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Sample records for plasmid pnvh99 qacg

  1. 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.

  2. 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

  3. 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 ...

  4. A Novel Plasmid, pSx1, Harboring a New Tn1696 Derivative from Extensively Drug-Resistant Shewanella xiamenensis Encoding OXA-416.

    Science.gov (United States)

    Yousfi, Khadidja; Touati, Abdelaziz; Lefebvre, Brigitte; Fournier, Éric; Côté, Jean-Charles; Soualhine, Hafid; Walker, Matthew; Bougdour, Djamila; Tremblay, Cécile; Bekal, Sadjia

    2017-06-01

    The whole genome sequencing of extensively drug-resistant Shewanella xiamenensis T17 isolated from hospital effluents in Algeria revealed the presence of a novel 268.4 kb plasmid designated pSx1, which carries several antibiotic-resistance genes in the novel Tn1696 derivative (Tn6297), in addition to the chromosomal blaOXA-48-like gene (blaOXA-416). The presence of the plasmid was confirmed by nuclease S1-PFGE analysis and transformation by electroporation into Escherichia coli DH10B. Tn6297 contains an In27 class 1 integron harboring the dfrA12-orfF-aadA2 array, msr(E) and mph(E) associated with IS26; a new efflux pump multidrug resistance composite transposon delimited by two ISEc29s; Tn-tet harboring tetR and tetA(C); a class 1 integron with the qacG gene cassette; qnrVC6 and dfrA23 associated with ISCR1; and a complex class 1 integron In4-like containing aacC1, aadA1, blaVEB-16, catA2, sul1Δ, cmlA9, tetR, tetA(G), aac(6')-II, and blaPSE-1. Its mer operon carries merB, but lacks merC, in contrast to Tn1696 and Tn21. This study represents the first characterization of a multidrug-resistant transposon and multidrug resistance plasmid in Shewanella and is the first report of blaOXA-416 in Algeria, providing evidence that Shewanella spp. could be an important reservoir and vehicle for drug resistance genes.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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...

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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...

  18. 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...

  19. 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.

  20. 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.

  1. 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...

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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-.

  12. 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 ...

  13. 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.

  14. 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...

  15. 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.

  16. 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.

  17. 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

  18. 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).

  19. 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...

  20. 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.

  1. 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.

  2. 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

  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 veter

  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. 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

  6. 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.

  7. 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...

  8. 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...

  9. 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...

  10. 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...

  11. 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

  12. 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...

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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...

  19. 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

  20. 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, ...

  1. 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.

  2. Plasmid transfer between bacteria in soil microcosms and the field

    Directory of Open Access Journals (Sweden)

    Eric Smit

    1997-01-01

    Full Text Available In ibis review factors influencing conjugal plasmid transfer between bacteria and the possible role of naturally occurring selftransmissible plasmide for the dissemination of recombinant DNA in soil will be discussed. In microcosm studies, plasmid transfer between various species of introduced bacteria has been detected. Moreover, plamid transfer to indigenous soil micoorganisms was observed. Soil is an oligotrophic environment and plasmid transfer occurred mainly under conditions which were nutritionally favourable for bacteria, such as in the plant rhizosphere and in the presence of clay minerais or added nutrients. Mobilizable plasmids, lacking the ability to transfer themselves, have been reported to be transferred in the presence of selftransmissible plasmids. A study comparing conjugal transfer in microcosme with those in the field revealed that the transfer rates found in microcosme and in the field were similar. Transfer of chromosomal DNA by plasmid RP4 could only be shown on filters and was not observed in soil. Transfer of plasmids carrying biodegradative genes appeared to be favoured in the presence of the compound that can be degraded. Evidence was found for the presence of naturally-occurring selftransmissible plasmids in bacteria in the rhizosphere which could mobilize recombinant plasmids.

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

    OpenAIRE

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

    1992-01-01

    This study examined the potential of bacteria from river epilithon to mobilize a recombinant catabolic plasmid, pD10, encoding 3-chlorobenzoate degradation and kanamycin resistance. Fifty-four mobilizing plasmids were exogenously isolated by triparental matings between strains of Pseudomonas putida and epilithic bacteria from the River Taff (South Wales, United Kingdom). Frequencies for mobilization ranged from 1.7 x 10(-8) to 4.5 x 10(-3) per recipient at 20 degrees C. The sizes of the mobil...

  4. Isolation of clinical strains of Pseudomonas aeruginosa harboring different plasmids.

    Science.gov (United States)

    Ranjbar, R; Owlia, P; Saderi, H; Bameri, Z; Izadi, M; Jonaidi, N; Morovvati, S

    2007-09-01

    Aim of this study was to investigate the presence of plasmids among the strains of P. aeruginosa isolated from clinically diagnosed cases in Tehran in 2006. A total of 38 strains of P. aeruginosa were isolated. With the exception of one isolate, all P. aeruginosa strains harbored at least one plasmid band. The electrophoretic analysis of plasmid DNAs showed different number of plasmid bands among the strains tested. The DNA band of 1.4 kbp was evident in 84.2% of the strains. Approximately 71 and 21% of the isolates harbored concomitantly two and three plasmids, respectively. Isolation of strains with diverse types of plasmids suggests the different cluster of P. aeruginosa might be disseminated during the current study period.

  5. Transformation of Haemophilus influenzae by plasmid RSF0885

    Energy Technology Data Exchange (ETDEWEB)

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

    1981-12-01

    Plasmid RSF0885, which conferred ampicillin resistance, transformed competent Haemophilus influenzae cells with low efficiency (maximun, less than 0.01%). As judged by competition experiments and uptake of radioactivity, plasmid RSF0885 deoxyribonucleic acid was taken up into competent H. influenzae cells several orders of magnitude less efficiently than H. influenzae chromosomal deoxyribonucleic acid. Plasmid RSF0885 transformed cells with even lower efficiency than could be accounted for by the low uptake. Transformation was not affected by rec-1 and rec-2 mutations in the recipient, and strains cured of the plasmid did not show increased transformation. Plasmid molecules cut once with a restriction enzyme that made blunt ends did not transform. Transformation was favored by the closed circular form of the plasmid.

  6. Conjugative botulinum neurotoxin-encoding plasmids in Clostridium botulinum.

    Directory of Open Access Journals (Sweden)

    Kristin M Marshall

    Full Text Available BACKGROUND: Clostridium botulinum produces seven distinct serotypes of botulinum neurotoxins (BoNTs. The genes encoding different subtype neurotoxins of serotypes A, B, F and several dual neurotoxin-producing strains have been shown to reside on plasmids, suggesting that intra- and interspecies transfer of BoNT-encoding plasmids may occur. The objective of the present study was to determine whether these C. botulinum BoNT-encoding plasmids are conjugative. METHODOLOGY/PRINCIPAL FINDINGS: C. botulinum BoNT-encoding plasmids pBotCDC-A3 (strain CDC-A3, pCLJ (strain 657Ba and pCLL (strain Eklund 17B were tagged with the erythromycin resistance marker (Erm using the ClosTron mutagenesis system by inserting a group II intron into the neurotoxin genes carried on these plasmids. Transfer of the tagged plasmids from the donor strains CDC-A3, 657Ba and Eklund 17B to tetracycline-resistant recipient C. botulinum strains was evaluated in mating experiments. Erythromycin and tetracycline resistant transconjugants were isolated from donor:recipient mating pairs tested. Transfer of the plasmids to the transconjugants was confirmed by pulsed-field gel electrophoresis (PFGE and Southern hybridizations. Transfer required cell-to-cell contact and was DNase resistant. This indicates that transfer of these plasmids occurs via a conjugation mechanism. CONCLUSIONS/SIGNIFICANCE: This is the first evidence supporting conjugal transfer of native botulinum neurotoxin-encoding plasmids in C. botulinum, and provides a probable mechanism for the lateral distribution of BoNT-encoding plasmids to other C. botulinum strains. The potential transfer of C. botulinum BoNT-encoding plasmids to other bacterial hosts in the environment or within the human intestine is of great concern for human pathogenicity and necessitates further characterization of these plasmids.

  7. Photonic plasmid stability of transformed Salmonella Typhimurium: A comparison of three unique plasmids

    Directory of Open Access Journals (Sweden)

    Lay Donald

    2009-07-01

    Full Text Available Abstract Background Acquiring a highly stable photonic plasmid in transformed Salmonella Typhimurium for use in biophotonic studies of bacterial tracking in vivo is critical to experimental paradigm development. The objective of this study was to determine stability of transformed Salmonella Typhimurium (S. typh-lux using three different plasmids and characterize their respective photonic properties. Results In presence of ampicillin (AMP, S. typh-lux with pCGLS-1, pAK1-lux and pXEN-1 plasmids exhibited 100% photon-emitting colonies over a 10-d study period. Photon emitters of S. typh-lux with pCGLS-1, pAK1-lux and pXEN-1 without AMP selection decreased over time (P 7 to 1 × 109 CFU, P 0.05; although photonic emissions across a range of bacterial concentrations were not different (1 × 104 to 1 × 106 CFU, P > 0.05. For very low density bacterial concentrations imaged in 96 well plates photonic emissions were positively correlated with bacterial concentration (P 3 to 1 × 105 CFU low to high were different in the 96-well plate format (P Conclusion These data characterize photon stability properties for S. typh-lux transformed with three different photon generating plasmids that may facilitate real-time Salmonella tracking using in vivo or in situ biophotonic paradigms.

  8. Bacteriophages Limit the Existence Conditions for Conjugative Plasmids

    Science.gov (United States)

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

    2015-01-01

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

  9. Plasmid genes required for microcin B17 production.

    Science.gov (United States)

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

    1985-09-01

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

  10. 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...

  11. [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.

  12. 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.

  13. [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.

  14. 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.

  15. Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha

    NARCIS (Netherlands)

    Faber, Klaas Nico; Swaving, Gert Jan; Faber, Folkert; Ab, Geert; Harder, Willem; Veenhuis, Marten; Haima, Pieter

    1992-01-01

    Using an optimized transformation protocol we have studied the possible interactions between transforming plasmid DNA and the Hansenula polymorpha genome. Plasmids consisting only of a pBR322 replicon, an antibiotic resistance marker for Escherichia coli and the Saccharomyces cerevisiae LEU2 gene we

  16. Genomic comparison of archaeal conjugative plasmids from Sulfolobus

    DEFF Research Database (Denmark)

    Greve, Bo Bjørn

    2004-01-01

    All of the known self-transmissable plasmids of the Archaea have been found in the genus Sulfolobus. To gain more insight into archaeal conjugative processes, four newly isolated self-transmissable plasmids, pKEF9, pHVE14, pARN3 and pARN4, were sequenced and subjected to a comparative sequence...

  17. 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.

  18. 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

  19. Examination of uropathogenic Escherichia coli strains conferring large plasmids

    Directory of Open Access Journals (Sweden)

    SUHARTONO

    2010-04-01

    Full Text Available Suhartono (2010 Examination of uropathogenic Escherichia coli strains conferring large plasmids. Biodiversitas 11: 59-64. Of major uropathogens, Escherichia coli has been widely known as a main pathogen of UTIs globally and has considerable medical and financial consequences. A strain of UPEC, namely E. coli ST131, confers a large plasmid encoding cephalosporinases (class C β-lactamase or AmpC that may be disseminated through horizontal transfer among bacterial populations. Therefore, it is worth examining such large plasmids by isolating, purifying, and digesting the plasmid with restriction enzymes. The examination of the large plasmids was conducted by isolating plasmid DNA visualized by agarose gel electrophoresis as well as by PFGE. The relationship of plasmids among isolates was carried out by HpaI restriction enzyme digestion. Of 36 isolates of E. coli ST 131, eight isolates possessed large plasmids, namely isolates 3, 9, 10, 12, 17, 18, 26 and 30 with the largest molecular size confirmed by agarose gel electrophoresis and PFGE was ~42kb and ~118kb respectively. Restriction enzyme analysis revealed that isolates 9, 10, 12, 17 and 18 have the common restriction patterns and those isolates might be closely related.

  20. Production and pharmaceutical formulation of plasmid DNA vaccines

    NARCIS (Netherlands)

    van der Heijden, I.

    2013-01-01

    Research leading to the thesis ‘Production and pharmaceutical formulation of plasmid DNA vaccines‘ can be divided into two parts. The first part describes the development of a Good Manufacturing Practice (GMP) compliant plasmid DNA production process of pDNA vaccines for the treatment of Human papil

  1. Production and pharmaceutical formulation of plasmid DNA vaccines

    NARCIS (Netherlands)

    van der Heijden, I.

    2013-01-01

    Research leading to the thesis ‘Production and pharmaceutical formulation of plasmid DNA vaccines‘ can be divided into two parts. The first part describes the development of a Good Manufacturing Practice (GMP) compliant plasmid DNA production process of pDNA vaccines for the treatment of Human papil

  2. 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.

  3. 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

  4. 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.

  5. Deciphering conjugative plasmid permissiveness in wastewater microbiomes

    DEFF Research Database (Denmark)

    Jacquiod, Samuel Jehan Auguste; Brejnrod, Asker Daniel; Milani, Stefan Morberg

    2017-01-01

    Wastewater treatment plants (WWTPs) are designed to robustly treat polluted water. They are characterized by ceaseless flows of organic, chemical and microbial matter, followed by treatment steps before environmental release. WWTPs are hotspots of horizontal gene transfer between bacteria via...... still remains largely uncharted. Furthermore, current in vitro methods used to assess conjugation in complex microbiomes do not include in situ behaviours of recipient cells, resulting in partial understanding of transfers. We investigated the in vitro conjugation capacities of WWTP microbiomes from...... diversity of recipient bacterial phyla for the plasmid was observed, especially in WWTP outlets. We also identified permissive bacteria potentially able to cross WWTPs and engage in conjugation before and after water treatment. Bacterial activity and lifestyle seem to influence conjugation extent...

  6. Expansion of the IncX plasmid family for improved identification and typing of novel plasmids in drug-resistant Enterobacteriaceae

    DEFF Research Database (Denmark)

    Johnson, Timothy J.; Bielak, Eliza Maria; Fortini, Daniela;

    2012-01-01

    and biofilm formation. Previous plasmid-based replicon typing procedures have indicated that the prevalence of IncX plasmids is low among members of the Enterobacteriaceae. However, examination of a number of IncX-like plasmid sequences and their occurrence in various organisms suggests that IncX plasmid...

  7. [Influence of spv plasmid genes group in Salmonella Enteritidis virulence for chickens. I. Occurrence of spv plasmid genes group in Salmonella Enteritidis large virulence plasmid].

    Science.gov (United States)

    Madajczak, Grzegorz; Binek, Marian

    2005-01-01

    Many Salmonella Enteritidis virulence factors are encoded by genes localized on plasmids, especially large virulence plasmid, in highly conserved fragment, they create spv plasmid gene group. The aims of realized researches were spv genes occurrence evaluation and composition analysis among Salmonella Enteritidis strains caused infection in chickens. Researches were realized on 107 isolates, where in every cases large virulence plasmid 59 kbp size were detected. Specific nucleotides sequences of spv genes (spvRABCD) were detected in 47.7% of isolates. In the rest of examined bacteria spv genes occurred variably. Most often extreme genes of spv group, like spvR and spvD were absent, what could indicate that factors encoded by them are not most important for Salmonella Enteritidis live and their expressed virulence.

  8. Sample displacement chromatography of plasmid DNA isoforms.

    Science.gov (United States)

    Černigoj, Urh; Martinuč, Urška; Cardoso, Sara; Sekirnik, Rok; Krajnc, Nika Lendero; Štrancar, Aleš

    2015-10-02

    Sample displacement chromatography (SDC) is a chromatographic technique that utilises different relative binding affinities of components in a sample mixture and has been widely studied in the context of peptide and protein purification. Here, we report a use of SDC to separate plasmid DNA (pDNA) isoforms under overloading conditions, where supercoiled (sc) isoform acts as a displacer of open circular (oc) or linear isoform. Since displacement is more efficient when mass transfer between stationary and mobile chromatographic phases is not limited by diffusion, we investigated convective interaction media (CIM) monoliths as stationary phases for pDNA isoform separation. CIM monoliths with different hydrophobicities and thus different binding affinities for pDNA (CIM C4 HLD, CIM-histamine and CIM-pyridine) were tested under hydrophobic interaction chromatography (HIC) conditions. SD efficiency for pDNA isoform separation was shown to be dependent on column selectivity for individual isoform, column efficiency and on ammonium sulfate (AS) concentration in loading buffer (binding strength). SD and negative mode elution often operate in parallel, therefore negative mode elution additionally influences the efficiency of the overall purification process. Optimisation of chromatographic conditions achieved 98% sc pDNA homogeneity and a dynamic binding capacity of over 1mg/mL at a relatively low concentration of AS. SDC was successfully implemented for the enrichment of sc pDNA for plasmid vectors of different sizes, and for separation of linear and and sc isoforms, independently of oc:sc isoform ratio, and flow-rate used. This study therefore identifies SDC as a promising new approach to large-scale pDNA purification, which is compatible with continuous, multicolumn chromatography systems, and could therefore be used to increase productivity of pDNA production in the future. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Permissiveness of soil microbial communities towards broad host range plasmids

    DEFF Research Database (Denmark)

    Klümper, Uli

    larger than previously assumed. I was able to show abundant plasmid transfer from the Gram negative donor strains to a wide diversity of Gram positive soil bacteria, formerly thought to constitute distinct clusters of gene transfer. Moreover, among the observed transconjugants, I identified a core super...... environmental factors that modulate plasmid transfer in soil microbial communities. In order to attain these goals, I developed a high-throughput method that enabled me to evaluate the permissiveness of bacterial communities towards introduced plasmids. This new approach is based on the introduction...... fraction of soil the bacteria (up to 1 in 10,000) were able to take up any of these broad host range conjugal plasmids. The transconjugal pools comprised 11 bacterial phyla. This finding indicates that the realized transfer range of broad host range plasmids in environmental microbial communities is much...

  10. Complex nature of enterococcal pheromone-responsive plasmids.

    Science.gov (United States)

    Wardal, Ewa; Sadowy, Ewa; Hryniewicz, Waleria

    2010-01-01

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

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

    Science.gov (United States)

    Kuwahara, S

    1978-09-01

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

  12. Bacterial Mitosis: ParM of Plasmid R1 Moves Plasmid DNA by an Actin-like Insertional Polymerization Mechanism

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Borch, Jonas; Dam, Mette

    2003-01-01

    Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system...

  13. 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.

  14. 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.

  15. In vitro replication of cyanobacterial plasmids from Synechocystis PCC 6803.

    Science.gov (United States)

    Yang, X; Daniell, H; McFadden, B

    1994-09-01

    Little knowledge of DNA replication in cyanobacteria is available. In this study, we report the development and characterization of an in vitro system for studies of replication of the endogenous plasmids from the unicellular cyanobacterium Synechocystis 6803. This system (fraction III) was isolated at high salt concentrations and partially purified on a heparin-agarose column. DNA polymerases in Synechocystis 6803 appeared to be associated with membranes and could be released by the addition of ammonium sulfate to 20% saturation. DNA synthesis in fraction III was dependent on the addition of cyanobacterial plasmids isolated from the same strain. The in vitro replication products consist mostly of the supercoiled form of the plasmids. Unlike replication of many Escherichia coli plasmids, replication of cyanobacterial plasmids did not require added ATP, was not inhibited by omission of the ribonucleotides, and was insensitive to the RNA polymerase inhibitor rifampicin and the gyrase inhibitor novobiocin, but was inhibited by ethidium bromide. These data suggest that RNA may not be involved in the initiation of replication of cyanobacterial plasmids from Synechocystis 6803. In addition, intermediates of replication have been detected by two-dimensional gel electrophoresis. Density labeling experiments also indicate that cyanobacterial plasmid synthesis in vitro occurs by a semiconservative replication.

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

    Science.gov (United States)

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

    1980-08-01

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

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

    Science.gov (United States)

    Baumgartner, A; Breer, C; Schopfer, K

    1989-04-05

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

  18. Effect of Plasmid Incompatibility on DNA Transfer to Streptococcus cremoris

    OpenAIRE

    Van Der Lelie, Daniel; Vossen, Jos M.B.M. van der; Venema, Gerard

    1988-01-01

    Several Streptococcus cremoris strains were used in protoplast transformation and interspecific protoplast fusion experiments with Streptococcus lactis and Bacillus subtilis, with pGKV110, pGKV21, and ΔpAMβ1 as the marker plasmids. ΔpAMβ1 is a 15.9-kilobase nonconjugative, deletion derivative of pAMβ1, which is considerably larger than the pGKV plasmids (approximately 4.5 kilobases). In general, ΔpAMβ1 was transferred more efficiently than the pGKV plasmids. Using electroporation, we were abl...

  19. Separation of plasmid DNA topoisomers by multimodal chromatography.

    Science.gov (United States)

    Silva-Santos, A Rita; Alves, Cláudia P A; Prazeres, Duarte Miguel F; Azevedo, Ana M

    2016-06-15

    The ability to analyze the distribution of topoisomers in a plasmid DNA sample is important when evaluating the quality of preparations intended for gene therapy and DNA vaccination or when performing biochemical studies on the action of topoisomerases and gyrases. Here, we describe the separation of supercoiled (sc) and open circular (oc) topoisomers by multimodal chromatography. A medium modified with the ligand N-benzyl-N-methyl ethanolamine and an elution scheme with increasing NaCl concentration are used to accomplish the baseline separation of sc and oc plasmid. The utility of the method is demonstrated by quantitating topoisomers in a purified plasmid sample. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Bacterial mitosis: ParM of plasmid R1 moves plasmid DNA by an actin-like insertional polymerization mechanism.

    Science.gov (United States)

    Møller-Jensen, Jakob; Borch, Jonas; Dam, Mette; Jensen, Rasmus B; Roepstorff, Peter; Gerdes, Kenn

    2003-12-01

    Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system act together to generate the force required for plasmid movement during segregation. ParR protein binds cooperatively to the centromeric parC DNA region, thereby forming a complex that interacts with the filament-forming actin-like ParM protein in an ATP-dependent manner, suggesting that plasmid movement is powered by insertional polymerization of ParM. Consistently, we find that segregating plasmids are positioned at the ends of extending ParM filaments. Thus, the process of R1 plasmid segregation in E. coli appears to be mechanistically analogous to the actin-based motility operating in eukaryotic cells. In addition, we find evidence suggesting that plasmid pairing is required for ParM polymerization.

  1. Influenza Plasmid DNA Vaccines: Progress and Prospects.

    Science.gov (United States)

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

    2015-01-01

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

  2. 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.

  3. Plasmid Conjugation in E. coli and Drug Resistance

    African Journals Online (AJOL)

    Prof. Ogunji

    respiratory infections etc) or prescribing the 'newest' antibiotics in the market when older “brands” may ..... influence an increase in mortality rate; high economic burden and longer hospital ... Conjugating plasmids into bacteria; Tri Parental.

  4. 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.

  5. Transfer of conjugative plasmids among bacteria under environmentally relevant conditions

    DEFF Research Database (Denmark)

    Musovic, Sanin

    at spredningskapacitet af en konjugerbare plasmid, der koder for kviksølv resistens via merA genet, finder sted under substrat begrænsede forhold til syntetisk bakterielt samfund. Plasmid overførsel var meget forhøjet ved kontinuert udsættelse af mikrokosms for en høj koncentration af kviksølv. De forskellige vækstrater...

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

    Science.gov (United States)

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

    2016-02-01

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

  7. 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.

  8. Plasmid copy number noise in monoclonal populations of bacteria

    Science.gov (United States)

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

    2010-01-01

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

  9. Construction and Use of Flow Cytometry Optimized Plasmid-Sensor Strains

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Oregaard, Gunnar; Sørensen, Søren Johannes;

    2009-01-01

    stability of the plasmid is high. The method presented here relies on a phenotypic (green fluorescence protein) marker, which is switched on if the host bacteria loses the residing plasmid. The incorporation of flow cytometry for single-cell detection and discrimination between plasmid-free and plasmid...

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

    Science.gov (United States)

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

    2012-01-01

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

  11. Survival and evolution of a large multidrug resistance plasmid in new clinical bacterial hosts

    DEFF Research Database (Denmark)

    Porse, Andreas; Schønning, Kristian; Munck, Christian

    2016-01-01

    of these plasmids within pathogenic hosts are poorly understood. Here we study plasmid-host adaptations following transfer of a 73 kb conjugative multidrug resistance plasmid to naïve clinical isolates of Klebsiella pneumoniae and Escherichia coli We use experimental evolution, mathematical modelling and population...... sequencing to show that the long-term persistence and molecular integrity of the plasmid is highly influenced by multiple factors within a 25 kb plasmid region constituting a host-dependent burden. In the E. coli hosts investigated here, improved plasmid stability readily evolves via IS26 mediated deletions...... of plasmid adaptation. While insertion sequences are well known to supply plasmids with adaptive traits, our findings suggest that they also play an important role in plasmid evolution by maintaining the plasticity necessary to alleviate plasmid-host constrains. Further, the observed evolutionary strategy...

  12. 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

  13. Altered Murine Tissue Colonization by Borrelia burgdorferi following Targeted Deletion of Linear Plasmid 17-Carried Genes

    OpenAIRE

    Casselli, Timothy; Tourand, Yvonne; Bankhead, Troy

    2012-01-01

    The causative agent of Lyme disease, Borrelia burgdorferi, possesses a segmented genome comprised of a single linear chromosome and upwards of 23 linear and circular plasmids. Much of what is known about plasmid-borne genes comes from studying laboratory clones that have spontaneously lost one or more plasmids during in vitro passage. Some plasmids, including the linear plasmid lp17, are never or rarely reported to be lost during routine culture; therefore, little is known about the requireme...

  14. 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

  15. 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

  16. Plasmid mediated antibiotic resistance in isolated bacteria from burned patients.

    Science.gov (United States)

    Beige, Fahimeh; Baseri Salehi, Majid; Bahador, Nima; Mobasherzadeh, Sina

    2015-01-01

    Nowadays, the treatment of burned patients is difficult because of the high frequency of infection with antibiotic resistance bacteria. This study was conducted to evaluate the level of antibiotic resistance in Gram-negative bacteria and its relation with the existence of plasmid. The samples were collected from two hundred twenty hospitalized burned patients in Isfahan burn hospital during a three-month period (March 2012 to June 2012). The samples were isolated and the Gram-negative bacteria were identified using phenotypic method and API 20E System. Antibiotic susceptibility and plasmid profile were determined by standard Agar disc diffusion and plasmid spin column extraction methods. Totally 117 Gram-negative bacteria were isolated, the most common were Pseudomonas aerugionsa (37.6%), P. fluorescens (25.6%), Acinetobacter baumanii (20/5%) and Klebsiella pneumoniae (7.6%), respectively. The isolates showed high frequency of antibiotic resistance against ceftazidime and co-amoxiclave (100%) and low frequency of antibiotic resistance against amikacin with (70%).The results indicated that 60% of the isolates harboured plasmid. On the other hand, the patients infected with A. baumanii and P. aeruginosa were cured (with 60% frequency) whereas, those infected with P. fluorescens were not cured. Hence, probably antibiotic resistance markers of A. baumanii and P. aeruginosa are plasmid mediated; however, P. fluorescens is chromosomally mediated. Based on our findings, P. aerugionsa is a major causative agent of wound infections and amikacin could be considered as a more effective antibiotic for treatment of the burned patients.

  17. 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.

  18. Functional amyloids as inhibitors of plasmid DNA replication

    Science.gov (United States)

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

    2016-01-01

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

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. Anion exchange purification of plasmid DNA using expanded bed adsorption.

    Science.gov (United States)

    Ferreira, G N; Cabral, J M; Prazeres, D M

    2000-01-01

    Recent developments in gene therapy with non-viral vectors and DNA vaccination have increased the demand for large amounts of pharmaceutical-grade plasmid DNA. The high viscosity of process streams is of major concern in the purification of plasmids, since it can cause high back pressures in column operations, thus limiting the throughput. In order to avoid these high back pressures, expanded bed anion exchange chromatography was evaluated as an alternative to fixed bed chromatography. A Streamline 25 column filled with 100 ml of Streamline QXL media, was equilibrated with 0.5 M NaCl in TE (10 mM Tris, 1 mM EDTA, pH = 8.0) buffer at an upward flow of 300 cmh-1, E. coli lysates (obtained from up to 3 liters of fermentation broth) were injected in the column. After washing out the unbound material, the media was allowed to sediment and the plasmid was eluted with 1 M NaCl in TE buffer at a downward flow of 120 cmh-1. Purification factors of 36 +/- 1 fold, 26 +/- 0.4 plasmid purity, and close to 100% yields were obtained when less than one settled column volume of plasmid feed was injected. However, both recovery yield and purity abruptly decreased when larger amounts were processed-values of 35 +/- 2 and 5 +/- 0.7 were obtained for the recovery yield and purity, respectively, when 250 ml of feedstock were processed. In these cases, gel clogging and expansion collapse were observed. The processing of larger volumes, thus larger plasmid quantities, was only possible by performing an isopropanol precipitation step prior to the chromatographic step. This step led to an enhancement of the purification step.

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

    Science.gov (United States)

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

    2010-02-01

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

  7. 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

  8. 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

  9. 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

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

    Science.gov (United States)

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

    2015-03-01

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

  11. Conjugal transfer of group B streptococcal plasmids and comobilization of Escherichia coli-Streptococcus shuttle plasmids to Lactobacillus plantarum.

    OpenAIRE

    1988-01-01

    The antibiotic resistance group B streptococcal plasmids, pIP501 and pVA797, were conjugally transferred from Streptococcus faecalis to Lactobacillus plantarum. The Escherichia coli-Streptococcus shuttle plasmids, pVA838 and pSA3, were mobilized from S. sanguis to L. plantarum by pVA797 via cointegrate formation. pVA838 readily resolved from pVA797 and was present in L. plantarum as deletion derivatives. The pVA797::pSA3 cointegrate failed to resolve in L. plantarum.

  12. 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.

  13. Genetic Characterization of ExPEC-Like Virulence Plasmids among a Subset of NMEC.

    Directory of Open Access Journals (Sweden)

    Bryon A Nicholson

    Full Text Available Neonatal Meningitis Escherichia coli (NMEC is one of the most common causes of neonatal bacterial meningitis in the US and elsewhere resulting in mortality or neurologic deficits in survivors. Large plasmids have been shown experimentally to increase the virulence of NMEC in the rat model of neonatal meningitis. Here, 9 ExPEC-like plasmids were isolated from NMEC and sequenced to identify the core and accessory plasmid genes of ExPEC-like virulence plasmids in NMEC and create an expanded plasmid phylogeny. Results showed sequenced virulence plasmids carry a strongly conserved core of genes with predicted functions in five distinct categories including: virulence, metabolism, plasmid stability, mobile elements, and unknown genes. The major functions of virulence-associated and plasmid core genes serve to increase in vivo fitness by adding multiple iron uptake systems to the genetic repertoire to facilitate NMEC's survival in the host's low iron environment, and systems to enhance bacterial resistance to host innate immunity. Phylogenetic analysis based on these core plasmid genes showed that at least two lineages of ExPEC-like plasmids could be discerned. Further, virulence plasmids from Avian Pathogenic E. coli and NMEC plasmids could not be differentiated based solely on the genes of the core plasmid genome.

  14. Pharmaceutical development of the plasmid DNA vaccine pDERMATT

    NARCIS (Netherlands)

    Quaak, S.G.L.

    2009-01-01

    The discovery of tumor specific antigens and self tolerance mechanisms against these antigens led to the assumption that antigens circulating at sufficient concentration levels could break this self tolerance mechanism and evoke immunological antitumor effects. pDERMATT (plasmid DNA encoding recombi

  15. 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.

  16. 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...

  17. Resistant plasmid profile analysis of multidrug resistant Escherichia ...

    African Journals Online (AJOL)

    Objectives: This study was carried out to determine the resistant plasmids of ... resistance pattern of micro-organisms to common an- tibiotics1 ... ment has necessitated the need for regular monitoring of antibiotics susceptibility trends to provide the basis for developing rational prescription programs, mak- ..... Paediatrics and.

  18. Pharmaceutical development of the plasmid DNA vaccine pDERMATT

    NARCIS (Netherlands)

    Quaak, S.G.L.

    2009-01-01

    The discovery of tumor specific antigens and self tolerance mechanisms against these antigens led to the assumption that antigens circulating at sufficient concentration levels could break this self tolerance mechanism and evoke immunological antitumor effects. pDERMATT (plasmid DNA encoding

  19. Use of plasmid DNA for induction of protective immunity

    DEFF Research Database (Denmark)

    Lorenzen, Niels

    2004-01-01

    Vaccines based on plasmid DNA have been tested for a number of fish pathogens but so far it is only in case of the rhabdoviruses, where the technology has been a real break through in vaccine research. Aspects of dose, time-course and mechanisms of protection, as well as practical use are discussed....

  20. Effects of maternal plasmid GHRH treatment on offspring growth

    Science.gov (United States)

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

  1. [Chromatographic separation of plasmid DNA by anion-exchange cryogel].

    Science.gov (United States)

    Guo, Yantao; Shen, Shaochuan; Yun, Junxian; Yao, Kejian

    2012-08-01

    Plasmid DNA (pDNA) is used as an important vector for gene therapy, and its wide application is restricted by the purity and yield. To obtain high-purity pDNA, a chromatographic method based on anion-exchange supermacroporous cryogel was explored. The anion-exchange cryogel was prepared by grafting diethylaminoethyl-dextran to the epoxide groups of polyacrylamide-based matrix and pUC19 plasmid was used as a target to test the method. The plasmid was transferred into Escherichia coli DH5alpha, cultivated, harvested and lysed. The obtained culture was centrifuged and the supernatant was used as the plasmid feedstock, which was loaded into the anion-exchange cryogel bed for chromatographic separation. By optimizing the pH of running buffer and the elution conditions, high-purity pDNA was obtained by elution with 0.5 mol/L sodium chloride solution at pH 6.6. Compared to the traditional methods for purification of pDNA, animal source enzymes and toxic reagents were not involved in the present separation process, ensuring the safety of both the purification operations and the obtained pDNA.

  2. 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.

  3. Tragedy of the commons among antibiotic resistance plasmids.

    Science.gov (United States)

    Smith, Jeff

    2012-04-01

    As social interactions are increasingly recognized as important determinants of microbial fitness, sociobiology is being enlisted to better understand the evolution of clinically relevant microbes and, potentially, to influence their evolution to aid human health. Of special interest are situations in which there exists a "tragedy of the commons," where natural selection leads to a net reduction in fitness for all members of a population. Here, I demonstrate the existence of a tragedy of the commons among antibiotic resistance plasmids of bacteria. In serial transfer culture, plasmids evolved a greater ability to superinfect already-infected bacteria, increasing plasmid fitness when evolved genotypes were rare. Evolved plasmids, however, fell victim to their own success, reducing the density of their bacterial hosts when they became common and suffering reduced fitness through vertical transmission. Social interactions can thus be an important determinant of evolution for the molecular endosymbionts of bacteria. These results also identify an avenue of evolution that reduces proliferation of both antibiotic resistance genes and their bacterial hosts. © 2012 The Author(s). Evolution© 2012 The Society for the Study of Evolution.

  4. 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

  5. Characterization of the Lactobacillus plantarum plasmid pCD033 and generation of the plasmid free strain L. plantarum 3NSH.

    Science.gov (United States)

    Heiss, Silvia; Grabherr, Reingard; Heinl, Stefan

    2015-09-01

    Lactobacillus plantarum CD033, a strain isolated from grass silage in Austria, harbors a 7.9 kb plasmid designated pCD033. Sequence analysis identified 14 open reading frames and 8 of these were supposed to be putative coding sequences. Gene annotation revealed no putative essential genes being plasmid encoded, but a plasmid addiction system based on a PemI/PemK-like toxin-antitoxin system, able to stabilize plasmid maintenance. Absence of a replication initiation protein, a double strand origin as well as a single strand origin on plasmid pCD033 suggests replication via a new type of theta mechanism, whereby plasmid replication is potentially initiated and regulated by non-coding RNA. Detailed examination of segregational stability of plasmid vectors consisting of pCD033-fragments, combined with a selection marker, resulted in definition of a stably maintained minimal replicon. A gene encoding a RepB/OrfX-like protein was found to be not essential for plasmid replication. Alignment of the amino acid sequence of this protein with related proteins unveiled a highly conserved amino acid motif (LLDQQQ). L. plantarum CD033 was cured of pCD033 resulting in the novel plasmid free strain L. plantarum 3NSH. Plasmid curing demonstrated that no essential features are provided by pCD033 under laboratory conditions.

  6. 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.

  7. Plasmid profiling of bacterial isolates from confined environments

    Science.gov (United States)

    van Houdt, Rob; Provoost, Ann; Coninx, Ilse; Leys, Natalie; Mergeay, Max

    Plasmid profiling of bacterial isolates from confined environments R. Van Houdt, I. Coninx, A. Provoost, N. Leys, and M. Mergeay Expertise group for Molecular and Cellular Biology, Institute for Environment, Health and Safety, Belgian Nuclear Research Centre (SCK•CEN), Boeretang 200, B-2400 Mol, Belgium. Human exploration of extreme and isolated hostile environments such as space requires special confined small volume habitats to protect and house the crew. However, human confinement in such small volume habitats has restrictions on waste disposal and personal hygiene and inevitably generates a particular community of microorganisms within the habitat. These microorganisms are mainly originating from the crew (skin, mucous membranes, upper respiratory tract, mouth, and gastrointestinal tract) but also include the residing environmental microorganisms. Earth-based confined habitats such as the Antarctic Research Station Concordia are used as test beds for long-duration spaceflights to study the physiologic and psychological adaptation to isolated environments. The dynamics of the environmental microbial population in such a test bed could render additional insights in assessing the potential health risks in long-duration space missions. Not only total bacterial contamination levels are important, but it is essential to identify also the predominant microbial taxa and their mobile genetic elements (MGE). These MGEs could be exchanged between bacteria by horizontal gene transfer and may alter the pathogenic potential since they often carry antibiotic resistance or more in general adaptation-enhancing traits. In this study several bacterial strains isolated in the Concordia research station were examined for their plasmid content. An optimized protocol for extraction of large plasmids showed the present of at least one plasmid in 50% of the strains. For all strains the minimal inhibitory concentration of a range of antibiotics was determined indicating resistance to

  8. Participation of the lytic replicon in bacteriophage P1 plasmid maintenance.

    OpenAIRE

    1989-01-01

    P1 bacteriophage carries at least two replicons: a plasmid replicon and a viral lytic replicon. Since the isolated plasmid replicon can maintain itself stably at the low copy number characteristic of intact P1 prophage, it has been assumed that this replicon is responsible for driving prophage replication. We provide evidence that when replication from the plasmid replicon is prevented, prophage replication continues, albeit at a reduced rate. The residual plasmid replication is due to incomp...

