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Sample records for incp-1 plasmid pkjk5

  1. A stable shuttle vector for Xylella fastidiosa based on an endogenous incP-1 plasmid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Xylella fastidiosa (Xf) strain RIV11 harbors a 25 kbp plasmid (pXFRIV11) belonging to the incP1 incompatibility group. Replication and stability factors of pXFRIV11 were identified and used to construct plasmids able to propagate in both Xf and Escherichia coli. Sequences required for replication i...

  2. Functional characterization of replication and stability factors of an incP-1 plasmid from Xylella fastidiosa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Xylella fastidiosa strain riv11 harbors a ~25 kbp plasmid (pXF-RIV11) belonging to the incP-1 incompatibility group. Replication and stability factors of pXF-RIV11 were identified and used to construct plasmids able to propagate in both X. fastidiosa and Escherichia coli. Replication in X. fastidi...

  3. Transferable antibiotic resistance plasmids from biogas plant digestates often belong to the IncP-1ε subgroup

    PubMed Central

    Wolters, Birgit; Kyselková, Martina; Krögerrecklenfort, Ellen; Kreuzig, Robert; Smalla, Kornelia

    2015-01-01

    Manure is known to contain residues of antibiotics administered to farm animals as well as bacteria carrying antibiotic resistance genes (ARGs). These genes are often located on mobile genetic elements. In biogas plants (BGPs), organic substrates such as manure and plant material are mixed and fermented in order to provide energy, and resulting digestates are used for soil fertilization. The fate of plasmid carrying bacteria from manure during the fermentation process is unknown. The present study focused on transferable antibiotic resistance plasmids from digestates of seven BGPs, using manure as a co-substrate, and their phenotypic and genotypic characterization. Plasmids conferring resistance to either tetracycline or sulfadiazine were captured by means of exogenous plasmid isolation from digestates into Pseudomonas putida KT2442 and Escherichia coli CV601 recipients, at transfer frequencies ranging from 10-5 to 10-7. Transconjugants (n = 101) were screened by PCR-Southern blot hybridization and real-time PCR for the presence of IncP-1, IncP-1ε, IncW, IncN, IncP-7, IncP-9, LowGC, and IncQ plasmids. While 61 plasmids remained unassigned, 40 plasmids belonged to the IncP-1ε subgroup. All these IncP-1ε plasmids were shown to harbor the genes tet(A), sul1, qacEΔ1, intI1, and integron gene cassette amplicons of different size. Further analysis of 16 representative IncP-1ε plasmids showed that they conferred six different multiple antibiotic resistance patterns and their diversity seemed to be driven by the gene cassette arrays. IncP-1ε plasmids displaying similar restriction and antibiotic resistance patterns were captured from different BGPs, suggesting that they may be typical of this environment. Our study showed that BGP digestates are a potential source of transferable antibiotic resistance plasmids, and in particular the broad host range IncP-1ε plasmids might contribute to the spread of ARGs when digestates are used as fertilizer. PMID:25653641

  4. Cultivation-Independent Screening Revealed Hot Spots of IncP-1, IncP-7 and IncP-9 Plasmid Occurrence in Different Environmental Habitats

    PubMed Central

    Dealtry, Simone; Ding, Guo-Chun; Weichelt, Viola; Dunon, Vincent; Schlüter, Andreas; Martini, María Carla; Papa, María Florencia Del; Lagares, Antonio; Amos, Gregory Charles Auton; Wellington, Elizabeth Margaret Helen; Gaze, William Hugo; Sipkema, Detmer; Sjöling, Sara; Springael, Dirk; Heuer, Holger; van Elsas, Jan Dirk; Thomas, Christopher; Smalla, Kornelia

    2014-01-01

    IncP-1, IncP-7 and IncP-9 plasmids often carry genes encoding enzymes involved in the degradation of man-made and natural contaminants, thus contributing to bacterial survival in polluted environments. However, the lack of suitable molecular tools often limits the detection of these plasmids in the environment. In this study, PCR followed by Southern blot hybridization detected the presence of plasmid-specific sequences in total community (TC-) DNA or fosmid DNA from samples originating from different environments and geographic regions. A novel primer system targeting IncP-9 plasmids was developed and applied along with established primers for IncP-1 and IncP-7. Screening TC-DNA from biopurification systems (BPS) which are used on farms for the purification of pesticide-contaminated water revealed high abundances of IncP-1 plasmids belonging to different subgroups as well as IncP-7 and IncP-9. The novel IncP-9 primer-system targeting the rep gene of nine IncP-9 subgroups allowed the detection of a high diversity of IncP-9 plasmid specific sequences in environments with different sources of pollution. Thus polluted sites are “hot spots” of plasmids potentially carrying catabolic genes. PMID:24587126

  5. Cultivation-independent screening revealed hot spots of IncP-1, IncP-7 and IncP-9 plasmid occurrence in different environmental habitats.

    PubMed

    Dealtry, Simone; Ding, Guo-Chun; Weichelt, Viola; Dunon, Vincent; Schlüter, Andreas; Martini, María Carla; Del Papa, María Florencia; Lagares, Antonio; Amos, Gregory Charles Auton; Wellington, Elizabeth Margaret Helen; Gaze, William Hugo; Sipkema, Detmer; Sjöling, Sara; Springael, Dirk; Heuer, Holger; van Elsas, Jan Dirk; Thomas, Christopher; Smalla, Kornelia

    2014-01-01

    IncP-1, IncP-7 and IncP-9 plasmids often carry genes encoding enzymes involved in the degradation of man-made and natural contaminants, thus contributing to bacterial survival in polluted environments. However, the lack of suitable molecular tools often limits the detection of these plasmids in the environment. In this study, PCR followed by Southern blot hybridization detected the presence of plasmid-specific sequences in total community (TC-) DNA or fosmid DNA from samples originating from different environments and geographic regions. A novel primer system targeting IncP-9 plasmids was developed and applied along with established primers for IncP-1 and IncP-7. Screening TC-DNA from biopurification systems (BPS) which are used on farms for the purification of pesticide-contaminated water revealed high abundances of IncP-1 plasmids belonging to different subgroups as well as IncP-7 and IncP-9. The novel IncP-9 primer-system targeting the rep gene of nine IncP-9 subgroups allowed the detection of a high diversity of IncP-9 plasmid specific sequences in environments with different sources of pollution. Thus polluted sites are "hot spots" of plasmids potentially carrying catabolic genes. PMID:24587126

  6. PemK toxin encoded by the Xylella fastidiosa IncP-1 plasmid pXF-RIV11 is a ribonuclease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Stable inheritance of the IncP-1 plasmid pXF-RIV11 in Xylella fastidiosa is conferred by the pemI/pemK plasmid addiction system. PemK serves as a toxin inhibiting bacterial growth; PemI is the corresponding antitoxin that blocks activity of PemK toxin by direct binding. Here, PemK toxin and PemI ant...

  7. Natural microbial communities supporting the transfer of the IncP-1β plasmid pB10 exhibit a higher initial content of plasmids from the same incompatibility group

    PubMed Central

    Bellanger, Xavier; Guilloteau, Hélène; Breuil, Bérengère; Merlin, Christophe

    2014-01-01

    Antibiotic resistance gene transfer mediated by plasmids is a matter of concern for public health, but permissive environments supporting plasmid dissemination are still quite difficult to identify. Lately, we have reported a molecular approach based on quantitative PCR (qPCR) to monitor the fate of the IncP-1β plasmid pB10 in natural microbial communities maintained in microcosms. Such plasmid transfer experiments were carried out with 13 different environmental matrices, and demonstrated that the transfer of the conjugative-proficient plasmid pB10 in complex environments is relatively rare and is strongly matrix dependent. An attempt to link the microbial community structure and the matrix permissiveness showed that TTGE analysis is not resolutive enough to point out common features among comparable communities supporting pB10 transfer. However, an estimation of the IncP-1α/IncP-1β plasmids abundance by qPCR demonstrated that pB10 transfer tends to be supported by environmental matrices exhibiting a higher content of IncP-1 plasmids. We suggest that the relative abundance of IncP-1 plasmids in a given microbial community reflects its permissiveness to the transfer of plasmids belonging to the same incompatibility group, which prevails over transfer limitation due to a phenomenon known as superinfection immunity. PMID:25505458

  8. Complete Nucleotide Sequence of IncP-1β Plasmid pDTC28 Reveals a Non-Functional Variant of the blaGES-Type Gene.

    PubMed

    Dang, Bingjun; Mao, Daqing; Luo, Yi

    2016-01-01

    Plasmid pDTC28 was isolated from the sediments of Haihe River using E. coli CV601 (gfp-tagged) as recipient and indigenous bacteria from the sediment as donors. This plasmid confers reduced susceptibility to tetracycline and sulfamethoxazole. The complete sequence of plasmid pDTC28 was 61,503 bp in length with an average G+C content of 64.09%. Plasmid pDTC28 belongs to the IncP-1β group by phylogenetic analysis. The backbones of plasmid pDTC28 and other IncP-1β plasmids are very classical and conserved, whereas the accessory regions of these plasmids are diverse. A blaGES-5-like gene was found on the accessory region, and this blaGES-5-like gene contained 18 silent mutations and 7 missense mutations compared with the blaGES-5 gene. The mutations resulted in 7 amino acid substitutions in GES-5 carbapenemase, causing the loss of function of the blaGES-5-like gene on plasmid pDTC28 against carbapenems and even β-lactams. The enzyme produced by the blaGES-5-like gene cassette may be a new variant of GES-type enzymes. Thus, the plasmid sequenced in this study will expand our understanding of GES-type β-lactamases and provide insights into the genetic platforms used for the dissemination of GES-type genes. PMID:27152950

  9. Complete Nucleotide Sequence of IncP-1β Plasmid pDTC28 Reveals a Non-Functional Variant of the blaGES-Type Gene

    PubMed Central

    Dang, Bingjun; Mao, Daqing; Luo, Yi

    2016-01-01

    Plasmid pDTC28 was isolated from the sediments of Haihe River using E. coli CV601 (gfp-tagged) as recipient and indigenous bacteria from the sediment as donors. This plasmid confers reduced susceptibility to tetracycline and sulfamethoxazole. The complete sequence of plasmid pDTC28 was 61,503 bp in length with an average G+C content of 64.09%. Plasmid pDTC28 belongs to the IncP-1β group by phylogenetic analysis. The backbones of plasmid pDTC28 and other IncP-1β plasmids are very classical and conserved, whereas the accessory regions of these plasmids are diverse. A blaGES-5-like gene was found on the accessory region, and this blaGES-5-like gene contained 18 silent mutations and 7 missense mutations compared with the blaGES-5 gene. The mutations resulted in 7 amino acid substitutions in GES-5 carbapenemase, causing the loss of function of the blaGES-5-like gene on plasmid pDTC28 against carbapenems and even β-lactams. The enzyme produced by the blaGES-5-like gene cassette may be a new variant of GES-type enzymes. Thus, the plasmid sequenced in this study will expand our understanding of GES-type β-lactamases and provide insights into the genetic platforms used for the dissemination of GES-type genes. PMID:27152950

  10. Effects of 100 years wastewater irrigation on resistance genes, class 1 integrons and IncP-1 plasmids in Mexican soil

    PubMed Central

    Jechalke, Sven; Broszat, Melanie; Lang, Friederike; Siebe, Christina; Smalla, Kornelia; Grohmann, Elisabeth

    2015-01-01

    Long-term irrigation with untreated wastewater can lead to an accumulation of antibiotic substances and antibiotic resistance genes in soil. However, little is known so far about effects of wastewater, applied for decades, on the abundance of IncP-1 plasmids and class 1 integrons which may contribute to the accumulation and spread of resistance genes in the environment, and their correlation with heavy metal concentrations. Therefore, a chronosequence of soils that were irrigated with wastewater from 0 to 100 years was sampled in the Mezquital Valley in Mexico in the dry season. The total community DNA was extracted and the absolute and relative abundance (relative to 16S rRNA genes) of antibiotic resistance genes (tet(W), tet(Q), aadA), class 1 integrons (intI1), quaternary ammonium compound resistance genes (qacE+qacEΔ1) and IncP-1 plasmids (korB) were quantified by real-time PCR. Except for intI1 and qacE+qacEΔ1 the abundances of selected genes were below the detection limit in non-irrigated soil. Confirming the results of a previous study, the absolute abundance of 16S rRNA genes in the samples increased significantly over time (linear regression model, p < 0.05) suggesting an increase in bacterial biomass due to repeated irrigation with wastewater. Correspondingly, all tested antibiotic resistance genes as well as intI1 and korB significantly increased in abundance over the period of 100 years of irrigation. In parallel, concentrations of the heavy metals Zn, Cu, Pb, Ni, and Cr significantly increased. However, no significant positive correlations were observed between the relative abundance of selected genes and years of irrigation, indicating no enrichment in the soil bacterial community due to repeated wastewater irrigation or due to a potential co-selection by increasing concentrations of heavy metals. PMID:25784901

  11. Analysis of defence systems and a conjugative IncP-1 plasmid in the marine polyaromatic hydrocarbons-degrading bacterium Cycloclasticus sp. 78-ME.

    PubMed

    Yakimov, Michail M; Crisafi, Francesca; Messina, Enzo; Smedile, Francesco; Lopatina, Anna; Denaro, Renata; Pieper, Dietmar H; Golyshin, Peter N; Giuliano, Laura

    2016-08-01

    Marine prokaryotes have evolved a broad repertoire of defence systems to protect their genomes from lateral gene transfer including innate or acquired immune systems and infection-induced programmed cell suicide and dormancy. Here we report on the analysis of multiple defence systems present in the genome of the strain Cycloclasticus sp. 78-ME isolated from petroleum deposits of the tanker 'Amoco Milford Haven'. Cycloclasticus are ubiquitous bacteria globally important in polyaromatic hydrocarbons degradation in marine environments. Two 'defence islands' were identified in 78-ME genome: the first harbouring CRISPR-Cas with toxin-antitoxin system, while the second was composed by an array of genes for toxin-antitoxin and restriction-modification proteins. Among all identified spacers of CRISPR-Cas system only seven spacers match sequences of phages and plasmids. Furthermore, a conjugative plasmid p7ME01, which belongs to a new IncP-1θ ancestral archetype without any accessory mobile elements was found in 78-ME. Our results provide the context to the co-occurrence of diverse defence mechanisms in the genome of Cycloclasticus sp. 78-ME, which protect the genome of this highly specialized PAH-degrader. This study contributes to the further understanding of complex networks established in petroleum-based microbial communities. PMID:27345842

  12. Conjugative transfer of a derivative of the IncP-1α plasmid RP4 and establishment of transconjugants in the indigenous bacterial community of poplar plants

    PubMed Central

    Ulrich, Andreas; Becker, Regina; Ulrich, Kristina; Ewald, Dietrich

    2015-01-01

    The persistence of traits introduced into the indigenous bacterial community of poplar plants was investigated using bioluminescence mediated by the luc gene. Three endophytic bacterial strains provided with the IncP-1α plasmid RP4-Tn-luc were used to inoculate poplar cuttings at different phenological stages. Screening of isolates by bioluminescence and real-time PCR detection of the luc gene revealed stable persistence for at least 10 weeks. Although the inoculated strains became established with a high population density after inoculation at leaf development (April) and senescence (October), the strains were suppressed by the indigenous bacteria at stem elongation (June). Transconjugants could be detected only at this phenological stage. Indigenous bacteria harbouring RP4-Tn-luc became established with densities ranging from 2 × 105 to 9 × 106 CFU g−1 fresh weight 3 and 10 weeks after inoculation. The increased colonization of the cuttings by indigenous bacteria at stem elongation seemed to strongly compete with the introduced strains. Otherwise, the phenological stage of the plants as well as the density of the indigenous recipients could serve as the driver for a more frequent conjugative plasmid transfer. A phylogenetic assignment of transconjugants indicated the transfer of RP4-Tn-luc into six genera of Proteobacteria, mainly Sphingomonas, Stenotrophomonas and Xanthomonas. PMID:26490946

  13. The Complete Sequences and Ecological Roles of Two IncP-1β Plasmids, pHB44 and pBS64, Isolated from the Mycosphere of Laccaria proxima

    PubMed Central

    Zhang, Miaozhi; Brons, Jolanda K.; van Elsas, Jan Dirk

    2016-01-01

    Two novel plasmids, coined pHB44 and pBS64, were recently found in Variovorax paradoxus strains HB44 and BS64 isolated from the mycosphere of Laccaria proxima, on two different sampling occasions. We here describe the full sequences of pHB44 and pBS64 and establish their evolutionary placement and ecological function. Both plasmids, unique for mycospheric V. paradoxus, were around 58 kb in size. They possessed, in a very similar fashion, three main plasmid backbone regions, which were predicted to be involved in plasmid replication, central control of maintenance, and conjugational transfer. Phylogenetic inference on the basis of seven selected and concatenated plasmid backbone genes provided solid evidence for the placement of the two plasmids in the IncP-1β1 group, with the recently isolated IncP-1β1 plasmid pMBUI8 as the closest relative. A comparative analysis of the sequences present in each of the recombinational hot spots (RHS) I to III across plasmids pHB44, pBS64, and pMBUI8 revealed the insertions found in plasmids pHB44 and pBS64 to be different from those of pMBUI8. Whereas, in the former two plasmids, RHS I and III were devoid of any major inserts, their RHS II regions contained inserts of 15,043 (pHB44) and 16,406 kb (pBS64), against about 9,3 kb for pMBUI8. Interestingly, these regions were highly similar across plasmids pHB44 and pBS64, and differed from that of pMBUI8. Closer inspection revealed the insert in the former plasmids to contain, next to transposases, an “mmf” gene cassette previously reported to encode metal “responsiveness” in the PromA plasmid pMOL98. Whereas the plasmid pHB44 RHS II contained the canonical mmf sequence, that in pBS64 contained, in addition, a “two-gene duplicated region” flanking the mmf C2 gene. In vitro experiments on the growth and survival of strains with or without plasmid pHB44 suggested this plasmid was involved in the binding and import of Fe3+ as well as V3+ ions into the host cells, thus

  14. Widespread occurrence of the tfd-II genes in soil bacteria revealed by nucleotide sequence analysis of 2,4-dichlorophenoxyacetic acid degradative plasmids pDB1 and p712.

    PubMed

    Kim, Dong-Uk; Kim, Min-Sun; Lim, Jong-Sung; Ka, Jong-Ok

    2013-05-01

    Variovorax sp. strain DB1 and Pseudomonas pickettii strain 712 are 2,4-dicholorophenoxy-acetic acid (2,4-D)-degrading bacteria, which were isolated from agricultural soils in Republic of Korea and USA, respectively. Each strain harbors a 2,4-D degradative plasmid and is able to utilize 2,4-D as the sole source of carbon for its growth. The 2,4-D degradative plasmid pDB1 of strain DB1 consisted of a 65,269-bp circular molecule with a G+C content of 66.23% and had 68 ORFs. The 2,4-D degradative plasmid p712 of strain 712 was composed of a 62,798-bp circular molecule with a 62.11% G+C content and had 62 ORFs. The plasmids pDB1 and p712 share significantly homologous 2,4-D degradative genes with high similarity to the tfdR, tfdB-II, tfdC-II, tfdD-II, tfdE-II, tfdF-II, tfdK and tfdA genes of plasmid pJP4 of Alcaligenes eutrophus isolated from Australia. In a phylogenetic analysis with trfA, traL, and trbA genes, pDB1 belonged to IncP-1β with pJP4, while p712 belonged to IncP-1ε with pKJK5 and pEMT3. The results indicated that, in spite of the differences in their backbone regions, the 2,4-D catabolic genes of the two plasmids were closely related and also related to the well-known 2,4-D degradative plasmid pJP4 even though all were isolated from different geographic regions. Other similarities in the genetic organization and the presence of IS1071 suggested that these catabolic genes may be on a transposable element, leading to widespread occurrence in soil bacteria. PMID:23376020

  15. Phylogeny of replication initiator protein TrfA reveals a highly divergent clade of incompatibility group P1 plasmids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Incompatibility group P-1 (incP-1) includes broad host range plasmids of Gram negative bacteria and are classified into five subgroups (alpha, beta, gamma, delta, and epsilon). The incP-1 replication module consists of the trfA gene, encoding the replication initiator protein TrfA, and the origin o...

  16. Diversification of broad host range plasmids correlates with the presence of antibiotic resistance genes.

    PubMed

    Li, Xiaobin; Wang, Yafei; Brown, Celeste J; Yao, Fei; Jiang, Yong; Top, Eva M; Li, Hui

    2016-01-01

    The IncP-1ε subgroup is a recently identified phylogenetic clade within IncP-1 plasmids, which plays an important role in the spread of antibiotic resistance and degradation of xenobiotic pollutants. Here, four IncP-1ε plasmids were exogenously captured from a petroleum-contaminated habitat in China and compared phylogenetically and genomically with previously reported IncP-1ε and other IncP-1 plasmids. The IncP-1ε plasmids can be clearly subdivided into two subclades, designated as ε-I and ε-II, based on phylogenetic analysis of backbone proteins TraI and TrfA. This was further supported by comparison of concatenated backbone genes. Moreover, the two subclades differed in the transposon types, phenotypes and insertion locations of the accessory elements. The accessory genes on ε-I plasmids were inserted between parA and traC, and harbored ISPa17 and Tn402-like transposon modules, typically carrying antibiotic resistance genes. In contrast, the accessory elements on ε-II plasmids were typically located between trfA and oriV, and contained IS1071, which was commonly inserted within the Tn501-like transposon, typically harboring a cluster of genes encoding mercury resistance and/or catabolic pathways. Our study is one of the first to compare IncP-1 plasmid genomes from China, expands the available collection of IncP-1ε plasmids and enhances our understanding of their diversity, biogeography and evolutionary history. PMID:26635412

  17. Functional identification of Xylella fastidiosa plasmid replication and stability factors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Xylella fastidiosa (Xf) strain RIV11 harbors a 25 kbp plasmid (pXFRIV11) belonging to the incP1 incompatibility group. Replication and stability factors of pXFRIV11 were identified and used to construct plasmids able to replicate in both Xf and Escherichia coli. Sequences required for replication i...

  18. Conjugative Plasmids of Neisseria gonorrhoeae

    PubMed Central

    Pachulec, Emilia; van der Does, Chris

    2010-01-01

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

  19. Piggery manure used for soil fertilization is a reservoir for transferable antibiotic resistance plasmids.

    PubMed

    Binh, Chu Thi Thanh; Heuer, Holger; Kaupenjohann, Martin; Smalla, Kornelia

    2008-10-01

    In this study, the prevalence and types of transferable antibiotic resistance plasmids in piggery manure were investigated. Samples from manure storage tanks of 15 farms in Germany were analysed, representing diverse sizes of herds, meat or piglet production. Antibiotic resistance plasmids from manure bacteria were captured in gfp-tagged rifampicin-resistant Escherichia coli and characterized. The occurrence of plasmid types was also detected in total community DNA by PCR and hybridization. A total of 228 transconjugants were captured from 15 manures using selective media supplemented with amoxicillin, sulfadiazine or tetracycline. The restriction patterns of 81 plasmids representing different antibiotic resistance patterns or different samples clustered into seven groups. Replicon probing revealed that 28 of the plasmids belonged to IncN, one to IncW, 13 to IncP-1 and 19 to the recently discovered pHHV216-like plasmids. The amoxicillin resistance gene bla-TEM was detected on 44 plasmids, and sulphonamide resistance genes sul1, sul2 and/or sul3 on 68 plasmids. Hybridization of replicon-specific sequences amplified from community DNA revealed that IncP-1 and pHHV216-like plasmids were detected in all manures, while IncN and IncW ones were less frequent. This study showed that 'field-scale' piggery manure is a reservoir of broad-host range plasmids conferring multiple antibiotic resistance genes. PMID:18557938

  20. Genetic organization of the catabolic plasmid pJP4 from Ralstonia eutropha JMP134 (pJP4) reveals mechanisms of adaptation to chloroaromatic pollutants and evolution of specialized chloroaromatic degradation pathways.

    PubMed

    Trefault, N; De la Iglesia, R; Molina, A M; Manzano, M; Ledger, T; Pérez-Pantoja, D; Sánchez, M A; Stuardo, M; González, B

    2004-07-01

    Ralstonia eutropha JMP134 (pJP4) is a useful model for the study of bacterial degradation of substituted aromatic pollutants. Several key degrading capabilities, encoded by tfd genes, are located in the 88 kb, self-transmissible, IncP-1 beta plasmid pJP4. The complete sequence of the 87,688 nucleotides of pJP4, encoding 83 open reading frames (ORFs), is reported. Most of the coding sequence corresponds to a well-conserved IncP-1 beta backbone and the previously reported tfd genes. In addition, we found hypothetical proteins putatively involved in the transport of aromatic compounds and short-chain fatty acid oxidation. ORFs related to mobile elements, including the Tn501-encoded mercury resistance determinants, an IS1071-based composite transposon and a cryptic class II transposon, are also present in pJP4. These mobile elements are inefficient in transposition and are located in two regions of pJP4 that are rich in remnants of lateral gene transfer events. pJP4 plasmid was able to capture chromosomal genes and form hybrid plasmids with the IncP-1 alpha plasmid RP4. These observations are integrated into a model for the evolution of pJP4, which reveals mechanisms of bacterial adaptation to degrade pollutants. PMID:15186344

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

    PubMed Central

    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. PMID:25389419

  2. Characterization of diverse 2,4-dichlorophenoxyacetic acid-degradative plasmids isolated from soil by complementation.

