivalent and pentavalent ions can inhibit many biochemical processes. Operons which encode arsenic resistance have been found in multicopy plasmids from both gram-positive and gram-negative bacteria. The resistance mechanism is encoded from a single operon which typically consists of an arsenite ion-inducible repressor that regulates expression of an arsenate reductase and inner membrane-associated arsenite export system. Using a lacZ transcriptional gene fusion library, we have identified an Escherichia coli operon whose expression is induced by cellular exposure to sodium arsenite at concentrations as low as 5 micrograms/liter. This chromosomal operon was cloned, sequenced, and found to consist of three cistrons which we named arsR, arsB, and arsC because of their strong homology to plasmid-borne ars operons. Mutants in the chromosomal ars operon were found to be approximately 10- to 100-fold more sensitive to sodium arsenate and arsenite exposure than wild-type E. coli, while wild-type E. coli that contained the operon cloned on a ColE1-based plasmid was found to be at least 2- to 10-fold more resistant to sodium arsenate and arsenite. Moreover, Southern blotting and high-stringency hybridization of this operon with chromosomal DNAs from a number of bacterial species showed homologous sequences among members of the family Enterobacteriaceae, and hybridization was detectable even in Pseudomonas aeruginosa. These results suggest that the chromosomal ars operon may be the evolutionary precursor of the plasmid-borne operon, as a multicopy plasmid location would allow the operon to be amplified and its products to confer increased resistance to this toxic metalloid.

  12. Presence and analysis of plasmids in human and animal associated arcobacter species.

    Science.gov (United States)

    Douidah, Laid; De Zutter, Lieven; Van Nieuwerburgh, Filip; Deforce, Dieter; Ingmer, Hanne; Vandenberg, Olivier; Van den Abeele, Anne-Marie; Houf, Kurt

    2014-01-01

    In this study, we report the screening of four Arcobacter species for the presence of small and large plasmids. Plasmids were present in 9.9% of the 273 examined strains. One Arcobacter cryaerophilus and four Arcobacter butzleri plasmids were selected for further sequencing. The size of three small plasmids isolated from A. butzleri and the one from A. cryaerophilus strains ranged between 4.8 and 5.1 kb, and the size of the large plasmid, isolated from A. butzleri, was 27.4 kbp. The G+C content of all plasmids ranged between 25.4% and 26.2%. A total of 95% of the large plasmid sequence represents coding information, which contrasts to the 20 to 30% for the small plasmids. Some of the open reading frames showed a high homology to putative conserved domains found in other related organisms, such as replication, mobilization and genes involved in type IV secretion system. The large plasmid carried 35 coding sequences, including seven genes in a contiguous region of 11.6 kbp that encodes an orthologous type IV secretion system found in the Wolinella succinogenes genome, Helicobacter pylori and Campylobacter jejuni plasmids, which makes this plasmid interesting for further exploration.

  13. Presence and analysis of plasmids in human and animal associated arcobacter species.

    Directory of Open Access Journals (Sweden)

    Laid Douidah

    Full Text Available In this study, we report the screening of four Arcobacter species for the presence of small and large plasmids. Plasmids were present in 9.9% of the 273 examined strains. One Arcobacter cryaerophilus and four Arcobacter butzleri plasmids were selected for further sequencing. The size of three small plasmids isolated from A. butzleri and the one from A. cryaerophilus strains ranged between 4.8 and 5.1 kb, and the size of the large plasmid, isolated from A. butzleri, was 27.4 kbp. The G+C content of all plasmids ranged between 25.4% and 26.2%. A total of 95% of the large plasmid sequence represents coding information, which contrasts to the 20 to 30% for the small plasmids. Some of the open reading frames showed a high homology to putative conserved domains found in other related organisms, such as replication, mobilization and genes involved in type IV secretion system. The large plasmid carried 35 coding sequences, including seven genes in a contiguous region of 11.6 kbp that encodes an orthologous type IV secretion system found in the Wolinella succinogenes genome, Helicobacter pylori and Campylobacter jejuni plasmids, which makes this plasmid interesting for further exploration.

  14. 我国D2-43病毒株PrM-E基因的复制型载体质粒DNA的免疫原性%The Immunogenicity of Replicative Virus Plasmid DNA Containing PrM-E Gene of Chinese D2-43 Virus Strain

    Institute of Scientific and Technical Information of China (English)

    陈水平; 秦鄂德; 于曼; 胡志君; 赵卫; 范宝昌; 王鹏程; 杨佩英

    2002-01-01

    观察含我国登革2型病毒株(D2-43)的PrM-E基因的复制型SFV(semliki forest virus)重组质粒DNA的免疫原性,为登革新型疫苗的研制提供依据.将PrM-E基因自T载体上切下,插入复制型SFV病毒载体质粒DNA中.将此重组质粒DNA以电穿孔法导入BHK21细胞,用间接免疫荧光法在感染细胞内可检测到登革2型病毒特异蛋白的表达.采用去除内毒素的质粒提取试剂盒制备重组质粒DNA,然后以不同剂量通过肌肉多点注射途径免疫Balb/c鼠,获得的鼠血清可与登革D2-43感染的C6/36抗原片起特异的抗原抗体反应.结果表明,含登革2型病毒PrM-E基因的复制型SFV病毒载体质粒DNA在Balb/c鼠中可诱导登革2型病毒特异抗体的产生,但抗体水平较低.%To study the immunogenicity of replicative plasmid DNA containing PrM-E gene of Chinese D2-43virus strain and lay a foundation for new vaccine of dengue, the PrM-E gene was cut from pGEM-T-ME with Apa I and Cla I and inserted into replicative semliki forest virus (SFV) vector. The recombinant plasmid DNA was electroporated into BHK21 cells, then expression products specific to dengue virus type 2 could be detected by IFA (indirect immunofluorescence assay) in transfected cells. Endotoxin-free plasmid DNA was prepared with EndoFree plasmid Maxi kit. Mice were immunized with different doses of the recombinant SFV plasmid DNA intramuscularly at multiple sites. The titres of antibody to dengue virus type 2 was measured by IFA. And sera with recombinant plasmid, could react with antigen of dengue 2 virus on microscope slides. The results showed that recombinant SFV plasmid DNA containing the PrM-E gene of D2-43 virus strain, could produce specific antibody to dengue 2 virus in mice, but the titre was low.

  15. BMP2和TGFβ3双基因真核表达载体的构建%Construction of bicistronic eukaryotic expression plasmid containing BMP2 and TGFβ3 genes

    Institute of Scientific and Technical Information of China (English)

    马小松; 刘金钊; 王昌耀; 王英振; 刘世海; 刘相萍

    2011-01-01

    [目的]构建骨形态发生蛋白BMP2和转化生长因子TGFβ3双基因真核表达载体.[方法]从人胚胎组织提取总RNA,反转录成cDNA,以pGEMT/BMP2和反转录的cDNA为模板,PCR扩增出BMP2和TGFβ3两个基因全长,将两个基因片段分别定向连入双基因真核表达载体pIRES;用酶切的方法筛选出阳性重组质粒,并进行测序鉴定.[结果]酶切鉴定证明已将BMP2和TGFβ3两个基因连入载体中,测序结果完全正确.[结论]成功构建pIRESBMP2/TGFβ3双基因真核表达载体.%[ Objective]To construct a bicistronic eukayotic expression plasmid consisting of BMP2 and TGFβ3 target genes.[ Method ] The DNA fragment of BMP2 and TGFβ3 genes were obtained from pGEMT/BMP2 plasmid and human embryonal tissue by PCR, respectively. They were inserted into bicistronic eukaryotic expression plasmid vector plRES. The inserted target genes in the plasmid were verified by restriction enzyme digestion and nucleotide sequencing. [ Result ] The direction and sequences of the new bicistronic eukaryotic expression plasmid pIRES - BMP2 -TGFβ3 were correct. [ Conclusion] The bicistronic eukaryotic expression plasmid has been constructed successfully.

  16. Bi(3n+1)Ti7Fe(3n-3)O(9n+11) Homologous Series: Slicing Perovskite Structure with Planar Interfaces Containing Anatase-like Chains.

    Science.gov (United States)

    Batuk, Dmitry; Tsirlin, Alexander A; Filimonov, Dmitry S; Zakharov, Konstantin V; Volkova, Olga S; Vasiliev, Alexander; Hadermann, Joke; Abakumov, Artem M

    2016-02-01

    The n = 3-6 members of a new perovskite-based homologous series Bi(3n+1)Ti7Fe(3n-3)O(9n+11) are reported. The crystal structure of the n = 3 Bi10Ti7Fe6O38 member is refined using a combination of X-ray and neutron powder diffraction data (a = 11.8511(2) Å, b = 3.85076(4) Å, c = 33.0722(6) Å, S.G. Immm), unveiling the partially ordered distribution of Ti(4+) and Fe(3+) cations and indicating the presence of static random displacements of the Bi and O atoms. All Bi(3n+1)Ti7Fe(3n-3)O(9n+11) structures are composed of perovskite blocks separated by translational interfaces parallel to the (001)p perovskite planes. The thickness of the perovskite blocks increases with n, while the atomic arrangement at the interfaces remains the same. The interfaces comprise chains of double edge-sharing (Fe,Ti)O6 octahedra connected to the octahedra of the perovskite blocks by sharing edges and corners. This configuration shifts the adjacent perovskite blocks relative to each other over a vector ½[110]p and creates S-shaped tunnels along the [010] direction. The tunnels accommodate double columns of the Bi(3+) cations, which stabilize the interfaces owing to the stereochemical activity of their lone electron pairs. The Bi(3n+1)Ti7Fe(3n-3)O(9n+11) structures can be formally considered either as intergrowths of perovskite modules and polysynthetically twinned modules of the Bi2Ti4O11 structure or as intergrowths of the 2D perovskite and 1D anatase fragments. Transmission electron microscopy (TEM) on Bi10Ti7Fe6O38 reveals that static atomic displacements of Bi and O inside the perovskite blocks are not completely random; they are cooperative, yet only short-range ordered. According to TEM, the interfaces can be laterally shifted with respect to each other over ±1/3a, introducing an additional degree of disorder. Bi10Ti7Fe6O38 is paramagnetic in the 1.5-1000 K temperature range due to dilution of the magnetic Fe(3+) cations with nonmagnetic Ti(4+). The n = 3, 4 compounds demonstrate a

  17. Plasmid interference for curing antibiotic resistance plasmids in vivo

    Science.gov (United States)

    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

  18. Homology, Analogy, and Ethology.

    Science.gov (United States)

    Beer, Colin G.

    1984-01-01

    Because the main criterion of structural homology (the principle of connections) does not exist for behavioral homology, the utility of the ethological concept of homology has been questioned. The confidence with which behavioral homologies can be claimed varies inversely with taxonomic distance. Thus, conjectures about long-range phylogenetic…

  19. Presence of Glycopeptide-Encoding Plasmids in Enterococcal Isolates from Food and Humans in Denmark

    DEFF Research Database (Denmark)

    Migura, Lourdes Garcia; Valenzuela, Antonio Jesus Sanchez; Jensen, Lars Bogø

    2011-01-01

    developed techniques for classification of plasmids. Replicons associated with sex pheromone-inducible plasmids were detected in all GR E. faecalis, whereas GR Enterococcus faecium contained plasmids known to be widely distributed among enterococci. vanA resistance is common in E. faecium isolates from meat...... and animals in Europe and is rarely found in E. faecalis. This article describes the first characterization of MGE from vanA mediated E. faecalis, thus linking this resistance genotype to pheromone responding plasmids....

  20. Human DNA contains sequences homologous to the 5'-non-coding region of hepatitis C virus: characterization with restriction endonucleases reveals individual varieties

    Institute of Scientific and Technical Information of China (English)

    Reinhard H Dennin; Jianer Wo

    2003-01-01

    Objective To investigate a 272 base pair section of the 5'-non-coding region of genomic DNA from the peripheral blood monounuclear cells of healthy hepatitis virus C (HCV)-negative human subjects (not patients). Results The suspected HCV-specific sequence was found in the DNA of each subject tested. The pre-PCR digestion assay reveals individual differences in their pattern of methylation, which may be due to possible epigenetic phenomena.Conclusions The results provide formal proof that these HCV-specific sequences are contained in the genomic or extra chromosomal target DNA, and probably belong to a new class of endogenous sequences.

  1. DNA sequence analysis of plasmids from multidrug resistant Salmonella enterica serotype Heidelberg isolates.

    Directory of Open Access Journals (Sweden)

    Jing Han

    Full Text Available Salmonella enterica serovar Heidelberg is among the most detected serovars in swine and poultry, ranks among the top five serotypes associated with human salmonellosis and is disproportionately associated with invasive infections and mortality in humans. Salmonella are known to carry plasmids associated with antimicrobial resistance and virulence. To identify plasmid-associated genes in multidrug resistant S. enterica serovar Heidelberg, antimicrobial resistance plasmids from five isolates were sequenced using the 454 LifeSciences pyrosequencing technology. Four of the isolates contained incompatibility group (Inc A/C multidrug resistance plasmids harboring at least eight antimicrobial resistance genes. Each of these strains also carried a second resistance plasmid including two IncFIB, an IncHI2 and a plasmid lacking an identified Inc group. The fifth isolate contained an IncI1 plasmid, encoding resistance to gentamicin, streptomycin and sulfonamides. Some of the IncA/C plasmids lacked the full concert of transfer genes and yet were able to be conjugally transferred, likely due to the transfer genes carried on the companion plasmids in the strains. Several non-IncA/C resistance plasmids also carried putative virulence genes. When the sequences were compared to previously sequenced plasmids, it was found that while all plasmids demonstrated some similarity to other plasmids, they were unique, often due to differences in mobile genetic elements in the plasmids. Our study suggests that Salmonella Heidelberg isolates harbor plasmids that co-select for antimicrobial resistance and virulence, along with genes that can mediate the transfer of plasmids within and among other bacterial isolates. Prevalence of such plasmids can complicate efforts to control the spread of S. enterica serovar Heidelberg in food animal and human populations.

  2. Effect of NADPH oxidase inhibitor-apocynin on the expression of Src homology-2 domain-containing phosphatase-1 (SHP-1 exposed renal ischemia/reperfusion injury in rats

    Directory of Open Access Journals (Sweden)

    Zhiming Li

    2015-01-01

    Full Text Available This study was designed to evaluate whether NADPH oxidase inhibitor (apocynin preconditioning induces expression of Src homology-2 domain-containing phosphatase-1 (SHP-1 to protect against renal ischemia/reperfusion (I/R injury (RI/RI in rats. Rats were pretreated with 50 mg/kg apocynin, then subjected to 45 min ischemia and 24 h reperfusion. The results indicated that apocynin preconditioning improved the recovery of renal function and nitroso-redox balance, reduced oxidative stress injury and inflammation damage, and upregulated expression of SHP-1 as compared to RI/RI group. Therefore our study demonstrated that apocynin preconditioning provided a protection to the kidney against I/R injury in rats partially through inducing expression of SHP-1.

  3. Src homology 2 domain-containing inositol-5-phosphatase and CCAAT enhancer-binding protein beta are targeted by miR-155 in B cells of Emicro-MiR-155 transgenic mice

    DEFF Research Database (Denmark)

    Costinean, Stefan; Sandhu, Sukhinder K; Pedersen, Irene M

    2009-01-01

    We showed that Emicro-MiR-155 transgenic mice develop acute lymphoblastic leukemia/high-grade lymphoma. Most of these leukemias start at approximately 9 months irrespective of the mouse strain. They are preceded by a polyclonal pre-B-cell proliferation, have variable clinical presentation......, are transplantable, and develop oligo/monoclonal expansion. In this study, we show that in these transgenic mice the B-cell precursors have the highest MiR-155 transgene expression and are at the origin of the leukemias. We determine that Src homology 2 domain-containing inositol-5-phosphatase (SHIP) and CCAAT...... a chain of events that leads to the accumulation of large pre-B cells and acute lymphoblastic leukemia/high-grade lymphoma....

  4. Association of Piebaldism, multiple café-au-lait macules, and intertriginous freckling: clinical evidence of a common pathway between KIT and sprouty-related, ena/vasodilator-stimulated phosphoprotein homology-1 domain containing protein 1 (SPRED1).

    Science.gov (United States)

    Chiu, Yvonne E; Dugan, Stefanie; Basel, Donald; Siegel, Dawn H

    2013-01-01

    Piebaldism is a rare genodermatosis caused by KIT mutations. We report the case of a 5-year-old boy who had the white forelock and leukoderma of piebaldism, but the presence of many café-au-lait macules and axillary and inguinal freckling complicated the diagnosis. Patients with similar cutaneous findings have been previously reported, and their disorder has been attributed to an overlap of piebaldism and neurofibromatosis type 1. Legius syndrome is a recently described syndrome caused by Sprouty-related, Ena/vasodilator-stimulated phosphoprotein homology-1 domain containing protein 1 (SPRED1) mutations that also has multiple café-au-lait macules and intertriginous freckling. Based on our current understanding of KIT and SPRED1 protein interactions, we propose that café-au-lait macules and freckling may be seen in some patients with piebaldism and does not necessarily represent coexistence of neurofibromatosis type 1.

  5. Construction of plasmid containing IVS-2-654 (C>T) single mutation of β-thalassemia gene by site-directed mutagenesis%定点诱变法构建β地中海贫血IVS-2-654(C>T)突变基因质粒

    Institute of Scientific and Technical Information of China (English)

    余玲玲; 田可港; 冯晶晶; 王慧燕; 郑晓群

    2012-01-01

    Objective To construct a plasmid containing IVS-2-654 (OT) single site mutation of p-thalassemia gene. Methods The plasmid DNA containing wild-type β-globin gene was used as PCR template. Plasmid containing IVS-2-654 (OT) mutation gene of β-globin was constructed by TA clone technology after site-directed mutagenesis of overlap extension PCR (OE-PCR). Results Direct DNA sequencing showed that the recombi-nant plasmid contained IVS-2-654 (OT) mutation gene of β-globin, which mutated from C to T at IVS-2-654 bp and the rest was completely identical with wide type. Conclusions The plasmid containing IVS-2-654 (C> T) mutation gene was successfully constructed, which laied the foundation for further screening studies to detect gene mutation of (Hhalassemia and other genetic diagnosis for this disease. Site-directed mutagenesisis of OE-PCR technique is simple and economic and worth of being promoted widely.%目的 构建含β地中海贫血(简称β地贫)IVS-2-654 (C>T)突变基因的质粒.方法 以含β珠蛋白野生型基因的质粒DNA为模板,采用重叠延伸PCR (OE-PCR)定点诱变后行TA克隆的方法构建含IVS-2-654 (C>T)突变基因的质粒.结果 双向测序结果表明:重组质粒的确含β地贫IVS-2-654 (C>T)突变基因,β珠蛋白IVS-2-654处的碱基已由C突变成T,其余序列与野生型完全相同,成功实现了定点诱变.结论 成功地构建了含β地贫IVS-2-654 (C>T)突变基因的质粒,进一步为该病基因诊断与筛查技术的研究奠定了实验基础;OE-PCR定点诱变法简便、经济,值得推广应用.

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

    Science.gov (United States)

    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.

  7. Novel plasmids and resistance phenotypes in Yersinia pestis: unique plasmid inventory of strain Java 9 mediates high levels of arsenic resistance.

    Directory of Open Access Journals (Sweden)

    Mark Eppinger

    Full Text Available 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.

  8. An RNA-binding complex involved in ribosome biogenesis contains a protein with homology to tRNA CCA-adding enzyme.

    Directory of Open Access Journals (Sweden)

    Jinzhong Lin

    2013-10-01

    Full Text Available A multitude of proteins and small nucleolar RNAs transiently associate with eukaryotic ribosomal RNAs to direct their modification and processing and the assembly of ribosomal proteins. Utp22 and Rrp7, two interacting proteins with no recognizable domain, are components of the 90S preribosome or the small subunit processome that conducts early processing of 18S rRNA. Here, we determine the cocrystal structure of Utp22 and Rrp7 complex at 1.97 Å resolution and the NMR structure of a C-terminal fragment of Rrp7, which is not visible in the crystal structure. The structure reveals that Utp22 surprisingly resembles a dimeric class I tRNA CCA-adding enzyme yet with degenerate active sites, raising an interesting evolutionary connection between tRNA and rRNA processing machineries. Rrp7 binds extensively to Utp22 using a deviant RNA recognition motif and an extended linker. Functional sites on the two proteins were identified by structure-based mutagenesis in yeast. We show that Rrp7 contains a flexible RNA-binding C-terminal tail that is essential for association with preribosomes. RNA-protein crosslinking shows that Rrp7 binds at the central domain of 18S rRNA and shares a neighborhood with two processing H/ACA snoRNAs snR30 and snR10. Depletion of snR30 prevents the stable assembly of Rrp7 into preribosomes. Our results provide insight into the evolutionary origin and functional context of Utp22 and Rrp7.

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

    Science.gov (United States)

    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. Copyright © 2017 American Society for Microbiology.

  10. An easy and versatile 2-step protocol for targeted modification and subcloning of DNA from bacterial artificial chromosomes using non-commercial plasmids

    Directory of Open Access Journals (Sweden)

    Hartwich Heiner

    2012-03-01

    Full Text Available Abstract Background Promoter-specific expression of foreign DNA in transgenic organisms often relies on bacterial artificial chromosomes (BACs. This approach requires modification and subcloning of BAC-DNA by recombineering technologies in Escherichia coli. Most current protocols rely on commercial kits or isolation of BACs, their transfer between different host strains, and their restriction. Findings In this report we present a 2-step protocol for efficient modification and subcloning of DNA from bacterial artificial chromosomes using the non-commercial plasmids pKM208 and pTP223, distributed from addgene.com. A targeting cassette was successfully integrated into a BAC and 42 kb of this construct were subcloned. Both a plasmid-derived substrate with longer homology arms and a PCR-generated substrate with short homology arms (50 bp were used for recombination. pKM208 and pTP223 contain all required genes for recombineering, but differ in their antibiotic resistance genes. This makes the system independent of the selection markers on the DNA molecules targeted for recombination. Conclusions The time and cost saving protocol presented here compares favorably to currently used systems. Using non-commercial plasmids, it allows targeted modification and cloning of large DNA (> 40 kb fragments in vivo without restriction and ligation. Furthermore, both steps are performed in the same host eliminating the need to isolate BAC DNA and to use different bacterial strains.

  11. Conjugal transfer of a virulence plasmid in the opportunistic intracellular actinomycete Rhodococcus equi.

    Science.gov (United States)

    Tripathi, V N; Harding, W C; Willingham-Lane, J M; Hondalus, M K

    2012-12-01

    Rhodococcus equi is a facultative intracellular, Gram-positive, soilborne actinomycete which can cause severe pyogranulomatous pneumonia with abscessation in young horses (foals) and in immunocompromised people, such as persons with AIDS. All strains of R. equi isolated from foals and approximately a third isolated from humans contain a large, ~81-kb plasmid which is essential for the intramacrophage growth of the organism and for virulence in foals and murine in vivo model systems. We found that the entire virulence plasmid could be transferred from plasmid-containing strains of R. equi (donor) to plasmid-free R. equi strains (recipient) at a high frequency and that plasmid transmission reestablished the capacity for intracellular growth in macrophages. Plasmid transfer required living cells and cell-to-cell contact and was unaffected by the presence of DNase, factors pointing to conjugation as the major means of genetic transfer. Deletion of a putative relaxase-encoding gene, traA, located in the proposed conjugative region of the plasmid, abolished plasmid transfer. Reversion of the traA mutation restored plasmid transmissibility. Finally, plasmid transmission to other Rhodococcus species and some additional related organisms was demonstrated. This is the first study showing a virulence plasmid transfer in R. equi, and it establishes a mechanism by which the virulence plasmid can move among bacteria in the soil.

  12. Combinatorial Floer Homology

    CERN Document Server

    de Silva, Vin; Salamon, Dietmar

    2012-01-01

    We define combinatorial Floer homology of a transverse pair of noncontractibe nonisotopic embedded loops in an oriented 2-manifold without boundary, prove that it is invariant under isotopy, and prove that it is isomorphic to the original Lagrangian Floer homology.

  13. Persistence Mechanisms of Conjugative Plasmids

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Hansen, Lars H.; Sørensen, Søren Johannes

    2009-01-01

    Are plasmids selfish parasitic DNA molecules or an integrated part of the bacterial genome? This chapter reviews the current understanding of the persistence mechanisms of conjugative plasmids harbored by bacterial cells and populations. The diversity and intricacy of mechanisms affecting...... the successful propagation and long-term continued existence of these extra-chromosomal elements is extensive. Apart from the accessory genetic elements that may provide plasmid-harboring cells a selective advantage, special focus is placed on the mechanisms conjugative plasmids employ to ensure their stable...... maintenance in the host cell. These importantly include the ability to self-mobilize in a process termed conjugative transfer, which may occur across species barriers. Other plasmid stabilizing mechanisms include the multimer resolution system, active partitioning, and post-segregational-killing of plasmid...

  14. The conjugative plasmid of a bean-nodulating Sinorhizobium fredii strain is assembled from sequences of two Rhizobium plasmids and the chromosome of a Sinorhizobium strain

    Directory of Open Access Journals (Sweden)

    Brom Susana

    2011-06-01

    Full Text Available Abstract Background Bean-nodulating Rhizobium etli originated in Mesoamerica, while soybean-nodulating Sinorhizobium fredii evolved in East Asia. S. fredii strains, such as GR64, have been isolated from bean nodules in Spain, suggesting the occurrence of conjugative transfer events between introduced and native strains. In R. etli CFN42, transfer of the symbiotic plasmid (pRet42d requires cointegration with the endogenous self-transmissible plasmid pRet42a. Aiming at further understanding the generation of diversity among bean nodulating strains, we analyzed the plasmids of S. fredii GR64: pSfr64a and pSfr64b (symbiotic plasmid. Results The conjugative transfer of the plasmids of strain GR64 was analyzed. Plasmid pSfr64a was self-transmissible, and required for transfer of the symbiotic plasmid. We sequenced pSfr64a, finding 166 ORFs. pSfr64a showed three large segments of different evolutionary origins; the first one presented 38 ORFs that were highly similar to genes located on the chromosome of Sinorhizobium strain NGR234; the second one harbored 51 ORFs with highest similarity to genes from pRet42d, including the replication, but not the symbiosis genes. Accordingly, pSfr64a was incompatible with the R. etli CFN42 symbiotic plasmid, but did not contribute to symbiosis. The third segment contained 36 ORFs with highest similarity to genes localized on pRet42a, 20 of them involved in conjugative transfer. Plasmid pRet42a was unable to substitute pSfr64a for induction of pSym transfer, and its own transfer was significantly diminished in GR64 background. The symbiotic plasmid pSfr64b was found to differ from typical R. etli symbiotic plasmids. Conclusions S. fredii GR64 contains a chimeric transmissible plasmid, with segments from two R. etli plasmids and a S. fredii chromosome, and a symbiotic plasmid different from the one usually found in R. etli bv phaseoli. We infer that these plasmids originated through the transfer of a symbiotic-conjugative-plasmid

  15. Plasmid DNA Manufacturing for Indirect and Direct Clinical Applications.

    Science.gov (United States)

    Schmeer, Marco; Buchholz, Tatjana; Schleef, Martin

    2017-10-01

    Plasmid DNA is currently gaining increasing importance for clinical research applications in gene therapy and genetic vaccination. For direct gene transfer into humans, good manufacturing practice (GMP)-grade plasmid DNA is mandatory. The same holds true if the drug substance contains a genetically modified cell, for example chimeric antigen receptor (CAR) T cells, where these cells as well as the contained plasmids are used. According to the responsible regulatory agencies, they have to be produced under full GMP. On the other hand, for GMP production of, for example, mRNA or viral vectors (lentiviral vectors, adeno-associated virus vectors, etc.), in many cases, High Quality Grade plasmid DNA is accepted as a starting material. The manufacturing process passes through different production steps. To ensure the right conditions are used for the plasmid, a pilot run must be conducted at the beginning. In this step, a followed upscaling with respect to reproducibility and influences on product quality is performed. Subsequently, a cell bank of the transformed productions strain is established and characterized. This cell bank is used for the cultivation process. After cell harvesting and lysis, several chromatography steps are conducted to receive a pure plasmid product. Depending on the respective required quality grade, the plasmid product is subject to several quality controls. The last step consists of formulation and filling of the product.

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

    Energy Technology Data Exchange (ETDEWEB)

    Benoit, T.G.; Wilson, G.R.; Bull, D.L.; Aronson, A.I. (Department of Agriculture, College Station, TX (USA))

    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. Remarkable stability of an instability-prone lentiviral vector plasmid in Escherichia coli Stbl3

    OpenAIRE

    Al-Allaf, Faisal A.; Tolmachov, Oleg E.; Zambetti, Lia Paola; Tchetchelnitski, Viktoria; Mehmet, Huseyin

    2012-01-01

    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 ...

  18. Effects of serum containing natural cerebrolysin on glucose-regulated protein 78 and CCAAT enhancer-binding protein homologous protein expression in neuronal PC12 cells following tunicamycin-induced endoplasmic reticulum stress

    Institute of Scientific and Technical Information of China (English)

    Zhengzhi Wu; Ming Li; Andrew C.J. HuangO; Xiuqing Jia; Yinghong Li; Manyin Chen

    2009-01-01

    BACKGROUND: Glucose-regulated protein 78 (GRP78), a marker of endoplasmic reticulum stress, can prolong cell survival. Alternatively, CCAAT enhancer-binding protein homologous protein (CHOP), a transcription factor specific for endopiasmic reticulum stress, can cause cell cycle arrest and cell apoptosis.OBJECTIVE: To study the protective effects of serum containing natural cerebrolysin on endoplasmic reticulum stress in tunicamycin-induced neuronal PC12 cells, and analyze the influence on GRP78 and CHOP expressions.DESIGN, TIME AND SETTING: A parallel controlled study was performed at the Institute of Integrated Western and Traditional Chinese Medicine, Shenzhen Hospital, Southern Medical University, between March 2006 and August 2008.MATERIALS: Adult Sprague-Dawley rats were perfused with natural Cerebrelysin aqueous extract (0.185 g/kg/d) to produce serum containing natural Cerebrolysin. Physiological saline was used to produce blank serum. PC12 cell line was provided by Shanghai Institute of Cell Biology, Chinese Academy of Science. Tunicamycin was provided by Sigma (St. Louis, USA), and natural Cerebrolysin, containing ginseng, rhizoma gastrodiae, and gingko leaf (1:2:2), by Shengzhen Institute of Integrated Western and Traditional Chinese Medicine.METHODS: PC12 cells were treated with DMEM culture media containing 10% blank serum (normal control group), tunicamycin (1μg/mL; model group), and 5%, 10%, and 15% serum containing natural cerebrolysin and tunicamycin (1μg/mL; low-, moderate-, and high-dose serum containing natural cerebrelysin groups), for 2 hours.MAIN OUTCOME MEASURES: PC12 cells were treated with tunicamycin for 48 hours after which apoptosis was measured using the TUNEL method to calculate apoptotic index. GRP78 expression was detected using immunocytochemistry. After 24 hours of treatment with tunicamycin, GRP78 and CHOP mRNA expressions were measured using RT-PCR.RESULTS: The apoptotic index and CHOP mRNA expression were in the model group

  19. Real Topological Cyclic Homology

    DEFF Research Database (Denmark)

    Høgenhaven, Amalie

    The main topics of this thesis are real topological Hochschild homology and real topological cyclic homology. If a ring or a ring spectrum is equipped with an anti-involution, then it induces additional structure on the topological Hochschild homology spectrum. The group O(2) acts on the spectrum......, where O(2) is the semi-direct product of T, the multiplicative group of complex number of modulus 1, by the group G=Gal(C/R). We refer to this O(2)-spectrum as the real topological Hochschild homology. This generalization leads to a G-equivariant version of topological cyclic homology, which we call...... real topological cyclic homology. The first part of the thesis computes the G-equivariant homotopy type of the real topological cyclic homology of spherical group rings at a prime p with anti-involution induced by taking inverses in the group. The second part of the thesis investigates the derived G...

  20. Phenotypic plasticity in bacterial plasmids.

    Science.gov (United States)

    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

  1. Plasmid Rolling-Circle Replication.

    Science.gov (United States)

    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.

  2. Isolation of T—DNA flanking plant DNA from T—DNA insertional embryo—lethal mutants of Arabidopsis thaliana by plasmid rescue technique

    Institute of Scientific and Technical Information of China (English)

    YAOXIAOLI; JIANGESUN; 等

    1996-01-01

    Three T-DNA insertional embryonic lethal mutants from NASC(The Nottingham Arabidopsis Stock Center) were first checked with their segregation ratio of abortive and normal seeds and the copy number of T-DNA insertion.The N4081 mutant has a segregation ratio of 1:3.04 in average and one T-DNA insertion site according to our assay.It was therefore chosen for further analysis.To isolate the joint fragment of T-DNA and plant DNA,the plasmid rescue technique was used.pEL-7,one of plasmids from left border of T-DNA,which contained pBR322 was selected from ampicillin plate.The T-DNA fragment of pEL-7 was checked by restriction enzyme analysis and Southern Blot.Restriction analysis confirmed the presence of known sites of EcoRI,PstI and PvuII on it.For confirming the presence of flanking plant DNA in this plasmid,pEL-7 DNA was labeled and hybridized with wild type and mutant plant DNA.The Southern Blot indicated the hybridization band in both of them.Furthermore,the junction of T-DNA/plant DNA was subcloned into bluescript SK+ and sequenced by Applied Biosystem 373A sequencer.The results showed the 822 bp fragment contained a 274 bp sequence,which is 99.6%homolog(273bp/274bp) to Ti plasmid pTi 15955,DNA.The bp of left 25 bp border repeat were also found in the juction of T-DNA and Plant DNA. Taken together,pEL-7 should coutain a joint fragment of T-DNA and flanking plant DNA.This plasmid DNA could be used for the isolation of plant gene,which will be helpful to elucidate the relationship between gene function and plant embryo development.

  3. Genomic and functional characterization of the modular broad-host-range RA3 plasmid, the archetype of the IncU group.

    Science.gov (United States)

    Kulinska, Anna; Czeredys, Magdalena; Hayes, Finbarr; Jagura-Burdzy, Grazyna

    2008-07-01

    IncU plasmids are a distinctive group of mobile elements with highly conserved backbone functions and variable antibiotic resistance gene cassettes. The IncU archetype is conjugative plasmid RA3, whose sequence (45,909 bp) shows it to be a mosaic, modular replicon with a class I integron different from that of other IncU replicons. Functional analysis demonstrated that RA3 possesses a broad host range and can efficiently self-transfer, replicate, and be maintained stably in alpha-, beta-, and gammaproteobacteria. RA3 contains 50 open reading frames clustered in distinct functional modules. The replication module encompasses the repA and repB genes embedded in long repetitive sequences. RepA, which is homologous to antitoxin proteins from alpha- and gammaproteobacteria, contains a Cro/cI-type DNA-binding domain present in the XRE family of transcriptional regulators. The repA promoter is repressed by RepA and RepB. The minireplicon encompasses repB and the downstream repetitive sequence r1/r2. RepB shows up to 80% similarity to putative replication initiation proteins from environmental plasmids of beta- and gammaproteobacteria, as well as similarity to replication proteins from alphaproteobacteria and Firmicutes. Stable maintenance functions of RA3 are most like those of IncP-1 broad-host-range plasmids and comprise the active partitioning apparatus formed by IncC (ParA) and KorB (ParB), the antirestriction protein KlcA, and accessory stability components KfrA and KfrC. The RA3 origin of transfer was localized experimentally between the maintenance and conjugative-transfer operons. The putative conjugative-transfer module is highly similar in organization and in its products to transfer regions of certain broad-host-range environmental plasmids.

  4. Lectures on functor homology

    CERN Document Server

    Touzé, Antoine

    2015-01-01

    This book features a series of lectures that explores three different fields in which functor homology (short for homological algebra in functor categories) has recently played a significant role. For each of these applications, the functor viewpoint provides both essential insights and new methods for tackling difficult mathematical problems. In the lectures by Aurélien Djament, polynomial functors appear as coefficients in the homology of infinite families of classical groups, e.g. general linear groups or symplectic groups, and their stabilization. Djament’s theorem states that this stable homology can be computed using only the homology with trivial coefficients and the manageable functor homology. The series includes an intriguing development of Scorichenko’s unpublished results. The lectures by Wilberd van der Kallen lead to the solution of the general cohomological finite generation problem, extending Hilbert’s fourteenth problem and its solution to the context of cohomology. The focus here is o...

  5. In Silico Detection and Typing of Plasmids using PlasmidFinder and Plasmid Multilocus Sequence Typing

    DEFF Research Database (Denmark)

    Carattoli, Alessandra; Zankari, Ea; García-Fernández, Aurora

    2014-01-01

    In the work presented here, we designed and developed two easy-to-use Web tools for in silico detection and characterization of whole-genome sequence (WGS) and whole-plasmid sequence data from members of the family Enterobacteriaceae. These tools will facilitate bacterial typing based on draft...... genomes of multidrug-resistant Enterobacteriaceae species by the rapid detection of known plasmid types. Replicon sequences from 559 fully sequenced plasmids associated with the family Enterobacteriaceae in the NCBI nucleotide database were collected to build a consensus database for integration...... sequences identified in the 559 fully sequenced plasmids. For plasmid multilocus sequence typing (pMLST) analysis, a database that is updated weekly was generated from www.pubmlst.org and integrated into a Web tool called pMLST. Both databases were evaluated using draft genomes from a collection...

  6. Lectures on knot homology

    CERN Document Server

    Nawata, Satoshi

    2015-01-01

    We provide various formulations of knot homology that are predicted by string dualities. In addition, we also explain the rich algebraic structure of knot homology which can be understood in terms of geometric representation theory in these formulations. These notes are based on lectures in the workshop "Physics and Mathematics of Link Homology" at Centre de Recherches Math\\'ematiques, Universit\\'e de Montr\\'eal.

  7. Nucleotide sequence analysis of the lactococcal EPS plasmid pNZ4000

    NARCIS (Netherlands)

    Kranenburg, van R.; Kleerebezem, M.; Vos, de W.M.

    2000-01-01

    The complete 42180-bp nucleotide sequence of the mobilization plasmid pNZ4000, coding for exopolysaccharide (EPS) production in Lactococcus lactis, was determined. This plasmid contains a region involved in EPS biosynthesis, four functional replicons, a region containing mobilization genes, and thre

  8. Construction and Identification of Plasmid pTA-TUB2

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    An about 1.40 Kb target gene fragment was yielded by PCR amplification with the plasmid pRB 129,which was identified by restriction enzyme digestion that the PCR product was TU B2 gene.The gene was digested by the restriction enzyme and was linked with pTA plasmid to construct pTA-TU B2 plasmid.The plasmid was transformed into Chaetomium spp.by PEG method and the transformation rate was 27/(2×105) and it is nine times higher than that of pRB 129.The transformants can grow on the PDA containing 1 000 μg*mL-1 carbendazim,which is 1 000 times higher than the original Chaetomium spp.The resistance was stable after 10 times transfer on non-selective medium.

  9. Two RNAs or DNAs May Artificially Fuse Together at a Short Homologous Sequence (SHS during Reverse Transcription or Polymerase Chain Reactions, and Thus Reporting an SHS-Containing Chimeric RNA Requires Extra Caution.

    Directory of Open Access Journals (Sweden)

    Bingkun Xie

    Full Text Available Tens of thousands of chimeric RNAs have been reported. Most of them contain a short homologous sequence (SHS at the joining site of the two partner genes but are not associated with a fusion gene. We hypothesize that many of these chimeras may be technical artifacts derived from SHS-caused mis-priming in reverse transcription (RT or polymerase chain reactions (PCR. We cloned six chimeric complementary DNAs (cDNAs formed by human mitochondrial (mt 16S rRNA sequences at an SHS, which were similar to several expression sequence tags (ESTs.These chimeras, which could not be detected with cDNA protection assay, were likely formed because some regions of the 16S rRNA are reversely complementary to another region to form an SHS, which allows the downstream sequence to loop back and anneal at the SHS to prime the synthesis of its complementary strand, yielding a palindromic sequence that can form a hairpin-like structure.We identified a 16S rRNA that ended at the 4th nucleotide(nt of the mt-tRNA-leu was dominant and thus should be the wild type. We also cloned a mouse Bcl2-Nek9 chimeric cDNA that contained a 5-nt unmatchable sequence between the two partners, contained two copies of the reverse primer in the same direction but did not contain the forward primer, making it unclear how this Bcl2-Nek9 was formed and amplified. Moreover, a cDNA was amplified because one primer has 4 nts matched to the template, suggesting that there may be many more artificial cDNAs than we have realized, because the nuclear and mt genomes have many more 4-nt than 5-nt or longer homologues. Altogether, the chimeric cDNAs we cloned are good examples suggesting that many cDNAs may be artifacts due to SHS-caused mis-priming and thus greater caution should be taken when new sequence is obtained from a technique involving DNA polymerization.

  10. One Step to Construct the Homologous Arm Vector Containing Multi-DNA Fragments%一步法快速构建多片段连接的同源臂载体

    Institute of Scientific and Technical Information of China (English)

    张金脉; 杨冠恒; 程艳; 黄英

    2013-01-01

    and the corre⁃sponding buffer were added into the reaction solution with 10~50 cycles of digestion and ligation to construct the homologous arm vector composed of multi-DNA fragments. The reaction was stopped at 72℃, and the ligated prod⁃ucts were transformed into the competent cells for the subsequent identification by enzyme digestion and PCR. pMD19-T was then optimized to eliminate the BsaⅠ site on it, and the racer gene fragment CMV-EGFP was in⁃serted into pHSG298-T vector containing resistant marker. The different combination of homologous arms were used to validated this methodology. Results: The results of enzyme digestion and PCR demonstrated that one step approach could successfully construct the homologous arm vector composed of multi-DNA fragments. Six different four-fragments composing vectors can be constructed using the optimized pMD19-T-O vector system within two days. Conclusion: The one step method for vector construction was an simple, feasible and reliable approach to connect multiple fragments seamlessly into one vector. This methodology can not only be used to construct some kinds of vector systems but also make precise point mutations. This approach was suitable for quick construction of gene targeting vector.