  9. 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....

  10. Enhanced brain targeting efficiency of intranasally administered plasmid DNA: an alternative route for brain gene therapy.

    Science.gov (United States)

    Han, In-Kwon; Kim, Mi Young; Byun, Hyang-Min; Hwang, Tae Sun; Kim, Jung Mogg; Hwang, Kwang Woo; Park, Tae Gwan; Jung, Woon-Won; Chun, Taehoon; Jeong, Gil-Jae; Oh, Yu-Kyoung

    2007-01-01

    Recently, nasal administration has been studied as a noninvasive route for delivery of plasmid DNA encoding therapeutic or antigenic genes. Here, we examined the brain targeting efficiency and transport pathways of intranasally administered plasmid DNA. Quantitative polymerase chain reaction (PCR) measurements of plasmid DNA in blood and brain tissues revealed that intranasally administered pCMVbeta (7.2 kb) and pN2/CMVbeta (14.1 kb) showed systemic absorption and brain distribution. Following intranasal administration, the beta-galactosidase protein encoded by these plasmids was significantly expressed in brain tissues. Kinetic studies showed that intranasally administered plasmid DNA reached the brain with a 2,595-fold higher efficiency than intravenously administered plasmid DNA did, 10 min post-dose. Over 1 h post-dose, the brain targeting efficiencies were consistently higher for intranasally administered plasmid DNA than for intravenously administered DNA. To examine how plasmid DNA enters the brain and moves to the various regions, we examined tissues from nine brain regions, at 5 and 10 min after intranasal or intravenous administration of plasmid DNA. Intravenously administered plasmid DNA displayed similar levels of plasmid DNA in the nine different regions, whereas, intranasally administered plasmid DNA exhibited different levels of distribution among the regions, with the highest plasmid DNA levels in the olfactory bulb. Moreover, plasmid DNA was mainly detected in the endothelial cells, but not in glial cells. Our results suggest that intranasally applied plasmid DNA may reach the brain through a direct route, possibly via the olfactory bulb, and that the nasal route might be an alternative method for efficiently delivering plasmid DNA to the brain.

  11. 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...

  12. Effect of plasmid pKM101 in ultraviolet irradiated uvr+ and uvr- Escherichia coli.

    Science.gov (United States)

    Slezáriková, V; Sedliaková, M; Andreeva, I V; Rusina OYu; Skavronskaya, A G

    1992-11-16

    The effect of plasmid pKM101 on UV irradiated excision proficient and excision deficient cells was investigated. The plasmid increased the survival of excision proficient cells while partially inhibiting thymine dimer excision. The frequency of mutations was almost unchanged. In excision deficient cells the effect of the plasmid on survival was less pronounced while cell mutability was increased. Our data indicate that the mucAB genes (carried by the plasmid) influence the two types of cells in a different way.

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

    DEFF Research Database (Denmark)

    Douidah, Laid; De Zutter, Lieven; Van Nieuwerburgh, Filip;

    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 sma...

  14. Occurrence and persistence of indigenous transconjugants carrying conjugative plasmids in soil.

    Science.gov (United States)

    Inoue, Daisuke; Soda, Satoshi; Tsutsui, Hirofumi; Yamazaki, Yuji; Murashige, Katsushi; Sei, Kazunari; Fujita, Masanori; Ike, Michihiko

    2009-09-01

    The transfer of the self-transmissible plasmids, RP4 and pJP4, from introduced bacteria to indigenous bacteria was examined in soil and slurry microcosms. The introduced plasmids persisted in indigenous transconjugants despite the low survival of introduced donors. The potential of the transconjugants for growth and conjugation affects the persistence of introduced plasmids in soil.

  15. Novel plasmid conferring kanamycin and tetracycline resistance in turkey-derived Campylobacter jejuni strain 11601MD

    Science.gov (United States)

    In Campylobacter spp., resistance to the antibiotics kanamycin and tetracycline is frequently associated with plasmid-borne genes. However, relatively few plasmids of Campylobacter jejuni have been fully characterized to date. A novel plasmid (p11601MD; 44,095 bp.) harboring tet(O) was identified in...

  16. Studies on the expression of plasmid-borne genes in the endosymbiotic state of Rhizobium leguminosarum

    NARCIS (Netherlands)

    Krol, A.J.M.

    1982-01-01

    The subject matter of the research reported in this thesis is the role of plasmid-borne genes of Rhizobium in symbiosis and nitrogen fixation. Plasmid DNA was isolated from Rhizobium leguminosarum strain PRE and the expression of plasmid DNA in nitrogen fixing nodules was investigated by hybridizati

  17. Mosaic structure and regulation of conjugal transfer of the Escherichia coli plasmid pRK100

    NARCIS (Netherlands)

    Starcic Erjavec, Marjanca

    2003-01-01

    Plasmids are extrachromosomal DNA elements that can be found in prokaryotic as well as in eukaryotic cells. They can vary in size and genetic make-up. The plasmid pRK100, which is the study subject of this thesis, is a large (145 kb) natural conjugative plasmid, which was isolated from an uropathoge

  18. 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.

  19. The evolution of a conjugative plasmid and its ability to increase bacterial fitness

    Science.gov (United States)

    Dionisio, F; Conceição, I.C; Marques, A.C.R; Fernandes, L; Gordo, I

    2005-01-01

    Conjugative plasmids are extra-chromosomal DNA elements that are capable of horizontal transmission and are found in many natural isolated bacteria. Although plasmids may carry beneficial genes to their bacterial host, they may also cause a fitness cost. In this work, we studied the evolution of the R1 plasmid and we found that, in spite of the R1 plasmid conferring an initial cost to its host, after 420 generations the cost disappeared in all five independent evolution experiments. In fact, in two of these five experiments evolved conjugative plasmids actually conferred a fitness advantage to their hosts. Furthermore, the relative fitness of the ancestral clone bearing one of the evolved plasmids is significantly higher than both the plasmid-free ancestral cells and the evolved cells carrying the evolved plasmid. Given that the R1 plasmid may spread among different species of enterobacteria, we wondered what the effect of the evolved plasmid would be inside Salmonella enterica cells. We found that the evolved plasmid is also able to dramatically increase the relative fitness of these cells. Our results suggest that even if general usage of antibiotics is halted, conjugative plasmids that have been selected with antibiotics in previous years can still persist among bacterial populations or even invade new strains. PMID:17148179

  20. A Bipolar Spindle of Antiparallel ParM Filaments Drives Bacterial Plasmid Segregation

    DEFF Research Database (Denmark)

    Gayathri, P; Fujii, T; Møller-Jensen, Jakob;

    2012-01-01

    To ensure their stable inheritance by daughter cells during cell division, bacterial low copy-number plasmids make simple DNA segregating machines that use an elongating protein filament between sister plasmids. In the ParMRC system of Escherichia coli R1 plasmid, ParM, an actin-like protein, forms...

  1. 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

  2. Determination of plasmid copy number reveals the total plasmid DNA amount is greater than the chromosomal DNA amount in Bacillus thuringiensis YBT-1520.

    Directory of Open Access Journals (Sweden)

    Chunying Zhong

    Full Text Available Bacillus thuringiensis is the most widely used bacterial bio-insecticide, and most insecticidal crystal protein-coding genes are located on plasmids. Most strains of B. thuringiensis harbor numerous diverse plasmids, although the plasmid copy numbers (PCNs of all native plasmids in this host and the corresponding total plasmid DNA amount remains unknown. In this study, we determined the PCNs of 11 plasmids (ranging from 2 kb to 416 kb in a sequenced B. thuringiensis subsp. kurstaki strain YBT-1520 using real-time qPCR. PCNs were found to range from 1.38 to 172, and were negatively correlated to plasmid size. The amount of total plasmid DNA (∼8.7 Mbp was 1.62-fold greater than the amount of chromosomal DNA (∼5.4 Mbp at the mid-exponential growth stage (OD(600 = 2.0 of the organism. Furthermore, we selected three plasmids with different sizes and replication mechanisms to determine the PCNs over the entire life cycle. We found that the PCNs dynamically shifted at different stages, reaching their maximum during the mid-exponential growth or stationary phases and remaining stable and close to their minimum after the prespore formation stage. The PCN of pBMB2062, which is the smallest plasmid (2062 bp and has the highest PCN of those tested, varied in strain YBT-1520, HD-1, and HD-136 (172, 115, and 94, respectively. These findings provide insight into both the total plasmid DNA amount of B. thuringiensis and the strong ability of the species to harbor plasmids.

  3. 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.

  4. Type 3 Fimbriae Encoded on Plasmids Are Expressed from a Unique Promoter without Affecting Host Motility, Facilitating an Exceptional Phenotype That Enhances Conjugal Plasmid Transfer

    DEFF Research Database (Denmark)

    Madsen, Jonas Stenlokke; Riber, Leise; Kot, Witold;

    2016-01-01

    on plasmids is systematically different, as MrkH, a c-di-GMP dependent transcriptional activator is not needed for strong expression of the fimbriae. MrkH is required for expression of type 3 fimbriae of the Klebsiella pneumoniae chromosome, wherefrom the fimbriae operon (mrkABCDF) of plasmids is believed...... to have originated. We find that mrkABCDFs of plasmids are highly expressed via a unique promoter that differs from the original Klebsiella promoter resulting in fundamental behavioral consequences. Plasmid associated mrkABCDFs did not influence the swimming behavior of the host, that hereby acquired...

  5. Coupling between the Basic Replicon and the Kis-Kid Maintenance System of Plasmid R1: Modulation by Kis Antitoxin Levels and Involvement in Control of Plasmid Replication

    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.

  6. 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 ...

  7. An Improved Method for Including Upper Size Range Plasmids in Metamobilomes

    DEFF Research Database (Denmark)

    Norman, Anders; Riber, Leise; Luo, Wenting

    2014-01-01

    cloning vector (pBR322), and a 56 Kbp conjugative plasmid (pKJK10), to represent lower- and upper plasmid size ranges, respectively. Subjecting a mixture of these plasmids to the overall isolation protocol revealed a 34-fold over-amplification of pBR322 after MDA. To address this bias, we propose......, as gene functions associated with these plasmids, such as conjugation, was exclusively encoded in the data output generated through the modified protocol. Thus, with the suggested modification, access to a large uncharacterized pool of accessory elements that reside on medium-to-large plasmids has been...

  8. Large plasmids of Escherichia coli and Salmonella encode highly diverse arrays of accessory genes on common replicon families.

    Science.gov (United States)

    Williams, Laura E; Wireman, Joy; Hilliard, Valda C; Summers, Anne O

    2013-01-01

    Plasmids are important in evolution and adaptation of host bacteria, yet we lack a comprehensive picture of their own natural variation. We used replicon typing and RFLP analysis to assess diversity and distribution of plasmids in the ECOR, SARA, SARB and SARC reference collections of Escherichia coli and Salmonella. Plasmids, especially large (≥30 kb) plasmids, are abundant in these collections. Host species and genotype clearly impact plasmid prevalence; plasmids are more abundant in ECOR than SAR, but, within ECOR, subgroup B2 strains have the fewest large plasmids. The majority of large plasmids have unique RFLP patterns, suggesting high variation, even within dominant replicon families IncF and IncI1. We found only four conserved plasmid types within ECOR, none of which are widely distributed. Within SAR, conserved plasmid types are primarily serovar-specific, including a pSLT-like plasmid in 13 Typhimurium strains. Conservation of pSLT contrasts with variability of other plasmids, suggesting evolution of serovar-specific virulence plasmids is distinct from that of most enterobacterial plasmids. We sequenced a conserved serovar Heidelberg plasmid but did not detect virulence or antibiotic resistance genes. Our data illustrate the high degree of natural variation in large plasmids of E. coli and Salmonella, even among plasmids sharing backbone genes.

  9. Cationic lipids delay the transfer of plasmid DNA to lysosomes.

    Science.gov (United States)

    Wattiaux, R; Jadot, M; Laurent, N; Dubois, F; Wattiaux-De Coninck, S

    1996-10-14

    Plasmid 35S DNA, naked or associated with different cationic lipid preparations was injected to rats. Subcellular distribution of radioactivity in the liver one hour after injection, was established by centrifugation methods. Results show that at that time, 35S DNA has reached lysosomes. On the contrary, when 35S DNA was complexed with lipids, radioactivity remains located in organelles whose distribution after differential and isopycnic centrifugation, is clearly distinct from that of arylsulfatase, lysosome marker enzyme. Injection of Triton WR 1339, a specific density perturbant of lysosomes, four days before 35S DNA injection causes a density decrease of radioactivity bearing structures, apparent one hour after naked 35S DNA injection but visible only after more than five hours, when 35S DNA associated with a cationic lipid is injected. These observations show that cationic lipids delay the transfer to lysosomes, of plasmid DNA taken up by the liver.

  10. 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.

  11. 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.

  12. A Rebeccamycin Analog Provides Plasmid-Encoded Niche Defense.

    Science.gov (United States)

    Van Arnam, Ethan B; Ruzzini, Antonio C; Sit, Clarissa S; Currie, Cameron R; Clardy, Jon

    2015-11-18

    Bacterial symbionts of fungus-growing ants occupy a highly specialized ecological niche and face the constant existential threat of displacement by another strain of ant-adapted bacteria. As part of a systematic study of the small molecules underlying this fraternal competition, we discovered an analog of the antitumor agent rebeccamycin, a member of the increasingly important indolocarbazole family. While several gene clusters consistent with this molecule's newly reported modification had previously been identified in metagenomic studies, the metabolite itself has been cryptic. The biosynthetic gene cluster for 9-methoxyrebeccamycin is encoded on a plasmid in a manner reminiscent of plasmid-derived peptide antimicrobials that commonly mediate antagonism among closely related Gram-negative bacteria.

  13. Dataset of plasmid DNA extraction using different magnetic nanoparticles (MNPs

    Directory of Open Access Journals (Sweden)

    H. Rahnama

    2016-12-01

    MNPs were characterized by energy dispersive spectroscopy (EDS and transmission electron microscopy (TEM. Finally, the overall efficiency of different MNPs (Fe3O4, Fe3O4/SiO2, Fe3O4/SiO2/TiO2 in plasmid DNA isolation was compared using gel electrophoresis analysis. The data supplied in this article supports the accompanying publication “Comparative study of three magnetic nano-particles (FeSO4, FeSO4/SiO2, FeSO4/SiO2/TiO2 in plasmid DNA extraction” (H. Rahnama, A. Sattarzadeh, F. Kazemi, N. Ahmadi, F. Sanjarian, Z. Zand, 2016 [1].

  14. Liquid-Crystalline Mesophases of Plasmid DNA in Bacteria

    Science.gov (United States)

    Reich, Ziv; Wachtel, Ellen J.; Minsky, Abraham

    1994-06-01

    Bacterial plasmids may often reach a copy number larger than 1000 per cell, corresponding to a total amount of DNA that may exceed the amount of DNA within the bacterial chromosome. This observation highlights the problem of cellular accommodation of large amounts of closed-circular nucleic acids, whose interwound conformation offers negligible DNA compaction. As determined by x-ray scattering experiments conducted on intact bacteria, supercoiled plasmids segregate within the cells into dense clusters characterized by a long-range order. In vitro studies performed at physiological DNA concentrations indicated that interwound DNA spontaneously forms liquid crystalline phases whose macroscopic structural properties are determined by the features of the molecular supercoiling. Because these features respond to cellular factors, DNA supercoiling may provide a sensitive regulatory link between cellular parameters and the packaging modes of interwound DNA in vivo.

  15. Current trends in separation of plasmid DNA vaccines: a review.

    Science.gov (United States)

    Ghanem, Ashraf; Healey, Robert; Adly, Frady G

    2013-01-14

    Plasmid DNA (pDNA)-based vaccines offer more rapid avenues for development and production if compared to those of conventional virus-based vaccines. They do not rely on time- or labour-intensive cell culture processes and allow greater flexibility in shipping and storage. Stimulating antibodies and cell-mediated components of the immune system are considered as some of the major advantages associated with the use of pDNA vaccines. This review summarizes the current trends in the purification of pDNA vaccines for practical and analytical applications. Special attention is paid to chromatographic techniques aimed at reducing the steps of final purification, post primary isolation and intermediate recovery, in order to reduce the number of steps necessary to reach a purified end product from the crude plasmid.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. Efficient transformation of Bacillus thuringiensis requires nonmethylated plasmid DNA.

    OpenAIRE

    Macaluso, A; Mettus, A M

    1991-01-01

    The transformation efficiency of Bacillus thuringiensis depends upon the source of plasmid DNA. DNA isolated from B. thuringiensis, Bacillus megaterium, or a Dam- Dcm- Escherichia coli strain efficiently transformed several B. thuringiensis strains, B. thuringiensis strains were grouped according to which B. thuringiensis backgrounds were suitable sources of DNA for transformation of other B. thuringiensis strains, suggesting that B. thuringiensis strains differ in DNA modification and restri...

  2. 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

  3. Growth dependence of conjugation explains limited plasmid invasion in biofilms: an individual‐based modelling study

    DEFF Research Database (Denmark)

    Merkey, Brian; Lardon, Laurent; Seoane, Jose Miguel;

    2011-01-01

    . By extending an individual‐based model of microbial growth and interactions to include the dynamics of plasmid carriage and transfer by individual cells, we were able to conduct in silico tests of this and other hypotheses on the dynamics of conjugal plasmid transfer in biofilms. For a generic model plasmid...... and scan speed) and spatial reach (EPS yield, conjugal pilus length) are more important for successful plasmid invasion than the recipients' growth rate or the probability of segregational loss. While this study identifies one factor that can limit plasmid invasion in biofilms, the new individual......Plasmid invasion in biofilms is often surprisingly limited in spite of the close contact of cells in a biofilm. We hypothesized that this poor plasmid spread into deeper biofilm layers is caused by a dependence of conjugation on the growth rate (relative to the maximum growth rate) of the donor...

  4. Differences in the stability of the plasmids of Yersinia pestis cultures in vitro: impact on virulence

    Directory of Open Access Journals (Sweden)

    TC Leal-Balbino

    2004-11-01

    Full Text Available Plasmid and chromosomal genes encode determinants of virulence for Yersinia pestis, the causative agent of plague. However, in vitro, Y. pestis genome is very plastic and several changes have been described. To evaluate the alterations in the plasmid content of the cultures in vitro and the impact of the alterations to their pathogenicity, three Y. pestis isolates were submitted to serial subculture, analysis of the plasmid content, and testing for the presence of characteristic genes in each plasmid of colonies selected after subculture. Different results were obtained with each strain. The plasmid content of one of them was shown to be stable; no apparent alteration was produced through 32 subcultures. In the other two strains, several alterations were observed. LD50 in mice of the parental strains and the derived cultures with different plasmid content were compared. No changes in the virulence plasmid content could be specifically correlated with changes in the LD50.

  5. Scaling-up recombinant plasmid DNA for clinical trial: current concern, solution and status.

    Science.gov (United States)

    Ismail, Ruzila; Allaudin, Zeenathul Nazariah; Lila, Mohd-Azmi Mohd

    2012-09-07

    Gene therapy and vaccines are rapidly developing field in which recombinant nucleic acids are introduced in mammalian cells for enhancement, restoration, initiation or silencing biochemical function. Beside simplicity in manipulation and rapid manufacture process, plasmid DNA-based vaccines have inherent features that make them promising vaccine candidates in a variety of diseases. This present review focuses on the safety concern of the genetic elements of plasmid such as propagation and expression units as well as their host genome for the production of recombinant plasmid DNA. The highlighted issues will be beneficial in characterizing and manufacturing plasmid DNA for save clinical use. Manipulation of regulatory units of plasmid will have impact towards addressing the safety concerns raised in human vaccine applications. The gene revolution with plasmid DNA by alteration of their plasmid and production host genetics will be promising for safe delivery and obtaining efficient outcomes.

  6. Partition-associated incompatibility caused by random assortment of pure plasmid clusters

    DEFF Research Database (Denmark)

    Ebersbach, Gitte; Sherratt, David J; Gerdes, Kenn;

    2005-01-01

    Summary Bacterial plasmids and chromosomes encode centromere-like partition loci that actively segregate DNA before cell division. The molecular mechanism behind DNA segregation in bacteria is largely unknown. Here we analyse the mechanism of partition-associated incompatibility for plasmid pB171......-lived pairing of plasmids. Instead, pure R1 and F foci were positioned along the length of the cell, and in a random order. Thus, our results raise the possibility that partition-mediated plasmid incompatibility is not caused by pairing of heterologous plasmids but instead by random positioning of pure plasmid...... clusters along the long axis of the cell. The strength of the incompatibility was correlated with the capability of the plasmids to compete for the mid-cell position....

  7. An insight of traditional plasmid curing in Vibrio species

    Directory of Open Access Journals (Sweden)

    Vengadesh eLetchumanan

    2015-07-01

    Full Text Available As the causative agent of foodborne related illness, Vibrio species causes a huge impact on the public health and management. Vibrio species is often associated with seafood as the latter plays a role as a vehicle to transmit bacterial infections. Hence, antibiotics are used not to promote growth but rather to prevent and treat bacterial infections. The extensive use of antibiotics in the aquaculture industry and environment has led to the emerging of antibiotic resistant strains. This phenomenon has triggered an alarming public health concern due to the increase number of pathogenic Vibrio strains that are resistant to clinically used antibiotics and is found in the environment. Antibiotic resistance and the genes location in the strains can be detected through plasmid curing assay. The results derived from plasmid curing assay is fast, cost effective, sufficient in providing insights and influence the antibiotic management policies in the aquaculture industry. This presentation aims in discussing and providing insights on various curing agents in Vibrio species. To our best of knowledge, this is a first review written discussing on plasmid curing in Vibrio species.

  8. 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.

  9. Competing ParA structures space bacterial plasmids equally over the nucleoid.

    Directory of Open Access Journals (Sweden)

    Robert Ietswaart

    2014-12-01

    Full Text Available Low copy number plasmids in bacteria require segregation for stable inheritance through cell division. This is often achieved by a parABC locus, comprising an ATPase ParA, DNA-binding protein ParB and a parC region, encoding ParB-binding sites. These minimal components space plasmids equally over the nucleoid, yet the underlying mechanism is not understood. Here we investigate a model where ParA-ATP can dynamically associate to the nucleoid and is hydrolyzed by plasmid-associated ParB, thereby creating nucleoid-bound, self-organizing ParA concentration gradients. We show mathematically that differences between competing ParA concentrations on either side of a plasmid can specify regular plasmid positioning. Such positioning can be achieved regardless of the exact mechanism of plasmid movement, including plasmid diffusion with ParA-mediated immobilization or directed plasmid motion induced by ParB/parC-stimulated ParA structure disassembly. However, we find experimentally that parABC from Escherichia coli plasmid pB171 increases plasmid mobility, inconsistent with diffusion/immobilization. Instead our observations favor directed plasmid motion. Our model predicts less oscillatory ParA dynamics than previously believed, a prediction we verify experimentally. We also show that ParA localization and plasmid positioning depend on the underlying nucleoid morphology, indicating that the chromosomal architecture constrains ParA structure formation. Our directed motion model unifies previously contradictory models for plasmid segregation and provides a robust mechanistic basis for self-organized plasmid spacing that may be widely applicable.

  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. Characterization of Multidrug-Resistant Escherichia coli by Plasmid Replicon Typing and Pulsed-Field Gel Electrophoresis

    Science.gov (United States)

    Background: Characterization of plasmids has particular clinical significance because genes encoding important traits such as antimicrobial resistance are frequently present in plasmids. Plasmid replicon typing is a multiplex PCR based method that can be used to classify 18 of the 26 known plasmid t...

  12. Type 3 Fimbriae Encoded on Plasmids Are Expressed from a Unique Promoter without Affecting Host Motility, Facilitating an Exceptional Phenotype That Enhances Conjugal Plasmid Transfer

    Science.gov (United States)

    Madsen, Jonas Stenløkke; Riber, Leise; Kot, Witold; Basfeld, Alrun; Burmølle, Mette; Hansen, Lars Hestbjerg; Sørensen, Søren Johannes

    2016-01-01

    Horizontal gene transfer (HGT), the transmission of genetic material to a recipient that is not the progeny of the donor, is fundamental in bacterial evolution. HGT is often mediated by mobile genetic elements such as conjugative plasmids, which may be in conflict with the chromosomal elements of the genome because they are independent replicons that may petition their own evolutionary strategy. Here we study differences between type 3 fimbriae encoded on wild type plasmids and in chromosomes. Using known and newly characterized plasmids we show that the expression of type 3 fimbriae encoded on plasmids is systematically different, as MrkH, a c-di-GMP dependent transcriptional activator is not needed for strong expression of the fimbriae. MrkH is required for expression of type 3 fimbriae of the Klebsiella pneumoniae chromosome, wherefrom the fimbriae operon (mrkABCDF) of plasmids is believed to have originated. We find that mrkABCDFs of plasmids are highly expressed via a unique promoter that differs from the original Klebsiella promoter resulting in fundamental behavioral consequences. Plasmid associated mrkABCDFs did not influence the swimming behavior of the host, that hereby acquired an exceptional phenotype being able to both actively swim (planktonic behavior) and express biofilm associated fimbriae (sessile behavior). We show that this exceptional phenotype enhances the conjugal transfer of the plasmid. PMID:27627107

  13. Type 3 fimbriae encoded on plasmids are expressed from a unique promoter without affecting host motility, facilitating an exceptional phenotype that enhances conjugal plasmid transfer

    DEFF Research Database (Denmark)

    Madsen, Jonas Stenløkke; Riber, Leise; Kot, Witold Piotr;

    2016-01-01

    of the genome because they are independent replicons that may petition their own evolutionary strategy. Here we study differences between type 3 fimbriae encoded on wild type plasmids and in chromosomes. Using known and newly characterized plasmids we show that the expression of type 3 fimbriae encoded...

  14. Conjugative transferability of the A/C plasmids from Salmonella enterica isolates that possess or lack blaCMY in the A/C plasmid backbone

    Science.gov (United States)

    The objective of this study was to gain a better understanding of the conjugative transfer of antimicrobial resistance plasmids from 205 Salmonella enterica strains, isolated from cattle to E. coli or Salmonella recipients. PCR-based replicon typing (PBRT) was used to type incompatibility plasmid r...

  15. Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis: isolation of a spontaneous mutant of Bacillus subtilis with enhanced transformability for Escherichia coli-propagated chimeric plasmid DNA.

    OpenAIRE

    Ostroff, G. R.; Pène, J. J.

    1983-01-01

    Hybrid plasmid DNA cloned in Escherichia coli undergoes deletions when returned to competent Bacillus subtilis, even in defined restriction and modification mutants of strain 168. We have isolated a mutant of B. subtilis MI112 which is stably transformed at high frequency by chimeric plasmid DNA propagated in E. coli.

  16. Expansion of a plasmid classification system for Gram-positive bacteria and determination of the diversity of plasmids in Staphylococcus aureus strains of human, animal, and food origins

    DEFF Research Database (Denmark)

    Lozano, C.; Garcia-Migura, L.; Aspiroz, C.

    2012-01-01

    An expansion of a previously described plasmid classification was performed and used to reveal the plasmid content of a collection of 92 Staphylococcus aureus strains of different origins. rep genes of other genera were detected in Staphylococcus. S1 pulsed-field gel electrophoresis (PFGE) hybrid...

  17. Analysis of plasmid diversity in 96 Rhodococcus equi strains isolated in Normandy (France) and sequencing of the 87-kb type I virulence plasmid.

    Science.gov (United States)

    Duquesne, Fabien; Hébert, Laurent; Sévin, Corinne; Breuil, Marie-France; Tapprest, Jackie; Laugier, Claire; Petry, Sandrine

    2010-10-01

    To characterize the potential epidemiological relationship between the origin of Rhodococcus equi strains and the type of their virulence plasmids, we performed a comparative analysis of virulence plasmid types encountered in 96 R. equi strains isolated from (1) autopsied horses, (2) organic samples (horse faeces, manure and straw) and (3) environmental samples. Our results revealed no clear epidemiological link between virulence plasmid type and the origin of R. equi strains isolated from horse-related environments. To understand this result, we determined the nucleotide sequence of the second most frequently isolated virulence plasmid type: a 87-kb type I (pVAPA116) plasmid and compared it with the previously sequenced (and most commonly encountered) 85-kb type I (pVAPA1037) plasmid. Our results show that the divergence between these two plasmids is mainly due to the presence of three allelic exchange loci, resulting in the deletion of two genes and the insertion of three genes in pVAPA116 compared with pVAPA1037. In conclusion, it appears that the divergence between the two sequenced rhodococcal virulence plasmids is not associated with the vap pathogenicity island and may result from an evolutionary process driven by a mobility-related invertase/resolvase invA-like gene. © 2010 ANSES. Journal compilation © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.

  18. 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.

  19. Insights into dynamics of mobile genetic elements in hyperthermophilic environments from five new Thermococcus plasmids.

    Directory of Open Access Journals (Sweden)

    Mart Krupovic

    Full Text Available Mobilome of hyperthermophilic archaea dwelling in deep-sea hydrothermal vents is poorly characterized. To gain insight into genetic diversity and dynamics of mobile genetic elements in these environments we have sequenced five new plasmids from different Thermococcus strains that have been isolated from geographically remote hydrothermal vents. The plasmids were ascribed to two subfamilies, pTN2-like and pEXT9a-like. Gene content and phylogenetic analyses illuminated a robust connection between pTN2-like plasmids and Pyrococcus abyssi virus 1 (PAV1, with roughly half of the viral genome being composed of genes that have homologues in plasmids. Unexpectedly, pEXT9a-like plasmids were found to be closely related to the previously sequenced plasmid pMETVU01 from Methanocaldococcus vulcanius M7. Our data suggests that the latter observation is most compatible with an unprecedented horizontal transfer of a pEXT9a-like plasmid from Thermococcales to Methanococcales. Gene content analysis revealed that thermococcal plasmids encode Hfq-like proteins and toxin-antitoxin (TA systems of two different families, VapBC and RelBE. Notably, although abundant in archaeal genomes, to our knowledge, TA and hfq-like genes have not been previously found in archaeal plasmids or viruses. Finally, the plasmids described here might prove to be useful in developing new genetic tools for hyperthermophiles.

  20. RK2 plasmid dynamics in Caulobacter crescentus cells--two modes of DNA replication initiation.

    Science.gov (United States)

    Wegrzyn, Katarzyna; Witosinska, Monika; Schweiger, Pawel; Bury, Katarzyna; Jenal, Urs; Konieczny, Igor

    2013-06-01

    Undisturbed plasmid dynamics is required for the stable maintenance of plasmid DNA in bacterial cells. In this work, we analysed subcellular localization, DNA synthesis and nucleoprotein complex formation of plasmid RK2 during the cell cycle of Caulobacter crescentus. Our microscopic observations showed asymmetrical distribution of plasmid RK2 foci between the two compartments of Caulobacter predivisional cells, resulting in asymmetrical allocation of plasmids to progeny cells. Moreover, using a quantitative PCR (qPCR) method, we estimated that multiple plasmid particles form a single fluorescent focus and that the number of plasmids per focus is approximately equal in both swarmer and predivisional Caulobacter cells. Analysis of the dynamics of TrfA-oriV complex formation during the Caulobacter cell cycle revealed that TrfA binds oriV primarily during the G1 phase, however, plasmid DNA synthesis occurs during the S and G2 phases of the Caulobacter cell cycle. Both in vitro and in vivo analysis of RK2 replication initiation in C. crescentus cells demonstrated that it is independent of the Caulobacter DnaA protein in the presence of the longer version of TrfA protein, TrfA-44. However, in vivo stability tests of plasmid RK2 derivatives suggested that a DnaA-dependent mode of plasmid replication initiation is also possible.

  1. Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity.

    Science.gov (United States)

    Münch, Karin M; Müller, Johannes; Wienecke, Sarah; Bergmann, Simone; Heyber, Steffi; Biedendieck, Rebekka; Münch, Richard; Jahn, Dieter

    2015-09-01

    During the past 2 decades, Bacillus megaterium has been systematically developed for the gram-per-liter scale production of recombinant proteins. The plasmid-based expression systems employed use a xylose-controlled promoter. Protein production analyses at the single-cell level using green fluorescent protein as a model product revealed cell culture heterogeneity characterized by a significant proportion of less productive bacteria. Due to the enormous size of B. megaterium, such bistable behavior seen in subpopulations was readily analyzed by time lapse microscopy and flow cytometry. Cell culture heterogeneity was not caused simply by plasmid loss: instead, an asymmetric distribution of plasmids during cell division was detected during the exponential-growth phase. Multicopy plasmids are generally randomly distributed between daughter cells. However, in vivo and in vitro experiments demonstrated that under conditions of strong protein production, plasmids are retained at one of the cell poles. Furthermore, it was found that cells with accumulated plasmids and high protein production ceased cell division. As a consequence, the overall protein production of the culture was achieved mainly by the subpopulation with a sufficient plasmid copy number. Based on our experimental data, we propose a model whereby the distribution of multicopy plasmids is controlled by polar fixation under protein production conditions. Thereby, cell lines with fluctuating plasmid abundance arise, which results in population heterogeneity. Our results provide initial insights into the mechanism of cellular heterogeneity during plasmid-based recombinant protein production in a Bacillus species.

  2. Insights into dynamics of mobile genetic elements in hyperthermophilic environments from five new Thermococcus plasmids.

    Science.gov (United States)

    Krupovic, Mart; Gonnet, Mathieu; Hania, Wajdi Ben; Forterre, Patrick; Erauso, Gaël

    2013-01-01

    Mobilome of hyperthermophilic archaea dwelling in deep-sea hydrothermal vents is poorly characterized. To gain insight into genetic diversity and dynamics of mobile genetic elements in these environments we have sequenced five new plasmids from different Thermococcus strains that have been isolated from geographically remote hydrothermal vents. The plasmids were ascribed to two subfamilies, pTN2-like and pEXT9a-like. Gene content and phylogenetic analyses illuminated a robust connection between pTN2-like plasmids and Pyrococcus abyssi virus 1 (PAV1), with roughly half of the viral genome being composed of genes that have homologues in plasmids. Unexpectedly, pEXT9a-like plasmids were found to be closely related to the previously sequenced plasmid pMETVU01 from Methanocaldococcus vulcanius M7. Our data suggests that the latter observation is most compatible with an unprecedented horizontal transfer of a pEXT9a-like plasmid from Thermococcales to Methanococcales. Gene content analysis revealed that thermococcal plasmids encode Hfq-like proteins and toxin-antitoxin (TA) systems of two different families, VapBC and RelBE. Notably, although abundant in archaeal genomes, to our knowledge, TA and hfq-like genes have not been previously found in archaeal plasmids or viruses. Finally, the plasmids described here might prove to be useful in developing new genetic tools for hyperthermophiles.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. Comparative genomics of the IncA/C multidrug resistance plasmid family.

    Science.gov (United States)

    Fricke, W Florian; Welch, Timothy J; McDermott, Patrick F; Mammel, Mark K; LeClerc, J Eugene; White, David G; Cebula, Thomas A; Ravel, Jacques

    2009-08-01

    Multidrug resistance (MDR) plasmids belonging to the IncA/C plasmid family are widely distributed among Salmonella and other enterobacterial isolates from agricultural sources and have, at least once, also been identified in a drug-resistant Yersinia pestis isolate (IP275) from Madagascar. Here, we present the complete plasmid sequences of the IncA/C reference plasmid pRA1 (143,963 bp), isolated in 1971 from the fish pathogen Aeromonas hydrophila, and of the cryptic IncA/C plasmid pRAx (49,763 bp), isolated from Escherichia coli transconjugant D7-3, which was obtained through pRA1 transfer in 1980. Using comparative sequence analysis of pRA1 and pRAx with recent members of the IncA/C plasmid family, we show that both plasmids provide novel insights into the evolution of the IncA/C MDR plasmid family and the minimal machinery necessary for stable IncA/C plasmid maintenance. Our results indicate that recent members of the IncA/C plasmid family evolved from a common ancestor, similar in composition to pRA1, through stepwise integration of horizontally acquired resistance gene arrays into a conserved plasmid backbone. Phylogenetic comparisons predict type IV secretion-like conjugative transfer operons encoded on the shared plasmid backbones to be closely related to a group of integrating conjugative elements, which use conjugative transfer for horizontal propagation but stably integrate into the host chromosome during vegetative growth. A hipAB toxin-antitoxin gene cluster found on pRA1, which in Escherichia coli is involved in the formation of persister cell subpopulations, suggests persistence as an early broad-spectrum antimicrobial resistance mechanism in the evolution of IncA/C resistance plasmids.

  8. 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.

  9. 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.