    PubMed Central

    Top, E M; Holben, W E; Forney, L J

    1995-01-01

    The diversity of 2,4-dichlorophenoxyacetic acid (2,4-D)-degradative plasmids in the microbial community of an agricultural soil was examined by complementation. This technique involved mixing a suitable Alcaligenes eutrophus (Rifr) recipient strain with the indigenous microbial populations extracted from soil. After incubation of this mixture, Rifr recipient strains which grow with 2,4-D as the only C source were selected. Two A. eutrophus strains were used as recipients: JMP228 (2,4-D-), which was previously derived from A. eutrophus JMP134 by curing of the 2,4-D-degradative plasmid pJP4, and JMP228 carrying pBH501aE (a plasmid derived from pJP4 by deletion of a large part of the tfdA gene which encodes the first step in the mineralization of 2,4-D). By using agricultural soil that had been treated with 2,4-D for several years, transconjugants were obtained with both recipients. However, when untreated control soil was used, no transconjugants were isolated. The various transconjugants had plasmids with seven different EcoRI restriction patterns. The corresponding plasmids are designated pEMT1 to pEMT7. Unlike pJP4, pEMT1 appeared not to be an IncP1 plasmid, but all the others (pEMT2 to pEMT7) belong to the IncP1 group. Hybridization with individual probes for the tfdA to tfdF genes of pJP4 demonstrated that all plasmids showed high degrees of homology to the tfdA gene. Only pEMT1 showed a high degree of homology to tfdB, tfdC, tfdD, tfdE, and tfdF, while the others showed only moderate degrees of homology to tfdB and low degrees of homology to tfdC.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7646006

  3. Comparison of Four Comamonas Catabolic Plasmids Reveals the Evolution of pBHB To Catabolize Haloaromatics

    PubMed Central

    Chen, Kai; Xu, Xihui; Zhang, Long; Gou, Zhenjiu; Li, Shunpeng

    2015-01-01

    Comamonas plasmids play important roles in shaping the phenotypes of their hosts and the adaptation of these hosts to changing environments, and understanding the evolutionary strategy of these plasmids is thus of great concern. In this study, the sequence of the 119-kb 3,5-dibromo-4-hydroxybenzonitrile-catabolizing plasmid pBHB from Comamonas sp. strain 7D-2 was studied and compared with those of three other Comamonas haloaromatic catabolic plasmids. Incompatibility group determination based on a phylogenetic analysis of 24 backbone gene proteins, as well as TrfA, revealed that these four plasmids all belong to the IncP-1β subgroup. Comparison of the four plasmids revealed a conserved backbone region and diverse genetic-load regions. The four plasmids share a core genome consisting of 40 genes (>50% similarities) and contain 12 to 50 unique genes each, most of which are xenobiotic-catabolic genes. Two functional reductive dehalogenase gene clusters are specifically located on pBHB, showing distinctive evolution of pBHB for haloaromatics. The higher catabolic ability of the bhbA2B2 cluster than the bhbAB cluster may be due to the transcription levels and the character of the dehalogenase gene itself rather than that of its extracytoplasmic binding receptor gene. The plasmid pBHB is riddled with transposons and insertion sequence (IS) elements, and ISs play important roles in the evolution of pBHB. The analysis of the origin of the bhb genes on pBHB suggested that these accessory genes evolved independently. Our work provides insights into the evolutionary strategies of Comamonas plasmids, especially into the adaptation mechanism employed by pBHB for haloaromatics. PMID:26682859

  4. Associations between multidrug resistance, plasmid content, and virulence potential among extraintestinal pathogenic and commensal Escherichia coli from humans and poultry.

    PubMed

    Johnson, Timothy J; Logue, Catherine M; Johnson, James R; Kuskowski, Michael A; Sherwood, Julie S; Barnes, H John; DebRoy, Chitrita; Wannemuehler, Yvonne M; Obata-Yasuoka, Mana; Spanjaard, Lodewijk; Nolan, Lisa K

    2012-01-01

    The emergence of plasmid-mediated multidrug resistance (MDR) among enteric bacteria presents a serious challenge to the treatment of bacterial infections in humans and animals. Recent studies suggest that avian Escherichia coli commonly possess the ability to resist multiple antimicrobial agents, and might serve as reservoirs of MDR for human extraintestinal pathogenic Escherichia coli (ExPEC) and commensal E. coli populations. We determined antimicrobial susceptibility profiles for 2202 human and avian E. coli isolates, then sought for associations among resistance profile, plasmid content, virulence factor profile, and phylogenetic group. Avian-source isolates harbored greater proportions of MDR than their human counterparts, and avian ExPEC had higher proportions of MDR than did avian commensal E. coli. MDR was significantly associated with possession of the IncA/C, IncP1-α, IncF, and IncI1 plasmid types. Overall, inferred virulence potential did not correlate with drug susceptibility phenotype. However, certain virulence genes were positively associated with MDR, including ireA, ibeA, fyuA, cvaC, iss, iutA, iha, and afa. According to the total dataset, isolates segregated significantly according to host species and clinical status, thus suggesting that avian and human ExPEC and commensal E. coli represent four distinct populations with limited overlap. These findings suggest that in extraintestinal E. coli, MDR is most commonly associated with plasmids, and that these plasmids are frequently found among avian-source E. coli from poultry production systems. PMID:21988401

  5. Increased Transfer of a Multidrug Resistance Plasmid in Escherichia coli Biofilms at the Air-Liquid Interface ▿

    PubMed Central

    Król, Jaroslaw E.; Nguyen, Hung Duc; Rogers, Linda M.; Beyenal, Haluk; Krone, Stephen M.; Top, Eva M.

    2011-01-01

    Although biofilms represent a common bacterial lifestyle in clinically and environmentally important habitats, there is scant information on the extent of gene transfer in these spatially structured populations. The objective of this study was to gain insight into factors that affect transfer of the promiscuous multidrug resistance plasmid pB10 in Escherichia coli biofilms. Biofilms were grown in different experimental settings, and plasmid transfer was monitored using laser scanning confocal microscopy and plate counting. In closed flow cells, plasmid transfer in surface-attached submerged biofilms was negligible. In contrast, a high plasmid transfer efficiency was observed in a biofilm floating at the air-liquid interface in an open flow cell with low flow rates. A vertical flow cell and a batch culture biofilm reactor were then used to detect plasmid transfer at different depths away from the air-liquid interface. Extensive plasmid transfer occurred only in a narrow zone near that interface. The much lower transfer frequencies in the lower zones coincided with rapidly decreasing oxygen concentrations. However, when an E. coli csrA mutant was used as the recipient, a thick biofilm was obtained at all depths, and plasmid transfer occurred at similar frequencies throughout. These results and data from separate aerobic and anaerobic matings suggest that oxygen can affect IncP-1 plasmid transfer efficiency, not only directly but also indirectly, through influencing population densities and therefore colocalization of donors and recipients. In conclusion, the air-liquid interface can be a hot spot for plasmid-mediated gene transfer due to high densities of juxtaposed donor and recipient cells. PMID:21642400

  6. Nucleotide Sequence of Plasmid pCNB1 from Comamonas Strain CNB-1 Reveals Novel Genetic Organization and Evolution for 4-Chloronitrobenzene Degradation▿

    PubMed Central

    Ma, Ying-Fei; Wu, Jian-Feng; Wang, Sheng-Yue; Jiang, Cheng-Ying; Zhang, Yun; Qi, Su-Wei; Liu, Lei; Zhao, Guo-Ping; Liu, Shuang-Jiang

    2007-01-01

    The nucleotide sequence of a new plasmid pCNB1 from Comamonas sp. strain CNB-1 that degrades 4-chloronitrobenzene (4CNB) was determined. pCNB1 belongs to the IncP-1β group and is 91,181 bp in length. A total of 95 open reading frames appear to be involved in (i) the replication, maintenance, and transfer of pCNB1; (ii) resistance to arsenate and chromate; and (iii) the degradation of 4CNB. The 4CNB degradative genes and arsenate resistance genes were located on an extraordinarily large transposon (44.5 kb), proposed as TnCNB1. TnCNB1 was flanked by two IS1071 elements and represents a new member of the composite I transposon family. The 4CNB degradative genes within TnCNB1 were separated by various truncated genes and genetic homologs from other DNA molecules. Genes for chromate resistance were located on another transposon that was similar to the Tn21 transposon of the class II replicative family that is frequently responsible for the mobilization of mercury resistance genes. Resistance to arsenate and chromate were experimentally confirmed, and transcriptions of arsenate and chromate resistance genes were demonstrated by reverse transcription-PCR. These results described a new member of the IncP-1β plasmid family, and the findings suggest that gene deletion and acquisition as well as genetic rearrangement of DNA molecules happened during the evolution of the 4CNB degradation pathway on pCNB1. PMID:17526790

  7. Effects of stress and other environmental factors on horizontal plasmid transfer assessed by direct quantification of discrete transfer events.

    PubMed

    Johnsen, Anders R; Kroer, Niels

    2007-03-01

    Selection pressure may affect the horizontal transfer of plasmids. The inability to distinguish between gene transfer and the growth of transconjugants complicates testing. We have developed a method that enables the quantification of discrete transfer events. It uses large numbers of replicate matings (192 or 384) in microtiter wells and the counting of transfer-positive and transfer-negative wells. We applied the method to study the transfer of the IncP1 plasmid pRO103 between Escherichia coli and Pseudomonas putida strains. pRO103 encodes resistance to mercury and tetracycline and partial degradation of 2,4-dichlorophenoxyacetic acid (2,4-D). The results showed positive correlation between transfer and donor metabolic activity, and an optimal temperature for transfer of 29 degrees C. On stimulation of donor activity, the optimal temperature was decreased to 24.5 degrees C. HgCl(2) above 1.0 microg L(-1) negatively affected transfer, whereas 2,4-D up to 0.3 mM had no effect. The negative effect of mercury was shown to be a result of stressing of the recipient. No effects of mercury on transfer could be detected by traditional filter mating. Thus, the method is superior to filter mating and, as the experimental design allows the manipulation of individual parameters, it is ideal for the assessment and comparison of effects of environmental factors on plasmid transfer. PMID:17100984

  8. Flexibility of KorA, a plasmid-encoded, global transcription regulator, in the presence and the absence of its operator.

    PubMed

    Rajasekar, Karthik V; Lovering, Andrew L; Dancea, Felician; Scott, David J; Harris, Sarah A; Bingle, Lewis E H; Roessle, Manfred; Thomas, Christopher M; Hyde, Eva I; White, Scott A

    2016-06-01

    The IncP (Incompatibility group P) plasmids are important carriers in the spread of antibiotic resistance across Gram-negative bacteria. Gene expression in the IncP-1 plasmids is stringently controlled by a network of four global repressors, KorA, KorB, TrbA and KorC interacting cooperatively. Intriguingly, KorA and KorB can act as co-repressors at varying distances between their operators, even when they are moved to be on opposite sides of the DNA. KorA is a homodimer with the 101-amino acid subunits, folding into an N-terminal DNA-binding domain and a C-terminal dimerization domain. In this study, we have determined the structures of the free KorA repressor and two complexes each bound to a 20-bp palindromic DNA duplex containing its consensus operator sequence. Using a combination of X-ray crystallography, nuclear magnetic resonance spectroscopy, SAXS and molecular dynamics calculations, we show that the linker between the two domains is very flexible and the protein remains highly mobile in the presence of DNA. This flexibility allows the DNA-binding domains of the dimer to straddle the operator DNA on binding and is likely to be important in cooperative binding to KorB. Unexpectedly, the C-terminal domain of KorA is structurally similar to the dimerization domain of the tumour suppressor p53. PMID:27016739

  9. Flexibility of KorA, a plasmid-encoded, global transcription regulator, in the presence and the absence of its operator

    PubMed Central

    Rajasekar, Karthik V.; Lovering, Andrew L.; Dancea, Felician; Scott, David J.; Harris, Sarah A.; Bingle, Lewis E.H.; Roessle, Manfred; Thomas, Christopher M.; Hyde, Eva I.; White, Scott A.

    2016-01-01

    The IncP (Incompatibility group P) plasmids are important carriers in the spread of antibiotic resistance across Gram-negative bacteria. Gene expression in the IncP-1 plasmids is stringently controlled by a network of four global repressors, KorA, KorB, TrbA and KorC interacting cooperatively. Intriguingly, KorA and KorB can act as co-repressors at varying distances between their operators, even when they are moved to be on opposite sides of the DNA. KorA is a homodimer with the 101-amino acid subunits, folding into an N-terminal DNA-binding domain and a C-terminal dimerization domain. In this study, we have determined the structures of the free KorA repressor and two complexes each bound to a 20-bp palindromic DNA duplex containing its consensus operator sequence. Using a combination of X-ray crystallography, nuclear magnetic resonance spectroscopy, SAXS and molecular dynamics calculations, we show that the linker between the two domains is very flexible and the protein remains highly mobile in the presence of DNA. This flexibility allows the DNA-binding domains of the dimer to straddle the operator DNA on binding and is likely to be important in cooperative binding to KorB. Unexpectedly, the C-terminal domain of KorA is structurally similar to the dimerization domain of the tumour suppressor p53. PMID:27016739

  10. Plasmids in Frankia sp.

    PubMed

    Normand, P; Simonet, P; Butour, J L; Rosenberg, C; Moiroud, A; Lalonde, M

    1983-07-01

    A method to achieve cell lysis and isolate Frankia sp. plasmid DNA was developed. A screening of Frankia sp. strains belonging to different host compatibility groups (Alnus sp., Elaeagnus sp., Ceanothus sp.) showed that, of 39 strains tested, 4 (strains Cp11, ARgN22d, ArI3, and EUN1f) possessed plasmids ranging in size from 7.1 to 32.2 kilobase pairs as estimated from agarose gel electrophoresis and electron microscopy. A total of 11 plasmids were detected. PMID:6863219

  11. Natural plasmids of filamentous fungi.

    PubMed Central

    Griffiths, A J

    1995-01-01

    Among eukaryotes, plasmids have been found in fungi and plants but not in animals. Most plasmids are mitochondrial. In filamentous fungi, plasmids are commonly encountered in isolates from natural populations. Individual populations may show a predominance of one type, but some plasmids have a global distribution, often crossing species boundaries. Surveys have shown that strains can contain more than one type of plasmid and that different types appear to be distributed independently. In crosses, plasmids are generally inherited maternally. Horizontal transmission is by cell contact. Circular plasmids are common only in Neurospora spp., but linear plasmids have been found in many fungi. Circular plasmids have one open reading frame (ORF) coding for a DNA polymerase or a reverse transcriptase. Linear plasmids generally have two ORFs, coding for presumptive DNA and RNA polymerases with amino acid motifs showing homology to viral polymerases. Plasmids often attain a high copy number, in excess of that of mitochondrial DNA. Linear plasmids have a protein attached to their 5' end, and this is presumed to act as a replication primer. Most plasmids are neutral passengers, but several linear plasmids integrate into mitochondrial DNA, causing death of the host culture. Inferred amino acid sequences of linear plasmid ORFs have been used to plot phylogenetic trees, which show a fair concordance with conventional trees. The circular Neurospora plasmids have replication systems that seem to be evolutionary intermediates between the RNA and the DNA worlds. PMID:8531891

  12. Sequences of Two Related Multiple Antibiotic Resistance Virulence Plasmids Sharing a Unique IS26-Related Molecular Signature Isolated from Different Escherichia coli Pathotypes from Different Hosts

    PubMed Central

    Venturini, Carola; Hassan, Karl A.; Roy Chowdhury, Piklu; Paulsen, Ian T.; Walker, Mark J.; Djordjevic, Steven P.

    2013-01-01

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

  13. Plasmids encoding therapeutic agents

    DOEpatents

    Keener, William K.

    2007-08-07

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

  14. Plasmid detection, characterization and ecology

    PubMed Central

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

    2015-01-01

    Plasmids are important vehicles for rapid adaptation of bacterial populations to changing environmental conditions. To reduce the cost of plasmid carriage, it is thought that only a fraction of a local population carries plasmids or is permissive to plasmid uptake. Plasmids provide various accessory traits which might be beneficial under particular conditions. The genetic variation generated by plasmid carriage within populations ensures the robustness towards environmental change. Plasmid-mediated gene transfer plays an important role not only in the mobilization and dissemination of antibiotic resistance genes but also in the spread of degradative pathways and pathogenicity determinants of pathogens. Here we summarize the state-of-the-art methods to study the occurrence, abundance and diversity of plasmids in environmental bacteria. Increasingly, cultivation independent total community DNA methods are being used to characterize and quantify the diversity and abundance of plasmids in relation to various biotic and abiotic factors. An improved understanding of the ecology of plasmids and their hosts is crucial in the development of intervention strategies for antibiotic resistance gene spread. We discuss the potentials and limitations of methods used to determine the host range of plasmids as the ecology of plasmids is tightly linked to their hosts. The recent advances in sequencing technologies provide an enormous potential for plasmid classification, diversity and evolution studies but numerous challenges still exist. PMID:26104560

  15. Nonconjugative Plasmids Encoding Sulfanilamide Resistance

    PubMed Central

    Mitsuhashi, Susumu; Inoue, Kunio; Inoue, Matsuhisa

    1977-01-01

    Nonconjugative plasmids encoding sulfanilamide (Sa) resistance were demonstrated at a high frequency in Shigella and Escherichia coli strains resistant to sulfanilamide. These Sa plasmids were all compatible with the standard plasmids used in compatibility testing. The sizes of seven Sa plasmids were measured by electron microscopy and ranged from 1.79 to 2.08 μm, corresponding to 3.5 to 3.9 megadaltons. Images PMID:334067

  16. Toxin plasmids of Clostridium perfringens.

    PubMed

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

    2013-06-01

    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

  17. Toxin Plasmids of Clostridium perfringens

    PubMed Central

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

    2013-01-01

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

  18. Replication of Staphylococcal Multiresistance Plasmids

    PubMed Central

    Firth, Neville; Apisiridej, Sumalee; Berg, Tracey; O'Rourke, Brendon A.; Curnock, Steve; Dyke, Keith G. H.; Skurray, Ronald A.

    2000-01-01

    Based on structural and functional properties, three groups of large staphylococcal multiresistance plasmids have been recognized, viz., the pSK1 family, pSK41-like conjugative plasmids, and β-lactamase–heavy-metal resistance plasmids. Here we describe an analysis of the replication functions of a representative of each of these plasmid groups. The replication initiation genes from the Staphylococcus aureus plasmids pSK1, pSK41, and pI9789::Tn552 were found to be related to each other and to the Staphylococcus xylosus plasmid pSX267 and are also related to rep genes of several plasmids from other gram-positive genera. Nucleotide sequence similarity between pSK1 and pI9789::Tn552 extended beyond their rep genes, encompassing upstream divergently transcribed genes, orf245 and orf256, respectively. Our analyses revealed that genes encoding proteins related to the deduced orf245 product are variously represented, in several types of organization, on plasmids possessing six seemingly evolutionarily distinct types of replication initiation genes and including both theta-mode and rolling-circle replicons. Construction of minireplicons and subsequent functional analysis demonstrated that orf245 is required for the segregational stability of the pSK1 replicon. In contrast, no gene equivalent to orf245 is evident on the conjugative plasmid pSK41, and a minireplicon encoding only the pSK41 rep gene was found to exhibit a segregational stability approaching that of the parent plasmid. Significantly, the results described establish that many of the large multiresistance plasmids that have been identified in clinical staphylococci, which were formerly presumed to be unrelated, actually utilize an evolutionarily related theta-mode replication system. PMID:10735859

  19. Phenotypic plasticity in bacterial plasmids.

    PubMed Central

    Turner, Paul E

    2004-01-01

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

  20. Plasmid acquisition in microgravity

    NASA Technical Reports Server (NTRS)

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

    1995-01-01

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

  1. Evolved plasmid-host interactions reduce plasmid interference cost.

    PubMed

    Yano, Hirokazu; Wegrzyn, Katarznya; Loftie-Eaton, Wesley; Johnson, Jenny; Deckert, Gail E; Rogers, Linda M; Konieczny, Igor; Top, Eva M

    2016-09-01

    Antibiotic selection drives adaptation of antibiotic resistance plasmids to new bacterial hosts, but the molecular mechanisms are still poorly understood. We previously showed that a broad-host-range plasmid was poorly maintained in Shewanella oneidensis, but rapidly adapted through mutations in the replication initiation gene trfA1. Here we examined if these mutations reduced the fitness cost of TrfA1, and whether this was due to changes in interaction with the host's DNA helicase DnaB. The strains expressing evolved TrfA1 variants showed a higher growth rate than those expressing ancestral TrfA1. The evolved TrfA1 variants showed a lower affinity to the helicase than ancestral TrfA1 and were no longer able to activate the helicase at the oriV without host DnaA. Moreover, persistence of the ancestral plasmid was increased upon overexpression of DnaB. Finally, the evolved TrfA1 variants generated higher plasmid copy numbers than ancestral TrfA1. The findings suggest that ancestral plasmid instability can at least partly be explained by titration of DnaB by TrfA1. Thus under antibiotic selection resistance plasmids can adapt to a novel bacterial host through partial loss of function mutations that simultaneously increase plasmid copy number and decrease unfavorably high affinity to one of the hosts' essential proteins. PMID:27121483

  2. Large plasmids of avian Escherichia coli isolates.

    PubMed

    Doetkott, D M; Nolan, L K; Giddings, C W; Berryhill, D L

    1996-01-01

    The plasmid DNA of 30 Escherichia coli isolates from chickens was extracted and examined using techniques designed to isolate large plasmids. This plasmid DNA was examined for the presence of certain known virulence-related genes including cvaC, traT, and some aerobactin-related sequences. Seventeen of the 30 isolates contained from one to four plasmids greater than 50 kb in size. Eleven of these 17 strains possessed plasmids greater than 100 kb in size. Therefore, E. coli isolates of chickens frequently contain large plasmids, and many of these plasmids are likely to contain virulence-related sequences. PMID:8980827

  3. Plasmid-mediated quinolone resistance

    PubMed Central

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

    2014-01-01

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

  4. Stability of Penicillinase Plasmids in Staphylococcus aureus

    PubMed Central

    Johnston, L. H.; Dyke, K. G. H.