  11. Degradative Plasmid and Heavy Metal Resistance Plasmid Naturally Coexist in Phenol and Cyanide Assimilating Bacteria

    Directory of Open Access Journals (Sweden)

    Bahig E.  Deeb

    2009-01-01

    Full Text Available Problem statement: Heavy metals are known to be powerful inhibitors of xenobiotics biodegradation activities. Alleviation the inhibitory effect of these metals on the phenol biodegradation activities in presence of heavy metals resistant plasmid was investigated. Approach: Combination of genetic systems of degradation of xenobiotic compound and heavy metal resistance was one of the approaches to the creation of polyfunctional strains for bioremediation of soil after co-contamination with organic pollutants and heavy metals. Results: A bacterial strain Pseudomonas putida PhCN (pPhCN1, pPhCN2 had been obtained. This bacterium contained two plasmids, a 120 Kb catabolic plasmid that encode for breakdown of phenol (pPhCN1 and pPhCN2 plasmid (100 Kb that code for cadmium and copper resistant. Cyanide assimilation by this bacterium was encoded by chromosomal genes. The inhibitory effect of cadmium (Cd2+ or copper (Cu2+ on the degradation of phenol and cyanide by P. putida strains PhCN and PhCN1 (contained pPhCN1 were investigated. The resistant strain PhCN showed high ability to degrade phenol and cyanide in presence of Cd2+ or Cu2+ comparing with the sensitive strain PhCN1. In addition, Cd2+ or Cu2+ was also found to exert a strong inhibitory effect on the C23O dioxygenase enzyme activity in the presence of cyanide as a nitrogen source. Conclusion: The presence of heavy metal resistance plasmid alleviated the inhibitory effect of metals on the phenol and cyanide assimilation by resistant strain.

  12. Excluded volume effect enhances the homology pairing of model chromosomes

    CERN Document Server

    Takamiya, Kazunori; Isami, Shuhei; Nishimori, Hiraku; Awazu, Akinori

    2015-01-01

    To investigate the structural dynamics of the homology pairing of polymers, we mod- eled the scenario of homologous chromosome pairings during meiosis in Schizosaccharomyces pombe, one of the simplest model organisms of eukaryotes. We consider a simple model consist- ing of pairs of homologous polymers with the same structures that are confined in a cylindrical container, which represents the local parts of chromosomes contained in an elongated nucleus of S. pombe. Brownian dynamics simulations of this model showed that the excluded volume effects among non-homological chromosomes and the transitional dynamics of nuclear shape serve to enhance the pairing of homologous chromosomes.

  13. Excluded volume effect enhances the homology pairing of model chromosomes

    Science.gov (United States)

    Takamiya, Kazunori; Yamamoto, Keisuke; Isami, Shuhei; Nishimori, Hiraku; Awazu, Akinori

    To investigate the structural dynamics of the homology pairing of polymers, we mod- eled the scenario of homologous chromosome pairings during meiosis in Schizosaccharomyces pombe, one of the simplest model organisms of eukaryotes. We consider a simple model consist- ing of pairs of homologous polymers with the same structures that are confined in a cylindrical container, which represents the local parts of chromosomes contained in an elongated nucleus of S. pombe. Brownian dynamics simulations of this model showed that the excluded volume effects among non-homological chromosomes and the transitional dynamics of nuclear shape serve to enhance the pairing of homologous chromosomes.

  14. Seifert fibered homology spheres with trivial Heegaard Floer homology

    OpenAIRE

    Eftekhary, Eaman

    2009-01-01

    We show that among Seifert fibered integer homology spheres, Poincare sphere (with either orientation) is the only non-trivial example which has trivial Heegaard Floer homology. Together with an earlier result, this shows that if an integer homology sphere has trivial Heegaard Floer homology, then it is a connected sum of a number of Poincare spheres and hyperbolic homology spheres.

  15. An interpretation of E_n-homology as functor homology

    OpenAIRE

    Livernet, Muriel; Richter, Birgit

    2009-01-01

    We prove that E_n-homology of non-unital commutative algebras can be described as functor homology when one considers functors from a certain category of planar trees with n levels. For different n these homology theories are connected by natural maps, ranging from Hochschild homology and its higher order versions to Gamma homology.

  16. Stem loop sequences specific to transposable element IS605 are found linked to lipoprotein genes in Borrelia plasmids.

    Directory of Open Access Journals (Sweden)

    Nicholas Delihas

    Full Text Available BACKGROUND: Plasmids of Borrelia species are dynamic structures that contain a large number of repetitive genes, gene fragments, and gene fusions. In addition, the transposable element IS605/200 family, as well as degenerate forms of this IS element, are prevalent. In Helicobacter pylori, flanking regions of the IS605 transposase gene contain sequences that fold into identical small stem loops. These function in transposition at the single-stranded DNA level. METHODOLOGY/PRINCIPAL FINDINGS: In work reported here, bioinformatics techniques were used to scan Borrelia plasmid genomes for IS605 transposable element specific stem loop sequences. Two variant stem loop motifs are found in the left and right flanking regions of the transposase gene. Both motifs appear to have dispersed in plasmid genomes and are found "free-standing" and phylogenetically conserved without the associated IS605 transposase gene or the adjacent flanking sequence. Importantly, IS605 specific stem loop sequences are also found at the 3' ends of lipoprotein genes (PFam12 and PFam60, however the left and right sequences appear to develop their own evolutionary patterns. The lipoprotein gene-linked left stem loop sequences maintain the IS605 stem loop motif in orthologs but only at the RNA level. These show mutations whereby variants fold into phylogenetically conserved RNA-type stem loops that contain the wobble non-Watson-Crick G-U base-pairing. The right flanking sequence is associated with the family lipoprotein-1 genes. A comparison of homologs shows that the IS605 stem loop motif rapidly dissipates, but a more elaborate secondary structure appears to develop in its place. CONCLUSIONS/SIGNIFICANCE: Stem loop sequences specific to the transposable element IS605 are present in plasmid regions devoid of a transposase gene and significantly, are found linked to lipoprotein genes in Borrelia plasmids. These sequences are evolutionarily conserved and/or structurally developed in

  17. Instability of multiple drug resistance plasmids in Salmonella typhimurium isolated from poultry.

    Science.gov (United States)

    Brown, D J; Threlfall, E J; Rowe, B

    1991-04-01

    Plasmids in five strains of Salmonella typhimurium resistant to ampicillin, chloramphenicol, gentamicin, neomycin/kanamycin, streptomycin, sulphonamides, tetracyclines and trimethoprim (ACGKSSuTTm), CGKSSuTTm, ACSSuT or CSSuT which had been isolated from poultry in the first 3 months of 1989 have been characterized and compared with plasmids in two strains of R-types ACGKSSuTTm and ASSuTTm isolated from two patients later in the year. With the exception of the human isolate of R-type ASSuTTm, all strains carried two non-conjugative plasmids, one coding for SSu and belonging to incompatibility group Q, and a second coding for multiple resistance and belonging to the FIme incompatibility group. The human isolate of R-type ASSuTTm did not carry the IncQ SSu plasmid but like the poultry isolates, carried a non-conjugative FIme plasmid. Restriction endonuclease digestion with the enzymes EcoR I, Pst I and Hind III demonstrated that the FIme plasmids from strains of different R-types showed a high degree of homology but exhibited numerous fragment size polymorphisms. The restriction digest fingerprint of plasmids in the human isolate of R-type ACGKSSuTTm was indistinguishable from a poultry isolate of the same R-type. Analysis of segregants of one of the poultry isolates of R-type ACGKSSuTTm demonstrated that resistance determinants could be rapidly lost from the FIme plasmid to give rise to a number of R-types and fingerprint patterns. Loss of tetracycline resistance from this plasmid appeared to be correlated with the integration of other plasmid-mediated resistances into the bacterial chromosome. Evidence is presented for the rapid loss of antimicrobial resistance determinants from a multiple resistance plasmid of the FIme incompatibility group in response to withdrawal of antibiotic selective pressure.

  18. Characterization of KfrA proteins encoded by a plasmid of Paenibacillus popilliae ATCC 14706T

    Directory of Open Access Journals (Sweden)

    Kazuhiro Iiyama

    2015-06-01

    Full Text Available A scaffold obtained from whole-genome shotgun sequencing of Paenibacillus popilliae ATCC 14706T shares partial homology with plasmids found in other strains of P. popilliae. PCR and sequencing for gap enclosure indicated that the scaffold originated from a 15,929-bp circular DNA. The restriction patterns of a plasmid isolated from P. popilliae ATCC 14706T were identical to those expected from the sequence; thus, this circular DNA was identified as a plasmid of ATCC 14706T and designated pPOP15.9. The plasmid encodes 17 putative open reading frames. Orfs 1, 5, 7, 8, and 9 are homologous to Orfs 11, 12, 15, 16, and 17, respectively. Orf1 and Orf11 are annotated as replication initiation proteins. Orf8 and Orf16 are homologs of KfrA, a plasmid-stabilizing protein in Gram-negative bacteria. Recombinant Orf8 and Orf16 proteins were assessed for the properties of KfrA. Indeed, they formed multimers and bound to inverted repeat sequences in upstream regions of both orf8 and orf16. A phylogenetic tree based on amino acid sequences of Orf8, Orf16 and Kfr proteins did not correlate with species lineage.

  19. Plasmid and chromosome partitioning: surprises from phylogeny

    DEFF Research Database (Denmark)

    Gerdes, Kenn; Møller-Jensen, Jakob; Bugge Jensen, Rasmus

    2000-01-01

    Plasmids encode partitioning genes (par) that are required for faithful plasmid segregation at cell division. Initially, par loci were identified on plasmids, but more recently they were also found on bacterial chromosomes. We present here a phylogenetic analysis of par loci from plasmids and chr...

  20. Geminiviruses: a tale of a plasmid becoming a virus

    Directory of Open Access Journals (Sweden)

    Krupovic Mart

    2009-05-01

    Full Text Available Abstract Background Geminiviruses (family Geminiviridae are small single-stranded (ss DNA viruses infecting plants. Their virion morphology is unique in the known viral world – two incomplete T = 1 icosahedra are joined together to form twinned particles. Geminiviruses utilize a rolling-circle mode to replicate their genomes. A limited sequence similarity between the three conserved motifs of the rolling-circle replication initiation proteins (RCR Reps of geminiviruses and plasmids of Gram-positive bacteria allowed Koonin and Ilyina to propose that geminiviruses descend from bacterial replicons. Results Phylogenetic and clustering analyses of various RCR Reps suggest that Rep proteins of geminiviruses share a most recent common ancestor with Reps encoded on plasmids of phytoplasmas, parasitic wall-less bacteria replicating both in plant and insect cells and therefore occupying a common ecological niche with geminiviruses. Capsid protein of Satellite tobacco necrosis virus was found to be the best template for homology-based structural modeling of the geminiviral capsid protein. Good stereochemical quality of the generated models indicates that the geminiviral capsid protein shares the same structural fold, the viral jelly-roll, with the vast majority of icosahedral plant-infecting ssRNA viruses. Conclusion We propose a plasmid-to-virus transition scenario, where a phytoplasmal plasmid acquired a capsid-coding gene from a plant RNA virus to give rise to the ancestor of geminiviruses.

  1. Minimax Rates for Homology Inference

    CERN Document Server

    Balakrishnan, Sivaraman; Sheehy, Don; Singh, Aarti; Wasserman, Larry

    2011-01-01

    Often, high dimensional data lie close to a low-dimensional submanifold and it is of interest to understand the geometry of these submanifolds. The homology groups of a manifold are important topological invariants that provide an algebraic summary of the manifold. These groups contain rich topological information, for instance, about the connected components, holes, tunnels and sometimes the dimension of the manifold. In this paper, we consider the statistical problem of estimating the homology of a manifold from noisy samples under several different noise models. We derive upper and lower bounds on the minimax risk for this problem. Our upper bounds are based on estimators which are constructed from a union of balls of appropriate radius around carefully selected points. In each case we establish complementary lower bounds using Le Cam's lemma.

  2. Dualities in Persistent (Co)Homology

    Energy Technology Data Exchange (ETDEWEB)

    de Silva, Vin; Morozov, Dmitriy; Vejdemo-Johansson, Mikael

    2011-09-16

    We consider sequences of absolute and relative homology and cohomology groups that arise naturally for a filtered cell complex. We establishalgebraic relationships between their persistence modules, and show that they contain equivalent information. We explain how one can use the existingalgorithm for persistent homology to process any of the four modules, and relate it to a recently introduced persistent cohomology algorithm. Wepresent experimental evidence for the practical efficiency of the latter algorithm.

  3. Dualities in Persistent (Co)Homology

    Energy Technology Data Exchange (ETDEWEB)

    de Silva, Vin; Morozov, Dmitriy; Vejdemo-Johansson, Mikael

    2011-09-16

    We consider sequences of absolute and relative homology and cohomology groups that arise naturally for a filtered cell complex. We establishalgebraic relationships between their persistence modules, and show that they contain equivalent information. We explain how one can use the existingalgorithm for persistent homology to process any of the four modules, and relate it to a recently introduced persistent cohomology algorithm. Wepresent experimental evidence for the practical efficiency of the latter algorithm.

  4. A New Shuttle Plasmid That Stably Replicates in Clostridium acetobutylicum.

    Science.gov (United States)

    Lee, Sang-Hyun; Kwon, Min-A; Choi, Sunwha; Kim, Sooah; Kim, Jungyeon; Shin, Yong-An; Kim, Kyoung Heon

    2015-10-01

    We have developed a new shuttle plasmid, designated as pLK1-MCS that can replicate in both Clostridium acetobutylicum and Escherichia coli, by combining the pUB110 and pUC19 plasmids. Plasmid pLK1-MCS replicated more stably than previously reported plasmids containing either the pIM13 or the pAMβ1 replicon in the absence of antibiotic selective pressure. The transfer frequency of pLK1-MCS into C. acetobutylicum was similar to the transfer frequency of other shuttle plasmids. We complemented C. acetobutylicum ML1 (that does not produce solvents such as acetone, butanol, and ethanol owing to loss of the megaplasmid pSOL1 harboring the adhE1-ctfAB-adc operon) by introducing pLK1-MCS carrying the adhE1-ctfAB-adc operon into C. acetobutylicum ML1. The transformed cells were able to resume anaerobic solvent production, indicating that the new shuttle plasmid has the potential for practical use in microbial biotechnology.

  5. On Galaxies and Homology

    OpenAIRE

    Novak, Gregory S.; Jonsson, Patrik; Primack, Joel R.; Cox, Thomas J.; Dekel, Avishai

    2012-01-01

    The definition of homology for single-component galaxies is clear, but for multi-component (luminous and dark matter) galaxies there is some ambiguity. We attempt to clarify the situation by carefully separating the different concepts of homology that have been used to date. We argue that the most useful definition is that a set of galaxies is homologous if they are the same in all respects up to a set of three dimensional scaling constants which may differ from one galaxy to the next. Noting...

  6. Plasmid curing and the loss of grip--the 65-kb replicon of Phaeobacter inhibens DSM 17395 is required for biofilm formation, motility and the colonization of marine algae.

    Science.gov (United States)

    Frank, Oliver; Michael, Victoria; Päuker, Orsola; Boedeker, Christian; Jogler, Christian; Rohde, Manfred; Petersen, Jörn

    2015-03-01

    Surface colonization is characteristic for a broad range of marine roseobacters and many strains have been isolated from biofilms, microbial mats and dinoflagellates. Phaeobacter inhibens DSM 17395, one of the best-studied representatives of the Roseobacter group, is an effective colonizer of marine surfaces, but the genetic basis of this trait is unknown. Based on the composition of its 65-kb RepA-I type plasmid that contains more than 20 genes for polysaccharide metabolism, including a rhamnose operon, which is required for O-antigen formation in Escherichia coli, it was hypothesized that this replicon was essential for surface attachment. Accordingly, a holistic approach was taken and the functional role of this extrachromosomal element in P. inhibens was investigated. Plasmid curing was performed with the homologous RepA-I replication system of Dinoroseobacter shibae DSM 16493(T). The Δ65-kb mutant completely lost its stickiness and could neither attach to artificial (glass, polystyrene) nor to natural surfaces (algae) and, consequently, its ability to form biofilms was impaired. Surprisingly, the mutant also lost the capacity for flagellar swimming motility required for surface colonization and the dispersal of biofilms. The data clearly showed that the 65-kb replicon of P. inhibens DSM 17395 was a genuine biofilm plasmid-mediating surface attachment. Homologous replicons are widely distributed among Rhodobacterales thus indicating the general importance of extrachromosomal elements for biofilm formation.

  7. MutS2 Promotes Homologous Recombination in Bacillus subtilis.

    Science.gov (United States)

    Burby, Peter E; Simmons, Lyle A

    2017-01-15

    Bacterial MutS proteins are subdivided into two families, MutS1 and MutS2. MutS1 family members recognize DNA replication errors during their participation in the well-characterized mismatch repair (MMR) pathway. In contrast to the well-described function of MutS1, the function of MutS2 in bacteria has remained less clear. In Helicobacter pylori and Thermus thermophilus, MutS2 has been shown to suppress homologous recombination. The role of MutS2 is unknown in the Gram-positive bacterium Bacillus subtilis In this work, we investigated the contribution of MutS2 to maintaining genome integrity in B. subtilis We found that deletion of mutS2 renders B. subtilis sensitive to the natural antibiotic mitomycin C (MMC), which requires homologous recombination for repair. We demonstrate that the C-terminal small MutS-related (Smr) domain is necessary but not sufficient for tolerance to MMC. Further, we developed a CRISPR/Cas9 genome editing system to test if the inducible prophage PBSX was the underlying cause of the observed MMC sensitivity. Genetic analysis revealed that MMC sensitivity was dependent on recombination and not on nucleotide excision repair or a symptom of prophage PBSX replication and cell lysis. We found that deletion of mutS2 resulted in decreased transformation efficiency using both plasmid and chromosomal DNA. Further, deletion of mutS2 in a strain lacking the Holliday junction endonuclease gene recU resulted in increased MMC sensitivity and decreased transformation efficiency, suggesting that MutS2 could function redundantly with RecU. Together, our results support a model where B. subtilis MutS2 helps to promote homologous recombination, demonstrating a new function for bacterial MutS2. Cells contain pathways that promote or inhibit recombination. MutS2 homologs are Smr-endonuclease domain-containing proteins that have been shown to function in antirecombination in some bacteria. We present evidence that B. subtilis MutS2 promotes recombination

  8. 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

    Directory of Open Access Journals (Sweden)

    Juan López-Villarejo

    2015-02-01

    Full Text Available 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.

  9. HOMOLOGY RIGIDITY OF GRASSMANNIANS

    Institute of Scientific and Technical Information of China (English)

    Li Fang; Duan Haibao

    2009-01-01

    Applying the theory of GrSbner basis to the Schubert presentation for the cohomology of Grassmannians [2], we extend the homology rigidity results known for the classical Grassmaniaas to the exceptional cases.

  10. Homology, convergence and parallelism.

    Science.gov (United States)

    Ghiselin, Michael T

    2016-01-05

    Homology is a relation of correspondence between parts of parts of larger wholes. It is used when tracking objects of interest through space and time and in the context of explanatory historical narratives. Homologues can be traced through a genealogical nexus back to a common ancestral precursor. Homology being a transitive relation, homologues remain homologous however much they may come to differ. Analogy is a relationship of correspondence between parts of members of classes having no relationship of common ancestry. Although homology is often treated as an alternative to convergence, the latter is not a kind of correspondence: rather, it is one of a class of processes that also includes divergence and parallelism. These often give rise to misleading appearances (homoplasies). Parallelism can be particularly hard to detect, especially when not accompanied by divergences in some parts of the body. © 2015 The Author(s).

  11. Sutures and contact homology I

    CERN Document Server

    Colin, Vincent; Honda, Ko; Hutchings, Michael

    2010-01-01

    We define a relative version of contact homology for contact manifolds with convex boundary, and prove basic properties of this relative contact homology. Similar considerations also hold for embedded contact homology.

  12. Plasmids and rickettsial evolution: insight from Rickettsia felis.

    Directory of Open Access Journals (Sweden)

    Joseph J Gillespie

    Full Text Available 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, pRFdelta, 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

  13. Exploring Antibiotic Resistance Genes and Metal Resistance Genes in Plasmid Metagenomes from Wastewater Treatment Plants

    Directory of Open Access Journals (Sweden)

    An-Dong eLi

    2015-09-01

    Full Text Available Plasmids operate as independent genetic elements in microorganism communities. Through horizontal gene transfer, they can provide their host microorganisms with important functions such as antibiotic resistance and heavy metal resistance. In this study, six metagenomic libraries were constructed with plasmid DNA extracted from influent, activated sludge and digested sludge of two wastewater treatment plants. Compared with the metagenomes of the total DNA extracted from the same sectors of the wastewater treatment plant, the plasmid metagenomes had significantly higher annotation rates, indicating that the functional genes on plasmids are commonly shared by those studied microorganisms. Meanwhile, the plasmid metagenomes also encoded many more genes related to defense mechanisms, including ARGs. Searching against an antibiotic resistance genes (ARGs database and a metal resistance genes (MRGs database revealed a broad-spectrum of antibiotic (323 out of a total 618 subtypes and metal resistance genes (23 out of a total 23 types on these plasmid metagenomes. The influent plasmid metagenomes contained many more resistance genes (both ARGs and MRGs than the activated sludge and the digested sludge metagenomes. Sixteen novel plasmids with a complete circular structure that carried these resistance genes were assembled from the plasmid metagenomes. The results of this study demonstrated that the plasmids in wastewater treatment plants could be important reservoirs for resistance genes, and may play a significant role in the horizontal transfer of these genes.

  14. Exploring antibiotic resistance genes and metal resistance genes in plasmid metagenomes from wastewater treatment plants.

    Science.gov (United States)

    Li, An-Dong; Li, Li-Guan; Zhang, Tong

    2015-01-01

    Plasmids operate as independent genetic elements in microorganism communities. Through horizontal gene transfer (HGT), they can provide their host microorganisms with important functions such as antibiotic resistance and heavy metal resistance. In this study, six metagenomic libraries were constructed with plasmid DNA extracted from influent, activated sludge (AS) and digested sludge (DS) of two wastewater treatment plants (WWTPs). Compared with the metagenomes of the total DNA extracted from the same sectors of the wastewater treatment plant, the plasmid metagenomes had significantly higher annotation rates, indicating that the functional genes on plasmids are commonly shared by those studied microorganisms. Meanwhile, the plasmid metagenomes also encoded many more genes related to defense mechanisms, including ARGs. Searching against an antibiotic resistance genes (ARGs) database and a metal resistance genes (MRGs) database revealed a broad-spectrum of antibiotic (323 out of a total 618 subtypes) and MRGs (23 out of a total 23 types) on these plasmid metagenomes. The influent plasmid metagenomes contained many more resistance genes (both ARGs and MRGs) than the AS and the DS metagenomes. Sixteen novel plasmids with a complete circular structure that carried these resistance genes were assembled from the plasmid metagenomes. The results of this study demonstrated that the plasmids in WWTPs could be important reservoirs for resistance genes, and may play a significant role in the horizontal transfer of these genes.

  15. Conservation of plasmids among Escherichia coli K1 isolates of diverse origins.

    Science.gov (United States)

    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.

  16. Conservation of plasmids among Escherichia coli K1 isolates of diverse origins.

    Science.gov (United States)

    Mercer, A A; Morelli, G; Heuzenroeder, M; Kamke, M; Achtman, M

    1984-01-01

    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. Images PMID:6094355

  17. A Site-Specific Integrative Plasmid Found in Pseudomonas aeruginosa Clinical Isolate HS87 along with A Plasmid Carrying an Aminoglycoside-Resistant Gene.

    Directory of Open Access Journals (Sweden)

    Dexi Bi

    Full Text Available Plasmids play critical roles in bacterial fitness and evolution of Pseudomonas aeruginosa. Here two plasmids found in a drug-resistant P. aeruginosa clinical isolate HS87 were completely sequenced. The pHS87b plasmid (11.2 kb carries phage-related genes and function-unknown genes. Notably, pHS87b encodes an integrase and has an adjacent tRNAThr-associated attachment site. A corresponding integrated form of pHS87b at the tRNAThr locus was identified on the chromosome of P. aeruginosa, showing that pHS87b is able to site-specifically integrate into the 3'-end of the tRNAThr gene. The pHS87a plasmid (26.8 kb displays a plastic structure containing a putative replication module, stability factors and a variable region. The RepA of pHS87a shows significant similarity to the replication proteins of pPT23A-family plasmids. pHS87a carries a transposon Tn6049, a truncated insertion sequence ΔIS1071 and a Tn402-like class 1 integron which contains an aacA4 cassette that may confer aminoglycoside resistance. Thus, pHS87b is a site-specific integrative plasmid whereas pHS87a is a plastic antibiotic resistance plasmid. The two native plasmids may promote the fitness and evolution of P. aeruginosa.

  18. New and Redesigned pRS Plasmid Shuttle Vectors for Genetic Manipulation of Saccharomycescerevisiae.

    Science.gov (United States)

    Chee, Mark K; Haase, Steven B

    2012-05-01

    We have constructed a set of 42 plasmid shuttle vectors based on the widely used pRS series for use in the budding yeast Saccharomyces cerevisiae and the bacterium Escherichia coli. This set of pRSII plasmids includes new shuttle vectors that can be used with histidine and adenine auxotrophic laboratory yeast strains carrying mutations in the genes HIS2 and ADE1, respectively. Our pRSII plasmids also include updated versions of commonly used pRS plasmids from which common restriction sites that occur within their yeast-selectable biosynthetic marker genes have been removed to increase the availability of unique restriction sites within their polylinker regions. Hence, our pRSII plasmids are a complete set of integrating, centromere and 2μ episomal plasmids with the biosynthetic marker genes ADE2, HIS3, TRP1, LEU2, URA3, HIS2, and ADE1 and a standardized selection of at least 16 unique restriction sites in their polylinkers. Additionally, we have expanded the range of drug selection options that can be used for PCR-mediated homologous replacement using pRS plasmid templates by replacing the G418-resistance kanMX4 cassette of pRS400 with MX4 cassettes encoding resistance to phleomycin, hygromycin B, nourseothricin, and bialaphos. Finally, in the process of generating the new plasmids, we have determined several errors in existing publicly available sequences for several commonly used yeast plasmids. Using our updated sequences, we constructed pRS plasmid backbones with a unique restriction site for inserting new markers to facilitate future expansion of the pRS series.

  19. Plasmid-mediated colistin resistance in Escherichia coli from the Arabian Peninsula

    Directory of Open Access Journals (Sweden)

    Ágnes Sonnevend

    2016-09-01

    Conclusions: This is the first report on the presence of the plasmid-coded mcr-1 gene in a variety of multi-resistant clinical isolates from the Arabian Peninsula indicating that several commonly used antibiotics can potentially facilitate the spread of mcr-1 carrying strains, or directly, mcr-1 containing plasmids.

  20. Effect of excessive cadmium chloride on the plasmids of E. coli HB 101 in vivo

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    After Escherichia coli HB101 with plasmid pWH58, pWH98, or pTBa5 were cultered respectively in amp LB broth which contained 50 mg/L CdCl2 constantly for 24 h, these plasmids were isolated from E. coli, and the effect of excessive CdCl2 on the E. coli HB101 and plasmid DNA was studied by surveying the growth of E. coli HB101 and plasmid, argarose gel electrophoresis and analysis of restriction fragment length polymorphism (RFLP) of plasmids, and plasmid transformation. The results showed that 50 mg/L CdCl2 treatment lagged the growth of E. coli HB101 for at least 4h, but after grown for 24h there were not significant differences in the growths of E. coli HB101s and the productions of plasmids between the treatment and control. These results implified that E. coli HB101 have induced adaptability to cadmium stress and excessive CdCl2 did not inhibit the replication and amp+ gene's expression of plasmid DNA in vivo of E. coli significantly. 50 mg/L CdCl2 treatment for 24 hours might cause the sequence's change of plasmid DNA, but could not lead to the random breakage of plasmid DNA strands. Moreover, after 50 mg/L of CdCl2 treatment in vivo the transformation activities of plasmid did not altered, implied excessive CdCl2 could not affect the superhelical structure of plasmid and also not break the loop of plasmid DNA evidently.

  1. Construction of eukaryotic expression plasmid containing human polymorphic epithelial mucin and granulocyte macrophage colony stimulating factor%人多形上皮黏蛋白与巨噬细胞集落刺激因子基因融合构建双基因多表位抗原的真核共表达质粒

    Institute of Scientific and Technical Information of China (English)

    袁时芳; 师长宏; 晏伟; 李南林; 吕勇刚; 王廷; 王岭; 张英起

    2008-01-01

    BACKGROUND: Previous studies demonstrated that construction of eoexpression plasmid containing multiple genes on the same vector could improve transfection and expression rates.OBJECTIVE: To construct eukaryotic expression plasmid pcDNA3.1 (+)-MUC1 -GM-CSF by human polymorphic epithclial mucin (MUC 1) and granulocyte macrophage colony stimulating factor.(GM-CSF) and to observe expression of recombinant plasmid in COS-7 cells.DESIGN,TIME AND SETTING: Gene recombination design,which was carried out in the Animal Central Laboratory,the Fourth Military University of Chinese PLA from September 2005 to December 2006.MATERIALS: Eukaryotic expression vector pcDNA3.1 (+) was presented by Pro.Taylor-Papadimitriou;pGEM-3zf()-GM-CSF plasmid,COS-7 cells,pUCI 8 vector,and E.coli DH5α were made in the center; female BALB/c mice were provided by Experimental Animal Center of the Fourth Military University of Chinese PLA.METHODS: Signal peptide was synthesized with encoded MUCI gene sections to obtain repeated sequence coneatemer after renaturation.Next,the accepted concatemer was cloned with GM-CSF following enzyme identification and sequencing analysis,and then they were put in eukaryotic expression vector pcDNA3.1(+) to construct eukaryotic coexpression plasmid pcDNA3.1 (+)-MUCI -GM-CSF in order to transform COS-7 cells.MAIN OUTCOME MEASURES: Gene expression was detected by indirect immunofluorescence and enzyme linked immunosorbent assay (ELISA).RESULTS: Enzyme identification and sequencing analysis showed that recombinant plasmid contained a fusion gene encompassing human MUC1 repeated sequence concatemer and GM-CSF; moreover,MUC1 expression was detected in COS-7 cells,while recombinant plasmid could induce the production of anti-GM-CSF antibody.CONCLUSION: The recombination between human MUC1 repeated sequence concatemer and GM-CSF gene successfully constructs eukaryotic coexpression plasmid.%背景:一些实验已经证明在同一载体上构建含多个基因的共表达

  2. Plasmid Profiles of Virulent Rhodococcus equi Strains Isolated from Infected Foals in Poland.

    Science.gov (United States)

    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.

  3. Singular (Lipschitz) homology and homology of integral currents

    OpenAIRE

    Riedweg, Christian; Schäppi, Daniel

    2009-01-01

    We compare the homology groups $H_n ^{IC}(X)$ of the chain complex of integral currents with compact support of a metric space $X$ with the singular Lipschitz homology $H^L_n (X)$ and with ordinary singular homology. If $X$ satisfies certain cone inequalities all these homology theories coincide. On the other hand, for the Hawaiian Earring the homology of integral currents differs from the singular Lipschitz homology and it differs also from the classical singular homology $H_n(X)$.

  4. Multiple Pathways of Plasmid DNA Transfer in Helicobacter pylori

    Science.gov (United States)

    Rohrer, Stefanie; Holsten, Lea; Weiss, Evelyn; Benghezal, Mohammed; Fischer, Wolfgang; Haas, Rainer

    2012-01-01

    Many Helicobacter pylori (Hp) strains carry cryptic plasmids of different size and gene content, the function of which is not well understood. A subgroup of these plasmids (e.g. pHel4, pHel12), contain a mobilisation region, but no cognate type IV secretion system (T4SS) for conjugative transfer. Instead, certain H. pylori strains (e.g. strain P12 carrying plasmid pHel12) can harbour up to four T4SSs in their genome (cag-T4SS, comB, tfs3, tfs4). Here, we show that such indigenous plasmids can be efficiently transferred between H. pylori strains, even in the presence of extracellular DNaseI eliminating natural transformation. Knockout of a plasmid-encoded mobA relaxase gene significantly reduced plasmid DNA transfer in the presence of DNaseI, suggesting a DNA conjugation or mobilisation process. To identify the T4SS involved in this conjugative DNA transfer, each individual T4SS was consecutively deleted from the bacterial chromosome. Using a marker-free counterselectable gene deletion procedure (rpsL counterselection method), a P12 mutant strain was finally obtained with no single T4SS (P12ΔT4SS). Mating experiments using these mutants identified the comB T4SS in the recipient strain as the major mediator of plasmid DNA transfer between H. pylori strains, both in a DNaseI-sensitive (natural transformation) as well as a DNaseI-resistant manner (conjugative transfer). However, transfer of a pHel12::cat plasmid from a P12ΔT4SS donor strain into a P12ΔT4SS recipient strain provided evidence for the existence of a third, T4SS-independent mechanism of DNA transfer. This novel type of plasmid DNA transfer, designated as alternate DNaseI-Resistant (ADR) mechanism, is observed at a rather low frequency under in vitro conditions. Taken together, our study describes for the first time the existence of three distinct pathways of plasmid DNA transfer between H. pylori underscoring the importance of horizontal gene transfer for this species. PMID:23029142

  5. Braid Floer homology

    Science.gov (United States)

    van den Berg, J. B.; Ghrist, R.; Vandervorst, R. C.; Wójcik, W.

    2015-09-01

    Area-preserving diffeomorphisms of a 2-disc can be regarded as time-1 maps of (non-autonomous) Hamiltonian flows on R / Z ×D2. The periodic flow-lines define braid (conjugacy) classes, up to full twists. We examine the dynamics relative to such braid classes and define a new invariant for such classes, the BRAID FLOER HOMOLOGY. This refinement of Floer homology, originally used for the Arnol'd Conjecture, yields a Morse-type forcing theory for periodic points of area-preserving diffeomorphisms of the 2-disc based on braiding. Contributions of this paper include (1) a monotonicity lemma for the behavior of the nonlinear Cauchy-Riemann equations with respect to algebraic lengths of braids; (2) establishment of the topological invariance of the resulting braid Floer homology; (3) a shift theorem describing the effect of twisting braids in terms of shifting the braid Floer homology; (4) computation of examples; and (5) a forcing theorem for the dynamics of Hamiltonian disc maps based on braid Floer homology.

  6. Co-resident plasmids travel together.

    Science.gov (United States)

    Gama, João Alves; Zilhão, Rita; Dionisio, Francisco

    2017-08-24

    Conjugative plasmids encode genes that enable them to transfer, by conjugation, from a given host cell to another cell. Conjugative transfer, despite being an important feature of conjugative plasmids, is not constitutive for most plasmids, the reason being that genes involved in horizontal transfer are mostly repressed. Only upon their transient de-repression are plasmids able to transfer horizontally. If host cells harbour multiple plasmids, their simultaneous transfer depends on simultaneous transient de-repression of all plasmids. If de-repression of different plasmids was random and independent events, simultaneous de-repression should be a rare event because the probability of simultaneous de-repression would be the product of the probabilities of de-repression of each plasmid. Some previous observations support this hypothesis, while others show that co-transfer of plasmids is more frequent than this reasoning indicates. Here, we show that co-transfer of multiple plasmids mainly results from non-independent events: the probability that all plasmids within a cell become de-repressed is much higher than if de-repression of plasmids genes were independent. We found a simple model for the probability of co-transfer: the plasmid having the lowest conjugation rates is the one who limits co-transfer. In this sense, cells receiving the plasmid with the lower transfer rate also receive the other plasmid. If de-repression happens simultaneously on co-resident plasmids, common cues may stimulate de-repression of distinct plasmids. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Construction of plant expression plasmid p2355-gtfB containing glucosyltransferase B of Streptococcus mutans%变异链球菌葡糖基转移酶植物表达质粒p2355-gtfB的构建

    Institute of Scientific and Technical Information of China (English)

    陈筑; 刘建国; 马欣荣; 张燕; 唐琳; 吴家媛; 张剑

    2012-01-01

    Objective To construct plant plasmid p2355-gtfB which contains multiple catalytic sites and antigen epitopes of glucosyltransferase of Streptococcus mutans. Methods Plasmid pcDNA3-gtfB was used as template to obtain the gtfB by PCR amplification. Then gtfB was inserted into inter-mediate vector pMD18-T, subcloned from the recombi-nant plasmid T-gtfB to plant expression vector p2355, then p2355-gtfB was transformed into Agrobacterium tumefaciens EHA105 by electroporation. Results Genetic segments 2. 9 kb and 3. 7 kb were obtained through restriction enzyme mapping analysis and DNA sequencing, and inserted gene 3 675 bp was shown in the construction recombinant plasmid p2355-gtfB through series analysis and detection. Conclusion The recombinant plasmid was constructed successfully. This research provided basis for further study on plants vaccine against dental caries.%目的 构建含变异链球菌葡糖基转移酶多个免疫显性抗原表位的植物表达质粒p2355-gtfB,为其转化植物研究奠定物质基础,为可食防龋疫苗研究提供条件.方法 以真核表达质粒pcDNA3-gtfB为模板,通过聚合酶链反应(polymerase chain reaction,PCR)扩增含编码变异链球菌葡糖基转移酶抗原表位的目的基因gtfB.将回收纯化的PCR产物经T-A克隆技术克隆于中间载体pMD18-T,双酶切鉴定插入方向后,将目的基因从中间载体释放,再克隆至高效的植物表达载体p2355,用电转化法转化根癌农杆菌EHA105,对重组质粒阳性克隆株进行筛选与鉴定.结果 通过对重组质粒T-gtfB进行酶切图谱分析,获得2.9kb和3.7 kb的2个片断,将重组质粒p2355-gtfB进行酶切、PCR及测序分析,显示p2355-gtfB中插入基因长度为3 675 bp,基因序列与双脱氧链终止法的测定结果相同,证明植物表达质粒p2355-gtfB构建成功,开放阅读框架正确.结论 本研究成功构建了含变异链球菌多个葡糖基转移酶免疫显性抗原表位编码

  8. The Chlamydophila felis plasmid is highly conserved.

    Science.gov (United States)

    Harley, Ross; Day, Sarinder; Di Rocco, Camillo; Helps, Chris

    2010-11-20

    The presence of a plasmid in the Chlamydiaceae is both species and strain specific. Knowledge of the prevalence of the plasmid in different Chlamydia species is important for future studies aiming to investigate the role of the plasmid in chlamydial biology and disease. Although strains of Chlamydophila felis with or without the plasmid have been identified, only a small number of laboratory-adapted strains have been analysed and the prevalence of the plasmid in field isolates has not been determined. This study aimed to determine the prevalence of the plasmid in C. felis-positive conjunctival and oropharyngeal clinical samples submitted for routine diagnosis of C. felis by real-time (Q)PCR. DNA extracts from four laboratory-adapted strains were also analysed. QPCR assays targeting regions of C. felis plasmid genes pCF01, pCF02 and pCF03 were developed for the detection of plasmid DNA. QPCR analysis of DNA extracts from C. felis-positive clinical samples found evidence of plasmid DNA in 591 of 595 samples representing 561 of 564 (99.5%) clinical cases. Plasmid DNA was also detected by QPCR in laboratory-adapted strains 1497V, K2487 and K2490, but not strain 905. We conclude that the plasmid is highly conserved in C. felis, and plasmid-deficient strains represent a rare but important population for future studies of chlamydial plasmid function.

  9. Gene Disruption by Homologous Recombination in the Xylella fastidiosa Citrus Variegated Chlorosis Strain

    Science.gov (United States)

    Gaurivaud, Patrice; Souza, Leonardo C. A.; Virgílio, Andrea C. D.; Mariano, Anelise G.; Palma, Renê R.; Monteiro, Patrícia B.

    2002-01-01

    Mutagenesis by homologous recombination was evaluated in Xylella fastidiosa by using the bga gene, coding for β-galactosidase, as a model. Integration of replicative plasmids by homologous recombination between the cloned truncated copy of bga and the endogenous gene was produced by one or two crossover events leading to β-galactosidase mutants. A promoterless chloramphenicol acetyltransferase gene was used to monitor the expression of the target gene and to select a cvaB mutant. PMID:12200328

  10. Copper Resistance Gene Homologs in Pathogenic and Saprophytic Bacterial Species from Tomato

    OpenAIRE

    Cooksey, Donald A.; Azad, Hamid R.; Cha, Jae-Soon; Lim, Chun-Keun

    1990-01-01

    Copper-resistant strains of Xanthomonas campestris pv. vesicatoria, Pseudomonas cichorii, Pseudomonas putida, Pseudomonas fluorescens, and a yellow Pseudomonas sp. were isolated from tomato plants or seeds. In Southern hybridizations, DNA from each strain showed homology with the copper resistance (cop) operon previously cloned from Pseudomonas syringae pv. tomato PT23. Homology was associated with plasmid and chromosomal DNA in X. compestris pv. vesicatoria, P. putida, and the yellow Pseudom...

  11. PLASMIDS FROM ANAEROCELLUM THERMOPHILUM AND USES THEREOF

    DEFF Research Database (Denmark)

    2003-01-01

    The present invention concerns the isolation of plasmids from extremely thermophilic anaerobic microorganisms and their use in genetic transformation of thermophilic and mesophilic microorganisms. More particular the invention concerns the use of thermostable plasmid vectors as tools for creating...

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

    Energy Technology Data Exchange (ETDEWEB)

    Guss, Adam M [ORNL; Olson, Daniel G. [Thayer School of Engineering at Dartmouth; Caiazza, Nicky [Mascoma Corporation; Lynd, Lee R [Thayer School of Engineering at Dartmouth

    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.