  10. Dual-Replicon Plasmids for Elimination of Resistant Plasmids%双复制子质粒消除耐药质粒的研究

    Institute of Scientific and Technical Information of China (English)

    莫冰; 余克花; 黎帆; 李蓉; 宋矿余

    2014-01-01

    Objective To observe the eliminating effect of dual-replicon plasmids on resistant plasmids.Methods The dual-replicon plasmids pKT230-oriV were constructed and transferred into bacteria with resistant plasmids pRK290 through the transformation and conjugational trans-fer pathways.The eliminating effect of dual-replicon plasmids on resistant plasmids was ob-served.Results The bacteria were cultured for 5 generations after the transfer of dual-replicon plasmids,and resistant plasmids pRK290 in bacteria were eliminated.Conclusion The conjuga-tional transfer of dual-replicon plasmids is a pathway to eliminate resistant plasmids.%目的:观察双复制子质粒对耐药质粒的消除作用。方法构建双复制子质粒 pKT230-oriV,并通过转化或接合转移途径转入带有 pRK290质粒的细菌中,观察双复制子质粒对耐药质粒的消除作用。结果转入双复制子质粒菌培养5代后,细菌中的 pRK290耐药质粒被消除。结论双复制子利用接合转移途径是消除耐药质粒的一个途径。

  11. 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.

  12. Plasmid DNA Supercoiling and Gyrase Activity in Escherichia coli Wild-Type and rpoS Stationary-Phase Cells

    Science.gov (United States)

    Reyes-Domínguez, Yazmid; Contreras-Ferrat, Gabriel; Ramírez-Santos, Jesús; Membrillo-Hernández, Jorge; Gómez-Eichelmann, M. Carmen

    2003-01-01

    Stationary-phase cells displayed a distribution of relaxed plasmids and had the ability to recover plasmid supercoiling as soon as nutrients became available. Preexisting gyrase molecules in these cells were responsible for this recovery. Stationary-phase rpoS cells showed a bimodal distribution of plasmids and failed to supercoil plasmids after the addition of nutrients, suggesting that rpoS plays a role in the regulation of plasmid topology during the stationary phase. PMID:12533486

  13. Multiple drug resistant carbapenemases producing Acinetobacter baumannii isolates harbours multiple R-plasmids

    Directory of Open Access Journals (Sweden)

    Rajagopalan Saranathan

    2014-01-01

    Full Text Available Background & objectives: The nosocomial human pathogen Acinetobacter baumannii has high propensity to develop resistance to antimicrobials and to become multidrug resistant (MDR, consequently complicating the treatment. This study was carried out to investigate the presence of resistant plasmids (R-plasmids among the clinical isolates of A. baumannii. In addition, the study was performed to check the presence of common β-lactamases encoding genes on these plasmids. Methods: A total of 55 clinical isolates of A. baumannii were included in the study and all were subjected to plasmid DNA isolation, followed by PCR to check the presence of resistance gene determinants such as blaOXA-23 , blaOXA-51, blaOXA-58 and blaIMP-1 on these plasmids that encode for oxacillinase (OXA and metallo-β-lactamase (MBL type of carbapenemases. Plasmid curing experiments were carried out on selected isolates using ethidium bromide and acridine orange as curing agents and the antibiotic resistance profiles were evaluated before and after curing. Results: All the isolates were identified as A. baumannii by 16SrDNA amplification and sequencing. Plasmid DNA isolated from these isolates showed the occurrence of multiple plasmids with size ranging from 500bp to ≥ 25 kb. The percentage of blaOXA-51 and blaOXA-23 on plasmids were found to be 78 and 42 per cent, respectively and 20 isolates (36% carried blaIMP-1 gene on plasmids. Significant difference was observed in the antibiograms of plasmid cured isolates when compared to their parental ones. The clinical isolates became susceptible to more than two antibiotic classes after curing of plasmids indicating plasmid borne resistance. Interpretation & conclusions: Our study determined the plasmid mediated resistance mechanisms and occurrence of different resistance genes on various plasmids isolated from MDR A. baumannii. The present findings showed the evidence for antibiotic resistance mediated through multiple plasmids in

  14. [Plasmid characteristics of naphthalene and salicylate biodegradation in Pseudomonas putida].

    Science.gov (United States)

    Zakharian, R A; Bakunin, K A; Gasparian, N S; Kocharian, Sh M; Arakelov, G M

    1980-01-01

    The object of this work was to study the physico-chemical and biological properties of DNAs of the biodegradation plasmids NAH and SAL. A comparative analysis of the physico-chemical parameters for these DNAs made it possible to detect a number of identical properties in them: the same sedimentation profile for covalently-closed circular DNA forms, 68--70 S; the molecular weight of ca. 50 MD; a roughly equal number of fragments (up to 23) was found when the DNAs of NAH and SAL were restricted by EcoRI endonuclease. The transformation of the plasmidless strain PpGI was done.

  15. pTAR-Encoded Proteins in Plasmid Partitioning

    OpenAIRE

    Kalnin, Kirill; Stegalkina, Svetlana; Yarmolinsky, Michael

    2000-01-01

    Partition cassettes, essential for the segregational stability of low-copy-number bacterial plasmids, typically encode two autoregulated proteins and an adjacent cis-acting centromere analog to which one or perhaps both proteins bind. The diminutive partition region of pTAR of Agrobacterium spp. was reported to be exceptional, encoding only a single protein, ParA (D. R. Gallie and C. I. Kado, J. Mol. Biol. 193:465–478, 1987). However, resequencing of the region revealed two small downstream g...

  16. 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...

  17. Brazilian purpuric fever caused by Haemophilus influenzae biogroup aegyptius strains lacking the 3031 plasmid.

    Science.gov (United States)

    Tondella, M L; Quinn, F D; Perkins, B A

    1995-01-01

    Brazilian purpuric fever (BPF) is a life-threatening pediatric infection caused by Haemophilus influenzae biogroup aegyptius (Hae), an organism formerly associated with only self-limited purulent conjunctivitis. Strains of Hae causing BPF have a 24-MDa plasmid with a specific AccI restriction pattern designated 3031. This plasmid was thought to code for a virulence factor because it had been detected only among Hae strains isolated from BPF cases or their contacts. From 3 typical BPF cases recently identified in São Paulo State, sterile-site Hae isolates were obtained; these isolates were similar to earlier BPF-associated Hae except they did not possess a 3031 plasmid. HindIII restricted chromosomal DNA from these strains was probed with purified 3031 plasmid DNA under high-stringency conditions. There was no evidence that 3031 plasmid DNA had become chromosomally integrated. It appears that the 3031 plasmid does not code for BPF-specific virulence factors.

  18. IncA/C plasmids: An emerging threat to human and animal health?

    Science.gov (United States)

    Johnson, Timothy J; Lang, Kevin S

    2012-01-01

    Incompatibility group IncA/C plasmids are large, low copy, theta-replicating plasmids that have been described in the literature for over 40 years. However, they have only recently been intensively studied on the genomic level because of their associations with the emergence of multidrug resistance in enteric pathogens of humans and animals. These plasmids are unique among other enterobacterial plasmids in many aspects, including their modular structure and gene content. While the IncA/C plasmid genome structure has now been well defined, many questions remain pertaining to their basic biological mechanisms of dissemination and regulation. Here, we discuss the history of IncA/C plasmids in light of our recent understanding of their population distribution, genomics, and effects on host bacteria.

  19. Long- term manure exposure increases soil bacterial community potential for plasmid uptake

    DEFF Research Database (Denmark)

    Musovic, Sanin; Klümper, Uli; Dechesne, Arnaud;

    2014-01-01

    Microbial communities derived from soils subject to different agronomic treatments were challenged with three broad host range plasmids, RP4, pIPO2tet and pRO101, via solid surface filter matings to assess their permissiveness. Approximately 1 in 10 000 soil bacterial cells could receive and main......Microbial communities derived from soils subject to different agronomic treatments were challenged with three broad host range plasmids, RP4, pIPO2tet and pRO101, via solid surface filter matings to assess their permissiveness. Approximately 1 in 10 000 soil bacterial cells could receive...... and maintain the plasmids. The community permissiveness increased up to 100% in communities derived from manured soil. While the plasmid transfer frequency was significantly influenced by both the type of plasmid and the agronomic treatment, the diversity of the transconjugal pools was purely plasmid dependent...

  20. Novel archaeal plasmid pAH1 and its interactions with the lipothrixvirus AFV1

    DEFF Research Database (Denmark)

    Basta, Tamara; Smyth, John; Forterre, Patrick

    2009-01-01

    to establish a system for studying plasmid-virus interactions we characterized the genome of pAH1 which closely resembles those of the Sulfolobus conjugative plasmids pARN3 and pARN4. pAH1 integrates site specifically into, and excises from, the host chromosome indicating a dynamic interaction with the latter....... Although nucleotide sequence comparisons revealed extensive intergenomic exchange during the evolution of archaeal conjugative plasmids, pAH1 was shown to be stably maintained suggesting that the host system is suitable for studying plasmid-virus interactions. AFV1 infection and propagation leads to a loss...... of the circular form of pAH1 and this effect correlates positively with the increase in the intracellular quantity of AFV1 DNA. We infer that the virus inhibits plasmid replication since no pAH1 degradation was observed. This mechanism of archaeal viral inhibition of plasmid propagation is not observed...

  1. Strategies and approaches in plasmidome studies—uncovering plasmid diversity disregarding of linear elements?

    Science.gov (United States)

    Dib, Julián R.; Wagenknecht, Martin; Farías, María E.; Meinhardt, Friedhelm

    2015-01-01

    The term plasmid was originally coined for circular, extrachromosomal genetic elements. Today, plasmids are widely recognized not only as important factors facilitating genome restructuring but also as vehicles for the dissemination of beneficial characters within bacterial communities. Plasmid diversity has been uncovered by means of culture-dependent or -independent approaches, such as endogenous or exogenous plasmid isolation as well as PCR-based detection or transposon-aided capture, respectively. High-throughput-sequencing made possible to cover total plasmid populations in a given environment, i.e., the plasmidome, and allowed to address the quality and significance of self-replicating genetic elements. Since such efforts were and still are rather restricted to circular molecules, here we put equal emphasis on the linear plasmids which—despite their frequent occurrence in a large number of bacteria—are largely neglected in prevalent plasmidome conceptions. PMID:26074886

  2. Characterization and comparative overview of complete sequences of the first plasmids of Pandoraea across clinical and non-clinical strains

    Directory of Open Access Journals (Sweden)

    Delicia Yong

    2016-10-01

    Full Text Available To date, information on plasmid analysis in Pandoraea spp. is scarce. To address the gap of knowledge on this, the complete sequences of eight plasmids from Pandoraea spp. namely Pandoraea faecigallinarum DSM 23572 (pPF72-1, pPF72-2, Pandoraea oxalativorans DSM 23570 (pPO70-1, pPO70-2, pPO70-3, pPO70-4, Pandoraea vervacti NS15 (pPV15 and Pandoraea apista DSM 16535 (pPA35 were studied for the first time in this study. The information on plasmid sequences in Pandoraea spp. is useful because these plasmid sequences did not match to any known plasmid sequence deposited in public databases. Replication genes were not identified in some plasmids, a situation that has led to the possibility of host interaction involvement. Some plasmids were also void of par genes and intriguingly, repA gene was also not discovered in these plasmids. This further leads to the hypothesis of host-plasmid interaction. Plasmid stabilization/stability protein-encoding genes were observed in some plasmids but were not established for participating in plasmid segregation. Toxin-antitoxin systems MazEF, VapBC, RelBE, YgiT-MqsR, HigBA and ParDE were identified across the plasmids and their presence would improve plasmid maintenance. Conjugation genes were identified portraying the conjugation ability amongst Pandoraea plasmids. Additionally, we found a shared region amongst some of the plasmids that consists of conjugation genes. The identification of genes involved in replication, segregation, toxin-antitoxin systems and conjugation, would aid the design of drugs to prevent the survival or transmission of plasmids carrying pathogenic properties. Additionally, genes conferring virulence and antibiotic resistance were identified among the plasmids. The observed features in the plasmids shed light on the Pandoraea spp. as opportunistic pathogens.

  3. 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

  4. Plasmid profiles and antibiotic susceptibility patterns of Staphylococcus aureus isolates from Nigeria.

    Science.gov (United States)

    Olukoya, D K; Asielue, J O; Olasupo, N A; Ikea, J K

    1995-06-01

    In an investigation into the problems of infections due to Staphylococcus aureus in Nigeria, 100 strains were isolated from various hospitals in Lagos. The strains were screened for the presence of plasmids and for susceptibility to antimicrobial agents. Plasmids were extracted by modification of the method of Takahashi and Nagono[1]. The plasmids were diverse in nature. The strains were found to be highly resistant to commonly prescribed antibiotics.

  5. Investigation of diversity of plasmids carrying the blaTEM-52 gene

    DEFF Research Database (Denmark)

    Bielak, Eliza Maria; Bergenholtz, Rikke D.; Jørgensen, Mikael Skaanning

    2011-01-01

    on various other plasmids belonging to IncA/C and IncL/M, while blaTEM-52c was found on IncN-like as well as on IncR plasmids. In the majority of cases (n = 21) the blaTEM-52 gene was located on a Tn3 transposon. Seven out of 10 blaTEM-52 plasmids tested in conjugation experiments were shown to be capable...

  6. Key features of mcr-1-bearing plasmids from Escherichia coli isolated from humans and food.

    Science.gov (United States)

    Zurfluh, Katrin; Nüesch-Inderbinen, Magdalena; Klumpp, Jochen; Poirel, Laurent; Nordmann, Patrice; Stephan, Roger

    2017-01-01

    Mcr-1-harboring Enterobacteriaceae are reported worldwide since their first discovery in 2015. However, a limited number of studies are available that compared full-length plasmid sequences of human and animal origins. In this study, mcr-1-bearing plasmids from seven Escherichia coli isolates recovered from patients (n = 3), poultry meat (n = 2) and turkey meat (n = 2) in Switzerland were further analyzed and compared. Isolates were characterized by multilocus sequence typing (MLST). The mcr-1-bearing plasmids were transferred by transformation into reference strain E. coli DH5α and MCR-1-producing transformants were selected on LB-agar supplemented with 2 mg/L colistin. Purified plasmids were then sequenced and compared. MLST revealed six distinct STs, illustrating the high clonal diversity among mcr-1-positive E. coli isolates of different origins. Two different mcr-1-positive plasmids were identified from a single E. coli ST48 human isolate. All other isolates possessed a single mcr-1 harboring plasmid. Transferable IncI2 (size ca. 60-61 kb) and IncX4 (size ca. 33-35 kb) type plasmids each bearing mcr-1 were found associated with human and food isolates. None of the mcr-1-positive IncI2 and IncX4 plasmids possessed any additional resistance determinants. Surprisingly, all but one of the sequenced mcr-1-positive plasmids lacked the ISApl1 element, which is a key element mediating acquisition of mcr-1 into various plasmid backbones. There is strong evidence that the food chain may be an important transmission route for mcr-1-bearing plasmids. Our data suggest that some "epidemic" plasmids rather than specific E. coli clones might be responsible for the spread of the mcr-1 gene along the food chain.

  7. 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.

  8. Occurrence of Plasmids in the Aromatic Degrading Bacterioplankton of the Baltic Sea

    OpenAIRE

    Ain Heinaru; Jaanis Juhanson; Eve Vedler; Eeva Heinaru; Jekaterina Jutkina

    2011-01-01

    Plasmids are mobile genetic elements that provide their hosts with many beneficial traits including in some cases the ability to degrade different aromatic compounds. To fulfill the knowledge gap regarding catabolic plasmids of the Baltic Sea water, a total of 209 biodegrading bacterial strains were isolated and screened for the presence of these mobile genetic elements. We found that both large and small plasmids are common in the cultivable Baltic Sea bacterioplankton and are particularly p...

  9. 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.

  10. Cefotaxime resistant Escherichia coli collected from a healthy volunteer; characterisation and the effect of plasmid loss.

    Directory of Open Access Journals (Sweden)

    Miranda Kirchner

    Full Text Available In this study 6 CTX-M positive E. coli isolates collected during a clinical study examining the effect of antibiotic use in a human trial were analysed. The aim of the study was to analyse these isolates and assess the effect of full or partial loss of plasmid genes on bacterial fitness and pathogenicity. A DNA array was utilised to assess resistance and virulence gene carriage. Plasmids were characterised by PCR-based replicon typing and addiction system multiplex PCR. A phenotypic array and insect virulence model were utilised to assess the effect of plasmid-loss in E. coli of a large multi-resistance plasmid. All six E. coli carrying bla CTX-M-14 were detected from a single participant and were identical by pulse field gel electrophoresis and MLST. Plasmid profiling and arrays indicated absence of a large multi-drug resistance (MDR F-replicon plasmid carrying blaTEM, aadA4, strA, strB, dfrA17/19, sul1, and tetB from one isolate. Although this isolate partially retained the plasmid it showed altered fitness characteristics e.g. inability to respire in presence of antiseptics, similar to a plasmid-cured strain. However, unlike the plasmid-cured or plasmid harbouring strains, the survival rate for Galleria mellonella infected by the former strain was approximately 5-times lower, indicating other possible changes accompanying partial plasmid loss. In conclusion, our results demonstrated that an apparently healthy individual can harbour bla CTX-M-14 E. coli strains. In one such strain, isolated from the same individual, partial absence of a large MDR plasmid resulted in altered fitness and virulence characteristics, which may have implications in the ability of this strain to infect and any subsequent treatment.

  11. Cefotaxime resistant Escherichia coli collected from a healthy volunteer; characterisation and the effect of plasmid loss.

    Science.gov (United States)

    Kirchner, Miranda; Abuoun, Manal; Mafura, Muriel; Bagnall, Mary; Hunt, Theresa; Thomas, Christopher; Weile, Jan; Anjum, Muna F

    2013-01-01

    In this study 6 CTX-M positive E. coli isolates collected during a clinical study examining the effect of antibiotic use in a human trial were analysed. The aim of the study was to analyse these isolates and assess the effect of full or partial loss of plasmid genes on bacterial fitness and pathogenicity. A DNA array was utilised to assess resistance and virulence gene carriage. Plasmids were characterised by PCR-based replicon typing and addiction system multiplex PCR. A phenotypic array and insect virulence model were utilised to assess the effect of plasmid-loss in E. coli of a large multi-resistance plasmid. All six E. coli carrying bla CTX-M-14 were detected from a single participant and were identical by pulse field gel electrophoresis and MLST. Plasmid profiling and arrays indicated absence of a large multi-drug resistance (MDR) F-replicon plasmid carrying blaTEM, aadA4, strA, strB, dfrA17/19, sul1, and tetB from one isolate. Although this isolate partially retained the plasmid it showed altered fitness characteristics e.g. inability to respire in presence of antiseptics, similar to a plasmid-cured strain. However, unlike the plasmid-cured or plasmid harbouring strains, the survival rate for Galleria mellonella infected by the former strain was approximately 5-times lower, indicating other possible changes accompanying partial plasmid loss. In conclusion, our results demonstrated that an apparently healthy individual can harbour bla CTX-M-14 E. coli strains. In one such strain, isolated from the same individual, partial absence of a large MDR plasmid resulted in altered fitness and virulence characteristics, which may have implications in the ability of this strain to infect and any subsequent treatment.

  12. Effects of medium composition on the production of plasmid DNA vector potentially for human gene therapy

    Institute of Scientific and Technical Information of China (English)

    XU Zhi-nan; SHEN Wen-he; CHEN Hao; CEN Pei-lin

    2005-01-01

    Plasmid vector is increasingly applied to gene therapy or gene vaccine. The production of plasmid pCMV-AP3 for cancer gene therapy was conducted in a modified MBL medium using a recombinant E. coli BL21 system. The effects of different MMBL components on plasmid yield, cell mass and specific plasmid DNA productivity were evaluated on shake-flask scale. The results showed that glucose was the optimal carbon source. High plasmid yield (58.3 mg/L) was obtained when 5.0 g/L glucose was added to MMBL. Glycerol could be chosen as a complementary carbon source because of the highest specific plasmid productivity (37.9 mg DNA/g DCW). After tests of different levels of nitrogen source and inorganic phosphate, a modified MMBL medium was formulated for optimal plasmid production. Further study showed that the initial acetate addition (less than 4.0 g/L) in MMBL improved plasmid production significantly, although it inhibited cell growth. The results will be useful for large-scale plasmid production using recombinant E. coli system.

  13. Modular construction of plasmids by parallel assembly of linear vector components.

    Science.gov (United States)

    Gao, XinZheng; Yan, Pu; Shen, Wentao; Li, Xiaoying; Zhou, Peng; Li, Yuenan

    2013-06-15

    Construction of plasmids is the basic and pivotal technology in molecular biology. The common method for constructing plasmids is to cut DNA fragments by restriction enzymes and then join the resulting fragments using ligase. We present here a modified Golden Gate cloning method for modular construction of plasmids. Unlike the original Golden Gate cloning system for cloning from entry vector to expression vector, this method can be used to construct plasmids immediately from linear DNA fragments. After polymerase chain reaction (PCR) amplification for flanking with BsaI sites, multiple linear DNA components (modules) can be parallel assembled into a circle plasmid by a single restriction-ligation reaction using the method. This method is flexible to construct different types of plasmids because the modules can be freely selected and assembled in any combination. This method was applied successfully to construct a prokaryotic expression plasmid from four modules and a plant expression plasmid from five modules (fragments). The results suggest that this method provides a simple and flexible platform for modular construction of plasmids.

  14. The evolution of collective restraint: policing and obedience among non-conjugative plasmids.

    Science.gov (United States)

    Kentzoglanakis, Kyriakos; García López, Diana; Brown, Sam P; Goldstein, Richard A

    2013-04-01

    The repression of competition by mechanisms of policing is now recognized as a major force in the maintenance of cooperation. General models on the evolution of policing have focused on the interplay between individual competitiveness and mutual policing, demonstrating a positive relationship between within-group diversity and levels of policing. We expand this perspective by investigating what is possibly the simplest example of reproductive policing: copy number control (CNC) among non-conjugative plasmids, a class of extra-chromosomal vertically transmitted molecular symbionts of bacteria. Through the formulation and analysis of a multi-scale dynamical model, we show that the establishment of stable reproductive restraint among plasmids requires the co-evolution of two fundamental plasmid traits: policing, through the production of plasmid-coded trans-acting replication inhibitors, and obedience, expressed as the binding affinity of plasmid-specific targets to those inhibitors. We explain the intrinsic replication instabilities that arise in the absence of policing and we show how these instabilities are resolved by the evolution of copy number control. Increasing levels of policing and obedience lead to improvements in group performance due to tighter control of local population size (plasmid copy number), delivering benefits both to plasmids, by reducing the risk of segregational loss and to the plasmid-host partnership, by increasing the rate of cell reproduction, and therefore plasmid vertical transmission.

  15. 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

  16. Postsymbiotic plasmid acquisition and evolution of the repA1-replicon in Buchnera aphidicola

    Science.gov (United States)

    Van Ham, Roeland C. H. J.; González-Candelas, Fernando; Silva, Francisco J.; Sabater, Beatriz; Moya, Andrés; Latorre, Amparo

    2000-01-01

    Buchnera aphidicola is an obligate, strictly vertically transmitted, bacterial symbiont of aphids. It supplies its host with essential amino acids, nutrients required by aphids but deficient in their diet of plant phloem sap. Several lineages of Buchnera show adaptation to their nutritional role in the form of plasmid-mediated amplification of key-genes involved in the biosynthesis of tryptophan (trpEG) and leucine (leuABCD). Phylogenetic analyses of these plasmid-encoded functions have thus far suggested the absence of horizontal plasmid exchange among lineages of Buchnera. Here, we describe three new Buchnera plasmids, obtained from species of the aphid host families Lachnidae and Pemphigidae. All three plasmids belong to the repA1 family of Buchnera plasmids, which is characterized by the presence of a repA1-replicon responsible for replication initiation. A comprehensive analysis of this family of plasmids unexpectedly revealed significantly incongruent phylogenies for different plasmid and chromosomally encoded loci. We infer from these incongruencies a case of horizontal plasmid transfer in Buchnera. This process may have been mediated by secondary endosymbionts, which occasionally undergo horizontal transmission in aphids. PMID:10984505

  17. Enhancing yields of low and single copy number plasmid DNAs from Escherichia coli cells.

    Science.gov (United States)

    Wood, Whitney N; Smith, Kyle D; Ream, Jennifer A; Kevin Lewis, L

    2017-02-01

    Many plasmids used for gene cloning and heterologous protein expression in Escherichia coli cells are low copy number or single copy number plasmids. The extraction of these types of plasmids from small bacterial cell cultures produces low DNA yields. In this study, we have quantitated yields of low copy and single copy number plasmid DNAs after growth of cells in four widely used broths (SB, SOC, TB, and 2xYT) and compared results to those obtained with LB, the most common E. coli cell growth medium. TB (terrific broth) consistently generated the greatest amount of plasmid DNA, in agreement with its ability to produce higher cell titers. The superiority of TB was primarily due to its high levels of yeast extract (24g/L) and was independent of glycerol, a unique component of this broth. Interestingly, simply preparing LB with similarly high levels of yeast extract (LB24 broth) resulted in plasmid yields that were equivalent to those of TB. By contrast, increasing ampicillin concentration to enhance plasmid retention did not improve plasmid DNA recovery. These experiments demonstrate that yields of low and single copy number plasmid DNAs from minipreps can be strongly enhanced using simple and inexpensive media. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. 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.

  19. Plasmid-Borne Antimicrobial Resistance of Staphylococcus aureus Isolated in a Hospital in Lisbon, Portugal.

    Science.gov (United States)

    Costa, Sofia Santos; Palma, Cláudia; Kadlec, Kristina; Fessler, Andrea T; Viveiros, Miguel; Melo-Cristino, José; Schwarz, Stefan; Couto, Isabel

    2016-12-01

    Plasmids play a key role in the genetic plasticity and survival of Staphylococcus aureus in challenging environments. Although many S. aureus plasmids have been described, still few studies portray the plasmid content of a given S. aureus population. The aim of this work was to characterize the plasmids carried by a collection of 53 S. aureus isolates collected in a large hospital in Lisbon, Portugal, and investigate their role in conferring resistance to several antimicrobial agents. Plasmids were present in 44 out of the 53 isolates and were grouped into eleven AccI restriction profiles. Plasmid curing of representative strains and comparison of antimicrobial susceptibility profiles between pairs of isogenic strains proved to be a valuable guidance tool in the identification of plasmid-located resistance genes. The plasmids harbored several resistance genes, namely blaZ (resistance to β-lactams), erm(C) (resistance to macrolides, lincosamides, and streptogramin B), cadA (resistance to cadmium and zinc), cadD (resistance to cadmium), and qacA and smr (resistance to biocides and dyes). This study demonstrates the impact of plasmids on the resistance properties of S. aureus, highlighting their role in the dissemination of antibiotic, heavy metal, and biocide resistance genes, and survival of this major pathogen in the hospital environment.

  20. Computational design and characterization of a temperature-sensitive plasmid replicon for gram positive thermophiles

    Directory of Open Access Journals (Sweden)

    Olson Daniel G

    2012-05-01

    Full Text Available Abstract Background Temperature-sensitive (Ts plasmids are useful tools for genetic engineering, but there are currently none compatible with the gram positive, thermophilic, obligate anaerobe, Clostridium thermocellum. Traditional mutagenesis techniques yield Ts mutants at a low frequency, and therefore requires the development of high-throughput screening protocols, which are also not available for this organism. Recently there has been progress in the development of computer algorithms which can predict Ts mutations. Most plasmids currently used for genetic modification of C. thermocellum are based on the replicon of plasmid pNW33N, which replicates using the RepB replication protein. To address this problem, we set out to create a Ts plasmid by mutating the gene coding for the RepB replication protein using an algorithm designed by Varadarajan et al. (1996 for predicting Ts mutants based on the amino-acid sequence of the protein. Results A library of 34 mutant plasmids was designed, synthesized and screened, resulting in 6 mutants which exhibited a Ts phenotype. Of these 6, the one with the most temperature-sensitive phenotype (M166A was compared with the original plasmid. It exhibited lower stability at 48°C and was completely unable to replicate at 55°C. Conclusions The plasmid described in this work could be useful in future efforts to genetically engineer C. thermocellum, and the method used to generate this plasmid may be useful for others trying to make Ts plasmids.

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

    Science.gov (United States)

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

    2012-01-01

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

  2. Degenerate primer MOB typing of multiresistant clinical isolates of E. coli uncovers new plasmid backbones.

    Science.gov (United States)

    Garcillán-Barcia, M Pilar; Ruiz del Castillo, Belén; Alvarado, Andrés; de la Cruz, Fernando; Martínez-Martínez, Luis

    2015-01-01

    Degenerate Primer MOB Typing is a PCR-based protocol for the classification of γ-proteobacterial transmissible plasmids in five phylogenetic relaxase MOB families. It was applied to a multiresistant E. coli collection, previously characterized by PCR-based replicon-typing, in order to compare both methods. Plasmids from 32 clinical isolates of multiresistant E. coli (19 extended spectrum beta-lactamase producers and 13 non producers) and their transconjugants were analyzed. A total of 95 relaxases were detected, at least one per isolate, underscoring the high potential of these strains for antibiotic-resistance transmission. MOBP12 and MOBF12 plasmids were the most abundant. Most MOB subfamilies detected were present in both subsets of the collection, indicating a shared mobilome among multiresistant E. coli. The plasmid profile obtained by both methods was compared, which provided useful data upon which decisions related to the implementation of detection methods in the clinic could be based. The phylogenetic depth at which replicon and MOB-typing classify plasmids is different. While replicon-typing aims at plasmid replication regions with non-degenerate primers, MOB-typing classifies plasmids into relaxase subfamilies using degenerate primers. As a result, MOB-typing provides a deeper phylogenetic depth than replicon-typing and new plasmid groups are uncovered. Significantly, MOB typing identified 17 plasmids and an integrative and conjugative element, which were not detected by replicon-typing. Four of these backbones were different from previously reported elements.

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

    OpenAIRE

    Setlow, J K; Cabrera-Juárez, E; Griffin, K

    1984-01-01

    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 pNov1. 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 th...

  4. 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

  5. A DNA polymerase mutation that suppresses the segregation bias of an ARS plasmid in Saccharomyces cerevisiae.

    Science.gov (United States)

    Houtteman, S W; Elder, R T

    1993-03-01

    Yeast autonomously replicating sequence (ARS) plasmids exhibit an unusual segregation pattern during mitosis. While the nucleus divides equally into mother and daughter cells, all copies of the ARS plasmid will often remain in the mother cell. A screen was designed to isolate mutations that suppress this segregation bias. A plasmid with a weak ARS (wARS) that displayed an extremely high segregation bias was constructed. When cells were grown under selection for the wARS plasmid, the resulting colonies grew slowly and had abnormal morphology. A spontaneous recessive mutation that restored normal colony morphology was identified. This mutation suppressed plasmid segregation bias, as indicated by the increased stability of the wARS plasmid in the mutant cells even though the plasmid was present at a lower copy number. An ARS1 plasmid was also more stable in mutant cells than in wild-type cells. The wild-type allele for this mutant gene was cloned and identified as POL delta (CDC2). This gene encodes DNA polymerase delta, which is essential for DNA replication. These results indicate that DNA polymerase delta plays some role in causing the segregation bias of ARS plasmids.

  6. High instability of a nematicidal Cry toxin plasmid in Bacillus thuringiensis.

    Science.gov (United States)

    Sheppard, Anna E; Nakad, Rania; Saebelfeld, Manja; Masche, Anna C; Dierking, Katja; Schulenburg, Hinrich

    2016-01-01

    In bacterial pathogens, virulence factors are often carried on plasmids and other mobile genetic elements, and as such, plasmid evolution is central in understanding pathogenicity. Bacillus thuringiensis is an invertebrate pathogen that uses plasmid-encoded crystal (Cry) toxins to establish infections inside the host. Our study aimed to quantify stability of two Cry toxin-encoding plasmids, BTI_23p and BTI_16p, under standard laboratory culturing conditions. These two plasmids are part of the genome of the B. thuringiensis strain MYBT18679, which is of particular interest because of its high pathogenicity towards nematodes. One of the plasmids, BTI_23p, was found to be highly unstable, with substantial loss occurring within a single growth cycle. Nevertheless, longer term experimental evolution in the absence of a host revealed maintenance of the plasmid at low levels in the bacterial populations. BTI_23p encodes two nematicidal Cry toxins, Cry21Aa2 and Cry14Aa1. Consistent with previous findings, loss of the plasmid abolished pathogenicity towards the nematode Caenorhabditis elegans, which could be rescued by addition of Cry21Aa2-expressing Escherichia coli. These results implicate BTI_23p as a plasmid that is required for successful infection, yet unstable when present at high frequency in the population, consistent with the role of Cry toxins as public goods.

  7. Cloning, sequencing, and sequence analysis of two novel plasmids from the thermophilic anaerobic bacterium Anaerocellum thermophilum

    DEFF Research Database (Denmark)

    Clausen, Anders; Mikkelsen, Marie Just; Schrøder, I.

    2004-01-01

    The nucleotide sequence of two novel plasmids isolated from the extreme thermophilic anaerobic bacterium Anaerocellum thermophilum DSM6725 (A. thermophilum), growing optimally at 70degreesC, has been determined. pBAS2 was found to be a 3653 bp plasmid with a GC content of 43%, and the sequence...... was found, but no single stranded intermediates, characteristic of rolling circle replication, were found on Southern blots. The larger plasmid, pBAL, was found to be a 8294 bp plasmid with a GC content of 39%. It revealed 17 ORFs, of which three showed similarity at the amino acid (aa) level to known...

  8. 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.

  9. The evolution of collective restraint: policing and obedience among non-conjugative plasmids.

    Directory of Open Access Journals (Sweden)

    Kyriakos Kentzoglanakis

    2013-04-01

    Full Text Available The repression of competition by mechanisms of policing is now recognized as a major force in the maintenance of cooperation. General models on the evolution of policing have focused on the interplay between individual competitiveness and mutual policing, demonstrating a positive relationship between within-group diversity and levels of policing. We expand this perspective by investigating what is possibly the simplest example of reproductive policing: copy number control (CNC among non-conjugative plasmids, a class of extra-chromosomal vertically transmitted molecular symbionts of bacteria. Through the formulation and analysis of a multi-scale dynamical model, we show that the establishment of stable reproductive restraint among plasmids requires the co-evolution of two fundamental plasmid traits: policing, through the production of plasmid-coded trans-acting replication inhibitors, and obedience, expressed as the binding affinity of plasmid-specific targets to those inhibitors. We explain the intrinsic replication instabilities that arise in the absence of policing and we show how these instabilities are resolved by the evolution of copy number control. Increasing levels of policing and obedience lead to improvements in group performance due to tighter control of local population size (plasmid copy number, delivering benefits both to plasmids, by reducing the risk of segregational loss and to the plasmid-host partnership, by increasing the rate of cell reproduction, and therefore plasmid vertical transmission.

  10. Plasmid profile in oral Fusobacterium nucleatum from humans and Cebus apella monkeys

    Directory of Open Access Journals (Sweden)

    Paula Marcia O.

    2003-01-01

    Full Text Available Fusobacterium nucleatum is a strict anaerobe and is indigenous of the human oral cavity. This organism is commonly recovered from different monomicrobial and mixed infections in humans and animals. In this study, the plasmid profile, the plasmid stability and the penicillin-resistance association in oral F. nucleatum isolated from periodontal patients, healthy subjects and Cebus apella monkeys were evaluated. Forty-five F. nucleatum strains from patients, 38 from healthy subjects and seven from C. apella were identified and analyzed. Plasmid extraction was performed in all the isolated strains. These elements were found in 26.7% strains from patients and one strain from C. apella. Strains from healthy subjects did not show any plasmid. Most of strains showed two plasmid bands ranging from 4 to 16 Kb, but digestions with endonucleases showed that they belonged to a single plasmid. The plasmid profile was similar and stable in human and monkey strains. Also, plasmids were classified into three groups according to size. Two strains were positive to beta-lactamase production and no plasmid DNA-hybridization with a beta-lactamase gene probe was observed, suggesting a chromosomal resistance.

  11. Quantifying and visualizing the transfer of exogenous plasmids to environmental microbial communities

    DEFF Research Database (Denmark)

    Dechesne, Arnaud

    2015-01-01

    of a community to take up exogenous plasmid should, however, be an important element affecting the fate of mobile genetic elements released in the environment. We have devised a method to evaluate the permissiveness of a bacterial community towards exogenous plasmids, both quantitatively (how many bacteria can......, our findings highlight the high potential for exogenous plasmids to be transferred to soil microbial communities and indicate that community permissiveness – as affected by environmental conditions- needs to be considered to predict the fate of plasmids in the environment....

  12. 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.

  13. pTAR-encoded proteins in plasmid partitioning.

    Science.gov (United States)

    Kalnin, K; Stegalkina, S; Yarmolinsky, M

    2000-04-01

    Partition cassettes, essential for the segregational stability of low-copy-number bacterial plasmids, typically encode two autoregulated proteins and an adjacent cis-acting centromere analog to which one or perhaps both proteins bind. The diminutive partition region of pTAR of Agrobacterium spp. was reported to be exceptional, encoding only a single protein, ParA (D. R. Gallie and C. I. Kado, J. Mol. Biol. 193:465-478, 1987). However, resequencing of the region revealed two small downstream genes, parB and orf-84, of which only parB was found to be essential for partitioning in A. tumefaciens. Purified ParA exhibited a weak ATPase activity that was modestly increased by nonspecific DNA. ParB bound in vitro to repeated sequences present in a region, parS, that possesses centromere and operator functions and within which we identified the primary transcription start site by primer extension. In certain respects the Par proteins behave normally in the foreign host Escherichia coli. In E. coli, as in A. tumefaciens, ParB repressed the partition operon; ParA, inactive alone, augmented this repression. Functional similarities between the partition system of pTAR and those of other plasmids and bacteria are prominent, despite differences in size, organization, and amino acid sequence.