    1971-01-01

    The isolation of mutants of Staphylococcus aureus that are affected in the stability of penicillinase plasmids is described. One mutation is plasmid borne and results in nonreplication of the plasmid at 42 C. A second type of mutation is host-borne and gives rise to instability of both mcrI and mcrII penicillinase plasmids but not a tetracycline-resistant plasmid. Images PMID:4105036

  5. A plasmid in Legionella pneumophila.

    PubMed Central

    Knudson, G B; Mikesell, P

    1980-01-01

    Sixteen strains from the six serogroups of Legionella pneumophila were examined for the presence of extrachromosomal genetic elements by a modified cleared lysate procedure, dye-buoyant centrifugation, and agarose gel electrophoresis. Two strains, Atlanta-1 and Atlanta-2 from serogroup II, each contained a plasmid of cryptic function with a molecular weight of ca. 30 megadaltons. Images Fig. 1 PMID:7429628

  6. Origin and Evolution of Rickettsial Plasmids

    PubMed Central

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

    2016-01-01

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

  7. Chromate resistance plasmid in Pseudomonas fluorescens.

    PubMed Central

    Bopp, L H; Chakrabarty, A M; Ehrlich, H L

    1983-01-01

    Chromate resistance of Pseudomonas fluorescens LB300, isolated from chromium-contaminated sediment in the upper Hudson River, was found to be plasmid specified. Loss of the plasmid (pLHB1) by spontaneous segregation or mitomycin C curing resulted in a simultaneous loss of chromate resistance. Subsequent transformation of such strains with purified pLHB1 plasmid DNA resulted in a simultaneous re-acquisition of the chromate resistance phenotype and the plasmid. When pLHB1 was transferred by conjugation to Escherichia coli, the plasmid still conferred chromate resistance. PMID:6309741

  8. Bacterial Plasmids in Antarctic Natural Microbial Assemblages

    PubMed Central

    Kobori, Hiromi; Sullivan, Cornelius W.; Shizuya, Hiroaki

    1984-01-01

    Samples of psychrophilic and psychrotrophic bacteria were collected from sea ice, seawater, sediments, and benthic or ice-associated animals in McMurdo Sound, Antarctica. A total of 155 strains were isolated and tested for the presence of plasmids by DNA agarose gel electrophoresis. Thirty-one percent of the isolates carried at least one kind of plasmid. Bacterial isolates taken from sediments showed the highest plasmid incidence (42%), and isolates from seawater showed the lowest plasmid incidence (20%). Plasmids were significantly more frequent in the strains which had been first isolated from low-nutrient media (46%) than in the strains which had been isolated from high-nutrient media (25%). Multiple forms of plasmids were observed in two-thirds of the plasmid-carrying strains. A majority of the plasmids detected were estimated to have a mass of 10 megadaltons or less. Among 48 plasmid-carrying strains, 7 showed antibiotic resistance. It is concluded that bacterial plasmids are ubiquitous in natural microbial assemblages of the pristine marine ecosystem of Antarctica. Images PMID:16346621

  9. Virulence Plasmids of Spore-Forming Bacteria.

    PubMed

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

    2014-12-01

    Plasmid-encoded virulence factors are important in the pathogenesis of diseases caused by spore-forming bacteria. Unlike many other bacteria, the most common virulence factors encoded by plasmids in Clostridium and Bacillus species are protein toxins. Clostridium perfringens causes several histotoxic and enterotoxin diseases in both humans and animals and produces a broad range of toxins, including many pore-forming toxins such as C. perfringens enterotoxin, epsilon-toxin, beta-toxin, and NetB. Genetic studies have led to the determination of the role of these toxins in disease pathogenesis. The genes for these toxins are generally carried on large conjugative plasmids that have common core replication, maintenance, and conjugation regions. There is considerable functional information available about the unique tcp conjugation locus carried by these plasmids, but less is known about plasmid maintenance. The latter is intriguing because many C. perfringens isolates stably maintain up to four different, but closely related, toxin plasmids. Toxin genes may also be plasmid-encoded in the neurotoxic clostridia. The tetanus toxin gene is located on a plasmid in Clostridium tetani, but the botulinum toxin genes may be chromosomal, plasmid-determined, or located on bacteriophages in Clostridium botulinum. In Bacillus anthracis it is well established that virulence is plasmid determined, with anthrax toxin genes located on pXO1 and capsule genes on a separate plasmid, pXO2. Orthologs of these plasmids are also found in other members of the Bacillus cereus group such as B. cereus and Bacillus thuringiensis. In B. thuringiensis these plasmids may carry genes encoding one or more insecticidal toxins. PMID:26104459

  10. Electroporation of plasmid DNA to swine muscle.

    PubMed

    Bodles-Brakhop, Angela M; Draghia-Akli, Ruxandra; Broderick, Kate; Khan, Amir S

    2011-01-01

    For plasmid-mediated gene therapy applications, a major limitation to scale up from rodents to large animals is the low expression level of injected plasmid DNA. The electroporation technique, which results in the passage of foreign material through the cell membrane, is one method that has been shown to be effective at improving local plasmid uptake and consequently, expression levels. Previous studies have determined that optimized electroporation parameters (such as electric field intensity, number of pulses, lag time between plasmid injections and electroporations, and optimal plasmid formulation conditions) are dependent on the target muscle type and individual species. Here, we provide a detailed protocol to optimize conditions for the successful intramuscular electroporation of plasmid DNA to swine, a large animal model. Our results suggest that the technique is safe and effective for veterinary applications. Furthermore, these results provide evidence for the feasibility of upcoming human applications. PMID:21194033

  11. PENICILLINASE PLASMID DNA FROM Staphylococcus aureus*

    PubMed Central

    Rush, Mark G.; Gordon, C. N.; Novick, Richard P.; Warner, Robert C.

    1969-01-01

    A penicillinase plasmid from Staphylococcus aureus and three of its derivatives, all previously identified as extrachromosomal genetic elements, have been isolated in high yield as circular duplex DNA molecules. The wild-type plasmid was found by contour-length measurements of electron micrographs to have a molecular weight of 18.6 × 106 daltons. Two plasmids with deletions encompassing six and eight of the eleven known plasmid cistrons had molecular weights of 16.4 × 106 and 15.3 × 106 daltons, respectively. This information was used to establish approximate physical distances for the genetic map. A high-frequency transducing element also derived from the plasmid had a molecular weight of approximately 24 × 106 daltons. Although each plasmid preparation appeared homogeneous by ultracentrifugal analysis, electron micrographs always revealed the presence of a low percentage of complex oligomeric forms, particularly circular and catenated dimers. Images PMID:5260933

  12. Homemade Site Directed Mutagenesis of Whole Plasmids

    PubMed Central

    Laible, Mark; Boonrod, Kajohn

    2009-01-01

    Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers. PMID:19488024

  13. Microwave effects on plasmid DNA

    SciTech Connect

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

    1987-05-01

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

  14. pLS101 plasmid vector

    DOEpatents

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

    1985-02-19

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

  15. pLS010 plasmid vector

    DOEpatents

    Lacks, Sanford A.; Balganesh, Tanjore S.

    1988-01-01

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

  16. Preparation of Saccharomyces cerevisiae expression plasmids.

    PubMed

    Drew, David; Kim, Hyun

    2012-01-01

    Expression plasmids for Saccharomyces cerevisiae offer a wide choice of vector copy number, promoters of varying strength and selection markers. These expression plasmids are usually shuttle vectors that can be propagated both in yeast and bacteria, making them useful in gene cloning. For heterologous production of membrane proteins, we used the green fluorescent protein (GFP) fusion technology which was previously developed in the Escherichia coli system. We designed an expression plasmid carrying an inducible GAL1 promoter, a gene encoding a membrane protein of interest and the GFP-octa-histidine sequence. Here we describe construction of multi-copy yeast expression plasmids by homologous recombination in S. cerevisiae. PMID:22454112

  17. Shifts in Abundance and Diversity of Mobile Genetic Elements after the Introduction of Diverse Pesticides into an On-Farm Biopurification System over the Course of a Year

    PubMed Central

    Dealtry, Simone; Holmsgaard, Peter N.; Dunon, Vincent; Jechalke, Sven; Ding, Guo-Chun; Krögerrecklenfort, Ellen; Heuer, Holger; Hansen, Lars H.; Springael, Dirk; Zühlke, Sebastian; Sørensen, Søren J.

    2014-01-01

    Biopurification systems (BPS) are used on farms to control pollution by treating pesticide-contaminated water. It is assumed that mobile genetic elements (MGEs) carrying genes coding for enzymes involved in degradation might contribute to the degradation of pesticides. Therefore, the composition and shifts of MGEs, in particular, of IncP-1 plasmids carried by BPS bacterial communities exposed to various pesticides, were monitored over the course of an agricultural season. PCR amplification of total community DNA using primers targeting genes specific to different plasmid groups combined with Southern blot hybridization indicated a high abundance of plasmids belonging to IncP-1, IncP-7, IncP-9, IncQ, and IncW, while IncU and IncN plasmids were less abundant or not detected. Furthermore, the integrase genes of class 1 and 2 integrons (intI1, intI2) and genes encoding resistance to sulfonamides (sul1, sul2) and streptomycin (aadA) were detected and seasonality was revealed. Amplicon pyrosequencing of the IncP-1 trfA gene coding for the replication initiation protein revealed high IncP-1 plasmid diversity and an increase in the abundance of IncP-1β and a decrease in the abundance of IncP-1ε over time. The data of the chemical analysis showed increasing concentrations of various pesticides over the course of the agricultural season. As an increase in the relative abundances of bacteria carrying IncP-1β plasmids also occurred, this might point to a role of these plasmids in the degradation of many different pesticides. PMID:24771027

  18. Natural plasmid transformation in Escherichia coli.

    PubMed

    Tsen, Suh-Der; Fang, Suh-Sen; Chen, Mei-Jye; Chien, Jun-Yi; Lee, Chih-Chun; Tsen, Darwin Han-Lin

    2002-01-01

    Although Escherichia coli does not have a natural transformation process, strains of E. coli can incorporate extracellular plasmids into cytoplasm 'naturally' at low frequencies. A standard method was developed in which stationary phase cells were concentrated, mixed with plasmids, and then plated on agar plates with nutrients which allowed cells to grow. Transformed cells could then be selected by harvesting cells and plating again on selective agar plates. Competence developed in the lag phase, but disappeared during exponential growth. As more plasmids were added to the cell suspension, the number of transformants increased, eventually reaching a plateau. Supercoiled monomeric or linear concatemeric DNA could transform cells, while linear monomeric DNA could not. Plasmid transformation was not related to conjugation and was recA-independent. Most of the E. coli strains surveyed had this process. All tested plasmids, except pACYC184, could transform E. coli. Insertion of a DNA fragment containing the ampicillin resistance gene into pACYC184 made the plasmid transformable. By inserting random 20-base-pair oligonucleotides into pACYC184 and selecting for transformable plasmids, a most frequent sequence was identified. This sequence resembled the bacterial interspersed medium repetitive sequence of E. coli, suggesting the existence of a recognition sequence. We conclude that plasmid natural transformation exists in E. coli. PMID:12065899

  19. CONSTRUCTION OF PLASMIDS FOR USE IN RISK ASSESSMENT RESEARCH

    EPA Science Inventory

    The report describes a series of selftransmissible and nonselftransmissible (cloning vector) plasmids constructed to compare results from different laboratory tests and plasmid systems. Plasmids were designed to overcome problems of reproducibility, confusion due to use of differ...

  20. Mini-F plasmid genes that couple host cell division to plasmid proliferation.

    PubMed Central

    Ogura, T; Hiraga, S

    1983-01-01

    A mechanism for stable maintenance of plasmids, besides the replication and partition mechanisms, has been found to be specified by genes of a mini-F plasmid. An oriC plasmid carrying both a mini-F segment necessary for partition [coordinates 46.4-49.4 kilobase pairs (kb) on the F map] and another segment (42.9-43.6 kb), designated ccd (coupled cell division), is more stably maintained than are oriC plasmids carrying only the partition segment; the stability is comparable to that of the parental mini-F plasmid. When replication of a plasmid carrying ccd is prevented and the plasmid copy number decreases, to as few as one per cell, host cell division is inhibited, but not increase of turbidity or chromosome replication. Appearance of plasmid-free segregants is therefore effectively prevented under such conditions. Experimental results suggest that reduction of the copy number of plasmids carrying the ccd region causes an inhibition of cell division and that the ccd region can be dissected into two functional regions; one (ccdB) inhibits cell division and the other (ccdA) releases the inhibition. The interplay of the ccdA and ccdB genes promotes stable plasmid maintenance by coupling host cell division to plasmid proliferation. PMID:6308648

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

    PubMed Central

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

    1992-01-01

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

  2. High-level plasmid-mediated gentamicin resistance and pheromone response of plasmids present in clinical isolates of Enterococcus faecalis.

    PubMed Central

    Shiojima, M; Tomita, H; Tanimoto, K; Fujimoto, S; Ike, Y

    1997-01-01

    Eleven pheromone-responding plasmids encoding erythromycin or gentamicin resistance were isolated from multiresistant clinical Enterococcus faecalis isolates. The plasmids were classified into six types with respect to their pheromone responses. The three erythromycin resistance plasmids responded to different pheromones. Of the eight gentamicin resistance plasmids, four plasmids responded to same pheromone. Southern hybridization studies showed that the genes involved in regulation of the pheromone response were conserved in the drug resistance plasmids. PMID:9056018

  3. PlasmID: a centralized repository for plasmid clone information and distribution

    PubMed Central

    Zuo, Dongmei; Mohr, Stephanie E.; Hu, Yanhui; Taycher, Elena; Rolfs, Andreas; Kramer, Jason; Williamson, Janice; LaBaer, Joshua

    2007-01-01

    The Plasmid Information Database (PlasmID; ) was developed as a community-based resource portal to facilitate search and request of plasmid clones shared with the Dana-Farber/Harvard Cancer Center (DF/HCC) DNA Resource Core. PlasmID serves as a central data repository and enables researchers to search the collection online using common gene names and identifiers, keywords, vector features, author names and PubMed IDs. As of October 2006, the repository contains >46 000 plasmids in 98 different vectors, including cloned cDNA and genomic fragments from 26 different species. Moreover, the clones include plasmid vectors useful for routine and cutting-edge techniques; functionally related sets of human cDNA clones; and genome-scale gene collections for Saccharomyces cerevisiae, Pseudomonas aeruginosa, Yersinia pestis, Francisella tularensis, Bacillus anthracis and Vibrio cholerae. Information about the plasmids has been fully annotated in adherence with a high-quality standard, and clone samples are stored as glycerol stocks in a state-of-the-art automated −80°C freezer storage system. Clone replication and distribution is highly automated to minimize human error. Infor-mation about vectors and plasmid clones, including downloadable maps and sequence data, is freely available online. Researchers interested in requesting clone samples or sharing their own plasmids with the repository can visit the PlasmID website for more information. PMID:17132831

  4. Generalized Transduction of Small Yersinia enterocolitica Plasmids

    PubMed Central

    Hertwig, Stefan; Popp, Andreas; Freytag, Barbara; Lurz, Rudi; Appel, Bernd

    1999-01-01

    To study phage-mediated gene transfer in Yersinia, the ability of Yersinia phages to transduce naturally occurring plasmids was investigated. The transduction experiments were performed with a temperate phage isolated from a pathogenic Yersinia enterocolitica strain and phage mixtures isolated from sewage. Small plasmids (4.3 and 5.8 kb) were transduced at a frequency of 10−5 to 10−7/PFU. However, we could not detect the transduction of any indigenous virulence plasmid (ca. 72 kb) in pathogenic Yersinia strains. Transductants obtained by infection with the temperate phage were lysogenic and harbored the phage genome in their chromosomes. PMID:10473387

  5. Plasmids as Tools for Containment.

    PubMed

    García, José L; Díaz, Eduardo

    2014-10-01

    Active containment systems are a major tool for reducing the uncertainty associated with the introduction of monocultures, genetically engineered or not, into target habitats for a large number of biotechnological applications (e.g., bioremediation, bioleaching, biopesticides, biofuels, biotransformations, live vaccines, etc.). While biological containment reduces the survival of the introduced organism outside the target habitat and/or upon completion of the projected task, gene containment strategies reduce the lateral spread of the key genetic determinants to indigenous microorganisms. In fundamental research, suicide circuits become relevant tools to address the role of gene transfer, mainly plasmid transfer, in evolution and how this transfer contributes to genome plasticity and to the rapid adaptation of microbial communities to environmental changes. Many lethal functions and regulatory circuits have been used and combined to design efficient containment systems. As many new genomes are being sequenced, novel lethal genes and regulatory elements are available, e.g., new toxin-antitoxin modules, and they could be used to increase further the current containment efficiencies and to expand containment to other organisms. Although the current containment systems can increase the predictability of genetically modified organisms in the environment, containment will never be absolute, due to the existence of mutations that lead to the appearance of surviving subpopulations. In this sense, orthogonal systems (xenobiology) appear to be the solution for setting a functional genetic firewall that will allow absolute containment of recombinant organisms. PMID:26104372

  6. Topological Behavior of Plasmid DNA

    PubMed Central

    Higgins, N. Patrick; Vologodskii, Alexander V.

    2015-01-01

    The discovery of the B-form structure of DNA by Watson and Crick led to an explosion of research on nucleic acids in the fields of biochemistry, biophysics, and genetics. Powerful techniques were developed to reveal a myriad of different structural conformations that change B-DNA as it is transcribed, replicated, and recombined and as sister chromosomes are moved into new daughter cell compartments during cell division. This article links the original discoveries of superhelical structure and molecular topology to non-B form DNA structure and contemporary biochemical and biophysical techniques. The emphasis is on the power of plasmids for studying DNA structure and function. The conditions that trigger the formation of alternative DNA structures such as left-handed Z-DNA, inter- and intra-molecular triplexes, triple-stranded DNA, and linked catenanes and hemicatenanes are explained. The DNA dynamics and topological issues are detailed for stalled replication forks and for torsional and structural changes on DNA in front of and behind a transcription complex and a replisome. The complex and interconnected roles of topoisomerases and abundant small nucleoid association proteins are explained. And methods are described for comparing in vivo and in vitro reactions to probe and understand the temporal pathways of DNA and chromosome chemistry that occur inside living cells. PMID:26104708

  7. Plasmid DNA fermentation strategies: influence on plasmid stability and cell physiology.

    PubMed

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

    2012-03-01

    In order to provide sufficient pharmaceutical-grade plasmid DNA material, it is essential to gain a comprehensive knowledge of the bioprocesses involved; so, the development of protocols and techniques that allow a fast monitoring of process performance is a valuable tool for bioprocess design. Regarding plasmid DNA production, the metabolic stress of the host strain as well as plasmid stability have been identified as two of the key parameters that greatly influence plasmid DNA yields. The present work describes the impact of batch and fed-batch fermentations using different C/N ratios and different feeding profiles on cell physiology and plasmid stability, investigating the potential of these two monitoring techniques as valuable tools for bioprocess development and design. The results obtained in batch fermentations showed that plasmid copy number values suffered a pronounced increase at the end of almost all fermentation conditions tested. Regarding fed-batch fermentations, the strategies with exponential feeding profiles, in contrast with those with constant feeding, showed higher biomass and plasmid yields, the maximum values obtained for these two parameters being 95.64 OD(600) and 344.3 mg plasmid DNA (pDNA)/L, respectively, when using an exponential feed rate of 0.2 h(-1). Despite the results obtained, cell physiology and plasmid stability monitoring revealed that, although higher pDNA overall yields were obtained, this fermentation exhibited lower plasmid stability and percentage of viable cells. In conclusion, this study allowed clarifying the bioprocess performance based on cell physiology and plasmid stability assessment, allowing improvement of the overall process and not only plasmid DNA yield and cell growth. PMID:22089386

  8. Expression Plasmids for Use in Candida glabrata

    PubMed Central

    Zordan, Rebecca E.; Ren, Yuxia; Pan, Shih-Jung; Rotondo, Giuseppe; Peñas, Alejandro De Las; Iluore, Joseph; Cormack, Brendan P.

    2013-01-01

    We describe a series of CEN/ARS episomal plasmids containing different Candida glabrata promoters, allowing for a range of constitutive or regulated expression of proteins in C. glabrata. The set of promoters includes three constitutive promoters (EGD2pr, HHT2pr, PDC1pr), two macrophage/phagocytosis-induced promoters (ACO2pr, LYS21pr), and one nutritionally regulated promoter (MET3pr). Each promoter was cloned into two plasmid backbones that differ in their selectable marker, URA3, or the dominant-selectable NAT1 gene, which confers resistance to the drug nourseothricin. Expression from the 12 resulting plasmids was assessed using GFP as a reporter and flow cytometry or quantitative reverse-transcription polymerase chain reaction to assess expression levels. Together this set of plasmids expands the toolkit of expression vectors available for use with C. glabrata. PMID:23934995

  9. SIMPLAS: A Simulation of Bacterial Plasmid Maintenance.

    ERIC Educational Resources Information Center

    Dunn, A.; And Others

    1988-01-01

    This article describes a computer simulation of bacterial physiology during growth in a chemostat. The program was designed to help students to appreciate and understand the related effects of parameters which influence plasmid persistence in bacterial populations. (CW)

  10. Shigella sonnei plasmids: evidence that a large plasmid is necessary for virulence.

    PubMed Central

    Sansonetti, P J; Kopecko, D J; Formal, S B

    1981-01-01

    Virulent form I Shigella sonnei strains contain a 120-megadalton plasmid that is absent in their form II derivatives, which are always avirulent and devoid of O side chains. In the present study, 165 biochemical and antibiotic traits were assessed, but no experimentally useful phenotype could be associated with this large form I plasmid. Therefore, the form I plasmids of several S. sonnei strains were tagged with the antibiotic resistance transposons Tn3, Tn5, or Tn10. Transposon-tagged form I plasmids were not self-transmissible, but could be mobilized by the plasmid R386. Form II S. sonnei transconjugants for the form I plasmid acquired both virulence and the ability to synthesize form I antigen, establishing that these properties are plasmid mediated. Further studies indicate that this 120-megadalton form I plasmid is physically unstable in any of several host bacteria and suggest that it is a member of the FI incompatibility group. Also, two commonly observed, small plasmids of S. sonnei, of 3.2 and 3.9 megadaltons, were shown to encode either colicin E1 production or resistance to streptomycin and sulfonamide, respectively. Images PMID:6271687

  11. Denitrification by Alcaligenes eutrophus is plasmid dependent.

    PubMed Central

    Römermann, D; Friedrich, B

    1985-01-01

    Curing of the hydrogenase-specifying megaplasmid pHG indigenous to strains of the facultative lithoautotrophic bacterium Alcaligenes eutrophus was correlated with a loss of denitrifying ability (Nitd). The retransfer of plasmid pHG1 reconstituted the Nitd phenotype. Plasmid-free mutants were still capable of converting some nitrate to nitrite, but they did not metabolize nitrite under anaerobic conditions. PMID:3886640

  12. Clostridium perfringens type A–E toxin plasmids

    PubMed Central

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

    2014-01-01

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

  13. Engineering large functional plasmids for biosafety.

    PubMed

    Cangelosi, Chris; Shank, Caroline; Santiago, Clayton; Wilson, James W

    2013-11-01

    Large bacterial plasmid constructs (generally 25-100 kb, but can be greater), such as those engineered with DNA encoding specific functions such as protein secretion or specialized metabolism, can carry antibiotic resistance genes and/or conjugation systems that typically must be removed before use in medical or environmental settings due to biosafety concerns. However, a convenient in vivo recombineering approach for intact large plasmids to sequentially remove multiple different genes using non-antibiotic selection methods is not described in the literature to our knowledge. We developed strategies and reagents for convenient removal of antibiotic resistance markers and conjugation genes while retaining non-antibiotic-based plasmid selection to increase practical utility of large engineered plasmids. This approach utilizes targeted lambda Red recombination of PCR products encoding the trpE and asd genes and as well as FLP/FRT-mediated marker removal. This is particularly important given that use of restriction enzymes with plasmids of this size is extremely problematic and often not feasible. This report provides the first example of the trpE gene/tryptophan prototrophy being used for recombineering selection. We applied this strategy to the plasmids R995+SPI-1 and R995+SPI-2 which encode cloned type III secretion systems to allow protein secretion and substrate delivery to eukaryotic cells. The resulting constructs are functional, stably maintained under conditions where the original constructs are unstable, completely defective for conjugative transfer, and transferred via electroporation. PMID:24055203

  14. Immobilization of plasmid DNA in bacterial ghosts.

    PubMed

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

    2005-02-16

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

  15. Curing Both Virulent Mega-Plasmids from Bacillus anthracis Wild-Type Strain A16 Simultaneously Using Plasmid Incompatibility.

    PubMed

    Wang, Dongshu; Gao, Zhiqi; Wang, Huagui; Feng, Erling; Zhu, Li; Liu, Xiankai; Wang, Hengliang

    2015-10-28

    Plasmid-cured derivative strains of Bacillus anthracis are frequently used in laboratory studies. Plasmid incompatibility, which does not increase the risk of chromosomal mutation, is a useful method for plasmid curing. However, in bacteria containing multiple plasmids, it often requires the sequential introduction of multiple, specific incompatibility plasmids. This lengthy process renders the traditional plasmid incompatibility method inefficient and mutation-prone. In this study, we successfully cured plasmids pXO1 and pXO2 from B. anthracis A16 simultaneously using only one recombinant incompatible plasmid, pKORT, to obtain a plasmid-free strain, designated A16DD. This method may also be useful for the simultaneous, one-step curing of multiple plasmids from other bacteria, including Bacillus thuringiensis and Yersinia pestis. PMID:26059513

  16. Inducible Escherichia coli fermentation for increased plasmid DNA production.

    PubMed

    Carnes, Aaron E; Hodgson, Clague P; Williams, James A

    2006-11-01

    Bacterial plasmids are the vectors of choice for DNA vaccines and short-term gene therapeutics. Growing plasmid DNA by microbial (Escherichia coli) fermentation is usually combined with alkaline lysis/chromatography methods of purification. To date, typical plasmid fermentation media and processes result in yields of 100-250 mg of plasmid DNA/l of culture medium, using standard high-copy pUC origin-containing plasmids. In order to address this initial and yield-limiting upstream step, we identified novel fermentation control parameters for fed-batch fermentation. The resulting fermentation strategies significantly increased specific plasmid yield with respect to cell mass while enhancing plasmid integrity and maintaining supercoiled DNA content. Fed-batch fermentation yield exceeding 1000 mg of plasmid DNA/l was obtained after reduction of plasmid-mediated metabolic burden during growth, and yields up to 1500 mg of plasmid DNA/l have been achieved with optimized plasmid backbones. Interestingly, by inducing high plasmid levels after sufficient biomass accumulation at low temperature and restricted growth, cells were able to tolerate significantly higher plasmid quantities than cells grown by conventional processes. This 5-10-fold increase in plasmid yield dramatically decreases plasmid manufacturing costs and improves the effectiveness of downstream purification by reducing the fraction of impurities. PMID:16819941

  17. Exploring the complex response to linuron of bacterial communities from biopurification systems by means of cultivation-independent methods.