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

    Directory of Open Access Journals (Sweden)

    Guss Adam M

    2012-05-01

    Full Text Available Abstract 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 pAMG205 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. Conclusions 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.

  14. Sequences of two related multiple antibiotic resistance virulence plasmids sharing a unique IS26-related molecular signature isolated from different Escherichia coli pathotypes from different hosts.

    Directory of Open Access Journals (Sweden)

    Carola Venturini

    Full Text Available Enterohemorrhagic Escherichia coli (EHEC and atypical enteropathogenic E. coli (aEPEC are important zoonotic pathogens that increasingly are becoming resistant to multiple antibiotics. Here we describe two plasmids, pO26-CRL125 (125 kb from a human O26:H- EHEC, and pO111-CRL115 (115kb from a bovine O111 aEPEC, that impart resistance to ampicillin, kanamycin, neomycin, streptomycin, sulfathiazole, trimethoprim and tetracycline and both contain atypical class 1 integrons with an identical IS26-mediated deletion in their 3´-conserved segment. Complete sequence analysis showed that pO26-CRL125 and pO111-CRL115 are essentially identical except for a 9.7 kb fragment, present in the backbone of pO26-CRL125 but absent in pO111-CRL115, and several indels. The 9.7 kb fragment encodes IncI-associated genes involved in plasmid stability during conjugation, a putative transposase gene and three imperfect repeats. Contiguous sequence identical to regions within these pO26-CRL125 imperfect repeats was identified in pO111-CRL115 precisely where the 9.7 kb fragment is missing, suggesting it may be mobile. Sequences shared between the plasmids include a complete IncZ replicon, a unique toxin/antitoxin system, IncI stability and maintenance genes, a novel putative serine protease autotransporter, and an IncI1 transfer system including a unique shufflon. Both plasmids carry a derivate Tn21 transposon with an atypical class 1 integron comprising a dfrA5 gene cassette encoding resistance to trimethoprim, and 24 bp of the 3´-conserved segment followed by Tn6026, which encodes resistance to ampicillin, kanymycin, neomycin, streptomycin and sulfathiazole. The Tn21-derivative transposon is linked to a truncated Tn1721, encoding resistance to tetracycline, via a region containing the IncP-1α oriV. Absence of the 5 bp direct repeats flanking Tn3-family transposons, indicates that homologous recombination events played a key role in the formation of this complex

  15. Comparative metagenomic analysis of plasmid encoded functions in the human gut microbiome

    Directory of Open Access Journals (Sweden)

    Marchesi Julian R

    2010-01-01

    Full Text Available Abstract Background Little is known regarding the pool of mobile genetic elements associated with the human gut microbiome. In this study we employed the culture independent TRACA system to isolate novel plasmids from the human gut microbiota, and a comparative metagenomic analysis to investigate the distribution and relative abundance of functions encoded by these plasmids in the human gut microbiome. Results Novel plasmids were acquired from the human gut microbiome, and homologous nucleotide sequences with high identity (>90% to two plasmids (pTRACA10 and pTRACA22 were identified in the multiple human gut microbiomes analysed here. However, no homologous nucleotide sequences to these plasmids were identified in the murine gut or environmental metagenomes. Functions encoded by the plasmids pTRACA10 and pTRACA22 were found to be more prevalent in the human gut microbiome when compared to microbial communities from other environments. Among the most prevalent functions identified was a putative RelBE toxin-antitoxin (TA addiction module, and subsequent analysis revealed that this was most closely related to putative TA modules from gut associated bacteria belonging to the Firmicutes. A broad phylogenetic distribution of RelE toxin genes was observed in gut associated bacterial species (Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria, but no RelE homologues were identified in gut associated archaeal species. We also provide indirect evidence for the horizontal transfer of these genes between bacterial species belonging to disparate phylogenetic divisions, namely Gram negative Proteobacteria and Gram positive species from the Firmicutes division. Conclusions The application of a culture independent system to capture novel plasmids from the human gut mobile metagenome, coupled with subsequent comparative metagenomic analysis, highlighted the unexpected prevalence of plasmid encoded functions in the gut microbial ecosystem. In

  16. Plasmid required for virulence of Agrobacterium tumefaciens

    Energy Technology Data Exchange (ETDEWEB)

    Watson, B.; Currier, T.C.; Gordon, M.P.; Chilton, M.D.; Nester, E.W.

    1975-07-01

    The irreversible loss of crown gall-inducing ability of Agrobacterium tumefaciens strain C-58 during growth at 37/sup 0/C is shown to be due to loss of a large plasmid (1.2 x 10/sup 8/ daltons). The gene responsible for this high rate of plasmid loss at elevated temperatures seems to be located on the plasmid. In addition, another spontaneous avirulent variant, A. tumefaciens strain IIBNV6, is shown to lack the virulence plasmid which its virulent sibling strain, IIBV7, possesses. Deoxyribonucleic acid reassociation measurements prove that the plasmid is eliminated, not integrated into the chromosome, in both of the avirulent derivatives. Transfer of virulence from donor strain C-58 to avirulent recipient strain A136 results from the transfer of a plasmid, which appears identical to the donor plasmid by deoxyribonucleic acid reassociation measurements. The transfer of virulence in another cross, K27 x A136, was also shown to result from the transfer of a large plasmid. These findings establish unequivocally that the large plasmid determines virulence. Two additional genetic determinants have been located on the virulence plasmid of A. tumefaciens strain C-58: the ability to utilize nopaline and sensitivity to a bacteriocin produced by strain 84. The latter trait can be exploited for selection of avirulent plasmid-free derivatives of strain C-58. The trait of nopaline utilization appears to be on the virulence plasmid also in strains IIBV7 and K27.

  17. Integration of a transfected gene into the genome of Babesia bovis occurs by legitimate homologous recombination mechanisms.

    Science.gov (United States)

    Suarez, Carlos E; Johnson, Wendell C; Herndon, David R; Laughery, Jacob M; Davis, William C

    2015-08-01

    This study examines the patterns of gene integration of gfp-bsd upon stable transfection into the T3Bo strain of Babesia bovis using a plasmid designed to integrate homologous sequences of the parasite's two identical ef-1α A and B genes. While the transfected BboTf-149-6 cell line displayed two distinct patterns of gene integration, clonal lines derived from this strain by cell sorting contained only single gfp-bsd insertions. Whole genome sequencing of two selected clonal lines, E9 and C6, indicated two distinct patterns of gfp-bsd insertion occurring by legitimate homologous recombination mechanisms: one into the expected ef-1α orf B, and another into the ef-1α B promoter. The data suggest that expression of the ef-1α orf B is not required for development of B. bovis in cultured erythrocyte stages. Use of legitimate homologous recombination mechanisms in transfected B. bovis supports the future use of transfection methods for developing efficient gene function assignment experiments using gene knockout techniques. Published by Elsevier B.V.

  18. Role of Plasmids in Lactobacillus brevis BSO 464 Hop Tolerance and Beer Spoilage

    Science.gov (United States)

    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

  19. Role of plasmids in Lactobacillus brevis BSO 464 hop tolerance and beer spoilage.

    Science.gov (United States)

    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.

  20. Gorenstein homological dimensions

    DEFF Research Database (Denmark)

    Holm, Henrik Granau

    2004-01-01

    In basic homological algebra, the projective, injective and 2at dimensions of modules play an important and fundamental role. In this paper, the closely related Gorenstein projective, Gorenstein injective and Gorenstein 2at dimensions are studied. There is a variety of nice results about Gorenstein...

  1. Gorenstein homological dimensions

    DEFF Research Database (Denmark)

    Holm, Henrik Granau

    2004-01-01

    In basic homological algebra, the projective, injective and 2at dimensions of modules play an important and fundamental role. In this paper, the closely related Gorenstein projective, Gorenstein injective and Gorenstein 2at dimensions are studied. There is a variety of nice results about Gorenstein...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  4. Molecular characterization and homologous overexpression of [FeFe]-hydrogenase in Clostridium tyrobutyricum JM1

    Energy Technology Data Exchange (ETDEWEB)

    Jo, Ji Hye [Biosciences Center, National Renewable Energy Laboratory, 1617 Cole Blvd, Golden, CO 80401-3393 (United States); Jeon, Che Ok [Department of Life Science, Chung-Ang University, Seoul 156-756 (Korea); Lee, Seung Yoon [K-water Research Institute, Korea Water Resources Corporation, 462-1, Jeonmin-dong, Yuseong-gu, Daejon 305-703 (Korea); Lee, Dae Sung [Department of Environmental Engineering, Kyungpook National University, 1370 Sankyuk-dong, Buk-gu, Daegu 702-701 (Korea); Park, Jong Moon [Department of Chemical Engineering, Pohang University of Science and Technology, San 31, Hyoja-dong, Nam-gu, Pohang, Gyeongbuk 790-784 (Korea)

    2010-02-15

    The H{sub 2}-evoving [FeFe]-hydrogenase in Clostridium tyrobutyricum JM1 was isolated to elucidate molecular characterization and modular structure of the hydrogenase. Then, homologous overexpression of the hydrogenase gene was for the first time performed to enhance hydrogen production. The hydA open reading frame (ORF) was 1734-bp, encodes 577 amino acids with a predicted molecular mass of 63,970 Da, and presents 80% and 75% identity at the amino acid level with the [FeFe]-hydrogenase genes of Clostridium kluyveri DSM 555 and Clostridium acetobutylicum ATCC 824, respectively. One histidine residue and 19 cysteine residues, known to fasten one [2Fe-2S] cluster, three [4Fe-4S] clusters and one H-cluster, were conserved in hydA of C. tyrobutyricum. A 2327-bp DNA region containing the ORF and the putative promoter region was amplified and subcloned into a pJIR418 shuttle vector. The gene transfer of the recombinant plasmid into C. tyrobutyricum JM1 was performed by a modified electrotransformation method. Homologous overexpression of the [FeFe]-hydrogenase gene resulted in a 1.7-fold and 1.5-fold increase in hydrogenase activity and hydrogen yield concomitant with the shift of metabolic pathway. (author)

  5. Characterization of an IncA/C Multidrug Resistance Plasmid in Vibrio alginolyticus.

    Science.gov (United States)

    Ye, Lianwei; Li, Ruichao; Lin, Dachuan; Zhou, Yuanjie; Fu, Aisi; Ding, Qiong; Chan, Edward Wai Chi; Yao, Wen; Chen, Sheng

    2016-05-01

    Cephalosporin-resistant Vibrio alginolyticus was first isolated from food products, with β-lactamases encoded by blaPER-1, blaVEB-1, and blaCMY-2 being the major mechanisms mediating their cephalosporin resistance. The complete sequence of a multidrug resistance plasmid, pVAS3-1, harboring the blaCMY-2 and qnrVC4 genes was decoded in this study. Its backbone exhibited genetic homology to known IncA/C plasmids recoverable from members of the family Enterobacteriaceae, suggesting its possible origin in Enterobacteriaceae. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  6. Characterization of plasmids in extensively drug-resistant acinetobacter strains isolated in India and Pakistan.

    Science.gov (United States)

    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.

  7. High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation.

    Science.gov (United States)

    Hoggard, Timothy; Liachko, Ivan; Burt, Cassaundra; Meikle, Troy; Jiang, Katherine; Craciun, Gheorghe; Dunham, Maitreya J; Fox, Catherine A

    2016-04-07

    The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid

  8. Origin and Evolution of Rickettsial Plasmids.

    Science.gov (United States)

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

    2016-01-01

    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. 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. Rickettsial plasmids had a complex evolution, starting with a vertical inheritance followed by a reductive evolution associated with increased complexity via horizontal gene transfer as well as

  9. 人胰岛素样生长因子1基因质粒载体的构建及其在人脐血源性神经干细胞中的表达%Construction of Plasmid Vector Containing Human Insulin-Like Growth Factor 1 and Its Expression in Human Umbilical Cord Blood-Derived Neural Stem Cells

    Institute of Scientific and Technical Information of China (English)

    王军; 吴值荣; 朱登纳; 高峰; 侯艳艳; 陈海; 王留霞

    2011-01-01

    Objective To construct the plasmid expression vector containing human insulin - like growth factor 1 ( IGF - 1 ), and examine the expression of IGF - 1 gene in human umbilical cord blood - derived neural stem cells(NSCs).Methods The IGF - 1 gene was extracted from the fetal liver via reverse tranacription polymerase chain reaction( RT - PCR) ,then the product of PCR and plasmid pcDNA3.1 were purified by gel extraction.After enzyme digestion of DNA restriction enzyme - BamH Ⅰ and Hind Ⅲ, purified IGF - 1 gene was cloned into expression plasmid vector pcDNA3.1 by T4 DNA Ligase.Recombinant plasmid was identified by DNA sequencing method and enzyme digestion of DNA restriction enzyme - BamH Ⅰ and Hind Ⅲ.Recombinant pcDNA3.1 - IGF - 1 and empty plasmid were transfected into human umbilical cord blood - derived NSCs through lipofectin transfection.After transfection, transfected umbilical cord blood - derived NSCs were filtered with neomycin( G418 ).The expression of IGF - 1 gene in the gene transfected umbilical cord blood - derived was examined by immunocytochemical method and RT - PCR.Results IGF - 1 gene was successfully extracted from the fetal liver.Recombinants peDNA3.1 -IGF - 1 was proved accurate by restriction enzyme digestion and sequencing.Recombinant was transfected into human umbilical cord blood -derived NSCs by liposome for 24 hours,then selection cell clones appeared after 2 weeks for G418 filtering.In umbilical cord blood -derived NSCs transfected by recombinant plasmid vector, the expression of IGF- 1 gene was successful, which was detected by immtmocyto-chemical method and the expression of IGF - 1 mRNA was also positive while the other was negative compared with controla,which was examined by RT - PCR.Conclusion IGF - 1 gene can expressed in umbilical cord blood - derived NSCs transfected by recombinant plasmid vector.%目的 构建人胰岛素样生长因子1(IGF-1)质粒表达载体,并观察重组体pcDNA3.1-IGF-1转染后的脐血

  10. Differentiation of IncL and IncM Plasmids Associated with the Spread of Clinically Relevant Antimicrobial Resistance.

    Directory of Open Access Journals (Sweden)

    Alessandra Carattoli

    Full Text Available blaOXA-48, blaNDM-1 and blaCTX-M-3 are clinically relevant resistance genes, frequently associated with the broad-host range plasmids of the IncL/M group. The L and M plasmids belong to two compatible groups, which were incorrectly classified together by molecular methods. In order to understand their evolution, we fully sequenced four IncL/M plasmids, including the reference plasmids R471 and R69, the recently described blaOXA-48-carrying plasmid pKPN-El.Nr7 from a Klebsiella pneumoniae isolated in Bern (Switzerland, and the blaSHV-5 carrying plasmid p202c from a Salmonella enterica from Tirana (Albania.Sequencing was performed using 454 Junior Genome Sequencer (Roche. Annotation was performed using Sequin and Artemis software. Plasmid sequences were compared with 13 fully sequenced plasmids belonging to the IncL/M group available in GenBank.Comparative analysis of plasmid genomes revealed two distinct genetic lineages, each containing one of the R471 (IncL and R69 (IncM reference plasmids. Conjugation experiments demonstrated that plasmids representative of the IncL and IncM groups were compatible with each other. The IncL group is constituted by the blaOXA-48-carrying plasmids and R471. The IncM group contains two sub-types of plasmids named IncM1 and IncM2 that are each incompatible.This work re-defines the structure of the IncL and IncM families and ascribes a definitive designation to the fully sequenced IncL/M plasmids available in GenBank.

  11. Homological stability for unordered configuration spaces

    DEFF Research Database (Denmark)

    Randal-Williams, Oscar

    2013-01-01

    This paper consists of two related parts. In the first part we give a self-contained proof of homological stability for the spaces C_n(M;X) of configurations of n unordered points in a connected open manifold M with labels in a path-connected space X, with the best possible integral stability range...

  12. Chlamydophila felis: plasmid detection in Italian isolates.

    Science.gov (United States)

    Di Francesco, Antonietta; Donati, Manuela; Salvatore, Daniela; Cevenini, Roberto; Di Paolo, Maria; Baldelli, Raffaella

    2010-04-01

    Plasmids have been detected in the majority of strains in the genus Chlamydia and in many Chlamydophila species. Previous studies showed that FP Pring and FP Cello Chlamydophila felis strains have an extrachromosomial plasmid, whereas the FP Baker strain does not. Azuma et al. recently sequenced the entire genomic DNA sequence of the Japanese Cp. felis strain Fe/C-56 and described a 7,552 base pair circular plasmid. In the present study a highly conserved plasmid gene was detected in 11 Italian Cp. felis isolates, showing 100% nucleotide identity with the plasmid gene of Fe/C-56 Cp. felis strain.

  13. Influence of Plasmid Type on the Replication of Rhodococcus equi in Host Macrophages.

    Science.gov (United States)

    Willingham-Lane, Jennifer M; Berghaus, Londa J; Giguère, Steeve; Hondalus, Mary K

    2016-01-01

    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

  14. Monopole Floer homology for rational homology 3-spheres

    OpenAIRE

    Froyshov, Kim A.

    2010-01-01

    We give a new construction of monopole Floer homology for $\\text{spin}^c$ rational homology $3$ -spheres. As applications, we define two invariants of certain $4$ -manifolds with $b_1=1$ and $b^+=0$ .

  15. 酵母双杂交诱饵表达载体pGBKT7-cide-3的构建、鉴定以及毒性、自激活效应的检测%Construction, identification and transformation of "bait plasmid" containing human cide-3 gene in yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    闵婕; 李青; 马楠; 康艳霞; 李娜

    2013-01-01

    Objective;To construct a bait vector containing human cide -3 gene in yeast two -hybrid system in order to screen the human liver cDNA library. Methods; The fragment including all exons of cide -3 gene was amplified by RT-PCR and was inserted into the multiple cloning sites of the shuttle vector pGBKT7. After confirmation with restricted endonuclease digestion and sequence analysis,by using PEG/LiAC method,the plasmid pGBKT7 - cide -3 was transformed into yeast AH109. The plasmid and protein isolated from the positive yeast AH109 clone was tested its expression,toxicity and transcriptional activation. Results:The human cide -3 cDNA was amplified. The restrictive endonuclease digestion and DNA sequencing proved that the plasmid pGBKT7 - cide - 3 was obtained. The yeast AH109 clone transformed with pGBKT7 - cide - 3 was screened out by the SD/ - Trp nutritional media. Sequence analysis revealed that the fragment was correctly inserted into pGBKT7 with a right reading frame and its expression in yeast was verified. Conclusion; The bait plasmid pGBKT7 -cide -3 constructed expresses correctly,and can not activate the transcription of reporter gene alone in yeast two - hybrid system.%目的:构建并转化酵母双杂交体系中“诱饵”载体pGBKT7-cide-3以便用于人肝cDNA文库的筛选.方法:用RT-PCR方法从人肝细胞株QZG中扩增cide-3基因全外显子片段,并定向克隆入细菌-酵母穿梭表达质粒pGBKT7中;用PEG/LiAC法将pGBKT7-cide-3转化感受态酵母菌AH109.提取转化酵母菌的质粒DNA和总蛋白进行鉴定.同时检测表达产物对报告基因的激活作用及对酵母株AH109的毒性.结果:成功扩增出人cide-3 cDNA片段,限制性内切酶酶切和DNA测序证实获得正确的重组质粒pGBKT7-cide-3;获得可在色氨酸缺陷培养基(SD/-Trp)上生长含pGBKT7-cide-3的酵母菌克隆.鉴定表明pG-BKT7-cide-3已转化入酵母菌AH109,无自主激活报告基因的能力及对酵母的生长无毒性.结

  16. Hundreds of circular novel plasmids and DNA elements identified in a rat cecum metamobilome.

    Directory of Open Access Journals (Sweden)

    Tue Sparholt Jørgensen

    Full Text Available Metagenomic approaches are widespread in microbiological research, but so far, the knowledge on extrachromosomal DNA diversity and composition has largely remained dependant on cultivating host organisms. Even with the emergence of metagenomics, complete circular sequences are rarely identified, and have required manual curation. We propose a robust in silico procedure for identifying complete small plasmids in metagenomic datasets from whole genome shotgun sequencing. From one very pure and exhaustively sequenced metamobilome from rat cecum, we identified a total of 616 circular sequences, 160 of which were carrying a gene with plasmid replication domain. Further homology analyses indicated that the majority of these plasmid sequences are novel. We confirmed the circularity of the complete plasmid candidates using an inverse-type PCR approach on a subset of sequences with 95% success, confirming the existence and length of discrete sequences. The implication of these findings is a broadened understanding of the traits of circular elements in nature and the possibility of massive data mining in existing metagenomic datasets to discover novel pools of complete plasmids thus vastly expanding the current plasmid database.

  17. TasA-tasB, a new putative toxin-antitoxin (TA system from Bacillus thuringiensis pGI1 plasmid is a widely distributed composite mazE-doc TA system

    Directory of Open Access Journals (Sweden)

    Mahillon Jacques

    2006-10-01

    Full Text Available Abstract Background Post-segregational killing systems are present in a large variety of microorganisms. When found on plasmids, they are described as addiction systems that act to maintain the plasmid during the partitioning of the cell. The plasmid to be maintained through the generations harbours a group of two genes, one coding for a stable toxin and the other coding for an unstable antitoxin that inhibits the effects of the toxin. If, during cell division, the plasmid is lost, the toxin and antitoxin proteins present in the cytosol cease to be newly expressed. The level of unstable antitoxin protein then rapidly decreases, leaving the toxin free to act on the cellular target, leading to cell death. Consequently, only cells harbouring the plasmid can survive. Results The pGI1 plasmid of Bacillus thuringiensis H1.1 harbours a group of two genes, one showing similarities with the Doc toxin of the phd-doc toxin-antitoxin system, potentially coding for a toxin-antitoxin system. Attempts were made to clone this putative system in the Gram-negative host Escherichia coli. The putative antitoxin tasA was easily cloned in E. coli. However, although several combinations of DNA fragment were used in the cloning strategy, only clones containing a mutation in the toxin gene could be recovered, suggesting a toxic activity of TasB. An exhaustive search was carried out in order to index genes homologous to those of the putative tasA-tasB system among microorganisms. This study revealed the presence of this system in great number and in a large variety of microorganisms, either as tasA-tasB homologues or in association with toxins (or antitoxins from other TA systems. Conclusion In this work, we showed that the pGI1 plasmid of B. thuringiensis H1.1 harbours genes resembling a toxin-antitoxin system, named tasA-tasB for thuringiensis addiction system. This system appeared to be functional but unregulated in E. coli. Bioinformatics studies showed that the tas

  18. Homological Methods in Equations of Mathematical Physics

    OpenAIRE

    Krasil'shchik, Joseph; Verbovetsky, Alexander

    1998-01-01

    These lecture notes are a systematic and self-contained exposition of the cohomological theories naturally related to partial differential equations: the Vinogradov C-spectral sequence and the C-cohomology, including the formulation in terms of the horizontal (characteristic) cohomology. Applications to computing invariants of differential equations are discussed. The lectures contain necessary introductory material on the geometric theory of differential equations and homological algebra.

  19. In Vivo Transmission of an IncA/C Plasmid in Escherichia coli Depends on Tetracycline Concentration, and Acquisition of the Plasmid Results in a Variable Cost of Fitness.

    Science.gov (United States)

    Johnson, Timothy J; Singer, Randall S; Isaacson, Richard E; Danzeisen, Jessica L; Lang, Kevin; Kobluk, Kristi; Rivet, Bernadette; Borewicz, Klaudyna; Frye, Jonathan G; Englen, Mark; Anderson, Janet; Davies, Peter R

    2015-05-15

    IncA/C plasmids are broad-host-range plasmids enabling multidrug resistance that have emerged worldwide among bacterial pathogens of humans and animals. Although antibiotic usage is suspected to be a driving force in the emergence of such strains, few studies have examined the impact of different types of antibiotic administration on the selection of plasmid-containing multidrug resistant isolates. In this study, chlortetracycline treatment at different concentrations in pig feed was examined for its impact on selection and dissemination of an IncA/C plasmid introduced orally via a commensal Escherichia coli host. Continuous low-dose administration of chlortetracycline at 50 g per ton had no observable impact on the proportions of IncA/C plasmid-containing E. coli from pig feces over the course of 35 days. In contrast, high-dose administration of chlortetracycline at 350 g per ton significantly increased IncA/C plasmid-containing E. coli in pig feces (P IncA/C plasmid to other indigenous E. coli hosts. There was no evidence of conjugal transfer of the IncA/C plasmid to bacterial species other than E. coli. In vitro competition assays demonstrated that bacterial host background substantially impacted the cost of IncA/C plasmid carriage in E. coli and Salmonella. In vitro transfer and selection experiments demonstrated that tetracycline at 32 μg/ml was necessary to enhance IncA/C plasmid conjugative transfer, while subinhibitory concentrations of tetracycline in vitro strongly selected for IncA/C plasmid-containing E. coli. Together, these experiments improve our knowledge on the impact of differing concentrations of tetracycline on the selection of IncA/C-type plasmids. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  20. 携带乙肝大包膜蛋白L基因与结核Esat6融合基因植物表达载体的构建及鉴定%Construction and Identification of the Expression Plasmid Containing Genes Encoding the Large Envelope Protein L of HBV and Esat-6 Gene of Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    李君武; 宫君原; 刘鑫; 叶秋萍; 黄清华

    2011-01-01

    To construction and identification of the expression plasmid containing genes encoding East6 gene of Mycobacterium tuberculosis and the large envelope protein L of HBV, and transform the recombinant vector into Agrobacterium tumefaciens LBA4404. The L and East-6 gene were amplied from pPICgK-L and Mycobacterium tuberculosis genome by PCR,then the fusion DNA fragment of L and East6 were amplied by Splicing by Overlap Extension were cloned into the vector pEGG. The combine fragment of promoter globulin-I and the target gene L-East6 which get from doubled enzymes digestion of the recombined plasmid pEG-G-L-Esat6 was inserted into the plant expression vector pCAMBIA1300 which contain the gene bar for herbicide resistance. Then transformed recombinant plasimid pCAMG-L- Esat6 into Agrobacterium tumerfaciens LBA4404. We have successfully constructed eukaryotic expression recombinantplasmid pCAMG-L-Esat6 and it is showed that the cloned sequence of L and Esat6 is correct by sequencing. We have successfully constructed expression plasmid containing genes encoding L protein of HBV and Esat6 gene and transformed into LBA4404,and lays a foundation for construction combined gene vaccine against HBV and MTB.%构建包含编码结核杆菌Esat6基因和乙肝病毒大包膜蛋白(L蛋白)基因的植物双元表达载体,并转化根癌农杆菌LBA4404.分别以质粒pPIC9K-L和结核杆菌基因组为模板进行PCR扩增,获得L和Esat6基因,然后运用部分重叠聚合酶链式反应扩增出L-Esat6融合基因片段,连接到有玉米特异性启动子globulin-1的pEGG载体上,将G-L-Esat6融合基因片段酶切下,连接到含有抗除草剂基因bar的双元表达载体pCAMBIAI300上,电击法将重组质粒转化到农杆菌LBA4404中.构建了真核表达重组质粒pCAMG-L-Esat6,测序分析表明,克隆的L和Esat6序列与NCBI上公布序列一致.成功构建与转化了包含编码乙肝病毒大包膜蛋白L基因和结核杆菌Esat6基因的植物表达载

  1. Three classes of plasmid (47-63 kb) carry the type B neurotoxin gene cluster of group II Clostridium botulinum.

    Science.gov (United States)

    Carter, Andrew T; Austin, John W; Weedmark, Kelly A; Corbett, Cindi; Peck, Michael W

    2014-08-01

    Pulsed-field gel electrophoresis and DNA sequence analysis of 26 strains of Group II (nonproteolytic) Clostridium botulinum type B4 showed that 23 strains carried their neurotoxin gene cluster on a 47-63 kb plasmid (three strains lacked any hybridization signal for the neurotoxin gene, presumably having lost their plasmid). Unexpectedly, no neurotoxin genes were found on the chromosome. This apparent constraint on neurotoxin gene transfer to the chromosome stands in marked contrast to Group I C. botulinum, in which neurotoxin gene clusters are routinely found in both locations. The three main classes of type B4 plasmid identified in this study shared different regions of homology, but were unrelated to any Group I or Group III plasmid. An important evolutionary aspect firmly links plasmid class to geographical origin, with one class apparently dominant in marine environments, whereas a second class is dominant in European terrestrial environments. A third class of plasmid is a hybrid between the other two other classes, providing evidence for contact between these seemingly geographically separated populations. Mobility via conjugation has been previously demonstrated for the type B4 plasmid of strain Eklund 17B, and similar genes associated with conjugation are present in all type B4 plasmids now described. A plasmid toxin-antitoxin system pemI gene located close to the neurotoxin gene cluster and conserved in each type B4 plasmid class may be important in understanding the mechanism which regulates this unique and unexpected bias toward plasmid-borne neurotoxin genes in Group II C. botulinum type B4.

  2. [Characterization of the transfer-related tra region of the conjugative plasmid p19 from a Bacillus subtilis soil strain].

    Science.gov (United States)

    Poluéktova, E U; Gagarina, E Iu; Nezametdinova, V Z; Shilovskiĭ, I P; Rodionova, S A; Prozorov, A A

    2010-01-01

    The nucleotide sequences of three DNA fragments (total size 30574 bp) of the plasmid p19 from the Bacillus subtilis 19 soil strain have been determined. Thirty open reading frames (ORFs) have been identified in these fragments. oriT of the plasmid has also been identified. As shown by the search for homologs of hypothetical protein products of these ORFs in databases, such homology exists for 18 ORFs. The protein products of nine ORFs can be assumed to have specific functions. Several ORFs were inactivated via insertional mutagenesis, and the conjugation capacity of the mutant plasmids was estimated. According to the data on homology of protein products and the results of ORF inactivation, regions of a total size of about 20 kb from the DNA fragments sequenced by us were inferred to belong to the tra region of p19. As follows from the analysis of the identified ORFs of the p19 tra region, it differs from the earlier described tra regions of other plasmids, irrespective of a certain similarity with the corresponding regions of plasmids of gram-positive bacteria from the genera Bacillus, Clostridium, and Listeria.

  3. Prevalence of ST1193 clone and IncI1/ST16 plasmid in E-coli isolates carrying blaCTX-M-55 gene from urinary tract infections patients in China.

    Science.gov (United States)

    Xia, Liang; Liu, Yang; Xia, Shu; Kudinha, Timothy; Xiao, Shu-Nian; Zhong, Nan-Shan; Ren, Guo-Sheng; Zhuo, Chao

    2017-03-24

    To study molecular epidemiology of CTX-M-55-carrying Escherichia coli isolates from urinary tract infections (UTIs) in China. 111 blaCTX-M-55-positive E.coli isolates from UTIs patients in China were studied. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to analyze the homologies among the strains. Conjugation experiments, S1nuclease PFGE and PCR analysis were performed to characterize plasmids harboring blaCTX-M-55 and their genetic environment. 111 isolates were clustered into 86 individual pulsotypes and three clusters by PFGE. Fifty-five (49.5%) of the isolates belonged to 8 STs. Most of the ST1193 isolates belonged to one PFGE cluster. Transconjugants (n = 45) derived from randomly selected blaCTX-M-55 donors (n = 58), were found to contain a single 90-kb conjugative plasmid, which mainly belonged to the IncI1 groups (34, 76%). Among the IncI1 plasmids, the blaCTX-M-55/IncI1/ST16 predominated (23/34, 68%). The blaTEM-1 and aac (3')-II genes were frequently detected on the IncI1 plasmids, and the insertion of ISEcp1 or IS26 was observed at the 48 bp or 45 bp upstream of the start codon of blaCTX-M-55 gene. The dissemination of blaCTX-M-55 gene among E. coli UTI isolates, appeared to be due to both the major clonal lineage of ST1193 and the horizontal transfer of epidemic plasmid IncI1/ST16.

  4. Prevalence of ST1193 clone and IncI1/ST16 plasmid in E-coli isolates carrying blaCTX-M-55 gene from urinary tract infections patients in China

    Science.gov (United States)

    Xia, Liang; Liu, Yang; Xia, Shu; Kudinha, Timothy; Xiao, Shu-nian; Zhong, Nan-shan; Ren, Guo-sheng; Zhuo, Chao

    2017-01-01

    To study molecular epidemiology of CTX-M-55-carrying Escherichia coli isolates from urinary tract infections (UTIs) in China. 111 blaCTX-M-55-positive E.coli isolates from UTIs patients in China were studied. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to analyze the homologies among the strains. Conjugation experiments, S1nuclease PFGE and PCR analysis were performed to characterize plasmids harboring blaCTX-M-55 and their genetic environment. 111 isolates were clustered into 86 individual pulsotypes and three clusters by PFGE. Fifty-five (49.5%) of the isolates belonged to 8 STs. Most of the ST1193 isolates belonged to one PFGE cluster. Transconjugants (n = 45) derived from randomly selected blaCTX-M-55 donors (n = 58), were found to contain a single 90-kb conjugative plasmid, which mainly belonged to the IncI1 groups (34, 76%). Among the IncI1 plasmids, the blaCTX-M-55/IncI1/ST16 predominated (23/34, 68%). The blaTEM-1 and aac (3′)-II genes were frequently detected on the IncI1 plasmids, and the insertion of ISEcp1 or IS26 was observed at the 48 bp or 45 bp upstream of the start codon of blaCTX-M-55 gene. The dissemination of blaCTX-M-55 gene among E. coli UTI isolates, appeared to be due to both the major clonal lineage of ST1193 and the horizontal transfer of epidemic plasmid IncI1/ST16. PMID:28338012

  5. Effects of Long-Term Storage on Plasmid Stability in Bacillus anthracis

    Science.gov (United States)

    Marston, Chung K.; Hoffmaster, Alex R.; Wilson, Kathy E.; Bragg, Sandra L.; Plikaytis, Brian; Brachman, Philip; Johnson, Scott; Kaufmann, Arnold F.; Popovic, Tanja

    2005-01-01

    The plasmid profiles of 619 cultures of Bacillus anthracis which had been isolated and stored between 1954 and 1989 were analyzed using the Laboratory Response Network real-time PCR assay targeting a chromosomal marker and both virulence plasmids (pXO1 and pXO2). The cultures were stored at ambient temperature on tryptic soy agar slants overlaid with mineral oil. When data were stratified by decade, there was a decreasing linear trend in the proportion of strains containing both plasmids with increased storage time (P < 0.001). There was no significant difference in the proportion of strains containing only pXO1 or strains containing only pXO2 (P = 0.25), but there was a statistical interdependence between the two plasmids (P = 0.004). Loss of viability of B. anthracis cultures stored on agar slants is also discussed. PMID:16332750

  6. Investigating homology between proteins using energetic profiles.

    Directory of Open Access Journals (Sweden)

    James O Wrabl

    2010-03-01

    Full Text Available Accumulated experimental observations demonstrate that protein stability is often preserved upon conservative point mutation. In contrast, less is known about the effects of large sequence or structure changes on the stability of a particular fold. Almost completely unknown is the degree to which stability of different regions of a protein is generally preserved throughout evolution. In this work, these questions are addressed through thermodynamic analysis of a large representative sample of protein fold space based on remote, yet accepted, homology. More than 3,000 proteins were computationally analyzed using the structural-thermodynamic algorithm COREX/BEST. Estimated position-specific stability (i.e., local Gibbs free energy of folding and its component enthalpy and entropy were quantitatively compared between all proteins in the sample according to all-vs.-all pairwise structural alignment. It was discovered that the local stabilities of homologous pairs were significantly more correlated than those of non-homologous pairs, indicating that local stability was indeed generally conserved throughout evolution. However, the position-specific enthalpy and entropy underlying stability were less correlated, suggesting that the overall regional stability of a protein was more important than the thermodynamic mechanism utilized to achieve that stability. Finally, two different types of statistically exceptional evolutionary structure-thermodynamic relationships were noted. First, many homologous proteins contained regions of similar thermodynamics despite localized structure change, suggesting a thermodynamic mechanism enabling evolutionary fold change. Second, some homologous proteins with extremely similar structures nonetheless exhibited different local stabilities, a phenomenon previously observed experimentally in this laboratory. These two observations, in conjunction with the principal conclusion that homologous proteins generally conserved

  7. Small, Enigmatic Plasmids of the Nosocomial Pathogen, Acinetobacter baumannii: Good, Bad, Who Knows?

    Directory of Open Access Journals (Sweden)

    Soo Sum Lean

    2017-08-01

    Full Text Available Acinetobacter baumannii is a Gram-negative nosocomial pathogen that has become a serious healthcare concern within a span of two decades due to its ability to rapidly acquire resistance to all classes of antimicrobial compounds. One of the key features of the A. baumannii genome is an open pan genome with a plethora of plasmids, transposons, integrons, and genomic islands, all of which play important roles in the evolution and success of this clinical pathogen, particularly in the acquisition of multidrug resistance determinants. An interesting genetic feature seen in majority of A. baumannii genomes analyzed is the presence of small plasmids that usually ranged from 2 to 10 kb in size, some of which harbor antibiotic resistance genes and homologs of plasmid mobilization genes. These plasmids are often overlooked when compared to their larger, conjugative counterparts that harbor multiple antibiotic resistance genes and transposable elements. In this mini-review, we will examine our current knowledge of these small A. baumannii plasmids and look into their genetic diversity and phylogenetic relationships. Some of these plasmids, such as the Rep-3 superfamily group and the pRAY-type, which has no recognizable replicase genes, are quite widespread among diverse A. baumannii clinical isolates worldwide, hinting at their usefulness to the lifestyle of this pathogen. Other small plasmids especially those from the Rep-1 superfamily are truly enigmatic, encoding only hypothetical proteins of unknown function, leading to the question of whether these small plasmids are “good” or “bad” to their host A. baumannii.

  8. Repetitive genomic sequences as a substrate for homologous integration in the Rhizopus oryzae genome.

    Science.gov (United States)

    Yuzbashev, Tigran V; Larina, Anna S; Vybornaya, Tatiana V; Yuzbasheva, Evgeniya Y; Gvilava, Ilia T; Sineoky, Sergey P

    2015-06-01

    The vast number of repetitive genomic elements was identified in the genome of Rhizopus oryzae. Such genomic repeats can be used as homologous regions for integration of plasmids. Here, we evaluated the use of two different repeats: the short (575 bp) rptZ, widely distributed (about 34 copies per genome) and the long (2053 bp) rptH, less prevalent (about 15 copies). The plasmid carrying rptZ integrated, but did so through a 2256-bp region of homology to the pyrG locus, a unique genomic sequence. Thus, the length of rptZ was below the minimal requirements for homologous strand exchange in this fungus. In contrast, rptH was used efficiently for homologous integration. The plasmid bearing this repeat integrated in multicopy fashion, with up to 25 copies arranged in tandem. The latter vector, pPyrG-H, could be a valuable tool for integration at homologous sequences, for such purposes as high-level expression of proteins. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  9. Identification of a Novel Conjugative Plasmid in Mycobacteria That Requires Both Type IV and Type VII Secretion

    KAUST Repository

    Ummels, R.

    2014-09-23

    Conjugative plasmids have been identified in a wide variety of different bacteria, ranging from proteobacteria to firmicutes, and conjugation is one of the most efficient routes for horizontal gene transfer. The most widespread mechanism of plasmid conjugation relies on different variants of the type IV secretion pathway. Here, we describe the identification of a novel type of conjugative plasmid that seems to be unique for mycobacteria. Interestingly, while this plasmid is efficiently exchanged between different species of slow-growing mycobacteria, including Mycobacterium tuberculosis, it could not be transferred to any of the fast-growing mycobacteria tested. Genetic analysis of the conjugative plasmid showed the presence of a locus containing homologues of three type IV secretion system components and a relaxase. In addition, a new type VII secretion locus was present. Using transposon insertion mutagenesis, we show that in fact both these secretion systems are essential for conjugation, indicating that this plasmid represents a new class of conjugative plasmids requiring two secretion machineries. This plasmid could form a useful new tool to exchange or introduce DNA in slow-growing mycobacteria. IMPORTANCE: Conjugative plasmids play an important role in horizontal gene transfer between different bacteria and, as such, in their adaptation and evolution. This effect is most obvious in the spread of antibiotic resistance genes. Thus far, conjugation of natural plasmids has been described only rarely for mycobacterial species. In fact, it is generally accepted that M. tuberculosis does not show any recent sign of horizontal gene transfer. In this study, we describe the identification of a new widespread conjugative plasmid that can also be efficiently transferred to M. tuberculosis. This plasmid therefore poses both a threat and an opportunity. The threat is that, through the acquisition of antibiotic resistance markers, this plasmid could start a rapid spread of

  10. Rabinowitz Floer homology: A survey

    CERN Document Server

    Albers, Peter

    2010-01-01

    Rabinowitz Floer homology is the semi-infinite dimensional Morse homology associated to the Rabinowitz action functional used in the pioneering work of Rabinowitz. Gradient flow lines are solutions of a vortex-like equation. In this survey article we describe the construction of Rabinowitz Floer homology and its applications to symplectic and contact topology, global Hamiltonian perturbations and the study of magnetic fields.