  14. Adsorption behavior of plasmid DNA onto perfusion chromatographic matrix

    Institute of Scientific and Technical Information of China (English)

    Miladys LIMONTA; Lourdes ZUMALACARREGUI; Dayana SOLER

    2012-01-01

    Anion exchange chromatography is the most popular chromatographic method for plasmid separa-tion.POROS RI 50 is a perfusion chromatographic support which is a reversed phase matrix and is an alterna-tive to conventional ones due to its mass transfer properties.The adsorption and elution of the pIDKE2 plasmidonto reversed phase POROS RI 50 was studied.Langmuir isotherm model was adjusted in order to get the max-imum adsorption capacity and the dissociation constant for POROS RI 50-plasmid DNA (pDNA) system.Break-through curves were obtained for volumetric flows between 0.69-3.33mL/min,given dynamic capacity up to2.3 times higher than those reported for ionic exchange matrix used during the purification process of plasmidswith similar size to that of pIDKE2.The efficiency was less than 45% for the flow conditions and initial concen-tration studied,which means that the support will not be operated under saturation circumstances.

  15. Using Plasmids as DNA Vaccines for Infectious Diseases.

    Science.gov (United States)

    Tregoning, John S; Kinnear, Ekaterina

    2014-12-01

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

  16. Plasmid Classification in an Era of Whole-Genome Sequencing: Application in Studies of Antibiotic Resistance Epidemiology

    Science.gov (United States)

    Orlek, Alex; Stoesser, Nicole; Anjum, Muna F.; Doumith, Michel; Ellington, Matthew J.; Peto, Tim; Crook, Derrick; Woodford, Neil; Walker, A. Sarah; Phan, Hang; Sheppard, Anna E.

    2017-01-01

    Plasmids are extra-chromosomal genetic elements ubiquitous in bacteria, and commonly transmissible between host cells. Their genomes include variable repertoires of ‘accessory genes,’ such as antibiotic resistance genes, as well as ‘backbone’ loci which are largely conserved within plasmid families, and often involved in key plasmid-specific functions (e.g., replication, stable inheritance, mobility). Classifying plasmids into different types according to their phylogenetic relatedness provides insight into the epidemiology of plasmid-mediated antibiotic resistance. Current typing schemes exploit backbone loci associated with replication (replicon typing), or plasmid mobility (MOB typing). Conventional PCR-based methods for plasmid typing remain widely used. With the emergence of whole-genome sequencing (WGS), large datasets can be analyzed using in silico plasmid typing methods. However, short reads from popular high-throughput sequencers can be challenging to assemble, so complete plasmid sequences may not be accurately reconstructed. Therefore, localizing resistance genes to specific plasmids may be difficult, limiting epidemiological insight. Long-read sequencing will become increasingly popular as costs decline, especially when resolving accurate plasmid structures is the primary goal. This review discusses the application of plasmid classification in WGS-based studies of antibiotic resistance epidemiology; novel in silico plasmid analysis tools are highlighted. Due to the diverse and plastic nature of plasmid genomes, current typing schemes do not classify all plasmids, and identifying conserved, phylogenetically concordant genes for subtyping and phylogenetics is challenging. Analyzing plasmids as nodes in a network that represents gene-sharing relationships between plasmids provides a complementary way to assess plasmid diversity, and allows inferences about horizontal gene transfer to be made. PMID:28232822

  17. IncHI2 Plasmids Are Predominant in Antibiotic-Resistant Salmonella Isolates

    Science.gov (United States)

    Chen, Wenyao; Fang, Tingzi; Zhou, Xiujuan; Zhang, Daofeng; Shi, Xianming; Shi, Chunlei

    2016-01-01

    The wide usage of antibiotics contributes to the increase in the prevalence of antibiotic-resistant Salmonella. Plasmids play a critical role in horizontal transfer of antibiotic resistance markers in Salmonella. This study aimed to screen and characterize plasmid profiles responsible for antibiotic resistance in Salmonella and ultimately to clarify the molecular mechanism of transferable plasmid-mediated antibiotic resistance. A total of 226 Salmonella isolates were examined for antimicrobial susceptibility by a disk diffusion method. Thirty-two isolates (14.2%) were resistant to at least one antibiotic. The presence of plasmid-mediated quinolone resistance (PMQR) genes and β-lactamase genes were established by PCR amplification. PCR-based replicon typing revealed that these 32 isolates represented seven plasmid incompatibility groups (IncP, HI2, A/C, FIIs, FIA, FIB, and I1), and the IncHI2 (59.4%) was predominant. Antibiotic resistance markers located on plasmids were identified through plasmid curing. Fifteen phenotypic variants were obtained with the curing efficiency of 46.9% (15/32). The cured plasmids mainly belong to the HI2 incompatibility group. The elimination of IncHI2 plasmids correlated with the loss of β-lactamase genes (blaOXA-1 and blaTEM-1) and PMQR genes (qnrA and aac(6′)-Ib-cr). Both IncHI2 and IncI1 plasmids in a S. enterica serovar Indiana isolate SJTUF 10584 were lost by curing. The blaCMY -2-carrying plasmid pS10584 from SJTUF 10584 was fully sequenced. Sequence analysis revealed that it possessed a plasmid scaffold typical for IncI1 plasmids with the unique genetic arrangement of IS1294-ΔISEcp1-blaCMY -2-blc-sugE-ΔecnR inserted into the colicin gene cia. These data suggested that IncHI2 was the major plasmid lineage contributing to the dissemination of antibiotic resistance in Salmonella and the activity of multiple mobile genetic elements may contribute to antibiotic resistance evolution and dissemination between different plasmid

  18. Genetic diversity and composition of a plasmid metagenome from a wastewater treatment plant.

    Science.gov (United States)

    Schlüter, Andreas; Krause, Lutz; Szczepanowski, Rafael; Goesmann, Alexander; Pühler, Alfred

    2008-08-31

    Plasmid metagenome nucleotide sequence data were recently obtained from wastewater treatment plant (WWTP) bacteria with reduced susceptibility to selected antimicrobial drugs by applying the ultrafast 454-sequencing technology. The sequence dataset comprising 36,071,493 bases (346,427 reads with an average read length of 104 bases) was analysed for genetic diversity and composition by using a newly developed bioinformatic pipeline based on assignment of environmental gene tags (EGTs) to protein families stored in the Pfam database. Short amino acid sequences deduced from the plasmid metagenome sequence reads were compared to profile hidden Markov models underlying Pfam. Obtained matches evidenced that many reads represent genes having predicted functions in plasmid replication, stability and plasmid mobility which indicates that WWTP bacteria harbour genetically stabilised and mobile plasmids. Moreover, the data confirm a high diversity of plasmids residing in WWTP bacteria. The mobile organic peroxide resistance plasmid pMAC from Acinetobacter baumannii was identified as reference plasmid for the most abundant replication module type in the sequenced sample. Accessory plasmid modules encode different transposons, insertion sequences, integrons, resistance and virulence determinants. Most of the matches to Transposase protein families were identified for transposases similar to the one of the chromate resistance transposon Tn5719. Noticeable are hits to beta-lactamase protein families which suggests that plasmids from WWTP bacteria encode different enzymes possessing beta-lactam-hydrolysing activity. Some of the sequence reads correspond to antibiotic resistance genes that were only recently identified in clinical isolates of human pathogens. EGT analysis thus proofed to be a very valuable method to explore genetic diversity and composition of the present plasmid metagenome dataset.

  19. DNASU plasmid and PSI:Biology-Materials repositories: resources to accelerate biological research.

    Science.gov (United States)

    Seiler, Catherine Y; Park, Jin G; Sharma, Amit; Hunter, Preston; Surapaneni, Padmini; Sedillo, Casey; Field, James; Algar, Rhys; Price, Andrea; Steel, Jason; Throop, Andrea; Fiacco, Michael; LaBaer, Joshua

    2014-01-01

    The mission of the DNASU Plasmid Repository is to accelerate research by providing high-quality, annotated plasmid samples and online plasmid resources to the research community through the curated DNASU database, website and repository (http://dnasu.asu.edu or http://dnasu.org). The collection includes plasmids from grant-funded, high-throughput cloning projects performed in our laboratory, plasmids from external researchers, and large collections from consortia such as the ORFeome Collaboration and the NIGMS-funded Protein Structure Initiative: Biology (PSI:Biology). Through DNASU, researchers can search for and access detailed information about each plasmid such as the full length gene insert sequence, vector information, associated publications, and links to external resources that provide additional protein annotations and experimental protocols. Plasmids can be requested directly through the DNASU website. DNASU and the PSI:Biology-Materials Repositories were previously described in the 2010 NAR Database Issue (Cormier, C.Y., Mohr, S.E., Zuo, D., Hu, Y., Rolfs, A., Kramer, J., Taycher, E., Kelley, F., Fiacco, M., Turnbull, G. et al. (2010) Protein Structure Initiative Material Repository: an open shared public resource of structural genomics plasmids for the biological community. Nucleic Acids Res., 38, D743-D749.). In this update we will describe the plasmid collection and highlight the new features in the website redesign, including new browse/search options, plasmid annotations and a dynamic vector mapping feature that was developed in collaboration with LabGenius. Overall, these plasmid resources continue to enable research with the goal of elucidating the role of proteins in both normal biological processes and disease.

  20. 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.

  1. 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

  2. Characterization and comparative analysis of antibiotic resistance plasmids isolated from a wastewater treatment plant.

    Directory of Open Access Journals (Sweden)

    Teddie O Rahube

    2014-10-01

    Full Text Available A wastewater treatment plant (WWTP is an environment high in nutrient concentration with diverse bacterial populations and can provide an ideal environment for the proliferation of mobile elements such as plasmids. WWTPs have also been identified as reservoirs for antibiotic resistance genes that are associated with human pathogens. The objectives of this study were to isolate and characterize self-transmissible or mobilizable resistance plasmids associated with effluent from wastewater treatment plant. An enrichment culture approach designed to capture plasmids conferring resistance to high concentrations of erythromycin was used to capture plasmids from an urban wastewater treatment plant servicing a population of ca. 210,000. DNA sequencing of the plasmids revealed diversity of plasmids represented by incompatibility groups IncU, col-E, IncFII and IncP-1β. Genes coding resistance to clinically relevant antibiotics (macrolide, tetracycline, beta-lactam, trimethoprim, chloramphenicol, sulphonamide, quaternary ammonium compounds and heavy metals were co-located on these plasmids, often within transposable and integrative mobile elements. Several of the plasmids were self-transmissible or mobilizable and could be maintained in the absence of antibiotic selection. The IncFII plasmid pEFC36a showed the highest degree of sequence identity to plasmid R1 which has been isolated in England more than fifty years ago from a patient suffering from a Salmonella infection. Functional conservation of key regulatory features of this F-like conjugation module were demonstrated by the finding that the conjugation frequency of pEFC36a could be stimulated by the positive regulator of plasmid R1 DNA transfer genes, TraJ.

  3. Characterization and comparative analysis of antibiotic resistance plasmids isolated from a wastewater treatment plant.

    Science.gov (United States)

    Rahube, Teddie O; Viana, Laia S; Koraimann, Günther; Yost, Christopher K

    2014-01-01

    A wastewater treatment plant (WWTP) is an environment high in nutrient concentration with diverse bacterial populations and can provide an ideal environment for the proliferation of mobile elements such as plasmids. WWTPs have also been identified as reservoirs for antibiotic resistance genes that are associated with human pathogens. The objectives of this study were to isolate and characterize self-transmissible or mobilizable resistance plasmids associated with effluent from WWTP. An enrichment culture approach designed to capture plasmids conferring resistance to high concentrations of erythromycin was used to capture plasmids from an urban WWTP servicing a population of ca. 210,000. DNA sequencing of the plasmids revealed diversity of plasmids represented by incompatibility groups IncU, col-E, IncFII and IncP-1β. Genes coding resistance to clinically relevant antibiotics (macrolide, tetracycline, beta-lactam, trimethoprim, chloramphenicol, sulphonamide), quaternary ammonium compounds and heavy metals were co-located on these plasmids, often within transposable and integrative mobile elements. Several of the plasmids were self-transmissible or mobilizable and could be maintained in the absence of antibiotic selection. The IncFII plasmid pEFC36a showed the highest degree of sequence identity to plasmid R1 which has been isolated in England more than 50 years ago from a patient suffering from a Salmonella infection. Functional conservation of key regulatory features of this F-like conjugation module were demonstrated by the finding that the conjugation frequency of pEFC36a could be stimulated by the positive regulator of plasmid R1 DNA transfer genes, TraJ.

  4. 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.

  5. 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.

  6. "Curing" of plasmid DNA in acetogen using microwave or applying an electric pulse improves cell growth and metabolite production as compared to the plasmid-harboring strain.

    Science.gov (United States)

    Berzin, Vel; Kiriukhin, Michael; Tyurin, Michael

    2013-03-01

    Plasmid-free acetogen Clostridium sp. MT962 electrotransformed with a small cryptic plasmid pMT351 was used to develop time- and cost-effective methods for plasmid elimination. Elimination of pMT351 restored production of acetate and ethanol to the levels of the plasmid-free strain with no dry cell weight changes. Destabilizing cell membrane via microwave at 2.45 GHz, or exposure to a single 12 ms square electric pulse at 35 kV cm⁻¹, eliminated pMT351 in 42-47 % of cells. Plasmid elimination with a single square electric pulse required 10 versus 0.1 J needed to introduce the same 3,202-bp plasmid into the cells as calculated per cell sample of Clostridium sp. MT962. Microwave caused visible changes in repPCR pattern and increased ethanol production at the expense of acetate. This is the first report on microwave of microwave ovens, wireless routers, and mobile devices causing chromosomal DNA aberrations in microbes along with carbon flux change.

  7. Regular cellular distribution of plasmids by oscillating and filament-forming ParA ATPase of plasmid pB171

    DEFF Research Database (Denmark)

    Ebersbach, Gitte; Ringgaard, Simon; Møller-Jensen, Jakob;

    2006-01-01

    with each other in a bacterial two-hybrid assay but do not interact with FtsZ, eight other essential cell division proteins or MreB actin. Based on these observations, we propose a simple model for how oscillating ParA filaments can mediate regular cellular distribution of plasmids. The model functions......Centromere-like loci from bacteria segregate plasmids to progeny cells before cell division. The ParA ATPase (a MinD homologue) of the par2 locus from plasmid pB171 forms oscillating helical structures over the nucleoid. Here we show that par2 distributes plasmid foci regularly along the length...... of the cell even in cells with many plasmids. In vitro, ParA binds ATP and ADP and has a cooperative ATPase activity. Moreover, ParA forms ATP-dependent filaments and cables, suggesting that ParA can provide the mechanical force for the observed regular distribution of plasmids. ParA and ParB interact...

  8. Regular Cellular Distribution of Plasmids by Oscillating and Filament-forming ParA ATPase of Plasmid pB171

    DEFF Research Database (Denmark)

    Ebersbach, Gitte; Ringgaard, Simon; Møller-Jensen, Jakob;

    2006-01-01

    with each other in a bacterial two-hybrid assay but do not interact with FtsZ, eight other essential cell division proteins or MreB actin. Based on these observations, we propose a simple model for how oscillating ParA filaments can mediate regular cellular distribution of plasmids. The model functions......Centromere-like loci from bacteria segregate plasmids to progeny cells before cell division. The ParA ATPase (a MinD homologue) of the par2 locus from plasmid pB171 forms oscillating helical structures over the nucleoid. Here we show that par2 distributes plasmid foci regularly along the length...... of the cell even in cells with many plasmids. In vitro, ParA binds ATP and ADP and has a cooperative ATPase activity. Moreover, ParA forms ATP-dependent filaments and cables, suggesting that ParA can provide the mechanical force for the observed regular distribution of plasmids. ParA and ParB interact...

  9. Application of a plasmid classification system to determine prevalence of replicon families among multidrug resistant enterococci

    Science.gov (United States)

    The presence and transfer of plasmids from commensal bacteria to more pathogenic bacteria may contribute to dissemination of antimicrobial resistance. However, prevalence of plasmids from commensal bacteria in food animals such as the enterococci remains largely unknown. In this study, the prevale...

  10. Diversity and stability of plasmids from glycopeptide resistant Enterococcus faecium isolated from pigs in Denmark

    DEFF Research Database (Denmark)

    Hasman, H.; Villadsen, A. G.; Aarestrup, Frank Møller

    2005-01-01

    was seen at the end of the 7-year period, coinciding with the ban in 1998 of the macrolide tylosin as growth promoter for pig production. The stability of the plasmid in its original host was compared with stability of the same plasmid in BM4105RF, when both strains were maintained in liquid cultures...

  11. Complete genome sequences of Incl1 Plasmids carrying extended-spectrum B-Lactamase genes

    NARCIS (Netherlands)

    Brouwer, M.S.M.; Bossers, A.; Harders, F.; Essen-Zandbergen, van A.; Mevius, D.J.; Smith, H.E.

    2014-01-01

    Extended spectrum beta-lactamases (ESBLs) confer resistance to clinically relevant antibiotics. Often, the resistance genes are carried by conjugative plasmids which are responsible for dissemination. Five IncI1 plasmids carrying ESBLs from commensal and clinical Escherichia coli isolates were compl

  12. CHARACTERIZATION OF SINGLE-STRAND ORIGINS OF CRYPTIC ROLLING-CIRCLE PLASMIDS FROM BACILLUS-SUBTILIS

    NARCIS (Netherlands)

    MEIJER, WJJ; VENEMA, G; BRON, S

    1995-01-01

    In this paper we describe the isolation and characterization of single strand origins (SSOs) of several cryptic Bacillus subtilis plasmids which use the rolling-circle mechanism of replication, The plasmids used in this study involved pTA1015, pTA1020, pTA1030, pTA1040, pTA1050 and pTA1060, The SSO

  13. Characterization of the Ac/Ds behaviour in transgenic tomato plants using plasmid rescue

    NARCIS (Netherlands)

    Rommens, Caius M.T.; Rudenko, George N.; Dijkwel, Paul P.; Haaren, Mark J.J. van; Ouwerkerk, Pieter B.F.; Blok, Karin M.; Nijkamp, H. John J.; Hille, Jacques

    1992-01-01

    We describe the use of plasmid rescue to facilitate studies on the behaviour of Ds and Ac elements in transgenic tomato plants. The rescue of Ds elements relies on the presence of a plasmid origin of replication and a marker gene selective in Escherichia coli within the element. The position within

  14. Single-molecule sequencing to track plasmid diversity of hospital-associated carbapenemase-producing Enterobacteriaceae.

    Science.gov (United States)

    Conlan, Sean; Thomas, Pamela J; Deming, Clayton; Park, Morgan; Lau, Anna F; Dekker, John P; Snitkin, Evan S; Clark, Tyson A; Luong, Khai; Song, Yi; Tsai, Yu-Chih; Boitano, Matthew; Dayal, Jyoti; Brooks, Shelise Y; Schmidt, Brian; Young, Alice C; Thomas, James W; Bouffard, Gerard G; Blakesley, Robert W; Mullikin, James C; Korlach, Jonas; Henderson, David K; Frank, Karen M; Palmore, Tara N; Segre, Julia A

    2014-09-17

    Public health officials have raised concerns that plasmid transfer between Enterobacteriaceae species may spread resistance to carbapenems, an antibiotic class of last resort, thereby rendering common health care-associated infections nearly impossible to treat. To determine the diversity of carbapenemase-encoding plasmids and assess their mobility among bacterial species, we performed comprehensive surveillance and genomic sequencing of carbapenem-resistant Enterobacteriaceae in the National Institutes of Health (NIH) Clinical Center patient population and hospital environment. We isolated a repertoire of carbapenemase-encoding Enterobacteriaceae, including multiple strains of Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, Enterobacter cloacae, Citrobacter freundii, and Pantoea species. Long-read genome sequencing with full end-to-end assembly revealed that these organisms carry the carbapenem resistance genes on a wide array of plasmids. K. pneumoniae and E. cloacae isolated simultaneously from a single patient harbored two different carbapenemase-encoding plasmids, indicating that plasmid transfer between organisms was unlikely within this patient. We did, however, find evidence of horizontal transfer of carbapenemase-encoding plasmids between K. pneumoniae, E. cloacae, and C. freundii in the hospital environment. Our data, including full plasmid identification, challenge assumptions about horizontal gene transfer events within patients and identify possible connections between patients and the hospital environment. In addition, we identified a new carbapenemase-encoding plasmid of potentially high clinical impact carried by K. pneumoniae, E. coli, E. cloacae, and Pantoea species, in unrelated patients and in the hospital environment. Copyright © 2014, American Association for the Advancement of Science.

  15. Comparative Genomics of the Conjugation Region of F-like Plasmids: Five Shades of F

    Science.gov (United States)

    Fernandez-Lopez, Raul; de Toro, Maria; Moncalian, Gabriel; Garcillan-Barcia, M. Pilar; de la Cruz, Fernando

    2016-01-01

    The F plasmid is the foremost representative of a large group of conjugative plasmids, prevalent in Escherichia coli, and widely distributed among the Enterobacteriaceae. These plasmids are of clinical relevance, given their frequent association with virulence determinants, colicins, and antibiotic resistance genes. Originally defined by their sensitivity to certain male-specific phages, IncF plasmids share a conserved conjugative system and regulatory circuits. In order to determine whether the genetic architecture and regulation circuits are preserved among these plasmids, we analyzed the natural diversity of F-like plasmids. Using the relaxase as a phylogenetic marker, we identified 256 plasmids belonging to the IncF/ MOBF12group, present as complete DNA sequences in the NCBI database. By comparative genomics, we identified five major groups of F-like plasmids. Each shows a particular operon structure and alternate regulatory systems. Results show that the IncF/MOBF12 conjugation gene cluster conforms a diverse and ancient group, which evolved alternative regulatory schemes in its adaptation to different environments and bacterial hosts. PMID:27891505

  16. Plasmid content of Erwinia amylovora in orchards in Washington and Oregon

    Science.gov (United States)

    We examined the plasmid content of a collection of 305 isolates of Erwinia amylovora from Washington and Oregon in the Pacific Northwest of the United States with PCR assays and RFLP. Nearly all isolates of E. amylovora carried plasmid pEA29, which is not found in other species of bacteria, but 4% ...

  17. A Simple and Inexpensive Method for Sending Binary Vector Plasmid DNA by Mail

    Science.gov (United States)

    We describe a simple cost-effective technique for the transport of plasmid DNA by mail. Our results demonstrate that common multipurpose printing paper is a satisfactory substrate and superior to the more absorbent 3MM chromatography paper for the transport of plasmid DNA through the U.S. first clas...

  18. 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.

  19. A classification system for plasmids from Enterococci and other Gram-positive bacteria

    DEFF Research Database (Denmark)

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

    2010-01-01

    A classification system for plasmids isolated from enterococci and other Gram-positive bacteria was developed based on 111 published plasmid sequences from enterococci and other Gram-positive bacteria; mostly staphylococci. Based on PCR amplification of conserved areas of the replication initiati...

  20. Diversity and stability of plasmids from glycopeptide resistant Enterococcus faecium isolated from pigs in Denmark

    DEFF Research Database (Denmark)

    Hasman, H.; Villadsen, A. G.; Aarestrup, Frank Møller

    2005-01-01

    was seen at the end of the 7-year period, coinciding with the ban in 1998 of the macrolide tylosin as growth promoter for pig production. The stability of the plasmid in its original host was compared with stability of the same plasmid in BM4105RF, when both strains were maintained in liquid cultures...

  1. An individual-based approach to explain plasmid invasion in bacterial populations

    DEFF Research Database (Denmark)

    Seoane, Jose Miguel; Yankelevich, Tatiana; Dechesne, Arnaud

    2011-01-01

    observe, however, that transient periods of elevated plasmid transfer in newly formed transconjugant cells are offset by unfavorable cell-to-cell contact mechanics, which ultimately precludes the pWWO TOL plasmid from fully invading tightly packed multicellular P. putida populations such as microcolonies...

  2. Complete Nucleotide Sequence of a Citrobacter freundii Plasmid Carrying KPC-2 in a Unique Genetic Environment

    Science.gov (United States)

    Yao, Yancheng; Imirzalioglu, Can; Hain, Torsten; Kaase, Martin; Gatermann, Soeren; Exner, Martin; Mielke, Martin; Hauri, Anja; Dragneva, Yolanta; Bill, Rita; Wendt, Constanze; Wirtz, Angela; Chakraborty, Trinad

    2014-01-01

    The complete and annotated nucleotide sequence of a 54,036-bp plasmid harboring a blaKPC-2 gene that is clonally present in Citrobacter isolates from different species is presented. The plasmid belongs to incompatibility group N (IncN) and harbors the class A carbapenemase KPC-2 in a unique genetic environment. PMID:25395635

  3. 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

  4. Characterization of plasmids that encode streptomycin-resistance in bacterial epiphytes of apple.

    Science.gov (United States)

    Huang, T C; Burr, T J

    1999-05-01

    Streptomycin resistance in strains of Pseudomonas syringae pv. papulans, Pantoea agglomerans and a yellow-pigmented, non-fluorescent Pseudomonas sp. (Py), isolated from apple orchards in New York and Washington states, is predominantly associated with strA-strB genes carried on conjugal plasmids (R plasmids). None of 128 resistant Erwinia amylovora strains from the eastern and western USA hybridized with a strA-strB probe, SMP3. Resistant Py strains transfered R plasmids to Ps. syringae pv. papulans and to Py in vitro at frequencies of 10(-1)-10(-2) per recipient cell whereas Ps. syringae pv. papulans transferred its plasmids at frequencies of 10(-2) to below detectable levels. Transfer of R plasmids to P. agglomerans was not detected and resistant P. agglomerans did not transfer their R plasmids to any recipients. R plasmids were found to be highly diverse as measured by DNA fingerprint analysis. Transfer-deficient transposon mutants of R plasmid pCPP519 were generated, and 3.9 kb EcoRI and 3.0 kb SmaI fragments that hybridized with a Tn5 probe were cloned and sequenced. The deduced amino acid sequences of the 3.9 kb fragment were similar to proteins involved in replication, nicking at oriT, and piliation in other bacteria.

  5. 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.

  6. Conjugal plasmid transfer (pAM beta 1) in Lactobacillus plantarum.

    OpenAIRE

    Shrago, A W; Chassy, B M; Dobrogosz, W J

    1986-01-01

    The streptococcal plasmid pAM beta 1 (erythromycin resistance) was transferred via conjugation from Streptococcus faecalis to Lactobacillus plantarum and was transferred among L. plantarum strains. Streptococcus sanguis Challis was transformed with pAM beta 1 isolated from these transconjugants, and transformants harboring intact pAM beta 1 could conjugate the plasmid back to L. plantarum.

  7. 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

  8. Genomic and Functional Characterization of qnr-Encoding Plasmids from Municipal Wastewater Biosolid Klebsiella pneumoniae Isolates.

    Science.gov (United States)

    Kaplan, Ella; Sela, Noa; Doron-Faigenboim, Adi; Navon-Venezia, Shiri; Jurkevitch, Edouard; Cytryn, Eddie

    2015-01-01

    Municipal wastewater treatment facilities are considered to be "hotspots" for antibiotic resistance, since they conjoin high densities of environmental and fecal bacteria with selective pressure in the form of sub-therapeutic concentrations of antibiotics. Discharged effluents and biosolids from these facilities can disseminate antibiotic resistant genes to terrestrial and aquatic environments, potentially contributing to the increasing global trend in antibiotic resistance. This phenomenon is especially pertinent when resistance genes are associated with mobile genetic elements such as conjugative plasmids, which can be transferred between bacterial phyla. Fluoroquinolones are among the most abundant antibiotic compounds detected in wastewater treatment facilities, especially in biosolids, where due to their hydrophobic properties they accumulate to concentrations that may exceed 40 mg/L. Although fluoroquinolone resistance is traditionally associated with mutations in the gyrA/topoisomerase IV genes, there is increasing evidence of plasmid-mediated quinolone resistance, which is primarily encoded on qnr genes. In this study, we sequenced seven qnr-harboring plasmids from a diverse collection of Klebsiella strains, isolated from dewatered biosolids from a large wastewater treatment facility in Israel. One of the plasmids, termed pKPSH-11XL was a large (185.4 kbp), multi-drug resistance, IncF-type plasmid that harbored qnrB and 10 additional antibiotic resistance genes that conferred resistance to five different antibiotic families. It was highly similar to the pKPN3-like plasmid family that has been detected in multidrug resistant clinical Klebsiella isolates. In contrast, the six additional plasmids were much smaller (7-9 Kbp) and harbored a qnrS -type gene. These plasmids were highly similar to each other and closely resembled pGNB2, a plasmid isolated from a German wastewater treatment facility. Comparative genome analyses of pKPSH-11XL and other pKPN3-like

  9. 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

  10. 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

  11. 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.

  12. Molecular relationship among fosfomycin-resistant plasmids and clinical impact of fosfomycin resistance.

    Science.gov (United States)

    Mendoza, M C; Teran, F J; Mendez, F J; Hardisson, C

    1988-10-01

    We have been carrying out a surveillance programme on plasmid-mediated fosfomycin resistance in our community over the last decade and have isolated and characterized several varieties of conjugative plasmids from different enterobacteriae. In this work we show that seven varieties of plasmids are related with the Inc M group, and carry the same For determinant which encodes a modifying enzyme. The comparative study on their R-phenotype, restriction analysis and DNA-DNA hybridization showed different degrees of molecular relationship among them. The spread of For-plasmids as well as the fosfomycin resistance by other mechanisms seems to be low in spite of the great For-plasmid diversity found.

  13. 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.

  14. Optimizing hyaluronidase dose and plasmid DNA delivery greatly improves gene electrotransfer efficiency in rat skeletal muscle

    DEFF Research Database (Denmark)

    Åkerström, Thorbjörn; Vedel, Kenneth; Needham Andersen, Josefine

    2015-01-01

    Transfection of rat skeletal muscle in vivo is a widely used research model. However, gene electrotransfer protocols have been developed for mice and yield variable results in rats. We investigated whether changes in hyaluronidase pre-treatment and plasmid DNA delivery can improve transfection...... efficiency in rat skeletal muscle. We found that pre-treating the muscle with a hyaluronidase dose suitable for rats (0.56. U/g b.w.) prior to plasmid DNA injection increased transfection efficiency by >200% whereas timing of the pre-treatment did not affect efficiency. Uniformly distributing plasmid DNA...... delivery across the muscle by increasing the number of plasmid DNA injections further enhanced transfection efficiency whereas increasing plasmid dose from 0.2 to 1.6. μg/g b.w. or vehicle volume had no effect. The optimized protocol resulted in ~80% (CI95%: 79-84%) transfected muscle fibers...

  15. Gyrase-dependent stabilization of pSC101 plasmid inheritance by transcriptionally active promoters.

    Science.gov (United States)

    Beaucage, S L; Miller, C A; Cohen, S N

    1991-09-01

    The pSC101 plasmid encodes a cis-acting genetic locus termed par that ensures the stable inheritance of plasmids in a population of dividing cells. In the absence of selection, par-defective plasmids are lost rapidly from the bacterial population. We report here that the stability of par-deleted pSC101 derivatives is restored by introducing certain adventitious bacterial promoters onto the plasmid. Stabilization requires active transcription from the inserted promoter and is affected by the site and orientation of the insertion, the length of the nascent transcript and DNA gyrase activity. While a promotor-associated overall increase in negative superhelicity of plasmid DNA was observed, stabilized inheritance appeared to be dependent on localized rather than generalized supercoiling. Our demonstration that promoter-induced DNA supercoiling can mimic the effects of the pSC101 par locus provides evidence that the previously reported superhelicity-generating effects of par are intrinsic to its function.

  16. Genome Stability of Lyme Disease Spirochetes: Comparative Genomics of Borrelia burgdorferi Plasmids

    Energy Technology Data Exchange (ETDEWEB)

    Casjens S. R.; Dunn J.; Mongodin, E. F.; Qiu, W.-G.; Luft, B. J.; Schutzer, S. E.; Gilcrease, E. B.; Huang, W. M.; Vujadinovic, M.; Aron, J. K.; Vargas, L. C.; Freeman, S.; Radune, D.; Weidman, J. F.; Dimitrov, G. I.; Khouri, H. M.; Sosa, J. E.; Halpin, R. A.; Fraser, C. M.

    2012-03-14

    Lyme disease is the most common tick-borne human illness in North America. In order to understand the molecular pathogenesis, natural diversity, population structure and epizootic spread of the North American Lyme agent, Borrelia burgdorferi sensu stricto, a much better understanding of the natural diversity of its genome will be required. Towards this end we present a comparative analysis of the nucleotide sequences of the numerous plasmids of B. burgdorferi isolates B31, N40, JD1 and 297. These strains were chosen because they include the three most commonly studied laboratory strains, and because they represent different major genetic lineages and so are informative regarding the genetic diversity and evolution of this organism. A unique feature of Borrelia genomes is that they carry a large number of linear and circular plasmids, and this work shows that strains N40, JD1, 297 and B31 carry related but non-identical sets of 16, 20, 19 and 21 plasmids, respectively, that comprise 33-40% of their genomes. We deduce that there are at least 28 plasmid compatibility types among the four strains. The B. burgdorferi {approx}900 Kbp linear chromosomes are evolutionarily exceptionally stable, except for a short {le}20 Kbp plasmid-like section at the right end. A few of the plasmids, including the linear lp54 and circular cp26, are also very stable. We show here that the other plasmids, especially the linear ones, are considerably more variable. Nearly all of the linear plasmids have undergone one or more substantial inter-plasmid rearrangements since their last common ancestor. In spite of these rearrangements and differences in plasmid contents, the overall gene complement of the different isolates has remained relatively constant.

  17. An extranuclear expression system for analysis of cytoplasmic promoters of yeast linear killer plasmids.

    Science.gov (United States)

    Schründer, J; Meinhardt, F

    1995-03-01

    Based on the cytoplasmically localized killer plasmids pGKL1 and pGKL2 of Kluyveromyces lactis two new linear hybrid plasmids were constructed which consist of pGKL1, into which in addition to the previously developed cytoplasmically expressible LEU2* selectable marker a glucose dehydrogenase-encoding bacterial gene (gdh A) has been integrated. One of the hybrid plasmids carries the bacterial gene preceded by an arbitrarily placed cytoplasmic promoter (upstream conserved sequence) in front of the coding region (pRKL121). The other plasmid was constructed in such a way that the ATG start codon of the gdh A gene was fused in frame to the ATG start codon of the killer plasmid's open reading frame 5 (pRKL122). The structures of both linear hybrid plasmids were confirmed by restriction analysis, Southern hybridization, and sequencing of the junction sites. Yeast strains carrying either of the plasmids expressed the glucose dehydrogenase gene; however, expression of the in phase fused gene was 40-fold higher compared to the arbitrarily placed cytoplasmic promoter. In general, an in phase fusion was not required for expression, but efficiency is dramatically enhanced when the 5' noncoding sequences in front of the heterologous genes are the same as those found on the native killer plasmids. The developed system can serve as a reporter for determining the efficiency of the different cytoplasmic promoters present on both linear plasmids. Hybrid plasmids were stably maintained without selective pressure in K. lactis and they were transferred and expressed also in Saccharomyces cerevisiae.

  18. 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.

  19. 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.

  20. Plasmid profile analysis in identification of epidemic strains of Salmonella enterica serovar Enteritidis

    Directory of Open Access Journals (Sweden)

    Miljković-Selimović Biljana

    2008-01-01

    Full Text Available Background/Aim. As illness caused by Sallmonella enterica serovar Enteritidis (S. Enteritidis occurs not only as sporadic cases but as outbreaks, to reveal the source and routes of spreading of infection it is necessary to identify epidemic strain by the use of some typing methods. To determine whether plasmid profile analysis, as genotyping method, could be applied for the investigation of epidemic strains, isolates of S. Enteritidis, recovered from patient's stools and food associated with outbreaks and those isolated from sporadic cases of diarrhea, were investigated. Methods. Investigation of antibiotic resistance was performed by Kirby - Bauer disc-diffusion method. Isolation of plasmid DNA was carried out by Birnboim and Dolly alkaline lysis method, modified by Ish-Horovitz. Results. Out of 276 izolates of S. Enteritidis 94 were isolated from patient's stools and food associated with outbreaks and 182 were isolated from sporadic cases of diarrhea. The presence of 12 plasmid profiles was established. An average correlation degree of plasmid profiles between the strains was 0.84, that implies high degree of similarity of plasmid profiles of epidemic and non- epidemic strains isolated at our geographic region for the given period of time. Conclusion. The strains of S. Enteritidis, isolated in outbreaks of enterocolitis as well as from spordic cases of diarrhea in the same period of time and at the same area, frequently exhibit the same plasmid profile characterized by a single plasmid of 38 MDa. Therefore, in most cases plasmid profile analysis is not valuable in the identification of epidemic strains of S. Enteritidis. However, for this purpose plasmid profile analysis could be used when drug-resistant strains of S. Enteritidis are isolated, as they often possess additional resistant plasmids what increases discrimination power of this method.