    PubMed

    Dealtry, Simone; Nour, Eman H; Holmsgaard, Peter N; Ding, Guo-Chun; Weichelt, Viola; Dunon, Vincent; Heuer, Holger; Hansen, Lars H; Sørensen, Søren J; Springael, Dirk; Smalla, Kornelia

    2016-02-01

    On-farm biopurification systems (BPSs) treat pesticide-contaminated wastewater at farms through biodegradation and sorption processes. However, information on the microbiota involved in pesticide removal in BPSs is scarce. Here we report on the response of BPS bacterial communities to the herbicide linuron (BPS(+)) compared with the control (BPS(-)) in a microcosm experiment. Both denaturing gradient gel electrophoresis (DGGE) and pyrosequencing of 16S rRNA gene fragments amplified from community DNA indicated shifts in the bacterial community after linuron application. Responding populations belonged to taxa that were previously reported from linuron degrading consortia cultivated from soil (Hyphomicrobiaceae, Comamonadaceae, Micrococcaceae). In addition, numerous taxa with increased relative abundance were identified that were previously not associated with linuron degradation. The relative abundance of IncP-1 korB copies increased in response to linuron application. Amplicon pyrosequencing of IncP-1 trfA genes revealed a high IncP-1 plasmid diversity and suggested that populations carrying IncP-1β plasmids increased in relative abundance. Transferable mercury resistance plasmids were exogenously captured from BPS(+)/BPS(-), and in three transconjugants from BPS(+) the gene hylA was detected. Our data suggest the existence of a multispecies linuron degrading bacterial food web and an involvement of IncP-1 plasmids in the adaptation of bacterial communities to pesticide pollution in BPSs. PMID:26705572

  18. In vivo visualization of type II plasmid segregation: bacterial actin filaments pushing plasmids

    PubMed Central

    Campbell, Christopher S.; Mullins, R. Dyche

    2007-01-01

    Type II par operons harness polymerization of the dynamically unstable actin-like protein ParM to segregate low-copy plasmids in rod-shaped bacteria. In this study, we use time-lapse fluorescence microscopy to follow plasmid dynamics and ParM assembly in Escherichia coli. Plasmids lacking a par operon undergo confined diffusion with a diffusion constant of 5 × 10−5 μm2/s and a confinement radius of 0.28 μm. Single par-containing plasmids also move diffusively but with a larger diffusion constant (4 × 10−4 μm2/s) and confinement radius (0.42 μm). ParM filaments are dynamically unstable in vivo and form spindles that link pairs of par-containing plasmids and drive them rapidly (3.1 μm/min) toward opposite poles of the cell. After reaching the poles, ParM filaments rapidly and completely depolymerize. After ParM disassembly, segregated plasmids resume diffusive motion, often encountering each other many times and undergoing multiple rounds of ParM-dependent segregation in a single cell cycle. We propose that in addition to driving segregation, the par operon enables plasmids to search space and find sister plasmids more effectively. PMID:18039937

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

    PubMed

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

    2016-10-10

    Antibiotic resistance is a serious problem in health care and is of widespread public concern. Conjugative plasmids are the most important vectors in the dissemination of antibiotic resistance genes. In this study, we determined the complete sequence of plasmid pNA6, a plasmid which was isolated from the sediments of Haihe River. This plasmid confers reduced susceptibility to ampicillin, erythromycin and sulfamethoxazole. The complete sequence of plasmid pNA6 was 52,210bp in length with an average G+C content of 52.70%. Plasmid pNA6 belongs to the IncU group by sequence queries against the GenBank database. This plasmid has a typical IncU backbone and shows the highest similarities with plasmid RA3 and plasmid pFBAOT6. Plasmid pNA6 carries a class 1 integron consisting of aacA4, ereA and dfrA1 genes. Moreover, plasmid pNA6 also harbors a blaTEM-1-containing complex structure which inserted into the replication region and maintenance region. This insertion site has never been found on other IncU plasmids. The sequencing of plasmid pNA6 will add new sequence information to IncU family plasmids and enhance our understanding of the plasticity of IncU family plasmids. PMID:27374151

  20. BioShuttle-mediated Plasmid Transfer

    PubMed Central

    Braun, Klaus; von Brasch, Leonie; Pipkorn, Ruediger; Ehemann, Volker; Jenne, Juergen; Spring, Herbert; Debus, Juergen; Didinger, Bernd; Rittgen, Werner; Waldeck, Waldemar

    2007-01-01

    An efficient gene transfer into target tissues and cells is needed for safe and effective treatment of genetic diseases like cancer. In this paper, we describe the development of a transport system and show its ability for transporting plasmids. This non-viral peptide-based BioShuttle-mediated transfer system consists of a nuclear localization address sequence realizing the delivery of the plasmid phNIS-IRES-EGFP coding for two independent reporter genes into nuclei of HeLa cells. The quantification of the transfer efficiency was achieved by measurements of the sodium iodide symporter activity. EGFP gene expression was measured with Confocal Laser Scanning Microscopy and quantified with biostatistical methods by analysis of the frequency of the amplitude distribution in the CLSM images. The results demonstrate that the “BioShuttle”-Technology is an appropriate tool for an effective transfer of genetic material carried by a plasmid. PMID:18026568

  1. Plasmid DNA from the acetotrophic methanogen Methanosarcina acetivorans

    SciTech Connect

    Sowers, K.R.; Gunsalus, R.P. )

    1988-10-01

    Nine acetotrophic and three methylotrophic strains of methane-producing bacteria were screened for the presence of plasmid DNA. Plasmids were detected in three marine isolates, including Methanosarcina acetivorans. All three plasmids appeared to be similar based on size and restriction site analyses. The plasmid from M. acetivorans, designated pC2A, was approximately 5.1 kilobase pairs in size and was estimated to be present in a low copy number of six plasmids per genome. Multimers were also observed. A restriction map was constructed. The function of this plasmid is cryptic.

  2. Electrotransfer of Plasmid Vector DNA into Muscle

    NASA Astrophysics Data System (ADS)

    Miyazaki, Satsuki; Miyazaki, Jun-Ichi

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

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

    PubMed Central

    Hill, K E; Weightman, A J; 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 mobilizing plasmids isolated ranged from 40 kb to over 200 kb, and 19 of 54 were found to encode mercury resistance. Plasmid-encoded resistance to tetracycline and streptomycin was also found but not resistance to UV light or various heavy metals. Eight plasmids of epilithic bacteria, analyzed by comparing restriction fragmentation patterns, showed significant differences between those isolated from different independent matings. Optimal temperatures for mobilization of pD10 were between 15 and 25 degrees C. Four mercury resistance plasmids were found to be broad host range, transferring mercury resistance and mobilizing pD10 readily to representative species of beta- and gamma-purple bacteria. In general, frequencies of pD10 mobilization by plasmids of epilithic bacteria were 2 to 3 orders of magnitude lower than conjugal transfer frequencies. Thus, there is a high potential for exchange of recombinant genes introduced into the epilithon by mobilization between a variety of bacterial species. Images PMID:1599248

  4. Spread of Plasmids Carrying Multiple GES Variants.

    PubMed

    Cuzon, Gaelle; Bogaerts, Pierre; Bauraing, Caroline; Huang, Te-Din; Bonnin, Rémy A; Glupczynski, Youri; Naas, Thierry

    2016-08-01

    Five GES-producing Enterobacteriaceae isolates that displayed an extended-spectrum β-lactamase (ESBL) phenotype harbored two GES variants: GES-7 ESBL and GES-6 carbapenemase. In all isolates, the two GES alleles were located on the same integron that was inserted into an 80-kb IncM1 self-conjugative plasmid. Whole-genome sequencing suggested in vivo horizontal gene transfer of the plasmid along with clonal diffusion of Enterobacter cloacae To our knowledge, this is the first description in Europe of clustered Enterobacteriaceae isolates carrying two GES β-lactamases, of which one has extended activity toward carbapenems. PMID:27216071

  5. Plasmid maintenance and protein overproduction in selective recycle bioreactors.

    PubMed

    Ogden, K L; Davis, R H

    1991-02-20

    A new plasmid construct has been used in conjunction with selective recycle to successfully maintain otherwise unstable plasmid-bearing E. coli cells in a continuous bioreactor and to produce significant amounts of the plasmid-encoded protein beta-lactamase. The plasmid is constructed so that pilin expression, which leads to bacterial flocculation, is under control of the tac operon. The plasmid-bearing cells are induced to flocculate in the separator, whereas cell growth and product synthesis occur in the main fermentation vessel without the inhibiting effects of pilin production. Selective recycle allows for the maintenance of the plasmid-bearing cells by separating flocculent, plasmid-bearing cells from nonflocculent, segregant cells in an inclined settler, and recycling only the plasmid-bearing cells to the reactor. As a result, product expression levels are maintained that are more than ten times the level achieved without selective recycle. All experimental data agree well with theoretical predictions. PMID:18597374

  6. Plasmid-protein relaxation complexes in Staphylococcus aureus.

    PubMed

    Novick, R

    1976-09-01

    Protein-deoxyribonucleic acid relaxation complexes have been demonstrated for six Staphylococcus aureus plasmids out of sixteen examined. Four of these encode stretomycin resistence, have molecular weights of about 2.7 x 10(6), and are isolated as supercoiled molecules that are virtally 100% relaxable by treatment with sodium dodecyl sulfate. It is probable that these four isolates represent a single widely disseminated plasmid species. The other two plasmids showing relaxation complexes have molecular weights of about 3 x 10(6) and encode chloramphenicol resistance. The complexes in these cases are unstable, and it has not been possible to induce more than 50% relaxation by any of the standard treatments. Ten other plasmids do not show detectable complexes. These include three penicillinase plasmids, four tetracycline-resistance plasmids, one plasmid carrying kanamycin-neomycin resistance, and finally, two chloramphenicol-resistance plasmids. PMID:956124

  7. DISTRIBUTION OF PLASMIDS IN GROUNDWATER BACTERIA

    EPA Science Inventory

    Bacteria isolated from groundwater aquifer core materials of pristine aquifers at Lula and Pickett, Oklahoma, and from a site with a history of aromatic hydrocarbon contamination and natural renovation located at Conroe, Texas, were screened for the presence of plasmid Deoxyribon...

  8. DYNAMICS OF PLASMID TRANSFER ON SURFACES

    EPA Science Inventory

    A protocol was developed to study the dynamics of growth and plasmid transfer in surface populations of bacteria. his method allows for quantitative estimates of cell population densities over time, as well as microscopic observations of colony growth and interactions. sing this ...

  9. Plasmid mediated enhancement of uv resistance in Streptococcus faecalis

    SciTech Connect

    Miehl, R.; Miller, M.; Yasbin, R.E.

    1980-01-01

    A 38.5-Mdal plasmid of Streptococcus faecalis subdp. zymogenes has been shown to enhance survival following uv irradiation. In addition, the presence of this plasmid increases the mutation frequencies following uv irradiation and enhanced W-reactivation. The data presented indicate that S. faecalis has an inducible error-prone repair system and that the plasmid enhances these repair functions.

  10. Compositional discordance between prokaryotic plasmids and host chromosomes

    PubMed Central

    van Passel, Mark WJ; Bart, Aldert; Luyf, Angela CM; van Kampen, Antoine HC; van der Ende, Arie

    2006-01-01

    Background Most plasmids depend on the host replication machinery and possess partitioning genes. These properties confine plasmids to a limited range of hosts, yielding a close and presumably stable relationship between plasmid and host. Hence, it is anticipated that due to amelioration the dinucleotide composition of plasmids is similar to that of the genome of their hosts. However, plasmids are also thought to play a major role in horizontal gene transfer and thus are frequently exchanged between hosts, suggesting dinucleotide composition dissimilarity between plasmid and host genome. We compared the dinucleotide composition of a large collection of plasmids with that of their host genomes to shed more light on this enigma. Results The dinucleotide frequency, coined the genome signature, facilitates the identification of putative horizontally transferred DNA in complete genome sequences, since it was found to be typical for a certain genome, and similar between related species. By comparison of the genome signature of 230 plasmid sequences with that of the genome of each respective host, we found that in general the genome signature of plasmids is dissimilar from that of their host genome. Conclusion Our results show that the genome signature of plasmids does not resemble that of their host genome. This indicates either absence of amelioration or a less stable relationship between plasmids and their host. We propose an indiscriminate lifestyle for plasmids preserving the genome signature discordance between these episomes and host chromosomes. PMID:16480495

  11. Plasmids captured in C. metallidurans CH34: defining the PromA family of broad-host-range plasmids.

    PubMed

    Van der Auwera, Géraldine A; Król, Jaroslaw E; Suzuki, Haruo; Foster, Brian; Van Houdt, Rob; Brown, Celeste J; Mergeay, Max; Top, Eva M

    2009-08-01

    The self-transmissible, broad-host-range (BHR) plasmid pMOL98 was previously isolated from polluted soil using a triparental plasmid capture approach and shown to possess a replicon similar to that of the BHR plasmids pSB102 and pIPO2. Here, complete sequence analysis and comparative genomics reveal that the 55.5 kb nucleotide sequence of pMOL98 shows extensive sequence similarity and synteny with the BHR plasmid family that now includes pIPO2, pSB102, pTER331, and pMRAD02. They share a plasmid backbone comprising replication, partitioning and conjugative transfer functions. Comparison of the variable accessory regions of these plasmids shows that the majority of natural transposons, as well as the mini-transposon used to mark the plasmids, are inserted in the parA locus. The transposon unique to pMOL98 appears to have inserted from the chromosome of the recipient strain used in the plasmid capture procedure. This demonstrates the necessity for careful screening of plasmids and host chromosomes to avoid mis-interpretation of plasmid genome content. The presence of very similar BHR plasmids with different accessory genes in geographically distinct locations suggests an important role in horizontal gene exchange and bacterial adaptation for this recently defined plasmid group, which we propose to name "PromA". PMID:19259779

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-12-01

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

  14. Plasmid-associated aggregation in Thermus thermophilus HB8

    SciTech Connect

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

    1990-01-01

    Thermus thermophilus HB8, a moderate thermophile, exhibits visible aggregation when growing on a rich broth. Strain HB8 also contains two cryptic plasmids. The authors isolated cured strains from HB8 and observed that loss of the 47-MDa plasmid was correlated with loss of aggregation. An enrichment procedure was developed for aggregating cells and used to demonstrate that aggregation was restored upon transformation of a cured strain with plasmid DNA. The aggregation phenotype of transformed cells was variably stable; most did not retain either the plasmid or the phenotype for prolonged periods of growth. Hybridization experiments using a partial sequence from the 47-MDa plasmid suggested the presence of a repeated DNA sequence on this plasmid and on the chromosome. This is the first report of a phenotype associated with a plasmid from a Thermus strain.

  15. Gene and cell survival: lessons from prokaryotic plasmid R1.

    PubMed

    de la Cueva-Méndez, Guillermo; Pimentel, Belén

    2007-05-01

    Plasmids are units of extrachromosomal genetic inheritance found in all kingdoms of life. They replicate autonomously and undergo stable propagation in their hosts. Despite their small size, plasmid replication and gene expression constitute a metabolic burden that compromises their stable maintenance in host cells. This pressure has driven the evolution of strategies to increase plasmid stability--a process accelerated by the ability of plasmids to transfer horizontally between cells and to exchange genetic material with their host and other resident episomal DNAs. These abilities drive the adaptability and diversity of plasmids and their host cells. Indeed, survival functions found in plasmids have chromosomal homologues that have an essential role in cellular responses to stress. An analysis of these functions in the prokaryotic plasmid R1, and of their intricate interrelationships, reveals remarkable overall similarities with other gene- and cell-survival strategies found within and beyond the prokaryotic world. PMID:17471262

  16. The 2 micron plasmid purloins the yeast cohesin complex

    PubMed Central

    Mehta, Shwetal; Yang, Xian Mei; Chan, Clarence S.; Dobson, Melanie J.; Jayaram, Makkuni; Velmurugan, Soundarapandian

    2002-01-01

    The yeast 2 micron plasmid achieves high fidelity segregation by coupling its partitioning pathway to that of the chromosomes. Mutations affecting distinct steps of chromosome segregation cause the plasmid to missegregate in tandem with the chromosomes. In the absence of the plasmid stability system, consisting of the Rep1 and Rep2 proteins and the STB DNA, plasmid and chromosome segregations are uncoupled. The Rep proteins, acting in concert, recruit the yeast cohesin complex to the STB locus. The periodicity of cohesin association and dissociation is nearly identical for the plasmid and the chromosomes. The timely disassembly of cohesin is a prerequisite for plasmid segregation. Cohesin-mediated pairing and unpairing likely provides a counting mechanism for evenly partitioning plasmids either in association with or independently of the chromosomes. PMID:12177044

  17. Plasmid R6K Replication Control

    PubMed Central

    Rakowski, Sheryl A.; Filutowicz, Marcin

    2013-01-01

    The focus of this minireview is the replication control of the 39.9-kb plasmid R6K and its derivatives. Historically, this plasmid was thought to have a narrow host range but more recent findings indicate that its derivatives can replicate in a variety of enteric and non-enteric bacterial species (Wild et al., 2004). In the four-plus decades since it was first described, R6K has proven to be an excellent model for studies of plasmid DNA replication. In part this is because of its similarities to other systems in which replication is activated and regulated by Rep protein and iteron-containing DNA. However its apparent idiosynchracies have also added to its significance (e.g., independent and co-dependent replication origins, and Rep dimers that stably bind iterons). Here, we survey the current state of knowledge regarding R6K replication and place individual regulatory elements into a proposed homeostatic model with implications for the biological significance of R6K and its multiple origins of replication. PMID:23474464

  18. Determination of Plasmid Segregational Stability in a Growing Bacterial Population.

    PubMed

    Kramer, M Gabriela

    2016-01-01

    Bacterial plasmids are extensively used as cloning vectors for a number of genes for academic and commercial purposes. Moreover, attenuated bacteria carrying recombinant plasmids expressing genes with anti-tumor activity have shown promising therapeutic results in animal models of cancer. Equitable plasmid distribution between daughter cells during cell division, i.e., plasmid segregational stability, depends on many factors, including the plasmid copy number, its replication mechanism, the levels of recombinant gene expression, the type of bacterial host, and the metabolic burden associated with all these factors. Plasmid vectors usually code for antibiotic-resistant functions, and, in order to enrich the culture with bacteria containing plasmids, antibiotic selective pressure is commonly used to eliminate plasmid-free segregants from the growing population. However, administration of antibiotics can be inconvenient for many industrial and therapeutic applications. Extensive ongoing research is being carried out to develop stably-inherited plasmid vectors. Here, I present an easy and precise method for determining the kinetics of plasmid loss or maintenance for every ten generations of bacterial growth in culture. PMID:26846807

  19. Properties of IncP-2 plasmids of Pseudomonas spp.

    PubMed Central

    Jacoby, G A; Sutton, L; Knobel, L; Mammen, P

    1983-01-01

    Thirty IncP-2 R plasmids from isolates of Pseudomonas spp. of diverse geographical origins were examined for the production of resistance properties. All the plasmids determined resistance to tellurite and all inhibited the propagation of certain DNA phages, although several patterns of phage inhibition were detected. Of the 30 plasmids, 29 determined resistance to streptomycin, 28 determined resistance to mercuric ion, and 24 determined resistance to sulfonamide. Resistance to other antibiotics, to compounds of arsenic, boron, or chromium, and to UV irradiation was less common. The degradative plasmid CAM also belonged to this group. When CAM was introduced into recipients carrying an IncP-2 R plasmid, recombinant plasmids were often formed in which antibiotic resistance and the ability to grow on camphor were transferred together to further recipients or were lost together in a strain in which IncP-2 plasmids were unstable. Such hybrid plasmid formation was rec dependent. CAM and other IncP-2 plasmids that determine UV light resistance demonstrated UV-enhanced, nonpolarized transfer of the Pseudomonas aeruginosa chromosome. By agarose gel electrophoresis, all IncP-2 R plasmids and CAM were ca. 300 X 10(6) in molecular weight. PMID:6638986

  20. Linear and Circular Plasmid Content in Borrelia burgdorferi Clinical Isolates

    PubMed Central

    Iyer, Radha; Kalu, Ogori; Purser, Joye; Norris, Steven; Stevenson, Brian; Schwartz, Ira

    2003-01-01

    The genome of Borrelia burgdorferi, the etiologic agent of Lyme disease, is composed of a linear chromosome and more than 20 linear and circular plasmids. Typically, plasmid content analysis has been carried out by pulsed-field gel electrophoresis and confirmed by Southern hybridization. However, multiple plasmids of virtually identical sizes (e.g., lp28 and cp32) complicate the interpretation of such data. The present study was undertaken to investigate the complete plasmid complements of B. burgdorferi clinical isolates cultivated from patients from a single region where early Lyme disease is endemic. A total of 21 isolates obtained from the skin biopsy or blood samples of Lyme disease patients were examined for their complete plasmid complements by Southern hybridization and plasmid-specific PCR analysis. All clinical isolates harbored at least six of the nine previously characterized cp32s. Fourteen isolates harbored all B31-like linear plasmids, and seven isolates simultaneously lacked lp56, lp38, and some segments of lp28-1. The distinctive plasmid profile observed in these seven isolates was specific to organisms that had ribosomal spacer type 2 and pulsed-field gel type A, which implies a clonal origin for this genotype. The presence of nearly identical complements of multiple linear and circular plasmids in all of the human isolates suggests that these plasmids may be particularly necessary for infection, adaptation, and/or maintenance in the infected host. PMID:12819050

  1. Characterization of toxin plasmids in Clostridium perfringens type C isolates.

    PubMed

    Gurjar, Abhijit; Li, Jihong; McClane, Bruce A

    2010-11-01

    Clostridium perfringens type C isolates cause enteritis necroticans in humans or necrotizing enteritis and enterotoxemia in domestic animals. Type C isolates always produce alpha toxin and beta toxin but often produce additional toxins, e.g., beta2 toxin or enterotoxin. Since plasmid carriage of toxin-encoding genes has not been systematically investigated for type C isolates, the current study used Southern blot hybridization of pulsed-field gels to test whether several toxin genes are plasmid borne among a collection of type C isolates. Those analyses revealed that the surveyed type C isolates carry their beta toxin-encoding gene (cpb) on plasmids ranging in size from ∼65 to ∼110 kb. When present in these type C isolates, the beta2 toxin gene localized to plasmids distinct from the cpb plasmid. However, some enterotoxin-positive type C isolates appeared to carry their enterotoxin-encoding cpe gene on a cpb plasmid. The tpeL gene encoding the large clostridial cytotoxin was localized to the cpb plasmids of some cpe-negative type C isolates. The cpb plasmids in most surveyed isolates were found to carry both IS1151 sequences and the tcp genes, which can mediate conjugative C. perfringens plasmid transfer. A dcm gene, which is often present near C. perfringens plasmid-borne toxin genes, was identified upstream of the cpb gene in many type C isolates. Overlapping PCR analyses suggested that the toxin-encoding plasmids of the surveyed type C isolates differ from the cpe plasmids of type A isolates. These findings provide new insight into plasmids of proven or potential importance for type C virulence. PMID:20823204

  2. Plasmid replicon typing of commensal and pathogenic Escherichia coli isolates.

    PubMed

    Johnson, Timothy J; Wannemuehler, Yvonne M; Johnson, Sara J; Logue, Catherine M; White, David G; Doetkott, Curt; Nolan, Lisa K

    2007-03-01

    Despite the critical role of plasmids in horizontal gene transfer, few studies have characterized plasmid relatedness among different bacterial populations. Recently, a multiplex PCR replicon typing protocol was developed for classification of plasmids occurring in members of the Enterobacteriaceae. Here, a simplified version of this replicon typing procedure which requires only three multiplex panels to identify 18 plasmid replicons is described. This method was used to screen 1,015 Escherichia coli isolates of avian, human, and poultry meat origin for plasmid replicon types. Additionally, the isolates were assessed for their content of several colicin-associated genes. Overall, a high degree of plasmid variability was observed, with 221 different profiles occurring among the 1,015 isolates examined. IncFIB plasmids were the most common type identified, regardless of the source type of E. coli. IncFIB plasmids occurred significantly more often in avian pathogenic E. coli (APEC) and retail poultry E. coli (RPEC) than in uropathogenic E. coli (UPEC) and avian and human fecal commensal E. coli isolates (AFEC and HFEC, respectively). APEC and RPEC were also significantly more likely than UPEC, HFEC, and AFEC to possess the colicin-associated genes cvaC, cbi, and/or cma in conjunction with one or more plasmid replicons. The results suggest that E. coli isolates contaminating retail poultry are notably similar to APEC with regard to plasmid profiles, with both generally containing multiple plasmid replicon types in conjunction with colicin-related genes. In contrast, UPEC and human and avian commensal E. coli isolates generally lack the plasmid replicons and colicin-related genes seen in APEC and RPEC, suggesting limited dissemination of such plasmids among these bacterial populations. PMID:17277222

  3. Plasmid Capture by the Bacillus thuringiensis Conjugative Plasmid pXO16▿

    PubMed Central

    Timmery, Sophie; Modrie, Pauline; Minet, Olivier; Mahillon, Jacques

    2009-01-01

    Conjugation, mobilization, and retromobilization are three related mechanisms of horizontal gene transfer in bacteria. They have been extensively studied in gram-negative species, where retromobilization, the capture of DNA from a recipient by a donor cell, was shown to result from two successive steps: the transfer of the conjugative plasmid from the donor to the recipient followed by the retrotransfer of the mobilizable plasmid to the donor. This successive model was established for gram-negative bacteria but was lacking experimental data from the gram-positive counterparts. In the present work, the mobilization and retromobilization abilities of the conjugative plasmid pXO16 from Bacillus thuringiensis subsp. israelensis were studied using the mobilizable plasmids pUB110 and pE194 and the “nonmobilizable” element pC194 lacking the mob and oriT features (all from Staphylococcus aureus). Experimental data suggested a successive model, since different retromobilization frequencies were observed between the small plasmids. More importantly, retromobilization was shown to be delayed by 50 and 150 min for pUB110 and pE194, respectively, compared to pXO16 conjugation. Natural liquid foods (cow milk, soy milk, and rice milk) were used to evaluate the putative ecological impact of these transfers. In cow and soy milk, conjugation, mobilization, and retromobilization were shown to occur at frequencies of 8.0 × 10−1, 1.0 × 10−2, and 1.2 × 10−4 transconjugants per recipient, respectively. These data are comparable to those obtained with LB medium and about 10-fold lower than in the case of rice milk. Taken together, these results emphasize the potential role of plasmid capture played by B. thuringiensis in natural environments. PMID:19181805

  4. Characterization of Plasmid pOR1 from Ornithobacterium rhinotracheale and Construction of a Shuttle Plasmid

    PubMed Central

    Jansen, Ruud; Chansiripornchai, Niwat; Gaastra, Wim; van Putten, Jos P. M.