  11. Nucleotide Sequences and Comparison of Two Large Conjugative Plasmids from Different Campylobacter species

    Science.gov (United States)

    2004-01-01

    conjugation share amino acid similarities to the T4SS of different Brucella species, but have the highest overall homology to the Mpf gene cluster in...rumen of sheep, cows and goats . Interestingly, repA, the three upstream ORFs of unknown function and the tet(O) gene in pCC31 and pTet have a G+C...subgroup of conjugative plasmids with extensive homology to pCC31 and pTet are relatively prevalent in clinical isolates of C. jejuni (i.e. eight out

  12. Quantifying and resolving multiple vector transformants in S. cerevisiae plasmid libraries

    Directory of Open Access Journals (Sweden)

    Gray Elizabeth C

    2009-11-01

    Full Text Available Abstract Background In addition to providing the molecular machinery for transcription and translation, recombinant microbial expression hosts maintain the critical genotype-phenotype link that is essential for high throughput screening and recovery of proteins encoded by plasmid libraries. It is known that Escherichia coli cells can be simultaneously transformed with multiple unique plasmids and thusly complicate recombinant library screening experiments. As a result of their potential to yield misleading results, bacterial multiple vector transformants have been thoroughly characterized in previous model studies. In contrast to bacterial systems, there is little quantitative information available regarding multiple vector transformants in yeast. Saccharomyces cerevisiae is the most widely used eukaryotic platform for cell surface display, combinatorial protein engineering, and other recombinant library screens. In order to characterize the extent and nature of multiple vector transformants in this important host, plasmid-born gene libraries constructed by yeast homologous recombination were analyzed by DNA sequencing. Results It was found that up to 90% of clones in yeast homologous recombination libraries may be multiple vector transformants, that on average these clones bear four or more unique mutant genes, and that these multiple vector cells persist as a significant proportion of library populations for greater than 24 hours during liquid outgrowth. Both vector concentration and vector to insert ratio influenced the library proportion of multiple vector transformants, but their population frequency was independent of transformation efficiency. Interestingly, the average number of plasmids born by multiple vector transformants did not vary with their library population proportion. Conclusion These results highlight the potential for multiple vector transformants to dominate yeast libraries constructed by homologous recombination. The

  13. Salmonella Typhimurium ST213 is associated with two types of IncA/C plasmids carrying multiple resistance determinants.

    Science.gov (United States)

    Wiesner, Magdalena; Calva, Edmundo; Fernández-Mora, Marcos; Cevallos, Miguel A; Campos, Freddy; Zaidi, Mussaret B; Silva, Claudia

    2011-01-11

    Salmonella Typhimurium ST213 was first detected in the Mexican Typhimurium population in 2001. It is associated with a multi-drug resistance phenotype and a plasmid-borne blaCMY-2 gene conferring resistance to extended-spectrum cephalosporins. The objective of the current study was to examine the association between the ST213 genotype and blaCMY-2 plasmids. The blaCMY-2 gene was carried by an IncA/C plasmid. ST213 strains lacking the blaCMY-2 gene carried a different IncA/C plasmid. PCR analysis of seven DNA regions distributed throughout the plasmids showed that these IncA/C plasmids were related, but the presence and absence of DNA stretches produced two divergent types I and II. A class 1 integron (dfrA12, orfF and aadA2) was detected in most of the type I plasmids. Type I contained all the plasmids carrying the blaCMY-2 gene and a subset of plasmids lacking blaCMY-2. Type II included all of the remaining blaCMY-2-negative plasmids. A sequence comparison of the seven DNA regions showed that both types were closely related to IncA/C plasmids found in Escherichia, Salmonella, Yersinia, Photobacterium, Vibrio and Aeromonas. Analysis of our Typhimurium strains showed that the region containing the blaCMY-2 gene is inserted between traA and traC as a single copy, like in the E. coli plasmid pAR060302. The floR allele was identical to that of Newport pSN254, suggesting a mosaic pattern of ancestry with plasmids from other Salmonella serovars and E. coli. Only one of the tested strains was able to conjugate the IncA/C plasmid at very low frequencies (10-7 to 10-9). The lack of conjugation ability of our IncA/C plasmids agrees with the clonal dissemination trend suggested by the chromosomal backgrounds and plasmid pattern associations. The ecological success of the newly emerging Typhimurium ST213 genotype in Mexico may be related to the carriage of IncA/C plasmids. We conclude that types I and II of IncA/C plasmids originated from a common ancestor and that the

  14. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp.

    Science.gov (United States)

    Bierman, M; Logan, R; O'Brien, K; Seno, E T; Rao, R N; Schoner, B E

    1992-07-01

    We have constructed cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. All vectors contain the 760-bp oriT fragment from the IncP plasmid, RK2. Transfer functions need to be supplied in trans by the E. coli donor strain. We have incorporated into these vectors selectable antibiotic-resistance markers (AmR, ThR, SpR) that function in Streptomyces spp. and other features that should allow for: (i) integration via homologous recombination between cloned DNA and the Streptomyces spp. chromosome, (ii) autonomous replication, or (iii) site-specific integration at the bacteriophage phi C31 attachment site. Shuttle cosmids for constructing genomic libraries and bacteriophage P1 cloning vector capable of accepting approx. 100-kb fragments are also described. A simple mating procedure has been developed for the conjugal transfer of these vectors from E. coli to Streptomyces spp. that involves plating of the donor strain and either germinated spores or mycelial fragments of the recipient strain. We have shown that several of these vectors can be introduced into Streptomyces fradiae, a strain that is notoriously difficult to transform by PEG-mediated protoplast transformation.

  15. The replication origin of a repABC plasmid

    Directory of Open Access Journals (Sweden)

    Cevallos Miguel A

    2011-06-01

    Full Text Available Abstract Background repABC operons are present on large, low copy-number plasmids and on some secondary chromosomes in at least 19 α-proteobacterial genera, and are responsible for the replication and segregation properties of these replicons. These operons consist, with some variations, of three genes: repA, repB, and repC. RepA and RepB are involved in plasmid partitioning and in the negative regulation of their own transcription, and RepC is the limiting factor for replication. An antisense RNA encoded between the repB-repC genes modulates repC expression. Results To identify the minimal region of the Rhizobium etli p42d plasmid that is capable of autonomous replication, we amplified different regions of the repABC operon using PCR and cloned the regions into a suicide vector. The resulting vectors were then introduced into R. etli strains that did or did not contain p42d. The minimal replicon consisted of a repC open reading frame under the control of a constitutive promoter with a Shine-Dalgarno sequence that we designed. A sequence analysis of repC revealed the presence of a large A+T-rich region but no iterons or DnaA boxes. Silent mutations that modified the A+T content of this region eliminated the replication capability of the plasmid. The minimal replicon could not be introduced into R. etli strain containing p42d, but similar constructs that carried repC from Sinorhizobium meliloti pSymA or the linear chromosome of Agrobacterium tumefaciens replicated in the presence or absence of p42d, indicating that RepC is an incompatibility factor. A hybrid gene construct expressing a RepC protein with the first 362 amino acid residues from p42d RepC and the last 39 amino acid residues of RepC from SymA was able to replicate in the presence of p42d. Conclusions RepC is the only element encoded in the repABC operon of the R. etli p42d plasmid that is necessary and sufficient for plasmid replication and is probably the initiator protein. The ori

  16. Persistence of Antibiotic Resistance Plasmids in Biofilms

    Science.gov (United States)

    2014-10-01

    plasmids* in*populations*of* Gram > negative *bacteria*grown*in*biofilms*and*well>mixed*liquid*cultures.** * Task2:*Characterize*the*evolution*of*plasmid...R.! Edwards.! 2005.! Overview! of! nosocomial! infections! caused! by! gramP negative ! bacilli .!Clin.!Infect.!Dis.!41:848P854.! LoftiePEaton,!W.,!A... negative ! interaction!between!one!of! its!chromosomal!segments!and!the!plasmid! by!simply!deleting!the!appropriate!chromosomal!segment.!! 7. None

  17. The role of FIS in the Rcd checkpoint and stable maintenance of plasmid ColE1.

    Science.gov (United States)

    Blaby, I K; Summers, D K

    2009-08-01

    Escherichia coli plasmid ColE1 lacks active partitioning, and copies are distributed randomly to daughter cells at division. The plasmid is maintained stably in the bacterial population as long as its copy number remains high. The accumulation of plasmid dimers and higher multimers depresses copy number, and is an important cause of multicopy plasmid instability. ColE1 dimers are restored to the monomeric state by site-specific recombination, which requires the host-encoded proteins XerCD, ArgR and PepA acting at the plasmid cer site. In addition, a 70 nt RNA expressed from the cer site of plasmid dimers delays the division of dimer-containing cells. Here, we report that the global regulator FIS binds to cer in a sequence-specific manner, close to the Rcd promoter (P(cer)). FIS is not required for plasmid dimer resolution, but is essential for repression of P(cer) in plasmid monomers. Repression also requires the XerCD recombinase, but not ArgR or PepA. We propose a model for monomer-dimer control of P(cer) in which the promoter is repressed in plasmid monomers by the concerted action of FIS and XerCD. Rcd transcription is triggered in plasmid dimers by the lifting of XerCD-mediated repression in the synaptic complex.

  18. TALEN-mediated knock-in via non-homologous end joining in the crustacean Daphnia magna.

    Science.gov (United States)

    Nakanishi, Takashi; Kato, Yasuhiko; Matsuura, Tomoaki; Watanabe, Hajime

    2016-11-07

    Transcription activator-like effector nucleases (TALENs) are versatile tools that enable the insertion of DNA into different organisms. Here, we confirmed TALEN-mediated knock-in via non-homologous end joining in the crustacean Daphnia magna, a model organism for ecological and toxicological genomics. We tested two different TALENs, ey1 TALEN and ey2 TALEN, both of which target the eyeless locus. The donor DNA plasmid, harbouring the H2B-GFP reporter gene, was designed to contain both TALEN target sites and was co-injected with each TALEN mRNA into eggs. The ey1 TALEN and ey2 TALEN constructs both resulted in H2B-GFP expression in Daphnia with a germline transmission efficiency of 3%. Of the three transgenic animals generated, two had donor DNA at the targeted genomic site, which suggested concurrent cleavage of the injected plasmid DNA and genome DNA. The availability of such tools that are capable of targeted knock-in of foreign genes will be extremely useful for advancing the knowledge of gene function and contribute to an increased understanding of functional genomics in Daphnia.

  19. 重组人凝血因子Ⅷ表达质粒的构建及其在HepG2细胞中的表达%Construction and expression of the recombinant plasmid containing BDDhFⅧ in HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    赵倩; 解金辉; 李双玉; 董磊; 种靖慧; 闫莉娜; 刘运德; 袁玉华

    2012-01-01

    Objective: To get stable cell line expressing B domain-deleted human FVU1 (BDDhFVIII) by constructing the eulunyotic expression plasmid. Methods: Eukaryotic expression plasmid containing BDDhFVIII was constructed and transfected into HepG2 cells via elec-troporation. The expression and purification of the target protein was detected by Western blot. Results: Results of enzyme digestion and sequence analysis demonstrated that the gene of BDDhFVIII was correctly inserted into the eukaryotic expression vector pcDNA4/v5-his. Western blot confirmed the successful expression of BDDhFVUl at the protein levels in HepG2 cells. Conclusion: Tne constructed eukaryotic expression vector was able to generate high level expression of human FVQI in HepG2 cells, thus could construct human blood coagulation FVHI stable cell line successfully.%目的:构建含有B区缺失型(△760aa-1639aa)人凝血因子ⅧⅢ(B domain-deleted human FⅧ,BDDhFⅧ)的真核表达质粒,转染HepG2细胞使其稳定表达人凝血因子Ⅷ.方法:将BDDhFⅧ基因片段插入pcDNA4/v5-his空载体中构建重组真核表达质粒,测序正确后电转入HepG2细胞,经Ni-NTA纯化,利用Western blot检测凝血因子Ⅷ在HepG2细胞中的表达,持续培养获得稳定表达BDDhFⅧ蛋白的细胞株.结果:经限制性酶切和测序鉴定均证实重组真核表达质粒pcDNA4/v5-his-BDDhFⅧ成功构建,在转染HepG2细胞后,Western blot检测证实人凝血因子Ⅷ可以在HepG2细胞中正确表达.结论:成功构建了人凝血因子Ⅷ的稳定细胞株,并能在HepG2细胞表达目的蛋白.

  20. Plasmid Profiles and Prevalence of Intermediately Virulent Rhodococcus equi from Pigs in Nakhonpathom Province, Thailand: Identification of a New Variant of the 70-kb Virulence Plasmid, Type 18

    Directory of Open Access Journals (Sweden)

    Chaithep Poolkhet

    2010-01-01

    Full Text Available The prevalence of intermediately virulent Rhodococcus equi isolates from pig submaxillary lymph nodes from four slaughterhouses in Nakhonpathom province, Thailand, was investigated. The isolates were tested for the presence of virulence plasmids and the 20-kDa virulence-associated protein antigen (VapB gene by PCR. Of the 734 submaxillary lymph nodes tested, 19 (2.6% produced positive cultures of R. equi. All 19 isolates were positive for the VapB gene and contained virulence plasmids that were identified as type 1 (six isolates, type 6 (two isolates, type 7 (one isolate, type 16 (two isolates, and a new variant (eight isolates. Based on the restriction digestion patterns of the plasmid DNAs, we tentatively designated the variant as type 18. Investigation of the prevalence and plasmid profiles of VapB-positive R. equi in pigs should be extended throughout Thailand to evaluate potential sources of zoonotic infections.

  1. The Plasmid Complement of the Cheese Isolate Lactococcus garvieae IPLA 31405 Revealed Adaptation to the Dairy Environment

    Science.gov (United States)

    Flórez, Ana Belén; Mayo, Baltasar

    2015-01-01

    Lactococcus garvieae is a lactic acid bacterium found in raw-milk dairy products as well as a range of aquatic and terrestrial environments. The plasmids in L. garvieae have received little attention compared to those of dairy Lactococcus lactis, in which the genes carried by these extrachromosomal elements are considered of adaptive value. The present work reports the sequencing and analysis of the plasmid complement of L. garvieae IPLA 31405, a strain isolated from a traditional, Spanish, starter-free cheese made from raw-milk. It consists of pLG9 and pLG42, of 9,124 and 42,240 nucleotides, respectively. Based on sequence and structural homology in the putative origin of replication (ori) region, pLG9 and pLG42 are predicted to replicate via a theta mechanism. Real-time, quantitative PCR showed the number of copies per chromosome equivalent of pLG9 and pLG42 to be around two and five, respectively. Sequence analysis identified eight complete open reading frames (orfs) in pLG9 and 36 in pLG42; these were organized into functional modules or cassettes containing different numbers of genes. These modules were flanked by complete or interrupted insertion sequence (IS)-like elements. Among the modules of pLG42 was a gene cluster encoding specific components of a phosphoenolpyruvate-phosphotransferase (PEP-PTS) system, including a phospho-β-galacosidase. The cluster showed a complete nucleotide identity respect to that in plasmids of L. lactis. Loss of pLG42 showed this to be involved in lactose assimilation. In the same plasmid, an operon encoding a type I restriction/modification (R/M) system was also identified. The specificity of this R/M system might be broadened by different R/M specificity subunits detected in pLG9 and in the bacterial chromosome. However, challenges of L. garvieae IPLA 31405 against L. lactis phages proved that the R/M system was not involved in phage resistance. Together, these results support the hypothesis that, as in L. lactis, pLG42

  2. Plasmid transfer systems in the rhizobia.

    Science.gov (United States)

    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.

  3. Plasmid Isolation in Legionella pneumophila and Legionella-like Organisms.

    Science.gov (United States)

    1980-08-22

    834. 14. Macrina, F. L., D. J. Kopecko, K. R. Jones, D. J. Ayers, and S. M. McCowen. 1978. A multiple plasmic-containing Escherichi coli strain...smaller 20 Mdal cryptic plasmid and was used as a control marker with the screening procedure. Escherichia coli V517 was supplied by E. M. Lederberg...Tris-borate buffer. This purified preparation was suitable for electrophoresis. Molecular weight estimates. Escherichia coli V517 was employed as an

  4. Characterization of the replication region of the Bacillus subtilis plasmid pLS20 : a novel type of replicon

    NARCIS (Netherlands)

    Meijer, WJJ; De Boer, AJ; van Tongeren, S; Venema, G; Bron, S

    1995-01-01

    A 3.1 kb fragment of the large (~55 kb) Bacillus subtilis plasmid pLS20 containing all the information for autonomous replication was cloned and sequenced. In contrast to the parental plasmid, derived minireplicons were unstably maintained. Using deletion analysis the fragment essential and sufficie

  5. Study on the construction of recombinant plasmid coexpressing newcastle disease virus F protein and chicken IL-2

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    This study investigated the protection against the ND in chickens by a recombinant DNA vaccine. A plasmid vector encoding NDV F protein, which is reqired for virus cell fusion and is important for vaccine induced immunity, was used as a model to study how DNA vaccines may be modulated by the simulaneous expression of chicken IL-2. The NDV D26 strain F gene with CMV promotor and BGH polyA signal sequence was amplified by PCR from eukaryotic plasmid pcDNA-F, which contains the full-length NDV F gene, and clond into reconstructed eukaryotic plasmid pcDNA-IL2, which contains chicken IL-2 gene. Restriction endonuclease cleavage and PCR amplification showed that a bicistronic plasmid encoding NDV F gene and chicken IL-2 separately was successfully constructed. Two-week-old SPF chickens were intramuscularly innoculated the recombinant plasmid. Antibody and lymphocyte proliferative assay showed that the humoral and cellular immunity of chickens vaccinated the recombinant plasmid greatly increased compared with those innoculated only plasmid expressing NDV F protein. Challenged with the lethal dose of NDV F48E9 strain, 72% chickens vaccinated recombinant plasmid were survived, and 30% chickens vaccinated plasmid expressing F protein were survived. These results proved the adjuvant effect of chicken IL-2, and further showed that the efficacy of a DNA vaccine can be greatly improved by simultaneous expression of IL-2.

  6. Comparative Sequence Analysis of Multidrug-Resistant IncA/C Plasmids from Salmonella enterica.

    Science.gov (United States)

    Hoffmann, Maria; Pettengill, James B; Gonzalez-Escalona, Narjol; Miller, John; Ayers, Sherry L; Zhao, Shaohua; Allard, Marc W; McDermott, Patrick F; Brown, Eric W; Monday, Steven R

    2017-01-01

    Determinants of multidrug resistance (MDR) are often encoded on mobile elements, such as plasmids, transposons, and integrons, which have the potential to transfer among foodborne pathogens, as well as to other virulent pathogens, increasing the threats these traits pose to human and veterinary health. Our understanding of MDR among Salmonella has been limited by the lack of closed plasmid genomes for comparisons across resistance phenotypes, due to difficulties in effectively separating the DNA of these high-molecular weight, low-copy-number plasmids from chromosomal DNA. To resolve this problem, we demonstrate an efficient protocol for isolating, sequencing and closing IncA/C plasmids from Salmonella sp. using single molecule real-time sequencing on a Pacific Biosciences (Pacbio) RS II Sequencer. We obtained six Salmonella enterica isolates from poultry, representing six different serovars, each exhibiting the MDR-Ampc resistance profile. Salmonella plasmids were obtained using a modified mini preparation and transformed with Escherichia coli DH10Br. A Qiagen Large-Construct kit™ was used to recover highly concentrated and purified plasmid DNA that was sequenced using PacBio technology. These six closed IncA/C plasmids ranged in size from 104 to 191 kb and shared a stable, conserved backbone containing 98 core genes, with only six differences among those core genes. The plasmids encoded a number of antimicrobial resistance genes, including those for quaternary ammonium compounds and mercury. We then compared our six IncA/C plasmid sequences: first with 14 IncA/C plasmids derived from S. enterica available at the National Center for Biotechnology Information (NCBI), and then with an additional 38 IncA/C plasmids derived from different taxa. These comparisons allowed us to build an evolutionary picture of how antimicrobial resistance may be mediated by this common plasmid backbone. Our project provides detailed genetic information about resistance genes in

  7. Selection of a multidrug resistance plasmid by sublethal levels of antibiotics and heavy metals.

    Science.gov (United States)

    Gullberg, Erik; Albrecht, Lisa M; Karlsson, Christoffer; Sandegren, Linus; Andersson, Dan I

    2014-10-07

    How sublethal levels of antibiotics and heavy metals select for clinically important multidrug resistance plasmids is largely unknown. Carriage of plasmids generally confers substantial fitness costs, implying that for the plasmid-carrying bacteria to be maintained in the population, the plasmid cost needs to be balanced by a selective pressure conferred by, for example, antibiotics or heavy metals. We studied the effects of low levels of antibiotics and heavy metals on the selective maintenance of a 220-kbp extended-spectrum β-lactamase (ESBL) plasmid identified in a hospital outbreak of Klebsiella pneumoniae and Escherichia coli. The concentrations of antibiotics and heavy metals required to maintain plasmid-carrying bacteria, the minimal selective concentrations (MSCs), were in all cases below (almost up to 140-fold) the MIC of the plasmid-free susceptible bacteria. This finding indicates that the very low antibiotic and heavy metal levels found in polluted environments and in treated humans and animals might be sufficiently high to maintain multiresistance plasmids. When resistance genes were moved from the plasmid to the chromosome, the MSC decreased, showing that MSC for a specific resistance conditionally depends on genetic context. This finding suggests that a cost-free resistance could be maintained in a population by an infinitesimally low concentration of antibiotic. By studying the effect of combinations of several compounds, it was observed that for certain combinations of drugs each new compound added lowered the minimal selective concentration of the others. This combination effect could be a significant factor in the selection of multidrug resistance plasmids/bacterial clones in complex multidrug environments. Importance: Antibiotic resistance is in many pathogenic bacteria caused by genes that are carried on large conjugative plasmids. These plasmids typically contain multiple antibiotic resistance genes as well as genes that confer resistance to

  8. A novel multidrug resistance plasmid isolated from an Escherichia coli strain resistant to aminoglycosides.

    Science.gov (United States)

    Sun, Hui; Li, Shasha; Xie, Zhijing; Yang, Fangfang; Sun, Yani; Zhu, Yanli; Zhao, Xiaomin; Jiang, Shijin

    2012-07-01

    Previous studies have reported several different plasmids that confer multidrug resistance (MDR) including resistance to aminoglycosides. In this study, we investigated the aminoglycoside resistance patterns for 224 Escherichia coli isolates from diseased chickens and ducks in China, characterized a novel MDR plasmid, and collected prevalence data on similar resistance plasmids. Antibiotic susceptibilities were determined using disc diffusion and the microdilution method. The plasmid pXZ was analysed by restriction fragment length polymorphism (RFLP) with EcoRI and SalI, and sequenced. The prevalence of similar resistance plasmids was assessed by multiplex PCR and by RFLP analysis. Among the 224 E. coli isolates, 189 (84.4%) were resistant to streptomycin, 125 (55.8%) were resistant to kanamycin, 116 (51.8%) were resistant to gentamicin, 106 (47.3%) were resistant to neomycin and 98 (43.8%) were resistant to amikacin. Among the 224 E. coli isolates, 17 contained a plasmid with the MDR-encoding region of pXZ, which showed high-level resistance to aminoglycosides (MICs of gentamicin and amikacin ≥ 512 mg/L). The plasmid pXZ was digested into five fragments by EcoRI and six fragments by SalI. The plasmid pXZ was a circular DNA molecule of 76635 bp with a 51.65% guanine + cytosine content and included four resistance genes (rmtB, fosA3, bla(TEM-1) and bla(CTX-M-24)). A novel MDR plasmid, pXZ, harbouring four resistance genes (rmtB, fosA3, bla(TEM-1) and bla(CTX-M)) was identified. To our knowledge, this is the first report of an aminoglycoside resistance plasmid harbouring the fosA3 gene.

  9. Field homology: a meaningful definition.

    Science.gov (United States)

    Cookson, K

    2001-02-01

    Field homology refers to populations of cells that derive from evolutionarily conserved regions of embryos but are distributed across sets of adult morphological structures that cannot be placed in one-to-one correspondance. The concept of field homology has proven especially attractive to comparative neurologists because it allows them to deal with the fact that sets of nuclei or nuclear subdivisions often cannot be compared on a one-to-one basis across phyletic groups. However, the concept of field homology has recently come under criticism. It has been argued that field homology is theoretically impossible because it requires sequences of developmental stages to be both evolutionarily conserved and evolutionarily modified. It has also been argued that field homology allows overly vague comparisons of adult morphological structures, fails to account for homologous structures that derive from non-homologous embryonic sources, and establishes overly rigid links between embryonic and adult morphology. All of these criticisms may be adequately addressed by explaining field homology in terms of differentiation. The present paper explains field homology in terms of differentiation using the amniote dorsal thalamus to illustrate major points. It is concluded that field homology is a meaningful concept when defined in terms of differentiation, applied to appropriate cases, and properly limited in its comparisons of adult structures.

  10. Rhythmical bimanual force production: homologous and non-homologous muscles.

    Science.gov (United States)

    Kennedy, Deanna M; Boyle, Jason B; Rhee, Joohyun; Shea, Charles H

    2015-01-01

    The experiment was designed to determine participants' ability to coordinate a bimanual multifrequency pattern of isometric forces using homologous or non-homologous muscles. Lissajous feedback was provided to reduce perceptual and attentional constraints. The primary purpose was to determine whether the activation of homologous and non-homologous muscles resulted in different patterns of distortions in the left limb forces that are related to the forces produced by the right limb. The task was to rhythmically produce a 1:2 pattern of isometric forces by exerting isometric forces on the left side force transducer with the left arm that was coordinated with the pattern of isometric forces produced on the right side force transducer with the right arm. The results indicated that participants were able to 'tune-in' a 1:2 coordination patterns using homologous (triceps muscles of the left and right limbs) and using non-homologous muscles (biceps left limb and triceps right limb) when provided Lissajous feedback. However, distinct but consistent and identifiable distortions in the left limb force traces were observed for both the homologous and non-homologous tasks. For the homologous task, the interference occurred in the left limb when the right limb was initiating and releasing force. For the non-homologous task, the interference in the left limb force occurred only when the right limb was releasing force. In both conditions, the interference appeared to continue from the point of force initiation and/or release to peak force velocity. The overall results are consistent with the notion that neural crosstalk manifests differently during the coordination of the limbs depending upon whether homologous or non-homologous muscles are activated.

  11. Exploring PFGE for Detecting Large Plasmids in Campylobacter jejuni and Campylobacter coli Isolated from Various Retail Meats

    Directory of Open Access Journals (Sweden)

    Daya Marasini

    2014-10-01

    Full Text Available Campylobacter spp. is one of the most prevalent bacterial pathogens in retail meat, particularly poultry, and is a leading cause of diarrhea in humans. Studies related to Campylobacter large plasmids are limited in the literature possibly due to difficulty in isolating them using available alkaline lysis methods. The objectives of this study were to determine the prevalence of plasmids, particularly large ones, in Campylobacter spp. isolated from various Oklahoma retail meats, and to explore PFGE (Pulsed Field Gel Electrophoresis as a tool in facilitating the detection of these plasmids. One hundred and eighty nine strains (94 Campylobacter jejuni and 95 Campylobacter coli were screened for the presence of plasmids using both alkaline lysis and PFGE. Plasmids were detected in 119/189 (63% using both methods. Most of the plasmids detected by alkaline lysis were smaller than 90 kb and only three were larger than 90 kb. Plasmids over 70 kb in size were detected in 33 more strains by PFGE of which 11 strains contained larger than 90 kb plasmids. Plasmids were more prevalent in Campylobacter coli (73.5% than in Campylobacter jejuni (52%. BglII restriction analysis of plasmids isolated from 102 isolates revealed 42 different restriction patterns. In conclusion, PFGE was able to detect large plasmids up to 180 Kb in Campylobacter spp. which might have been missed if the alkaline lysis method was solely used. Campylobacter spp. isolated from retail meats harbor a diverse population of plasmids with variable sizes. To our knowledge, this is the first study to use PFGE to detect large plasmids in Campylobacter.

  12. Complete genetic analysis of plasmids carrying mcr-1 and other resistance genes in an Escherichia coli isolate of animal origin.

    Science.gov (United States)

    Li, Ruichao; Xie, Miaomiao; Lv, Jingzhang; Wai-Chi Chan, Edward; Chen, Sheng

    2017-03-01

    To investigate the genetic features of three plasmids recovered from an MCR-1 and ESBL-producing Escherichia coli strain, HYEC7, and characterize the transmission mechanism of mcr-1 . The genetic profiles of three plasmids were determined by PCR, S1-PFGE, Southern hybridization and WGS analysis. The ability of the mcr-1 -bearing plasmid to undergo conjugation was also assessed. The mcr-1 -bearing transposon Tn 6330 was characterized by PCR and DNA sequencing. Complete sequences of three plasmids were obtained. A non-conjugative phage P7-like plasmid, pHYEC7- mcr1 , was found to harbour the mcr-1 -bearing transposon Tn 6330 , which could be excised from the plasmid by generating a circular intermediate harbouring mcr-1 and the IS Apl1 element. The insertion of the circular intermediate into another plasmid, pHYEC7-IncHI2, could form pHNSHP45-2, the original IncHI2-type mcr-1 -carrying plasmid that was reported. The third plasmid, pHYEC7-110, harboured two replicons, IncX1 and IncFIB, and comprised multiple antimicrobial resistance mobile elements, some of which were shared by pHYEC7-IncHI2. The Tn 6330 element located in the phage-like plasmid pHYEC7- mcr1 could be excised from the plasmid and formed a circular intermediate that could be integrated into plasmids containing the IS Apl1 element. This phenomenon indicated that Tn 6330 is a key element responsible for widespread dissemination of mcr-1 among various types of plasmids and bacterial chromosomes. The dissemination rate of such an element may be further enhanced upon translocation into phage-like vectors, which may also be transmitted via transduction events.

  13. Homology, homoplasy, novelty, and behavior.

    Science.gov (United States)

    Hall, Brian K

    2013-01-01

    Richard Owen coined the modern definition of homology in 1843. Owen's conception of homology was pre-evolutionary, nontransformative (homology maintained basic plans or archetypes), and applied to the fully formed structures of animals. I sketch out the transition to an evolutionary approach to homology in which all classes of similarity are interpreted against the single branching tree of life, and outline the evidence for the application of homology across all levels and features of the biological hierarchy, including behavior. Owen contrasted homology with analogy. While this is not incorrect it is a pre-evolutionary contrast. Lankester [Lankester [1870] Journal of Natural History, 6 (31), 34-43] proposed homoplasy as the class of homology applicable to features formed by independent evolution. Today we identify homology, convergence, parallelism, and novelties as patterns of evolutionary change. A central issue in homology [Owen [1843] Lectures on comparative anatomy and physiology of the invertebrate animals, delivered at the Royal College of Surgeons in 1843. London: Longman, Brown, Green & Longmans] has been whether homology of features-the "same" portion of the brain in different species, for example-depends upon those features sharing common developmental pathways. Owen did not require this criterion, although he observed that homologues often do share developmental pathways (and we now know, often share gene pathways). A similar situation has been explored in the study of behavior, especially whether behaviors must share a common structural, developmental, neural, or genetic basis to be classified as homologous. However, and importantly, development and genes evolve. As shown with both theory and examples, morphological and behavioral features of the phenotype can be homologized as structural or behavioral homologues, respectively, even when their developmental or genetic bases differ (are not homologous). Copyright © 2012 Wiley Periodicals, Inc.

  14. A Phage-Like IncY Plasmid Carrying the mcr-1 Gene in Escherichia coli from a Pig Farm in China.

    Science.gov (United States)

    Zhang, Chunping; Feng, Yuqing; Liu, Fei; Jiang, Hui; Qu, Zhina; Lei, Meng; Wang, Jianfeng; Zhang, Bing; Hu, Yongfei; Ding, Jiabo; Zhu, Baoli

    2017-03-01

    We report here a new type of plasmid that carries the mcr-1 gene, the pMCR-1-P3 plasmid, harbored in an Escherichia coli strain isolated from a pig farm in China. pMCR-1-P3 belongs to the IncY incompatibility group and is a phage-like plasmid that contains a large portion of phage-related sequences. The backbone of this plasmid is different from that of other mcr-1-carrying plasmids reported previously. Copyright © 2017 American Society for Microbiology.

  15. Nascent DNA synthesis during homologous recombination is synergistically promoted by the rad51 recombinase and DNA homology.

    Science.gov (United States)

    Mundia, Maureen M; Desai, Vatsal; Magwood, Alissa C; Baker, Mark D

    2014-05-01

    In this study, we exploited a plasmid-based assay that detects the new DNA synthesis (3' extension) that accompanies Rad51-mediated homology searching and strand invasion steps of homologous recombination to investigate the interplay between Rad51 concentration and homology length. Mouse hybridoma cells that express endogenous levels of Rad51 display an approximate linear increase in the frequency of 3' extension for homology lengths of 500 bp to 2 kb. At values below ∼500 bp, the frequency of 3' extension declines markedly, suggesting that this might represent the minimal efficient processing segment for 3' extension. Overexpression of wild-type Rad51 stimulated the frequency of 3' extension by ∼3-fold for homology lengths homology was >2 kb, 3' extension frequency increased by as much as 10-fold. Excess wild-type Rad51 did not increase the average 3' extension tract length. Analysis of cell lines expressing N-terminally FLAG-tagged Rad51 polymerization mutants F86E, A89E, or F86E/A89E established that the 3' extension process requires Rad51 polymerization activity. Mouse hybridoma cells that have reduced Brca2 (Breast cancer susceptibility 2) due to stable expression of small interfering RNA show a significant reduction in 3' extension efficiency; expression of wild-type human BRCA2, but not a BRCA2 variant devoid of BRC repeats 1-8, rescues the 3' extension defect in these cells. Our results suggest that increased Rad51 concentration and homology length interact synergistically to promote 3' extension, presumably as a result of enhanced Brca2-mediated Rad51 polymerization.

  16. Trans activation of plasmid-borne promoters by adenovirus and several herpes group viruses.

    OpenAIRE

    Everett, R D; Dunlop, M

    1984-01-01

    This paper describes experiments to test the ability of a number of viruses of the Herpes group, and also Adenovirus-2 and SV40, to activate transcription from the Herpes simplex virus-1 glycoprotein D and the rabbit beta-globin promoters. Plasmids containing these genes were transfected into HeLa cells which were then infected with various viruses. Transcriptional activation in trans of the plasmid-borne promoters was monitored by quantitative S1 nuclease analysis of total cytoplasmic RNA is...

  17. Plasmid loss and changes within the chromosomal DNA of Streptomyces reticuli.

    OpenAIRE

    Schrempf, H

    1982-01-01

    The sporulating wild-type strain of Streptomyces reticuli, which produces a melanin pigment and the macrolide leucomycin, contains plasmid DNA of 48 to 49 megadaltons. Plasmidless variants had an altered secondary metabolism and a changed antibiotic resistance pattern. By using a new colony hybridization technique developed for streptomycetes, it could be shown that plasmidless variants could be transformed with the wild-type plasmid DNA, which, however, is quickly lost from regenerated mycel...

  18. Transposition of Tn5096 from a temperature-sensitive transducible plasmid in Streptomyces spp.

    Science.gov (United States)

    McHenney, M A; Baltz, R H

    1991-09-01

    Transposon Tn5096 was inserted into a derivative of the temperature-sensitive plasmid pMT660 containing the bacteriophage FP43 pac site. The resulting plasmid, pRHB126, was transduced by FP43 into several Streptomyces species. Tn5096 transposed from pRHB126 into different sites in the genomes of Streptomyces ambofaciens, Streptomyces cinnamonensis, Streptomyces coelicolor A3(2), Streptomyces fradiae, Streptomyces griseofuscus, and Streptomyces thermotolerans.

  19. Transposition of Tn5096 from a temperature-sensitive transducible plasmid in Streptomyces spp.

    OpenAIRE

    McHenney, M A; Baltz, R H

    1991-01-01

    Transposon Tn5096 was inserted into a derivative of the temperature-sensitive plasmid pMT660 containing the bacteriophage FP43 pac site. The resulting plasmid, pRHB126, was transduced by FP43 into several Streptomyces species. Tn5096 transposed from pRHB126 into different sites in the genomes of Streptomyces ambofaciens, Streptomyces cinnamonensis, Streptomyces coelicolor A3(2), Streptomyces fradiae, Streptomyces griseofuscus, and Streptomyces thermotolerans.

  20. Computational Homology for Software Validation

    Science.gov (United States)

    2015-03-01

    COMPUTATIONAL HOMOLOGY FOR SOFTWARE VALIDATION SYRACUSE UNIVERSITY MARCH 2015 FINAL TECHNICAL REPORT APPROVED FOR PUBLIC RELEASE; DISTRIBUTION...COVERED (From - To) SEP 2011 – SEP 2014 4. TITLE AND SUBTITLE COMPUTATIONAL HOMOLOGY FOR SOFTWARE VALIDATION 5a. CONTRACT NUMBER FA8750-11-2-0275 5b...verdict as to whether the application of persistent homology to the problem of obtaining objective signatures that would indicate the present or absence

  1. Grid diagrams and Khovanov homology

    DEFF Research Database (Denmark)

    Droz, Jean-Marie; Wagner, Emmanuel

    2009-01-01

    We explain how to compute the Jones polynomial of a link from one of its grid diagrams and we observe a connection between Bigelow’s homological definition of the Jones polynomial and Kauffman’s definition of the Jones polynomial. Consequently, we prove that the Maslov grading on the Seidel......–Smith symplectic link invariant coincides with the difference between the homological grading on Khovanov homology and the Jones grading on Khovanov homology. We give some evidence for the truth of the Seidel–Smith conjecture....

  2. Exploration and exploitation of homologous series of bis(acrylamidoalkanes containing pyridyl and phenyl groups: β-sheet versus two-dimensional layers in solid-state photochemical [2 + 2] reactions

    Directory of Open Access Journals (Sweden)

    Mousumi Garai

    2015-09-01

    Full Text Available The homologous series of phenyl and pyridyl substituted bis(acrylamidoalkanes have been synthesized with the aim of systematic analysis of their crystal structures and their solid-state [2 + 2] reactivities. The changes in the crystal structures with respect to a small change in the molecular structure, that is by varying alkyl spacers between acrylamides and/or by varying the end groups (phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl on the C-terminal of the amide, were analyzed in terms of hydrogen-bonding interference (N—H...Npy versus N—H...O=C and network geometries. In this series, a greater tendency towards the formation of N—H...O hydrogen bonds (β-sheets and two-dimensional networks over N—H...N hydrogen bonds was observed. Among all the structures seven structures were found to have the required alignments of double bonds for the [2 + 2] reaction such that the formations of single dimer, double dimer and polymer are facilitated. However, only four structures were found to exhibit such a solid-state [2 + 2] reaction to form a single dimer and polymers. The two-dimensional hydrogen-bonding layer via N—H...O hydrogen bonds was found to promote solid-state [2 + 2] photo-polymerization in a single-crystal-to-single-crystal manner. Such two-dimensional layers were encountered only when the spacer between acryl amide moieties is butyl. Only four out of the 16 derivatives were found to form hydrates, two each from 2-pyridyl and 4-pyridyl derivatives. The water molecules in these structures govern the hydrogen-bonding networks by the formation of an octameric water cluster and one-dimensional zigzag water chains. The trends in the melting points and densities were also analyzed.

  3. Homological stability for unordered configuration spaces

    DEFF Research Database (Denmark)

    Randal-Williams, Oscar

    2013-01-01

    This paper consists of two related parts. In the first part we give a self-contained proof of homological stability for the spaces C_n(M;X) of configurations of n unordered points in a connected open manifold M with labels in a path-connected space X, with the best possible integral stability range...... of 2* \\leq n. Along the way we give a new proof of the high connectivity of the complex of injective words. If the manifold has dimension at least three, we show that in rational homology the stability range may be improved to * \\leq n. In the second part we study to what extent the homology...... of the spaces C_n(M) can be considered stable when M is a closed manifold. In this case there are no stabilisation maps, but one may still ask if the dimensions of the homology groups over some field stabilise with n. We prove that this is true when M is odd-dimensional, or when the field is F_2 or Q...

  4. Plasmid-Mediated Quinolone Resistance (PMQR) Genes and Class 1 Integrons in Quinolone-Resistant Marine Bacteria and Clinical Isolates of Escherichia coli from an Aquacultural Area.

    Science.gov (United States)

    Tomova, Alexandra; Ivanova, Larisa; Buschmann, Alejandro H; Godfrey, Henry P; Cabello, Felipe C

    2017-06-23

    Antimicrobial usage in aquaculture selects for antimicrobial-resistant microorganisms in the marine environment. The relevance of this selection to terrestrial animal and human health is unclear. Quinolone-resistance genes qnrA, qnrB, and qnrS were chromosomally located in four randomly chosen quinolone-resistant marine bacteria isolated from an aquacultural area with heavy quinolone usage. In quinolone-resistant uropathogenic clinical isolates of Escherichia coli from a coastal area bordering the same aquacultural region, qnrA was chromosomally located in two E. coli isolates, while qnrB and qnrS were located in small molecular weight plasmids in two other E. coli isolates. Three quinolone-resistant marine bacteria and three quinolone-resistant E. coli contained class 1 integrons but without physical association with PMQR genes. In both marine bacteria and uropathogenic E. coli, class 1 integrons had similar co-linear structures, identical gene cassettes, and similarities in their flanking regions. In a Marinobacter sp. marine isolate and in one E. coli clinical isolate, sequences immediately upstream of the qnrS gene were homologous to comparable sequences of numerous plasmid-located qnrS genes while downstream sequences were different. The observed commonality of quinolone resistance genes and integrons suggests that aquacultural use of antimicrobials might facilitate horizontal gene transfer between bacteria in diverse ecological locations.

  5. Effect of low temperature on stability of theta-type plasmids in Carnobacterium maltaromaticum.

    Science.gov (United States)

    Bohaychuk, Valerie M; van Belkum, Marco J; Stiles, Michael E; McMullen, Lynn M

    2008-03-01

    The heterologous production of useful peptides such as bacteriocins by lactic acid bacteria (LAB) has been studied for use in the biopreservation of foods. Recombinant plasmids can suffer drawbacks such as segregational instability affecting the production of these peptides in certain environments such as absence of selective pressure or low temperature. The link between growth temperature characteristics of parental strains and stability of theta-type plasmids at a low temperature was investigated. The growth of four parental strains at 4 degrees C and stability of five derivative theta-type plasmids transformed into Carnobacterium maltaromaticum UAL26 at 25 and 4 degrees C were determined. Two plasmids (pCD11 and pCaT) derived from psychrotrophic LAB and plasmid, pHW800, from Enterococcus faecium 226 with unknown growth temperature characteristics, had excellent stability when strains were grown at 4 degrees C. Plasmids (pTRKH2 and pUCB820) derived from LAB that did not grow at refrigeration temperatures were not stable at 4 degrees C. When a DNA fragment from pCD11 containing 22-bp repeats, a putative replication initiation site, and the gene for the RepA protein was inserted into pTRKH2, the resulting derivative plasmid was 100% stable at 4 degrees C.