  1. The large universal Pantoea plasmid LPP-1 plays a major role in biological and ecological diversification.

    Science.gov (United States)

    De Maayer, Pieter; Chan, Wai-Yin; Blom, Jochen; Venter, Stephanus N; Duffy, Brion; Smits, Theo H M; Coutinho, Teresa A

    2012-11-15

    Pantoea spp. are frequently isolated from a wide range of ecological niches and have various biological roles, as plant epi- or endophytes, biocontrol agents, plant-growth promoters or as pathogens of both plant and animal hosts. This suggests that members of this genus have undergone extensive genotypic diversification. One means by which this occurs among bacteria is through the acquisition and maintenance of plasmids. Here, we have analyzed and compared the sequences of a large plasmid common to all sequenced Pantoea spp. The Large PantoeaPlasmids (LPP-1) of twenty strains encompassing seven different Pantoea species, including pathogens and endo-/epiphytes of a wide range of plant hosts as well as insect-associated strains, were compared. The LPP-1 plasmid sequences range in size from ~281 to 794 kb and carry between 238 and 750 protein coding sequences (CDS). A core set of 46 proteins, encompassing 2.2% of the total pan-plasmid (2,095 CDS), conserved among all LPP-1 plasmid sequences, includes those required for thiamine and pigment biosynthesis. Phylogenetic analysis reveals that these plasmids have arisen from an ancestral plasmid, which has undergone extensive diversification. Analysis of the proteins encoded on LPP-1 also showed that these plasmids contribute to a wide range of Pantoea phenotypes, including the transport and catabolism of various substrates, inorganic ion assimilation, resistance to antibiotics and heavy metals, colonization and persistence in the host and environment, pathogenesis and antibiosis. LPP-1 is universal to all Pantoea spp. whose genomes have been sequenced to date and is derived from an ancestral plasmid. LPP-1 encodes a large array of proteins that have played a major role in the adaptation of the different Pantoea spp. to their various ecological niches and their specialization as pathogens, biocontrol agents or benign saprophytes found in many diverse environments.

  2. The large universal Pantoea plasmid LPP-1 plays a major role in biological and ecological diversification

    Directory of Open Access Journals (Sweden)

    De Maayer Pieter

    2012-11-01

    Full Text Available Abstract Background Pantoea spp. are frequently isolated from a wide range of ecological niches and have various biological roles, as plant epi- or endophytes, biocontrol agents, plant-growth promoters or as pathogens of both plant and animal hosts. This suggests that members of this genus have undergone extensive genotypic diversification. One means by which this occurs among bacteria is through the acquisition and maintenance of plasmids. Here, we have analyzed and compared the sequences of a large plasmid common to all sequenced Pantoea spp. Results and discussion The Large PantoeaPlasmids (LPP-1 of twenty strains encompassing seven different Pantoea species, including pathogens and endo-/epiphytes of a wide range of plant hosts as well as insect-associated strains, were compared. The LPP-1 plasmid sequences range in size from ~281 to 794 kb and carry between 238 and 750 protein coding sequences (CDS. A core set of 46 proteins, encompassing 2.2% of the total pan-plasmid (2,095 CDS, conserved among all LPP-1 plasmid sequences, includes those required for thiamine and pigment biosynthesis. Phylogenetic analysis reveals that these plasmids have arisen from an ancestral plasmid, which has undergone extensive diversification. Analysis of the proteins encoded on LPP-1 also showed that these plasmids contribute to a wide range of Pantoea phenotypes, including the transport and catabolism of various substrates, inorganic ion assimilation, resistance to antibiotics and heavy metals, colonization and persistence in the host and environment, pathogenesis and antibiosis. Conclusions LPP-1 is universal to all Pantoea spp. whose genomes have been sequenced to date and is derived from an ancestral plasmid. LPP-1 encodes a large array of proteins that have played a major role in the adaptation of the different Pantoea spp. to their various ecological niches and their specialization as pathogens, biocontrol agents or benign saprophytes found in many diverse

  3. Genome stability of Lyme disease spirochetes: comparative genomics of Borrelia burgdorferi plasmids.

    Directory of Open Access Journals (Sweden)

    Sherwood R Casjens

    Full Text Available Lyme disease is the most common tick-borne human illness in North America. In order to understand the molecular pathogenesis, natural diversity, population structure and epizootic spread of the North American Lyme agent, Borrelia burgdorferi sensu stricto, a much better understanding of the natural diversity of its genome will be required. Towards this end we present a comparative analysis of the nucleotide sequences of the numerous plasmids of B. burgdorferi isolates B31, N40, JD1 and 297. These strains were chosen because they include the three most commonly studied laboratory strains, and because they represent different major genetic lineages and so are informative regarding the genetic diversity and evolution of this organism. A unique feature of Borrelia genomes is that they carry a large number of linear and circular plasmids, and this work shows that strains N40, JD1, 297 and B31 carry related but non-identical sets of 16, 20, 19 and 21 plasmids, respectively, that comprise 33-40% of their genomes. We deduce that there are at least 28 plasmid compatibility types among the four strains. The B. burgdorferi ∼900 Kbp linear chromosomes are evolutionarily exceptionally stable, except for a short ≤20 Kbp plasmid-like section at the right end. A few of the plasmids, including the linear lp54 and circular cp26, are also very stable. We show here that the other plasmids, especially the linear ones, are considerably more variable. Nearly all of the linear plasmids have undergone one or more substantial inter-plasmid rearrangements since their last common ancestor. In spite of these rearrangements and differences in plasmid contents, the overall gene complement of the different isolates has remained relatively constant.

  4. 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 ).

  5. Characterization of multidrug-resistant Escherichia coli by antimicrobial resistance profiles, plasmid replicon typing, and pulsed-field gel electrophoresis

    Science.gov (United States)

    Aim: Plasmid characterization has particular clinical importance because genes encoding significant traits including antimicrobial resistance are frequently carried on plasmids. The objective of this study was to examine the distribution of multidrug resistance (MDR) in Escherichia coli in relation ...

  6. Characterization and restriction analysis of the P sex factor and the cryptic plasmid of Vibrio cholerae strain V58.

    Science.gov (United States)

    Bartowsky, E J; Morelli, G; Kamke, M; Manning, P A

    1987-07-01

    The P plasmid of Vibrio cholerae is a derepressed sex factor restricted to V. cholerae and has been shown to express surface exclusion. We have isolated the plasmids of strain V58 and have found that in addition to P, two further cryptic plasmids are also present. P has a size of 68 kb as determined by both electron microscopy and restriction endonuclease analysis. These other plasmids are 34 and 4.7 kb in size. Restriction maps of P and the larger cryptic plasmid have been determined. It has been demonstrated that P differs from the standard Inc group test plasmids and also expresses a surface exclusion system. The ability of the type Inc plasmids to be transferred to V. cholerae by either liquid or filter matings and the stability of these plasmids in V. cholerae have also been examined.

  7. Extended Function of Plasmid Partition Genes: the Sop System of Linear Phage-Plasmid N15 Facilitates Late Gene Expression▿

    Science.gov (United States)

    Ravin, Nikolai V.; Rech, Jérôme; Lane, David

    2008-01-01

    The mitotic stability of the linear plasmid-prophage N15 of Escherichia coli depends on a partition system closely related to that of the F plasmid SopABC. The two Sop systems are distinguished mainly by the arrangement of their centromeric SopB-binding sites, clustered in F (sopC) and dispersed in N15 (IR1 to IR4). Because two of the N15 inverted repeat (IR) sites are located close to elements presumed (by analogy with phage λ) to regulate late gene expression during the lytic growth of N15, we asked whether Sop partition functions play a role in this process. In N15, a putative Q antiterminator gene is located 6 kb upstream of the probable major late promoter and two intrinsic terminator-like sequences, in contrast to λ, where the Q gene is adjacent to the late promoter. Northern hybridization and lacZ reporter activity confirmed the identity of the N15 late promoter (p52), demonstrated antiterminator activity of the Q analogue, and located terminator sequences between p52 and the first open reading frame. Following prophage induction, N15 mutated in IR2 (downstream from gene Q) or IR3 (upstream of p52) showed a pronounced delay in lysis relative to that for wild-type N15. Expression of ir3−-p52::lacZ during N15 wild-type lytic growth was strongly reduced relative to the equivalent ir3+ fusion. The provision of Q protein and the IR2 and SopAB proteins in trans to ir3+-p52::lacZ increased expression beyond that seen in the absence of any one of these factors. These results indicate that the N15 Sop system has a dual role: partition and regulation of late gene transcription during lytic growth. PMID:18359814

  8. Extended function of plasmid partition genes: the Sop system of linear phage-plasmid N15 facilitates late gene expression.

    Science.gov (United States)

    Ravin, Nikolai V; Rech, Jérôme; Lane, David

    2008-05-01

    The mitotic stability of the linear plasmid-prophage N15 of Escherichia coli depends on a partition system closely related to that of the F plasmid SopABC. The two Sop systems are distinguished mainly by the arrangement of their centromeric SopB-binding sites, clustered in F (sopC) and dispersed in N15 (IR1 to IR4). Because two of the N15 inverted repeat (IR) sites are located close to elements presumed (by analogy with phage lambda) to regulate late gene expression during the lytic growth of N15, we asked whether Sop partition functions play a role in this process. In N15, a putative Q antiterminator gene is located 6 kb upstream of the probable major late promoter and two intrinsic terminator-like sequences, in contrast to lambda, where the Q gene is adjacent to the late promoter. Northern hybridization and lacZ reporter activity confirmed the identity of the N15 late promoter (p52), demonstrated antiterminator activity of the Q analogue, and located terminator sequences between p52 and the first open reading frame. Following prophage induction, N15 mutated in IR2 (downstream from gene Q) or IR3 (upstream of p52) showed a pronounced delay in lysis relative to that for wild-type N15. Expression of ir3(-)-p52::lacZ during N15 wild-type lytic growth was strongly reduced relative to the equivalent ir3(+) fusion. The provision of Q protein and the IR2 and SopAB proteins in trans to ir3(+)-p52::lacZ increased expression beyond that seen in the absence of any one of these factors. These results indicate that the N15 Sop system has a dual role: partition and regulation of late gene transcription during lytic growth.

  9. Influence of Single Base Change in Shine-Dalgarno Sequence on the Stability of B.Subtilis Plasmid PSM604

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    B.Subtilis expression plasmids generally require a stringent Shine-Dalgarno Sequence(SDS). Site-directed-mutagenesis was explored to change the Shine-Dalgarno Sequence from AAAAATGGGG (mutant type) to AAAAAGGGGG (wild type) in recombinant plasmid PSM604. The single base substitution made the plasmid with wild SDS unstable in structure and segregation. The interaction of SDS with subtilisin leader sequence of PSM604 might be responsible for the instability of plasmid.

  10. Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR.

    Science.gov (United States)

    Plotka, Magdalena; Wozniak, Mateusz; Kaczorowski, Tadeusz

    2017-01-01

    Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within Escherichia coli DH5α cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a

  11. Factors affecting plasmid production in Escherichia coli from a resource allocation standpoint

    Directory of Open Access Journals (Sweden)

    Cunningham Drew S

    2009-05-01

    Full Text Available Abstract Background Plasmids are being reconsidered as viable vector alternatives to viruses for gene therapies and vaccines because they are safer, non-toxic, and simpler to produce. Accordingly, there has been renewed interest in the production of plasmid DNA itself as the therapeutic end-product of a bioprocess. Improvement to the best current yields and productivities of such emerging processes would help ensure economic feasibility on the industrial scale. Our goal, therefore, was to develop a stoichiometric model of Escherichia coli metabolism in order to (1 determine its maximum theoretical plasmid-producing capacity, and to (2 identify factors that significantly impact plasmid production. Results Such a model was developed for the production of a high copy plasmid under conditions of batch aerobic growth on glucose minimal medium. The objective of the model was to maximize plasmid production. By employing certain constraints and examining the resulting flux distributions, several factors were determined that significantly impact plasmid yield. Acetate production and constitutive expression of the plasmid's antibiotic resistance marker exert negative effects, while low pyruvate kinase (Pyk flux and the generation of NADPH by transhydrogenase activity offer positive effects. The highest theoretical yield (592 mg/g resulted under conditions of no marker or acetate production, nil Pyk flux, and the maximum allowable transhydrogenase activity. For comparison, when these four fluxes were constrained to wild-type values, yields on the order of tens of mg/g resulted, which are on par with the best experimental yields reported to date. Conclusion These results suggest that specific plasmid yields can theoretically reach 12 times their current experimental maximum (51 mg/g. Moreover, they imply that abolishing Pyk activity and/or transhydrogenase up-regulation would be useful strategies to implement when designing host strains for plasmid

  12. Genomic and functional characterization of qnr-encoding plasmids from municipal wastewater biosolid Klebsiella pneumoniae isolates

    Directory of Open Access Journals (Sweden)

    Ella eKaplan

    2015-12-01

    Full Text Available Municipal wastewater treatment facilities are considered to be hotspots for antibiotic resistance since they conjoin high densities of environmental and fecal bacteria with selective pressure in the form of sub-therapeutic concentrations of antibiotics. Discharged effluents and biosolids from these facilities can disseminate antibiotic resistant genes to terrestrial and aquatic environments, potentially contributing to the increasing global trend in antibiotic resistance. This phenomenon is especially pertinent when resistance genes are associated with mobile genetic elements such as conjugative plasmids, which can be transferred between bacterial phyla. Fluoroquinolones are among the most abundant antibiotic compounds detected in wastewater treatment facilities, especially in biosolids, where due to their hydrophobic properties they accumulate to concentrations that may exceed 40 mg/L. Although fluoroquinolone resistance is traditionally associated with mutations in the gyrA/topoisomerase IV genes, there is increasing evidence of plasmid-mediated quinolone resistance, which is primarily encoded on qnr genes. In this study, we sequenced seven qnr-harboring plasmids from a diverse collection of Klebsiella strains, isolated from dewatered biosolids from a large wastewater treatment facility in Israel. One of the plasmids, termed pKPSH-11XL was a large (185.4 kbp, multi-drug resistance, IncF-type plasmid that harbored qnrB and 10 additional antibiotic resistance genes that conferred resistance to 5 different antibiotic families. It was highly similar to the pKPN3-like plasmid family that has been detected in multidrug resistant clinical Klebsiella isolates. In contrast, the six additional plasmids were much smaller (7-9Kbp and harbored a qnrS -type gene. These plasmids were highly similar to each other and closely resembled pGNB2, a plasmid isolated from a German wastewater treatment facility. Comparative genome analyses of pKPSH-11XL and other p

  13. PLASMID PROFILES AND PHAGE TYPES OF SALMONELLA-TYPHIMURIUM ISOLATED FROM SUCCESSIVE FLOCKS OF CHICKENS ON 3 PARENT STOCK FARMS

    DEFF Research Database (Denmark)

    Baggesen, Dorte Lau; Olsen, J. E.; Bisgaard, M.

    1992-01-01

    for restriction enzyme analysis and colony hybridization. The main phage type on each of the three farms was 110. Plasmid profiling, however, allowed further subtyping. All but three isolates carried the serotype-specific virulence-associated plasmid. Restriction enzyme analysis showed variations in this plasmid...

  14. A comparison of the growth responses following intramuscular GHRH plasmid administration versus daily growth hormone injections in young pigs

    Science.gov (United States)

    The efficacy of daily porcine growth hormone (GH) injections versus plasmid-driven porcine GH-releasing hormone (pGHRH) production to promote growth was assessed. Ten-day-old piglets were injected intramuscularly with 0.1, 1, or 3 mg pGHRH, or a control plasmid followed by electroporation. Plasmid c...

  15. Characterization of Antimicrobial Resistance Dissemination across Plasmid Communities Classified by Network Analysis

    Directory of Open Access Journals (Sweden)

    Akifumi Yamashita

    2014-04-01

    Full Text Available The global clustering of gene families through network analysis has been demonstrated in whole genome, plasmid, and microbiome analyses. In this study, we carried out a plasmidome network analysis of all available complete bacterial plasmids to determine plasmid associations. A blastp clustering search at 100% aa identity cut-off and sharing at least one gene between plasmids, followed by a multilevel community network analysis revealed that a surprisingly large number of the plasmids were connected by one largest connected component (LCC, with dozens of community sub-groupings. The LCC consisted mainly of Bacilli and Gammaproteobacteria plasmids. Intriguingly, horizontal gene transfer (HGT was noted between different phyla (i.e., Staphylococcus and Pasteurellaceae, suggesting that Pasteurellaceae can acquire antimicrobial resistance (AMR genes from closely contacting Staphylococcus spp., which produce the external supplement of V-factor (NAD. Such community network analysis facilitate displaying possible recent HGTs like a class 1 integron, str and tet resistance markers between communities. Furthermore, the distribution of the Inc replicon type and AMR genes, such as the extended-spectrum ß-lactamase (ESBL CTX-M or the carbapenemases KPC NDM-1, implies that such genes generally circulate within limited communities belonging to typical bacterial genera. Thus, plasmidome network analysis provides a remarkable discriminatory power for plasmid-related HGT and evolution.

  16. 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.

  17. 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.

  18. Epsilon-toxin plasmids of Clostridium perfringens type D are conjugative.

    Science.gov (United States)

    Hughes, Meredith L; Poon, Rachael; Adams, Vicki; Sayeed, Sameera; Saputo, Juliann; Uzal, Francisco A; McClane, Bruce A; Rood, Julian I

    2007-11-01

    Isolates of Clostridium perfringens type D produce the potent epsilon-toxin (a CDC/U.S. Department of Agriculture overlap class B select agent) and are responsible for several economically significant enterotoxemias of domestic livestock. It is well established that the epsilon-toxin structural gene, etx, occurs on large plasmids. We show here that at least two of these plasmids are conjugative. The etx gene on these plasmids was insertionally inactivated using a chloramphenicol resistance cassette to phenotypically tag the plasmid. High-frequency conjugative transfer of the tagged plasmids into the C. perfringens type A strain JIR325 was demonstrated, and the resultant transconjugants were shown to act as donors in subsequent mating experiments. We also demonstrated the transfer of "unmarked" native epsilon-toxin plasmids into strain JIR325 by exploiting the high transfer frequency. The transconjugants isolated in these experiments expressed functional epsilon-toxin since their supernatants had cytopathic effects on MDCK cells and were toxic in mice. Using the widely accepted multiplex PCR approach for toxin genotyping, these type A-derived transconjugants were genotypically type D. These findings have significant implications for the C. perfringens typing system since it is based on the toxin profile of each strain. Our study demonstrated the fluid nature of the toxinotypes and their dependence upon the presence or absence of toxin plasmids, some of which have for the first time been shown to be conjugative.

  19. Epsilon-Toxin Plasmids of Clostridium perfringens Type D Are Conjugative▿ †

    Science.gov (United States)

    Hughes, Meredith L.; Poon, Rachael; Adams, Vicki; Sayeed, Sameera; Saputo, Juliann; Uzal, Francisco A.; McClane, Bruce A.; Rood, Julian I.

    2007-01-01

    Isolates of Clostridium perfringens type D produce the potent epsilon-toxin (a CDC/U.S. Department of Agriculture overlap class B select agent) and are responsible for several economically significant enterotoxemias of domestic livestock. It is well established that the epsilon-toxin structural gene, etx, occurs on large plasmids. We show here that at least two of these plasmids are conjugative. The etx gene on these plasmids was insertionally inactivated using a chloramphenicol resistance cassette to phenotypically tag the plasmid. High-frequency conjugative transfer of the tagged plasmids into the C. perfringens type A strain JIR325 was demonstrated, and the resultant transconjugants were shown to act as donors in subsequent mating experiments. We also demonstrated the transfer of “unmarked” native ɛ-toxin plasmids into strain JIR325 by exploiting the high transfer frequency. The transconjugants isolated in these experiments expressed functional ɛ-toxin since their supernatants had cytopathic effects on MDCK cells and were toxic in mice. Using the widely accepted multiplex PCR approach for toxin genotyping, these type A-derived transconjugants were genotypically type D. These findings have significant implications for the C. perfringens typing system since it is based on the toxin profile of each strain. Our study demonstrated the fluid nature of the toxinotypes and their dependence upon the presence or absence of toxin plasmids, some of which have for the first time been shown to be conjugative. PMID:17720791

  20. Plasmid as a measure of microbial degradation capacity for 2,4-dichlorophenoxyacetic acid.

    Science.gov (United States)

    Chong, Nyuk-Min; Chang, Hung-Wei

    2009-02-01

    The purpose of this research was to pursuit the quantification of microbial degradation capacity for 2,4-dichlorophenoxyacetic acid (2,4-D) by detecting and quantifying a prominent 2,4-D degradation encoding plasmid. Batch reactor acclimation, de-acclimation, and re-acclimation tests were conducted during which periods the courses of 2,4-D dissipation and plasmid evolution were quantitatively measured. Pure cultures of bacterial strains were detected to give rise to a plasmid approximately the size of 90 kb after acclimation. The 90 kb plasmid content of Arthrobacter sp. increased when degradation occurred after acclimation, with a rate that corresponded closely to the degradation rate. During de-acclimation, plasmid content declined exponentially at a half-life of approximately 3.5 days. Re-acclimation saw a renewed induction of plasmid, but substrate consumption limited the rise of plasmid to a level much lower than after the first acclimation. This research recommends a method for measuring the microbial degradation capability for a xenobiotic.

  1. 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.

  2. TOL plasmid transfer during bacterial conjugation in vitro and rhizoremediation of oil compounds in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Jussila, Minna M. [Department of Applied Chemistry and Microbiology, Viikki Biocenter, P.O. Box 56 (Viikinkaari 9), FI-00014 University of Helsinki, Helsinki (Finland)]. E-mail: minna.m.jussila@helsinki.fi; Zhao, Ji [Department of Applied Chemistry and Microbiology, Viikki Biocenter, P.O. Box 56 (Viikinkaari 9), FI-00014 University of Helsinki, Helsinki (Finland); Suominen, Leena [Department of Applied Chemistry and Microbiology, Viikki Biocenter, P.O. Box 56 (Viikinkaari 9), FI-00014 University of Helsinki, Helsinki (Finland); Lindstroem, Kristina [Department of Applied Chemistry and Microbiology, Viikki Biocenter, P.O. Box 56 (Viikinkaari 9), FI-00014 University of Helsinki, Helsinki (Finland)

    2007-03-15

    Molecular profiling methods for horizontal transfer of aromatics-degrading plasmids were developed and applied during rhizoremediation in vivo and conjugations in vitro. pWW0 was conjugated from Pseudomonas to Rhizobium. The xylE gene was detected both in Rhizobium galegae bv. officinalis and bv. orientalis, but it was neither stably maintained in orientalis nor functional in officinalis. TOL plasmids were a major group of catabolic plasmids among the bacterial strains isolated from the oil-contaminated rhizosphere of Galega orientalis. A new finding was that some Pseudomonas migulae and Pseudomonas oryzihabitans strains harbored a TOL plasmid with both pWW0- and pDK1-type xylE gene. P. oryzihabitans 29 had received the archetypal TOL plasmid pWW0 from Pseudomonas putida PaW85. As an application for environmental biotechnology, the biodegradation potential of oil-polluted soil and the success of bioremediation could be estimated by monitoring changes not only in the type and amount but also in transfer of degradation plasmids. - Horizontal transfer of degradation plasmids in the oil-contaminated rhizosphere reveals the dynamic nature of the intrinsic biodegradation potential.

  3. Accurate determination of plasmid copy number of flow-sorted cells using droplet digital PCR.

    Science.gov (United States)

    Jahn, Michael; Vorpahl, Carsten; Türkowsky, Dominique; Lindmeyer, Martin; Bühler, Bruno; Harms, Hauke; Müller, Susann

    2014-06-17

    Many biotechnological processes rely on the expression of a plasmid-based target gene. A constant and sufficient number of plasmids per cell is desired for efficient protein production. To date, only a few methods for the determination of plasmid copy number (PCN) are available, and most of them average the PCN of total populations disregarding heterogeneous distributions. Here, we utilize the highly precise quantification of DNA molecules by droplet digital PCR (ddPCR) and combine it with cell sorting using flow cytometry. A duplex PCR assay was set up requiring only 1000 sorted cells for precise determination of PCN. The robustness of this method was proven by thorough optimization of cell sorting, cell disruption, and PCR conditions. When non plasmid-harboring cells of Pseudomonas putida KT2440 were spiked with different dilutions of the expression plasmid pA-EGFP_B, a PCN from 1 to 64 could be accurately detected. As a proof of principle, induced cultures of P. putida KT2440 producing an EGFP-fused model protein by means of the plasmid pA-EGFP_B were investigated by flow cytometry and showed two distinct subpopulations, fluorescent and nonfluorescent cells. These two subpopulations were sorted for PCN determination with ddPCR. A remarkably diverging plasmid distribution was found within the population, with nonfluorescent cells showing a much lower PCN (≤1) than fluorescent cells (PCN of up to 5) under standard conditions.

  4. Low-dose plasmid DNA treatment increases plasma vasopressin and regulates blood pressure in experimental endotoxemia

    Directory of Open Access Journals (Sweden)

    Malardo Thiago

    2012-11-01

    Full Text Available Abstract Background Although plasmid DNA encoding an antigen from pathogens or tumor cells has been widely studied as vaccine, the use of plasmid vector (without insert as therapeutic agent requires further investigation. Results Here, we showed that plasmid DNA (pcDNA3 at low doses inhibits the production of IL-6 and TNF-α by lipopolysaccharide (LPS-stimulated macrophage cell line J774. These findings led us to evaluate whether plasmid DNA could act as an anti-inflammatory agent in a Wistar rat endotoxemia model. Rats injected simultaneously with 1.5 mg/kg of LPS and 10 or 20 μg of plasmid DNA had a remarkable attenuation of mean arterial blood pressure (MAP drop at 2 hours after treatment when compared with rats injected with LPS only. The beneficial effect of the plasmid DNA on MAP was associated with decreased expression of IL-6 in liver and increased concentration of plasma vasopressin (AVP, a known vasoconstrictor that has been investigated in hemorrhagic shock management. No difference was observed in relation to nitric oxide (NO production. Conclusion Our results demonstrate for the first time that plasmid DNA vector at low doses presents anti-inflammatory property and constitutes a novel approach with therapeutic potential in inflammatory diseases.

  5. 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.

  6. 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

  7. Inheritance of Mitochondrial DNA and Plasmids in the Ascomycetous Fungus, Epichloe Typhina

    OpenAIRE

    Chung, K. R.; Leuchtmann, A.; Schardl, C. L.

    1996-01-01

    We analyzed the inheritance of mitochondrial DNA (mtDNA) species in matings of the grass symbiont Epichloe typhina. Eighty progeny were analyzed from a cross in which the maternal (stromal) parent possessed three linear plasmids, designated Callan-a (7.5 kb), Aubonne-a (2.1 kb) and Bergell (2.0 kb), and the paternal parent had one plasmid, Aubonne-b (2.1 kb). Maternal transmission of all plasmids was observed in 76 progeny; two progeny possessed Bergell and Callan-a, but had the maternal Aubo...

  8. 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

  9. Microbial degradation of pyridine using Pseudomonas sp. and isolation of plasmid responsible for degradation.

    Science.gov (United States)

    Mohan, S Venkata; Sistla, Srinivas; Guru, R Kumar; Prasad, K Krishna; Kumar, C Suresh; Ramakrishna, S V; Sarma, P N

    2003-01-01

    Pseudomonas (PI2) capable of degrading pyridine was isolated from the mixed population of the activated sludge unit which was being used for treating complex effluents, the strain was characterized. Aerobic degradation of pyridine was studied with the isolated strain and the growth parameters were evaluated. Pyridine degradation was further conformed by chromatography (HPLC) analysis. The process parameters like biomass growth and dissolved oxygen consumption were monitored during pyridine degradation. In order to conform with the plasmid capability to degrade pyridine, the requisite plasmid was isolated and transferred to DH 5alpha Escherichia coli. The subsequent biodegradation studies revealed the ability of the transformed plasmid capability to degrade the pyridine.

  10. Plasmid Transfer into the Homoacetogen Acetobacterium woodii by Electroporation and Conjugation

    OpenAIRE

    Strätz, Michael; Sauer, Uwe; Kuhn, Anita; Dürre, Peter

    1994-01-01

    Shuttle vectors (pMS3 and pMS4) which replicated in Escherichia coli and in gram-positive Acetobacterium woodii were constructed by ligating the replication origin of plasmid pAMβ1 with the E. coli cloning vector pUC19 and the tetM gene of streptococcal transposon Tn916. Electrotransformation of A. woodii was achieved at frequencies of 4.5 × 103 transformants per μg of plasmid DNA. For conjugal plasmid transfer, the mobilizable shuttle vector pKV12 was constructed by cloning the tetM gene int...

  11. Screening large numbers of recombinant plasmids: modifications and additions to alkaline lysis for greater efficiency

    Institute of Scientific and Technical Information of China (English)

    XU Yibing; N.V. CHANDRASEKHARAN; Daniel L. SIMMONS

    2006-01-01

    Selecting bacteria transformed with recombinant plasmid is a laborious step in gene cloning experiments. This selection process is even more tedious when large numbers of clones need to be screened. We describe here modifications to the ultra fast plasmid preparation method described previously by Law and Crickmore. The modified method is coupled to an efficient PCR step to rapidly determine orientation of the inserts. Compared to traditional methods of analysis requiring growth of overnight cultures, plasmid isolation and restriction enzyme digestion to determine orientation this procedure allows for the analysis and storage of a large number of recombinants within a few hours.

  12. Underexpression of Ap from R-Plasmids in Fast-Growing Rhizobium Species

    Science.gov (United States)

    Sikka, Virendra K.; Kumar, Sushil

    1984-01-01

    The presence of the plasmid RP1 in the cells of Rhizobium leguminosarum strains Rld1, 300, and 248, R. phaseoli 1233, R. trifolii strains T1 and 6661, and R. meliloti 4013 was found to appreciably increase bacterial resistance toward kanamycin and tetracycline but not toward ampicillin. The presence of 16 other R-plasmids in R. leguminosarum was also found to either not increase or only marginally increase bacterial resistance toward ampicillin. It appears now that underexpression of the plasmid-specified ampicillin function is common to most fast- and slow-growing rhizobia. PMID:16346686

  13. Structural analysis of the ParR/parC plasmid partition complex

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Ringgaard, Simon; Mercogliano, Christopher P

    2007-01-01

    Accurate DNA partition at cell division is vital to all living organisms. In bacteria, this process can involve partition loci, which are found on both chromosomes and plasmids. The initial step in Escherichia coli plasmid R1 partition involves the formation of a partition complex between the DNA...... and biochemical experiments support a structural arrangement in which the centromere-like parC DNA is wrapped around a ParR protein scaffold. This structure holds implications for how ParM polymerization drives active DNA transport during plasmid partition....

  14. Specific structural probing of plasmid-coded ribosomal RNAs from Escherichia coli

    DEFF Research Database (Denmark)

    Aagaard, C; Rosendahl, G; Dam, M

    1991-01-01

    The preferred method for construction and in vivo expression of mutagenised Escherichia coli ribosomal RNAs (rRNAs) is via high copy number plasmids. Transcription of wild-type rRNA from the seven chromosomal rrn operons in strains harbouring plasmid-coded mutant rRNAs leads to a heterogeneous...... ribosome population, which consequently hinders direct probing of mutant rRNAs. Here, we describe how nonconserved helical regions of plasmid-coded rRNA have been altered in a manner that preserves their secondary structures while creating new sites for primer extension of mutant rRNAs. This facilitates...

  15. Isolation of plasmid from the blue-green alga Spirulina platensis

    Science.gov (United States)

    Qin, Song; Tong, Shun; Zhang, Peijun; Tseng, C. K.

    1993-09-01

    CCC plasmid was isolated from an economically important blue-green alga — Spirulina platensis (1.7×106 dalton from the S6 strain and 1.2×106 dalton from the F3 strain) using a rapid method based on ultrasonic disruption of algal cells and alkaline removal of chromosomal DNA. The difference in the molecular weight of the CCC DNAs from the two strains differing in form suggests that plasmid may be related with the differentiation of algal form. This modified method, which does not use any lysozyme, is a quick and effective method of plasmid isolation, especially for filamentous blue-green algae.

  16. Structures of actin-like ParM filaments show architecture of plasmid-segregating spindles

    OpenAIRE

    Bharat, Tanmay A. M.; Murshudov, Garib N.; Sachse, Carsten; Löwe, Jan

    2015-01-01

    Active segregation of E. coli low-copy number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments 1-6 . ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments 3,7-9 . Growing ParMRC spindles push sister plasmids to the cell poles 9,10 . Here, using modern electron cryomicroscopy methods we have investigated the structures and arrangements of ParM filaments in vitro an...

  17. Separation of topological forms of plasmid DNA by anion-exchange HPLC: shifts in elution order of linear DNA.

    Science.gov (United States)

    Smith, Clara R; DePrince, Randolph B; Dackor, Jennifer; Weigl, Debra; Griffith, Jack; Persmark, Magnus

    2007-07-01

    We sought to establish a single anion-exchange HPLC method for the separation of linear, open circular and supercoiled plasmid topoisomers using purified topoisomeric forms of three plasmids (3.0, 5.5 and 7.6 kb). However, finding one condition proved elusive as the topoisomer elution order was determined to depend on salt gradient slope. The observed change in selectivity increased with plasmid size and was most pronounced for the linear form. Indeed, the elution order of the linear 7.6 kb plasmid was reversed relative to the supercoiled form. This observation may have implications for methods used in quality control of plasmid DNA.

  18. Pathogenicity of Vibrio anguillarum serogroup O1 strains compared to plasmids, outer membrane protein profiles and siderophore production

    DEFF Research Database (Denmark)

    Pedersen, K.; Gram, Lone; Austin, D.A.;

    1997-01-01

    The virulence of 18 strains of Vibrio anguillarum serogroup 01 was compared to plasmid content, expression of siderophores and outer membrane proteins. All strains, irrespective of plasmid content, produced siderophores and inducible outer membrane proteins under iron-limited conditions. Only...... strains that carried the 67 kbp virulence plasmid or derivatives of it produced the outer membrane protein, OM2. All virulent strains harboured the 67 kbp plasmid or derivatives of it, indicating its importance for virulence. However, some strains carrying the virulence plasmid or a derivative of it...

  19. Atypical Enteropathogenic Escherichia coli Secretes Plasmid Encoded Toxin

    Directory of Open Access Journals (Sweden)

    Rita C. Ruiz

    2014-01-01

    Full Text Available Plasmid encoded toxin (Pet is a serine protease originally described in enteroaggregative Escherichia coli (EAEC prototype strain 042 whose entire characterization was essentially obtained from studies performed with the purified toxin. Here we show that Pet is not exclusive to EAEC. Atypical enteropathogenic Escherichia coli (aEPEC strains, isolated from diarrhea cases, express Pet and its detection in supernatants of infected HEp-2 cells coincides with the appearance of cell damage, which, in turn, were similar to those described with purified Pet. Pet secretion and the cytotoxic effects are time and culture medium dependent. In presence of DMEM supplemented with tryptone cell rounding and detachment were observed after just 5 h of incubation with the bacteria. In the absence of tryptone, the cytotoxic effects were detected only after 24 h of infection. We also show that, in addition to the prototype EAEC, other pet+ EAEC strains, also isolated from diarrhea cases, induce cellular damage in the same degree as the aEPEC. The cytotoxic effects of EAEC and aEPEC strains were significantly reduced in the presence of a serine protease inhibitor or anti-Pet IgG serum. Our results show a common aspect between the aEPEC and EAEC and provide the first evidence pointing to a role of Pet in aEPEC pathogenesis.

  20. Complexation Between Cationic Diblock Copolymers and Plasmid DNA

    Science.gov (United States)

    Jung, Seyoung; Reineke, Theresa; Lodge, Timothy

    Deoxyribonucleic acids (DNA), as polyanions, can spontaneously bind with polycations to form polyelectrolyte complexes. When the polycation is a diblock copolymer with one cationic block and one uncharged hydrophilic block, the polyelectrolyte complexes formed with plasmid DNA (pDNA) are often colloidally stable, and show great promise in the field of polymeric gene therapy. While the resulting properties (size, stability, and toxicity to biological systems) of the complexes have been studied for numerous cationic diblocks, the fundamentals of the pDNA-diblock binding process have not been extensively investigated. Herein, we report how the cationic block content of a diblock influences the pDNA-diblock interactions. pDNA with 7164 base pairs and poly(2-deoxy-2-methacrylamido glucopyranose)-block-poly(N-(2-aminoethyl) methacrylamide) (PMAG-b-PAEMA) are used as the model pDNA and cationic diblock, respectively. To vary the cationic block content, two PMAG-b-PAEMA copolymers with similar PMAG block lengths but distinct PAEMA block lengths and a PAEMA homopolymer are utilized. We show that the enthalpy change from pDNA-diblock interactions is dependent on the cationic diblock composition, and is closely associated with both the binding strength and the pDNA tertiary structure.

  1. Properties of the arsenate reductase of plasmid R773.

    Science.gov (United States)

    Gladysheva, T B; Oden, K L; Rosen, B P

    1994-06-14

    Resistance to toxic oxyanions in Escherichia coli is conferred by the ars operon carried on plasmid R773. The gene products of this operon catalyze extrusion of antimonials and arsenicals from cells of E. coli, thus providing resistance to those toxic oxyanions. In addition, resistance to arsenate is conferred by the product of the arsC gene. In this report, purified ArsC protein was shown to catalyze reduction of arsenate to arsenite. The enzymatic activity of the ArsC protein required glutaredoxin as a source of reducing equivalents. Other reductants, including glutathione and thioredoxin, were not effective electron donors. A spectrophotometric assay was devised in which arsenate reduction was coupled to NADPH oxidation. The results obtained with the coupled assay corresponded to those found by direct reduction of radioactive arsenate to arsenite. The only substrate of the reaction was arsenate (Km = 8 mM); other oxyanions including phosphate, sulfate, and antimonate were not reduced. Phosphate and sulfate were weak inhibitors, while the product, arsenite, was a stronger inhibitor (Ki = 0.1 mM). Arsenate reductase activity exhibited a pH optimum of 6.3-6.8. These results indicate that the ArsC protein is a novel reductase, and elucidation of its enzymatic mechanism should be of interest.