    2004-01-01

    The bacterium Ornithobacterium rhinotracheale has been recognized as an emerging pathogen in poultry since about 10 years ago. Knowledge of this bacterium and its mechanisms of virulence is still very limited. Here we report the development of a transformation system that enables genetic modification of O. rhinotracheale. The system is based on a cryptic plasmid, pOR1, that was derived from an O. rhinotracheale strain of serotype K. Sequencing indicated that the plasmid consisted of 14,787 nucleotides. Sequence analysis revealed one replication origin and several rep genes that control plasmid replication and copy number, respectively. In addition, pOR1 contains genes with similarity to a heavy-metal-transporting ATPase, a TonB-linked siderophore receptor, and a laccase. Reverse transcription-PCR demonstrated that these genes were transcribed. Other putative open reading frames exhibited similarities with a virulence-associated protein in Actinobacillus actinomycetemcomitans and a number of genes coding for proteins with unknown function. An Escherichia coli-O. rhinotracheale shuttle plasmid (pOREC1) was constructed by cloning the replication origin and rep genes from pOR1 and the cfxA gene from Bacteroides vulgatus, which codes for resistance to the antibiotic cefoxitin, into plasmid pGEM7 by using E. coli as a host. pOREC1 was electroporated into O. rhinotracheale and yielded cefoxitin-resistant transformants. The pOREC1 isolated from these transformants was reintroduced into E. coli, demonstrating that pOREC1 acts as an independent replicon in both E. coli and O. rhinotracheale, fulfilling the criteria for a shuttle plasmid that can be used for transformation, targeted mutagenesis, and the construction of defined attenuated vaccine strains. PMID:15466524

  5. Sequence of two plasmids from Clostridium perfringens chicken necrotic enteritis isolates and comparison with C. perfringens conjugative plasmids.

    PubMed

    Parreira, Valeria R; Costa, Marcio; Eikmeyer, Felix; Blom, Jochen; Prescott, John F

    2012-01-01

    Twenty-six isolates of Clostridium perfringens of different MLST types from chickens with necrotic enteritis (NE) (15 netB-positive) or from healthy chickens (6 netB-positive, 5 netB-negative) were found to contain 1-4 large plasmids, with most netB-positive isolates containing 3 large and variably sized plasmids which were more numerous and larger than plasmids in netB-negative isolates. NetB and cpb2 were found on different plasmids consistent with previous studies. The pathogenicity locus NELoc1, which includes netB, was largely conserved in these plasmids whereas NeLoc3, present in the cpb2 containing plasmids, was less well conserved. A netB-positive and a cpb2-positive plasmid were likely to be conjugative, and the plasmids were completely sequenced. Both plasmids possessed the intact tcp conjugative region characteristic of C. perfringens conjugative plasmids. Comparative genomic analysis of nine CpCPs, including the two plasmids described here, showed extensive gene rearrangements including pathogenicity locus and accessory gene insertions around rather than within the backbone region. The pattern that emerges from this analysis is that the major toxin-containing regions of the variety of virulence-associated CpCPs are organized as complex pathogenicity loci. How these different but related CpCPs can co-exist in the same host has been an unanswered question. Analysis of the replication-partition region of these plasmids suggests that this region controls plasmid incompatibility, and that CpCPs can be grouped into at least four incompatibility groups. PMID:23189158

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

    SciTech Connect

    Mathis, J.N.

    1983-01-01

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

  7. Community-wide plasmid gene mobilization and selection

    PubMed Central

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

    2013-01-01

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

  8. Identification of plasmid partition function in coryneform bacteria

    SciTech Connect

    Kurusu, Yasurou; Satoh, Yukie; Inui, Masayuki; Kohama, Keiko; Kobayashi, Miki; Terasawa, Masato; Yukawa, Hideaki )

    1991-03-01

    The authors have identified and characterized a partition function that is required for stable maintenance of plasmids in the coryneform bacteria Brevibacterium flavum MJ233 and Corynebacterium glutamicum ATCC 31831. This function is localized to a HindIII-NspV fragment (673 bp) adjacent to the replication region of the plasmid, named pBY503, from Brevibacterium stationis IFO 12144. The function was independent of copy number control and was not associated directly with plasmid replication functions. This fragment was able to stabilize the unstable plasmids in cis but not in trans.

  9. Analysis of Genetic Toggle Switch Systems Encoded on Plasmids

    NASA Astrophysics Data System (ADS)

    Loinger, Adiel; Biham, Ofer

    2009-08-01

    Genetic switch systems with mutual repression of two transcription factors, encoded on plasmids, are studied using stochastic methods. The plasmid copy number is found to strongly affect the behavior of these systems. More specifically, the average time between spontaneous switching events quickly increases with the number of plasmids. It was shown before that for a single copy encoded on the chromosome, the exclusive switch is more stable than the general switch. Here we show that when the switch is encoded on a sufficiently large number of plasmids, the situation is reversed and the general switch is more stable than the exclusive switch. These predictions can be tested experimentally using methods of synthetic biology.

  10. Ultrasensitive plasmid mapping by high performance capillary electrophoresis.

    PubMed

    Maschke, H E; Frenz, J; Belenkii, A; Karger, B L; Hancock, W S

    1993-01-01

    This paper compares high performance capillary electrophoresis (HPCE) and conventional slab electrophoresis in mapping of four closely related plasmids with three different restriction enzymes. The plasmids express full length and truncated forms of a growth factor receptor oncogene product and were digested with HpaII, HaeIII and RsaI. The resulting oligonucleotide fragments were under 2000 base pairs in length, a size well suited to separation by HPCE with linear polyacrylamide as a sieving matrix. Plasmid mapping is an essential tool in biotechnology both for the design of an expression system and for monitoring the stability of the expression system during fermentation. HPCE can yield much higher resolution of oligonucleotides than attainable in conventional agarose gel electrophoretic procedures for plasmid mapping. In the examples described here, the HpaII digests provided the surest identification of individual plasmids in the HPCE analysis and could discriminate among all four plasmids. In conventional slab electrophoresis, however, the RsaI digests provided the best discrimination, although two of the plasmids in this system yielded essentially identical electrophoretic patterns. Hence the optimal restriction enzyme for plasmid mapping applications with HPCE may differ from that selected on the basis of conventional slab gel analysis, and the former technique can provide higher discrimination among related plasmids. The advantages of the HPCE format with respect to speed, low sample consumption and resolution are described. PMID:8354236

  11. Impact of plasmid quality on lipoplex-mediated transfection.

    PubMed

    De La Vega, Jonathan; Braak, Bas Ter; Azzoni, Adriano R; Monteiro, Gabriel A; Prazeres, Duarte Miguel F

    2013-11-01

    This work investigates the impact of quality attributes (impurity content, plasmid charge, and compactness) of plasmid DNA isolated with different purification methodologies on the characteristics of lipoplexes prepared thereof (size, zeta potential, stability) and on their ability to transfect mammalian cells. A 3.7 kb plasmid with a green fluorescence protein (GFP) reporter gene, Lipofectamine®-based liposomes, and Chinese Hamster Ovary (CHO) cells were used as models. The plasmid was purified by hydrophobic interaction chromatography (HIC)/gel filtration, and with three commercial kits, which combine the use of chaotropic salts with silica membranes/glass fiber fleeces. The HIC-based protocol delivered a plasmid with the smallest hydrodynamic diameter (144 nm) and zeta potential (-46.5 mV), which is virtually free from impurities. When formulated with Lipofectamine®, this plasmid originated the smallest (146 nm), most charged (+13 mV), and most stable lipoplexes. In vitro transfection experiments further showed that these lipoplexes performed better in terms of plasmid uptake (∼500,000 vs. ∼100,000-200,000 copy number/cell), transfection efficiency (50% vs. 20%-40%), and GFP expression levels (twofold higher) when compared with lipoplexes prepared with plasmids isolated using commercial kits. Overall our observations highlight the potential impact that plasmid purification methodologies can have on the outcome of gene transfer experiments and trials. PMID:23996350

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

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

    2014-07-16

    Growing ornamental fish industry is associated with public health concerns including extensive antibiotic use accompanied by increasing antibiotic resistance. The aim of this study was to analyze Aeromonas isolates from imported tropical ornamental fish and coldwater koi carps bred in the Czech Republic to assess the potential risk of ornamental fish as a source of plasmid-mediated quinolone resistance genes (PMQR) and antibiotic resistance plasmids. A collection of Aeromonas spp. with reduced susceptibility to ciprofloxacin (MIC ≥ 0.05 mg/L) was selected for the detection of PMQR genes. Isolates harbouring PMQR genes were further analyzed for the additional antibiotic resistance, integron content, clonality, biofilm production and transferability of PMQR genes by conjugation and transformation. Comparative analysis of plasmids carrying PMQR genes was performed. Fifteen (19%, n=80) isolates from koi carps and 18 (24%, n=76) isolates from imported ornamental fish were positive for qnrS2, aac(6')-Ib-cr or qnrB17 genes. PMQR-positive isolates from imported ornamental fish showed higher MIC levels to quinolones, multiresistance and diverse content of antibiotic resistance genes and integrons compared to the isolates from the carps. Related IncU plasmids harbouring qnrS2 and aac(6')-Ib-cr genes were found in Aeromonas spp. from imported ornamental fish and koi carps from various geographical areas. Ornamental fish may represent a potential source of multiresistant bacteria and mobile genetic elements for the environment and for humans. PMID:24629900

  15. Plasmids for heterologous expression in Pasteurella haemolytica.

    PubMed

    Fedorova, N D; Highlander, S K

    1997-02-28

    New cloning and expression vectors that replicate both in Pasteurella haemolytica and in Escherichia coli were constructed based on a native sulfonamide (SuR) and streptomycin (SmR) resistant plasmid of P. haemolytica called pYFC1. Each shuttle vector includes an MCS and a selectable antibiotic resistance marker that is expressed in both organisms. Plasmid pNF2176 carries the P. haemolytica ROB-1 beta-lactamase gene (blaP, ApR) and pNF2214 carries the Tn903 aph3 kanamycin resistance (KmR) element. The expression vector, pNF2176, was created by placing the MCS downstream of the sulfonamide gene promoter (PsulII) on pYFC1; this was used to clone and express the promoterless Tn9 chloramphenicol resistance gene (cat, CmR) in P. haemolytica (pNF2200). A promoter-probe vector (pNF2283) was constructed from pNF2200 by deleting PsulII. PMID:9074498

  16. CONJUGAL TRANSFER AT NATURAL POPULATION DENSITIES IN A MICROCOSM SIMULATING AN ESTUARINE ENVIRONMENT

    EPA Science Inventory

    Estuarine microcosms were used to follow conjugal transfer of a broad host range IncP1 plasmid from a Pseudomonas putida donor to indigenous bacteria. onor cells were added at a concentration similar to the natural abundance of bacteria in the water column (10 6/mi). ransfer was ...

  17. An oligonucleotide microarray to characterize multidrug resistant plasmids

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  18. Purification of large plasmids with methacrylate monolithic columns.

    PubMed

    Krajnc, Nika Lendero; Smrekar, Franci; Cerne, Jasmina; Raspor, Peter; Modic, Martina; Krgovic, Danijela; Strancar, Ales; Podgornik, Ales

    2009-08-01

    The rapid evolution of gene therapy and DNA vaccines results in an increasing interest in producing large quantities of pharmaceutical grade plasmid DNA. Most current clinical trials involve plasmids of 10 kb or smaller in size, however, future requirements for multigene vectors including extensive control regions may require the production of larger plasmids, e. g., 20 kb and bigger. The objective of this study was to examine certain process conditions for purification of large plasmids with the size of up to 93 kb. Since there is a lack of knowledge about production and purification of bigger plasmid DNA, cell lysis and storage conditions were investigated. The impact of chromatographic system and methacrylate monolithic column on the degradation of plasmid molecules under nonbinding conditions at different flow rates was studied. Furthermore, capacity measurements varying salt concentration in loading buffer were performed and the capacities up to 13 mg of plasmid per mL of the monolithic column were obtained. The capacity flow independence in the range from 130 to 370 cm/h was observed. Using high resolution monolithic column the separation of linear and supercoiled isoforms of large plasmids was obtained. Last but not least, since the baseline separation of RNA and pDNA was achieved, the one step purification on larger CIM DEAE 8 mL tube monolithic column was performed and the fractions were analyzed by CIM analytical monolithic columns. PMID:19598166

  19. Rapid compensatory evolution promotes the survival of conjugative plasmids

    PubMed Central

    Harrison, Ellie; Dytham, Calvin; Hall, James P. J.; Guymer, David; Spiers, Andrew J.; Paterson, Steve; Brockhurst, Michael A.

    2016-01-01

    ABSTRACT Conjugative plasmids play a vital role in bacterial adaptation through horizontal gene transfer. Explaining how plasmids persist in host populations however is difficult, given the high costs often associated with plasmid carriage. Compensatory evolution to ameliorate this cost can rescue plasmids from extinction. In a recently published study we showed that compensatory evolution repeatedly targeted the same bacterial regulatory system, GacA/GacS, in populations of plasmid-carrying bacteria evolving across a range of selective environments. Mutations in these genes arose rapidly and completely eliminated the cost of plasmid carriage. Here we extend our analysis using an individual based model to explore the dynamics of compensatory evolution in this system. We show that mutations which ameliorate the cost of plasmid carriage can prevent both the loss of plasmids from the population and the fixation of accessory traits on the bacterial chromosome. We discuss how dependent the outcome of compensatory evolution is on the strength and availability of such mutations and the rate at which beneficial accessory traits integrate on the host chromosome. PMID:27510852

  20. Inc A/C Plasmids in Multidrug resistant Salmonella enterica

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteria plasmids are fragments of extra-chromosomal double stranded deoxyribonucleic acid (DNA) that can contain a variety of genes beneficial to the survival of the host bacteria. Classification and tracking of plasmids is beneficial because they are potentially a medium of horizontal gene transf...

  1. Horizontal gene transfer of stress resistance genes through plasmid transport.

    PubMed

    Shoeb, Erum; Badar, Uzma; Akhter, Jameela; Shams, Hina; Sultana, Maria; Ansari, Maqsood A

    2012-03-01

    The horizontal gene transfer of plasmid-determined stress tolerance was achieved under lab conditions. Bacterial isolates, Enterobacter cloacae (DGE50) and Escherichia coli (DGE57) were used throughout the study. Samples were collected from contaminated marine water and soil to isolate bacterial strains having tolerance against heavy metals and antimicrobial agents. We have demonstrated plasmid transfer, from Amp(+)Cu(+)Zn(-) strain (DGE50) to Amp(-)Cu(-)Zn(+) strain (DGE57), producing Amp(+)Cu(+)Zn(+) transconjugants (DGE(TC50→57)) and Amp(+)Cu(-)Zn(+) transformants (DGE(TF50→57)). DGE57 did not carry any plasmid, therefore, it can be speculated that zinc tolerance gene in DGE57 is located on chromosome. DGE50 was found to carry three plasmids, out of which two were transferred through conjugation into DGE57, and only one was transferred through transformation. Plasmid transferred through transformation was one out of the two transferred through conjugation. Through the results of transformation it was revealed that the genes of copper and ampicillin tolerance in DGE50 were located on separate plasmids, since only ampicillin tolerance genes were transferred through transformation as a result of one plasmid transfer. By showing transfer of plasmids under lab conditions and monitoring retention of respective phenotype via conjugation and transformation, it is very well demonstrated how multiple stress tolerant strains are generated in nature. PMID:22805823

  2. Linear plasmids in plant mitochondria: peaceful coexistences or malicious invasions?

    PubMed

    Handa, Hirokazu

    2008-01-01

    Plant mitochondria contain small extrachromosomal DNAs in addition to a large and complex main mitochondrial genome. These molecules can be regarded as extrachromosomal replicons or plasmids, of which there are two forms, circular and linear. Linear mitochondrial plasmids are present in many fungi and in some plants, but they seem to be absent from most animal cells. They usually have a common structural feature, called an invertron, that is characterized by the presence of terminal inverted repeats and proteins covalently attached to their 5 termini. Linear mitochondrial plasmids possess one to six ORFs that can encode unknown proteins but often code for the DNA and RNA polymerases. Although the functions of most linear plasmids in plant mitochondria are unknown, some plasmids may be associated with mitochondrial genome rearrangements and may have phenotypic effects due to their integration into mitochondrial genome. The Brassica 11.6-kb plasmid, one of the linear mitochondrial plasmids in plants, shows a non-maternal inheritance, in contrast to mitochondrial genomes. The origin of these plasmids is still a mystery, but indirect evidence indicates the possibility of horizontal transfer from fungal mitochondria. In this review, the main features of these unique DNAs present in plant mitochondria are described. PMID:18326073

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

    PubMed

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

    2010-11-01

    Biotechnical production processes often operate with plasmid-based expression systems in well-established prokaryotic and eukaryotic hosts such as Escherichia coli or Saccharomyces cerevisiae, respectively. Genetically engineered organisms produce important chemicals, biopolymers, biofuels and high-value proteins like insulin. In those bioprocesses plasmids in recombinant hosts have an essential impact on productivity. Plasmid-free cells lead to losses in the entire product recovery and decrease the profitability of the whole process. Use of antibiotics in industrial fermentations is not an applicable option to maintain plasmid stability. Especially in pharmaceutical or GMP-based fermentation processes, deployed antibiotics must be inactivated and removed. Several plasmid addiction systems (PAS) were described in the literature. However, not every system has reached a full applicable state. This review compares most known addiction systems and is focusing on biotechnical applications. PMID:21255361

  4. Cloning and analysis of a large plasmid pBMB165 from Bacillus thuringiensis revealed a novel plasmid organization.

    PubMed

    Wang, Yueying; Peng, Donghai; Dong, Zhaoxia; Zhu, Lei; Guo, Suxia; Sun, Ming

    2013-01-01

    In this study, we report a rapid cloning strategy for large native plasmids via a contig linkage map by BAC libraries. Using this method, we cloned a large plasmid pBMB165 from Bacillus thuringiensis serovar tenebrionis strain YBT-1765. Complete sequencing showed that pBMB165 is 77,627 bp long with a GC-content of 35.36%, and contains 103 open reading frames (ORFs). Sequence analysis and comparison reveals that pBMB165 represents a novel plasmid organization: it mainly consists of a pXO2-like replicon and mobile genetic elements (an inducible prophage BMBTP3 and a set of transposable elements). This is the first description of this plasmid organization pattern, which may result from recombination events among the plasmid replicon, prophage and transposable elements. This plasmid organization reveals that the prophage BMBTP3 may use the plasmid replicon to maintain its genetic stability. Our results provide a new approach to understanding co-evolution between bacterial plasmids and bacteriophage. PMID:24312580

  5. Plasmid Copy Number Determination by Quantitative Polymerase Chain Reaction.

    PubMed

    Anindyajati; Artarini, A Anita; Riani, Catur; Retnoningrum, Debbie S

    2016-01-01

    Recombinant therapeutic proteins are biopharmaceutical products that develop rapidly for years. Recombinant protein production in certain hosts requires vector expression harboring the gene encoding the corresponding protein. Escherichia coli is the prokaryote organism mostly used in recombinant protein production, commonly using a plasmid as the expression vector. Recombinant protein production is affected by plasmid copy number harboring the encoded gene, hence the determination of plasmid copy number also plays an important role in establishing a recombinant protein production system. On the industrial scale, a low copy number of plasmids are more suitable due to their better stability. In the previous study we constructed pCAD, a plasmid derived from the low copy number pBR322 plasmid. This study was aimed to confirm pCAD's copy number by quantitative polymerase chain reaction (qPCR). Plasmid copy number was determined by comparing the quantification signal from the plasmid to those from the chromosome. Copy number was then calculated by using a known copy number plasmid as a standard. Two pairs of primers, called tdk and ori, were designed for targeting a single gene tdk in the chromosome and a conserved domain in the plasmid's ori, respectively. Primer quality was analyzed in silico using PrimerSelect DNASTAR and PraTo software prior to in vitro evaluation on primer specificity and efficiency as well as optimization of qPCR conditions. Plasmid copy number determination was conducted on E. coli lysates harboring each plasmid, with the number of cells ranging from 10(2)-10(5) cells/μL. Cells were lysed by incubation at 95ºC for 10 minutes, followed by immediate freezing at -4°C. pBR322 plasmid with the copy number of ~19 copies/cell was used as the standard, while pJExpress414-sod plasmid possessing the high copy number pUC ori was also determined to test the method being used. In silico analysis based on primer-primer and primer-template interactions showed

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

    PubMed Central

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

    2016-01-01

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

  7. Plasmid incidence in bacteria from deep subsurface sediments

    SciTech Connect

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

    1988-12-01

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

  8. Fractional precipitation of plasmid DNA from lysate by CTAB.

    PubMed

    Lander, Russel J; Winters, Michael A; Meacle, Francis J; Buckland, Barry C; Lee, Ann L

    2002-09-30

    Preparative-scale purification of plasmid DNA has been attempted by diverse methods, including precipitation with solvents, salts, and detergents and chromatography with ion-exchange, reversed-phase, and size-exclusion columns. Chromatographic methods such as hydrophobic interaction chromatography (HIC), reversed phase chromatography (RPC), and size exclusion chromatography (SEC) are the only effective means of eliminating the closely related relaxed and denatured forms of plasmid as well as endotoxin to acceptable levels. However, the anticipated costs of manufacturing-scale chromatography are high due to (a) large projected volumes of the high-dosage therapeutic molecule and (b) restricted loading of the large plasmid molecule in the pores of expensive resins. As an alternative to chromatography, we show herein that precipitation with the cationic detergent, cetyltrimethylammonium bromide (CTAB), is effective for selective precipitation of plasmid DNA from proteins, RNA, and endotoxin. Moreover, CTAB affords novel selectivity by removal of host genomic DNA and even the more closely related relaxed and denatured forms of plasmid as earlier, separate fractions. Finally, plasmid that has been precipitated by CTAB can be purified by selectively dissolving under conditions of controlled salt concentration. The selectivity mechanism is most likely based upon conformational differences among the several forms of DNA. As such, CTAB precipitation provides an ideal nonchromatographic capture step for the manufacture of plasmid DNA. PMID:12209800

  9. Investigation of plasmid-induced growth defect in Pseudomonas putida.

    PubMed

    Mi, Jia; Sydow, Anne; Schempp, Florence; Becher, Daniela; Schewe, Hendrik; Schrader, Jens; Buchhaupt, Markus

    2016-08-10

    Genetic engineering in bacteria mainly relies on the use of plasmids. But despite their pervasive use for physiological studies as well as for the design and optimization of industrially used production strains, only limited information about plasmid induced growth defects is available for different replicons and organisms. Here, we present the identification and characterization of such a phenomenon for Pseudomonas putida transformants carrying the pBBR1-derived plasmid pMiS1. We identified the kanamycin resistance gene and the transcription factor encoding rhaR gene to be causal for the growth defect in P. putida. In contrast, this effect was not observed in Escherichia coli. The plasmid-induced growth defect was eliminated after introduction of a mutation in the plasmid-encoded rep gene, thus enabling construction of the non-toxic variant pMiS4. GFP reporters construct analyses and qPCR experiments revealed a distinctly lowered plasmid copy number for pMiS4, which is probably the reason for alleviation of the growth defect by this mutation. Our work expands the knowledge about plasmid-induced growth defects and provides a useful low-copy pBBR1 replicon variant. PMID:27287537

  10. Plasmid incidence in bacteria from deep subsurface sediments.

    PubMed

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

    1988-12-01

    Bacteria were isolated from deep terrestrial subsurface sediments underlying the coastal plain of South Carolina. A total of 163 isolates from deep sediments, surface soil, and return drill muds were examined for plasmid DNA content and resistance to the antibiotics penicillin, ampicillin, carbenicillin, streptomycin, kanamycin, and tetracycline. MICs of Cu, Cr, and Hg for each isolate were also determined. The overall frequency of plasmid occurrence in the subsurface bacteria was 33%. Resistance was most frequent to penicillin (70% of all isolates), ampicillin (49%), and carbenicillin (32%) and was concluded to be related to the concentrations of the individual antibiotics in the disks used for assaying resistance and to the production of low levels of beta-lactamase. The frequencies of resistance to penicillin and ampicillin were significantly greater for isolates bearing plasmids than for plasmidless isolates; however, resistance was not transferable to penicillin-sensitive Escherichia coli. Hybridization of subsurface bacterial plasmids and chromosomal DNA with a whole-TOL-plasmid (pWWO) probe revealed some homology of subsurface bacterial plasmid and chromosomal DNAs, indicating a potential for those bacteria to harbor catabolic genes on plasmids or chromosomes. The incidences of antibiotic resistance and MICs of metals for subsurface bacteria were significantly different from those for drill mud bacteria, ruling out the possibility that bacteria from sediments were derived from drill muds. PMID:16347789

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

    PubMed Central

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

    1977-01-01

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

  12. Sociobiological Control of Plasmid Copy Number in Bacteria

    PubMed Central

    Watve, Mukta M.; Dahanukar, Neelesh; Watve, Milind G.