  6. Use of FabV-Triclosan Plasmid Selection System for Efficient Expression and Production of Recombinant Proteins in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Syed A Ali

    Full Text Available Maintenance of recombinant plasmid vectors in host bacteria relies on the presence of selection antibiotics in the growth media to suppress plasmid -free segregants. However, presence of antibiotic resistance genes and antibiotics themselves is not acceptable in several applications of biotechnology. Previously, we have shown that FabV-Triclosan selection system can be used to select high and medium copy number plasmid vectors in E. coli. Here, we have extended our previous work and demonstrated that expression vectors containing FabV can be used efficiently to express heterologous recombinant proteins in similar or better amounts in E. coli host when compared with expression vectors containing β-lactamase. Use of small amount of non-antibiotic Triclosan as selection agent in growth medium, enhanced plasmid stability, applicability in various culture media, and compatibility with other selection systems for multiple plasmid maintenance are noteworthy features of FabV-Triclosan selection system.

  7. Remarks on Khovanov Homology and the Potts Model

    CERN Document Server

    Kauffman, Louis H

    2009-01-01

    This paper is about Khovanov homology and its relationships with statistical mechanics models such as the Ising model and the Potts model. The paper gives a relatively self-contained introduction to Khovanov homology, and also to a reformulation of the Potts model in terms of a bracket state sum expansion on a knot diagram K(G) related to a planar graph G via the medial construction. We consider the original Khovanov homology and also the homology defined by Stosic via the dichromatic polynomial, and examine those values of the Potts model where the partition function can be expressed in terms of homological Euler characteristics. These points occur at imaginary temperature, and consequences of this phenomenon will be studied in subsequent work. This paper is dedicated to Oleg Viro on his 60-th birthday.

  8. Replisome Assembly at Bacterial Chromosomes and Iteron Plasmids

    Directory of Open Access Journals (Sweden)

    Katarzyna Ewa Wegrzyn

    2016-08-01

    Full Text Available 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.

  9. Plasmids and packaging cell lines for use in phage display

    Science.gov (United States)

    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. Polymerase chain reaction-based gene removal from plasmids

    Directory of Open Access Journals (Sweden)

    Vishnu Vardhan Krishnamurthy

    2015-09-01

    Full Text Available This data article contains supplementary figures and methods to the research article entitled, “Multiplex gene removal by two-step polymerase chain reactions” (Krishnamurthy et al., Anal. Biochem., 2015, doi:http://dx.doi.org/10.1016/j.ab.2015.03.033, which presents a restriction-enzyme free method to remove multiple DNA segments from plasmids. Restriction-free cloning methods have dramatically improved the flexibility and speed of genetic manipulation compared to conventional assays based on restriction enzyme digestion (Lale and Valla, 2014. DNA Cloning and Assembly Methods, vol. 1116. Here, we show the basic scheme and characterize the success rate for single and multiplex gene removal from plasmids. In addition, we optimize experimental conditions, including the amount of template, multiple primers mixing, and buffers for DpnI treatment, used in the one-pot reaction for multiplex gene removal.

  11. Replication efficiency of rolling-circle replicon-based plasmids derived from porcine circovirus 2 in eukaryotic cells.

    Science.gov (United States)

    Faurez, Florence; Dory, Daniel; Henry, Aurélie; Bougeard, Stéphanie; Jestin, André

    2010-04-01

    In this study, a method was developed to measure replication rates of rolling-circle replicon-based plasmids in eukaryotic cells. This method is based on the discriminative quantitation of MboI-resistant, non-replicated input plasmids and DpnI-resistant, replicated plasmids. To do so, porcine circovirus type 2 (PCV2) replicon-based plasmids were constructed. These plasmids contained the PCV2 origin of replication, the PCV2 Rep promoter and the PCV2 Rep gene. The results show that the replication rate depends on the length of the PCV2 replicon-based plasmid and not on the respective position of the Rep promoter and the promoter of the gene of interest that encodes the enhanced green fluorescent protein (eGFP). In all cases, it was necessary to add the Rep gene encoded by a plasmid and cotransfected as a replication booster. This method can evaluate the replication potential of replicon-based plasmids quickly and is thereby a promising tool for the development of plasmids for vaccine purposes.

  12. Relative Homological Algebra Volume 1

    CERN Document Server

    2011-01-01

    This is the second revised edition of an introduction to contemporary relative homological algebra. It supplies important material essential to understand topics in algebra, algebraic geometry and algebraic topology. Each section comes with exercises providing practice problems for students as well as additional important results for specialists. The book is also suitable for an introductory course in commutative and ordinary homological algebra.

  13. Evolving the Concept of Homology

    Science.gov (United States)

    Naples, Virginia L.; Miller, Jon S.

    2009-01-01

    Understanding homology is fundamental to learning about evolution. The present study shows an exercise that can be varied in complexity, for which students compile research illustrating the fate of homologous fish skull elements, and assemble a mural to serve as a learning aid. The skull of the most primitive living Actinopterygian (bony fish),…

  14. Genomic comparison of Escherichia coli O104:H4 isolates from 2009 and 2011 reveals plasmid, and prophage heterogeneity, including shiga toxin encoding phage stx2.

    Directory of Open Access Journals (Sweden)

    Sanaa A Ahmed

    Full Text Available In May of 2011, an enteroaggregative Escherichia coli O104:H4 strain that had acquired a Shiga toxin 2-converting phage caused a large outbreak of bloody diarrhea in Europe which was notable for its high prevalence of hemolytic uremic syndrome cases. Several studies have described the genomic inventory and phylogenies of strains associated with the outbreak and a collection of historical E. coli O104:H4 isolates using draft genome assemblies. We present the complete, closed genome sequences of an isolate from the 2011 outbreak (2011C-3493 and two isolates from cases of bloody diarrhea that occurred in the Republic of Georgia in 2009 (2009EL-2050 and 2009EL-2071. Comparative genome analysis indicates that, while the Georgian strains are the nearest neighbors to the 2011 outbreak isolates sequenced to date, structural and nucleotide-level differences are evident in the Stx2 phage genomes, the mer/tet antibiotic resistance island, and in the prophage and plasmid profiles of the strains, including a previously undescribed plasmid with homology to the pMT virulence plasmid of Yersinia pestis. In addition, multiphenotype analysis showed that 2009EL-2071 possessed higher resistance to polymyxin and membrane-disrupting agents. Finally, we show evidence by electron microscopy of the presence of a common phage morphotype among the European and Georgian strains and a second phage morphotype among the Georgian strains. The presence of at least two stx2 phage genotypes in host genetic backgrounds that may derive from a recent common ancestor of the 2011 outbreak isolates indicates that the emergence of stx2 phage-containing E. coli O104:H4 strains probably occurred more than once, or that the current outbreak isolates may be the result of a recent transfer of a new stx2 phage element into a pre-existing stx2-positive genetic background.

  15. Paracoccus denitrificans possesses two BioR homologs having a role in regulation of biotin metabolism.

    Science.gov (United States)

    Feng, Youjun; Kumar, Ritesh; Ravcheev, Dmitry A; Zhang, Huimin

    2015-08-01

    Recently, we determined that BioR, the GntR family of transcription factor, acts as a repressor for biotin metabolism exclusively distributed in certain species of α-proteobacteria, including the zoonotic agent Brucella melitensis and the plant pathogen Agrobacterium tumefaciens. However, the scenario is unusual in Paracoccus denitrificans, another closely related member of the same phylum α-proteobacteria featuring with denitrification. Not only does it encode two BioR homologs Pden_1431 and Pden_2922 (designated as BioR1 and BioR2, respectively), but also has six predictive BioR-recognizable sites (the two bioR homolog each has one site, whereas the two bio operons (bioBFDAGC and bioYB) each contains two tandem BioR boxes). It raised the possibility that unexpected complexity is present in BioR-mediated biotin regulation. Here we report that this is the case. The identity of the purified BioR proteins (BioR1 and BioR2) was confirmed with LC-QToF-MS. Phylogenetic analyses combined with GC percentage raised a possibility that the bioR2 gene might be acquired by horizontal gene transfer. Gel shift assays revealed that the predicted BioR-binding sites are functional for the two BioR homologs, in much similarity to the scenario seen with the BioR site of A. tumefaciens bioBFDAZ. Using the A. tumefaciens reporter system carrying a plasmid-borne LacZ fusion, we revealed that the two homologs of P. denitrificans BioR are functional repressors for biotin metabolism. As anticipated, not only does the addition of exogenous biotin stimulate efficiently the expression of bioYB operon encoding biotin transport/uptake system BioY, but also inhibits the transcription of the bioBFDAGC operon resembling the de novo biotin synthetic pathway. EMSA-based screening failed to demonstrate that the biotin-related metabolite is involved in BioR-DNA interplay, which is consistent with our former observation with Brucella BioR. Our finding defined a complex regulatory network for biotin

  16. HomologMiner: looking for homologous genomic groups in whole genomes.

    Science.gov (United States)

    Hou, Minmei; Berman, Piotr; Hsu, Chih-Hao; Harris, Robert S

    2007-04-15

    Complex genomes contain numerous repeated sequences, and genomic duplication is believed to be a main evolutionary mechanism to obtain new functions. Several tools are available for de novo repeat sequence identification, and many approaches exist for clustering homologous protein sequences. We present an efficient new approach to identify and cluster homologous DNA sequences with high accuracy at the level of whole genomes, excluding low-complexity repeats, tandem repeats and annotated interspersed repeats. We also determine the boundaries of each group member so that it closely represents a biological unit, e.g. a complete gene, or a partial gene coding a protein domain. We developed a program called HomologMiner to identify homologous groups applicable to genome sequences that have been properly marked for low-complexity repeats and annotated interspersed repeats. We applied it to the whole genomes of human (hg17), macaque (rheMac2) and mouse (mm8). Groups obtained include gene families (e.g. olfactory receptor gene family, zinc finger families), unannotated interspersed repeats and additional homologous groups that resulted from recent segmental duplications. Our program incorporates several new methods: a new abstract definition of consistent duplicate units, a new criterion to remove moderately frequent tandem repeats, and new algorithmic techniques. We also provide preliminary analysis of the output on the three genomes mentioned above, and show several applications including identifying boundaries of tandem gene clusters and novel interspersed repeat families. All programs and datasets are downloadable from www.bx.psu.edu/miller_lab.

  17. Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway

    DEFF Research Database (Denmark)

    Lee, Jae Seong; Beuchert Kallehauge, Thomas; Pedersen, Lasse Ebdrup

    2015-01-01

    gene integration into site-specific loci in CHO cells using CRISPR/Cas9 genome editing system and compatible donor plasmid harboring a gene of interest (GOI) and short homology arms. This strategy has enabled precise insertion of a 3.7-kb gene expression cassette at defined loci in CHO cells following...

  18. Hydrocarbon mineralization in sediments and plasmid incidence in sediment bacteria from the campeche bank.

    Science.gov (United States)

    Leahy, J G; Somerville, C C; Cunningham, K A; Adamantiades, G A; Byrd, J J; Colwell, R R

    1990-06-01

    Rates of degradation of radiolabeled hydrocarbons and incidence of bacterial plasmid DNA were investigated in sediment samples collected from the Campeche Bank, Gulf of Mexico, site of an offshore oil field containing several petroleum platforms. Overall rates of mineralization of [C]hexadecane and [C]phenanthrene measured for sediments were negligible; <1% of the substrate was converted to CO(2) in all cases. Low mineralization rates are ascribed to nutrient limitations and to lack of adaptation by microbial communities to hydrocarbon contaminants. Plasmid frequency data for sediment bacteria similarly showed no correlation with proximity to the oil field, but, instead, showed correlation with water column depth at each sampling site. Significant differences between sites were observed for proportion of isolates carrying single or multiple plasmids and mean number of plasmids per isolate, each of which increased as a function of depth.

  19. Comparative investigations of Klebsiella species of clinical origin: plasmid patterns, biochemical reactions, antibiotic resistances and serotypes.

    Science.gov (United States)

    Podschun, R; Heineken, P; Ullmann, U; Sonntag, H G

    1986-09-01

    A total of 124 K. pneumoniae and 52 K. oxytoca isolates obtained from clinical specimens was investigated for plasmid patterns, biochemical reactions, antibiotic resistances and serotypes regarding to the distribution and relationships of these characters. A great diversity of plasmid patterns, bio/serotypes and resistance patterns was revealed. About 90% of strains contained plasmid DNA and up to seven plasmid bands per isolate could be shown. For K. pneumoniae, serotype 7 and for K. oxytoca, type 55 were most common. In general, little difference between both species was found and characters were similarly distributed. With respect to the site of isolation, serotype 7 was predominating in K. pneumoniae strains from the respiratory tract. Highly multiple-resistant organism were found in the largest number in specimens from the urogenital tract, in the lowest in specimens from wounds. Extensive statistical analyses did not detect any relationship among the characters investigated.

  20. Rapid isolation of plasmid DNA by LiCl-ethidium bromide treatment and gel filtration.

    Science.gov (United States)

    Kondo, T; Mukai, M; Kondo, Y

    1991-10-01

    We established a simple and rapid plasmid DNA purification method. Crude plasmid DNA preparations are treated with 4 M LiCl in the presence of 0.6 mg/ml ethidium bromide to precipitate RNA and proteins contained in the DNA preparations. After removal of RNA and protein precipitates, the supernatant is filtered through a Sepharose CL6B column to remove low-molecular-weight contaminants. This procedure takes only 30 min and provides pure plasmid DNA preparations that consist mainly of covalently closed circular plasmid DNA but have no detectable RNA and protein. The purified DNA preparations are susceptible to various six- and four-base-recognition restriction endonucleases, T4 DNA ligase, the Klenow fragment of DNA polymerase I, and T7 and Taq DNA polymerase. Since no special equipment is needed for this purification method, 20 or more samples of microgram to milligram levels can be treated in parallel.

  1. Plasmid-mediated colistin resistance in Escherichia coli from the Arabian Peninsula.

    Science.gov (United States)

    Sonnevend, Ágnes; Ghazawi, Akela; Alqahtani, Manaf; Shibl, Atef; Jamal, Wafa; Hashmey, Rayhan; Pal, Tibor

    2016-09-01

    Searching for the presence of the mcr-1 gene in colistin resistant Enterobacteriaceae in countries of the Arabian Peninsula. Seventy-five independent, colistin resistant Enterobacteriaceae strains isolated from clinical cases in Bahrain, Kuwait, Oman, Saudi Arabia and the United Arab Emirates were tested by PCR for the mcr-1 gene. mcr-1 positive strains were genotyped, and their antibiotic susceptibility was established. The mcr-1 containing plasmids were mobilized into Escherichia coli K-12 and their sequence was determined. Four E. coli isolates (two from Bahrain, one from Saudi Arabia and one from the United Arab Emirates) were identified carrying the mcr-1 gene on conjugative plasmids. They belonged to global multidrug resistant E. coli clones, i.e. ST648, ST224, ST68 and ST131, respectively. One strain carried the blaNDM-1 carbapenemase gene. Three strains carried mcr-1 on IncI2 type plasmids, one of them also harboring a blaCTX-M-64 gene. In the fourth strain mcr-1 was located on a 240kb IncHI2 plasmid co-harboring 13 other resistance genes. This is the first report on the presence of the plasmid-coded mcr-1 gene in a variety of multi-resistant clinical isolates from the Arabian Peninsula indicating that several commonly used antibiotics can potentially facilitate the spread of mcr-1 carrying strains, or directly, mcr-1 containing plasmids. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  2. Transfer of an indigenous plasmid of Rhizobium loti to other rhizobia and Agrobacterium tumefaciens.

    Science.gov (United States)

    Pankhurst, C E; Broughton, W J; Wieneke, U

    1983-08-01

    Rhizobium loti strains NZP2037 and NZP2213 were each found to contain a single large plasmid: pRlo2037a (240 MDal) and pRlo2213a (120 MDal), respectively. Plasmid DNA present in crude cell lysates of each strain and purified pRlo2037a DNA did not hybridize with pID1, a recombinant plasmid containing part of the nitrogen fixation (nif) region of R. meliloti, indicating that nif genes were not present on these plasmids. The transposon Tn5 was inserted into pRlo2037a and this plasmid was then transferred into R. leguminosarum, R. meliloti and Agrobacterium tumefaciens. All transconjugants failed to nodulate Lotus pedunculatus, suggesting that the ability to nodulate this legume was also not carried on pRlo2037a. Transfer of pRlo2037a to R. loti strain NZP2213 did not alter the Nod+ Fix- phenotype of this strain for L. pedunculatus. Determinants for flavolan resistance, believed to be necessary for effective nodulation of L. pedunculatus, were not carried on pRlo2037a. These data suggest that nodulation, nitrogen fixation and flavolan resistance genes are not present on the large plasmid in R. loti strain NZP2037.

  3. Integration host factor is required for replication of pYGK-derived plasmids in Aggregatibacter actinomycetemcomitans.

    Science.gov (United States)

    Torres-Escobar, Ascención; Juárez-Rodríguez, María D; Demuth, Donald R

    2014-08-01

    In this study, we show that integration host factor protein (IHF) is required for replication of pYGK plasmids in Aggregatibacter actinomycetemcomitans. YGK plasmids were not replicated in A. actinomycetemcomitans strains lacking either the α- or β- subunit of IHF. However, the deletion mutants were complemented, and plasmid replication was restored when the promoter region and gene for either ihfA or ihfB was cloned into pYGK. We also identified two motifs that resemble the consensus IHF-binding site in a 813-bp fragment containing the pYGK origin of replication. Using electrophoretic mobility shift assays, purified IHFα-IHFβ protein complex was shown to bind to probes containing either of these motifs. To our knowledge, this is the first report showing that plasmid replication is IHF-dependent in the family Pasteurellaceae. In addition, using site-direct mutagenesis, the XbaI and KpnI restriction sites in the suicide vector pJT1 were modified to generate plasmid pJT10. The introduction of these new unique sites in pJT10 facilitates the transfer of transcriptional or translational lacZ fusion constructs for the generation of single-copy chromosomal insertion of the reporter construct. Plasmid pJT10 and its derivatives will be useful for genetic studies in Aggregatibacter (Actinobacillus) and probably other genera of Pasteurellaceae, including Haemophilus, Pasteurella, and Mannheimia.

  4. Relative rates of homologous and nonhomologous recombination in transfected DNA.

    OpenAIRE

    Roth, D B; Wilson, J H

    1985-01-01

    Both homologous and nonhomologous recombination events occur at high efficiency in DNA molecules transfected into mammalian cells. Both types of recombination occur with similar overall efficiencies, as measured by an endpoint assay, but their relative rates are unknown. In this communication, we measure the relative rates of homologous and nonhomologous recombination in DNA transfected into monkey cells. This measurement is made by using a linear simian virus 40 genome that contains a 131-ba...

  5. Characterization of the Complete Nucleotide Sequences of IncA/C2 Plasmids Carrying In809-Like Integrons from Enterobacteriaceae Isolates of Wildlife Origin.

    Science.gov (United States)

    Papagiannitsis, Costas C; Kutilova, Iva; Medvecky, Matej; Hrabak, Jaroslav; Dolejska, Monika

    2017-09-01

    A total of 18 Enterobacteriaceae (17 from gulls and 1 from a clinical sample) collected from Australia, carrying IncA/C plasmids with the IMP-encoding In809-like integrons, were studied. Seven plasmids, being representatives of different origins, plasmid sizes, replicon combinations, and resistance genes, were completely sequenced. Plasmid pEc158, identified in a clinical Escherichia coli ST752 isolate, showed extensive similarity to type 2 IncA/C2 plasmids. pEc158 carried none of the blaCMY-2-like region or ARI-B and ARI-A regions, while it contained a hybrid transposon structure. The six remaining plasmids, which were of wildlife origin, were highly similar to each other and probably were fusion derivatives of type 1 and type 2 A/C2 plasmids. The latter plasmids contained an ARI-B region and hybrid transposon structures. In all plasmids, hybrid transposon structures containing In809-like integrons were inserted 3,434 bp downstream of the rhs2 start codon. In all cases, the one outermost 38-bp inverted repeat (IR) of the transposon was associated with the Tn1696 tnp module, while the other outermost 38-bp IR of the transposon was associated with either a Tn6317-like module or a Tn21 mer module. However, the internal structure of the transposon and the resistance genes were different in each plasmid. These findings indicated that, for the specific periods of time and settings, different IncA/C2 plasmid types carrying In809-like elements circulated among isolates of wildlife and clinical origins. Additionally, they provided the basis for speculations regarding the reshuffling of IncA/C2 plasmids with In809-like integrons and confirmed the rapid evolution of IncA/C2 plasmid lineages. Copyright © 2017 American Society for Microbiology.

  6. Plasmid typing of Shigella sonnei epidemic strains and molecular relationship of their R-plasmids.

    Science.gov (United States)

    Mendoza, M C; Gonzalez, A J; Mendez, F J; Hardisson, C

    1988-06-01

    We conducted a surveillance program on epidemic and/or endemic Shigella strains in Asturias (Spain), their frequency and dispersion in our community, and their R-plasmids. We analyzed initial isolates of Shigella sonnei from two epidemic outbreaks using antibiotic resistance patterns and plasmid profile analysis as epidemiological markers. We found that the 2 outbreaks were caused by different S. sonnei strains, which respectively carried one and two R-plasmids together with other plasmids. The molecular relationship among these and three other R-plasmids from two S. sonnei strains isolated during a previous outbreak, were studied by restriction enzyme analysis and DNA-DNA hybridizations. We were able to establish different levels of relationship among the six R-plasmids.

  7. Rapid recombination among transfected plasmids, chimeric episome formation and trans gene expression in Plasmodium falciparum.

    Science.gov (United States)

    Kadekoppala, M; Cheresh, P; Catron, D; Ji, D D; Deitsch, K; Wellems, T E; Seifert, H S; Haldar, K

    2001-02-01

    Although recombination is known to be important to generating diversity in the human malaria parasite P. falciparum, the low efficiencies of transfection and the fact that integration of transfected DNA into chromosomes is observed only after long periods (typically 12 weeks or more) have made it difficult to genetically manipulate the blood stages of this major human pathogen. Here we show that co-transfection of a P. falciparum line with two plasmids, one expressing a green fluorescent protein (gfp) reporter and the other expressing a drug resistance marker (Tgdhfr-ts M23), allowed selection of a population in which about approximately 30% of the parasites produce GFP. In these GFP-producing parasites, the transfected plasmids had recombined into chimeric episomes as large as 20 kb and could be maintained under drug pressure for at least 16 weeks. Our data suggest that chimera formation occurs early (detected by 7--14 days) and that it involves homologous recombination favored by presence of the same P. falciparum 5'hrp3 UTR promoting transcription from each plasmid. This indicates the presence of high levels of homologous recombination activity in blood stage parasites that can be used to drive rapid recombination of newly introduced DNA, study mechanisms of recombination, and introduce genes for trans expression in P. falciparum.

  8. Cereulide synthetase gene cluster from emetic Bacillus cereus: Structure and location on a mega virulence plasmid related to Bacillus anthracis toxin plasmid pXO1

    Directory of Open Access Journals (Sweden)

    Wagner Martin

    2006-03-01

    Full Text Available Abstract Background Cereulide, a depsipeptide structurally related to valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Recently, it has been shown that this toxin is produced by a nonribosomal peptide synthetase (NRPS, but its exact genetic organization and biochemical synthesis is unknown. Results The complete sequence of the cereulide synthetase (ces gene cluster, which encodes the enzymatic machinery required for the biosynthesis of cereulide, was dissected. The 24 kb ces gene cluster comprises 7 CDSs and includes, besides the typical NRPS genes like a phosphopantetheinyl transferase and two CDSs encoding enzyme modules for the activation and incorporation of monomers in the growing peptide chain, a CDS encoding a putative hydrolase in the upstream region and an ABC transporter in the downstream part. The enzyme modules responsible for incorporation of the hydroxyl acids showed an unusual structure while the modules responsible for the activation of the amino acids Ala and Val showed the typical domain organization of NRPS. The ces gene locus is flanked by genetic regions with high homology to virulence plasmids of B. cereus, Bacillus thuringiensis and Bacillus anthracis. PFGE and Southern hybridization showed that the ces genes are restricted to emetic B. cereus and indeed located on a 208 kb megaplasmid, which has high similarities to pXO1-like plasmids. Conclusion The ces gene cluster that is located on a pXO1-like virulence plasmid represents, beside the insecticidal and the anthrax toxins, a third type of B. cereus group toxins encoded on megaplasmids. The ces genes are restricted to emetic toxin producers, but pXO1-like plasmids are also present in emetic-like strains. These data might indicate the presence of an ancient plasmid in B. cereus which has acquired different virulence genes over time. Due to the unusual structure of the hydroxyl acid incorporating enzyme modules of Ces

  9. Identification of two replicons in phage-plasmid P4.

    Science.gov (United States)

    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. Prevalence and molecular characterization of plasmid- mediated ...

    African Journals Online (AJOL)

    lactamase genes among nosocomial Staphylococcus aureus drug resistance isolates in Taiwan. .... Table 2: Plasmid profiles of the clinical antibiotic-resistant pathogens. Strain. Profile .... Madec J. Characterization of clinical canine methicillin-.

  11. Homology theory on algebraic varieties

    CERN Document Server

    Wallace, Andrew H

    1958-01-01

    Homology Theory on Algebraic Varieties, Volume 6 deals with the principles of homology theory in algebraic geometry and includes the main theorems first formulated by Lefschetz, one of which is interpreted in terms of relative homology and another concerns the Poincaré formula. The actual details of the proofs of these theorems are introduced by geometrical descriptions, sometimes aided with diagrams. This book is comprised of eight chapters and begins with a discussion on linear sections of an algebraic variety, with emphasis on the fibring of a variety defined over the complex numbers. The n

  12. Compositional Homology and Creative Thinking

    Directory of Open Access Journals (Sweden)

    Salvatore Tedesco

    2015-05-01

    Full Text Available The concept of homology is the most solid theoretical basis elaborated by the morphological thinking during its history. The enucleation of some general criteria for the interpretation of homology is today a fundamental tool for life sciences, and for restoring their own opening to the question of qualitative innovation that arose so powerfully in the original Darwinian project. The aim of this paper is to verify the possible uses of the concept of compositional homology in order to provide of an adequate understanding of the dynamics of creative thinking.

  13. Plasmid and chromosome segregation in prokaryotes

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Bugge Jensen, Rasmus; Gerdes, Kenn

    2000-01-01

    Recent major advances in the understanding of prokaryotic DNA segregation have been achieved by using fluorescence microscopy to visualize the localization of cellular components. Plasmids and bacterial chromosomes are partitioned in a highly dynamic fashion, suggesting the presence of a mitotic......-like apparatus in prokaryotes. The identification of chromosomal homologues of the well-characterized plasmid partitioning genes indicates that there could be a general mechanism of bacterial DNA partitioning. Udgivelsesdato: July 1...

  14. Curing of plasmid pXO1 from Bacillus anthracis using plasmid incompatibility.

    Directory of Open Access Journals (Sweden)

    Xiankai Liu

    Full Text Available The large plasmid pXO1 encoding the anthrax toxin is important for the virulence of Bacillus anthracis. It is essential to cure pXO1 from B. anthracis to evaluate its role in the pathogenesis of anthrax infection. Because conventional methods for curing plasmids (e.g., curing agents or growth at elevated temperatures can induce mutations in the host chromosomal DNA, we developed a specific and reliable method to eliminate pXO1 from B. anthracis using plasmid incompatibility. Three putative replication origins of pXO1 were inserted into a temperature-sensitive plasmid to generate three incompatible plasmids. One of the three plasmids successfully eliminated the large plasmid pXO1 from B. anthracis vaccine strain A16R and wild type strain A16. These findings provided additional information about the replication/partitioning of pXO1 and demonstrated that introducing a small incompatible plasmid can generate plasmid-cured strains of B. anthracis without inducing spontaneous mutations in the host chromosome.

  15. Multiple plasmid interference - Pledging allegiance to my enemy's enemy.

    Science.gov (United States)

    Gama, João Alves; Zilhão, Rita; Dionisio, Francisco

    2017-08-24

    As shown in the previous article, two distinct conjugative plasmids sometimes interact within bacterial cells, implicating changes of transfer rates. In most cases of interactions within bacteria, the transfer of one of the plasmids decreases. Less frequently, the transfer rate of one of the plasmids increases. Here we analyse what happens if three distinct conjugative plasmids colonize the same bacterial cell. Our aim is to understand how interactions between two plasmids affect the transfer rate of the third plasmid. After showing that plasmids interact in 59 out of 84 possible interactions we show that, with some exceptions, if the transfer rate of a plasmid decreases in the presence of a second plasmid, a decrease is also observed in the presence of a third plasmid. Moreover, if the conjugation rate of a plasmid increases in the presence of another, an increase is also observed if there is a third plasmid in the cell. Both types of interactions are mostly independent of the third plasmid's identity, even if sometimes the third plasmid quantitatively distorts the interaction of the other two plasmids. There is a bias towards negative intensifying interactions, which provide good news concerning the spread conjugative plasmids encoding antibiotic-resistance genes and virulence factors. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Development of a gene transfer system for curing of plasmids in the marine fish pathogen Vibrio salmonicida.

    Science.gov (United States)

    Valla, S; Frydenlund, K; Coucheron, D H; Haugan, K; Johansen, B; Jørgensen, T; Knudsen, G; Strøm, A

    1992-01-01

    All reported natural isolates of the marine fish pathogen Vibrio salmonicida contain plasmids, and in another marine fish pathogen, Vibrio anguillarum, it has been shown that a plasmid is important for expression of virulence by the organism. To study the function of the plasmids in V. salmonicida, we developed a gene transfer system based on the plasmid RSF1010 replicon. The gene transfer system was used to construct a plasmid-free strain, and this strain was found to behave similarly to the wild type in a fish pathogenicity test based on intraperitoneal injection of the bacteria. We were unable to detect any other phenotypic differences between the two strains. It could therefore be concluded that at least in the V. salmonicida strain tested, extrachromosomal DNA is not required for expression of virulence. Images PMID:1622274

  17. Dynamics of elimination of plasmids and expression of VEGF121 gene transfected into human mesenchymal stem cells by different methods.

    Science.gov (United States)

    Smirnikhina, S A; Lavrov, A V; Bochkov, N P

    2011-05-01

    We compared two methods of transfection (lipofection and electroporation) with plasmid containing VEGF121 gene in four cultures of mesenchymal stem cells from the human adipose tissue. The efficacy of transfection after 1 day, the dynamics of plasmid elimination after 3, 6, 9 days, and expression of the target gene were evaluated. Transfection by both methods failed in one of 4 cultures. Analysis of the plasmid elimination dynamics showed that the content of plasmids introduced by both methods decreased by 30-69% in all cultures by day 3 and then remained unchanged from day 3 to day 9. The expression of the target gene did not correlate with the content of plasmids in cells and varied by 2-10 times in control cells and cells transfected by both methods. Fluctuation of VEGF121 expression was not related to methylation.

  18. The muc+ gene of plasmid pKM101 prevents respiration shutoff in far ultraviolet-irradiated Salmonella typhimurium.

    Science.gov (United States)

    Swenson, P A

    1981-01-01

    The plasmid PKM101 is known to protect Escherichia coli and Salmonella typhimurium against killing by far UV irradiation and to enhance UV-induced mutagenesis. The muc+ gene of the plasmid is responsible for both of these effects. This paper shows that respiration of S. typhimurium shuts off about an hour after UV irradiation and that pKM101 prevents the shutoff. Plasmids which contained Tn5 translocatable elements, either in (and having produced a muc mutation) or flanking the muc+ gene, have been introduced into S. typhimurium. The muc mutant plasmid, which does not protect its host against UV killing and does not enhance UV induced mutagenesis, also does not protect against UV induced respiration shutoff. Likewise, plasmids in which the Tn5 translocatable elements flank the muc+ gene protect against shutoff of respiration. Thus the muc+ gene of pKM101 is responsible for protection against UV induced shutoff of respiration in S. typhimurium.

  19. A PHF8 homolog in C. elegans promotes DNA repair via homologous recombination.

    Directory of Open Access Journals (Sweden)

    Changrim Lee

    Full Text Available PHF8 is a JmjC domain-containing histone demethylase, defects in which are associated with X-linked mental retardation. In this study, we examined the roles of two PHF8 homologs, JMJD-1.1 and JMJD-1.2, in the model organism C. elegans in response to DNA damage. A deletion mutation in either of the genes led to hypersensitivity to interstrand DNA crosslinks (ICLs, while only mutation of jmjd-1.1 resulted in hypersensitivity to double-strand DNA breaks (DSBs. In response to ICLs, JMJD-1.1 did not affect the focus formation of FCD-2, a homolog of FANCD2, a key protein in the Fanconi anemia pathway. However, the dynamic behavior of RPA-1 and RAD-51 was affected by the mutation: the accumulations of both proteins at ICLs appeared normal, but their subsequent disappearance was retarded, suggesting that later steps of homologous recombination were defective. Similar changes in the dynamic behavior of RPA-1 and RAD-51 were seen in response to DSBs, supporting a role of JMJD-1.1 in homologous recombination. Such a role was also supported by our finding that the hypersensitivity of jmjd-1.1 worms to ICLs was rescued by knockdown of lig-4, a homolog of Ligase 4 active in nonhomologous end-joining. The hypersensitivity of jmjd-1.1 worms to ICLs was increased by rad-54 knockdown, suggesting that JMJD-1.1 acts in parallel with RAD-54 in modulating chromatin structure. Indeed, the level of histone H3 Lys9 tri-methylation, a marker of heterochromatin, was higher in jmjd-1.1 cells than in wild-type cells. We conclude that the histone demethylase JMJD-1.1 influences homologous recombination either by relaxing heterochromatin structure or by indirectly regulating the expression of multiple genes affecting DNA repair.

  20. Impact of homologous and non-homologous recombination in the genomic evolution of Escherichia coli.

    Science.gov (United States)

    Didelot, Xavier; Méric, Guillaume; Falush, Daniel; Darling, Aaron E

    2012-06-19

    Escherichia coli is an important species of bacteria that can live as a harmless inhabitant of the guts of many animals, as a pathogen causing life-threatening conditions or freely in the non-host environment. This diversity of lifestyles has made it a particular focus of interest for studies of genetic variation, mainly with the aim to understand how a commensal can become a deadly pathogen. Many whole genomes of E. coli have been fully sequenced in the past few years, which offer helpful data to help understand how this important species evolved. We compared 27 whole genomes encompassing four phylogroups of Escherichia coli (A, B1, B2 and E). From the core-genome we established the clonal relationships between the isolates as well as the role played by homologous recombination during their evolution from a common ancestor. We found strong evidence for sexual isolation between three lineages (A+B1, B2, E), which could be explained by the ecological structuring of E. coli and may represent on-going speciation. We identified three hotspots of homologous recombination, one of which had not been previously described and contains the aroC gene, involved in the essential shikimate metabolic pathway. We also described the role played by non-homologous recombination in the pan-genome, and showed that this process was highly heterogeneous. Our analyses revealed in particular that the genomes of three enterohaemorrhagic (EHEC) strains within phylogroup B1 have converged from originally separate backgrounds as a result of both homologous and non-homologous recombination. Recombination is an important force shaping the genomic evolution and diversification of E. coli, both by replacing fragments of genes with an homologous sequence and also by introducing new genes. In this study, several non-random patterns of these events were identified which correlated with important changes in the lifestyle of the bacteria, and therefore provide additional evidence to explain the

  1. Fivebranes and 3-manifold homology

    CERN Document Server

    Gukov, Sergei; Vafa, Cumrun

    2016-01-01

    Motivated by physical constructions of homological knot invariants, we study their analogs for closed 3-manifolds. We show that fivebrane compactifications provide a universal description of various old and new homological invariants of 3-manifolds. In terms of 3d/3d correspondence, such invariants are given by the Q-cohomology of the Hilbert space of partially topologically twisted 3d N=2 theory T[M_3] on a Riemann surface with defects. We demonstrate this by concrete and explicit calculations in the case of monopole/Heegaard Floer homology and a 3-manifold analog of Khovanov-Rozansky link homology. The latter gives a categorification of Chern-Simons partition function. Some of the new key elements include the explicit form of the S-transform and a novel connection between categorification and a previously mysterious role of Eichler integrals in Chern-Simons theory.

  2. Fivebranes and 3-manifold homology

    Science.gov (United States)

    Gukov, Sergei; Putrov, Pavel; Vafa, Cumrun

    2017-07-01

    Motivated by physical constructions of homological knot invariants, we study their analogs for closed 3-manifolds. We show that fivebrane compactifications provide a universal description of various old and new homological invariants of 3-manifolds. In terms of 3d/3d correspondence, such invariants are given by the Q-cohomology of the Hilbert space of partially topologically twisted 3d N=2 theory T[ M 3] on a Riemann surface with defects. We demonstrate this by concrete and explicit calculations in the case of monopole/Heegaard Floer homology and a 3-manifold analog of Khovanov-Rozansky link homology. The latter gives a categorification of Chern-Simons partition function. Some of the new key elements include the explicit form of the S-transform and a novel connection between categorification and a previously mysterious role of Eichler integrals in Chern-Simons theory.

  3. Clostridium perfringens type A–E toxin plasmids

    Science.gov (United States)

    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

  4. Characterization of a cfr-Carrying Plasmid from Porcine Escherichia coli That Closely Resembles Plasmid pEA3 from the Plant Pathogen Erwinia amylovora.

    Science.gov (United States)

    Zhang, Rongmin; Sun, Bin; Wang, Yang; Lei, Lei; Schwarz, Stefan; Wu, Congming

    2015-11-02

    The multiresistance gene cfr was found in two porcine Escherichia coli isolates, one harboring it on the conjugative 33,885-bp plasmid pFSEC-01, the other harboring it in the chromosomal DNA. Sequence analysis of pFSEC-01 revealed that a 6,769-bp fragment containing the cfr gene bracketed by two IS26 elements was inserted into a plasmid closely related to pEA3 from the plant pathogen Erwinia amylovora, suggesting that pFSEC-01 may be transferred between different bacterial genera of both animal and plant origin.

  5. Type 3 fimbriae, encoded by the conjugative plasmid pOLA52, enhance biofilm formation and transfer frequencies in Enterobacteriaceae strains

    DEFF Research Database (Denmark)

    Burmølle, Mette; Bahl, Martin Iain; Jensen, Lars Bogø

    2008-01-01

    biofilm-deficient mutants, which all mapped within a putative operon with high homology to the mrkABCDF operon of Klebsiella pneumoniae, where these genes are responsible for type 3 fimbriae expression, attachment to surfaces and biofilm formation. Biofilm formation in microtitre plates and in urinary......, resulting in enhanced conjugation frequencies and biofilm formation of the plasmid-harbouring strain....

  6. Utilization of an unstable plasmid and the I-SceI endonuclease to generate routine markerless deletion mutants in Francisella tularensis

    Science.gov (United States)

    Horzempa, Joseph; Shanks, Robert M.Q.; Brown, Matthew J.; Russo, Brian C.; O’Dee, Dawn M.; Nau, Gerard J.

    2011-01-01

    We engineered an efficient system to make Francisella tularensis deletion mutations using an unstable, poorly maintained plasmid to enhance the likelihood of homologous recombination. For counterselection, we adapted a strategy using I-SceI, which causes a double-stranded break in the integrated suicide vector, forcing a second recombination to mediate allelic replacement. PMID:19879904

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

    Science.gov (United States)

    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).

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

    Science.gov (United States)

    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.

  9. The sudden dominance of blaCTX-M harbouring plasmids in Shigella spp. Circulating in Southern Vietnam.

    Directory of Open Access Journals (Sweden)

    Nhu Thi Khanh Nguyen

    2010-06-01

    Full Text Available Plasmid mediated antimicrobial resistance in the Enterobacteriaceae is a global problem. The rise of CTX-M class extended spectrum beta lactamases (ESBLs has been well documented in industrialized countries. Vietnam is representative of a typical transitional middle income country where the spectrum of infectious diseases combined with the spread of drug resistance is shifting and bringing new healthcare challenges.We collected hospital admission data from the pediatric population attending the hospital for tropical diseases in Ho Chi Minh City with Shigella infections. Organisms were cultured from all enrolled patients and subjected to antimicrobial susceptibility testing. Those that were ESBL positive were subjected to further investigation. These investigations included PCR amplification for common ESBL genes, plasmid investigation, conjugation, microarray hybridization and DNA sequencing of a bla(CTX-M encoding plasmid.We show that two different bla(CTX-M genes are circulating in this bacterial population in this location. Sequence of one of the ESBL plasmids shows that rather than the gene being integrated into a preexisting MDR plasmid, the bla(CTX-M gene is located on relatively simple conjugative plasmid. The sequenced plasmid (pEG356 carried the bla(CTX-M-24 gene on an ISEcp1 element and demonstrated considerable sequence homology with other IncFI plasmids.The rapid dissemination, spread of antimicrobial resistance and changing population of Shigella spp. concurrent with economic growth are pertinent to many other countries undergoing similar development. Third generation cephalosporins are commonly used empiric antibiotics in Ho Chi Minh City. We recommend that these agents should not be considered for therapy of dysentery in this setting.

  10. Mechanistic basis of plasmid-specific DNA binding of the F plasmid regulatory protein, TraM.

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Mette Burmølle

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  13. Transcriptional regulation is affected by subnuclear targeting of reporter plasmids to PML nuclear bodies.