  2. Plasmid DNA gene therapy by electroporation: principles and recent advances.

    Science.gov (United States)

    Murakami, Tatsufumi; Sunada, Yoshihide

    2011-12-01

    Simple plasmid DNA injection is a safe and feasible gene transfer method, but it confers low transfection efficiency and transgene expression. This non-viral gene transfer method is enhanced by physical delivery methods, such as electroporation and the use of a gene gun. In vivo electroporation has been rapidly developed over the last two decades to deliver DNA to various tissues or organs. It is generally considered that membrane permeabilization and DNA electrophoresis play important roles in electro-gene transfer. Skeletal muscle is a well characterized target tissue for electroporation, because it is accessible and allows for long-lasting gene expression ( > one year). Skin is also a target tissue because of its accessibility and immunogenicity. Numerous studies have been performed using in vivo electroporation in animal models of disease. Clinical trials of DNA vaccines and immunotherapy for cancer treatment using in vivo electroporation have been initiated in patients with melanoma and prostate cancer. Furthermore, electroporation has been applied to DNA vaccines for infectious diseases to enhance immunogenicity, and the relevant clinical trials have been initiated. The gene gun approach is also being applied for the delivery of DNA vaccines against infectious diseases to the skin. Here, we review recent advances in the mechanism of in vivo electroporation, and summarize the findings of recent preclinical and clinical studies using this technology.

  3. plasmid in Saccharomyces species and in Saccharomyces cerevisiae.

    Science.gov (United States)

    Strope, Pooja K; Kozmin, Stanislav G; Skelly, Daniel A; Magwene, Paul M; Dietrich, Fred S; McCusker, John H

    2015-12-01

    We determined that extrachromosomal 2μ plasmid was present in 67 of the Saccharomyces cerevisiae 100-genome strains; in addition to variation in the size and copy number of 2μ, we identified three distinct classes of 2μ. We identified 2μ presence/absence and class associations with populations, clinical origin and nuclear genotypes. We also screened genome sequences of S. paradoxus, S. kudriavzevii, S. uvarum, S. eubayanus, S. mikatae, S. arboricolus and S. bayanus strains for both integrated and extrachromosomal 2μ. Similar to S. cerevisiae, we found no integrated 2μ sequences in any S. paradoxus strains. However, we identified part of 2μ integrated into the genomes of some S. uvarum, S. kudriavzevii, S. mikatae and S. bayanus strains, which were distinct from each other and from all extrachromosomal 2μ. We identified extrachromosomal 2μ in one S. paradoxus, one S. eubayanus, two S. bayanus and 13 S. uvarum strains. The extrachromosomal 2μ in S. paradoxus, S. eubayanus and S. cerevisiae were distinct from each other. In contrast, the extrachromosomal 2μ in S. bayanus and S. uvarum strains were identical with each other and with one of the three classes of S. cerevisiae 2μ, consistent with interspecific transfer.

  4. R-plasmid transfer in a wastewater treatment plant.

    Science.gov (United States)

    Mach, P A; Grimes, D J

    1982-12-01

    Enteric bacteria have been examined for their ability to transfer antibiotic resistance in a wastewater treatment plant. Resistant Salmonella enteritidis, Proteus mirabilis, and Escherichia coli were isolated from clinical specimens and primary sewage effluent. Resistance to ampicillin, chloramphenicol, streptomycin, sulfadiazine, and tetracycline was demonstrated by spread plate and tube dilution techniques. Plasmid mediation of resistance was shown by ethidium bromide curing, agarose gel electrophoresis, and direct cell transfer. Each donor was mated with susceptible E. coli and Shigella sonnei. Mating pairs (and recipient controls) were suspended in unchlorinated primary effluent that had been filtered and autoclaved. Suspensions were added to membrane diffusion chambers which were then placed in the primary and secondary setting tanks of the wastewater treatment plant. Resistant recombinants were detected by replica plating nutrient agar master plates onto xylose lysine desoxycholate agar plates that contained per milliliter of medium 10 micrograms of ampicillin, 30 micrograms of chloramphenicol, 10 micrograms of streptomycin, 100 micrograms of sulfadiazine, or 30 micrograms of tetracycline. Mean transfer frequencies for laboratory matings were 2.1 X 10(-3). In situ matings for primary and secondary settling resulted in frequencies of 4.9 X 10(-5) and 7.5 X 10(-5), respectively. These values suggest that a significant level of resistance transfer occurs in wastewater treatment plants in the absence of antibiotics as selective agents.

  5. Evidence for plasmid-encoded virulence factors in the phytopathogenic bacterium Clavibacter michiganensis subsp. michiganensis NCPPB382.

    Science.gov (United States)

    Meletzus, D; Bermphol, A; Dreier, J; Eichenlaub, R

    1993-01-01

    The tomato pathogen Clavibacter michiganensis subsp. michiganensis NCPPB382, which causes bacterial wilt, harbors two plasmids pCM1 (27.5 kb) and pCM2 (72 kb). After curing of the plasmids, bacterial derivatives were still proficient in the ability to colonize the host plant and in the production of exopolysaccharides but exhibited a reduced virulence. When one of the two plasmids is lost, there is a significant delay in the development of wilting symptoms after infection and a plasmid-free derivative is not able to induce disease symptoms. By cloning of restriction fragments of both plasmids in the plasmid-free strain CMM100, two DNA fragments which restored the virulent phenotype were identified. Further analysis suggested that a fragment of plasmid pCM1 encodes an endocellulase which is involved in the expression of the pathogenic phenotype. Images PMID:8458855

  6. 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

  7. 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.

  8. 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.

  9. Construction of the plasmid-free strain for human growth hormone production.

    Science.gov (United States)

    Schulga, A A; Mechev, P V; Kirpichnikov, M P; Skryabin, K G; Deyev, S M

    2016-01-01

    The E. coli strain, overproducing human growth hormone (hGH) was made by integration of the hGH gene under the control of T7 promoter into the chromosomal LacZ gene of BL21(DE3) via lambda Red recombineering. The strain gave higher productivity (50 mg·L(-1)·OD550(-1)) and better growth characteristics than the corresponding strain in which the same hGH expression cassette was placed in a plasmid. The protein produced by the plasmid-free strain was purified and characterized to be hGH. The results demonstrates that a plasmid-free recombinant strain having a single-copy gene expression cassette in the chromosome could provide better gene activity regulation, higher productivity, superior growth characteristics, as well as more stringent control of the gene sequence invariance than a plasmid-based strain.

  10. TOL plasmid carriage enhances biofilm formation and increases extracellular DNA content in Pseudomonas putida KT2440

    DEFF Research Database (Denmark)

    D'Alvise, Paul; Sjoholm, O.R.; Yankelevich, T.;

    2010-01-01

    : TOL carriage leads to more extracellular DNA (eDNA) in pellicles and biofilms. Pellicles were dissolved by DNase I treatment. Enhanced cell lysis due to plasmid carriage was ruled out as the mechanism for eDNA release. We report, for the first time, that carriage of a conjugative plasmid leads......Adherent growth of Pseudomonas putida KT2440 with and without the TOL plasmid (pWWO) at the solid-liquid and air-liquid interface was examined. We compared biofilm formation on glass in flow cells, and assayed pellicle (air-liquid interface biofilm) formation in stagnant liquid cultures by confocal...... laser scanning microscopy. The TOL-carrying strains formed pellicles and thick biofilms, whereas the same strains without the plasmid displayed little adherent growth. Microscopy using fluorescent nucleic acid-specific stains revealed differences in the production of extracellular polymeric substances...

  11. 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.

  12. Antimicrobial Susceptibility and Plasmid Replicon Typing of Salmonella enterica serovar Kentucky isolates recovered from Broilers

    Science.gov (United States)

    Salmonella Kentucky has become the predominate serotype recovered from broiler slaughter in the United States and the prevalence of antimicrobial resistance (AMR) has increased dramatically in this serotype. Relationships between AMR, genotype, and plasmid replicon types were characterized for 600 ...

  13. Novel assay to measure the plasmid mobilizing potential of mixed microbial communities

    DEFF Research Database (Denmark)

    Klümper, Uli; Droumpali, Ariadni; Dechesne, Arnaud;

    2014-01-01

    compare the transfer frequency of a mobilizable plasmid to that of a mobilizing and conjugal plasmid measured for a model strain and for the assayed community. With Pseudomonas putida carrying the gfp-tagged mobilizable RSF1010 plasmid as donor strain, we conducted solid surface mating experiments...... community for RP4 (at 1.16x10-4 transconjugants per recipient (T/R)) was similar to that previously measured for soil microbial communities. RSF1010 was mobilized by the model community at a frequency of 1.16x10-5 T/R, only one order of magnitude lower than its permissiveness to RP4. This mobilization...... frequency is unexpectedly high considering that (i) mobilization requires the presence of mobilizing conjugal plasmids within the permissive fraction of the recipients; (ii) in pure culture experiments with P. putida retromobilization of RSF1010 through RP4 only took place in approximately half...

  14. PLASMID DNA DAMAGE CAUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITIN

    Science.gov (United States)

    PLASMID DNA DAMAGE CAOUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITINABSTRACT Both dimethylarsinic acid (DMA(V)) and dimethylarsinous acid (DMA(III)) release iron from human liver ferritin (HLF) with or without the presence of ascorbic acid. ...

  15. Improvement of PCR reaction conditions for site-directed mutagenesis of big plasmids

    Institute of Scientific and Technical Information of China (English)

    Bogdan MUNTEANU; Mario BRAUN; Kajohn BOONROD

    2012-01-01

    QuickChange mutagenesis is the method of choice for site-directed mutagenesis (SDM) of target sequences in a plasmid.It can be applied successfully to small plasmids (up to 10 kb).However,this method cannot efficiently mutate bigger plasmids.Using KOD Hot Start polymerase in combination with high performance liquid chromatography (HPLC) purified primers,we were able to achieve SDM in big plasmids (up to 16 kb) involving not only a single base change but also multiple base changes.Moreover,only six polymerase chain reaction (PCR) cycles and 0.5 μl of polymerase (instead of 18 PCR cycles and 1.0 μl of enzyme In the standard protocol) were sufficient for the reaction.

  16. Nitrogen fixation by Klebsiella pneumoniae is inhibited by certain multicopy hybrid nif plasmids.

    Science.gov (United States)

    Riedel, G E; Brown, S E; Ausubel, F M

    1983-01-01

    In our studies of nif gene regulation, we have observed that certain hybrid nif plasmids drastically inhibit the expression of the chromosomal nif genes of Klebsiella pneumonia. Wild-type (Nif+) K. pneumoniae strains that acquire certain hybrid nif plasmids also acquire the Nif- phenotype; these strains lose 90 to 99% of all detectable nitrogen fixation activity and grow poorly (or not at all) on solid media with N2 as the sole nitrogen source. We describe experiments which defined this inhibition of the Nif+ phenotype by hybrid nif plasmids and identify and characterize four nif DNA regions associated with this inhibition. We show that plasmids carrying these nif regions could recombine with, but not complement, nif chromosomal mutations. Our results suggest that inhibition of the Nif+ phenotype will provide a useful bioassay for some of the factors that mediate nif gene expression.

  17. New Plasmid Tools for Genetic Analysis of Actinobacilus pleuropneumoniae and Other Pasteurellaceae

    National Research Council Canada - National Science Library

    Janine T Bossé; Andrew L Durham; Andrew N Rycroft; J Simon Kroll; Paul R Langford

    2009-01-01

      We have generated a set of plasmids, based on the mobilizable shuttle vector pMIDG100, which can be used as tools for genetic manipulation of Actinobacillus pleuropneumoniae and other members of the Pasteurellaceae...

  18. Development of Lactobacillus plantarum LL441 and its plasmid-cured derivatives in cheese.

    Science.gov (United States)

    Delgado, Susana; Mayo, Baltasar

    2003-04-01

    A wild Lactobacillus plantarum strain and two of its plasmid-cured derivatives were separately used as adjunct cultures in the manufacture of a Gouda-like traditional Spanish cheese. The wild strain, LL441, harbours seven plasmids and produces a lantibiotic-like bacteriocin. The LL441-B2 derivative has lost plasmids of 40 and 80 kb and the bacteriocin-producing capability. The LL441-B11 derivative has lost in addition a 70 kb plasmid encoding active alpha- and beta-galactosidases. All three strains could be used as adjunct cultures as none of the technological and biochemical parameters of the cheeses was affected. Both the wild-type and the two derivatives were recovered from experimental cheeses up to 30 days after manufacture at similar rates of nearly 20%. Thus, the phenotypic traits under examination were not essential for L. plantarum to grow into the cheese matrix.

  19. 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

  20. Plasmid metagenomics reveals multiple antibiotic resistance gene classes among the gut microbiomes of hospitalised patients

    DEFF Research Database (Denmark)

    Jitwasinkul, Tossawan; Suriyaphol, Prapat; Tangphatsornruang, Sithichoke

    2016-01-01

    Antibiotic resistance genes are rapidly spread between pathogens and the normal flora, with plasmids playing an important role in their circulation. This study aimed to investigate antibiotic resistance plasmids in the gut microbiome of hospitalised patients. Stool samples were collected from seven...... sequences (using >80% alignment length as the cut-off), and ResFinder was used to classify the antibiotic resistance gene pools. Plasmid replicon modules were used for plasmid typing. Forty-six genes conferring resistance to several classes of antibiotics were identified in the stool samples. Several...... antibiotic resistance genes were shared by the patients; interestingly, most were reported previously in food animals and healthy humans. Four antibiotic resistance genes were found in the healthy subject. One gene (aph3-III) was identified in the patients and the healthy subject and was related...

  1. Optimizing hyaluronidase dose and plasmid DNA delivery greatly improves gene electrotransfer efficiency in rat skeletal muscle

    DEFF Research Database (Denmark)

    Åkerström, Thorbjörn; Vedel, Kenneth; Needham Andersen, Josefine;

    2015-01-01

    delivery across the muscle by increasing the number of plasmid DNA injections further enhanced transfection efficiency whereas increasing plasmid dose from 0.2 to 1.6. μg/g b.w. or vehicle volume had no effect. The optimized protocol resulted in ~80% (CI95%: 79-84%) transfected muscle fibers......Transfection of rat skeletal muscle in vivo is a widely used research model. However, gene electrotransfer protocols have been developed for mice and yield variable results in rats. We investigated whether changes in hyaluronidase pre-treatment and plasmid DNA delivery can improve transfection...... with a homogenous distribution. We also show that transfection was stable over five weeks of regular exercise or inactivity. Our findings show that species-specific plasmid DNA delivery and hyaluronidase pre-treatment greatly improves transfection efficiency in rat skeletal muscle....

  2. 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.

  3. Ordering the mob: Insights into replicon and MOB typing schemes from analysis of a curated dataset of publicly available plasmids.

    Science.gov (United States)

    Orlek, Alex; Phan, Hang; Sheppard, Anna E; Doumith, Michel; Ellington, Matthew; Peto, Tim; Crook, Derrick; Walker, A Sarah; Woodford, Neil; Anjum, Muna F; Stoesser, Nicole

    2017-03-09

    Plasmid typing can provide insights into the epidemiology and transmission of plasmid-mediated antibiotic resistance. The principal plasmid typing schemes are replicon typing and MOB typing, which utilize variation in replication loci and relaxase proteins respectively. Previous studies investigating the proportion of plasmids assigned a type by these schemes ('typeability') have yielded conflicting results; moreover, thousands of plasmid sequences have been added to NCBI in recent years, without consistent annotation to indicate which sequences represent complete plasmids. Here, a curated dataset of complete Enterobacteriaceae plasmids from NCBI was compiled, and used to assess the typeability and concordance of in silico replicon and MOB typing schemes. Concordance was assessed at hierarchical replicon type resolutions, from replicon family-level to plasmid multilocus sequence type (pMLST)-level, where available. We found that 85% and 65% of the curated plasmids could be replicon and MOB typed, respectively. Overall, plasmid size and the number of resistance genes were significant independent predictors of replicon and MOB typing success. We found some degree of non-concordance between replicon families and MOB types, which was only partly resolved when partitioning plasmids into finer-resolution groups (replicon and pMLST types). In some cases, non-concordance was attributed to ambiguous boundaries between MOBP and MOBQ types; in other cases, backbone mosaicism was considered a more plausible explanation. β-lactamase resistance genes tended not to show fidelity to a particular plasmid type, though some previously reported associations were supported. Overall, replicon and MOB typing schemes are likely to continue playing an important role in plasmid analysis, but their performance is constrained by the diverse and dynamic nature of plasmid genomes.

  4. 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.

  5. 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

  6. 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

  7. 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...

  8. 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.

  9. Abundance and diversity of plasmid-associated genes among clinical isolates of Enterococcus faecalis.

    Science.gov (United States)

    Wardal, Ewa; Gawryszewska, Iwona; Hryniewicz, Waleria; Sadowy, Ewa

    2013-11-01

    Enterococcus faecalis, a normal compound of the human intestinal microbiome, plays an important role in hospital-acquired infections. Plasmids make a significant contribution to the acquisition of the novel traits such as antimicrobial resistance and virulence by this pathogen. The study investigated the plasmid content and the diversity of plasmid-associated genes in a group of 152 hospital isolates of E. faecalis. The majority of plasmids visualized by pulsed-field gel electrophoresis of S1 nuclease-digested DNA fell into the range of 50-100 kb. PCR-based screening allowed detection of genes of the rep1(pIP501), rep2(pRE25), rep4(pMBB1), rep6(pS86), rep7(pT181), rep8(pAM373), rep9(pAD1/pTEF2/pCF10), rep10(pIM13) and rep13(pC194) families in 29 different combinations. The par and ω-ε-ζ plasmid stabilization systems were ubiquitous (45 isolates, 29.6% and 88 isolates, 57.9%, respectively), while the axe-txe system was not found. The asa1 gene homologues encoding aggregation substance characteristic for the pAD1 and related group of pheromone-responsive plasmids were present in 106 isolates. A variety of sequence variants, including novel ones, of genes associated with pheromone-responsive plasmids, such as rep8(pAM373), rep9(pAD1/pTEF2/pCF10), par, and asa1 were observed. In conclusion, there is a big and only partially characterized pool of diverse plasmids in clinical E. faecalis.

  10. 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...

  11. Broad-Host-Range IncP-1 plasmids and their resistance potential

    Directory of Open Access Journals (Sweden)

    Magdalena ePopowska

    2013-03-01

    Full Text Available The plasmids of the incompatibility group IncP-1, also called IncP, as extrachromosomal genetic elements can transfer and replicate virtually in all Gram-negative bacteria. They are composed of backbone genes that encode a variety of essential functions and accessory genes that have implications for human health and environmental bioremediation. Broad-host-range IncP plasmids are known to spread genes between distinct phylogenetic groups of bacteria. These genes often code for resistances to a broad spectrum of antibiotics, heavy metals and quaternary ammonium compounds used as disinfectants. The backbone of these plasmids carries modules that enable them to effectively replicate, move to a new host via conjugative transfer and to be stably maintained in bacterial cells. The adaptive, resistance and virulence genes are mainly located on mobile genetic elements integrated between the functional plasmid backbone modules. Environmental studies have demonstrated the wide distribution of IncP-like replicons in manure, soils and wastewater treatment plants. They also are present in strains of pathogenic or opportunistic bacteria, which can be a cause for concern, because they may encode multiresistance. Their broad distribution suggests that IncP plasmids play a crucial role in bacterial adaptation by utilizing horizontal gene transfer. This review summarizes the variety of genetic information and physiological functions carried by IncP plasmids, which can contribute to the spread of antibiotic and heavy metal resistance while also mediating the process of bioremediation of pollutants. Due to the location of the resistance genes on plasmids with a broad host range and the presence of transposons carrying these genes it seems that the spread of these genes would be possible and quite hazardous in infection control. Future studies are required to determine the level of risk of the spread of resistance genes located on these plasmids.

  12. 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.

  13. 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.

  14. Modular construction of plasmids through ligation-free assembly of vector components with oligonucleotide linkers.

    Science.gov (United States)

    Vroom, Jonathan A; Wang, Clifford L

    2008-06-01

    We have developed a modular method of plasmid construction that can join multiple DNA components in a single reaction. A nicking enzyme is used to create 5' and 3' overhangs on PCR-generated DNA components. Without the use of ligase or restriction enzymes, components are joined using oligonucleotide linkers that recognize the overhangs. By specifying the sequences of the linkers, desired components can be assembled in any combination and order to generate different plasmid vectors.

  15. Broad host range plasmids can invade an unexpectedly diverse fraction of a soil bacterial community

    DEFF Research Database (Denmark)

    Klümper, Uli; Riber, Leise; Dechesne, Arnaud

    2014-01-01

    range of IncP- and IncPromA-type broad host range plasmids from three proteobacterial donors to a soil bacterial community. We identified transfer to many different recipients belonging to 11 different bacterial phyla. The prevalence of transconjugants belonging to diverse Gram-positive Firmicutes...... bacteria and can, therefore, directly connect large proportions of the soil bacterial gene pool. This finding reinforces the evolutionary and medical significances of these plasmids....

  16. Transposon Insertion Reveals pRM, a Plasmid of Rickettsia monacensis▿

    OpenAIRE

    Baldridge, Gerald D.; Burkhardt, Nicole Y.; Felsheim, Roderick F.; Timothy J Kurtti; Munderloh, Ulrike G.

    2007-01-01

    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 m...

  17. 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.

  18. 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.

  19. Penicillinase-producing plasmid types in Neisseria gonorrhoeae clinical isolates from Australia.

    Science.gov (United States)

    Whiley, David; Trembizki, Ella; Buckley, Cameron; Freeman, Kevin; Lawrence, Andrew; Limnios, Athena; Pearson, Julie; Smith, Helen; Stevens, Kerrie; Lahra, Monica M

    2014-12-01

    Penicillinase-producing Neisseria gonorrhoeae (PPNG) carrying the blaTEM-135 gene is of particular concern, as it is considered a stepping stone toward resistance to extended-spectrum cephalosporins. Here, we sought to characterize plasmid types and the occurrence of the blaTEM-135 gene for N. gonorrhoeae clinical isolates from Australia. We found that blaTEM-135 was prevalent in Australian PPNG and was detected on all three major plasmid types.

  20. Arsenic efflux governed by the arsenic resistance determinant of Staphylococcus aureus plasmid pI258.

    OpenAIRE

    Bröer, S; Ji, G.; Bröer, A; Silver, S

    1993-01-01

    The arsenic resistance operon of Staphylococcus aureus plasmid pI258 determined lowered net cellular uptake of 73As by an active efflux mechanism. Arsenite was exported from the cells; intracellular arsenate was first reduced to arsenite and then transported out of the cells. Resistant cells showed lower accumulation of 73As originating from both arsenate and arsenite. Active efflux from cells loaded with arsenite required the presence of the plasmid-determined arsB gene. Efflux of arsenic or...

  1. 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).

  2. Second generation sequencing for elucidating the diversity of bacteria and plasmids in soil

    DEFF Research Database (Denmark)

    Holmsgaard, Peter Nikolai

    and increased the relative abundance of Gammaproteobacteria. Additionally, a high abundance of IncP-1, IncP-7, IncP-9, IncQ and IncW plasmids and class I and II integrons were detected. Amplicon pyrosequencing of the IncP-1 plasmids showed an increase in relative abundance of the IncP-1β and decrease of IncP-1ε...

  3. Serious overestimation in quantitative PCR by circular (supercoiled plasmid standard: microalgal pcna as the model gene.

    Directory of Open Access Journals (Sweden)

    Yubo Hou

    Full Text Available Quantitative real-time PCR (qPCR has become a gold standard for the quantification of nucleic acids and microorganism abundances, in which plasmid DNA carrying the target genes are most commonly used as the standard. A recent study showed that supercoiled circular confirmation of DNA appeared to suppress PCR amplification. However, to what extent to which different structural types of DNA (circular versus linear used as the standard may affect the quantification accuracy has not been evaluated. In this study, we quantitatively compared qPCR accuracies based on circular plasmid (mostly in supercoiled form and linear DNA standards (linearized plasmid DNA or PCR amplicons, using proliferating cell nuclear gene (pcna, the ubiquitous eukaryotic gene, in five marine microalgae as a model gene. We observed that PCR using circular plasmids as template gave 2.65-4.38 more of the threshold cycle number than did equimolar linear standards. While the documented genome sequence of the diatom Thalassiosira pseudonana shows a single copy of pcna, qPCR using the circular plasmid as standard yielded an estimate of 7.77 copies of pcna per genome whereas that using the linear standard gave 1.02 copies per genome. We conclude that circular plasmid DNA is unsuitable as a standard, and linear DNA should be used instead, in absolute qPCR. The serious overestimation by the circular plasmid standard is likely due to the undetected lower efficiency of its amplification in the early stage of PCR when the supercoiled plasmid is the dominant template.

  4. Isolation of Plasmids in Legionella pneumophila and Legionella-Like Organisms

    Science.gov (United States)

    1981-03-01

    INtF1(-r1iN ANt11;MM NITY. St.?? NAI 1 1271 2. Voj . NO .j toNO TES (Isolation of Plasmids in Legionella pneumophila and Legionella -Like Organisms...Agarose gel electrophoresis was employed to screen nine strains of Legionella - like bacteria and one strain of Legionella pneumophila for the presence of...similar to sideration of the ubiquitous nature of plasmid but genetically distinct front Legionella pneumophila elements. These results indicate that

  5. 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.

  6. Fabrication of Size-Tunable Metallic Nanoparticles Using Plasmid DNA as a Biomolecular Reactor.

    Science.gov (United States)

    Samson, Jacopo; Piscopo, Irene; Yampolski, Alex; Nahirney, Patrick; Parpas, Andrea; Aggarwal, Amit; Saleh, Raihan; Drain, Charles Michael

    2011-10-21

    Plasmid DNA can be used as a template to yield gold, palladium, silver, and chromium nanoparticles of different sizes based on variations in incubation time at 70 °C with gold phosphine complexes, with the acetates of silver or palladium, or chromium acetylacetonate. The employment of mild synthetic conditions, minimal procedural steps, and aqueous solvents makes this method environmentally greener and ensures general feasibility. The use of plasmids exploits the capabilities of the biotechnology industry as a source of nanoreactor materials.

  7. TEM-1-encoding small plasmids impose dissimilar fitness costs on Haemophilus influenzae and Haemophilus parainfluenzae.

    Science.gov (United States)

    Søndergaard, Annette; Lund, Marianne; Nørskov-Lauritsen, Niels

    2015-12-01

    Only two beta-lactamases, TEM-1 and ROB-1, have been observed in Haemophilus influenzae, while four different TEM but no ROB enzymes have been found in Haemophilus parainfluenzae. In order to investigate the mechanisms behind the dissemination of small beta-lactamase-encoding plasmids in H. influenzae and H. parainfluenzae, we assessed the fitness cost of three TEM-1- (pPN223, pA1209, pA1606), one TEM-15- (pSF3) and one ROB-1-bearing (pB1000) plasmid when expressed in either bacterial species. All plasmids were stable in H. influenzae and H. parainfluenzae except pB1000, which showed on average (sample mean) 76% curing in H. parainfluenzae after 5  days of subculture. Competition assays between isogenic strains with and without plasmid showed no competitive disadvantage of pPN223 and pA1606 in H. influenzae, or of pA1209 in H. parainfluenzae. In contrast, pSF3 and pB1000 were associated with significant competitive disadvantages in both species. Some of the competitive disadvantages may be related to differences in plasmid copy number and mRNA expression of the beta-lactamase genes, as revealed by quantitative PCR analysis. In conclusion, plasmids encoding TEM beta-lactamases isolated from H. influenzae and H. parainfluenzae can be stably transferred between species. The fast curing of pB1000 in H. parainfluenzae observed in this study correlates to the fact that ROB-1 has never been reported for this species. TEM-1-encoding plasmids are associated with the lowest level of fitness cost, but different TEM-1 plasmids confer different levels of fitness cost on the two hosts.

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

    Science.gov (United States)

    Chiang, Chen-Li; Sung, Ching-Shan

    2006-07-01

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

  9. Role of TLR3 in the immunogenicity of replicon plasmid-based vaccines.

    Science.gov (United States)

    Diebold, S S; Schulz, O; Alexopoulou, L; Leitner, W W; Flavell, R A; Reis e Sousa, C

    2009-03-01

    Replicon plasmids encoding an alphavirus RNA replicase constitute an alternative to conventional DNA plasmids with promise for DNA vaccination in humans. Replicase activity amplifies the levels of transgene mRNA through a copying process involving double-stranded (ds) RNA intermediates, which contribute to vaccine immunogenicity by activating innate antiviral responses. Toll-like receptor 3 (TLR3) is a dsRNA innate immune receptor expressed by antigen-presenting dendritic cells (DCs). Here, we test the hypothesis that TLR3 is necessary for the immunogenicity of replicon plasmid-based DNA vaccines. We show that mouse CD8 alpha(+) DC phagocytose dying replicon plasmid-transfected cells in vitro and are activated in a TLR3-dependent manner by dsRNA present within those cells. However, we find that cytotoxic T-cell responses to a replicon plasmid intramuscular vaccine are not diminished in the absence of TLR3 in vivo. Our results underscore the potential role of TLR3 in mediating immune activation by dsRNA-bearing replicon plasmid-transfected cells and indicate that other innate sensing pathways can compensate for TLR3 absence in vivo.

  10. 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.

  11. 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.

  12. Development of a novel plasmid as a shuttle vector for heterologous gene expression in Mycoplasma yeatsii.

    Science.gov (United States)

    Kent, Bethany N; Foecking, Mark F; Calcutt, Michael J

    2012-10-01

    A circular plasmid, pMyBK1, was detected in Mycoplasma yeatsii strain GIH(T). Analysis of the sequence of the 3432-bp replicon identified two predicted open reading frames (ORFs), one with sequence similarity to multiple plasmid mobilization proteins and one that matches only to hypothetical ORFs encoded by integrated chromosomal elements in the sequenced genomes of two Mycoplasma species. Shuttle vectors were constructed in Escherichia coli which could be introduced into M. yeatsii at high efficiency (10(4)-10(5) per μg DNA) by electroporation. Independent deletion analysis of the two ORFs disclosed that whereas mob was dispensable, orf2 was necessary for plasmid replication or maintenance. The absence of plasmid-encoded database matches for ORF2 indicates that pMyBK1 represents a novel plasmid family. One shuttle vector was used to demonstrate heterologous expression of the Mycoplasma fermentans malp gene and was stable during multiple passages. The host-plasmid system described has potential application for genetic manipulation in a genus for which few replicative vectors are available.

  13. Altered murine tissue colonization by Borrelia burgdorferi following targeted deletion of linear plasmid 17-carried genes.

    Science.gov (United States)

    Casselli, Timothy; Tourand, Yvonne; Bankhead, Troy

    2012-05-01

    The causative agent of Lyme disease, Borrelia burgdorferi, possesses a segmented genome comprised of a single linear chromosome and upwards of 23 linear and circular plasmids. Much of what is known about plasmid-borne genes comes from studying laboratory clones that have spontaneously lost one or more plasmids during in vitro passage. Some plasmids, including the linear plasmid lp17, are never or rarely reported to be lost during routine culture; therefore, little is known about the requirement of these conserved plasmids for infectivity. In this study, the effects of deleting regions of lp17 were examined both in vitro and in vivo. A mutant strain lacking the genes bbd16 to bbd25 showed no deficiency in the ability to establish infection or disseminate to the bloodstream of mice; however, colonization of peripheral tissues was delayed. Despite the ability to colonize ear, heart, and joint tissues, this mutant exhibited a defect in bladder tissue colonization for up to 56 days postinfection. This phenotype was not observed in immunodeficient mice, suggesting that bladder colonization by the mutant strain was inhibited by an adaptive immune-based mechanism. Moreover, the mutant displayed increased expression of outer surface protein C in vitro, which was correlated with the absence of the gene bbd18. To our knowledge, this is the first report involving genetic manipulation of lp17 in an infectious clone of B. burgdorferi and reveals for the first time the effects of lp17 gene deletion during murine infection by the Lyme disease spirochete.

  14. Position effect on expression of dsd genes cloned onto multicopy plasmids.

    Science.gov (United States)

    Carothers, A M; Heincz, M C; McFall, E

    1980-04-01

    In the D-serine deaminase system of Escherichia coli, which is regulated by positive control, we have fouand a complete lack of trans activation in vivo with multicopy dsd hybrid plasmids. A PLASmid carrying the regulatory gene, dsdC+, did not promote expression of chromosomal dsdCO+A+ loci, nor did a chromosomal dsdC+ gene promote expression of plasmid-borne dsdC delta O+A+ (dsd regulatory gene negative) restriction fragments. However, hybrid plasmids that comprise the entire dsd system (dsdC+O+A+) are highly inducible for the enzyme. These dsd hybrid plasmid deoxyribonucleic acids functioned well as templates in the in vitro coupled transcription-translation system. In vitro-synthesized dsdC+ protein promoted expression of the dsdA+ operation efficiently. Exogenously purified dsdC+ protein also activated expression of several dsdC delta O+A+ plasmid deoxyribonucleic acid templates in vitro. An explanation that reconciles these results with previous dominance studies is presented.

  15. 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.

  16. A one-step miniprep for the isolation of plasmid DNA and lambda phage particles.

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    George Lezin

    Full Text Available Plasmid DNA minipreps are fundamental techniques in molecular biology. Current plasmid DNA minipreps use alkali and the anionic detergent SDS in a three-solution format. In addition, alkali minipreps usually require additional column-based purification steps and cannot isolate other extra-chromosomal elements, such as bacteriophages. Non-ionic detergents (NIDs have been used occasionally as components of multiple-solution plasmid DNA minipreps, but a one-step approach has not been developed. Here, we have established a one-tube, one-solution NID plasmid DNA miniprep, and we show that this approach also isolates bacteriophage lambda particles. NID minipreps are more time-efficient than alkali minipreps, and NID plasmid DNA performs better than alkali DNA in many downstream applications. In fact, NID crude lysate DNA is sufficiently pure to be used in digestion and sequencing reactions. Microscopic analysis showed that the NID procedure fragments E. coli cells into small protoplast-like components, which may, at least in part, explain the effectiveness of this approach. This work demonstrates that one-step NID minipreps are a robust method to generate high quality plasmid DNA, and NID approaches can also isolate bacteriophage lambda particles, outperforming current standard alkali-based minipreps.

  17. Genetic diversity of Xanthomonas axonopodis pv. citri based on plasmid profile and pulsed field gel electrophoresis

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    Carvalho Flávia Maria de Souza

    2005-01-01

    Full Text Available Xanthomonas axonopodis pv. citri strains that cause disease in citrus were investigated by pulsed field and plasmid profile analysis. For the first method, genomic DNA was digested by the rare-cutting enzymes Xba I and Vsp I. The strains evaluated were collected in seven different States of Brazil and in Argentina, Bolivia, Paraguay and Uruguay. Genetic variability was found among strains of X. axonopodis pv. citri from different geographical areas Argentina, Bolivia and Uruguay, with similarities varying from 0.62 to 0.83. However, the strains collected in Brazil, despite being from different States, have shown a genetic similarity ranging from 0.83 to 1.00. Cluster analysis showed a relationship between genomic similarity and geographical origin of the strains. Plasmids were observed in all strains, with a total of five different plasmids, with sizes between 57.7 and 83.0 kilobases. The 72.6 kb plasmid was the most frequent, present in 15 out of 22 strains, while the 68.1 kb plasmid was observed in two strains only. Although the plasmid diversity detected in the present study was not very great, the X. axonopodis pv. citri strains evaluated showed a considerable degree of diversity with regard to this extrachromosomal genetic element.

  18. 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.

  19. 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.

  20. Plasmid Profiling and Curing of Lactobacillus Strains Isolated from Fermented Milk for Probiotic Applications

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    B. Lavanya

    2011-04-01

    Full Text Available In this study, the antimicrobial susceptibilities and presence of plasmids in 7 probiotics strains which had been isolated from the fermented milk were determined. Resistance to 8 commonly used antibiotics $- lactans (penicillin, ampicilin, gram positive spectrum (vanomycin, broad spectrum (rifampin, trimethoprim and aminogycosides (kanamycin, streptomycin, and bacitracin was assessed by disk diffusion. Among these strains 20, 20, 60, 70, 90 and 100% were found to be exhibit a significant degree of resistance to kanamycin, trimetroprim, rifampicin, kanamycin, amphicilin and penicillin respectively. Further, plasmid profile and curing of plasmid were performed for the seven isolates. Analysis of the plasmid profiles of the 7 cured derivatives revealed loss of plasmids except 2 strains where curing was partially effective. All the strains lost penicillin resistance after curing indicating that plasmids encodes for resistance character. However, vanomycin resistance is not lost upon curing which indicates that such resistance is usually intrinsic (chromosomally encoded and not transmissible. Finally, the antimicrobial susceptibility after curing was done to check the safety aspect of the isolates for their application as probiotics and among the 7 strains, 5 were proved to be potent probiotics.