    2010-01-01

    All genes critical for plasmid replication regulation are located on the plasmid rather than on the host chromosome. It is possible therefore that there can be copy-up “cheater” mutants. In spite of this possibility, low copy number plasmids appear to exist stably in host populations. We examined this paradox using a multilevel selection model. Simulations showed that, a slightly higher copy number mutant could out-compete the wild type. Consequently, another mutant with still higher copy number could invade the first invader. However, the realized benefit of increasing intra-host fitness was saturating whereas that of inter-host fitness was exponential. As a result, above a threshold, intra-host selection was overcompensated by inter-host selection and the low copy number wild type plasmid could back invade a very high copy number plasmid. This led to a rock-paper-scissor (RPS) like situation that allowed the coexistence of plasmids with varied copy numbers. Furthermore, another type of cheater that had lost the genes required for conjugation but could hitchhike on a conjugal plasmid, could further reduce the advantage of copy-up mutants. These sociobiological interactions may compliment molecular mechanisms of replication regulation in stabilizing the copy numbers. PMID:20195362

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

    PubMed

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

    2016-01-01

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

  14. Molecular classification of IncP-9 naphthalene degradation plasmids

    SciTech Connect

    Izmalkova, T.Y.; Mavrodi, D.V.; Sokolov, S.L.; Kosheleva, I.A.; Smalla, K.; Thomas, C.M.; Boronin, A.M.

    2006-07-15

    A large collection of naphthalene-degrading fluorescent Pseudomonas strains isolated from sites contaminated with coal tar and crude oil was screened for the presence of IncP-9 plasmids. Seventeen strains were found to carry naphthalene catabolic plasmids ranging in size from 83 to 120kb and were selected for further study. Results of molecular genotyping revealed that 15 strains were closely related to P. putida, one to P. fluorescens, and one to P. aeruginosa. All catabolic plasmids found in these strains, with the exception of pBS216, pSN11, and p8909N-1, turned out to belong to IncP-9 {beta}-subgroup. Plasmids pBS216, pSN11, and p8909N-1 were identified as members of IncP-9 {delta}-subgroup. One plasmid, pBS2, contains fused replicons of IncP-9 {beta} and IncP-7 groups. RFLP analyses of the naphthalene catabolic plasmids revealed that organisation of the replicon correlates well with the overall plasmid structure. Comparative PCR studies with conserved oligonucleotide primers indicated that genes for key enzymes of naphthalene catabolism are highly conserved among all studied plasmids. Three bacterial strains, P. putida BS202, P. putida BS3701, and P. putida BS3790, were found to have two different salicylate hydroxylase genes one of which has no similarity to the 'classic' enzyme encoded by nahG gene. Discovery of a large group of plasmid with unique nahR suggested that the regulatory loop may also represent a variable part of the pathway for catabolism of naphthalene in fluorescent Pseudomonas spp.

  15. Separation of plasmid DNA topoisomers by multimodal chromatography.

    PubMed

    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. PMID:27033004

  16. Characterization of ampicillin resistance plasmids from Haemophilus ducreyi.

    PubMed Central

    Totten, P A; Handsfield, H H; Peters, D; Holmes, K K; Falkow, S

    1982-01-01

    Seven strains of Haemophilus ducreyi from diverse geographic origins were analyzed for their plasmid content. All strains were multiply resistant, but only resistance to ampicillin was transferred to Escherichia coli by transformation. The H. ducreyi plasmids encoding for ampicillin resistance were 7.4, 5.7, and 3.6 megadaltons and encoded for part or all of TnA, and ampicillin transposon. The relatedness of these plasmids was examined by restriction endonuclease digestion and DNA-DNA homology with isolated DNA fragments from TnA. Images PMID:6282212

  17. Photoinduced silver nanoparticles/nanorings on plasmid DNA scaffolds.

    PubMed

    Liu, Jianhua; Zhang, Xiaoliang; Yu, Mei; Li, Songmei; Zhang, Jindan

    2012-01-23

    Biological scaffolds are being actively explored for the synthesis of nanomaterials with novel structures and unexpected properties. Toroidal plasmid DNA separated from the Bacillus host is applied as a sacrificial mold for the synthesis of silver nanoparticles and nanorings. The photoirradiation method is applied to reduce Ag(I) on the plasmid. The nanoparticles are obtained by varying the concentration of the Ag(I) ion solution and the exposure time of the plasmid-Ag(I) complex under UV light at 254 nm and room temperature. It is found that the plasmid serves not only as a template but also as a reductant to drive the silver nucleation and deposition. The resulting nanoparticles have a face-centered cubic (fcc) crystal structure and 20-30 nm average diameter. The detailed mechanism is discussed, and other metals or alloys could also be synthesized with this method. PMID:22102552

  18. A series of template plasmids for Escherichia coli genome engineering.

    PubMed

    Deb, Shalini S; Reshamwala, Shamlan M S; Lali, Arvind M

    2016-06-01

    Metabolic engineering strategies often employ multi-copy episomal vectors to overexpress genes. However, chromosome-based overexpression is preferred as it avoids the use of selective pressure and reduces metabolic burden on the cell. We have constructed a series of template plasmids for λ Red-mediated Escherichia coli genome engineering. The template plasmids allow construction of genome integrating cassettes that can be used to integrate single copies of DNA sequences at predetermined sites or replace promoter regions. The constructed cassettes provide flexibility in terms of expression levels achieved and antibiotics used for selection, as well as allowing construction of marker-free strains. The modular design of the template plasmids allows replacement of genetic parts to construct new templates. Gene integration and promoter replacement using the template plasmids are illustrated. PMID:27071533

  19. [New low-copy plasmid in cyanobacterium Anabaena variabilis].

    PubMed

    Mardanov, A V; Beletskiĭ, A V; Gumerov, V M; Karbysheva, E A; Mikheeva, L E

    2013-08-01

    Complete genome sequencing was performed for Anabaena variabilis ATCC 29413 from the collection of the Chair of Genetics, Department of Biology, Moscow State University, Russia. In addition to known plasmids A, B, and C, a new circular low-copy plasmid was detected and named D. It was also sequenced completely and found to have 27051 bp. The plasmid contained the parA and parB genes of the partition system, two genes that encode replication proteins, a gene for site-specific recombinase, atype-I restriction-modification system, and several genes with unknown functions. Analysis by PCR revealed the presence of plasmid D in two epiphytic strains from Vietnam, i.e., Anabaena sp. 182 and Anabaena sp. 281, as well as in Anabaena sp. V5 and A. azollae (Newton's isolate). PMID:25474879

  20. [New low-copy plasmid in cyanobacterium Anabaena variabilis].

    PubMed

    2013-08-01

    Complete genome sequencing was performed for Anabaena variabilis ATCC 29413 from the collection of the Chair of Genetics, Department of Biology, Moscow State University, Russia. In addition to known plasmids A, B, and C, a new circular low-copy plasmid was detected and named D. It was also sequenced completely and found to have 27051 bp. The plasmid contained the parA and parB genes of the partition system, two genes that encode replication proteins, a gene for site-specific recombinase, atype-I restriction-modification system, and several genes with unknown functions. Analysis by PCR revealed the presence of plasmid D in two epiphytic strains from Vietnam, i.e., Anabaena sp. 182 and Anabaena sp. 281, as well as in Anabaena sp. V5 and A. azollae (Newton's isolate). PMID:25508658

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  2. Transfer of plasmids by conjugation in Streptococcus pneumoniae

    SciTech Connect

    Smith, M.D.; Shoemaker, N.B.; Burdett, V.; Guild, W.R.

    1980-01-01

    Transfer of resistance plasmids occurred by conjugation in Streptococcus pneumoniae (pneumococcus) similiarly to the process in other streptococcal groups. The 20-megadalton plasmid pIP501 mediated its own DNase-resistant transfer by filter mating and mobilized the 3.6-megadalton non-self-transmissible pMV158. Pneumococcal strains acted as donors or as recipients for intraspecies transfers and for interspecific transfers with Streptococcus faecalis. Transfer-deficient mutants of pIP501 have been found.

  3. A novel chromatographic procedure for purification of bacterial plasmids.

    PubMed

    Bywater, M; Bywater, R; Hellman, L

    1983-07-01

    A new, rapid procedure for purifying bacterial plasmids with high recovery is described. The sequence of operations consists essentially of treatment with alkali, ribonuclease, and proteinase K, followed by chisam extraction and gel filtration on Sephacryl S-1000, and finally a precipitation step using isopropanol at room temperature. The method gives rather good yields of plasmid DNA of high purity, and lends itself to scaling up. PMID:6312836

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

    PubMed Central

    Williams, Julia J.; Hergenrother, Paul J.

    2008-01-01

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

  5. Automated Filtration-Based High-Throughput Plasmid Preparation System

    PubMed Central

    Itoh, Masayoshi; Kitsunai, Tokuji; Akiyama, Junichi; Shibata, Kazuhiro; Izawa, Masaki; Kawai, Jun; Tomaru, Yasuhiro; Carninci, Piero; Shibata, Yuko; Ozawa, Yasuhiro; Muramatsu, Masami; Okazaki, Yasushi; Hayashizaki, Yoshihide

    1999-01-01

    Current methods of plasmid preparation do not allow for large capacity automated processing. We have developed an automated high-throughput system that prepares plasmid DNA for large-scale sequencing. This system is based on our previously reported filtration method. In this method, cell harvesting, alkaline lysis, and plasmid purification occur in a single 96-well microtiter plate from which sequence-ready DNA samples are collected. The plates are designed to allow all reagents to be injected from above the wells and the spent reagents to be aspirated from below. This design has enabled us to build a linear process plasmid preparation system consisting of an automated filter plate stacker and a 21-stage automated plasmid preparator. The 96-well plates used are outfitted with glass-filters that trap Escherichia coli before the plates are stacked in the automated stacker. The plates move from the stacker to each of the 21 stages of the preparator. At specific stages, various reagents or chemicals are injected into the wells from above. Finally, the plates are collected in the second stacker. The optimal throughput of the preparator is 40,000 samples in 17.5 hr. Here, we describe a pilot experiment preparing 15,360 templates in 160 specially designed 96-well glass-filter plates. The prepared plasmids were subjected to restriction digestion, DNA sequencing, and transcriptional sequencing. PMID:10330126

  6. Plasmid-free T7-based Escherichia coli expression systems.

    PubMed

    Striedner, Gerald; Pfaffenzeller, Irene; Markus, Luchner; Nemecek, Sabine; Grabherr, Reingard; Bayer, Karl

    2010-03-01

    In order to release host cells from plasmid-mediated increases in metabolic load and high gene dosages, we developed a plasmid-free, T7-based E. coli expression system in which the target gene is site-specifically integrated into the genome of the host. With this system, plasmid-loss, a source of instability for conventional expression systems, was eliminated. At the same time, system leakiness, a challenging problem with recombinant systems, was minimized. The efficiency of the T7 RNA polymerase compensates for low gene dosage and provides high rates of recombinant gene expression without fatal consequences to host metabolism. Relative to conventional pET systems, this system permits improved process stability and increases the host cell's capacity for recombinant gene expression, resulting in higher product yields. The stability of the plasmid-free system was proven in chemostat cultivation for 40 generations in a non-induced and for 10 generations in a fully induced state. For this reason plasmid-free systems benefit the development of continuous production processes with E. coli. However, time and effort of the more complex cloning procedure have to be considered in relation to the advantages of plasmid-free systems in upstream-processing. PMID:19891007

  7. Functional analysis of the yeast plasmid partition locus STB

    PubMed Central

    Murray, James A. H.; Cesareni, Gianni

    1986-01-01

    Derivatives of the yeast 2μ plasmid with the cis-acting locus STB (also called REP3) are stably maintained if two plasmid-encoded proteins are present in trans. There are conflicting reports of both the extent of STB and its possible involvement in plasmid partition or copy number control. We have resolved the controversy by constructing 2µ derivatives with a conditional STB function, and showing that when STB is inactivated plasmids become concentrated in a small fraction of the population although the total number of plasmids remains unaltered. Moreover we show that STB consists of two functionally distinct domains which we call STB-proximal and STB-distal relative to the origin of replication. Although STB-proximal is sufficient for proper partitioning, this function is severely disrupted by active transcription from neighbouring sequences. STB-distal is important to protect STB-proximal and ORI from such transcription, and can be effeciently replaced by a 94-bp terminator fragment in an orientation-dependent manner. We find that STB-distal contains an additional element which depresses transcription from upstream promoters. We also describe the phenomenon of replicaton inhibition which we believe can exlain the anomalous instability of some yeast plasmids. ImagesFig. 4.Fig. 5.Fig. 6.Fig. 7. PMID:16453734

  8. Plasmid Carriage and the Serum Sensitivity of Enterobacteria

    PubMed Central

    Taylor, Peter W.; Hughes, Colin

    1978-01-01

    The carriage of a range of plasmids by rough, serum-sensitive laboratory strains of Escherichia coli made no difference to their reactivity in human serum as determined by two methods. Plasmid-carrying enterobacteria isolated from polluted river water gave a variety of responses to serum. Smooth E. coli river isolate C8 was killed by serum but only after a delay of 1 h, and curing of antibiotic resistance and colicin determinants from this strain led to a small but significant increase in serum sensitivity. Plasmids from eight strains were transferred by conjugation to a cured derivative of C8 (C8−NalR), and in six cases a significant increase in the serum resistance of the progeny was observed. Plasmid-mediated enhancement of resistance was particularly marked with plasmids R1 and NR1, and a round of replication mutant of NR1 conferred greater resistance than did the normal R factor. However, R1 and NR1 were unable to modify the serum response of a cured strain (P21−NalR) derived from promptly serum-sensitive isolate P21. These findings suggest that lipopolysaccharide O-side chains, the cell surface components responsible for the delay in serum killing, are essential for the expression of plasmid factors that modify sensitivity to serum. Examination of K(A)− variants of two isolates indicated that the K(A) antigen has only a marginal effect on the serum response. PMID:365738

  9. Dye affinity cryogels for plasmid DNA purification.

    PubMed

    Çimen, Duygu; Yılmaz, Fatma; Perçin, Işık; Türkmen, Deniz; Denizli, Adil

    2015-11-01

    The aim of this study is to prepare megaporous dye-affinity cryogel discs for the purification of plasmid DNA (pDNA) from bacterial lysate. Poly(hydroxyethyl methacrylate) [PHEMA] cryogel discs were produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) redox pair in an ice bath. Cibacron Blue F3GA was used as an affinity ligand (loading amount: 68.9μmol/g polymer). The amount of pDNA adsorbed onto the PHEMA-Cibacron Blue F3GA cryogel discs first increased and then reached a plateau value (i.e., 32.5mg/g cryogel) at 3.0mg/mL pDNA concentration. Compared with the PHEMA cryogel (0.11mg/g cryogel), the pDNA adsorption capacity of the PHEMA-Cibacron Blue F3GA cryogel (32.4mg/g polymer) was improved significantly due to the Cibacron Blue 3GA immobilization onto the polymeric matrix. pDNA adsorption amount decreased from 11.7mg/g to 1.1mg/g with the increasing of NaCl concentration. The maximum pDNA adsorption was achieved at 4°C. The overall recovery of pDNA was calculated as 90%. The PHEMA-Cibacron Blue F3GA cryogel discs could be used five times without decreasing the pDNA adsorption capacity significantly. The results show that the PHEMA-Cibacron Blue F3GA cryogel discs promise high selectivity for pDNA. PMID:26249596

  10. Relationship between pNG2, an Emr plasmid in Corynebacterium diphtheriae, and plasmids in aerobic skin coryneforms.

    PubMed Central

    Schiller, J; Strom, M; Groman, N; Coyle, M

    1983-01-01

    Erythromycin-resistant (Emr) coryneforms from cutaneous lesions and erythromycin-susceptible (Ems) coryneforms from normal skin sites were screened for plasmids. Approximately one-third of the 40 isolates carried one or more plasmids ranging in mass from 2.5 to 36 megadaltons, all exhibiting different restriction enzyme digest patterns. In contrast, only Corynebacterium diphtheriae strains comprising a single cohort of apparently identical Emr, pNG2-carrying isolates have been identified as plasmid carriers. Homology was demonstrated between pNG2 and a number of fragments in restriction enzyme digests of plasmids from both Emr and Ems skin coryneforms under high-stringency conditions. However, none was detected between pNG2 and the genomic or plasmid DNAs of Emr staphylococci or streptococci isolated concurrently with the Emr coryneforms. One coryneform plasmid, pNG34, exhibited extensive homology with pNG2, and many comigrating fragments were observed. Very little relationship was observed between C. diphtheriae and the skin coryneforms when their genomic DNAs were hybridized. The origin and presence of pNG2 in Emr C. diphtheriae is discussed in relation to these findings. Images PMID:6318665

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

    NASA Astrophysics Data System (ADS)

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

    1999-01-01

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

  12. Plasmids and Rickettsial Evolution: Insight from Rickettsia felis

    PubMed Central

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

    2007-01-01

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

  13. Rapid alkaline extraction method for the isolation of plasmid DNA

    SciTech Connect

    Birnboim, H.C.

    1983-01-01

    Plasmids are double-stranded circular DNA molecules that have the property of self-replication, independent of chromosomal DNA. Although the presence of a plasmid in a bacterial cell may be detected genetically as a change in phenotype, often it is necessary to isolate plasmid DNA for molecular studies, such as size determination, restriction enzyme mapping, and nucleotide sequencing, or for the construction of new hybrid plasmids. The degree of purification required will depend upon the intended use. Less purified plasmid DNA is often satisfactory for recombinant DNA studies, and a large number of shorter and simpler methods have been developed. This chapter describes one such method that uses an alkaline extraction step. It is rapid enough to be used as a screening method, permitting 50-100 or more samples to be extracted in a few hours. The DNA is sufficiently pure to be digestible by restriction enzymes, an important advantage for screening. A preparative version that allows isolation of larger quantities of more highly purified material is also described.

  14. Functional amyloids as inhibitors of plasmid DNA replication.

    PubMed

    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

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

    NASA Astrophysics Data System (ADS)

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

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

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

    SciTech Connect

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

    2012-01-01

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

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

    PubMed

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

    2016-01-01

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

  18. Functional amyloids as inhibitors of plasmid DNA replication

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  20. Conjugative plasmids: vessels of the communal gene pool

    PubMed Central

    Norman, Anders; Hansen, Lars H.; Sørensen, Søren J.

    2009-01-01

    Comparative whole-genome analyses have demonstrated that horizontal gene transfer (HGT) provides a significant contribution to prokaryotic genome innovation. The evolution of specific prokaryotes is therefore tightly linked to the environment in which they live and the communal pool of genes available within that environment. Here we use the term supergenome to describe the set of all genes that a prokaryotic ‘individual’ can draw on within a particular environmental setting. Conjugative plasmids can be considered particularly successful entities within the communal pool, which have enabled HGT over large taxonomic distances. These plasmids are collections of discrete regions of genes that function as ‘backbone modules’ to undertake different aspects of overall plasmid maintenance and propagation. Conjugative plasmids often carry suites of ‘accessory elements’ that contribute adaptive traits to the hosts and, potentially, other resident prokaryotes within specific environmental niches. Insight into the evolution of plasmid modules therefore contributes to our knowledge of gene dissemination and evolution within prokaryotic communities. This communal pool provides the prokaryotes with an important mechanistic framework for obtaining adaptability and functional diversity that alleviates the need for large genomes of specialized ‘private genes’. PMID:19571247

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

    PubMed

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

    1977-03-01

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

  2. Mitochondrial DNAs and plasmids as taxonomic characteristics in Trichoderma viride.

    PubMed Central

    Meyer, R J

    1991-01-01

    Mitochondrial DNA (mtDNA) was purified from 12 isolates of the Trichoderma viride aggregate and found to be, on the average, 32.7 kb in size. Plasmids were present in the mtDNA preparations from 8 of 12 strains of T. viride examined. Plasmids in four of the strains produced ladderlike banding patterns on gels, and these plasmids were studied in detail. The ladderlike patterns were produced by single molecules that were supercoiled to various degrees. Plasmids from two of the strains do not have homology with the mtDNA but do have a limited amount of homology with each other. No phenotype could be associated with the presence of a plasmid. Restriction endonuclease digestion of the mtDNAs produced patterns in which the presence or absence of certain fragments correlated with the classification of the strains into T. viride group I or II. Phenetic cluster analysis and parsimony analysis of the fragment patterns produced groups that corresponded to T. viride groups I and II. The fragment patterns were very diverse, with nearly all strains having a unique pattern. However, two strains of T. viride group I from widely different geographical locations did have identical restriction patterns for all the enzymes used in this study. This result indicates that it may not be possible to use mtDNA restriction patterns alone to identify Trichoderma strains. Images PMID:1768099

  3. Evolution of genes on the Salmonella Virulence plasmid phylogeny revealed from sequencing of the virulence plasmids of S. enterica serotype Dublin and comparative analysis.

    PubMed

    Chu, Chishih; Feng, Ye; Chien, An-Chi; Hu, Songnian; Chu, Chi-Hong; Chiu, Cheng-Hsun

    2008-11-01

    Salmonella enterica serotype Dublin harbors an approximately 80-kb virulence plasmid (pSDV), which mediates systemic infection in cattle. There are two types of pSDV: one is pSDVu (pOU1113) in strain OU7025 and the other pSDVr (pOU1115) in OU7409 (SD Lane) and many clinical isolates. Sequence analysis showed that pSDVr was a recombinant plasmid (co-integrate) of pSDVu and a plasmid similar to a 35-kb indigenous plasmid (pOU1114) of S. Dublin. Most of the F-transfer region in pSDVu was replaced by a DNA segment from the pOU1114-like plasmid containing an extra replicon and a pilX operon encoding for a type IV secretion system to form pSDVr. We reconstructed the particular evolutionary history of the seven virulence plasmids of Salmonella by comparative sequence analysis. The whole evolutionary process might begin with two different F-like plasmids (IncFI and IncFII), which then incorporated the spv operon and fimbriae operon from the chromosome to form the primitive virulence plasmids. Subsequently, these plasmids descended by deletion from a relatively large plasmid to smaller ones, with some recombination events occurring over time. Our results suggest that the phylogeny of virulence plasmids as a result of frequent recombination provides the opportunity for rapid evolution of Salmonella in response to the environmental cues. PMID:18718522

  4. Proton-induced direct and indirect damage of plasmid DNA.

    PubMed

    Vyšín, Luděk; Pachnerová Brabcová, Kateřina; Štěpán, Václav; Moretto-Capelle, Patrick; Bugler, Beatrix; Legube, Gaelle; Cafarelli, Pierre; Casta, Romain; Champeaux, Jean Philippe; Sence, Martine; Vlk, Martin; Wagner, Richard; Štursa, Jan; Zach, Václav; Incerti, Sebastien; Juha, Libor; Davídková, Marie

    2015-08-01

    Clustered DNA damage induced by 10, 20 and 30 MeV protons in pBR322 plasmid DNA was investigated. Besides determination of strand breaks, additional lesions were detected using base excision repair enzymes. The plasmid was irradiated in dry form, where indirect radiation effects were almost fully suppressed, and in water solution containing only minimal residual radical scavenger. Simultaneous irradiation of the plasmid DNA in the dry form and in the solution demonstrated the contribution of the indirect effect as prevalent. The damage composition slightly differed when comparing the results for liquid and dry samples. The obtained data were also subjected to analysis concerning different methodological approaches, particularly the influence of irradiation geometry, models used for calculation of strand break yields and interpretation of the strand breaks detected with the enzymes. It was shown that these parameters strongly affect the results. PMID:26007308

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

    PubMed

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

    2014-12-01

    Recent genome analysis of Erwinia amylovora, the causal agent of fire blight disease on Rosaceae, has shown that the chromosome is highly conserved among strains and that plasmids are the principal source of genomic diversity. A new circular plasmid, pEA68, was found in E. amylovora strain 692 (LMG 28361), isolated in Poland from Sorbus (mountain ash) with fire blight symptoms. Annotation of the 68,763-bp IncFIIa-type plasmid revealed that it contains 79 predicted CDS, among which two operons (tra, pil) are associated with mobility. The plasmid is maintained stably in E. amylovora and does not possess genes associated with antibiotic resistance or known virulence genes. Curing E. amylovora strain 692 of pEA68 did not influence its virulence in apple shoots nor amylovoran synthesis. Of 488 strains of E. amylovora from seventeen countries, pEA68 was only found in two additional strains from Belgium. Although the spread of pEA68 is currently limited to Europe, pEA68 comprises, together with pEA72 and pEA78 both found in North America, a new plasmid family that spans two continents. PMID:25178659

  6. Genetic recombination of bacterial plasmid DNA: effect of RecF pathway mutations on plasmid recombination in Escherichia coli.