    Science.gov (United States)

    Block, Gregory J; Eskiw, Christopher H; Dellaire, Graham; Bazett-Jones, David P

    2006-12-01

    Whereas the PML protein has been reported to have both transcriptional coactivator and corepressor potential, the contribution of the PML nuclear body (PML NB) itself to transcriptional regulation is not well understood. Here we demonstrate that plasmid DNA artificially tethered to PML or the PML NB-targeting domain of Sp100 is preferentially localized to PML NBs. Using the tethering technique, we targeted a simian virus 40 promoter-driven luciferase reporter plasmid to PML NBs, resulting in the repression of the transgene transcriptional activity. Conversely, the tethering of a cytomegalovirus promoter-containing reporter plasmid resulted in activation. Targeting a minimal eukaryotic promoter did not affect its activity. The expression of targeted promoters could be modulated by altering the cellular concentration of PML NB components, including Sp100 and isoforms of the PML protein. Finally, we demonstrate that ICP0, the promiscuous herpes simplex virus transactivator, increases the level of transcriptional activation of plasmid DNA tethered to the PML NB. We conclude that when PML NB components are artificially tethered to reporter plasmids, the PML NB contributes to the regulation of the tethered DNA in a promoter-dependent manner. Our findings demonstrate that transient transcription assays are sensitive to the subnuclear localization of the transgene plasmid.

  14. Genomics of high molecular weight plasmids isolated from an on-farm biopurification system.

    Science.gov (United States)

    Martini, María C; Wibberg, Daniel; Lozano, Mauricio; Torres Tejerizo, Gonzalo; Albicoro, Francisco J; Jaenicke, Sebastian; van Elsas, Jan Dirk; Petroni, Alejandro; Garcillán-Barcia, M Pilar; de la Cruz, Fernando; Schlüter, Andreas; Pühler, Alfred; Pistorio, Mariano; Lagares, Antonio; Del Papa, María F

    2016-06-20

    The use of biopurification systems (BPS) constitutes an efficient strategy to eliminate pesticides from polluted wastewaters from farm activities. BPS environments contain a high microbial density and diversity facilitating the exchange of information among bacteria, mediated by mobile genetic elements (MGEs), which play a key role in bacterial adaptation and evolution in such environments. Here we sequenced and characterized high-molecular-weight plasmids from a bacterial collection of an on-farm BPS. The high-throughput-sequencing of the plasmid pool yielded a total of several Mb sequence information. Assembly of the sequence data resulted in six complete replicons. Using in silico analyses we identified plasmid replication genes whose encoding proteins represent 13 different Pfam families, as well as proteins involved in plasmid conjugation, indicating a large diversity of plasmid replicons and suggesting the occurrence of horizontal gene transfer (HGT) events within the habitat analyzed. In addition, genes conferring resistance to 10 classes of antimicrobial compounds and those encoding enzymes potentially involved in pesticide and aromatic hydrocarbon degradation were found. Global analysis of the plasmid pool suggest that the analyzed BPS represents a key environment for further studies addressing the dissemination of MGEs carrying catabolic genes and pathway assembly regarding degradation capabilities.

  15. Molecular characterization of Syrian date palm cultivars using plasmid-like DNA markers.

    Science.gov (United States)

    Haider, N; Nabulsi, I

    2012-02-01

    Date palm (Phoenix dactylifera L.) is one of the most important domesticated fruit trees in the Near East and North African countries. This tree has been, for several decades, in serious threat of being completely destroyed by the "Bayoud" disease caused by Fusarium oxysporum f. sp. albedinis. In this study, 18 Syrian date palm cultivars and four male trees were analyzed according to the identity of mitochondrial plasmid-like DNAs. A PCR strategy that employs plasmid-like DNAs-specific primer pair was used. These primers amplify a product of either 373-bp or 265-bp that corresponds to the S-(Bayoud-susceptible) or the R-plasmid (Bayoud-resistant), respectively. Generated data revealed that only six cultivars ('Medjool', 'Ashrasi', 'Gish Rabi', 'Khineze', and yellow- and red-'Kabkab') have the S-plasmid, suggesting their susceptibility to the fusariosis, while the remaining 12 cultivars and the four male trees contain the R-plasmid, suggesting their resistance to the fusariosis. The PCR process applied here has been proved efficient for the rapid screening for the presence of the S and R DNAs in Syrian date palm. PCR markers developed in this study could be useful for the screening of date palm lines growing in the field. The availability of such diagnostic tool for plasmid characterization in date palm would also be of great importance in establishing propagation and breeding programs of date palm in Syria.

  16. Processing of Nonconjugative Resistance Plasmids by Conjugation Nicking Enzyme of Staphylococci

    Energy Technology Data Exchange (ETDEWEB)

    Pollet, Rebecca M.; Ingle, James D.; Hymes, Jeff P.; Eakes, Thomas C.; Eto, Karina Yui; Kwong, Stephen M.; Ramsay, Joshua P.; Firth, Neville; Redinbo, Matthew R.; Christie, P. J.

    2016-01-04

    Antimicrobial resistance inStaphylococcus aureuspresents an increasing threat to human health. This resistance is often encoded on mobile plasmids, such as pSK41; however, the mechanism of transfer of these plasmids is not well understood. In this study, we first examine key protein-DNA interactions formed by the relaxase enzyme, NES, which initiates and terminates the transfer of the multidrug resistance plasmid pSK41. Two loops on the NES protein, hairpin loops 1 and 2, form extensive contacts with the DNA hairpin formed at theoriTregion of pSK41, and here we establish that these contacts are essential for proper DNA cleavage and religation by the full 665-residue NES proteinin vitro. Second, pSK156 and pCA347 are nonconjugativeStaphylococcus aureusplasmids that contain sequences similar to theoriTregion of pSK41 but differ in the sequence predicted to form a DNA hairpin. We show that pSK41-encoded NES is able to bind, cleave, and religate theoriTsequences of these nonconjugative plasmidsin vitro. Although pSK41 could mobilize a coresident plasmid harboring its cognateoriT, it was unable to mobilize plasmids containing the pSK156 and pCA347 variantoriTmimics, suggesting that an accessory protein like that previously shown to confer specificity in the pWBG749 system may also be involved in transmission of plasmids containing a pSK41-likeoriT. These data indicate that the conjugative relaxase intransmechanism recently described for the pWBG749 family of plasmids also applies to the pSK41 family of plasmids, further heightening the potential significance of this mechanism in the horizontal transfer of staphylococcal plasmids.

    IMPORTANCEUnderstanding the

  17. Relative rates of homologous and nonhomologous recombination in transfected DNA.

    Science.gov (United States)

    Roth, D B; Wilson, J H

    1985-05-01

    Both homologous and nonhomologous recombination events occur at high efficiency in DNA molecules transfected into mammalian cells. Both types of recombination occur with similar overall efficiencies, as measured by an endpoint assay, but their relative rates are unknown. In this communication, we measure the relative rates of homologous and nonhomologous recombination in DNA transfected into monkey cells. This measurement is made by using a linear simian virus 40 genome that contains a 131-base-pair duplication at its termini. Once inside the cell, this molecule must circularize to initiate lytic infection. Circularization can occur either by direct, nonhomologous end-joining or by homologous recombination within the duplicated region. Although the products of the two recombination pathways are different, they are equally infectious. Since homologous and nonhomologous recombination processes are competing for the same substrate, the relative amounts of the products of each pathway should reflect the relative rates of homologous and nonhomologous recombination. Analysis of individual recombinant genomes from 164 plaques indicates that the rate of circularization by nonhomologous recombination is 2- to 3-fold higher than the rate of homologous recombination. The assay system described here may prove to be useful for testing procedures designed to influence the relative rates of homologous and nonhomologous recombination.

  18. Co-evolution of genomes and plasmids within Chlamydia trachomatis and the emergence in Sweden of a new variant strain

    Directory of Open Access Journals (Sweden)

    Skilton Rachel J

    2009-05-01

    Full Text Available Abstract Background Chlamydia trachomatis is the most common cause of sexually transmitted infections globally and the leading cause of preventable blindness in the developing world. There are two biovariants of C. trachomatis: 'trachoma', causing ocular and genital tract infections, and the invasive 'lymphogranuloma venereum' strains. Recently, a new variant of the genital tract C. trachomatis emerged in Sweden. This variant escaped routine diagnostic tests because it carries a plasmid with a deletion. Failure to detect this strain has meant it has spread rapidly across the country provoking a worldwide alert. In addition to being a key diagnostic target, the plasmid has been linked to chlamydial virulence. Analysis of chlamydial plasmids and their cognate chromosomes was undertaken to provide insights into the evolutionary relationship between chromosome and plasmid. This is essential knowledge if the plasmid is to be continued to be relied on as a key diagnostic marker, and for an understanding of the evolution of Chlamydia trachomatis. Results The genomes of two new C. trachomatis strains were sequenced, together with plasmids from six C. trachomatis isolates, including the new variant strain from Sweden. The plasmid from the new Swedish variant has a 377 bp deletion in the first predicted coding sequence, abolishing the site used for PCR detection, resulting in negative diagnosis. In addition, the variant plasmid has a 44 bp duplication downstream of the deletion. The region containing the second predicted coding sequence is the most highly conserved region of the plasmids investigated. Phylogenetic analysis of the plasmids and chromosomes are fully congruent. Moreover this analysis also shows that ocular and genital strains diverged from a common C. trachomatis progenitor. Conclusion The evolutionary pathways of the chlamydial genome and plasmid imply that inheritance of the plasmid is tightly linked with its cognate chromosome. These data

  19. Identification of dfrA14 in two distinct plasmids conferring trimethoprim resistance in Actinobacillus pleuropneumoniae.

    Science.gov (United States)

    Bossé, Janine T; Li, Yanwen; Walker, Stephanie; Atherton, Tom; Fernandez Crespo, Roberto; Williamson, Susanna M; Rogers, Jon; Chaudhuri, Roy R; Weinert, Lucy A; Oshota, Olusegun; Holden, Matt T G; Maskell, Duncan J; Tucker, Alexander W; Wren, Brendan W; Rycroft, Andrew N; Langford, Paul R

    2015-08-01

    The objective of this study was to determine the distribution and genetic basis of trimethoprim resistance in Actinobacillus pleuropneumoniae isolates from pigs in England. Clinical isolates collected between 1998 and 2011 were tested for resistance to trimethoprim and sulphonamide. The genetic basis of trimethoprim resistance was determined by shotgun WGS analysis and the subsequent isolation and sequencing of plasmids. A total of 16 (out of 106) A. pleuropneumoniae isolates were resistant to both trimethoprim (MIC >32 mg/L) and sulfisoxazole (MIC ≥256 mg/L), and a further 32 were resistant only to sulfisoxazole (MIC ≥256 mg/L). Genome sequence data for the trimethoprim-resistant isolates revealed the presence of the dfrA14 dihydrofolate reductase gene. The distribution of plasmid sequences in multiple contigs suggested the presence of two distinct dfrA14-containing plasmids in different isolates, which was confirmed by plasmid isolation and sequencing. Both plasmids encoded mobilization genes, the sulphonamide resistance gene sul2, as well as dfrA14 inserted into strA, a streptomycin-resistance-associated gene, although the gene order differed between the two plasmids. One of the plasmids further encoded the strB streptomycin-resistance-associated gene. This is the first description of mobilizable plasmids conferring trimethoprim resistance in A. pleuropneumoniae and, to our knowledge, the first report of dfrA14 in any member of the Pasteurellaceae. The identification of dfrA14 conferring trimethoprim resistance in A. pleuropneumoniae isolates will facilitate PCR screens for resistance to this important antimicrobial. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.

  20. Characterization of plasmids in a human clinical strain of Lactococcus garvieae.

    Directory of Open Access Journals (Sweden)

    Mónica Aguado-Urda

    Full Text Available The present work describes the molecular characterization of five circular plasmids found in the human clinical strain Lactococcus garvieae 21881. The plasmids were designated pGL1-pGL5, with molecular sizes of 4,536 bp, 4,572 bp, 12,948 bp, 14,006 bp and 68,798 bp, respectively. Based on detailed sequence analysis, some of these plasmids appear to be mosaics composed of DNA obtained by modular exchange between different species of lactic acid bacteria. Based on sequence data and the derived presence of certain genes and proteins, the plasmid pGL2 appears to replicate via a rolling-circle mechanism, while the other four plasmids appear to belong to the group of lactococcal theta-type replicons. The plasmids pGL1, pGL2 and pGL5 encode putative proteins related with bacteriocin synthesis and bacteriocin secretion and immunity. The plasmid pGL5 harbors genes (txn, orf5 and orf25 encoding proteins that could be considered putative virulence factors. The gene txn encodes a protein with an enzymatic domain corresponding to the family actin-ADP-ribosyltransferases toxins, which are known to play a key role in pathogenesis of a variety of bacterial pathogens. The genes orf5 and orf25 encode two putative surface proteins containing the cell wall-sorting motif LPXTG, with mucin-binding and collagen-binding protein domains, respectively. These proteins could be involved in the adherence of L. garvieae to mucus from the intestine, facilitating further interaction with intestinal epithelial cells and to collagenous tissues such as the collagen-rich heart valves. To our knowledge, this is the first report on the characterization of plasmids in a human clinical strain of this pathogen.

  1. Complete sequence of a full-length DNA and molecular characterization of one plasmid from chinaberry (Melia azedarach Z) witches'-broom phytoplasma%苦楝丛枝植原体质粒的测定与分子特征

    Institute of Scientific and Technical Information of China (English)

    宋传生; 林彩丽; 田国忠; 赵文军; 朱水芳; 牟海青; 胡佳续; 王曦茁; 郭民伟

    2011-01-01

    [ Objective ] To clone plasmid from chinaberry witches'-broom phytoplasma and analyse its molecular characterization. [Methods] Fragments of one plasmid (pCWBFq) in chinaberry witches'-broom phytoplasma-Fuqing strain ( CWBFq) were amplified with primer pairs which were designed according to plasmid sequences published on NCBI. Transmembrane domain and subcellular localization predictions of proteins encoded by the plasmid pCWBFq as well as phylogenetic analysis among the plasmid sequences were completed by using bioinformatic softwares. Southern blot analysis was performed to detect the plasmids existed in CWBFq and several other phytoplasmas with the pCWBFq repA probe. [ Results] One complete plasmid was sequenced from CWBFq. pCWBFq comprised 4446 bp and had a nucleotide content of 73. 5% A + T and encoded six proteins. Protein P2, P3, P4 and P5 of pCWBFq contained 3,2,1 and 2 tranmembrane domains respectively, and their predicted signal peptide values were 0.989, 0.505, 0.918 and 0.914 respectively. Homologous comparison showed that RepA homology between pCWBFq and other phytoplasmas was between 9. 6% -85. 6% , however, the homology of different SSB proteins was between 74. 0% - 89. 4% . Southern blotting withpCWBFq repA probe confirmed the existence of the plasmids in CWBFq. In addition, The hybridizations occurred with paulownia witches'-broom phytoplasma-Nanyang strain ( PaWBNy ) , periwinkle virescence phytoplasma-Hainan stanin (PeVHn) , chinaberry witches'-broom phytoplasma-Fuzhou strain ( CWBFz) and mulberry dwarf phytoplasma -Puyang strain (MDPy) , whereas, no hybridizarions occurred with jujube witches'-broom phytoplasma-Beijing strain ( JWBBj) , cherry lethal yellows phytoplasma-Xichang strain (CLYXc) and Bischofia polycarpa witches'-broom phytoplasma-Nanchang strain ( BiWBNc ). [ Conclusion ] The plasmid encoded a replication associated protein ( RepA) and a single-stranded DNA binding protein ( SSB) , which were for the replication of plasmid. Four

  2. TALEN-mediated homologous recombination in Daphnia magna.

    Science.gov (United States)

    Nakanishi, Takashi; Kato, Yasuhiko; Matsuura, Tomoaki; Watanabe, Hajime

    2015-12-17

    Transcription Activator-Like Effector Nucleases (TALENs) offer versatile tools to engineer endogenous genomic loci in various organisms. We established a homologous recombination (HR)-based knock-in using TALEN in the crustacean Daphnia magna, a model for ecological and toxicological genomics. We constructed TALENs and designed the 67 bp donor insert targeting a point deletion in the eyeless mutant that shows eye deformities. Co-injection of the TALEN mRNA with donor DNA into eggs led to the precise integration of the donor insert in the germ line, which recovered eye deformities in offspring. The frequency of HR events in the germ line was 2% by using both plasmid and single strand oligo DNA with 1.5 kb and 80 nt homology to the target. Deficiency of ligase 4 involved in non-homologous end joining repair did not increase the HR efficiency. Our data represent efficient HR-based knock-in by TALENs in D. magna, which is a promising tool to understand Daphnia gene functions.

  3. Complete Sequence of a Full-length DNA and Molecular Characterization of one Plasmid from Periwinkle Little Leaf Phytoplasma%长春花小叶病植原体质粒DNA克隆及其分子特征

    Institute of Scientific and Technical Information of China (English)

    郑文虎; 车海彦; 符瑞益; 杨毅; 罗大全

    2012-01-01

    为了测定长春花小叶病植原体质粒及分析其分子特征,扩增长春花小叶病植原体质粒片段,然后进行反向扩增,拼接得到质粒全序列,且预测质粒编码蛋白的跨膜区、亚细胞定位等.结果表明,获得的质粒全长为4 102bp,A+T含量为75.18%,编码5个蛋白,且将该质粒命名为pPLLHn-1.其中ORF的2、3、4编码蛋白分别含有2、1、2个跨膜区,ORF3的信号肽(Singnal peptide,SP)的信号值为0.948,ORF3编码的氨基酸序列与洋葱黄化植原体质粒EcOYW1参与质粒拷贝数控制的蛋白(Copy number control protein)具有97%的同源性.此结果说明长春花小叶病植原体质粒pPLLHn-1编码的5个蛋白中,除与质粒复制有关的RepA和SSB外,另外3个均为跨膜蛋白,根据分泌蛋白的特征分析初步推断ORF3蛋白为分泌蛋白.%DNA fragments of the plasmid (pPLLHn-l)in Periwinkle little leaf phytoplasma Hainan 9train (PLL-Hn)were amplified with primer pairs which were designed according to plasmid sequences published on NCBI. Transmembrane domain and subcellular localization predictions of proteins encoded by the plasmid among the pPLLHn-1 plasmid sequences were completed by using bioinformatic softwares. The plasmid comprised 4 102 bp and had a nucleotide content of 75.18% A+T and encoded five ORFs. ORF2, ORF3 and ORF4 of pPLLHn-1 contained 2, 1 and 2 tranmetnbrane domains respectively, and signal peptide values of ORF3 was 0.948. Homologous comparison showed that Cop homology between ORF3 and the Cop of EcOYWl was 97%. Two ORFs of the pPLLHn-1 plasmid encoded a replication associated protein (Rep A) and a single-stranded DNA binding protein(SSB), respectively. Other three putative proteins encoded by the plasmid were predicted to contain one or more transmembrane domains respectively. According to the characteristics of secreted proteins, ORF3 protein was concluded a secreted protein.

  4. Plasmids in the driving seat: The regulatory RNA Rcd gives plasmid ColE1 control over division and growth of its E. coli host.

    Science.gov (United States)

    Gaimster, Hannah; Summers, David

    2015-03-01

    Regulation by non-coding RNAs was found to be widespread among plasmids and other mobile elements of bacteria well before its ubiquity in the eukaryotic world was suspected. As an increasing number of examples was characterised, a common mechanism began to emerge. Non-coding RNAs, such as CopA and Sok from plasmid R1, or RNAI from ColE1, exerted regulation by refolding the secondary structures of their target RNAs or modifying their translation. One regulatory RNA that seemed to swim against the tide was Rcd, encoded within the multimer resolution site of ColE1. Required for high fidelity maintenance of the plasmid in recombination-proficient hosts, Rcd was found to have a protein target, elevating indole production by stimulating tryptophanase. Rcd production is up-regulated in dimer-containing cells and the consequent increase in indole is part of the response to the rapid accumulation of dimers by over-replication (known as the dimer catastrophe). It is proposed that indole simultaneously inhibits cell division and plasmid replication, stopping the catastrophe and allowing time for the resolution of dimers to monomers. The idea of a plasmid-mediated cell division checkpoint, proposed but then discarded in the 1980s, appears to be enjoying a revival. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Object-oriented persistent homology

    Science.gov (United States)

    Wang, Bao; Wei, Guo-Wei

    2016-01-01

    Persistent homology provides a new approach for the topological simplification of big data via measuring the life time of intrinsic topological features in a filtration process and has found its success in scientific and engineering applications. However, such a success is essentially limited to qualitative data classification and analysis. Indeed, persistent homology has rarely been employed for quantitative modeling and prediction. Additionally, the present persistent homology is a passive tool, rather than a proactive technique, for classification and analysis. In this work, we outline a general protocol to construct object-oriented persistent homology methods. By means of differential geometry theory of surfaces, we construct an objective functional, namely, a surface free energy defined on the data of interest. The minimization of the objective functional leads to a Laplace-Beltrami operator which generates a multiscale representation of the initial data and offers an objective oriented filtration process. The resulting differential geometry based object-oriented persistent homology is able to preserve desirable geometric features in the evolutionary filtration and enhances the corresponding topological persistence. The cubical complex based homology algorithm is employed in the present work to be compatible with the Cartesian representation of the Laplace-Beltrami flow. The proposed Laplace-Beltrami flow based persistent homology method is extensively validated. The consistence between Laplace-Beltrami flow based filtration and Euclidean distance based filtration is confirmed on the Vietoris-Rips complex for a large amount of numerical tests. The convergence and reliability of the present Laplace-Beltrami flow based cubical complex filtration approach are analyzed over various spatial and temporal mesh sizes. The Laplace-Beltrami flow based persistent homology approach is utilized to study the intrinsic topology of proteins and fullerene molecules. Based on a

  6. Object-oriented Persistent Homology.

    Science.gov (United States)

    Wang, Bao; Wei, Guo-Wei

    2016-01-15

    Persistent homology provides a new approach for the topological simplification of big data via measuring the life time of intrinsic topological features in a filtration process and has found its success in scientific and engineering applications. However, such a success is essentially limited to qualitative data classification and analysis. Indeed, persistent homology has rarely been employed for quantitative modeling and prediction. Additionally, the present persistent homology is a passive tool, rather than a proactive technique, for classification and analysis. In this work, we outline a general protocol to construct object-oriented persistent homology methods. By means of differential geometry theory of surfaces, we construct an objective functional, namely, a surface free energy defined on the data of interest. The minimization of the objective functional leads to a Laplace-Beltrami operator which generates a multiscale representation of the initial data and offers an objective oriented filtration process. The resulting differential geometry based object-oriented persistent homology is able to preserve desirable geometric features in the evolutionary filtration and enhances the corresponding topological persistence. The cubical complex based homology algorithm is employed in the present work to be compatible with the Cartesian representation of the Laplace-Beltrami flow. The proposed Laplace-Beltrami flow based persistent homology method is extensively validated. The consistence between Laplace-Beltrami flow based filtration and Euclidean distance based filtration is confirmed on the Vietoris-Rips complex for a large amount of numerical tests. The convergence and reliability of the present Laplace-Beltrami flow based cubical complex filtration approach are analyzed over various spatial and temporal mesh sizes. The Laplace-Beltrami flow based persistent homology approach is utilized to study the intrinsic topology of proteins and fullerene molecules. Based on a

  7. The semaphorontic view of homology.

    Science.gov (United States)

    Havstad, Joyce C; Assis, Leandro C S; Rieppel, Olivier

    2015-11-01

    The relation of homology is generally characterized as an identity relation, or alternatively as a correspondence relation, both of which are transitive. We use the example of the ontogenetic development and evolutionary origin of the gnathostome jaw to discuss identity and transitivity of the homology relation under the transformationist and emergentist paradigms respectively. Token identity and consequent transitivity of homology relations are shown to be requirements that are too strong to allow the origin of genuine evolutionary novelties. We consequently introduce the concept of compositional identity that is grounded in relations prevailing between parts (organs and organ systems) of a whole (organism). We recognize an ontogenetic identity of parts within a whole throughout the sequence of successive developmental stages of those parts: this is an intra-organismal character identity maintained throughout developmental trajectory. Correspondingly, we recognize a phylogenetic identity of homologous parts within two or more organisms of different species: this is an inter-species character identity maintained throughout evolutionary trajectory. These different dimensions of character identity--ontogenetic (through development) and phylogenetic (via shared evolutionary history)--break the transitivity of homology relations. Under the transformationist paradigm, the relation of homology reigns over the entire character (-state) transformation series, and thus encompasses the plesiomorphic as well as the apomorphic condition of form. In contrast, genuine evolutionary novelties originate not through transformation of ancestral characters (-states), but instead through deviating developmental trajectories that result in alternate characters. Under the emergentist paradigm, homology is thus synonymous with synapomorphy. © 2015 The Authors. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution Published by Wiley Periodicals, Inc.

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

    OpenAIRE

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

    2011-01-01

    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 ant...

  9. Transduction of plasmid DNA in Streptomyces spp. and related genera by bacteriophage FP43.

    Science.gov (United States)

    McHenney, M A; Baltz, R H

    1988-05-01

    A segment (hft) of bacteriophage FP43 DNA cloned into plasmid pIJ702 mediated high-frequency transduction of the resulting plasmid (pRHB101) by FP43 in Streptomyces griseofuscus. The transducing particles contained linear concatemers of plasmid DNA. Lysates of FP43 prepared on S. griseofuscus containing pRHB101 also transduced many other Streptomyces species, including several that restrict plaque formation by FP43 and at least two that produce restriction endonucleases that cut pRHB101 DNA. Transduction efficiencies in different species were influenced by the addition of anti-FP43 antiserum to the transduction plates, the temperature for cell growth before transduction, the multiplicity of infection, and the host on which the transducing lysate was prepared. FP43 lysates prepared on S. griseofuscus(pRHB101) also transduced species of Streptoverticillium, Chainia, and Saccharopolyspora.

  10. Transposon insertion reveals pRM, a plasmid of Rickettsia monacensis.

    Science.gov (United States)

    Baldridge, Gerald D; Burkhardt, Nicole Y; Felsheim, Roderick F; Kurtti, Timothy J; Munderloh, Ulrike G

    2007-08-01

    Until the recent discovery of pRF in Rickettsia felis, the obligate intracellular bacteria of the genus Rickettsia (Rickettsiales: Rickettsiaceae) were thought not to possess plasmids. We describe pRM, a plasmid from Rickettsia monacensis, which was detected by pulsed-field gel electrophoresis and Southern blot analyses of DNA from two independent R. monacensis populations transformed by transposon-mediated insertion of coupled green fluorescent protein and chloramphenicol acetyltransferase marker genes into pRM. Two-dimensional electrophoresis showed that pRM was present in rickettsial cells as circular and linear isomers. The 23,486-nucleotide (31.8% G/C) pRM plasmid was cloned from the transformant populations by chloramphenicol marker rescue of restriction enzyme-digested transformant DNA fragments and PCR using primers derived from sequences of overlapping restriction fragments. The plasmid was sequenced. Based on BLAST searches of the GenBank database, pRM contained 23 predicted genes or pseudogenes and was remarkably similar to the larger pRF plasmid. Two of the 23 genes were unique to pRM and pRF among sequenced rickettsial genomes, and 4 of the genes shared by pRM and pRF were otherwise found only on chromosomes of R. felis or the ancestral group rickettsiae R. bellii and R. canadensis. We obtained pulsed-field gel electrophoresis and Southern blot evidence for a plasmid in R. amblyommii isolate WB-8-2 that contained genes conserved between pRM and pRF. The pRM plasmid may provide a basis for the development of a rickettsial transformation vector.

  11. Characterization of the plasmid encoded virulence region pat-1 of phytopathogenic Clavibacter michiganensis subsp. michiganensis.

    Science.gov (United States)

    Dreier, J; Meletzus, D; Eichenlaub, R

    1997-03-01

    The tomato pathogen Clavibacter michiganensis subsp. michiganensis NCPPB382, causing bacterial wilt and canker, harbors two plasmids, pCM1 (27.5 kb) and pCM2 (72 kb), carrying genes involved in virulence. The region of plasmid pCM2 encoding the pathogenicity locus pat-1 was mapped by deletion analysis and complementation studies to a 1.5-kb Bg/II/SmaI DNA fragment. Introduction of the pat-1 region into endophytic, plasmid-free isolates of C. michiganensis subsp. michiganensis converted these bacteria into virulent pathogens. Based on the nucleotide sequence of the pat-1 region, an open reading frame (ORF1) can be predicted, coding for a protein of 280 amino acids and 29.7 kDa with homology to serine proteases. Introduction of a frame-shift mutation in ORF1 leads to a loss of the pathogenic phenotype. Northern (RNA) hybridizations identified an 1.5-knt transcript of the pat-1 structural gene. The site of transcription initiation was mapped by primer extension and a typical -10/-35 region was located with significant homology to the consensus Escherichia coli sigma 70 and Bacillus subtilis sigma 43 promoters. Downstream of the pat-1 structural gene, a peculiar repetitive sequence motif (pat-1rep) is located, consisting of 20 direct tandem repeats preceded by a run of 14 guanosine residues. DNA sequences homologous to pat-1rep were isolated and characterized from four virulent C. michiganensis subsp. michiganensis strains exhibiting a high extent of structural conservation. The deletion of this repetitive sequence reduced virulence significantly but did not lead to a complete loss of the virulence phenotype.

  12. All IncP-1 plasmid subgroups, including the novel e subgroup, are prevalent in the influent of a Danish wastewater treatment plant

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Burmølle, Mette; Meisner, Annelein

    2009-01-01

    IncP-1 plasmids. A neighbor-joining tree, based on a multiple alignment of 72 obtained sequences together with homologous sequences of previously published IncP-1 plasmids, revealed that all established subgroups of IncP-1 plasmids, a, ß, ¿ and d, were present in the wastewater treatment plant......The presence and diversity of IncP-1 plasmids in the influent of a Danish wastewater treatment plant was studied by PCR amplification of the trfA gene in community DNA followed by sequencing. Three sets of PCR primers were designed to amplify a 281 bp fragment of trfA from all currently sequenced...

  13. Prokaryotic homologs of Argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements

    Directory of Open Access Journals (Sweden)

    van der Oost John

    2009-08-01

    Full Text Available Abstract Background In eukaryotes, RNA interference (RNAi is a major mechanism of defense against viruses and transposable elements as well of regulating translation of endogenous mRNAs. The RNAi systems recognize the target RNA molecules via small guide RNAs that are completely or partially complementary to a region of the target. Key components of the RNAi systems are proteins of the Argonaute-PIWI family some of which function as slicers, the nucleases that cleave the target RNA that is base-paired to a guide RNA. Numerous prokaryotes possess the CRISPR-associated system (CASS of defense against phages and plasmids that is, in part, mechanistically analogous but not homologous to eukaryotic RNAi systems. Many prokaryotes also encode homologs of Argonaute-PIWI proteins but their functions remain unknown. Results We present a detailed analysis of Argonaute-PIWI protein sequences and the genomic neighborhoods of the respective genes in prokaryotes. Whereas eukaryotic Ago/PIWI proteins always contain PAZ (oligonucleotide binding and PIWI (active or inactivated nuclease domains, the prokaryotic Argonaute homologs (pAgos fall into two major groups in which the PAZ domain is either present or absent. The monophyly of each group is supported by a phylogenetic analysis of the conserved PIWI-domains. Almost all pAgos that lack a PAZ domain appear to be inactivated, and the respective genes are associated with a variety of predicted nucleases in putative operons. An additional, uncharacterized domain that is fused to various nucleases appears to be a unique signature of operons encoding the short (lacking PAZ pAgo form. By contrast, almost all PAZ-domain containing pAgos are predicted to be active nucleases. Some proteins of this group (e.g., that from Aquifex aeolicus have been experimentally shown to possess nuclease activity, and are not typically associated with genes for other (putative nucleases. Given these observations, the apparent extensive

  14. A monopole homology for integral homology 3-spheres

    OpenAIRE

    Li, Weiping

    2014-01-01

    To an integral homology 3-sphere Y, we assign a well-defined {\\mathbb Z}-graded (monopole) homology MH*(Y, Ih(Q; h0)) whose construction in principle follows from the instanton Floer theory with the dependence of the spectral flow Ih(Q; h0), where Q is the unique U(1)-reducible monopole of the Seiberg-Witten equation on Y and h0 is a reference perturbation datum. The definition uses the moduli space of monopoles on Y \\times {\\mathbb R} introduced by Seiberg-Witten in studying smooth ...

  15. A conserved virulence plasmidic region contributes to the virulence of the multiresistant Escherichia coli meningitis strain S286 belonging to phylogenetic group C.

    Directory of Open Access Journals (Sweden)

    Chloé Lemaître

    Full Text Available Recent isolation of the non-K1 Escherichia coli neonatal meningitis strain S286, belonging to phylogroup C, which is closely related to major group B1, and producing an extended-spectrum beta-lactamase, encouraged us to seek the genetic determinants responsible for its virulence. We show that S286 belongs to the sequence O type ST23O78 and harbors 4 large plasmids. The largest one, pS286colV (~120 kb, not related to resistance, contains genes characteristic of a Conserved Virulence Plasmidic (CVP region initially identified in B2 extra-intestinal avian pathogenic E. coli (APEC strains and in the B2 neonatal meningitis E. coli strain S88. The sequence of this CVP region has a strong homology (98% with that of the recently sequenced plasmid pChi7122-1 of the O78 APEC strain Chi7122. A CVP plasmid-cured variant of S286 was less virulent than the wild type strain in a neonatal rat sepsis model with a significant lower level of bacteremia at 24 h (4.1 ± 1.41 versus 2.60 ± 0.16 log CFU/ml, p = 0.001 and mortality. However, the mortality in the model of adult mice was comparable between wild type and variant indicating that pS286colV is not sufficient by itself to fully explain the virulence of S286. Gene expression analysis of pS286colV in iron depleted environment was very close to that of pS88, suggesting that genes of CVP region may be expressed similarly in two very different genetic backgrounds (group C versus group B2. Screening a collection of 178 human A/B1 extraintestinal pathogenic E. coli (ExPEC strains revealed that the CVP region is highly prevalent (23% and MLST analysis indicated that these CVP positive strains belong to several clusters and mostly to phylogroup C. The virulence of S286 is explained in part by the presence of CVP region and this region has spread in different clusters of human A/B1 ExPEC, especially in group C.

  16. A conserved virulence plasmidic region contributes to the virulence of the multiresistant Escherichia coli meningitis strain S286 belonging to phylogenetic group C.

    Science.gov (United States)

    Lemaître, Chloé; Mahjoub-Messai, Farah; Dupont, Damien; Caro, Valérie; Diancourt, Laure; Bingen, Edouard; Bidet, Philippe; Bonacorsi, Stéphane

    2013-01-01

    Recent isolation of the non-K1 Escherichia coli neonatal meningitis strain S286, belonging to phylogroup C, which is closely related to major group B1, and producing an extended-spectrum beta-lactamase, encouraged us to seek the genetic determinants responsible for its virulence. We show that S286 belongs to the sequence O type ST23O78 and harbors 4 large plasmids. The largest one, pS286colV (~120 kb), not related to resistance, contains genes characteristic of a Conserved Virulence Plasmidic (CVP) region initially identified in B2 extra-intestinal avian pathogenic E. coli (APEC) strains and in the B2 neonatal meningitis E. coli strain S88. The sequence of this CVP region has a strong homology (98%) with that of the recently sequenced plasmid pChi7122-1 of the O78 APEC strain Chi7122. A CVP plasmid-cured variant of S286 was less virulent than the wild type strain in a neonatal rat sepsis model with a significant lower level of bacteremia at 24 h (4.1 ± 1.41 versus 2.60 ± 0.16 log CFU/ml, p = 0.001) and mortality. However, the mortality in the model of adult mice was comparable between wild type and variant indicating that pS286colV is not sufficient by itself to fully explain the virulence of S286. Gene expression analysis of pS286colV in iron depleted environment was very close to that of pS88, suggesting that genes of CVP region may be expressed similarly in two very different genetic backgrounds (group C versus group B2). Screening a collection of 178 human A/B1 extraintestinal pathogenic E. coli (ExPEC) strains revealed that the CVP region is highly prevalent (23%) and MLST analysis indicated that these CVP positive strains belong to several clusters and mostly to phylogroup C. The virulence of S286 is explained in part by the presence of CVP region and this region has spread in different clusters of human A/B1 ExPEC, especially in group C.

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

    OpenAIRE

    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; Paul. B. Rainey; Saunders, Nigel J.; Seeger, Kathy

    2007-01-01

    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 contain...

  18. Homology of locally semialgebraic spaces

    CERN Document Server

    Delfs, Hans

    1991-01-01

    Locally semialgebraic spaces serve as an appropriate framework for studying the topological properties of varieties and semialgebraic sets over a real closed field. This book contributes to the fundamental theory of semialgebraic topology and falls into two main parts. The first dealswith sheaves and their cohomology on spaces which locally look like a constructible subset of a real spectrum. Topics like families of support, homotopy, acyclic sheaves, base-change theorems and cohomological dimension are considered. In the second part a homology theory for locally complete locally semialgebraic spaces over a real closed field is developed, the semialgebraic analogue of classical Bore-Moore-homology. Topics include fundamental classes of manifolds and varieties, Poincare duality, extensions of the base field and a comparison with the classical theory. Applying semialgebraic Borel-Moore-homology, a semialgebraic ("topological") approach to intersection theory on varieties over an algebraically closed field of ch...

  19. Mobile insertion cassette elements found in small non-transmissible plasmids in Proteeae may explain qnrD mobilization.

    Directory of Open Access Journals (Sweden)

    Thomas Guillard

    Full Text Available qnrD is a plasmid mediated quinolone resistance gene from unknown origin, recently described in Enterobacteriaceae. It encodes a pentapeptide repeat protein 36-60% different from the other Qnr (A, B, C, S and VC. Since most qnrD-positive strains were described as strains belonging to Proteus or Providencia genera, we hypothesized that qnrD originated in Proteeae before disseminating to other enterobacterial species. We screened 317 strains of Proteeae for qnrD and its genetic support by PCR. For all the seven qnrD-positive strains (4 Proteus mirabilis, 1 Proteus vulgaris and 2 Providencia rettgeri the gene was carried onto a small non-transmissible plasmid, contrarily to other qnr genes that are usually carried onto large multi-resistant plasmids. Nucleotide sequences of the qnrD-bearing plasmids were 96% identical. Plasmids contained 3 ORFs apart from qnrD and belonged to an undescribed incompatibility group. Only one plasmid, in P. vulgaris, was slightly different with a 1,568-bp insertion between qnrD and its promoter, leading to absence of quinolone resistance. We sought for similar plasmids in 15 reference strains of Proteeae, but which were tested negative for qnrD, and found a 48% identical plasmid (pVERM in Providencia vermicola. In order to explain how qnrD could have been inserted into such native plasmid, we sought for gene mobilization structures. qnrD was found to be located within a mobile insertion cassette (mic element which sequences are similar to one mic also found in pVERM. Our conclusions are that (i the small non-transmissible qnrD-plasmids described here may result from the recombination between an as-yet-unknown progenitor of qnrD and pVERM, (ii these plasmids are maintained in Proteeae being a qnrD reservoir (iii the mic element may explain qnrD mobilization from non-transmissible plasmids to mobilizable or conjugative plasmids from other Enterobacteriaceae, (iv they can recombined with larger multiresistant plasmids

  20. The Salmonella genomic island 1 is specifically mobilized in trans by the IncA/C multidrug resistance plasmid family.

    Science.gov (United States)

    Douard, Gregory; Praud, Karine; Cloeckaert, Axel; Doublet, Benoît

    2010-12-20

    The Salmonella genomic island 1 (SGI1) is a Salmonella enterica-derived integrative mobilizable element (IME) containing various complex multiple resistance integrons identified in several S. enterica serovars and in Proteus mirabilis. Previous studies have shown that SGI1 transfers horizontally by in trans mobilization in the presence of the IncA/C conjugative helper plasmid pR55. Here, we report the ability of different prevalent multidrug resistance (MDR) plasmids including extended-spectrum β-lactamase (ESBL) gene-carrying plasmids to mobilize the multidrug resistance genomic island SGI1. Through conjugation experiments, none of the 24 conjugative plasmids tested of the IncFI, FII, HI2, I1, L/M, N, P incompatibility groups were able to mobilize SGI1 at a detectable level (transfer frequency IncA/C incompatibility group. Several conjugative IncA/C MDR plasmids as well as the sequenced IncA/C reference plasmid pRA1 of 143,963 bp were shown to mobilize in trans SGI1 from a S. enterica donor to the Escherichia coli recipient strain. Depending on the IncA/C plasmid used, the conjugative transfer of SGI1 occurred at frequencies ranging from 10(-3) to 10(-6) transconjugants per donor. Of particular concern, some large IncA/C MDR plasmids carrying the extended-spectrum cephalosporinase bla(CMY-2) gene were shown to mobilize in trans SGI1. The ability of the IncA/C MDR plasmid family to mobilize SGI1 could contribute to its spread by horizontal transfer among enteric pathogens. Moreover, the increasing prevalence of IncA/C plasmids in MDR S. enterica isolates worldwide has potential implications for the epidemic success of the antibiotic resistance genomic island SGI1 and its close derivatives.