  1. Therapeutic low-intensity red laser for herpes labialis on plasmid survival and bacterial transformation.

    Science.gov (United States)

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

    2013-05-01

    A low-intensity laser is used in treating herpes labialis based on the biostimulative effect, albeit the photobiological basis is not well understood. In this work experimental models based on Escherichia coli cultures and plasmids were used to evaluate effects of low-intensity red laser on DNA at fluences for treatment of herpes labialis. To this end, survival and transformation efficiency of plasmids in E. coli AB1157 (wild type), BH20 (fpg/mutM(-)) and BW9091 (xthA(-)), content of the supercoiled form of plasmid DNA, as well as nucleic acids and protein content from bacterial cultures exposed to the laser, were evaluated. The data indicate low-intensity red laser: (i) alters the survival of plasmids in wild type, fpg/mutM(-) and xthA(-)E. coli cultures depending of growth phase, (ii) alters the content of the supercoiled form of plasmids in the wild type and fpg/mutM(-)E. coli cells, (iii) alters the content of nucleic acids and proteins in wild type E. coli cells, (iv) alters the transformation efficiency of plasmids in wild type and fpg/mutM(-)E. coli competent cells. These data could be used to understand positive effects of low-intensity lasers on herpes labialis treatment.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Intramuscular electroporation of a P1A-encoding plasmid vaccine delays P815 mastocytoma growth.

    Science.gov (United States)

    Vandermeulen, Gaëlle; Uyttenhove, Catherine; De Plaen, Etienne; Van den Eynde, Benoît J; Préat, Véronique

    2014-12-01

    This study aimed to construct DNA vaccines encoding the mouse P1A tumor antigen and to generate a protective immune response against the P815 mastocytoma, as a model for vaccines against human MAGE-type tumor antigens. DNA vaccines were constructed and delivered to mice by intramuscular electroporation before tumor challenge. Immunization with a plasmid coding for the full-length P1A significantly delayed tumor growth and mice survived at least 10 days longer than untreated controls. 10% of the mice completely rejected the P815 tumors while 50% of them showed a regression phase followed by tumor regrowth. Mice immunized by electroporation of a P1A(35-43) minigene-encoding plasmid failed to reject tumor and even delay tumor growth. The P1A(35-43)-encoding plasmid was modified and helper epitope sequences were inserted. However, these modified plasmids were not able to improve the response against P815 mastocytoma. Consistent with these results, a 12-fold higher CTL activity was observed when the plasmid coding for full-length P1A was delivered as compared to the plasmid encoding the P1A(35-43) epitope. Our results demonstrated that electroporation is an efficient method to deliver DNA vaccines against P815 and suggested the superiority of full-length as compared to minigene constructs for DNA vaccines.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. Effects of different replicons in conjugative plasmids on transformation efficiency, plasmid stability, gene expression and n-butanol biosynthesis in Clostridium tyrobutyricum.

    Science.gov (United States)

    Yu, Mingrui; Du, Yinming; Jiang, Wenyan; Chang, Wei-Lun; Yang, Shang-Tian; Tang, I-Ching

    2012-01-01

    Clostridium tyrobutyricum ATCC 25755 can produce butyric acid, acetic acid, and hydrogen as the main products from various carbon sources. In this study, C. tyrobutyricum was used as a host to produce n-butanol by expressing adhE2 gene under the control of a native thiolase promoter using four different conjugative plasmids (pMTL82151, 83151, 84151, and 85151) each with a different replicon (pBP1 from C. botulinum NCTC2916, pCB102 from C. butyricum, pCD6 from Clostridium difficile, and pIM13 from Bacillus subtilis). The effects of different replicons on transformation efficiency, plasmid stability, adhE2 expression and aldehyde/alcohol dehydrogenase activities, and butanol production by different mutants of C. tyrobutyricum were investigated. Among the four plasmids and replicons studied, pMTL82151 with pBP1 gave the highest transformation efficiency, plasmid stability, gene expression, and butanol biosynthesis. Butanol production from various substrates, including glucose, xylose, mannose, and mannitol were then investigated with the best mutant strain harboring adhE2 in pMTL82151. A high butanol titer of 20.5 g/L with 0.33 g/g yield and 0.32 g/L h productivity was obtained with mannitol as the substrate in batch fermentation with pH controlled at ~6.0.

  12. Effects of different replicons in conjugative plasmids on transformation efficiency, plasmid stability, gene expression and n-butanol biosynthesis in Clostridium tyrobutyricum

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Mingrui; Du, Yinming; Jiang, Wenyan; Chang, Wei-Lun; Yang, Shang-Tian [Ohio State Univ., Columbus, OH (United States). William G. Lowrie Dept. of Chemical and Biomolecular Engineering; Tang, I-Ching [Bioprocessing Innovative Company, Dublin, OH (United States)

    2012-01-15

    Clostridium tyrobutyricum ATCC 25755 can produce butyric acid, acetic acid, and hydrogen as the main products from various carbon sources. In this study, C. tyrobutyricum was used as a host to produce n-butanol by expressing adhE2 gene under the control of a native thiolase promoter using four different conjugative plasmids (pMTL82151, 83151, 84151, and 85151) each with a different replicon (pBP1 from C. botulinum NCTC2916, pCB102 from C. butyricum, pCD6 from Clostridium difficile, and pIM13 from Bacillus subtilis). The effects of different replicons on transformation efficiency, plasmid stability, adhE2 expression and aldehyde/alcohol dehydrogenase activities, and butanol production by different mutants of C. tyrobutyricum were investigated. Among the four plasmids and replicons studied, pMTL82151 with pBP1 gave the highest transformation efficiency, plasmid stability, gene expression, and butanol biosynthesis. Butanol production from various substrates, including glucose, xylose, mannose, and mannitol were then investigated with the best mutant strain harboring adhE2 in pMTL82151. A high butanol titer of 20.5 g/L with 0.33 g/g yield and 0.32 g/L h productivity was obtained with mannitol as the substrate in batch fermentation with pH controlled at {proportional_to}6.0. (orig.)

  13. Characterization of pPCP1 Plasmids in Yersinia pestis Strains Isolated from the Former Soviet Union

    Directory of Open Access Journals (Sweden)

    Chythanya Rajanna

    2010-01-01

    Full Text Available Complete sequences of 9.5-kb pPCP1 plasmids in three Yersinia pestis strains from the former Soviet Union (FSU were determined and compared with those of pPCP1 plasmids in three well-characterized, non-FSU Y. pestis strains (KIM, CO92, and 91001. Two of the FSU plasmids were from strains C2614 and C2944, isolated from plague foci in Russia, and one plasmid was from strain C790 from Kyrgyzstan. Sequence analyses identified four sequence types among the six plasmids. The pPCP1 plasmids in the FSU strains were most genetically related to the pPCP1 plasmid in the KIM strain and least related to the pPCP1 plasmid in Y. pestis 91001. The FSU strains generally had larger pPCP1 plasmid copy numbers compared to strain CO92. Expression of the plasmid's pla gene was significantly (P≤.05 higher in strain C2944 than in strain CO92. Given pla's role in Y. pestis virulence, this difference may have important implications for the strain's virulence.

  14. 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.

  15. 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.

  16. 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.

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

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    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.

  18. ColE1-Plasmid Production in Escherichia coli: Mathematical Simulation and Experimental Validation

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    Freudenau, Inga; Lutter, Petra; Baier, Ruth; Schleef, Martin; Bednarz, Hanna; Lara, Alvaro R.; Niehaus, Karsten

    2015-01-01

    Plasmids have become very important as pharmaceutical gene vectors in the fields of gene therapy and genetic vaccination in the past years. In this study, we present a dynamic model to simulate the ColE1-like plasmid replication control, once for a DH5α-strain carrying a low copy plasmid (DH5α-pSUP 201-3) and once for a DH5α-strain carrying a high copy plasmid (DH5α-pCMV-lacZ) by using ordinary differential equations and the MATLAB software. The model includes the plasmid replication control by two regulatory RNA molecules (RNAI and RNAII) as well as the replication control by uncharged tRNA molecules. To validate the model, experimental data like RNAI- and RNAII concentration, plasmid copy number (PCN), and growth rate for three different time points in the exponential phase were determined. Depending on the sampled time point, the measured RNAI- and RNAII concentrations for DH5α-pSUP 201-3 reside between 6 ± 0.7 and 34 ± 7 RNAI molecules per cell and 0.44 ± 0.1 and 3 ± 0.9 RNAII molecules per cell. The determined PCNs averaged between 46 ± 26 and 48 ± 30 plasmids per cell. The experimentally determined data for DH5α-pCMV-lacZ reside between 345 ± 203 and 1086 ± 298 RNAI molecules per cell and 22 ± 2 and 75 ± 10 RNAII molecules per cell with an averaged PCN of 1514 ± 1301 and 5806 ± 4828 depending on the measured time point. As the model was shown to be consistent with the experimentally determined data, measured at three different time points within the growth of the same strain, we performed predictive simulations concerning the effect of uncharged tRNA molecules on the ColE1-like plasmid replication control. The hypothesis is that these tRNA molecules would have an enhancing effect on the plasmid production. The in silico analysis predicts that uncharged tRNA molecules would indeed increase the plasmid DNA production. PMID:26389114

  19. ColE1-plasmid production in Escherichia coli: Mathematical Simulation and Experimental Validation

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    Inga eFreudenau

    2015-09-01

    Full Text Available Plasmids have become very important as pharmaceutical gene vectors in the fields of gene therapy and genetic vaccination in the last years. In this study, we present a dynamic model to simulate the ColE1-like plasmid replication control, once for a DH5α-strain carrying a low copy plasmid (DH5α-pSUP 201-3 and once for a DH5α-strain carrying a high copy plasmid (DH5α-pCMV-lacZ by using ordinary differential equations (ODE and the MATLAB software. The model includes the plasmid replication control by two regulatory RNA molecules (RNAI and RNAII as well as the replication control by uncharged tRNA molecules. To validate the model, experimental data like RNAI- and RNAII concentration, plasmid copy number (PCN, and growth rate for three different time points in the exponential phase were determined. Depending on the sampled time point, the measured RNAI and RNAII concentrations for DH5α-pSUP 201-3 reside between 6 ±0.7 to 34 ±7 RNAI molecules per cell and 0.44 ±0.1 to 3 ±0.9 RNAII molecules per cell. The determined plasmid copy numbers (PCN averaged between 46 ±26 to 48 ±30 plasmids per cell. The experimentally determined data for DH5α-pCMV-lacZ reside between 345 ±203 to 1086 ±298 RNAI molecules per cell and 22 ±2 to 75 ±10 RNAII molecules per cell with an averaged PCN of 1514 ±1301 to 5806 ±4828 depending on the measured time point. As the model was shown to be consistent with the experimentally determined data, measured at three different time points within the growth of the same strain, we performed predictive simulations concerning the effect of uncharged tRNA molecules on the ColE1-like plasmid replication control. The hypothesis is that these tRNA molecules would have an enhancing effect on the plasmid production. The in silico analysis predicts that uncharged tRNA molecules would indeed increase the pDNA production.

  20. An Enterobacter plasmid as a new genetic background for the transposon Tn1331

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    Alavi MR

    2011-11-01

    Full Text Available Mohammad R Alavi1,2, Vlado Antonic2, Adrien Ravizee1, Peter J Weina3, Mina Izadjoo1,2, Alexander Stojadinovic21Division of Wound Biology and Translational Research, Armed Forces Institute of Pathology and American Registry of Pathology, Washington DC, 2Combat Wound Initiative Program, Walter Reed Army Medical Center, Washington DC, 3The Walter Reed Army Institute of Research, Silver Spring, MD, USABackground: Genus Enterobacter includes important opportunistic nosocomial pathogens that could infect complex wounds. The presence of antibiotic resistance genes in these microorganisms represents a challenging clinical problem in the treatment of these wounds. In the authors’ screening of antibiotic-resistant bacteria from complex wounds, an Enterobacter species was isolated that harbors antibiotic-resistant plasmids conferring resistance to Escherichia coli. The aim of this study was to identify the resistance genes carried by one of these plasmids.Methods: The plasmids from the Enterobacter isolate were propagated in E. coli and one of the plasmids, designated as pR23, was sequenced by the Sanger method using fluorescent dye-terminator chemistry on a genetic analyzer. The assembled sequence was annotated by search of the GenBank database.Results: Plasmid pR23 is composed of the transposon Tn1331 and a backbone plasmid that is identical to the plasmid pPIGDM1 from Enterobacter agglomerans. The multidrug-resistance transposon Tn1331, which confers resistance to aminoglycoside and beta lactam antibiotics, has been previously isolated only from Klebsiella. The Enterobacter plasmid pPIGDM1, which carries a ColE1-like origin of replication and has no apparent selective marker, appears to provide a backbone for propagation of Tn1331 in Enterobacter. The recognition sequence of Tn1331 transposase for insertion into pPIGDM1 is the pentanucleotide TATTA, which occurs only once throughout the length of this plasmid.Conclusion: Transposition of Tn1331 into

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

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    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

  2. 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.

  3. 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.

  4. Increasing plasmid transformation efficiency of natural spizizen method in Bacillus Subtilis by a cell permeable peptide

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    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.

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

    Science.gov (United States)

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

    2009-03-18

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

  6. Molecular Diversity and Plasmid Analysis of KPC-Producing Escherichia coli.

    Science.gov (United States)

    Chavda, Kalyan D; Chen, Liang; Jacobs, Michael R; Bonomo, Robert A; Kreiswirth, Barry N

    2016-07-01

    The emergence and spread of Klebsiella pneumoniae carbapenemase (KPC) among Enterobacteriaceae presents a major public health threat to the world. Although not as common as in K. pneumoniae, KPC is also found in Escherichia coli strains. Here, we genetically characterized 9 carbapenem-resistant E. coli strains isolated from six hospitals in the United States and completely sequenced their blaKPC-harboring plasmids. The nine strains were isolated from different geographical locations and belonged to 8 different E. coli sequence types. Seven blaKPC-harboring plasmids belonged to four different known incompatibility groups (IncN, -FIA, -FIIK2, and -FIIK1) and ranged in size from ∼16 kb to ∼241 kb. In this analysis, we also identified two plasmids that have novel replicons: (i) pBK28610, which is similar to p34978-3 with an insertion of Tn4401b, and (ii) pBK31611, which does not have an apparent homologue in the GenBank database. Moreover, we report the emergence of a pKP048-like plasmid, pBK34397, in E. coli in the United States. Meanwhile, we also found examples of interspecies spread of blaKPC plasmids, as pBK34592 is identical to pBK30683, isolated from K. pneumoniae In addition, we discovered examples of acquisition (pBK32602 acquired an ∼46-kb fragment including a novel replication gene, along with Tn4401b and other resistance genes) and/or loss (pKpQIL-Ec has a 14.5-kb deletion compared to pKpQIL-10 and pBK33689) of DNA, demonstrating the plasticity of these plasmids and their rapid evolution in the clinic. Overall, our study shows that the spread of blaKPC-producing E. coli is largely due to horizontal transfer of blaKPC-harboring plasmids and related mobile elements into diverse genetic backgrounds.

  7. 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.

  8. Effects of a recombinant gene expression on ColE1-like plasmid segregation in Escherichia coli

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    Ivanov Ivan

    2011-03-01

    Full Text Available Abstract Background Segregation of expression plasmids leads to loss of recombinant DNA from transformed bacterial cells due to the irregular distribution of plasmids between the daughter cells during cell division. Under non-selective conditions this segregational instability results in a heterogeneous population of cells, where the non-productive plasmid-free cells overgrow the plasmid-bearing cells thus decreasing the yield of recombinant protein. Amongst the factors affecting segregational plasmid instability are: the plasmid design, plasmid copy-number, host cell genotype, fermentation conditions etc. This study aims to investigate the influence of transcription and translation on the segregation of recombinant plasmids designed for constitutive gene expression in Escherichia coli LE392 at glucose-limited continuous cultivation. To this end a series of pBR322-based plasmids carrying a synthetic human interferon-gamma (hIFNγ gene placed under the control of different regulatory elements (promoter and ribosome-binding sites were used as a model. Results Bacterial growth and product formation kinetics of transformed E. coli LE392 cells cultivated continuously were described by a structured kinetic model proposed by Lee et al. (1985. The obtained results demonstrated that both transcription and translation efficiency strongly affected plasmid segregation. The segregation of plasmid having a deleted promoter did not exceed 5% after 190 h of cultivation. The observed high plasmid stability was not related with an increase in the plasmid copy-number. A reverse correlation between the yield of recombinant protein (as modulated by using different ribosome binding sites and segregational plasmid stability (determined by the above model was also observed. Conclusions Switching-off transcription of the hIFNγ gene has a stabilising effect on ColE1-like plasmids against segregation, which is not associated with an increase in the plasmid copy

  9. 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.

  10. Effects of the presence of ColE1 plasmid DNA in Escherichia coli on the host cell metabolism

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    Węgrzyn Alicja

    2006-11-01

    Full Text Available Abstract Background Although understanding of physiological interactions between plasmid DNA and its host is important for vector design and host optimization in many biotechnological applications, to our knowledge, global studies on plasmid-host interactions have not been performed to date even for well-characterized plasmids. Results Escherichia coli cells, either devoid of plasmid DNA or bearing plasmid pOri1 (with a single ColE1 replication origin or plasmid pOri2 (with double ColE1 replication origins, were cultured in a chemostat. We used a combination of metabolic flux analysis, DNA microarray and enzyme activity analysis methods to explore differences in the metabolism between these strains. We found that the presence of plasmids significantly influenced various metabolic pathways in the host cells, e.g. glycolysis, the tricarboxylic acid (TCA cycle and the pentose phosphate (PP pathway. Expression of rpiA, a gene coding for ribose-5-phosphate isomerase A, was considerably decreased in E. coli carrying a high copy number plasmid relative to E. coli carrying a low copy number plasmid and plasmid-free E. coli. The rpiA gene was cloned into an expression vector to construct plasmid pETrpiA. Following induction of pETrpiA-bearing E. coli, which harbored either pOri1 or pOri2, with isopropyl-β-D-thiogalactopyranoside (IPTG, the copy number of pOri1 and pOri2 was sigificantly higher than that measured in a host devoid of pETrpiA. Conclusion The presence of plasmids can significantly influence some metabolic pathways in the host cell. We believe that the results of detailed metabolic analysis may be useful in optimizing host strains, vectors and cultivation conditions for various biotechnological purposes.

  11. 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.

  12. In situ NIR spectroscopy monitoring of plasmid production processes effect of producing strain, medium composition and the cultivation strategy

    OpenAIRE

    Lopes, Marta B.; Gonçalves, Geisa A. L.; Felício-Silva, Daniel; Prather, Kristala L. J.; Monteiro, Gabriel; Prazeres, Duarte M. F.; Calado, Cecília Ribeiro da Cruz

    2015-01-01

    BACKGROUNDWhile the pharmaceutical industry keeps an eye on plasmid DNA production for new generation gene therapies, real-time monitoring techniques for plasmid bioproduction are as yet unavailable. This work shows the possibility of in situ monitoring of plasmid production in Escherichia coli cultures using a near infrared (NIR) fiber optic probe. RESULTSPartial least squares (PLS) regression models based on the NIR spectra were developed for predicting bioprocess critical variables su...

  13. Origin-of-transfer sequences facilitate mobilisation of non-conjugative antimicrobial-resistance plasmids in Staphylococcus aureus

    Science.gov (United States)

    O'Brien, Frances G.; Yui Eto, Karina; Murphy, Riley J. T.; Fairhurst, Heather M.; Coombs, Geoffrey W.; Grubb, Warren B.; Ramsay, Joshua P.

    2015-01-01

    Staphylococcus aureus is a common cause of hospital, community and livestock-associated infections and is increasingly resistant to multiple antimicrobials. A significant proportion of antimicrobial-resistance genes are plasmid-borne, but only a minority of S. aureus plasmids encode proteins required for conjugative transfer or Mob relaxase proteins required for mobilisation. The pWBG749 family of S. aureus conjugative plasmids can facilitate the horizontal transfer of diverse antimicrobial-resistance plasmids that lack Mob genes. Here we reveal that these mobilisable plasmids carry copies of the pWBG749 origin-of-transfer (oriT) sequence and that these oriT sequences facilitate mobilisation by pWBG749. Sequences resembling the pWBG749 oriT were identified on half of all sequenced S. aureus plasmids, including the most prevalent large antimicrobial-resistance/virulence-gene plasmids, pIB485, pMW2 and pUSA300HOUMR. oriT sequences formed five subfamilies with distinct inverted-repeat-2 (IR2) sequences. pWBG749-family plasmids encoding each IR2 were identified and pWBG749 mobilisation was found to be specific for plasmids carrying matching IR2 sequences. Specificity of mobilisation was conferred by a putative ribbon-helix-helix-protein gene smpO. Several plasmids carried 2–3 oriT variants and pWBG749-mediated recombination occurred between distinct oriT sites during mobilisation. These observations suggest this relaxase-in trans mechanism of mobilisation by pWBG749-family plasmids is a common mechanism of plasmid dissemination in S. aureus. PMID:26243776

  14. Prevalence of plasmid-mediated quinolone resistance determinants in Enterobacteriaceae strains isolated in North-East Italy.

    Science.gov (United States)

    Kocsis, B; Mazzariol, A; Kocsis, E; Koncan, R; Fontana, R; Cornaglia, G

    2013-02-01

    We investigated the prevalence of plasmid-mediated quinolone resistance genes in 756 clinical isolates of Enterobacteriaceae originating from Microbiology Diagnostic Laboratories of North-East Italy. Five point zero two percent of isolates carried a qnr determinant while the aac(6')-Ib-cr determinant was detected in 9·25% of isolates. We also investigated the association between the plasmid-mediated quinolone resistance and the beta-lactamase genes, and characterized the plasmids carrying these determinants of resistance.

  15. Colonization of the respiratory tract by a virulent strain of avian Escherichia coli requires carriage of a conjugative plasmid.

    Science.gov (United States)

    Ginns, C A; Benham, M L; Adams, L M; Whithear, K G; Bettelheim, K A; Crabb, B S; Browning, G F

    2000-03-01

    The E3 strain of E. coli was isolated in an outbreak of respiratory disease in broiler chickens, and experimental aerosol exposure of chickens to this strain induced disease similar to that seen in the field. In order to establish whether the virulent phenotype of this strain was associated with carriage of particular plasmids, four plasmid-cured derivatives, each lacking two or more of the plasmids carried by the wild-type strain, were assessed for virulence. Virulence was found to be associated with one large plasmid, pVM01. Plasmid pVM01 was marked by introduction of the transposon TnphoA, carrying kanamycin resistance, and was then cloned by transformation of E. coli strain DH5alpha. The cloned plasmid was then reintroduced by conjugation into an avirulent plasmid-cured derivative of strain E3 which lacked pVM01. The conjugant was shown to be as virulent as the wild-type strain E3, establishing that this plasmid is required for virulence following aerosol exposure. This virulence plasmid conferred expression of a hydroxamate siderophore, but not colicins, on both strain E3 and strain DH5alpha. Carriage of this plasmid was required for strain E3 to colonize the respiratory tracts of chickens but was not necessary for colonization of the gastrointestinal tract. However, the virulence plasmid did not confer virulence, or the capacity to colonize the respiratory tract, on strain DH5alpha. Thus, these studies have established that infection of chickens with E. coli strain E3 by the respiratory route is dependent on carriage of a conjugative virulence plasmid, which confers the capacity to colonize specifically the respiratory tract and which also carries genes for expression of a hydroxymate siderophore. These findings will facilitate identification of the specific genes required for virulence in these pathogens.

  16. Plasmid profiling and antibiotics resisitance of Escherichia coli strains isolated from Mytilus galloprovincialis and seawater

    Directory of Open Access Journals (Sweden)

    Cumhur Avşar

    2014-09-01

    Full Text Available Objective: To investigate plasmid DNA profiles and the antibiotic resistance of a total of 41 strains of Escherichia coli (E. coli isolated from seawater and mussel collected from 15 different sampling stations in Sinop, Turkey. Methods: Most probable number technique was used for detection of E. coli. Antibiotic susceptibilities of the isolates were determined by the disc diffusion method. Plasmid DNA of the strains was extracted by the alkaline lyses procedure. Results: According to morphological and physiological properties, it was determined that the isolates belonged to E. coli species. Antibiotic susceptibility of the strains was determined against seven standard drugs using disc diffusion method. All isolates were resistant to bacitracin (100%, novobiocin (100%, ampicillin (12.5%, tetracycline (7.5%, ceftazidime (5% and imipenem (2.5%, respectively, whereas the strains were susceptible to polymyxin B (100%. The multiple antibiotic resistance values for the strains were found in range from 0.28 to 0.57. In addition, plasmid DNA analyses results confirmed that 22 strains harbored a single or more than two plasmids sized approximately between 24.500 to 1.618 bp. The high-size plasmid (14.700 bp was observed as common in 21 of all strains. Conclusions: As a result, our study indicated that the presence of antibiotic resistant E. coli strains in seawater and mussel might be potential risk for public health issue.

  17. Protein-Nanocrystal Conjugates Support a Single Filament Polymerization Model in R1 Plasmid Segregation

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Charina L.; Claridge, Shelley A.; Garner, Ethan C.; Alivisatos, A. Paul; Mullins, R. Dyche

    2008-07-15

    To ensure inheritance by daughter cells, many low-copy number bacterial plasmids, including the R1 drug-resistance plasmid, encode their own DNA segregation systems. The par operon of plasmid R1 directs construction of a simple spindle structure that converts free energy of polymerization of an actin-like protein, ParM, into work required to move sister plasmids to opposite poles of rod-shaped cells. The structures of individual components have been solved, but little is known about the ultrastructure of the R1 spindle. To determine the number of ParM filaments in a minimal R1 spindle, we used DNA-gold nanocrystal conjugates as mimics of the R1 plasmid. Wefound that each end of a single polar ParM filament binds to a single ParR/parC-gold complex, consistent with the idea that ParM filaments bind in the hollow core of the ParR/parC ring complex. Our results further suggest that multifilament spindles observed in vivo are associated with clusters of plasmidssegregating as a unit.

  18. 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.

  19. 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.

  20. Identification of putative DnaN-binding motifs in plasmid replication initiation proteins.

    Science.gov (United States)

    Dalrymple, Brian P; Kongsuwan, Kritaya; Wijffels, Gene

    2007-01-01

    Recently the plasmid RK2 replication initiation protein, TrfA, has been shown to bind to the beta subunit of DNA Polymerase III (DnaN) via a short pentapeptide with the consensus QL[S/D]LF. A second consensus peptide, the hexapeptide QLxLxL, has also been demonstrated to mediate binding to DnaN. Here we describe the results of a comprehensive survey of replication initiation proteins encoded by bacterial plasmids to identify putative DnaN-binding sites. Both pentapeptide and hexapeptide motifs have been identified in a number of families of replication initiation proteins. The distribution of sites is sporadic and closely related families of proteins may differ in the presence, location, or type of putative DnaN-binding motif. Neither motif has been identified in replication initiation proteins encoded by plasmids that replicate via rolling circles or strand displacement. The results suggest that the recruitment of DnaN to the origin of replication of a replisome by plasmid replication initiation proteins is not generally required for plasmid replication, but that in some cases it may be beneficial for efficiency of replication initiation.

  1. Optimization of a plasmid electroporation protocol for Aeromonas salmonicida subsp. salmonicida.

    Science.gov (United States)

    Dallaire-Dufresne, Stéphanie; Emond-Rheault, Jean-Guillaume; Attéré, Sabrina A; Tanaka, Katherine H; Trudel, Mélanie V; Frenette, Michel; Charette, Steve J

    2014-03-01

    Aeromonas salmonicida subsp. salmonicida is a major fish pathogen. Molecular tools are required to study the virulence and genomic stability of this bacterium. An efficient electroporation-mediated transformation protocol for A. salmonicida subsp. salmonicida would make genetic studies faster and easier. In the present study, we designed the 4.1-kb pSDD1 plasmid as a tool for optimizing an electroporation protocol for A. salmonicida subsp. salmonicida. We systematically tested the electroporation conditions to develop a protocol that generates the maximum number of transformants. Under these optimal conditions (25 kV/cm, 200 Ω, 25 μF), we achieved an electroporation efficiency of up to 1×10(5) CFU/μg DNA. The electroporation protocol was also tested using another plasmid of 10.6-kb and three different strains of A. salmonicida subsp. salmonicida. The strains displayed significant differences in their electro-transformation competencies. Strain 01-B526 was the easiest to electroporate, especially with the pSDD1 plasmid. This plasmid was stably maintained in the 01-B526 transformants, as were the native plasmids, but could be easily cured by removing the selection conditions. This is the first efficient electroporation protocol reported for A. salmonicida subsp. salmonicida, and offers new possibilities for studying this bacterium.

  2. Incidence of plasmid and antibiotic resistance in psychrotrophic bacteria isolated from Antarctic sponges

    Directory of Open Access Journals (Sweden)

    Vivia Bruni

    2011-04-01

    Full Text Available A total of 297 bacterial strains were isolated from five Antarctic sponge species and tested by agarose gel electrophoresis for the presence of plasmid molecules. At least one kind of plasmid was carried by 69 isolates (about 23%. The disc diffusion susceptibility test was used to assay the resistance of plasmid-harbouring bacteria towards 11 antibiotics. A multiple resistance was observed for the 72% of strains, among which the 33% were resistant to only two antibiotics. Bacteria showed a high degree of resistance towards O/129 (71%, tetracycline (42% and nalidixic acid (25%, whereas any isolate showed resistance to gentamicin. The 16S rDNA sequencing revealed that plasmid-harbouring strains were mainly affiliated to the Gammaproteobacteria (81%, whereas the other detected phylogenetic groups (i.e. Firmicutes, Alphaproteobacteria, Actinobacteria and CFB group of Bacteroidetes were less abundant, each representing between 1% and 6% of the total isolates. The present study will contribute to the poor and fragmentary knowledge on plasmid incidence in natural microbial populations. In addition, monitoring antibiotic resistance in bacteria from remote areas, such as Antarctica, could also be a useful tool to evaluate the impact of anthropic pressure.

  3. 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.

  4. 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.

  5. Plasmid selection in Escherichia coli using an endogenous essential gene marker

    Directory of Open Access Journals (Sweden)

    Good Liam

    2008-08-01

    Full Text Available Abstract Background Antibiotic resistance genes are widely used for selection of recombinant bacteria, but their use risks contributing to the spread of antibiotic resistance. In particular, the practice is inappropriate for some intrinsically resistant bacteria and in vaccine production, and costly for industrial scale production. Non-antibiotic systems are available, but require mutant host strains, defined media or expensive reagents. An unexplored concept is over-expression of a host essential gene to enable selection in the presence of a chemical inhibitor of the gene product. To test this idea in E. coli, we used the growth essential target gene fabI as the plasmid-borne marker and the biocide triclosan as the selective agent. Results The new cloning vector, pFab, enabled selection by triclosan at 1 μM. Interestingly, pFab out-performed the parent pUC19-ampicillin system in cell growth, plasmid stability and plasmid yield. Also, pFab was toxic to host cells in a way that was reversed by triclosan. Therefore, pFab and triclosan are toxic when used alone but in combination they enhance growth and plasmid production through a gene-inhibitor interaction. Conclusion The fabI-triclosan model system provides an alternative plasmid selection method based on essential gene over-expression, without the use of antibiotic-resistance genes and conventional antibiotics.

  6. 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.

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

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    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.

  8. Temperature Depended Role of Shigella flexneri Invasion Plasmid on the Interaction with Acanthamoeba castellanii

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    Amir Saeed

    2012-01-01

    Full Text Available Shigella flexneri is a Gram-negative bacterium causing the diarrhoeal disease shigellosis in humans. The virulence genes required for invasion are clustered on a large 220 kb plasmid encoding type three secretion system (TTSS apparatus and virulence factors such as adhesions and invasion plasmid antigens (Ipa. The bacterium is transmitted by contaminated food, water, or from person to person. Acanthamoebae are free-living amoebae (FLA which are found in diverse environments and isolated from various water sources. Different bacteria interact differently with FLA since Francisella tularensis, Vibrio cholerae, Shigella sonnei, and S. dysenteriae are able to grow inside A. castellanii. In contrast, Pseudomonas aeruginosa induces both necrosis and apoptosis to kill A. castellanii. The aim of this study is to examine the role of invasion plasmid of S. flexneri on the interaction with A. castellanii at two different temperatures. A. castellanii in the absence or presence of wild type, IpaB mutant, or plasmid-cured strain S. flexneri was cultured at 30∘C and 37∘C and the interaction was analysed by viable count of both bacteria and amoebae, electron microscopy, flow cytometry, and statistical analysis. The outcome of the interaction was depended on the temperature since the growth of A. castellanii was inhibited at 30∘C, and A. castellanii was killed by invasion plasmid mediated necrosis at 37∘C.

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

    Science.gov (United States)

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

    2013-06-01

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

  10. The pPSU Plasmids for Generating DNA Molecular Weight Markers.

    Science.gov (United States)

    Henrici, Ryan C; Pecen, Turner J; Johnston, James L; Tan, Song

    2017-05-26

    Visualizing nucleic acids by gel electrophoresis is one of the most common techniques in molecular biology, and reference molecular weight markers or ladders are commonly used for size estimation. We have created the pPSU1 & pPSU2 pair of molecular weight marker plasmids which produce both 100 bp and 1 kb DNA ladders when digested with two common restriction enzymes. The 100 bp ladder fragments have been optimized to migrate appropriately on both agarose and native polyacrylamide, unlike many currently available DNA ladders. Sufficient plasmid DNA can be isolated from 100 ml E. coli cultures for the two plasmids to produce 100 bp or 1 kb ladders for 1000 gels. As such, the pPSU1 and pPSU2 plasmids provide reference fragments from 50 to 10000 bp at a fraction of the cost of commercial DNA ladders. The pPSU1 and pPSU2 plasmids are available without licensing restrictions to nonprofit academic users, affording freely available high-quality, low-cost molecular weight standards for molecular biology applications.

  11. 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.

  12. Isolation and plasmid characterization of carbapenemase (IMP-4) producing Salmonella enterica Typhimurium from cats

    Science.gov (United States)

    Abraham, Sam; O’Dea, Mark; Trott, Darren J.; Abraham, Rebecca J.; Hughes, David; Pang, Stanley; McKew, Genevieve; Cheong, Elaine Y. L.; Merlino, John; Saputra, Sugiyono; Malik, Richard; Gottlieb, Thomas

    2016-01-01

    Carbapenem-resistant Enterobacteriaceae (CRE) are a pressing public health issue due to limited therapeutic options to treat such infections. CREs have been predominantly isolated from humans and environmental samples and they are rarely reported among companion animals. In this study we report on the isolation and plasmid characterization of carbapenemase (IMP-4) producing Salmonella enterica Typhimurium from a companion animal. Carbapenemase-producing S. enterica Typhimurium carrying blaIMP-4 was identified from a systemically unwell (index) cat and three additional cats at an animal shelter. All isolates were identical and belonged to ST19. Genome sequencing revealed the acquisition of a multidrug-resistant IncHI2 plasmid (pIMP4-SEM1) that encoded resistance to nine antimicrobial classes including carbapenems and carried the blaIMP-4-qacG-aacA4-catB3 cassette array. The plasmid also encoded resistance to arsenic (MIC-150 mM). Comparative analysis revealed that the plasmid pIMP4-SEM1 showed greatest similarity to two blaIMP-8 carrying IncHI2 plasmids from Enterobacter spp. isolated from humans in China. This is the first report of CRE carrying a blaIMP-4 gene causing a clinical infection in a companion animal, with presumed nosocomial spread. This study illustrates the broader community risk entailed in escalating CRE transmission within a zoonotic species such as Salmonella, and in a cycle that encompasses humans, animals and the environment. PMID:27767038

  13. Chromosomal context and replication properties of ARS plasmids in Schizosaccharomyces pombe

    Indian Academy of Sciences (India)

    Aditya S Pratihar; Vishnu P Tripathi; Mukesh P Yadav; Dharani D Dubey

    2015-12-01

    Short, specific DNA sequences called as Autonomously Replicating Sequence (ARS) elements function as plasmid as well as chromosomal replication origins in yeasts. As compared to ARSs, different chromosomal origins vary greatly in their efficiency and timing of replication probably due to their wider chromosomal context. The two Schizosaccharomyces pombe ARS elements, ars727 and ars2OO4, represent two extremities in their chromosomal origin activity - ars727 is inactive and late replicating, while ars2OO4 is a highly active, early-firing origin. To determine the effect of chromosomal context on the activity of these ARS elements, we have cloned them with their extended chromosomal context as well as in the context of each other in both orientations and analysed their replication efficiency by ARS and plasmid stability assays. We found that these ARS elements retain their origin activity in their extended/altered context. However, deletion of a 133-bp region of the previously reported ars727-associated late replication enforcing element (LRE) caused advancement in replication timing of the resulting plasmid. These results confirm the role of LRE in directing plasmid replication timing and suggest that the plasmid origin efficiency of ars2OO4 or ars727 remains unaltered by the extended chromosomal context.

  14. Plasmid-Mediated Dimethoate Degradation by Bacillus licheniformis Isolated From a Fresh Water Fish Labeo rohita

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    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.

  15. 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

  16. Biofilm models for the food industry: hot spots for plasmid transfer?

    Science.gov (United States)

    Van Meervenne, Eva; De Weirdt, Rosemarie; Van Coillie, Els; Devlieghere, Frank; Herman, Lieve; Boon, Nico

    2014-04-01

    Biofilms represent a substantial problem in the food industry, with food spoilage, equipment failure, and public health aspects to consider. Besides, biofilms may be a hot spot for plasmid transfer, by which antibiotic resistance can be disseminated to potential foodborne pathogens. This study investigated biomass and plasmid transfer in dual-species (Pseudomonas putida and Escherichia coli) biofilm models relevant to the food industry. Two different configurations (flow-through and drip-flow) and two different inoculation procedures (donor-recipient and recipient-donor) were tested. The drip-flow configuration integrated stainless steel coupons in the setup while the flow-through configuration included a glass flow cell and silicone tubing. The highest biomass density [10 log (cells cm-²)] was obtained in the silicone tubing when first the recipient strain was inoculated. High plasmid transfer ratios, up to 1/10 (transconjugants/total bacteria), were found. Depending on the order of inoculation, a difference in transfer efficiency between the biofilm models could be found. The ease by which the multiresistance plasmid was transferred highlights the importance of biofilms in the food industry as hot spots for the acquisition of multiresistance plasmids. This can impede the treatment of foodborne illnesses if pathogens acquire this multiresistance in or from the biofilm.