    PubMed Central

    Kolodner, R; Fishel, R A; Howard, M

    1985-01-01

    Tn5 insertion mutations in the recN gene, and in what appears to be a new RecF pathway gene designated recO and mapping at approximately 55.4 min on the standard genetic map, were isolated by screening Tn5 insertion mutations that cotransduced with tyrA. The recO1504::Tn5 mutation decreased the frequency of recombination during Hfr-mediated crosses and increased the susceptibility to killing by UV irradiation and mitomycin C when present in a recB recC sbcB background, but only increased the sensitivity to killing by UV irradiation when present in an otherwise Rec+ background. The effects of these and other RecF pathway mutations on plasmid recombination were tested. Mutations in the recJ, recO, and ssb genes, when present in otherwise Rec+ E. coli strains, decreased the frequency of plasmid recombination, whereas the lexA3, recAo281, recN, and ruv mutations had no effect on plasmid recombination. Tn5 insertion mutations in the lexA gene increased the frequency of plasmid recombination. These data indicate that plasmid recombination events in wild-type Escherichia coli strains are catalyzed by a recombination pathway that is related to the RecF recombination pathway and that some component of this pathway besides the recA gene product is regulated by the lexA gene product. PMID:2993230

  7. The development of plasmid-free strains of Agrobacterium tumefaciens by using incompatibility with a Rhizobium meliloti plasmid to eliminate pAtC58.

    PubMed

    Hynes, M F; Simon, R; Pühler, A

    1985-03-01

    Agrobacterium tumefaciens strains LBA275 and LBA290 were cured of their cryptic plasmid pAtC58 by the introduction of the Rhizobium meliloti plasmid pRme41a, which is incompatible with pAtC58. pRme41a and pTiC58, the resident Ti plasmid of LBA275, were subsequently eliminated by growth at supraoptimal temperature (40 degrees C). The resulting plasmid-free Agrobacterium strains, UBAPF1 and UBAPF2, have proved extremely useful for the study of Rhizobium plasmids. The loss of the cryptic plasmid pAtC58 has no effect on the tumor-forming ability of the Agrobacterium strains; when the Ti plasmid is present, normal tumors are formed on Kalanchoe daigremontiana. PMID:4001194

  8. Complete Sequence of a blaKPC-Harboring Cointegrate Plasmid Isolated from Escherichia coli

    PubMed Central

    Chavda, Kalyan D.; Chen, Liang; Jacobs, Michael R.; Rojtman, Albert D.; Bonomo, Robert A.

    2015-01-01

    Horizontal transfer of blaKPC-harboring plasmids contributes significantly to the inter- and intraspecies spread of Klebsiella pneumoniae carbapenemase (KPC). Here we report the complete nucleotide sequence of a blaKPC-harboring IncFIA plasmid, pBK32533, from Escherichia coli. pBK32533 is a cointegrate plasmid comprising of a 72-kb sequence identical to that of the nonconjugative pBK30661 plasmid plus an additional 170-kb element that harbors the genes for plasmid transfer. pBK32533 demonstrates how blaKPC can be spread from a nonconjugative plasmid through cointegration. PMID:25753632

  9. Cloning and expression of a plasmid-linked pediocin determinant trait of Pediococcus acidilactici F.

    PubMed

    Osmanağaoğlu, O; Beyatli, Y; Gündüz, U

    2000-01-01

    Plasmid DNA from Pediococcus acidilactici F was prepared by lysozyme-mutanolysin method and purified by cesium chloride-ethidium bromide (CsCl-EtBr) density gradient ultracentrifugation. Agarose gel electrophoresis of plasmid DNA and plasmid-curing experiments suggested that bacteriocin activity was harboured on a small plasmid of approximately 9.1 kb (kilobasepair) in Pediococcus acidilactici F. Plasmid encoding bacteriocin production in P. acidilactici F was examined for restriction enzyme cleavage patterns and its map has been constructed. An Escherichia coli strain transformed with the recombinant plasmid, pQE322, produced and, most probably, secreted pediocin F. PMID:10746198

  10. Involvement of Linear Plasmids in Aerobic Biodegradation of Vinyl Chloride

    SciTech Connect

    BRIGMON, ROBINL.

    2004-06-14

    Pseudomonas putida strain AJ and Ochrobactrum strain TD were isolated from hazardous waste sites based on their ability to use vinyl chloride (VC) as a sole source of carbon and energy under aerobic conditions. Strains AJ and TD also use ethene and ethylene oxide as growth substrates. Strain AJ contained a linear megaplasmid (approximately 260 kb) when grown on VC or ethene, but no circular plasmids. While growing on ethylene oxide, the size of the linear plasmid in strain AJ decreased to approximately 100 kb, although its ability to use VC as a substrate was retained. The linear plasmids in strain AJ were cured and its ability to consume VC, ethene, and ethylene oxide was lost following growth on a rich substrate (Luria-Bertani broth) through at least three transfers. Strain TD contained three linear plasmids, ranging in size from approximately 100 kb to 320 kb, when growing on VC or ethene. As with strain AJ, the linear plasmids in strain TD were cured following growth on Luria -Bertani broth and its ability to consume VC and ethene was lost. Further analysis of these linear plasmids may help reveal the pathway for VC biodegradation in strains AJ and TD and explain why this process occurs at many but not all sites where groundwater is contaminated with chloroethenes. Metabolism of VC and ethene by strains AJ and TD is initiated by an alkene monooxygenase. Their yields during growth on VC (0.15-0.20 mg total suspended solids per mg VC) are similar to the yields reported for other isolates i.e., Mycobacterium sp., Nocardioides sp., and Pseudomonas sp.

  11. Effects of maternal plasmid GHRH treatment on offspring growth.

    PubMed

    Khan, Amir S; Bodles-Brakhop, Angela M; Fiorotto, Marta L; Draghia-Akli, Ruxandra

    2010-02-23

    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 HV-GHRH (n=4) plasmid. At birth, half of each litter was cross-fostered (treated to controls and controls to treated only). Piglets from plasmid-injected sows were heavier at birth (HV-GHRH, 1.65+/-0.07kg; Wt-GHRH, 1.46+/-0.05kg vs. Controls, 1.27+/-0.03kg; P>or=0.001) and at weaning (Wt-GHRH, 6.01+/-0.21kg and HV-GHRH, 6.34+/-0.15kg vs. Controls, 5.37+/-0.14kg; P>or=0.02, respectively). Control piglets cross-fostered to plasmid-injected sows grew faster to weaning (Wt-GHRH, 5.61+/-0.15kg and HV-GHRH, 5.70+/-0.29kg vs. Controls, 5.08+/-0.22kg; P>0.05, respectively). Piglets from plasmid-injected sows that suckled on control sows were larger than control piglets on control sows (Wt-GHRH, 5.93+/-0.20kg and HV-GHRH, 6.2+/-0.19kg vs. Controls, 5.08+/-0.22kg; P>0.05, respectively), but smaller than their littermates left on their treated mothers. The observed improvements were maintained until the end of the study when the offspring were 170-day-old. The results suggest that the improved growth of offspring of GHRH plasmid-treated sows pre-weaning is attributable to improved maternal performance, while after weaning the effects on the pituitary component are relevant. PMID:20188245

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

    PubMed

    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. PMID:10840595

  13. Plasmid vector with temperature-controlled gene expression

    SciTech Connect

    Kravchenko, V.V.; Yamshchikov, V.F.; Pletnev, A.G.

    1986-02-01

    In plasmid pBR327, a fragment 169 b.p. long including promotor p/sub 3/ of the bla gene has been deleted. The deletional derivative so obtained (pSP2) has been used to construct a recombinant plasmid bearing a fragment of phage lambda DNA with the p/sub R/ promotor and the gene of the temperature-sensitive repressor cI. It has been shown that the plasmid vector so constructed (pCE119) with promotor cR performs repressor-cI-controlled transcription of the bla gene, as a result of which induction for an hour at 42/sup 0/C leads to an almost 100-fold increase in the amount of product of the bla gene as compared with that at 32/sup 0/C. The possibility of the use of plasmid cPE119 for the expression of other genes has been demonstrated for the case of the semisynthetic ..beta..-galactosidase gene of E. coli. In this case, on induction of the cells with recombinant plasmid pCEZ12 for 3 hours at 42/sup 0/C, a 300-fold increase in the amount of active ..beta..-galactosidase, as compared with that at 32/sup 0/C, was observed. It is important to point out that under these conditions (at 42/sup 0/C), at least 99% of the cells containing the plasmid retain the phenotype lacZ/sup +/, which indicates the stability of the proposed vector system

  14. Genetic transformation of a clinical (genital tract), plasmid-free isolate of Chlamydia trachomatis: engineering the plasmid as a cloning vector.

    PubMed

    Wang, Yibing; Kahane, Simona; Cutcliffe, Lesley T; Skilton, Rachel J; Lambden, Paul R; Persson, Kenneth; Bjartling, Carina; Clarke, Ian N

    2013-01-01

    Our study had three objectives: to extend the plasmid-based transformation protocol to a clinical isolate of C. trachomatis belonging to the trachoma biovar, to provide "proof of principle" that it is possible to "knock out" selected plasmid genes (retaining a replication competent plasmid) and to investigate the plasticity of the plasmid. A recently developed, plasmid-based transformation protocol for LGV isolates of C. trachomatis was modified and a plasmid-free, genital tract C. trachomatis isolate from Sweden (SWFP-) was genetically transformed. Transformation of this non-LGV C. trachomatis host required a centrifugation step, but the absence of the natural plasmid removed the need for plaque purification of transformants. Transformants expressed GFP, were penicillin resistant and iodine stain positive for accumulated glycogen. The transforming plasmid did not recombine with the host chromosome. A derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene was engineered. CDS5 encodes pgp3, a protein secreted from the inclusion into the cell cytoplasm. This plasmid (pCDS5KO) was used to transform C. trachomatis SWFP-, and established that pgp3 is dispensable for plasmid function. The work shows it is possible to selectively delete segments of the chlamydial plasmid, and this is the first step towards a detailed molecular dissection of the role of the plasmid. The 3.6 kb β-galactosidase cassette was inserted into the deletion site of CDS5 to produce plasmid placZ-CDS5KO. Transformants were penicillin resistant, expressed GFP and stained for glycogen. In addition, they expressed β-galactosidase showing that the lacZ cassette was functional in C. trachomatis. An assay was developed that allowed the visualisation of individual inclusions by X-gal staining. The ability to express active β-galactosidase within chlamydial inclusions is an important advance as it allows simple, rapid assays to measure directly chlamydial infectivity without the need for

  15. Separation of plasmid DNA isoforms using centrifugal ultrafiltration.

    PubMed

    Borujeni, Ehsan Espah; Zydney, Andrew L

    2012-07-01

    Centrifugal ultrafiltration is a well-established method for concentrating and purifying DNA. Here, we describe the use of centrifugal ultrafiltration for the separation of plasmid DNA isoforms based on differences in elongational flexibility of the supercoiled, open-circular, and linear plasmids. Transmission of each isoform is minimal below a critical value of the filtration velocity, which is directly related to the magnitude of the centrifugal speed and the system geometry. A discontinuous diafiltration process was used to enrich the desired isoform, as determined by agarose gel electrophoresis. The simplicity and efficacy of this membrane-based separation are attractive for multiple applications requiring the use of separated DNA isoforms. PMID:22780319

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

    PubMed

    Nicholson, Bryon A; West, Aaron C; Mangiamele, Paul; Barbieri, Nicolle; Wannemuehler, Yvonne; Nolan, Lisa K; Logue, Catherine M; Li, Ganwu

    2016-01-01

    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. PMID:26800268

  17. Plasmid profiles, restriction fragment length polymorphisms and O-serotypes among Vibrio anguillarum isolates.

    PubMed Central

    Pedersen, K.; Tiainen, T.; Larsen, J. L.

    1996-01-01

    A total of 279 Vibrio anguillarum strains were serotyped and examined for plasmid content. Plasmids were subjected to digestion with restriction enzymes. Most strains belonged to serogroup O1 (39%) and O2 (16%). In total 164 strains (53%) carried plasmids. Of the O1 and O2 isolates, 92% and 30%, respectively, carried one or more plasmids. Restriction fragment length polymorphism (RFLP) analysis of plasmid DNA indicated that plasmids belonged to several groups. Each group seemed to be restricted to a single O-serovar. The largest group was the pJM1-like plasmids among most serovar O1 strains. Most of these plasmids were about 67 kb like the pJM1 plasmid, but various derivatives ranged from 26-77 kb. RFLP studies of the 67 kb plasmids revealed 17 different restriction patterns. Some patterns were dominant among European strains whereas others were dominant among North American strains. The results confirmed the applicability of O-serotyping together with plasmid profile and restriction analysis of plasmids for typing of V. anguillarum. They also indicated that plasmids among strains which belonged to the traditional fish pathogenic serogroups, O1 and O2, showed more homology than did strains from most other serogroups, that were usually non-pathogenic, environmental bacteria. Images Fig. 1 Fig. 2 Fig. 3 PMID:8972671

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

    PubMed Central

    Nicholson, Bryon A.; West, Aaron C.; Mangiamele, Paul; Barbieri, Nicolle; Wannemuehler, Yvonne; Nolan, Lisa K.; Logue, Catherine M.; Li, Ganwu

    2016-01-01

    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. PMID:26800268

  19. IMP-1 encoded by a novel Tn402-like class 1 integron in clinical Achromobacter xylosoxidans, China

    PubMed Central

    Chen, Zhenhong; Fang, Haihong; Wang, Li; Sun, Fengjun; Wang, Yong; Yin, Zhe; Yang, Huiying; Yang, Wenhui; Wang, Jie; Xia, Peiyuan; Zhou, Dongsheng; Liu, Changting

    2014-01-01

    Achromobacter xylosoxidans strain A22732 is isolated from a pneumonia patient in China and produces carbapenemases OXA-114e and IMP-1, which are encoded by chromosome and plasmid, respectively, and confer resistance to multiple ß-lactam antibiotics including carbapenems. The blaIMP-1 gene together with aacA7 and orfE is captured by a novel Tn402-like class 1 integron in a conjugative IncP-1ß plasmid. In addition to the intrinsic integron promoter PcW, there is still a blaIMP-1 gene cassette-specific promoter. This is the first report of carbapenemase-encoding IncP-1ß plasmid in clinical bacterial isolate. PMID:25428613

  20. USE OF A NOVEL PLASMID TO MONITOR THE FATE OF A GENETICALLY ENGINEERED PSEUDOMONAS PUTIDA STRAIN

    EPA Science Inventory

    Plasmid pSI30 was constructed to increase the sensitivity of detection of a genetically engineered microorganism (GEM) and its recombinant DNA in environmental samples. his broad host-range, mobilizable plasmid contained chlorocatechol (clc) degradative genes, antibiotic resistan...

  1. Nucleotide sequence of a small cryptic plasmid from Acidithiobacillus ferrooxidans strain A-6

    SciTech Connect

    F. Roberto

    2003-10-01

    A 2.1 kb cryptic plasmid from Acidithiobacillus ferrooxidans strain A-6 was isolated and cloned into the E. coli vector plasmid, pUC128. The cloned plasmid was mapped by restriction enzyme fragment analysis and subsequently sequenced. At this time over half the plasmid sequence has been determined and compared to sequences in the GenBank nucleotide and protein sequence databases. Much of the plasmid remains cryptic, but substantial nucleotide and protein sequence similarities have been observed to the putative replication protein, RepA, of the small cryptic plasmids pAYS and pAYL found in the ammonia-oxidizing Nitrosomonas sp. Strain ENI-11. These results suggest an entirely new class of plasmid is maintained in at least one strain of Acidithiobacillus ferrooxidans and other acidophilic bacteria, and raises interesting questions about the origin of this plasmid in acidic environments.

  2. Conservation of plasmids among plant-pathogenic Pseudomonas syringae isolates of diverse origins.

    PubMed

    von Bodman, S B; Shaw, P D

    1987-05-01

    Thirty isolates of Pseudomonas syringae pv. tabaci, pv. angulata (pathogens on tobacco), pv. coronafaciens, and pv. striafaciens (pathogens on oats) were examined for plasmid DNAs. The strains were obtained from plants throughout the world, some over 50 years ago. Of the 22 tobacco pathogens, 16 contain predominantly one type of plasmid, the pJP27.00 type. The remaining six tobacco-specific strains do not harbor detectable plasmids. The oat pathogens contain one, two, or three plasmids. DNA homology studies indicate that the plasmid DNAs are highly conserved. More importantly, the plasmids harbored by strains isolated from one host plant are conserved most stringently; e.g., the plasmids from the tobacco pathogens are, with one exception, indistinguishable by restriction endonuclease digestion and Southern hybridization. There is also extensive homology among plasmids indigenous to the oat-specific P. syringae pv. coronafaciens and pv. striafaciens strains. PMID:3628554

  3. A Time-Efficient and User-Friendly Method for Plasmid DNA Restriction Analysis.

    ERIC Educational Resources Information Center

    LaBanca, Frank; Berg, Claire M.

    1998-01-01

    Describes an experiment in which plasmid DNA is digested with restriction enzymes that cleave the plasmid either once or twice. The DNA is stained, loaded on a gel, electrophoresed, and viewed under normal laboratory conditions during electrophoresis. (DDR)

  4. Description of a 2,683-base-pair plasmid containing qnrD in two Providencia rettgeri isolates.

    PubMed

    Guillard, Thomas; Cambau, Emmanuelle; Neuwirth, Catherine; Nenninger, Thomas; Mbadi, Aurore; Brasme, Lucien; Vernet-Garnier, Véronique; Bajolet, Odile; de Champs, Christophe

    2012-01-01

    qnr genes are plasmid-mediated quinolone resistance genes mainly harbored on large conjugative multiresistant plasmids. The qnrD gene was recently observed in Salmonella enterica on a small nonconjugative plasmid (p2007057). We describe two strains of Providencia rettgeri harboring qnrD on nonconjugative plasmids. The plasmids were 99% identical, with 2,683 bp and four open reading frames, including qnrD, but exhibited only 53% identity with the plasmid found in S. enterica. PMID:21986831

  5. Autonomous plasmid-like replication of a conjugative transposon

    PubMed Central

    Lee, Catherine A.; Babic, Ana; Grossman, Alan D.

    2010-01-01

    Summary Integrative and conjugative elements (ICEs), a.k.a. conjugative transposons, are mobile genetic elements involved in many biological processes, including pathogenesis, symbiosis, and the spread of antibiotic resistance. Unlike conjugative plasmids that are extra-chromosomal and replicate autonomously, ICEs are integrated in the chromosome and replicate passively during chromosomal replication. It is generally thought that ICEs do not replicate autonomously. We found that when induced, Bacillus subtilis ICEBs1 undergoes autonomous plasmid-like replication. Replication was unidirectional, initiated from the ICEBs1 origin of transfer, oriT, and required the ICEBs1-encoded relaxase NicK. Replication also required several host proteins needed for chromosomal replication, but did not require the replicative helicase DnaC or the helicase loader protein DnaB. Rather, replication of ICEBs1 required the helicase PcrA that is required for rolling circle replication of many plasmids. Transfer of ICEBs1 from the donor required PcrA, but did not require replication, indicating that PcrA, and not DNA replication, facilitates unwinding of ICEBs1 DNA for horizontal transfer. Although not needed for horizontal transfer, replication of ICEBs1 was needed for stability of the element. We propose that autonomous plasmid-like replication is a common property of ICEs and contributes to the stability and maintenance of these mobile genetic elements in bacterial populations. PMID:19943900

  6. Analysis of plasmid deletional instability in Bacillus subtilis.

    PubMed Central

    Hahn, J; Dubnau, D

    1985-01-01

    Using a model system, we have studied deletion formation in Bacillus subtilis. When the staphylococcal plasmids pSA2100 (7.1 kilobases) and pUB110 (4.5 kilobases) were ligated to one another at their unique XbaI sites and transformed into either rec+ or recE4 strains of B. subtilis, an intramolecular recombination event usually occurred. Two plasmids, one of 2.6 kilobases and the other of 9.0 kilobases, were consistently isolated and shown by restriction enzyme analysis to be derived by recombination occurring in the pSA2100-pUB110 cointegrate. Analysis of the sequence of the junctions of the recombinant plasmids and of the crossover regions of the parental plasmids suggested that a reciprocal, conservative, intramolecular recombination event had occurred between short 18-base-pair homologous sequences that were oriented as direct repeats and bounded by regions of dyad symmetry. Evidence is presented that the above illegitimate recombination event is biased to occur intramolecularly and that randomly chosen direct repeats of either 22 or 29 base pairs are not sufficient to support recombination. The recombination event occurs in recA1, recB2, recD3, recE5, recL16, recM13, polA59, polA13, uvr-22, uvr-13, and stb mutants of B. subtilis and does not require that the competent state be established. Images PMID:3922940

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

    PubMed

    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. PMID:23185899

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

    PubMed Central

    Krupovic, Mart; Ravantti, Janne J; Bamford, Dennis H

    2009-01-01

    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. PMID:19460138

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

    SciTech Connect

    Cooksey, D.A. )

    1990-01-01

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

  10. Effects of maternal plasmid GHRH treatment on offspring growth

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  11. [Transfer of plasmid beta-lactamases in enterobacteria].