  1. Distribution of small native plasmids in Streptococcus pyogenes in India.

    Science.gov (United States)

    Bergmann, René; Nerlich, Andreas; Chhatwal, Gursharan S; Nitsche-Schmitz, D Patric

    2014-05-01

    Complete characterization of a Streptococcus pyogenes population from a defined geographic region comprises information on the plasmids that circulate in these bacteria. Therefore, we determined the distribution of small plasmids (pyogenes isolates from India, where diversity of strains and incidence rates of S. pyogenes infections are high. The collection comprised 77 emm-types. For plasmid detection and discrimination, we developed PCRs for different plasmid replication initiation protein genes, the putative repressor gene copG and bacteriocin genes dysA and scnM57. Plasmid distribution was limited to 13 emm-types. Co-detection analysis using aforementioned PCRs revealed four distinct plasmid sub-types, two of which were previously unknown. Representative plasmids pA852 and pA996 of the two uncharacterized plasmid sub-types were sequenced. These two plasmids could be assigned to the pMV158 and the pC194/pUB110 family of rolling-circle plasmids, respectively. The majority of small plasmids found in India belonged to the two newly characterized sub-types, with pA852- and pA996-like plasmids amounting to 42% and 22% of all detected plasmids, respectively. None of the detected plasmids coded for a known antibiotic resistance gene. Instead, all of the four plasmid sub-types carried known or potential bacteriocin genes. These genes may have influence on the evolutionary success of certain S. pyogenes genotypes. Notably, pA852-like plasmids were found in all isolates of the most prevalent emm-type 11.0. Together, a priori fitness of this genotype and increased fitness due to the acquired plasmids may have rendered type emm11.0 successful and caused the prevalence of pA852-like plasmids in India.

  2. Rapid Construction of EGFP Labled Recombinant Adenovirus Containing hVEGF165 and Its Expression in Haematopoietic Cells

    Institute of Scientific and Technical Information of China (English)

    仲照东; 邹萍; 黄士昂; 胡中波; 刘凌波; 卢运萍

    2003-01-01

    By using AdEasy system, which is based on the homologous recombination in bacteria, an EGFP labeled recombinant adenovirus vector containing hVEGF165 was constructed quickly and efficiently expressed in mouse haematopoietic cells. First, hVEGF165 coding sequence was subcloned into shuttle plasmid pAdTrack-CMV, then cotransformed with adenoviral backbone vector pAdEasy-1 into E. coli strain BJ5183. The recombinant adenoviral plasmid was transfected into HEK293 cells to assembly replication-defective adenovirus Ad-EGFP/hVEGF165. The expression of EGFP could be easily detected. The rate of EGFP positive mouse bone marrow mononuclear cells by flow cytometric analysis was 27.3 % (MOI= 100), and the expression of hVEGF165 protein in the supernatant was (1385+332) pg/106 cells. These results suggest that the construction of adenovirus vector by homologous recombination in bacteria features high efficiency and simplicity. The prepared high titer AdEGFP/hVEGF165 can be used an efficient helpful vector to infect hematopoietic cells.

  3. Complete DNA sequence and gene analysis of the virulence plasmid pCP301 of Shigella flexneri 2a

    Institute of Scientific and Technical Information of China (English)

    张继瑜; 刘红; 张笑兵; 杨剑; 杨帆; 杨国威; 沈岩; 侯云德; 金奇

    2003-01-01

    The complete nucleotide sequence and organization of the large virulence plasmid pCP301 (termed by us) of Shigella flexneri 2a strain 301 were determined and analyzed. The result showed that the entire DNA sequence of pCP301 is composed of 221618 bp which form a circular plasmid. Sequence analysis identified 272 open reading frames (ORFs), among which, 194 correspond to the proteins described previously, 61 have low identity (<60%) to known proteins and the rest 17 have no regions of significant homology with proteins in database. The genes of pCP301 mainly include the genes associated with bacterial virulence, the genes associated with regulation and the genes relating to plasmid maintenance, stability and DNA metabolism. Insertion sequence (IS) elements are 68 kb in length and account for 30 percent of complete sequence of the plasmid which indicates that gene multiple rearrangements of the pCP301 have taken place in Shigella flexneri evolution history. The research result is helpful for interpreting the pathogenesis of Shigella, as well as the genetics and evolution of the plasmid.

  4. Toward the identification of a type I toxin-antitoxin system in the plasmid DNA of dairy Lactobacillus rhamnosus.

    Science.gov (United States)

    Folli, Claudia; Levante, Alessia; Percudani, Riccardo; Amidani, Davide; Bottazzi, Stefania; Ferrari, Alberto; Rivetti, Claudio; Neviani, Erasmo; Lazzi, Camilla

    2017-09-21

    Plasmids carry genes that give bacteria beneficial traits and allow them to survive in competitive environments. In many cases, they also harbor toxin-antitoxin (TA) systems necessary for plasmid maintenance. TA systems are generally characterized by a stable "toxin", a protein or peptide capable of killing the cell upon plasmid loss and by an unstable "antitoxin", a protein or a non-coding RNA that inhibits toxin activity. Here we report data toward the identification of a RNA-regulated TA system in the plasmid DNA of L. rhamnosus isolated from cheese. The proposed TA system comprises two convergently transcribed RNAs: a toxin RNA encoding a 29 amino acid peptide named Lpt and an antitoxin non-coding RNA. Both toxin and antitoxin RNAs resulted upregulated under conditions mimicking cheese ripening. The toxicity of the Lpt peptide was demonstrated in E. coli by cloning the Lpt ORF under the control of an inducible promoter. Bioinformatics screening of the bacterial nucleotide database, shows that regions homologous to the Lpt TA locus are widely distributed in the Lactobacillus genus, particularly within the L. casei group, suggesting a relevant role of TA systems in plasmid maintenance of cheese microbiota.

  5. Characterization of a novel type of MLSB resistance plasmid from Staphylococcus saprophyticus carrying a constitutively expressed erm(C) gene.

    Science.gov (United States)

    Hauschild, Tomasz; Lüthje, Petra; Schwarz, Stefan

    2006-06-15

    An erm(C)-carrying plasmid of unusual size and restriction map, designated pSES22, was identified in a Staphylococcus saprophyticus strain and sequenced completely. Constitutive expression of the erm(C) gene from pSES22 is based on a novel 22-bp tandem duplication in the erm(C) translational attenuator. Comparative analysis of the deduced Erm(C) amino acid sequence revealed that Erm(C) from pSES22 - together with an Erm(C) methylase from S. hyicus - represented a separate branch in the homology tree of Erm(C) methylases. Structural comparisons showed that plasmid pSES22 differed distinctly from all other completely sequenced erm(C)-carrying resistance plasmids. However, pSES22 was similar to several members of a diverse group of small plasmids, all of which carried closely related plasmid backbones consisting of the genes repU and pre/mob, but differed in their resistance genes.

  6. Homology group on manifolds and their foldings

    Directory of Open Access Journals (Sweden)

    M. Abu-Saleem

    2010-03-01

    Full Text Available In this paper, we introduce the definition of the induced unfolding on the homology group. Some types of conditional foldings restricted on the elements of the homology groups are deduced. The effect of retraction on the homology group of a manifold is dicussed. The unfolding of variation curvature of manifolds on their homology group are represented. The relations between homology group of the manifold and its folding are deduced.

  7. MIP1, a new yeast gene homologous to the rat mitochondrial intermediate peptidase gene, is required for oxidative metabolism in Saccharomyces cerevisiae.

    Science.gov (United States)

    Isaya, G; Miklos, D; Rollins, R A

    1994-08-01

    Cleavage of amino-terminal octapeptides, F/L/IXXS/T/GXXXX, by mitochondrial intermediate peptidase (MIP) is typical of many mitochondrial precursor proteins imported to the matrix and the inner membrane. We previously described the molecular characterization of rat liver MIP (RMIP) and indicated a putative homolog in the sequence predicted from gene YCL57w of yeast chromosome III. A new yeast gene, MIP1, has now been isolated by screening a Saccharomyces cerevisiae genomic library with an RMIP cDNA probe. MIP1 predicts a protein of 772 amino acids (YMIP), which is 54% similar and 31% identical to RMIP and includes a putative 37-residue mitochondrial leader peptide. RMIP and YMIP contain the sequence LFHEMGHAM HSMLGRT, which includes a zinc-binding motif, HEXXH, while the predicted YCL57w protein contains a comparable sequence with a lower degree of homology. No obvious biochemical phenotype was observed in a chromosomally disrupted ycl57w mutant. In contrast, a mip1 mutant was unable to grow on nonfermentable substrates, while a mip1 ycl57w double disruption did not result in a more severe phenotype. The mip1 mutant exhibited defects of complexes III and IV of the respiratory chain, caused by failure to carry out the second MIP-catalyzed cleavage of the nuclear-encoded precursors for cytochrome oxidase subunit IV (CoxIV) and the iron-sulfur protein (Fe-S) of the bc1 complex to mature proteins. In vivo, intermediate-size CoxIV was accumulated in the mitochondrial matrix, while intermediate-size Fe-S was targeted to the inner membrane. Moreover, mip1 mitochondrial fractions failed to carry out maturation of the human ornithine transcarbamylase intermediate (iOTC), specifically cleaved by RMIP. A CEN plasmid-encoded YMIP protein restored normal MIP activity along with respiratory competence. Thus, YMIP is a functional homolog of RMIP and represents a new component of the yeast mitochondrial import machinery.

  8. [Construction of Frankia genomic libraries and isolation of clones homologous to nodulation genes from Rhizobium leguminosarum].

    Science.gov (United States)

    Cui, Y H; Qin, M; Wang, Y L; Ding, J; Ma, Q S

    1990-01-01

    High molecular genomic DNAs were isolated by using the lysozyme plus achromopeptidase system from Frankia strains At4, Ccol and Hr16, the root nodule endophytes of Alnus, Casuarina and Hippophae respectively, and used to construct genomic libraries in pLAFR1, a broad host range cosmid vector within many gram-negative hosts. The genomic libraries were screened by in situ colony hybridization to identify clones homologous to common nodulation genes of Rhizobium leguminosarum, based on the sequence homology of EcoRI-digested Frankia total DNA to nodABC from Rhizobium meliloti. Several clones showing relatively strong hybridization were found, the recombinant plasmid was isolated, and their homology with Rhizobium nodulation genes was confirmed by spot hybridization. Further work on these positive clones is now underway.

  9. Klebsiella pneumoniae multiresistance plasmid pMET1: similarity with the Yersinia pestis plasmid pCRY and integrative conjugative elements.

    Directory of Open Access Journals (Sweden)

    Alfonso J C Soler Bistué

    Full Text Available BACKGROUND: Dissemination of antimicrobial resistance genes has become an important public health and biodefense threat. Plasmids are important contributors to the rapid acquisition of antibiotic resistance by pathogenic bacteria. PRINCIPAL FINDINGS: The nucleotide sequence of the Klebsiella pneumoniae multiresistance plasmid pMET1 comprises 41,723 bp and includes Tn1331.2, a transposon that carries the bla(TEM-1 gene and a perfect duplication of a 3-kbp region including the aac(6'-Ib, aadA1, and bla(OXA-9 genes. The replication region of pMET1 has been identified. Replication is independent of DNA polymerase I, and the replication region is highly related to that of the cryptic Yersinia pestis 91001 plasmid pCRY. The potential partition region has the general organization known as the parFG locus. The self-transmissible pMET1 plasmid includes a type IV secretion system consisting of proteins that make up the mating pair formation complex (Mpf and the DNA transfer (Dtr system. The Mpf is highly related to those in the plasmid pCRY, the mobilizable high-pathogenicity island from E. coli ECOR31 (HPI(ECOR31, which has been proposed to be an integrative conjugative element (ICE progenitor of high-pathogenicity islands in other Enterobacteriaceae including Yersinia species, and ICE(Kp1, an ICE found in a K. pneumoniae strain causing primary liver abscess. The Dtr MobB and MobC proteins are highly related to those of pCRY, but the endonuclease is related to that of plasmid pK245 and has no significant homology with the protein of similar function in pCRY. The region upstream of mobB includes the putative oriT and shares 90% identity with the same region in the HPI(ECOR31. CONCLUSIONS: The comparative analyses of pMET1 with pCRY, HPI(ECOR31, and ICE(Kp1 show a very active rate of genetic exchanges between Enterobacteriaceae including Yersinia species, which represents a high public health and biodefense threat due to transfer of multiple resistance

  10. Molecular analysis of the replication origin of the Lactococcus lactis plasmid pCI305

    NARCIS (Netherlands)

    Foley, S; Bron, S; Venema, G; Daly, C; Fitzgerald, GF

    1996-01-01

    The replication origin region, ori, of the Lactococcus lactis subsp. lactis plasmid pCI305 contains three-and-one-half directly repeated 22-bp sequences and two inverted repeat sequences, IR1 and IR2. These inverted repeat sequences overlap the promoter of the repB gene, which encodes a protein (Rep

  11. Transgene organisation in potato after particle bombardment-mediated (co-) transformation using plasmids and gene cassettes

    NARCIS (Netherlands)

    Romano, A.; Raemakers, C.J.J.M.; Bernardi, J.; Visser, R.G.F.; Mooibroek, A.

    2003-01-01

    Protocols for efficient co-transformation of potato internodes with genes contained in separate plasmids or gene cassettes (i.e., linear PCR fragments comprising a promoter-gene-terminator) using particle bombardment were established. Twenty-eight out of 62 (45%) and 11 out of 65 (17%) plants transf

  12. Dutch patients, retail chicken meat and poultry share the same ESBL genes, plasmids and strains.

    Science.gov (United States)

    Leverstein-van Hall, M A; Dierikx, C M; Cohen Stuart, J; Voets, G M; van den Munckhof, M P; van Essen-Zandbergen, A; Platteel, T; Fluit, A C; van de Sande-Bruinsma, N; Scharinga, J; Bonten, M J M; Mevius, D J

    2011-06-01

    Intestinal carriage of extended-spectrum beta-lactamase (ESBL) -producing bacteria in food-producing animals and contamination of retail meat may contribute to increased incidences of infections with ESBL-producing bacteria in humans. Therefore, distribution of ESBL genes, plasmids and strain genotypes in Escherichia coli obtained from poultry and retail chicken meat in the Netherlands was determined and defined as 'poultry-associated' (PA). Subsequently, the proportion of E. coli isolates with PA ESBL genes, plasmids and strains was quantified in a representative sample of clinical isolates. The E. coli were derived from 98 retail chicken meat samples, a prevalence survey among poultry, and 516 human clinical samples from 31 laboratories collected during a 3-month period in 2009. Isolates were analysed using an ESBL-specific microarray, sequencing of ESBL genes, PCR-based replicon typing of plasmids, plasmid multi-locus sequence typing (pMLST) and strain genotyping (MLST). Six ESBL genes were defined as PA (bla(CTX-M-1) , bla(CTX-M-2) , bla(SHV-2) , bla(SHV-12) , bla(TEM-20) , bla(TEM-52) ): 35% of the human isolates contained PA ESBL genes and 19% contained PA ESBL genes located on IncI1 plasmids that were genetically indistinguishable from those obtained from poultry (meat). Of these ESBL genes, 86% were bla(CTX-M-1) and bla(TEM-52) genes, which were also the predominant genes in poultry (78%) and retail chicken meat (75%). Of the retail meat samples, 94% contained ESBL-producing isolates of which 39% belonged to E. coli genotypes also present in human samples. These findings are suggestive for transmission of ESBL genes, plasmids and E. coli isolates from poultry to humans, most likely through the food chain.

  13. Stress responses and replication of plasmids in bacterial cells

    Directory of Open Access Journals (Sweden)

    Wegrzyn Alicja

    2002-05-01

    Full Text Available Abstract Plasmids, DNA (or rarely RNA molecules which replicate in cells autonomously (independently of chromosomes as non-essential genetic elements, play important roles for microbes grown under specific environmental conditions as well as in scientific laboratories and in biotechnology. For example, bacterial plasmids are excellent models in studies on regulation of DNA replication, and their derivatives are the most commonly used vectors in genetic engineering. Detailed mechanisms of replication initiation, which is the crucial process for efficient maintenance of plasmids in cells, have been elucidated for several plasmids. However, to understand plasmid biology, it is necessary to understand regulation of plasmid DNA replication in response to different environmental conditions in which host cells exist. Knowledge of such regulatory processes is also very important for those who use plasmids as expression vectors to produce large amounts of recombinant proteins. Variable conditions in large-scale fermentations must influence replication of plasmid DNA in cells, thus affecting the efficiency of recombinant gene expression significantly. Contrary to extensively investigated biochemistry of plasmid replication, molecular mechanisms of regulation of plasmid DNA replication in response to various environmental stress conditions are relatively poorly understood. There are, however, recently published studies that add significant data to our knowledge on relations between cellular stress responses and control of plasmid DNA replication. In this review we focus on plasmids derived from bacteriophage λ that are among the best investigated replicons. Nevertheless, recent results of studies on other plasmids are also discussed shortly.

  14. Mediators of homologous DNA pairing.

    Science.gov (United States)

    Zelensky, Alex; Kanaar, Roland; Wyman, Claire

    2014-10-09

    Homologous DNA pairing and strand exchange are at the core of homologous recombination. These reactions are promoted by a DNA-strand-exchange protein assembled into a nucleoprotein filament comprising the DNA-pairing protein, ATP, and single-stranded DNA. The catalytic activity of this molecular machine depends on control of its dynamic instability by accessory factors. Here we discuss proteins known as recombination mediators that facilitate formation and functional activation of the DNA-strand-exchange protein filament. Although the basics of homologous pairing and DNA-strand exchange are highly conserved in evolution, differences in mediator function are required to cope with differences in how single-stranded DNA is packaged by the single-stranded DNA-binding protein in different species, and the biochemical details of how the different DNA-strand-exchange proteins nucleate and extend into a nucleoprotein filament. The set of (potential) mediator proteins has apparently expanded greatly in evolution, raising interesting questions about the need for additional control and coordination of homologous recombination in more complex organisms. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

  15. Homological Type of Geometric Transitions

    CERN Document Server

    Rossi, Michele

    2010-01-01

    The present paper gives an account and quantifies the change in topology induced by small and type II geometric transitions, by introducing the notion of the \\emph{homological type} of a geometric transition. The obtained results agree with, and go further than, most results and estimates, given to date by several authors, both in mathematical and physical literature.

  16. Homological stability of diffeomorphism groups

    DEFF Research Database (Denmark)

    Berglund, Alexander; Madsen, Ib Henning

    2013-01-01

    In this paper we prove a stability theorem for block diffeomorphisms of 2d -dimensional manifolds that are connected sums of S d ×S d . Combining this with a recent theorem of S. Galatius and O. Randal-Williams and Morlet’s lemma of disjunction, we determine the homology of the classifying space ...

  17. The contribution of homology arms to nuclease-assisted genome engineering.

    Science.gov (United States)

    Baker, Oliver; Tsurkan, Sarah; Fu, Jun; Klink, Barbara; Rump, Andreas; Obst, Mandy; Kranz, Andrea; Schröck, Evelin; Anastassiadis, Konstantinos; Stewart, A Francis

    2017-07-27

    Designer nucleases like CRISPR/Cas9 enable fluent site-directed damage or small mutations in many genomes. Strategies for their use to achieve more complex tasks like regional exchanges for gene humanization or the establishment of conditional alleles are still emerging. To optimize Cas9-assisted targeting, we measured the relationship between targeting frequency and homology length in targeting constructs using a hypoxanthine-guanine phosphoribosyl-transferase assay in mouse embryonic stem cells. Targeting frequency with supercoiled plasmids improved steeply up to 2 kb total homology and continued to increase with even longer homology arms, thereby implying that Cas9-assisted targeting efficiencies can be improved using homology arms of 1 kb or greater. To humanize the Kmt2d gene, we built a hybrid mouse/human targeting construct in a bacterial artificial chromosome by recombineering. To simplify the possible outcomes, we employed a single Cas9 cleavage strategy and best achieved the intended 42 kb regional exchange with a targeting construct including a very long homology arm to recombine ∼42 kb away from the cleavage site. We recommend the use of long homology arm targeting constructs for accurate and efficient complex genome engineering, particularly when combined with the simplifying advantages of using just one Cas9 cleavage at the genome target site. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. Plasmids of psychrophilic and psychrotolerant bacteria and their role in adaptation to cold environments.

    Science.gov (United States)

    Dziewit, Lukasz; Bartosik, Dariusz

    2014-01-01

    Extremely cold environments are a challenge for all organisms. They are mostly inhabited by psychrophilic and psychrotolerant bacteria, which employ various strategies to cope with the cold. Such harsh environments are often highly vulnerable to the influence of external factors and may undergo frequent dynamic changes. The rapid adjustment of bacteria to changing environmental conditions is crucial for their survival. Such "short-term" evolution is often enabled by plasmids-extrachromosomal replicons that represent major players in horizontal gene transfer. The genomic sequences of thousands of microorganisms, including those of many cold-active bacteria have been obtained over the last decade, but the collected data have yet to be thoroughly analyzed. This report describes the results of a meta-analysis of the NCBI sequence databases to identify and characterize plasmids of psychrophilic and psychrotolerant bacteria. We have performed in-depth analyses of 66 plasmids, almost half of which are cryptic replicons not exceeding 10 kb in size. Our analyses of the larger plasmids revealed the presence of numerous genes, which may increase the phenotypic flexibility of their host strains. These genes encode enzymes possibly involved in (i) protection against cold and ultraviolet radiation, (ii) scavenging of reactive oxygen species, (iii) metabolism of amino acids, carbohydrates, nucleotides and lipids, (iv) energy production and conversion, (v) utilization of toxic organic compounds (e.g., naphthalene), and (vi) resistance to heavy metals, metalloids and antibiotics. Some of the plasmids also contain type II restriction-modification systems, which are involved in both plasmid stabilization and protection against foreign DNA. Moreover, approx. 50% of the analyzed plasmids carry genetic modules responsible for conjugal transfer or mobilization for transfer, which may facilitate the spread of these replicons among various bacteria, including across species boundaries.

  19. Increasing plasmid transformation efficiency of natural spizizen method in Bacillus Subtilis by a cell permeable peptide

    Directory of Open Access Journals (Sweden)

    Mehrdad Moosazadeh Moghaddam

    2013-01-01

    Full Text Available Introduction: Some of bacterial species are able to uptake DNA molecule from environment, the yield of this process depends on some conditions such as plasmid size and host type. In the case of Bacillus subtilis, DNA uptake has low efficacy. Using Spizizen minimal medium is common method in plasmid transformation into B. subtilis, but rate of this process is not suitable and noteworthy. The aim of this study was investigation of novel method for improvement of DNA transformation into B. subtilis based on CM11 cationic peptide as a membrane permeable agent.Materials and methods: In this study, for optimization of pWB980 plasmid transformation into B. subtilis, the CM11 cationic peptide was used. For this purpose, B. subtilis competent cell preparation in the present of different concentration of peptide was implemented by two methods. In the first method, after treatment of bacteria with different amount of peptide for 14h, plasmid was added. In the second method, several concentration of peptide with plasmid was exposed to bacteria simultaneously. Bacteria that uptake DNA were screened on LB agar medium containing kanamycin. The total transformed bacteria per microgram of DNA was calculated and compared with the control.Results: Plasmid transformation in best conditions was 6.5 folds higher than the control. This result was statistically significant (P value <0.001.Discussion and conclusion: This study showed that CM11 cationic peptide as a membrane permeable agent was able to increase plasmid transformation rate into B. subtilis. This property was useful for resolution of low transformation efficacy.

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

    Science.gov (United States)

    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.

  1. DNA polymerase beta can substitute for DNA polymerase I in the initiation of plasmid DNA replication.

    OpenAIRE

    1995-01-01

    We previously demonstrated that mammalian DNA polymerase beta can substitute for DNA polymerase I of Escherichia coli in DNA replication and in base excision repair. We have now obtained genetic evidence suggesting that DNA polymerase beta can substitute for E. coli DNA polymerase I in the initiation of replication of a plasmid containing a pMB1 origin of DNA replication. Specifically, we demonstrate that a plasmid with a pMB1 origin of replication can be maintained in an E. coli polA mutant ...

  2. Expression of Hepatitis B Virus Surface Antigen Gene in Cultured Cells by Using Recombinant Plasmid Vectors

    OpenAIRE

    Siddiqui, Aleem

    1983-01-01

    By using a new host-vector system, expression of the gene coding for hepatitis B surface antigen has been studied. A subgenomic fragment of cloned hepatitis B viral DNA was inserted into the plasmid vector pSV010. Transfection of COS cells with the recombinant plasmid vector containing hepatitis sequences leads to the synthesis of hepatitis B surface antigen, which is released in the culture medium in the form of 22-nm particles similar to those found in the sera of hepatitis carriers.

  3. Plasmid mediated quinolone resistance in Enterobacteriaceae

    NARCIS (Netherlands)

    Veldman, K.T.; LS Klinisch Onderzoek Wagenaar

    2014-01-01

    This thesis describes the occurrence of Plasmid Mediated Quinolone Resistance (PMQR) in Salmonella and E. coli from The Netherlands and other European countries. Furthermore, the genetic background of these genes was characterized. Fluoroquinolones are widely used antibiotics in both human and

  4. Plasmid mediated quinolone resistance in Enterobacteriaceae

    NARCIS (Netherlands)

    Veldman, K.T.; LS Klinisch Onderzoek Wagenaar

    2014-01-01

    This thesis describes the occurrence of Plasmid Mediated Quinolone Resistance (PMQR) in Salmonella and E. coli from The Netherlands and other European countries. Furthermore, the genetic background of these genes was characterized. Fluoroquinolones are widely used antibiotics in both human and veter

  5. Plasmid mediated quinolone resistance in Enterobacteriaceae

    NARCIS (Netherlands)

    Veldman, K.T.; LS Klinisch Onderzoek Wagenaar

    2014-01-01

    This thesis describes the occurrence of Plasmid Mediated Quinolone Resistance (PMQR) in Salmonella and E. coli from The Netherlands and other European countries. Furthermore, the genetic background of these genes was characterized. Fluoroquinolones are widely used antibiotics in both human and veter

  6. A Type III protein-RNA toxin-antitoxin system from Bacillus thuringiensis promotes plasmid retention during spore development.

    Science.gov (United States)

    Short, Francesca L; Monson, Rita E; Salmond, George P C

    2015-01-01

    Members of the Bacillus cereus sensu lato group of bacteria often contain multiple large plasmids, including those encoding virulence factors in B. anthracis. Bacillus species can develop into spores in response to stress. During sporulation the genomic content of the cell is heavily compressed, which could result in counterselection of extrachromosomal genomic elements, unless they have robust stabilization and segregation systems. Toxin-antitoxin (TA) systems are near-ubiquitous in prokaryotes and have multiple biological roles, including plasmid stabilization during vegetative growth. Here, we have shown that a Type III TA system, based on an RNA antitoxin and endoribonuclease toxin, from plasmid pAW63 in Bacillus thuringiensis serovar kurstaki HD-73 can dramatically promote plasmid retention in populations undergoing sporulation and germination, and we provide evidence that this occurs through the post-segregational killing of plasmid-free forespores. Our findings show how an extremely common genetic module can be used to ensure plasmid maintenance during stress-induced developmental transitions, with implications for plasmid dynamics in B. cereus s.l. bacteria.

  7. The porcine circovirus type 1 capsid gene promoter improves antigen expression and immunogenicity in a HIV-1 plasmid vaccine

    Directory of Open Access Journals (Sweden)

    Burger Marieta

    2011-02-01

    Full Text Available Abstract Background One of the promising avenues for development of vaccines against Human immunodeficiency virus type 1 (HIV-1 and other human pathogens is the use of plasmid-based DNA vaccines. However, relatively large doses of plasmid must be injected for a relatively weak response. We investigated whether genome elements from Porcine circovirus type 1 (PCV-1, an apathogenic small ssDNA-containing virus, had useful expression-enhancing properties that could allow dose-sparing in a plasmid vaccine. Results The linearised PCV-1 genome inserted 5' of the CMV promoter in the well-characterised HIV-1 plasmid vaccine pTHgrttnC increased expression of the polyantigen up to 2-fold, and elicited 3-fold higher CTL responses in mice at 10-fold lower doses than unmodified pTHgrttnC. The PCV-1 capsid gene promoter (Pcap alone was equally effective. Enhancing activity was traced to a putative composite host transcription factor binding site and a "Conserved Late Element" transcription-enhancing sequence previously unidentified in circoviruses. Conclusions We identified a novel PCV-1 genome-derived enhancer sequence that significantly increased antigen expression from plasmids in in vitro assays, and improved immunogenicity in mice of the HIV-1 subtype C vaccine plasmid, pTHgrttnC. This should allow significant dose sparing of, or increased responses to, this and other plasmid-based vaccines. We also report investigations of the potential of other circovirus-derived sequences to be similarly used.

  8. DNA sequence analysis of the composite plasmid pTC conferring virulence and antimicrobial resistance for porcine enterotoxigenic Escherichia coli.

    Science.gov (United States)

    Fekete, Péter Z; Brzuszkiewicz, Elzbieta; Blum-Oehler, Gabriele; Olasz, Ferenc; Szabó, Mónika; Gottschalk, Gerhard; Hacker, Jörg; Nagy, Béla

    2012-01-01

    In this study the plasmid pTC, a 90 kb self-conjugative virulence plasmid of the porcine enterotoxigenic Escherichia coli (ETEC) strain EC2173 encoding the STa and STb heat-stable enterotoxins and tetracycline resistance, has been sequenced in two steps. As a result we identified five main distinct regions of pTC: (i) the maintenance region responsible for the extreme stability of the plasmid, (ii) the TSL (toxin-specific locus comprising the estA and estB genes) which is unique and characteristic for pTC, (iii) a Tn10 transposon, encoding tetracycline resistance, (iv) the tra (plasmid transfer) region, and (v) the colE1-like origin of replication. It is concluded that pTC is a self-transmissible composite plasmid harbouring antibiotic resistance and virulence genes. pTC belongs to a group of large conjugative E. coli plasmids represented by NR1 with a widespread tra backbone which might have evolved from a common ancestor. This is the first report of a completely sequenced animal ETEC virulence plasmid containing an antimicrobial resistance locus, thereby representing a selection advantage for spread of pathogenicity in the presence of antimicrobials leading to increased disease potential. Copyright © 2011. Published by Elsevier GmbH.

  9. Plasmids spread very fast in heterogeneous bacterial communities.

    Science.gov (United States)

    Dionisio, Francisco; Matic, Ivan; Radman, Miroslav; Rodrigues, Olivia R; Taddei, François

    2002-01-01

    Conjugative plasmids can mediate gene transfer between bacterial taxa in diverse environments. The ability to donate the F-type conjugative plasmid R1 greatly varies among enteric bacteria due to the interaction of the system that represses sex-pili formations (products of finOP) of plasmids already harbored by a bacterial strain with those of the R1 plasmid. The presence of efficient donors in heterogeneous bacterial populations can accelerate plasmid transfer and can spread by several orders of magnitude. Such donors allow millions of other bacteria to acquire the plasmid in a matter of days whereas, in the absence of such strains, plasmid dissemination would take years. This "amplification effect" could have an impact on the evolution of bacterial pathogens that exist in heterogeneous bacterial communities because conjugative plasmids can carry virulence or antibiotic-resistance genes. PMID:12524329

  10. Disruption of an ADE6 Homolog of Ustilago maydis

    Science.gov (United States)

    Ustilago maydis secretes iron-binding compounds during times of iron depletion. A putative homolog of the Sacharromyces cereviseae ADE6 and Escherichia coli purL genes was identified near a multigenic complex, which contains two genes sid1 and sid2 involved in a siderophore biosynthetic pathway. The...

  11. Endogenous mutagenesis in recombinant sulfolobus plasmids.

    Science.gov (United States)

    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.

  12. Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes.

    Science.gov (United States)

    McDaniel, Jonathan R; Mackay, J Andrew; Quiroz, Felipe García; Chilkoti, Ashutosh

    2010-04-12

    This paper reports a new strategy, recursive directional ligation by plasmid reconstruction (PRe-RDL), to rapidly clone highly repetitive polypeptides of any sequence and specified length over a large range of molecular weights. In a single cycle of PRe-RDL, two halves of a parent plasmid, each containing a copy of an oligomer, are ligated together, thereby dimerizing the oligomer and reconstituting a functional plasmid. This process is carried out recursively to assemble an oligomeric gene with the desired number of repeats. PRe-RDL has several unique features that stem from the use of type IIs restriction endonucleases: first, PRe-RDL is a seamless cloning method that leaves no extraneous nucleotides at the ligation junction. Because it uses type IIs endonucleases to ligate the two halves of the plasmid, PRe-RDL also addresses the major limitation of RDL in that it abolishes any restriction on the gene sequence that can be oligomerized. The reconstitution of a functional plasmid only upon successful ligation in PRe-RDL also addresses two other limitations of RDL: the significant background from self-ligation of the vector observed in RDL, and the decreased efficiency of ligation due to nonproductive circularization of the insert. PRe-RDL can also be used to assemble genes that encode different sequences in a predetermined order to encode block copolymers or append leader and trailer peptide sequences to the oligomerized gene.

  13. Characterization of Plasmid pPO1 from the Hyperacidophile Picrophilus oshimae

    Directory of Open Access Journals (Sweden)

    Angel Angelov

    2011-01-01

    Full Text Available Picrophilus oshimae and Picrophilus torridus are free-living, moderately thermophilic and acidophilic organisms from the lineage of Euryarchaeota. With a pH optimum of growth at pH 0.7 and the ability to even withstand molar concentrations of sulphuric acid, these organisms represent the most extreme acidophiles known. So far, nothing is known about plasmid biology in these hyperacidophiles. Also, there are no genetic tools available for this genus. We have mobilized the 7.6 Kbp plasmid from P. oshimae in E. coli by introducing origin-containing transposons and described the plasmid in terms of its nucleotide sequence, copy number in the native host, mode of replication, and transcriptional start sites of the encoded ORFs. Plasmid pPO1 may encode a restriction/modification system in addition to its replication functions. The information gained from the pPO1 plasmid may prove useful in developing a cloning system for this group of extreme acidophiles.

  14. Mechanism of DNA Segregation in Prokaryotes: Replicon Pairing by parC of Plasmid R1

    Science.gov (United States)

    Jensen, Rasmus Bugge; Lurz, Rudi; Gerdes, Kenn

    1998-07-01

    Prokaryotic chromosomes and plasmids encode partitioning systems that are required for DNA segregation at cell division. The systems are thought to be functionally analogous to eukaryotic centromeres and to play a general role in DNA segregation. The parA system of plasmid R1 encodes two proteins ParM and ParR, and a cis-acting centromere-like site denoted parC. The ParR protein binds to parC in vivo and in vitro. The ParM protein is an ATPase that interacts with ParR specifically bound to parC. Using electron microscopy, we show here that parC mediates efficient pairing of plasmid molecules. The pairing requires binding of ParR to parC and is stimulated by the ParM ATPase. The ParM mediated stimulation of plasmid pairing is dependent on ATP hydrolysis by ParM. Using a ligation kinetics assay, we find that ParR stimulates ligation of parC-containing DNA fragments. The rate-of-ligation was increased by wild type ParM protein but not by mutant ParM protein deficient in the ATPase activity. Thus, two independent assays show that parC mediates pairing of plasmid molecules in vitro. These results are consistent with the proposal that replicon pairing is part of the mechanism of DNA segregation in prokaryotes.

  15. Structures of actin-like ParM filaments show architecture of plasmid-segregating spindles.

    Science.gov (United States)

    Bharat, Tanmay A M; Murshudov, Garib N; Sachse, Carsten; Löwe, Jan

    2015-07-02

    Active segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments. Growing ParMRC spindles push sister plasmids to the cell poles. Here, using modern electron cryomicroscopy methods, we investigate the structures and arrangements of ParM filaments in vitro and in cells, revealing at near-atomic resolution how subunits and filaments come together to produce the simplest known mitotic machinery. To understand the mechanism of dynamic instability, we determine structures of ParM filaments in different nucleotide states. The structure of filaments bound to the ATP analogue AMPPNP is determined at 4.3 Å resolution and refined. The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions. Also using electron cryomicroscopy, we reconstruct ParM doublets forming antiparallel spindles. Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.

  16. Plasmid-Mediated Dimethoate Degradation by Bacillus licheniformis Isolated From a Fresh Water Fish Labeo rohita

    Directory of Open Access Journals (Sweden)

    Manisha Deb Mandal

    2005-01-01

    Full Text Available The Bacillus licheniformis strain isolated from the intestine of Labeo rohita by an enrichment technique showed capability of utilizing dimethoate as the sole source of carbon. The bacterium rapidly utilized dimethoate beyond 0.6 mg/mL and showed prolific growth in a mineral salts medium containing 0.45 mg/mL dimethoate. The isolated B licheniformis exhibited high level of tolerance of dimethoate (3.5 mg/mL in nutrient broth, while its cured mutant did not tolerate dimethoate beyond 0.45 mg/mL and it was unable to utilize dimethoate. The wild B licheniformis strain transferred dimethoate degradation property to E coli C600 (Nar, F− strain. The transconjugant harbored a plasmid of the same molecular size (approximately 54 kb as that of the donor plasmid; the cured strain was plasmid less. Thus a single plasmid of approximately 54 kb was involved in dimethoate degradation. Genes encoding resistance to antibiotic and heavy metal were also located on the plasmid.

  17. Plasmid-Mediated Dimethoate Degradation by Bacillus licheniformis Isolated From a Fresh Water Fish Labeo rohita

    Science.gov (United States)

    2005-01-01

    The Bacillus licheniformis strain isolated from the intestine of Labeo rohita by an enrichment technique showed capability of utilizing dimethoate as the sole source of carbon. The bacterium rapidly utilized dimethoate beyond 0.6 mg/mL and showed prolific growth in a mineral salts medium containing 0.45 mg/mL dimethoate. The isolated B licheniformis exhibited high level of tolerance of dimethoate (3.5 mg/mL) in nutrient broth, while its cured mutant did not tolerate dimethoate beyond 0.45 mg/mL and it was unable to utilize dimethoate. The wild B licheniformis strain transferred dimethoate degradation property to E coli C600 (Nar, F−) strain. The transconjugant harbored a plasmid of the same molecular size (approximately 54 kb) as that of the donor plasmid; the cured strain was plasmid less. Thus a single plasmid of approximately 54 kb was involved in dimethoate degradation. Genes encoding resistance to antibiotic and heavy metal were also located on the plasmid. PMID:16192686

  18. Conjugative transfer of an IncA/C plasmid-borne blaCMY-2 gene through genetic re-arrangements with an IncX1 plasmid.

    Science.gov (United States)

    Wiesner, Magdalena; Fernández-Mora, Marcos; Cevallos, Miguel A; Zavala-Alvarado, Crispín; Zaidi, Mussaret B; Calva, Edmundo; Silva, Claudia

    2013-11-21

    Our observation that in the Mexican Salmonella Typhimurium population none of the ST19 and ST213 strains harbored both the Salmonella virulence plasmid (pSTV) and the prevalent IncA/C plasmid (pA/C) led us to hypothesize that restriction to horizontal transfer of these plasmids existed. We designed a conjugation scheme using ST213 strain YU39 as donor of the blaCMY-2 gene (conferring resistance to ceftriaxone; CRO) carried by pA/C, and two E. coli lab strains (DH5α and HB101) and two Typhimurium ST19 strains (SO1 and LT2) carrying pSTV as recipients. The aim of this study was to determine if the genetic background of the different recipient strains affected the transfer frequencies of pA/C. YU39 was able to transfer CRO resistance, via a novel conjugative mechanism, to all the recipient strains although at low frequencies (10-7 to 10-10). The presence of pSTV in the recipients had little effect on the conjugation frequency. The analysis of the transconjugants showed that three different phenomena were occurring associated to the transfer of blaCMY-2: 1) the co-integration of pA/C and pX1; 2) the transposition of the CMY region from pA/C to pX1; or 3) the rearrangement of pA/C. In addition, the co-lateral mobilization of a small (5 kb) ColE1-like plasmid was observed. The transconjugant plasmids involving pX1 re-arrangements (either via co-integration or ISEcp1-mediated transposition) obtained the capacity to conjugate at very high levels, similar to those found for pX1 (10-1). Two versions of the region containing blaCMY-2 were found to transpose to pX1: the large version was inserted into an intergenic region located where the "genetic load" operons are frequently inserted into pX1, while the short version was inserted into the stbDE operon involved in plasmid addiction system. This is the first study to report the acquisition of an extended spectrum cephalosporin (ESC)-resistance gene by an IncX1 plasmid. We showed that the transfer of the YU39 blaCMY-2 gene

  19. Plasmid-mediated tetracycline resistance in Haemophilus ducreyi.

    OpenAIRE

    Albritton, W L; Maclean, I W; Slaney, L A; Ronald, A. R.; Deneer, H G

    1984-01-01

    Clinical isolates of Haemophilus ducreyi were shown to be resistant to tetracycline. Resistance was associated in some strains with a 30-megadalton plasmid capable of transferring resistance in conjugative matings with other strains of H. ducreyi and other species of Haemophilus. Restriction endonuclease digestion patterns suggest a relationship between H. ducreyi plasmids and other tetracycline resistance plasmids in Haemophilus. The presence of plasmid-mediated resistance to the tetracyclin...

  20. Plasmid DNA entry into postmitotic nuclei of primary rat myotubes.

    OpenAIRE

    Dowty, M E; Williams, P.; G. Zhang; Hagstrom, J E; Wolff, J A

    1995-01-01

    These studies were initiated to elucidate the mechanism of DNA nuclear transport in mammalian cells. Biotin- or gold-labeled plasmid and plasmid DNA expression vectors for Escherichia coli beta-galactosidase or firefly luciferase were microinjected into the cytoplasm of primary rat myotubes in culture. Plasmid DNA was expressed in up to 70% of the injected myotubes, which indicates that it entered intact, postmitotic nuclei. The nuclear transport of plasmid DNA occurred through the nuclear po...

  1. Replication of plasmids in gram-negative bacteria.

    OpenAIRE

    1989-01-01

    Replication of plasmid deoxyribonucleic acid (DNA) is dependent on three stages: initiation, elongation, and termination. The first stage, initiation, depends on plasmid-encoded properties such as the replication origin and, in most cases, the replication initiation protein (Rep protein). In recent years the understanding of initiation and regulation of plasmid replication in Escherichia coli has increased considerably, but it is only for the ColE1-type plasmids that significant biochemical d...