  17. Nanofabrication and characterization of PVA-organofiller/Ag nanocoatings on pMAD plasmids

    Science.gov (United States)

    Erdonmez, D.; Mosayyebi, S.; Erkan, K.; Salimi, K.; Nagizade, N.; Saglam, N.; Rzayev, Z. M. O.

    2014-11-01

    Nowadays, the most important problem in microbial researches is bacterial resistance which is carried out by DNA plasmids against antibacterial agents. The effect of antibacterial nanoparticles on bacteria is remarkable, but studies on the interactions of these particles with plasmids do not search or there are no adequate studies. We proposed that the nanoparticles, which are disrupted the self-assembled structure of plasmids, may decrease the resistance of bacteria, and therefore, increase the activity of utilized antibacterial agents. In this work, we synthesized polymer nanofiber webs samples by electrospinning technique from pure water solution of nanocomposites with different contents of silver nanoparticles, and surface morphology of nanofibers composites were characterized by SEM microscopy. Their interactions with pMAD DNA plasmids were investigated. It was demonstrated that the synthesized Ag-carrying nanohybrid composites with higher surface contacted areas were significantly inhibited the activity of plasmid DNA against bacterial resistance. Agreeing with obtained results, synthesized nanofiber coatings can be recommended for the widely applications in nanobiotechnology, nanomedicine, and bioengineering processing.

  18. 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.

  19. Molecular mechanism of immune response induced by foreign plasmid DNA after oral administration in mice

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To study immune response induced by foreign plasmid DNA after oral administration in mice.METHODS: Mice were orally administered with 200 μg of plasmid pcDNA3 once and spleen was isolated 4 h and 18 h after administration. Total RNA was extracted from spleen and gene expression profile of BALB/c mice spleen was analyzed by using Affymetrix oligonucleotide GeneChip. Functional cluster analysis was conducted by GenMAPP software.RESULTS: At 4 h and 18 h after oral plasmid pcDNA3 administration, a number of immune-related genes,including cytokine and cytokine receptors, chemokines and chemokine receptor, complement molecule,proteasome, histocompatibility molecule, lymphocyte antigen complex and apoptotic genes, were up-regulated. Moreover, MAPPFinder results also showed that numerous immune response processes were up-regulated. In contrast, the immunoglobulin genes were down-regulated.CONCLUSION: Foreign plasmid DNA can modulate the genes expression related to immune system via the gastrointestinal tract, and further analysis of the related immune process may help understand the molecular mechanisms of immune response induced by foreign plasmid via the gastrointestinal tract.

  20. Development and application of a general plasmid reference material for GMO screening.

    Science.gov (United States)

    Wu, Yuhua; Li, Jun; Wang, Yulei; Li, Xiaofei; Li, Yunjing; Zhu, Li; Li, Jun; Wu, Gang

    The use of analytical controls is essential when performing GMO detection through screening tests. Additionally, the presence of taxon-specific sequences is analyzed mostly for quality control during GMO detection. In this study, 11 commonly used genetic elements involving three promoters (P-35S, P-FMV35S and P-NOS), four marker genes (Bar, NPTII, HPT and Pmi), and four terminators (T-NOS, T-35S, T-g7 and T-e9), together with the reference gene fragments from six major crops of maize, soybean, rapeseed, rice, cotton and wheat, were co-integrated into the same single plasmid to construct a general reference plasmid pBI121-Screening. The suitability test of pBI121-Screening plasmid as reference material indicated that the non-target sequence on the pBI121-Screening plasmid did not affect the PCR amplification efficiencies of screening methods and taxon-specific methods. The sensitivity of screening and taxon-specific assays ranged from 5 to 10 copies of pBI121-Screening plasmid, meeting the sensitivity requirement of GMO detection. The construction of pBI121-Screening solves the lack of a general positive control for screening tests, thereby reducing the workload and cost of preparing a plurality of the positive control. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Plasmid Mediated Antibiotic and Heavy Metal Resistance in Bacillus Strains Isolated From Soils in Rize, Turkey

    Directory of Open Access Journals (Sweden)

    Elif SEVİM

    2015-09-01

    Full Text Available Fifteen Bacillus strains which were isolated from soil samples were examined for resistance to 17 different antibiotics (ampicillin, methicillin, erythromycin, norfloxacin, cephalotine, gentamycin, ciprofloxacin, streptomycin, tobramycin, chloramphenicol, trimethoprim-sulfamethoxazole, tetracycline, vancomycin, oxacilin, neomycin, kanamycin and, novabiocin and to 10 different heavy metals (copper, lead, cobalt, chrome, iron, mercury, zinc, nickel, manganese and, cadmium and for the presence of plasmid DNA. A total of eleven strains (67% were resistant to at least one antibiotic. The most common resistance was observed against methicillin and oxacillin. The most resistance strains were found as Bacillus sp. B3 and Bacillus sp. B11. High heavy metal resistance against copper, chromium, zinc, iron and nickel was detected, but mercury and cobalt resistance was not detected, except for 3 strains (B3, B11, and B12 which showed mercury resistance. It has been determined that seven Bacillus strains have plasmids. The isolated plasmids were transformed into the Bacillus subtilis W168 and it was shown that heavy metal and antibiotic resistance determinants were carried on these plasmids. These results showed that there was a correlation between plasmid content and resistance for both antibiotic and heavy metal resistance

  2. Plasmid profiling and antibiotics resisitance of Escherichia coli strains isolated from Mytilus galloprovincialis and seawater

    Institute of Scientific and Technical Information of China (English)

    Cumhur Avşar; İsmet Berber

    2014-01-01

    Objective: To investigate plasmid DNA profiles and the antibiotic resistance of a total of 41 strains of Escherichia coli (E. coli) isolated from seawater and mussel collected from 15 different sampling stations in Sinop, Turkey. Methods: Most probable number technique was used for detection of E. coli. Antibiotic susceptibilities of the isolates were determined by the disc diffusion method. Plasmid DNA of the strains was extracted by the alkaline lyses procedure.Results:According to morphological and physiological properties, it was determined that the isolates belonged to E. coli species. Antibiotic susceptibility of the strains was determined against seven standard drugs using disc diffusion method. All isolates were resistant to bacitracin (100%), novobiocin (100%), ampicillin (12.5%), tetracycline (7.5%), ceftazidime (5%) and imipenem (2.5%), respectively, whereas the strains were susceptible to polymyxin B (100%). The multiple antibiotic resistance values for the strains were found in range from 0.28 to 0.57. In addition, plasmid DNA analyses results confirmed that 22 strains harbored a single or more than two plasmids sized approximately between 24.500 to 1.618 bp. The high-size plasmid (14.700 bp) was observed as common in 21 of all strains.Conclusions:As a result, our study indicated that the presence of antibiotic resistant E. coli strains in seawater and mussel might be potential risk for public health issue.

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

    Science.gov (United States)

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

    2013-07-01

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

  4. 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.

  5. Toxin Kid uncouples DNA replication and cell division to enforce retention of plasmid R1 in Escherichia coli cells.

    Science.gov (United States)

    Pimentel, Belén; Nair, Radhika; Bermejo-Rodríguez, Camino; Preston, Mark A; Agu, Chukwuma A; Wang, Xindan; Bernal, Juan A; Sherratt, David J; de la Cueva-Méndez, Guillermo

    2014-02-18

    Worldwide dissemination of antibiotic resistance in bacteria is facilitated by plasmids that encode postsegregational killing (PSK) systems. These produce a stable toxin (T) and a labile antitoxin (A) conditioning cell survival to plasmid maintenance, because only this ensures neutralization of toxicity. Shortage of antibiotic alternatives and the link of TA pairs to PSK have stimulated the opinion that premature toxin activation could be used to kill these recalcitrant organisms in the clinic. However, validation of TA pairs as therapeutic targets requires unambiguous understanding of their mode of action, consequences for cell viability, and function in plasmids. Conflicting with widespread notions concerning these issues, we had proposed that the TA pair kis-kid (killing suppressor-killing determinant) might function as a plasmid rescue system and not as a PSK system, but this remained to be validated. Here, we aimed to clarify unsettled mechanistic aspects of Kid activation, and of the effects of this for kis-kid-bearing plasmids and their host cells. We confirm that activation of Kid occurs in cells that are about to lose the toxin-encoding plasmid, and we show that this provokes highly selective restriction of protein outputs that inhibits cell division temporarily, avoiding plasmid loss, and stimulates DNA replication, promoting plasmid rescue. Kis and Kid are conserved in plasmids encoding multiple antibiotic resistance genes, including extended spectrum β-lactamases, for which therapeutic options are scarce, and our findings advise against the activation of this TA pair to fight pathogens carrying these extrachromosomal DNAs.

  6. Protein Structure Initiative Material Repository: an open shared public resource of structural genomics plasmids for the biological community

    Science.gov (United States)

    Cormier, Catherine Y.; Mohr, Stephanie E.; Zuo, Dongmei; Hu, Yanhui; Rolfs, Andreas; Kramer, Jason; Taycher, Elena; Kelley, Fontina; Fiacco, Michael; Turnbull, Greggory; LaBaer, Joshua

    2010-01-01

    The Protein Structure Initiative Material Repository (PSI-MR; http://psimr.asu.edu) provides centralized storage and distribution for the protein expression plasmids created by PSI researchers. These plasmids are a resource that allows the research community to dissect the biological function of proteins whose structures have been identified by the PSI. The plasmid annotation, which includes the full length sequence, vector information and associated publications, is stored in a freely available, searchable database called DNASU (http://dnasu.asu.edu). Each PSI plasmid is also linked to a variety of additional resources, which facilitates cross-referencing of a particular plasmid to protein annotations and experimental data. Plasmid samples can be requested directly through the website. We have also developed a novel strategy to avoid the most common concern encountered when distributing plasmids namely, the complexity of material transfer agreement (MTA) processing and the resulting delays this causes. The Expedited Process MTA, in which we created a network of institutions that agree to the terms of transfer in advance of a material request, eliminates these delays. Our hope is that by creating a repository of expression-ready plasmids and expediting the process for receiving these plasmids, we will help accelerate the accessibility and pace of scientific discovery. PMID:19906724

  7. 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.

  8. 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

  9. Immune Responses in Mice Injected with gD Plasmid DNA of Infectious Bovine Rhinotracheitis Virus

    Institute of Scientific and Technical Information of China (English)

    LI Ji-chang; TONG Guang-zhi; QIU Hua-ji

    2004-01-01

    The gene encoding gD of isolate Luojing of infectious bovine rhinotracheitis virus (IBRV)was amplified,sequenced, and cloned into plasmid pcDNA 3.1, resulting in a recombinant pcDNA-gD. Groups of BALB/c mice were injected with 100 μ g of plasmid only or together with liposome. After immunization, serum samples were collected from mice every 2 weeks for a 10-week period and tested for protein-specific antibody with enzyme-linked immunosorbent assay(ELISA). It was showed that the plasmid encoding IBRV glycopretein D developed gene-specific antibody. This report indicates the potential of DNA injection as a method of vaccination.

  10. 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.

  11. 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.

  12. TOL Plasmid Carriage Enhances Biofilm Formation and Increases Extracellular DNA Content in Pseudomonas Putida KT2440

    DEFF Research Database (Denmark)

    Smets, Barth F.; D'Alvise, Paul; Yankelovich, T.;

    of extracellular polymeric substances: TOL carriage leads to more extracellular DNA (eDNA) in pellicles and biofilms. Pellicles were dissolved by DNAse I treatment. eDNA was observed as ominous fibrous structures. Quantitative analysis of live and dead cells in static cultures was performed by flow cytometry......Adherent growth of Pseudomonas putida KT2440 with and without the TOL plasmid (pWWO) at the solid-liquid and air-liquid interface was examined. We compared biofilm formation on glass in flow cells, and assayed pellicle (air-liquid interface biofilm) formation in stagnant liquid cultures by confocal...... combined with specific cytostains; release of cytoplasmic material was assayed by a β-glucosidase assay. Enhanced cell lysis due to plasmid carriage was ruled out as the mechanism for eDNA release. We report, for the first time, that carriage of a conjugative plasmid leads to increased biofilm formation...

  13. First report on vertical transmission of a plasmid DNA in freshwater prawn, Macrobrachium rosenbergii.

    Science.gov (United States)

    Chowdhury, Labrechai Mog; Gireesh-Babu, P; Pavan-Kumar, A; Suresh Babu, P P; Chaudhari, Aparna

    2014-09-01

    Outbreak of WSSV disease is one of the major stumbling blocks in shrimp aquaculture. DNA vaccines have shown potential for mass scale vaccination owing to their stability, cost effectiveness and easy maintenance. Development of economically feasible delivery strategies remains to be a major challenge. This study demonstrates vertical transmission of a plasmid DNA in a decapod Macrobrachium rosenbergii for the first time. Females at three different maturation stages (immature, matured and berried) and mature males were injected with a plasmid DNA and allowed to spawn with untreated counterparts. Using specific primers the plasmid DNA could be amplified from the offspring of all groups except that of berried females. For this confirmation genomic DNA was isolated from 3 pools of 10 post larvae in each group. This presents an ideal strategy to protect young ones at zero stress.

  14. [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.

  15. Kinetic Properties of Four Plasmid-Mediated AmpC β-Lactamases

    Science.gov (United States)

    Bauvois, Cédric; Ibuka, Akiko Shimizu; Celso, Almeida; Alba, Jimena; Ishii, Yoshikazu; Frère, Jean-Marie; Galleni, Moreno

    2005-01-01

    The heterologous production in Escherichia coli, the purification, and the kinetic characterization of four plasmid-encoded class C β-lactamases (ACT-1, MIR-1, CMY-2, and CMY-1) were performed. Except for their instability, these enzymes are very similar to the known chromosomally encoded AmpC β-lactamases. Their kinetic parameters did not show major differences from those obtained for the corresponding chromosomal enzymes. However, the Km values of CMY-2 for cefuroxime, cefotaxime, and oxacillin were significantly decreased compared to those of the chromosomal AmpC enzymes. Finally, the susceptibility patterns of different E. coli hosts producing a plasmid- or a chromosome-encoded class C enzyme toward β-lactam antibiotics are mainly due to the overproduction of the β-lactamase in the periplasmic space of the bacteria rather than to a specific catalytic profile of the plasmid-encoded β-lactamases. PMID:16189104

  16. Bioaugmentation of DDT-contaminated soil by dissemination of the catabolic plasmid pDOD.

    Science.gov (United States)

    Gao, Chunming; Jin, Xiangxiang; Ren, Jingbei; Fang, Hua; Yu, Yunlong

    2015-01-01

    A plasmid transfer-mediated bioaugmentation method for the enhancement of dichlorodiphenyltrichloroethane (DDT) degradation in soil was developed using the catabolic plasmid pDOD from Sphingobacterium sp. D-6. The pDOD plasmid could be transferred to soil bacteria, such as members of Cellulomonas, to form DDT degraders and thus accelerate DDT degradation. The transfer efficiency of pDOD was affected by the donor, temperature, moisture, and soil type. Approximately 50.7% of the DDT in the contaminated field was removed 210 days after the application of Escherichia coli TG I (pDOD-gfp). The results suggested that seeding pDOD into soil is an effective bioaugmentation method for enhancing the degradation of DDT.

  17. Transfer of plasmid-mediated resistance to tetracycline in pathogenic bacteria from fish and aquaculture environments.

    Science.gov (United States)

    Guglielmetti, Elena; Korhonen, Jenni M; Heikkinen, Jouni; Morelli, Lorenzo; von Wright, Atte

    2009-04-01

    The transferability of a large plasmid that harbors a tetracycline resistance gene tet(S), to fish and human pathogens was assessed using electrotransformation and conjugation. The plasmid, originally isolated from fish intestinal Lactococcus lactis ssp. lactis KYA-7, has potent antagonistic activity against the selected recipients (Lactococcus garvieae and Listeria monocytogenes), preventing conjugation. Therefore the tetracycline resistance determinant was transferred via electroporation to L. garvieae. A transformant clone was used as the donor in conjugation experiments with three different L. monocytogenes strains. To our knowledge, this is the first study showing the transfer of an antibiotic resistance plasmid from fish-associated lactic bacteria to L. monocytogenes, even if the donor L. garvieae was not the original host of the tetracycline resistance but experimentally created by electroporation. These results demonstrate that the antibiotic resistance genes in the fish intestinal bacteria have the potential to spread both to fish and human pathogens, posing a risk to aquaculture and consumer safety.

  18. Novobiocin resistance marker in Haemophilus influenzae that is not expressed on a plasmid

    Energy Technology Data Exchange (ETDEWEB)

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

    1982-09-01

    The plasmid pNov2, carrying a cloned chromosomal marker conferring resistance to at least 2.5 ..mu..g of novobiocin per ml, was constructed with a new Haemophilus influenzae cloning vehicle, pDM2. The novobiocin marker of pNov2 was not normally expressed, but in Rec/sup +/ cells approximately one in 10/sup 4/ cells in a culture of a transformant became novobiocin resistant, a frequency about four orders of magnitude higher than the spontaneous mutation frequency. Variants of such cells that had lost the plasmid were also novobiocin resistant. Since Rec/sup -/ cultures bearing pNov2 showed novobiocin resistance only at the normal mutation frequency, the authors concluded that the Rec/sup +/ novobiocin-resistant transformants arose because of a rare recombination between plasmid and chromosome. Evidence is presented that novobiocin sensitivity is dominant over this particular novobiocin resistance marker.

  19. Isolation of a minireplicon of the plasmid pG6303 of Lactobacillus plantarum G63 and characterization of the plasmid-encoded Rep replication protein

    Indian Academy of Sciences (India)

    Jing Fan; Xuedong Xi; Yan Huang; Zhongli Cui

    2015-06-01

    A cryptic 10.0-kb plasmid pG6303 from a multiplasmid-containing Lactobacillus plantarum G63 was studied. The analysis of replicon was facilitated by the construction of shuttle vectors and electrotransformation into L. plantarum. The pG6303 replicon included (i) an open reading frame encoding the putative Rep replication initiation protein; and (ii) the putative origin of replication. The Rep protein was expressed as a fusion with the hexa-histidine (His) at its C-terminal end and purified by Ni-affinity chromatography. The electrophoretic mobility shift assays in pG6303 showed that the purified Rep protein specifically bound from 5582 to 5945 bp, differing from the putative origin of replication of pG6303. We speculate that pG6303 replication is a new mode of plasmid replication.

  20. Defining the Role of ATP Hydrolysis in Mitotic Segregation of Bacterial Plasmids

    Science.gov (United States)

    Ah-Seng, Yoan; Rech, Jérôme; Lane, David; Bouet, Jean-Yves

    2013-01-01

    Hydrolysis of ATP by partition ATPases, although considered a key step in the segregation mechanism that assures stable inheritance of plasmids, is intrinsically very weak. The cognate centromere-binding protein (CBP), together with DNA, stimulates the ATPase to hydrolyse ATP and to undertake the relocation that incites plasmid movement, apparently confirming the need for hydrolysis in partition. However, ATP-binding alone changes ATPase conformation and properties, making it difficult to rigorously distinguish the substrate and cofactor roles of ATP in vivo. We had shown that mutation of arginines R36 and R42 in the F plasmid CBP, SopB, reduces stimulation of SopA-catalyzed ATP hydrolysis without changing SopA-SopB affinity, suggesting the role of hydrolysis could be analyzed using SopA with normal conformational responses to ATP. Here, we report that strongly reducing SopB-mediated stimulation of ATP hydrolysis results in only slight destabilization of mini-F, although the instability, as well as an increase in mini-F clustering, is proportional to the ATPase deficit. Unexpectedly, the reduced stimulation also increased the frequency of SopA relocation over the nucleoid. The increase was due to drastic shortening of the period spent by SopA at nucleoid ends; average speed of migration per se was unchanged. Reduced ATP hydrolysis was also associated with pronounced deviations in positioning of mini-F, though time-averaged positions changed only modestly. Thus, by specifically targeting SopB-stimulated ATP hydrolysis our study reveals that even at levels of ATPase which reduce the efficiency of splitting clusters and the constancy of plasmid positioning, SopB still activates SopA mobility and plasmid positioning, and sustains near wild type levels of plasmid stability. PMID:24367270

  1. 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.

  2. Protection from ischemic heart injury by a vigilant heme oxygenase-1 plasmid system.

    Science.gov (United States)

    Tang, Yao Liang; Tang, Yi; Zhang, Y Clare; Qian, Keping; Shen, Leping; Phillips, M Ian

    2004-04-01

    Although human heme oxygenase-1 (hHO-1) could provide a useful approach for cellular protection in the ischemic heart, constitutive overexpression of hHO-1 may lead to unwanted side effects. To avoid this, we designed a hypoxia-regulated hHO-1 gene therapy system that can be switched on and off. This vigilant plasmid system is composed of myosin light chain-2v promoter and a gene switch that is based on an oxygen-dependent degradation domain from the hypoxia inducible factor-1-alpha. The vector can sense ischemia and switch on the hHO-1 gene system, specifically in the heart. In an in vivo experiment, the vigilant hHO-1 plasmid or saline was injected intramyocardially into myocardial infarction mice or sham operation mice. After gene transfer, expression of hHO-1 was only detected in the ischemic heart treated with vigilant hHO-1 plasmids. Masson trichrome staining showed significantly fewer fibrotic areas in vigilant hHO-1 plasmids-treated mice compared with saline control (43.0%+/-4.8% versus 62.5%+/-3.3%, PhHO-1 expression in peri-infarct border areas, concomitant with higher Bcl-2 levels and lower Bax, Bak, and caspase 3 levels in the ischemic myocardium compared with saline control. By use of a cardiac catheter, heart from vigilant hHO-1 plasmids-treated mice showed improved recovery of contractile and diastolic performance after myocardial infarction compared with saline control. This study documents the beneficial regulation and therapeutic potential of vigilant plasmid-mediated hHO-1 gene transfer. This novel gene transfer strategy can provide cardiac-specific protection from future repeated bouts of ischemic injury.

  3. Defining the role of ATP hydrolysis in mitotic segregation of bacterial plasmids.

    Directory of Open Access Journals (Sweden)

    Yoan Ah-Seng

    Full Text Available Hydrolysis of ATP by partition ATPases, although considered a key step in the segregation mechanism that assures stable inheritance of plasmids, is intrinsically very weak. The cognate centromere-binding protein (CBP, together with DNA, stimulates the ATPase to hydrolyse ATP and to undertake the relocation that incites plasmid movement, apparently confirming the need for hydrolysis in partition. However, ATP-binding alone changes ATPase conformation and properties, making it difficult to rigorously distinguish the substrate and cofactor roles of ATP in vivo. We had shown that mutation of arginines R36 and R42 in the F plasmid CBP, SopB, reduces stimulation of SopA-catalyzed ATP hydrolysis without changing SopA-SopB affinity, suggesting the role of hydrolysis could be analyzed using SopA with normal conformational responses to ATP. Here, we report that strongly reducing SopB-mediated stimulation of ATP hydrolysis results in only slight destabilization of mini-F, although the instability, as well as an increase in mini-F clustering, is proportional to the ATPase deficit. Unexpectedly, the reduced stimulation also increased the frequency of SopA relocation over the nucleoid. The increase was due to drastic shortening of the period spent by SopA at nucleoid ends; average speed of migration per se was unchanged. Reduced ATP hydrolysis was also associated with pronounced deviations in positioning of mini-F, though time-averaged positions changed only modestly. Thus, by specifically targeting SopB-stimulated ATP hydrolysis our study reveals that even at levels of ATPase which reduce the efficiency of splitting clusters and the constancy of plasmid positioning, SopB still activates SopA mobility and plasmid positioning, and sustains near wild type levels of plasmid stability.

  4. A new and improved host-independent plasmid system for RK2-based conjugal transfer.

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    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.

  5. Plasmid metagenomics reveals multiple antibiotic resistance gene classes among the gut microbiomes of hospitalised patients.

    Science.gov (United States)

    Jitwasinkul, Tossawan; Suriyaphol, Prapat; Tangphatsornruang, Sithichoke; Hansen, Martin Asser; Hansen, Lars Hestbjerg; Sørensen, Søren Johannes; Permpikul, Chairat; Rongrungruang, Yong; Tribuddharat, Chanwit

    2016-09-01

    Antibiotic resistance genes are rapidly spread between pathogens and the normal flora, with plasmids playing an important role in their circulation. This study aimed to investigate antibiotic resistance plasmids in the gut microbiome of hospitalised patients. Stool samples were collected from seven inpatients at Siriraj Hospital (Bangkok, Thailand) and were compared with a sample from a healthy volunteer. Plasmids from the gut microbiomes extracted from the stool samples were subjected to high-throughput DNA sequencing (GS Junior). Newbler-assembled DNA reads were categorised into known and unknown sequences (using >80% alignment length as the cut-off), and ResFinder was used to classify the antibiotic resistance gene pools. Plasmid replicon modules were used for plasmid typing. Forty-six genes conferring resistance to several classes of antibiotics were identified in the stool samples. Several antibiotic resistance genes were shared by the patients; interestingly, most were reported previously in food animals and healthy humans. Four antibiotic resistance genes were found in the healthy subject. One gene (aph3-III) was identified in the patients and the healthy subject and was related to that in cattle. Uncommon genes of hospital origin such as blaTEM-124-like and fosA, which confer resistance to extended-spectrum β-lactams and fosfomycin, respectively, were identified. The resistance genes did not match the patients' drug treatments. In conclusion, several plasmid types were identified in the gut microbiome; however, it was difficult to link these to the antibiotic resistance genes identified. That the antibiotic resistance genes came from hospital and community environments is worrying.

  6. 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.

  7. Complete nucleotide sequence of the self-transmissible TOL plasmid pD2RT provides new insight into arrangement of toluene catabolic plasmids

    DEFF Research Database (Denmark)

    Jutkina, Jekaterina; Hansen, Lars Hestbjerg; Li, Lili

    2013-01-01

    In the present study we report the complete nucleotide sequence of the toluene catabolic plasmid pD2RT of Pseudomonas migulae strain D2RT isolated from Baltic Sea water. The pD2RT is 129,894 base pairs in size with an average G+ C content of 53.75%. A total of 135 open reading frames (ORFs) were ...

  8. Co-evolution of genomes and plasmids within Chlamydia trachomatis and the emergence in Sweden of a new variant strain

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    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

  9. An abyssal mobilome: viruses, plasmids and vesicles from deep-sea hydrothermal vents.

    Science.gov (United States)

    Lossouarn, Julien; Dupont, Samuel; Gorlas, Aurore; Mercier, Coraline; Bienvenu, Nadege; Marguet, Evelyne; Forterre, Patrick; Geslin, Claire

    2015-12-01

    Mobile genetic elements (MGEs) such as viruses, plasmids, vesicles, gene transfer agents (GTAs), transposons and transpovirions, which collectively represent the mobilome, interact with cellular organisms from all three domains of life, including those thriving in the most extreme environments. While efforts have been made to better understand deep-sea vent microbial ecology, our knowledge of the mobilome associated with prokaryotes inhabiting deep-sea hydrothermal vents remains limited. Here we focus on the abyssal mobilome by reviewing accumulating data on viruses, plasmids and vesicles associated with thermophilic and hyperthermophilic Bacteria and Archaea present in deep-sea hydrothermal vents.

  10. Metal stressors consistently modulate bacterial conjugal plasmid uptake potential in a phylogenetically conserved manner

    DEFF Research Database (Denmark)

    Klümper, Uli; Dechesne, Arnaud; Riber, Leise

    2017-01-01

    The environmental stimulants and inhibitors of conjugal plasmid transfer in microbial communities are poorly understood. Specifically, it is not known whether exposure to stressors may cause a community to alter its plasmid uptake ability. We assessed whether metals (Cu, Cd, Ni, Zn) and one metal...... that community permissiveness is sensitive to metal(loid) stress in a manner that is both partially consistent across stressors and phylogenetically conserved.The ISME Journal advance online publication, 2 August 2016; doi:10.1038/ismej.2016.98....

  11. Fabrication of Size-Tunable Metallic Nanoparticles Using Plasmid DNA as a Biomolecular Reactor

    Directory of Open Access Journals (Sweden)

    Charles Michael Drain

    2011-10-01

    Full Text Available Plasmid DNA can be used as a template to yield gold, palladium, silver, and chromium nanoparticles of different sizes based on variations in incubation time at 70 °C with gold phosphine complexes, with the acetates of silver or palladium, or chromium acetylacetonate. The employment of mild synthetic conditions, minimal procedural steps, and aqueous solvents makes this method environmentally greener and ensures general feasibility. The use of plasmids exploits the capabilities of the biotechnology industry as a source of nanoreactor materials.

  12. Microevolutionary events involving narrow host plasmids influences local fixation of vancomycin-resistance in Enterococcus populations.

    Directory of Open Access Journals (Sweden)

    Ana R Freitas

    Full Text Available Vancomycin-resistance in enterococci (VRE is associated with isolates within ST18, ST17, ST78 Enterococcus faecium (Efm and ST6 Enterococcus faecalis (Efs human adapted lineages. Despite of its global spread, vancomycin resistance rates in enterococcal populations greatly vary temporally and geographically. Portugal is one of the European countries where Tn1546 (vanA is consistently found in a variety of environments. A comprehensive multi-hierarchical analysis of VRE isolates (75 Efm and 29 Efs from Portuguese hospitals and aquatic surroundings (1996-2008 was performed to clarify the local dynamics of VRE. Clonal relatedness was established by PFGE and MLST while plasmid characterization comprised the analysis of known relaxases, rep initiator proteins and toxin-antitoxin systems (TA by PCR-based typing schemes, RFLP comparison, hybridization and sequencing. Tn1546 variants were characterized by PCR overlapping/sequencing. Intra- and inter-hospital dissemination of Efm ST18, ST132 and ST280 and Efs ST6 clones, carrying rolling-circle (pEFNP1/pRI1 and theta-replicating (pCIZ2-like, Inc18, pHTβ-like, two pRUM-variants, pLG1-like, and pheromone-responsive plasmids was documented. Tn1546 variants, mostly containing ISEf1 or IS1216, were located on plasmids (30-150 kb with a high degree of mosaicism and heterogeneous RFLP patterns that seem to have resulted from the interplay between broad host Inc18 plasmids (pIP501, pRE25, pEF1, and narrow host RepA_N plasmids (pRUM, pAD1-like. TAs of Inc18 (ω-ε-ζ and pRUM (Axe-Txe plasmids were infrequently detected. Some plasmid chimeras were persistently recovered over years from different clonal lineages. This work represents the first multi-hierarchical analysis of VRE, revealing a frequent recombinatorial diversification of a limited number of interacting clonal backgrounds, plasmids and transposons at local scale. These interactions provide a continuous process of parapatric clonalization driving a full

  13. Persistence of Free Plasmid DNA in Soil Monitored by Various Methods, Including a Transformation Assay

    Science.gov (United States)

    Romanowski, Gerd; Lorenz, Michael G.; Sayler, Gary; Wackernagel, Wilfried

    1992-01-01

    The persistence and stability of free plasmid pUC8-ISP DNA introduced into 10-g samples of various soils and kept at 23°C were monitored over a period of 60 days. The soils were sampled at a plant science farm and included a loamy sand soil (no. 1), a clay soil (no. 2), and a silty clay soil (no. 3). Four different methods allowed monitoring of (i) the production of acid-soluble radioactive material from [3H]thymidine-labeled plasmid DNA, (ii) the decrease of hybridizing nucleotide sequences in slot blot analysis, (iii) the loss of plasmid integrity measured by Southern hybridization, and (iv) the decay of the biological activity as determined by transformation of Ca2+-treated Escherichia coli cells with the DNA extracted from soil. Acid-soluble material was not produced within the first 24 h but then increased to 45% (soil no. 1), 27% (soil no. 2), and 77% (soil no. 3) until the end of incubation. A quite parallel loss of material giving a slot blot hybridization signal was observed. Southern hybridization indicated that after 1 h in the soils, plasmid DNA was mostly in the form of circular and full-length linear molecules but that, depending on the soil type, after 2 to 5 days full-length plasmid molecules were hardly detectable. The transforming activity of plasmid DNA reextracted from the soils followed inactivation curves over 2 to 4 orders of magnitude and dropped below the detection limit after 10 days. The inactivation was slower in soil no. 2 (28.2-h half-life time of the transforming activity of a plasmid molecule) than in soils no. 3 (15.1 h) and no. 1 (9.1 h). The studies provide data on the persistence of free DNA molecules in natural bacterial soil habitats. The data suggest that plasmid DNA may persist long enough to be available for uptake by competent recipient cells in situ. Images PMID:16348772

  14. Design of expanded bed supports for the recovery of plasmid DNA by anion exchange adsorption

    DEFF Research Database (Denmark)

    Theodossiou, Irini; Søndergaard, M.; Thomas, Owen R. T.

    2001-01-01

    In this study we detail the rational design of new chromatographic adsorbents tailored for the capture of plasmid DNA. Features present on current chromatographic supports that can significantly enhance plasmid binding capacity have been identified in packed bed chromatography experiments...... and blueprints for improved expanded bed adsorbents have been put forward. The characterisation and testing of small (20-40 mum) high density (>3.7 g cm(-3)) pellicular expanded bed materials functionalised with various anion exchange structures is presented. In studies with calf thymus DNA, dynamic binding...

  15. 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...

  16. 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.

  17. 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 ...

  18. Microevolutionary Events Involving Narrow Host Plasmids Influences Local Fixation of Vancomycin-Resistance in Enterococcus Populations

    Science.gov (United States)

    Freitas, Ana R.; Novais, Carla; Tedim, Ana P.; Francia, María Victoria; Baquero, Fernando; Peixe, Luísa; Coque, Teresa M.

    2013-01-01

    Vancomycin-resistance in enterococci (VRE) is associated with isolates within ST18, ST17, ST78 Enterococcus faecium (Efm) and ST6 Enterococcus faecalis (Efs) human adapted lineages. Despite of its global spread, vancomycin resistance rates in enterococcal populations greatly vary temporally and geographically. Portugal is one of the European countries where Tn1546 (vanA) is consistently found in a variety of environments. A comprehensive multi-hierarchical analysis of VRE isolates (75 Efm and 29 Efs) from Portuguese hospitals and aquatic surroundings (1996–2008) was performed to clarify the local dynamics of VRE. Clonal relatedness was established by PFGE and MLST while plasmid characterization comprised the analysis of known relaxases, rep initiator proteins and toxin-antitoxin systems (TA) by PCR-based typing schemes, RFLP comparison, hybridization and sequencing. Tn1546 variants were characterized by PCR overlapping/sequencing. Intra- and inter-hospital dissemination of Efm ST18, ST132 and ST280 and Efs ST6 clones, carrying rolling-circle (pEFNP1/pRI1) and theta-replicating (pCIZ2-like, Inc18, pHTβ-like, two pRUM-variants, pLG1-like, and pheromone-responsive) plasmids was documented. Tn1546 variants, mostly containing ISEf1 or IS1216, were located on plasmids (30–150 kb) with a high degree of mosaicism and heterogeneous RFLP patterns that seem to have resulted from the interplay between broad host Inc18 plasmids (pIP501, pRE25, pEF1), and narrow host RepA_N plasmids (pRUM, pAD1-like). TAs of Inc18 (ω-ε-ζ) and pRUM (Axe-Txe) plasmids were infrequently detected. Some plasmid chimeras were persistently recovered over years from different clonal lineages. This work represents the first multi-hierarchical analysis of VRE, revealing a frequent recombinatorial diversification of a limited number of interacting clonal backgrounds, plasmids and transposons at local scale. These interactions provide a continuous process of parapatric clonalization driving a full

  19. Construction and identification of helper plasmids of newcastle disease virus Italien strain

    Directory of Open Access Journals (Sweden)

    Zhen REN

    2012-07-01

    Full Text Available Objective Newcastle disease virus (NDV is a naturally oncolytic virus that has been shown to be safe and effective for cancer therapy. NDV virions possess a non-segmented negative-sense single-stranded RNA genome which contains six genes encoding the nucleocapsid protein (NP, phosphoprotein (P, large polymerase protein (L, matrix protein, fusion protein, and hemagglutinin-neuraminidase. The ribonucleoprotein (RNP complex consisting of the genomic RNA and the three proteins NP, P, and L are the active template for transcription and replication of the viral genome. The purpose of this study was to construct the expression plasmids of NP, P and L genes of NDV Italien strain in which phage T7 promoter was a transcription promoter for the aim of generation of recombinant NDV. Methods NP, P and L genes were cloned from the genome RNA of NDV Italien followed by introduction into the downstream of T7 promoter and internal ribosome entry sites to construct the expression plasmids of NP, P and L, respectively. Expression of exogenous gene in BSR-T7/5 cells which constitutively express phage T7 RNA polymerase and transfected with plasmids of NP and P was detected by indirect immunofluorescence assay. The function of NP, P and L proteins expressed by constructed plasmids to facilitate the genomic RNA to form RNP complex was tested using minigenome of NDV Italien carrying firefly luciferase as a reporter gene. Results The expression plasmids of NP, P and L genes were confirmed by DNA sequencing. Using the indirect immunofluorescence assay, we detected the expression of viral NP and P proteins in BSR-T7/5 cells. When the helper plasmids were co-transfected with NDV minigenome plasmid, the expression of firefly luciferase was more significant compared with the control group (P < 0.001. Conclusion The helper plasmids of NDV Italien strain using T7 promoter as a transcription promoter has been constructed successfully, and it provides a basis for the

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

    OpenAIRE

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

    1991-01-01

    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,...