  2. Humoral Immune Response Elicited by Plasmid DNA Containing HGV E2 Gene Fragment%含庚型肝炎病毒E2基因片段质粒DNA能诱发相当强烈的体液免疫应答

    Institute of Scientific and Technical Information of China (English)

    Fethia Ben Yebdri; Abderrahmane AAZAZ; 叶凯; 马辉文; 童立恒

    2004-01-01

    研究了庚型肝炎病毒E2(HGV E2)基因片段作为DNA疫苗的可行性.将来自于质粒pThioHis-E2编码HGV E2的基因片段(559bp)亚克隆到质粒pCMV-S中,使之和HBsAg基因位于同一阅读框,形成重组质粒pCMV-S-E2.用纯化的质pCMV-S-E2 DNA注射到昆明小鼠后腿四头肌中来免疫小鼠,同进用pCMV-S作为对照.间隔14天再加强一次免疫.在加强免疫后8天眼眶取血.用E2-GST融合蛋白作为固定化抗原,通过ELISA检测受试小鼠的体液免疫应答.结果表明,用质粒pCMV-S-E DNA免疫的小鼠可以产生很强的体液免疫应答.%In order to study the feasibility of E2 gene fragment of hepatitis virus G(HGV)as a component of DNA vaccine against the hepatitis virus G infection,a 559bp DNA fragment encoding HGV E2 was cloned into plasmid pCMV-S from pThioHis-E2 in the same reading frame with HBsAg gene to form a recombinant plasmid named pCMV-S-E2.BALB/c mice of Kunming strain were immunized with purified plasmid DNA of pCMV-S-E2 by intra-muscularly inoculation.The immunizations were boosted twice at an interval of 14 days.The whole blood was collected from mice orbit on the day-8 after the last boost.Mice sera were screened by ELISA to determine the humoral immune response using E2-GST fusion protein as the immobilized antigen and the sera from mice immunized with pCMVS as control.The result indicated that the immunization with plasmid DNA of pCMV-S-E2 could induce quite strong humoral immune response.

  3. Role of the virulence plasmid pR99 and the metalloprotease Vvp in resistance of Vibrio vulnificus serovar E to eel innate immunity.

    Science.gov (United States)

    Valiente, E; Lee, C T; Lamas, J; Hor, L; Amaro, C

    2008-01-01

    Vibrio vulnificus biotype 2 serovar E (VSE) is a bacterial pathogen that produces a haemorrhagic septicaemia called vibriosis in eels. Its ability to grow in blood is conferred by a recently described virulence plasmid [Lee CT, Amaro C, Wu KM, Valiente E, Chang YF, Tsai SF, et al. A common virulence plasmid in biotype 2 Vibrio vulnificus and its dissemination aided by a conjugal plasmid. Journal of Bacteriology, submitted for publication.]. In this study, we analyzed the role of this plasmid together with the role played by the metalloprotease (Vvp) in the interaction between bacteria and eel innate immunity. To this end, we compared and statistically analyzed the differences in resistance to serum and mucus factors (complement, selected antimicrobial peptides, transferrin and lysozyme) and also to phagocytosis/opsonophagocytosis between one VSE strain and its derivatives: a plasmid-cured strain and a vvp-deficient mutant. The wild-type and the metalloprotease-deficient strains were resistant to both the bactericidal action of fresh serum and the phagocytosis and opsonophagocytosis by eel phagocytes, confirming that Vvp is not involved in resistance to eel innate immunity. In contrast, the cured strain was sensitive to both the bactericidal action of eel serum activated by the alternative pathway and phagocytosis/opsonophagocytosis. Since no plasmid-encoded ORF, with homology to known genes, is related to the resistance to innate immunity [Lee CT, Amaro C, Wu KM, Valiente E, Chang YF, Tsai SF, et al. A common virulence plasmid in biotype 2 Vibrio vulnificus and its dissemination aided by a conjugal plasmid. Journal of Bacteriology, submitted for publication.], this function could be codified by one or more new genes. Further studies are underway to characterize the plasmid-encoded system responsible for V. vulnificus resistance to the innate immune system of eels.

  4. Plasmid Segregation: Spatial Awareness at the Molecular Level

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Gerdes, Kenn

    2007-01-01

    In bacteria, low-copy number plasmids ensure their stable inheritance by partition loci (par), which actively distribute plasmid replicates to each side of the cell division plane. Using time-lapse fluorescence microscopic tracking of segregating plasmid molecules, a new study provides novel insi...

  5. Cloning of Two Bacteriocin Genes from a Lactococcal Bacteriocin Plasmid

    NARCIS (Netherlands)

    Belkum, Marco J. van; Hayema, Bert Jan; Geis, Arnold; Kok, Jan; Venema, Gerard

    1989-01-01

    Lactococcus lactis subsp. cremoris 9B4 plasmid p9B4-6 (60 kilobases [kb]), which specifies bacteriocin production and immunity, was analyzed with restriction endonucleases, and fragments of this plasmid were cloned into shuttle vectors based on the broad-host-range plasmid pWVO1. Two regions on p9B4

  6. Multilocus sequence typing of IncN plasmids

    DEFF Research Database (Denmark)

    García-Fernández, Aurora; Villa, Laura; Moodley, Arshnee

    2011-01-01

    categorization of IncN plasmids. METHODS: Twelve fully sequenced IncN plasmids available at GenBank were analysed in silico for selecting the loci for the IncN-specific pMLST. A total of 58 plasmids originating from different reservoirs (human, pig, poultry, cattle and horses) and geographic regions (Italy...

  7. Comparative genomics of multidrug resistance-encoding IncA/C plasmids from commensal and pathogenic Escherichia coli from multiple animal sources.

    Science.gov (United States)

    Fernández-Alarcón, Claudia; Singer, Randall S; Johnson, Timothy J

    2011-01-01

    Incompatibility group A/C (IncA/C) plasmids have received recent attention for their broad host range and ability to confer resistance to multiple antimicrobial agents. Due to the potential spread of multidrug resistance (MDR) phenotypes from foodborne pathogens to human pathogens, the dissemination of these plasmids represents a public health risk. In this study, four animal-source IncA/C plasmids isolated from Escherichia coli were sequenced and analyzed, including isolates from commercial dairy cows, pigs and turkeys in the U.S. and Chile. These plasmids were initially selected because they either contained the floR and tetA genes encoding for florfenicol and tetracycline resistance, respectively, and/or the bla(CMY-2) gene encoding for extended spectrum β-lactamase resistance. Overall, sequence analysis revealed that each of the four plasmids retained a remarkably stable and conserved backbone sequence, with differences observed primarily within their accessory regions, which presumably have evolved via horizontal gene transfer events involving multiple modules. Comparison of these plasmids with other available IncA/C plasmid sequences further defined the core and accessory elements of these plasmids in E. coli and Salmonella. Our results suggest that the bla(CMY-2) plasmid lineage appears to have derived from an ancestral IncA/C plasmid type harboring floR-tetAR-strAB and Tn21-like accessory modules. Evidence is mounting that IncA/C plasmids are widespread among enteric bacteria of production animals and these emergent plasmids have flexibility in their acquisition of MDR-encoding modules, necessitating further study to understand the evolutionary mechanisms involved in their dissemination and stability in bacterial populations.

  8. IncP-1ε Plasmids are Important Vectors of Antibiotic Resistance Genes in Agricultural Systems: Diversification Driven by Class 1 Integron Gene Cassettes.

    Science.gov (United States)

    Heuer, Holger; Binh, Chu T T; Jechalke, Sven; Kopmann, Christoph; Zimmerling, Ute; Krögerrecklenfort, Ellen; Ledger, Thomas; González, Bernardo; Top, Eva; Smalla, Kornelia

    2012-01-01

    The role of broad-host range IncP-1ε plasmids in the dissemination of antibiotic resistance in agricultural systems has not yet been investigated. These plasmids were detected in total DNA from all of 16 manure samples and in arable soil based on a novel 5'-nuclease assay for real-time PCR. A correlation between IncP-1ε plasmid abundance and antibiotic usage was revealed. In a soil microcosm experiment the abundance of IncP-1ε plasmids was significantly increased even 127 days after application of manure containing the antibiotic compound sulfadiazine, compared to soil receiving only manure, only sulfadiazine, or water. Fifty IncP-1ε plasmids that were captured in E. coli CV601gfp from bacterial communities of manure and arable soil were characterized by PCR and hybridization. All plasmids carried class 1 integrons with highly varying sizes of the gene cassette region and the sul1 gene. Three IncP-1ε plasmids captured from soil bacteria and one from manure were completely sequenced. The backbones were nearly identical to that of the previously described IncP-1ε plasmid pKJK5. The plasmids differed mainly in the composition of a Tn402-like transposon carrying a class 1 integron with varying gene cassettes, IS1326, and in three of the plasmids the tetracycline resistance transposon Tn1721 with various truncations. Diverse Beta- and Gammaproteobacteria were revealed as hosts of one of the IncP-1ε plasmids in soil microcosms. Our data suggest that IncP-1ε plasmids are important vectors for horizontal transfer of antibiotic resistance in agricultural systems.

  9. IncP-1ε plasmids are important vectors of antibiotic resistance genes in agricultural systems: diversification driven by class 1 integron gene cassettes

    Directory of Open Access Journals (Sweden)

    Holger eHeuer

    2012-01-01

    Full Text Available The role of broad host range IncP-1ε plasmids in the dissemination of antibiotic resistance in agricultural systems has not yet been investigated. These plasmids were detected in total DNA from all of 16 manure samples and in arable soil based on a novel 5’-nuclease assay for real time PCR. A correlation between IncP-1ε plasmid abundance and antibiotic usage was revealed. In a soil microcosm experiment the abundance of IncP-1ε plasmids was significantly increased even 127 days after application of manure containing the antibiotic compound sulfadiazine, compared to soil receiving only manure, only sulfadiazine, or water. Fifty IncP-1ε plasmids that were captured in E. coli CV601gfp from bacterial communities of manure and arable soil were characterized by PCR and hybridisation. All plasmids carried class 1 integrons with highly varying sizes of the gene cassette region and the sul1 gene. Three IncP-1ε plasmids captured from soil bacteria and one from manure were completely sequenced. The backbones were nearly identical to that of the previously described IncP-1ε plasmid pKJK5. The plasmids differed mainly in the composition of a Tn402-like transposon carrying a class 1 integron with varying gene cassettes, IS1326, and in three of the plasmids the tetracycline resistance transposon Tn1721 with various truncations. Diverse Beta- and Gammaproteobacteria were revealed as hosts of one of the IncP-1ε plasmids in soil microcosms. Our data suggest that IncP-1ε plasmids are important vectors for horizontal transfer of antibiotic resistance in agricultural systems.

  10. Impact of homologous and non-homologous recombination in the genomic evolution of Escherichia coli

    Directory of Open Access Journals (Sweden)

    Didelot Xavier

    2012-06-01

    Full Text Available Abstract Background Escherichia coli is an important species of bacteria that can live as a harmless inhabitant of the guts of many animals, as a pathogen causing life-threatening conditions or freely in the non-host environment. This diversity of lifestyles has made it a particular focus of interest for studies of genetic variation, mainly with the aim to understand how a commensal can become a deadly pathogen. Many whole genomes of E. coli have been fully sequenced in the past few years, which offer helpful data to help understand how this important species evolved. Results We compared 27 whole genomes encompassing four phylogroups of Escherichia coli (A, B1, B2 and E. From the core-genome we established the clonal relationships between the isolates as well as the role played by homologous recombination during their evolution from a common ancestor. We found strong evidence for sexual isolation between three lineages (A+B1, B2, E, which could be explained by the ecological structuring of E. coli and may represent on-going speciation. We identified three hotspots of homologous recombination, one of which had not been previously described and contains the aroC gene, involved in the essential shikimate metabolic pathway. We also described the role played by non-homologous recombination in the pan-genome, and showed that this process was highly heterogeneous. Our analyses revealed in particular that the genomes of three enterohaemorrhagic (EHEC strains within phylogroup B1 have converged from originally separate backgrounds as a result of both homologous and non-homologous recombination. Conclusions Recombination is an important force shaping the genomic evolution and diversification of E. coli, both by replacing fragments of genes with an homologous sequence and also by introducing new genes. In this study, several non-random patterns of these events were identified which correlated with important changes in the lifestyle of the bacteria, and

  11. Construtcion of Neisseria Gonorrhoeae Porin B Plasmid Recombinant and Its Expression in E.coli

    Institute of Scientific and Technical Information of China (English)

    SONG Qifa; LIAO Fang; YE Siying; CUI Bing; XIONG Ping

    2005-01-01

    Summary: A prokaryotic expression recombinant plasmid pET-PIB to express porin B (PIB) of Neisseria gonorrhoeae in E.coli DE3 was constructed in order to provide a basis of research in detection, prophylactic and therapeutic vaccine against the pathogen infection. The gene encoding PIB was amplified by PCR from Neisseria gonorrhoeae and cloned into prokaryotic expression plasmid pET-28a(+) to construct a pET-PIB recombinant, which was verified by restriction endonuclease and DNA sequencing. Protein PIB was expressed in E.coli DE3 induced with IPTG. The antigenicity of the expressed protein was evaluated by indirect ELISA. Rabbits were immunized with the protein and serum was collected after immunization. To assess the immunogenicity of the protein, the titer of serum to protein PIB was determined by ELISA. DNA sequence analysis showed that the nucleic acid sequence of PIB gene was 99.28 % of homology compared with that (NGPIB18) published in GenBank. A 41 kD fused protein was detected by SDS-PAGE and was proven to have reactivity with anti-PIB polyclonal antibody from mouse. A polyclonal antibody to PIB of 1:4000 titer determined by indirect ELISA was obtained from rabbit immunized with the purified product. Recombinant plasmid encoding PIB of Neisseria gonorrhoeae was constructed. Protein PIB with antigenicity and immunogenicity was successfully expressed.

  12. Mimivirus reveals Mre11/Rad50 fusion proteins with a sporadic distribution in eukaryotes, bacteria, viruses and plasmids

    Directory of Open Access Journals (Sweden)

    Ogata Hiroyuki

    2011-09-01

    Full Text Available Abstract Background The Mre11/Rad50 complex and the homologous SbcD/SbcC complex in bacteria play crucial roles in the metabolism of DNA double-strand breaks, including DNA repair, genome replication, homologous recombination and non-homologous end-joining in cellular life forms and viruses. Here we investigated the amino acid sequence of the Mimivirus R555 gene product, originally annotated as a Rad50 homolog, and later shown to have close homologs in marine microbial metagenomes. Results Our bioinformatics analysis revealed that R555 protein sequence is constituted from the fusion of an N-terminal Mre11-like domain with a C-terminal Rad50-like domain. A systematic database search revealed twelve additional cases of Mre11/Rad50 (or SbcD/SbcC fusions in a wide variety of unrelated organisms including unicellular and multicellular eukaryotes, the megaplasmid of a bacterium associated to deep-sea hydrothermal vents (Deferribacter desulfuricans and the plasmid of Clostridium kluyveri. We also showed that R555 homologs are abundant in the metagenomes from different aquatic environments and that they most likely belong to aquatic viruses. The observed phyletic distribution of these fusion proteins suggests their recurrent creation and lateral gene transfers across organisms. Conclusions The existence of the fused version of protein sequences is consistent with known functional interactions between Mre11 and Rad50, and the gene fusion probably enhanced the opportunity for lateral transfer. The abundance of the Mre11/Rad50 fusion genes in viral metagenomes and their sporadic phyletic distribution in cellular organisms suggest that viruses, plasmids and transposons played a crucial role in the formation of the fusion proteins and their propagation into cellular genomes.

  13. Comparative genomic hybridization analysis shows different epidemiology of chromosomal and plasmid-borne cpe-carrying Clostridium perfringens type A.

    Directory of Open Access Journals (Sweden)

    Päivi Lahti

    Full Text Available Clostridium perfringens, one of the most common causes of food poisonings, can carry the enterotoxin gene, cpe, in its chromosome or on a plasmid. C. perfringens food poisonings are more frequently caused by the chromosomal cpe-carrying strains, while the plasmid-borne cpe-positive genotypes are more commonly found in the human feces and environmental samples. Different tolerance to food processing conditions by the plasmid-borne and chromosomal cpe-carrying strains has been reported, but the reservoirs and contamination routes of enterotoxin-producing C. perfringens remain unknown. A comparative genomic hybridization (CGH analysis with a DNA microarray based on three C. perfringens type A genomes was conducted to shed light on the epidemiology of C. perfringens food poisonings caused by plasmid-borne and chromosomal cpe-carrying strains by comparing chromosomal and plasmid-borne cpe-positive and cpe-negative C. perfringens isolates from human, animal, environmental, and food samples. The chromosomal and plasmid-borne cpe-positive C. perfringens genotypes formed two distinct clusters. Variable genes were involved with myo-inositol, ethanolamine and cellobiose metabolism, suggesting a new epidemiological model for C. perfringens food poisonings. The CGH results were complemented with growth studies, which demonstrated different myo-inositol, ethanolamine, and cellobiose metabolism between the chromosomal and plasmid-borne cpe-carrying strains. These findings support a ubiquitous occurrence of the plasmid-borne cpe-positive strains and their adaptation to the mammalian intestine, whereas the chromosomal cpe-positive strains appear to have a narrow niche in environments containing degrading plant material. Thus the epidemiology of the food poisonings caused by two populations appears different, the plasmid-borne cpe-positive strains probably contaminating foods via humans and the chromosomal strains being connected to plant material.

  14. Sutured Floer homology and hypergraphs

    CERN Document Server

    Juhász, András; Rasmussen, Jacob

    2011-01-01

    By applying Seifert's algorithm to a special alternating diagram of a link L, one obtains a Seifert surface F of L. We show that the support of the sutured Floer homology of the sutured manifold complementary to F is affine isomorphic to the set of lattice points given as hypertrees in a certain hypergraph that is naturally associated to the diagram. This implies that the Floer groups in question are supported in a set of Spin^c structures that are the integer lattice points of a convex polytope. This property has an immediate extension to Seifert surfaces arising from homogeneous link diagrams (including all alternating and positive diagrams). In another direction, together with work in progress of the second author and others, our correspondence suggests a method for computing the "top" coefficients of the HOMFLY polynomial of a special alternating link from the sutured Floer homology of a Seifert surface complement for a certain dual link.

  15. Bacteriophages limit the existence conditions for conjugative plasmids.

    Science.gov (United States)

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

    2015-06-02

    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. Conjugative plasmids are infectious loops of DNA capable of transmitting DNA between bacterial cells and between species. Because plasmids often carry extra genes that allow bacteria to live in otherwise-inhospitable environments, their dynamics are central to understanding bacterial adaptive evolution. The plasmid-bacterium interaction has typically been studied in isolation, but in natural bacterial communities, bacteriophages, viruses that infect bacteria, are ubiquitous. Using experiments, mathematical models, and computer simulations we show that bacteriophages drive plasmid dynamics through their ecological and evolutionary effects on bacteria and ultimately

  16. Assembly and sorting of homologous BAC contigs in allotetraploid cotton genomes

    Science.gov (United States)

    Upland cotton (G. hirsutum) is a diploidized allopolyploid species containing At and Dt sub-genomes that have partial homology. Assembly and sorting of homologous BAC contigs into their subgenomes and further to individual chromosomes are of both great interest and great challenge for genome-wide i...

  17. A new method for rapidly generating gene-targeting vectors by engineering BACs through homologous recombination in bacteria.

    Science.gov (United States)

    Cotta-de-Almeida, Vinicius; Schonhoff, Susan; Shibata, Tomoyuki; Leiter, Andrew; Snapper, Scott B

    2003-09-01

    Generating knockout mice is still an expensive and highly time-consuming process. Target construct generation, the first labor-intensive step in this process, requires the manipulation of large fragments of DNA and numerous, and often cumbersome, cloning steps. Here we show the development of a rapid approach for generating targeting constructs that capitalizes on efficient homologous recombination between linear DNA fragments and circular plasmids in Escherichia coli ("recombineering"), the availability of bacterial artificial chromosomes (BACs), and the accessibility of the sequence of the mouse genome. Employing recombineering, we demonstrate with only 1-2 template plasmids, short homologies (40-50bp) between donor and target DNA, and one subcloning step that we can efficiently manipulate BACs in situ to generate a complicated targeting vector. This procedure avoids the need to construct or screen genomic libraries and permits the generation of most standard, conditional, or knock-in targeting vectors, often within two weeks.

  18. Deep homology: a view from systematics.

    Science.gov (United States)

    Scotland, Robert W

    2010-05-01

    Over the past decade, it has been discovered that disparate aspects of morphology - often of distantly related groups of organisms - are regulated by the same genetic regulatory mechanisms. Those discoveries provide a new perspective on morphological evolutionary change. A conceptual framework for exploring these research findings is termed 'deep homology'. A comparative framework for morphological relations of homology is provided that distinguishes analogy, homoplasy, plesiomorphy and synapomorphy. Four examples - three from plants and one from animals - demonstrate that homologous developmental mechanisms can regulate a range of morphological relations including analogy, homoplasy and examples of uncertain homology. Deep homology is part of a much wider range of phenomena in which biological (genes, regulatory mechanisms, morphological traits) and phylogenetic levels of homology can both be disassociated. Therefore, to understand homology, precise, comparative, independent statements of both biological and phylogenetic levels of homology are necessary.

  19. E. coli recA gene improves gene targeted homologous recombination in Mycoplasma hyorhinis.

    Science.gov (United States)

    Ishag, Hassan Z A; Xiong, Qiyan; Liu, Maojun; Feng, Zhixin; Shao, Guoqing

    2017-05-01

    Mycoplasma hyorhinis is an opportunistic pathogen of pigs. Recently, it has been shown to transform cell cultures, increasing the attention of the researchers. Studies on the pathogenesis require specific genetic tool that is not yet available for the pathogen. To address this limitation, we constructed two suicide plasmids pGEMT-tetM/LR and pGEMT-recA-tetM/LR having a tetracycline resistance marker flanked by two hemolysin gene arms. The latter plasmid encodes an E. coli recA, a gene involved in DNA recombination, repair and maintenance of DNA. Using inactivation of the hemolysin gene, which results in a detectable and measurable phenotype, we found that each plasmid can disrupt the hemolysin gene of M. hyorhinis through a double cross-over homologous recombination. However, inclusion of the E. coli recA gene in the construct resulted in 9-fold increase in the frequency of hemolysin gene mutants among the screened tetracycline resistance colonies. The resultant hemolysin mutant strain lacks the ability to lyse mouse bed blood cells (RBC) when tested in vitro (p<0.001). The host-plasmid system described in this study, has applications for the genetic manipulation of this pathogen and potentially other mycoplasmas.

  20. Transfer of a gonococcal beta-lactamase plasmid to conjugation-deficient Neisseria cinerea strains by transformation.

    Science.gov (United States)

    Genco, C A; Clark, V L

    1988-12-01

    We have previously shown that some strains of Neisseria cinerea can serve as recipients in conjugation (Con+) with Neisseria gonorrhoeae while others cannot (Con-). To determine if a replication defect contributes to the inability of certain strains of N. cinerea to serve as recipients in conjugation, we attempted to introduce a naturally occurring gonococcal beta-lactamase plasmid into N. cinerea by transformation. Various methods were employed, and all proved unsuccessful. Since specific sequences are required for DNA uptake in transformation of N. gonorrhoeae, we constructed a number of hybrid plasmids containing N. cinerea chromosomal DNA inserted into the N. gonorrhoeae/Escherichia coli beta-lactamase shuttle vector, pLES2. When nine randomly selected plasmids with inserts were used to transform an N. cinerea strain which did not accept the gonococcal beta-lactamase plasmid by conjugation, transformants were observed with four of the hybrid plasmids. The presence of one of the hybrid plasmids, pCAG9, in transformants was confirmed by agarose gel electrophoresis, Southern hybridization, and beta-lactamase production. When an N. gonorrhoeae donor strain containing pCAG9 was used in conjugation with several N. cinerea strains, only those strains that were previously shown to act as recipients could accept and maintain pCAG9. The ability of pCAG9 and the other three hybrid plasmids to transform Con- strains demonstrates that the beta-lactamase plasmid can replicate in Con- strains, and, therefore, the Con- phenotype is due to a block in some other stage of the conjugation process.

  1. Homology requirements for recombination in Escherichia coli.

    OpenAIRE

    Watt, V M; Ingles, C J; Urdea, M S; Rutter, W J

    1985-01-01

    The DNA sequence homology required for recombination in Escherichia coli has been determined by measuring the recombination frequency between insulin DNA in a miniplasmid pi VX and a homologous sequence in a bacteriophage lambda vector. A minimum of approximately equal to 20 base pairs in a completely homologous segment is required for significant recombination. There is an exponential increase in the frequency of recombination when the length of homologous DNA is increased from 20 base pairs...

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

    Directory of Open Access Journals (Sweden)

    Val F Lanza

    2014-12-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  4. Lagrangian Quantum Homology for Lagrangian cobordism

    OpenAIRE

    Singer, Berit

    2015-01-01

    We extend the definition of Lagrangian quantum homology to monotone Lagrangian cobordism and establish its general algebraic properties. In particular we develop a relative version of Lagrangian quantum homology associated to a cobordism relative to a part of its boundary and study relations of this invariant to the ambient quantum homology.

  5. Discrete homology theory for metric spaces

    NARCIS (Netherlands)

    H. Barcelo (Hélène); V. Capraro (Valerio); J. A. White; H. Barcelo (Hélène)

    2014-01-01

    htmlabstractWe define and study a notion of discrete homology theory for metric spaces. Instead of working with simplicial homology, our chain complexes are given by Lipschitz maps from an n n -dimensional cube to a fixed metric space. We prove that the resulting homology theory satisfies a

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

    Science.gov (United States)

    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.

  7. Permissiveness of soil microbial communities towards broad host range plasmids

    DEFF Research Database (Denmark)

    Klümper, Uli

    at high frequencies from diverse donors, I showed plasmid or donor dependence of plasmid transfer to other species. Additionally, environmental factors like stress also impact the permissiveness of phylogenetic groups towards plasmids. The developed method and results increase our ability to predict......Horizontal transfer of mobile genetic elements facilitates adaptive and evolutionary processes in bacteria. Among the known mobile genetic elements, plasmids can confer their hosts with accessory adaptive traits, such as antibiotic or heavy metal resistances, or additional metabolic pathways...... and the extent of bacterial phyla permissive towards plasmid receipt are largely unknown. Historically, methods exploring the underlying genetic and environmental factors of plasmid transfer have been heavily reliant on cultivation and expression of plasmid encoded phenotypes. This has provided an incomplete...

  8. Genetic characterization of mcr-1-bearing plasmids to depict molecular mechanisms underlying dissemination of the colistin resistance determinant.

    Science.gov (United States)

    Li, Ruichao; Xie, Miaomiao; Zhang, Jinfei; Yang, Zhiqiang; Liu, Lizhang; Liu, Xiaobo; Zheng, Zhiwei; Chan, Edward Wai-Chi; Chen, Sheng

    2017-02-01

    To analyse and compare mcr-1-bearing plasmids from animal Escherichia coli isolates, and to investigate potential mechanisms underlying dissemination of mcr-1. Ninety-seven ESBL-producing E. coli strains isolated from pig farms in China were screened for the mcr-1 gene. Fifteen mcr-1-positive strains were subjected to molecular characterization and bioinformatic analysis of the mcr-1-bearing plasmids that they harboured. Three major types of mcr-1-bearing plasmids were recovered: IncX4 (∼33 kb), IncI2 (∼60 kb) and IncHI2 (∼216-280 kb), among which the IncX4 and IncI2 plasmids were found to harbour the mcr-1 gene only, whereas multiple resistance elements including blaCTX-M, blaCMY, blaTEM, fosA, qnrS, floR and oqxAB were detected, in various combinations, alongside mcr-1 in the IncHI2 plasmids. The profiles of mcr-1-bearing plasmids in the test strains were highly variable, with coexistence of two mcr-1-bearing plasmids being common. However, the MIC of colistin was not affected by the number of mcr-1-carrying plasmids harboured. Comparative analysis of the plasmids showed that they contained an mcr-1 gene cassette with varied structures (mcr-1-orf, ISApl1-mcr-1-orf and Tn6330), with the IncHI2 type being the most active in acquiring foreign resistance genes. A novel transposon, Tn6330, with the structure ISApl1-mcr-1-orf-ISApl1 was found to be the key element mediating translocation of mcr-1 into various plasmid backbones through formation of a circular intermediate. The mcr-1 gene can be disseminated via multiple mobile elements including Tn6330, its circular intermediate and plasmids harbouring such elements. It is often co-transmitted with other resistance determinants through IncHI2 plasmids. The functional mechanism of Tn6330, a typical composite transposon harbouring mcr-1, should be further investigated. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For

  9. Characteristics of plasmids in multi-drug-resistant Enterobacteriaceae isolated during prospective surveillance of a newly opened hospital in Iraq.

    Directory of Open Access Journals (Sweden)

    Xiao-Zhe Huang

    Full Text Available BACKGROUND: Gram-negative multidrug-resistant (MDR bacteria are major causes of nosocomial infections, and antibiotic resistance in these organisms is often plasmid mediated. Data are scarce pertaining to molecular mechanisms of antibiotic resistance in resource constrained areas such as Iraq. METHODOLOGY/PRINCIPAL FINDINGS: In this study, all MDR Enterobacteriaceae (n = 38 and randomly selected non-MDR counterparts (n = 41 isolated from patients, healthcare workers and environmental surfaces in a newly opened hospital in Iraq were investigated to characterize plasmids found in these isolates and determine their contribution to antibiotic resistance. Our results demonstrated that MDR E. coli and K. pneumoniae isolates harbored significantly more (≥ 3 plasmids compared to their non-MDR counterparts, which carried ≤ 2 plasmids (p<0.01. Various large plasmids (~52 to 100 kb from representative isolates were confirmed to contain multiple resistance genes by DNA microarray analysis. Aminoglycoside (acc, aadA, aph, strA/B, and ksgA, β-lactam (bla(TEM1, bla(AMPC, bla(CTX-M-15, bla(OXA-1, bla(VIM-2 and bla(SHV, sulfamethoxazole/trimethoprim (sul/dfr, tetracycline (tet and chloramphenicol (cat resistance genes were detected on these plasmids. Additionally, multiple plasmids carrying multiple antibiotic resistance genes were found in the same host strain. Genetic transfer-associated genes were identified on the plasmids from both MDR and non-MDR isolates. Seven plasmid replicon types (FII, FIA, FIB, B/O, K, I1 and N were detected in the isolates, while globally disseminated IncA/C and IncHI1 plasmids were not detected in these isolates. CONCLUSIONS/SIGNIFICANCE: This is the first report of the characteristics of the plasmids found in Enterobacteriaceae isolated following the opening of a new hospital in Iraq. The information provided here furthers our understanding of the mechanisms of drug resistance in this specific region and their evolutionary

  10. Modeling sRNA-Regulated Plasmid Maintenance

    Science.gov (United States)

    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

  11. Modeling sRNA-regulated Plasmid Maintenance

    CERN Document Server

    Gong, Chen Chris

    2016-01-01

    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, ...

  12. PDBalert: automatic, recurrent remote homology tracking and protein structure prediction

    Directory of Open Access Journals (Sweden)

    Söding Johannes

    2008-11-01

    Full Text Available Abstract Background During the last years, methods for remote homology detection have grown more and more sensitive and reliable. Automatic structure prediction servers relying on these methods can generate useful 3D models even below 20% sequence identity between the protein of interest and the known structure (template. When no homologs can be found in the protein structure database (PDB, the user would need to rerun the same search at regular intervals in order to make timely use of a template once it becomes available. Results PDBalert is a web-based automatic system that sends an email alert as soon as a structure with homology to a protein in the user's watch list is released to the PDB database or appears among the sequences on hold. The mail contains links to the search results and to an automatically generated 3D homology model. The sequence search is performed with the same software as used by the very sensitive and reliable remote homology detection server HHpred, which is based on pairwise comparison of Hidden Markov models. Conclusion PDBalert will accelerate the information flow from the PDB database to all those who can profit from the newly released protein structures for predicting the 3D structure or function of their proteins of interest.

  13. A new and improved host-independent plasmid system for RK2-based conjugal transfer.

    Directory of Open Access Journals (Sweden)

    Trine Aakvik Strand

    Full Text Available Bacterial conjugation is a process that is mediated either by a direct cell-to-cell junction or by formation of a bridge between the cells. It is often used to transfer DNA constructs designed in Escherichia coli to recipient bacteria, yeast, plants and mammalian cells. Plasmids bearing the RK2/RP4 origin of transfer (oriT are mostly mobilized using the E. coli S17-1/SM10 donor strains, in which transfer helper functions are provided from a chromosomally integrated RP4::Mu. We have observed that large plasmids were occasionally modified after conjugal transfer when using E. coli S17-1 as a donor. All modified plasmids had increased in size, which most probably was a result of co-transfer of DNA from the chromosomally located oriT. It has earlier also been demonstrated that the bacteriophage Mu is silently transferred to recipient cells by these donor strains, and both occurrences are very likely to lead to mutations within the recipient DNA. Here we report the construction of a new biological system addressing both the above mentioned problems in which the transfer helper functions are provided by a plasmid lacking a functional oriT. This system is compatible with all other replicons commonly used in conjugation experiments and further enables the use of diverse bacterial strains as donors. Plasmids containing large inserts were successfully conjugated and the plasmid modifications observed when E. coli S17-1 was used as donor were eliminated by the use of the new host-independent vector system.

  14. Characterization of the replication, transfer, and plasmid/lytic phage cycle of the Streptomyces plasmid-phage pZL12.

    Science.gov (United States)

    Zhong, Li; Cheng, Qiuxiang; Tian, Xinli; Zhao, Liqian; Qin, Zhongjun

    2010-07-01

    We report here the isolation and recombinational cloning of a large plasmid, pZL12, from endophytic Streptomyces sp. 9R-2. pZL12 comprises 90,435 bp, encoding 112 genes, 30 of which are organized in a large operon resembling bacteriophage genes. A replication locus (repA) and a conjugal transfer locus (traA-traC) were identified in pZL12. Surprisingly, the supernatant of a 9R-2 liquid culture containing partially purified phage particles infected 9R-2 cured of pZL12 (9R-2X) to form plaques, and a phage particle (phiZL12) was observed by transmission electron microscopy. Major structural proteins (capsid, portal, and tail) of phiZL12 virions were encoded by pZL12 genes. Like bacteriophage P1, linear phiZL12 DNA contained ends from a largely random pZL12 sequence. There was also a hot end sequence in linear phiZL12. phiZL12 virions efficiently infected only one host, 9R-2X, but failed to infect and form plaques in 18 other Streptomyces strains. Some 9R-2X spores rescued from lysis by infection of phiZL12 virions contained a circular pZL12 plasmid, completing a cycle comprising autonomous plasmid pZL12 and lytic phage phiZL12. These results confirm pZL12 as the first example of a plasmid-phage in Streptomyces.

  15. Virtual Khovanov homology using cobordisms

    DEFF Research Database (Denmark)

    Tubbenhauer, Daniel

    2014-01-01

    We give a geometric interpretation of the Khovanov complex for virtual links. Geometric interpretation means that we use a cobordism structure like D. Bar-Natan, but we allow non orientable cobordisms. Like D. Bar-Natans geometric complex our construction should work for virtual tangles too....... This geometric complex allows, in contrast to the geometric version of V. Turaev and P. Turner, a direct extension of the classical Khovanov complex (h=t=0) and of the variant of Lee (h=0,t=1). Furthermore we give a classification of all unoriented TQFTs which can be used to define virtual link homologies...

  16. Virtual Khovanov homology using cobordisms

    DEFF Research Database (Denmark)

    Tubbenhauer, Daniel

    2014-01-01

    We give a geometric interpretation of the Khovanov complex for virtual links. Geometric interpretation means that we use a cobordism structure like D. Bar-Natan, but we allow non orientable cobordisms. Like D. Bar-Natans geometric complex our construction should work for virtual tangles too....... This geometric complex allows, in contrast to the geometric version of V. Turaev and P. Turner, a direct extension of the classical Khovanov complex (h=t=0) and of the variant of Lee (h=0,t=1). Furthermore we give a classification of all unoriented TQFTs which can be used to define virtual link homologies...

  17. Changing plasmid types responsible for extended-spectrum cephalosporin resistance in Escherichia coli O157:H7 in the USA, 1996-2009.

    Science.gov (United States)

    Folster, J P; Pecic, G; Stroika, S; Rickert, R; Whichard, J M

    2014-06-01

    Escherichia coli O157 is a major cause of food-borne illness. Plasmids are genetic elements that mobilise antimicrobial resistance determinants, including blaCMY β-lactamases that confer resistance to extended-spectrum cephalosporins (ESCs). ESCs are important for treating a variety of infections. IncA/C plasmids are found among diverse sources, including cattle, the principal source of E. coli O157 infections in humans. IncI1 plasmids are common among E. coli and Salmonella from poultry and other avian sources. To broaden our understanding of the reservoirs of blaCMY, the types of plasmids carrying blaCMY among E. coli O157 were determined. From 1996 to 2009, 3742 E. coli O157 isolates were tested. Eleven isolates (0.29%) were ceftriaxone-resistant and had a blaCMY-2-containing plasmid. All four isolates submitted before 2001 as well as a single 2001 isolate had blaCMY encoded on IncA/C plasmids, whilst all five isolates submitted after 2001 and a single 2001 isolate had blaCMY carried on IncI1 plasmids. The IncI1 plasmids were ST2, ST20 and ST23. We conclude that cephalosporin resistance among E. coli O157:H7 is due to plasmid-encoded blaCMY genes and that plasmid types appear to have shifted from IncA/C to IncI1. This shift suggests either a change in plasmid type among animal reservoirs or that the organism has expanded into avian reservoirs. More analysis of human, retail meat and food animal isolates is necessary to broaden our understanding of the antimicrobial resistance determinants of ESC resistance among E. coli O157. Published by Elsevier Ltd.

  18. Changing plasmid types responsible for extended spectrum cephalosporin resistance in Escherichia coli O157:H7 in the United States, 1996-2009.

    Science.gov (United States)

    Folster, J P; Pecic, G; Stroika, S; Rickert, R; Whichard, J

    2014-06-01

    Escherichia coli O157 is a major cause of foodborne illness. Plasmids are genetic elements that mobilize antimicrobial resistance determinants including blaCMY β-lactamases that confer resistance to extended-spectrum cephalosporins (ESC). ESCs are important for treating a variety of infections. IncA/C plasmids are found among diverse sources, including cattle, the principal source of E. coli O157 infections in humans. IncI1 plasmids are common among E. coli and Salmonella from poultry and other avian sources. To broaden our understanding of reservoirs of blaCMY, we determined the types of plasmids carrying blaCMY among E. coli O157. From 1996 to 2009, 3742 E. coli O157 isolates were tested. Eleven (0.29%) were ceftriaxone resistant and had a blaCMY-2-containing plasmid. All four isolates submitted before 2001 and a single 2001 isolate had blaCMY encoded on IncA/C plasmids, while all five isolates submitted after 2001 and a single 2001 isolate had blaCMY carried on IncI1 plasmids. The IncI1 plasmids were ST2, ST20, and ST23. We conclude that cephalosporin resistance among E. coli O157:H7 is due to plasmid-encoded blaCMY genes and that plasmid types appear to have shifted from IncA/C to IncI1. This shift suggests either a change in plasmid type among animal reservoirs or that the organism has expanded into avian reservoirs. More analysis of human, retail meat, and food animal isolates is necessary to broaden our understanding of the antimicrobial resistance determinants of ESC resistance among E. coli O157.

  19. A Transmissible Plasmid-Borne Pathogenicity Island Confers Piscibactin Biosynthesis in the Fish Pathogen Photobacterium damselae subsp. piscicida

    Science.gov (United States)

    Rivas, Amable J.; Balado, Miguel; Fuentes-Monteverde, Juan Carlos; Rodríguez, Jaime; Jiménez, Carlos; Lemos, Manuel L.; Waldor, Matthew K.

    2015-01-01

    The fish pathogen Photobacterium damselae subsp. piscicida produces the siderophore piscibactin. A gene cluster that resembles the Yersinia high-pathogenicity island (HPI) encodes piscibactin biosynthesis. Here, we report that this HPI-like cluster is part of a hitherto-uncharacterized 68-kb plasmid dubbed pPHDP70. This plasmid lacks homologs of genes that mediate conjugation, but we found that it could be transferred at low frequencies from P. damselae subsp. piscicida to a mollusk pathogenic Vibrio alginolyticus strain and to other Gram-negative bacteria, likely dependent on the conjugative functions of the coresident plasmid pPHDP60. Following its conjugative transfer, pPHDP70 restored the capacity of a vibrioferrin mutant of V. alginolyticus to grow under low-iron conditions, and piscibactin became detectable in its supernatant. Thus, pPHDP70 appears to harbor all the genes required for piscibactin biosynthesis and transport. P. damselae subsp. piscicida strains cured of pPHDP70 no longer produced piscibactin, had impaired growth under iron-limited conditions, and exhibited markedly decreased virulence in fish. Collectively, our findings highlight the importance of pPHDP70, with its capacity for piscibactin-mediated iron acquisition, in the virulence of P. damselae subsp. piscicida. Horizontal transmission of this plasmid-borne piscibactin synthesis gene cluster in the marine environment may facilitate the emergence of new pathogens. PMID:26092457

  20. CARTOGRAPHIE DU PLASMIDE pSU100, PLASMIDE CRYPTIQUE DE LACTOBACILLUS CASEI

    Directory of Open Access Journals (Sweden)

    F BENSALAH

    2003-06-01

    Ce plasmide appelé pSU100 a été cloné dans le vecteur de transformation pUC18 au site EcoRI chez E. coli JM103. Les profils électrophorétiques de restriction obtenus par des digestions simples, doubles et triples sous l’action de 33 endonucléases, ont contribué à l’élaboration d’une carte de restriction de ce plasmide. Cinq sites uniques ont été identifiés, ainsi que d’autres sites doubles et multiples. Une étude préliminaire du rôle physiologique de ce plasmide a permis de déceler une résistance à la kanamycine.

  21. <