    PubMed

    Umaran, A; Garaizar, J; Gallego, L; Colom, K; Cisterna, R

    1989-04-01

    The aim of the present study was to determine which types of beta-lactamases codified by plasmids are transferred by conjugation from several species of enterobacteria. To this end, 352 strains of ampicillin-resistant enterobacteria from clinical samples from the Hospital Civil of Bilbao were evaluated. Their beta-lactamase activity and their capacity to transfer this capacity by conjugation were evaluated. The several types of plasmidic beta-lactamases in the strains that conjugated and in their respective transconjugants were characterized by analytic isoelectric approach, and also the sensitivity of these stains to 20 beta-lactamic antibiotics and the size of their plasmids. Twenty different types were detected, with a clear predominance of TEM 1. Type TEM 2 was found in 19% of the strains which conjugated, and much less commonly the types SHV 1, HMS 1 and a beta-lactamase of an approximate pl of 4.9 were found. The transfer of these beta-lactamases is mediated by a great variety of plasmids and is associated with variable levels of resistance to penicillins and unstable cephalosporins. The presence of betalactamases with activity on the more stable cephalosporins has not been detected. PMID:2490696

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

    PubMed Central

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

    2015-01-01

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

  13. The replication origin of a repABC plasmid

    PubMed Central

    2011-01-01

    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 oriV of this plasmid resides

  14. Thioredoxin from Streptomyces aureofaciens controls coiling of plasmid DNA.

    PubMed

    Golubnitchaya-Labudova, O; Horecka, T; Kapalla, M; Perecko, D; Kutejova, E; Lubec, G

    1998-01-01

    A number of potential functions of thioredoxin have been proposed in literature, including a role for DNA replication. The aim of our study was to investigate the effects of thioredoxin from Streptomyces aureofaciens (Trx S.a.) on plasmid DNA. Trx S.a. was incubated with plasmid forms and the incubation product(s) characterized on agarose gels. To compare Trx activity with enzymes with known DNA modifying activities, topoisomerase I, II (gyrase) and T4 DNA ligase were incubated with plasmid DNA in parallel. For the demonstration of nick removal a PCR technique was used. Trx S.a. bound non-specifically to plasmid DNA relaxing supercoiled circle closed form (CCC form) with subsequent formation of the circle closed form (CC form) as a major product. The amplification of a specific DNA template, possible only after nick removal, took place following incubation with Trx. The effect of topoisomerase I on plasmid DNA resembled Trx S.a. activity. We propose the following mechanism for CCC relaxation: Binding of Trx leads to a break of one strand and CC is formed by stepwise relaxation, ending with nick removal. The concomitant finding of open circle form (OC form) generation after incubation with Trx may indicate the generation of an intermediate due to the postulated strand break at initiation. This control of coiling may play a role in the DNA replication machinery, providing CC as a readily available substrate for DNA polymerases. In addition, Trx may serve in DNA repair mechanisms by its nonspecific binding to DNA and nick removing activity. PMID:9449230

  15. Plasmid copy number underlies adaptive mutability in bacteria.

    PubMed

    Sano, Emiko; Maisnier-Patin, Sophie; Aboubechara, John Paul; Quiñones-Soto, Semarhy; Roth, John R

    2014-11-01

    The origin of mutations under selection has been intensively studied using the Cairns-Foster system, in which cells of an Escherichia coli lac mutant are plated on lactose and give rise to 100 Lac+ revertants over several days. These revertants have been attributed variously to stress-induced mutagenesis of nongrowing cells or to selective improvement of preexisting weakly Lac+ cells with no mutagenesis. Most revertant colonies (90%) contain stably Lac+ cells, while others (10%) contain cells with an unstable amplification of the leaky mutant lac allele. Evidence is presented that both stable and unstable Lac+ revertant colonies are initiated by preexisting cells with multiple copies of the F'lac plasmid, which carries the mutant lac allele. The tetracycline analog anhydrotetracycline (AnTc) inhibits growth of cells with multiple copies of the tetA gene. Populations with tetA on their F'lac plasmid include rare cells with an elevated plasmid copy number and multiple copies of both the tetA and lac genes. Pregrowth of such populations with AnTc reduces the number of cells with multiple F'lac copies and consequently the number of Lac+ colonies appearing under selection. Revertant yield is restored rapidly by a few generations of growth without AnTc. We suggest that preexisting cells with multiple F'lac copies divide very little under selection but have enough energy to replicate their F'lac plasmids repeatedly until reversion initiates a stable Lac+ colony. Preexisting cells whose high-copy plasmid includes an internal lac duplication grow under selection and produce an unstable Lac+ colony. In this model, all revertant colonies are initiated by preexisting cells and cannot be stress induced. PMID:25173846

  16. GeneGuard: A modular plasmid system designed for biosafety.

    PubMed

    Wright, Oliver; Delmans, Mihails; Stan, Guy-Bart; Ellis, Tom

    2015-03-20

    Synthetic biology applications in biosensing, bioremediation, and biomining envision the use of engineered microbes beyond a contained laboratory. Deployment of such microbes in the environment raises concerns of unchecked cellular proliferation or unwanted spread of synthetic genes. While antibiotic-resistant plasmids are the most utilized vectors for introducing synthetic genes into bacteria, they are also inherently insecure, acting naturally to propagate DNA from one cell to another. To introduce security into bacterial synthetic biology, we here took on the task of completely reformatting plasmids to be dependent on their intended host strain and inherently disadvantageous for others. Using conditional origins of replication, rich-media compatible auxotrophies, and toxin-antitoxin pairs we constructed a mutually dependent host-plasmid platform, called GeneGuard. In this, replication initiators for the R6K or ColE2-P9 origins are provided in trans by a specified host, whose essential thyA or dapA gene is translocated from a genomic to a plasmid location. This reciprocal arrangement is stable for at least 100 generations without antibiotic selection and is compatible for use in LB medium and soil. Toxin genes ζ or Kid are also employed in an auxiliary manner to make the vector disadvantageous for strains not expressing their antitoxins. These devices, in isolation and in concert, severely reduce unintentional plasmid propagation in E. coli and B. subtilis and do not disrupt the intended E. coli host's growth dynamics. Our GeneGuard system comprises several versions of modular cargo-ready vectors, along with their requisite genomic integration cassettes, and is demonstrated here as an efficient vector for heavy-metal biosensors. PMID:24847673

  17. Plasmid profiling of bacterial isolates from confined environments

    NASA Astrophysics Data System (ADS)

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

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

  18. Plasmid Mediated Antibiotic Resistance in Isolated Bacteria From Burned Patients

    PubMed Central

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

    2014-01-01

    Background: Nowadays, the treatment of burned patients is difficult because of the high frequency of infection with antibiotic resistance bacteria. Objectives: This study was conducted to evaluate the level of antibiotic resistance in Gram-negative bacteria and its relation with the existence of plasmid. Materials and Methods: 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. Results: 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. Conclusions: 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. PMID:25789121

  19. Chromosome and Plasmids of the Tick-Borne Relapsing Fever Agent Borrelia hermsii

    PubMed Central

    2016-01-01

    The zoonotic pathogen Borrelia hermsii bears its multiple paralogous genes for variable antigens on several linear plasmids. Application of combined long-read and short-read next-generation sequencing provided complete sequences for antigen-encoding plasmids as well as other linear and circular plasmids and the linear chromosome of the genome. PMID:27284141

  20. Generation and Molecular Characterization of New Temperature-Sensitive Plasmids Intended for Genetic Engineering of Pasteurellaceae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Temperature-sensitive (TS) plasmids were generated through chemical mutagenesis of a derivative of the streptomycin resistance parent plasmid pD70, isolated from Mannheimia hemolytica serotype 1. Three TS plasmids which failed to replicate at or above 42°C in M. hemolytica but which were fully func...

  1. Mix and match of KPC-2 encoding plasmids in Enterobacteriaceae-comparative genomics.

    PubMed

    Chmelnitsky, Inna; Shklyar, Maya; Leavitt, Azita; Sadovsky, Evgeniya; Navon-Venezia, Shiri; Ben Dalak, Maayan; Edgar, Rotem; Carmeli, Yehuda

    2014-06-01

    We performed comparative sequence analysis of 3 blaKPC-2 encoding plasmids to examine evolution of these plasmids and their dissemination. We found that all of them have an IncN replicon with a newly determined IncN plasmid sequence type (ST), ST15. The 2 Klebsiella pneumoniae (KPN) plasmids also harbor an IncF2A1-B1- replicon. The blaKPC-2 is located in the Tn4401c transposon with a newly discovered mutation in the P2 promoter. Screening of the 27 additional blaKPC-2 carrying plasmids from Enterobacter cloacae, Escherichia coli (EC), and K. pneumoniae showed that: all KPN and EC plasmids are IncN plasmids belonging to ST15; 4/7 KPN and 1/6 EC plasmids contain an additional IncF2A1-B1- replicon; all Enterobacter plasmids belong to neither IncN nor IncF2A1-B1- replicon plasmids; 6/7 KPN and 2/5 EC plasmids carry the mutated P2 promoter. Study of the blaKPC-2 environment, transposon, pMLST, and Inc group suggests transposon and plasmid inter- and intra-species dissemination and evolution. PMID:24743043

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  3. Chromosome and Plasmids of the Tick-Borne Relapsing Fever Agent Borrelia hermsii.

    PubMed

    Barbour, Alan G

    2016-01-01

    The zoonotic pathogen Borrelia hermsii bears its multiple paralogous genes for variable antigens on several linear plasmids. Application of combined long-read and short-read next-generation sequencing provided complete sequences for antigen-encoding plasmids as well as other linear and circular plasmids and the linear chromosome of the genome. PMID:27284141

  4. The 2 micrometer plasmid stability system: analyses of the interactions among plasmid- and host-encoded components.

    PubMed

    Velmurugan, S; Ahn, Y T; Yang, X M; Wu, X L; Jayaram, M

    1998-12-01

    The stable inheritance of the 2 micrometer plasmid in a growing population of Saccharomyces cerevisiae is dependent on two plasmid-encoded proteins (Rep1p and Rep2p), together with the cis-acting locus REP3 (STB). In this study we demonstrate that short carboxy-terminal deletions of Rep1p and Rep2p severely diminish their normal capacity to localize to the yeast nucleus. The nuclear targeting, as well as their functional role in plasmid partitioning, can be restored by the addition of a nuclear localization sequence to the amino or the carboxy terminus of the shortened Rep proteins. Analyses of deletion derivatives of the Rep proteins by using the in vivo dihybrid genetic test in yeast, as well as by glutathione S-transferase fusion trapping assays in vitro demonstrate that the amino-terminal portion of Rep1p (ca. 150 amino acids long) is responsible for its interactions with Rep2p. In a monohybrid in vivo assay, we have identified Rep1p, Rep2p, and a host-encoded protein, Shf1p, as being capable of interacting with the STB locus. The Shf1 protein expressed in Escherichia coli can bind with high specificity to the STB sequence in vitro. In a yeast strain deleted for the SHF1 locus, a 2 micrometer circle-derived plasmid shows relatively poor stability. PMID:9819432

  5. The 2μm Plasmid Stability System: Analyses of the Interactions among Plasmid- and Host-Encoded Components

    PubMed Central

    Velmurugan, Soundarapandian; Ahn, Yong-Tae; Yang, Xian-Mei; Wu, Xu-Li; Jayaram, Makkuni

    1998-01-01

    The stable inheritance of the 2μm plasmid in a growing population of Saccharomyces cerevisiae is dependent on two plasmid-encoded proteins (Rep1p and Rep2p), together with the cis-acting locus REP3 (STB). In this study we demonstrate that short carboxy-terminal deletions of Rep1p and Rep2p severely diminish their normal capacity to localize to the yeast nucleus. The nuclear targeting, as well as their functional role in plasmid partitioning, can be restored by the addition of a nuclear localization sequence to the amino or the carboxy terminus of the shortened Rep proteins. Analyses of deletion derivatives of the Rep proteins by using the in vivo dihybrid genetic test in yeast, as well as by glutathione S-transferase fusion trapping assays in vitro demonstrate that the amino-terminal portion of Rep1p (ca. 150 amino acids long) is responsible for its interactions with Rep2p. In a monohybrid in vivo assay, we have identified Rep1p, Rep2p, and a host-encoded protein, Shf1p, as being capable of interacting with the STB locus. The Shf1 protein expressed in Escherichia coli can bind with high specificity to the STB sequence in vitro. In a yeast strain deleted for the SHF1 locus, a 2μm circle-derived plasmid shows relatively poor stability. PMID:9819432

  6. Plasmid vectors for Xylella fastidiosa utilizing a toxin-antitoxin system for plasmid stability in the absence of antibiotic selection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacte...

  7. Role of the 85-Kilobase Plasmid and Plasmid-Encoded Virulence-Associated Protein A in Intracellular Survival and Virulence of Rhodococcus equi

    PubMed Central

    Giguère, Steeve; Hondalus, Mary K.; Yager, Julie A.; Darrah, Patricia; Mosser, David M.; Prescott, John F.

    1999-01-01

    Rhodococcus equi is a facultative intracellular pathogen of macrophages and a cause of pneumonia in young horses (foals) and immunocompromised people. Isolates of R. equi from pneumonic foals typically contain large, 85- or 90-kb plasmids encoding a highly immunogenic virulence-associated protein (VapA). The objective of this study was to determine the role of the 85-kb plasmid and VapA in the intracellular survival and virulence of R. equi. Clinical isolates containing the plasmid and expressing VapA efficiently replicated within mouse macrophages in vitro, while plasmid-cured derivatives of these organisms did not multiply intracellularly. An isolate harboring the large plasmid also replicated in the tissues of experimentally infected mice, whereas its plasmid-cured derivative was rapidly cleared. All foals experimentally infected with a plasmid-containing clinical isolate developed severe bronchopneumonia, whereas the foals infected with its plasmid-cured derivative remained asymptomatic and free of visible lung lesions. By day 14 postinfection, lung bacterial burdens had increased considerably in foals challenged with the plasmid-containing clinical isolate. In contrast, bacteria could no longer be cultured from the lungs of foals challenged with the isogenic plasmid-cured derivative. A recombinant, plasmid-cured derivative expressing wild-type levels of VapA failed to replicate in macrophages and remained avirulent for both mice and foals. These results show that the 85-kb plasmid of R. equi is essential for intracellular replication within macrophages and for development of disease in the native host, the foal. However, expression of VapA alone is not sufficient to restore the virulence phenotype. PMID:10377138

  8. Parallel compensatory evolution stabilizes plasmids across the parasitism-mutualism continuum.

    PubMed

    Harrison, Ellie; Guymer, David; Spiers, Andrew J; Paterson, Steve; Brockhurst, Michael A

    2015-08-01

    Plasmids drive genomic diversity in bacteria via horizontal gene transfer [1, 2]; nevertheless, explaining their survival in bacterial populations is challenging [3]. Theory predicts that irrespective of their net fitness effects, plasmids should be lost: when parasitic (costs outweigh benefits), plasmids should decline due to purifying selection [4-6], yet under mutualism (benefits outweigh costs), selection favors the capture of beneficial accessory genes by the chromosome and loss of the costly plasmid backbone [4]. While compensatory evolution can enhance plasmid stability within populations [7-15], the propensity for this to occur across the parasitism-mutualism continuum is unknown. We experimentally evolved Pseudomonas fluorescens and its mercury resistance mega-plasmid, pQBR103 [16], across an environment-mediated parasitism-mutualism continuum. Compensatory evolution stabilized plasmids by rapidly ameliorating the cost of plasmid carriage in all environments. Genomic analysis revealed that, in both parasitic and mutualistic treatments, evolution repeatedly targeted the gacA/gacS bacterial two-component global regulatory system while leaving the plasmid sequence intact. Deletion of either gacA or gacS was sufficient to completely ameliorate the cost of plasmid carriage. Mutation of gacA/gacS downregulated the expression of ∼17% of chromosomal and plasmid genes and appears to have relieved the translational demand imposed by the plasmid. Chromosomal capture of mercury resistance accompanied by plasmid loss occurred throughout the experiment but very rarely invaded to high frequency, suggesting that rapid compensatory evolution can limit this process. Compensatory evolution can explain the widespread occurrence of plasmids and allows bacteria to retain horizontally acquired plasmids even in environments where their accessory genes are not immediately useful. PMID:26190075

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

    NASA Astrophysics Data System (ADS)

    Mudgett, John Stuart

    1987-09-01

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

  10. Complete sequence of three plasmids from Bacillus thuringiensis INTA-FR7-4 environmental isolate and comparison with related plasmids from the Bacillus cereus group.

    PubMed

    Amadio, Ariel F; Benintende, Graciela B; Zandomeni, Rubén O

    2009-11-01

    Bacillus thuringiensis is an insect pathogen used worldwide as a bioinsecticide. It belongs to the Bacillus cereus sensu lato group as well as Bacillus anthracis and B. cereus. Plasmids from this group of organisms have been implicated in pathogenicity as they carry the genes responsible for different types of diseases that affect mammals and insects. Some plasmids, like pAW63 and pBT9727, encode a functional conjugation machinery allowing them to be transferred to a recipient cell. They also share extensive homology with the non-functional conjugation apparatus of pXO2 from B. anthracis. In this study we report the complete sequence of three plasmids from an environmental B. thuringiensis isolate from Argentina, obtained by a shotgun sequencing method. We obtained the complete nucleotide sequence of plasmids pFR12 (12,095bp), pFR12.5 (12,459bp) and pFR55 (55,712bp) from B. thuringiensis INTA-FR7-4. pFR12 and pFR12.5 were classified as cryptic as they do not code for any obvious functions besides replication and mobilization. Both small plasmids were classified as RCR plasmids due to similarities with the replicases they encode. Plasmid pFR55 showed a structural organization similar to that observed for plasmids pAW63, pBT9727 and pXO2. pFR55 also shares a tra region with these plasmids, containing genes related to T4SS and conjugation. A comparison between pFR55 and conjugative plasmids led to the postulation that pFR55 is a conjugative plasmid. Genes related to replication functions in pFR55 are different to those described for plasmids with known complete sequences. pFR55 is the first completely sequenced plasmid with a replication machinery related to that of ori44. The analysis of the complete sequence of plasmids from an environmental isolate of B. thuringiensis permitted the identification of a near complete conjugation apparatus in pFR55, resembling those of plasmids pAW63, pBT9727 and pXO2. The availability of this sequence is a step forward in the study

  11. [Plasmids of streptomycetes strains isolated from soils of Ukraine with different anthropogenic loading].

    PubMed

    Luk'ianchuk, V V; Polishchuk, L V; Matseliukh, B P

    2010-01-01

    Screening of plasmid DNA was carried out among 94 streptomycetes cultures which were isolated from the samples of Ukrainian soils with different anthropogenic contamination. Seventeen streptomycetes strains containing plasmid DNA were found. It is established that some cultures contain more than one plasmid (Streptomyces sp.M15, S.sp.T8, S.sp.T19). Depending on a molecular sizes the found plasmids were divided in 2 groups: 3 kb-15 kb, and 30 kb-70 kb. Research of a few morphological and physiological properties of plasmid strains of streptomycetes was carried out. The paper is presented in Ukrainian. PMID:21117293

  12. Presence and Analysis of Plasmids in Human and Animal Associated Arcobacter Species

    PubMed Central

    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. PMID:24465575

  13. Plasmid analysis of Shigella dysenteriae type 1 isolates obtained from widely scattered geographical locations.

    PubMed Central

    Haider, K; Kay, B A; Talukder, K A; Huq, M I

    1988-01-01

    Plasmid profiles and antimicrobial susceptibility patterns of 343 strains of Shigella dysenteriae type 1, obtained from 18 different geographical locations, were analyzed. Three plasmids, with molecular sizes of 140, 6, and 2 megadaltons (MDa), were present in 94, 98, and 96%, respectively, of the 343 strains isolated during either epidemic or nonepidemic periods from 1965 to 1987. In addition to these plasmids, 83% of the strains harbored a 4-MDa plasmid and 25% harbored a 20-MDa plasmid. Various plasmid profiles were observed in which the 140-, 6-, and 2-MDa plasmids occurred commonly, irrespective of the place of isolation and drug resistance pattern of the strains. Certain profiles showed significant association with drug resistance patterns. These findings suggest that three plasmids, of molecular sizes 140, 6, and 2 MDa, are unique to S. dysenteriae type 1 strains and may indicate the global spread of a pathogenic bacterial clone. Additionally, these core plasmids, plus plasmids of various other sizes, could be used to identify emerging subclones which are causing both epidemic and sporadic disease. Thus, plasmid profiles of S. dysenteriae type 1 strains can be used to monitor possible pandemic strains as well as individual epidemic strains. Images PMID:3053762

  14. Asymmetrical Inheritance of Plasmids Depends on Dynamic Cellular Geometry and Volume Exclusion Effects

    PubMed Central

    Marquez-Lago, Tatiana T.

    2015-01-01

    The asymmetrical inheritance of plasmid DNA, as well as other cellular components, has been shown to be involved in replicative aging. In Saccharomyces cerevisiae, there is an ongoing debate regarding the mechanisms underlying this important asymmetry. Currently proposed models suggest it is established via diffusion, but differ on whether a diffusion barrier is necessary or not. However, no study so far incorporated key aspects to segregation, such as dynamic morphology changes throughout anaphase or plasmids size. Here, we determine the distinct effects and contributions of individual cellular variability, plasmid volume and moving boundaries in the asymmetric segregation of plasmids. We do this by measuring cellular nuclear geometries and plasmid diffusion rates with confocal microscopy, subsequently incorporating this data into a growing domain stochastic spatial simulator. Our modelling and simulations confirms that plasmid asymmetrical inheritance does not require an active barrier to diffusion, and provides a full analysis on plasmid size effects. PMID:26468952

  15. Analysis of plasmids in nosocomial strains of multiple-antibiotic-resistant Staphylococcus aureus.

    PubMed Central

    Lyon, B R; May, J W; Skurray, R A

    1983-01-01

    Nosocomial infections caused by Staphylococcus aureus strains resistant to methicillin and multiple antibiotics have reached epidemic proportions in Melbourne, Australia, over the past 5 years. Plasmid analysis of representative clinical isolates demonstrated the presence of three classes of plasmid DNA in most strains. Resistance to gentamicin, kanamycin, and tobramycin was usually mediated by an 18-megadalton plasmid but could also be encoded by a related 22-megadalton plasmid. Two distinguishable plasmids of 3 megadaltons each endowed resistance to chloramphenicol, and the third class consisted of small plasmids, each approximately 1 megadalton in size, with no attributable function. An extensive array of resistance determinants, including some which have usually been associated with a plasmid locus, were found to exist on the chromosome. Evidence that resistance to gentamicin, kanamycin, and tobramycin is chromosomally encoded in some clinical isolates suggests that this determinant may have undergone genetic translocation onto the staphylococcal chromosome. Images PMID:6311086

  16. Asymmetrical Inheritance of Plasmids Depends on Dynamic Cellular Geometry and Volume Exclusion Effects.

    PubMed

    Denton, Jai A; Ghosh, Atiyo; Marquez-Lago, Tatiana T

    2015-01-01

    The asymmetrical inheritance of plasmid DNA, as well as other cellular components, has been shown to be involved in replicative aging. In Saccharomyces cerevisiae, there is an ongoing debate regarding the mechanisms underlying this important asymmetry. Currently proposed models suggest it is established via diffusion, but differ on whether a diffusion barrier is necessary or not. However, no study so far incorporated key aspects to segregation, such as dynamic morphology changes throughout anaphase or plasmids size. Here, we determine the distinct effects and contributions of individual cellular variability, plasmid volume and moving boundaries in the asymmetric segregation of plasmids. We do this by measuring cellular nuclear geometries and plasmid diffusion rates with confocal microscopy, subsequently incorporating this data into a growing domain stochastic spatial simulator. Our modelling and simulations confirms that plasmid asymmetrical inheritance does not require an active barrier to diffusion, and provides a full analysis on plasmid size effects. PMID:26468952

  17. Replisome Assembly at Bacterial Chromosomes and Iteron Plasmids

    PubMed Central

    Wegrzyn, Katarzyna E.; Gross, Marta; Uciechowska, Urszula; Konieczny, Igor

    2016-01-01

    The proper initiation and occurrence of DNA synthesis depends on the formation and rearrangements of nucleoprotein complexes within the origin of DNA replication. In this review article, we present the current knowledge on the molecular mechanism of replication complex assembly at the origin of bacterial chromosome and plasmid replicon containing direct repeats (iterons) within the origin sequence. We describe recent findings on chromosomal and plasmid replication initiators, DnaA and Rep proteins, respectively, and their sequence-specific interactions with double- and single-stranded DNA. Also, we discuss the current understanding of the activities of DnaA and Rep proteins required for replisome assembly that is fundamental to the duplication and stability of genetic information in bacterial cells. PMID:27563644

  18. Replisome Assembly at Bacterial Chromosomes and Iteron Plasmids.

    PubMed

    Wegrzyn, Katarzyna E; Gross, Marta; Uciechowska, Urszula; Konieczny, Igor

    2016-01-01

    The proper initiation and occurrence of DNA synthesis depends on the formation and rearrangements of nucleoprotein complexes within the origin of DNA replication. In this review article, we present the current knowledge on the molecular mechanism of replication complex assembly at the origin of bacterial chromosome and plasmid replicon containing direct repeats (iterons) within the origin sequence. We describe recent findings on chromosomal and plasmid replication initiators, DnaA and Rep proteins, respectively, and their sequence-specific interactions with double- and single-stranded DNA. Also, we discuss the current understanding of the activities of DnaA and Rep proteins required for replisome assembly that is fundamental to the duplication and stability of genetic information in bacterial cells. PMID:27563644

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

    DOEpatents

    Bradbury, Andrew M.

    2012-07-24

    The invention relates to a novel phagemid display system for packaging phagemid DNA into phagemid particles which completely avoids the use of helper phage. The system of the invention incorporates the use of bacterial packaging cell lines which have been transformed with helper plasmids containing all required phage proteins but not the packaging signals. The absence of packaging signals in these helper plasmids prevents their DNA from being packaged in the bacterial cell, which provides a number of significant advantages over the use of both standard and modified helper phage. Packaged phagemids expressing a protein or peptide of interest, in fusion with a phage coat protein such as g3p, are generated simply by transfecting phagemid into the packaging cell line.

  20. Spheroplast formation and plasmid isolation from Rhodococcus spp.

    PubMed

    Assaf, N A; Dick, W A

    1993-12-01

    The genus Rhodococcus comprises aerobic gram-positive actinomycetes that show considerable morphological and metabolic diversity and are known to be involved in the development of plant diseases and degradation of environmental pollutants. We describe a method for cell lysis and large plasmid DNA isolation from Rhodococcus by creating lysozyme susceptible cells by predigestion with the enzyme mutanolysin. Mutanolysin action resulted in the liberation of reducing sugars and free amino acids from the peptidoglycan layers of the cell wall. A 1-h predigestion with mutanolysin followed by a 0.5-h incubation with lysozyme resulted in spheroplast formation. Complete lysis of cells and efficient isolation of intact large plasmid DNA (108 kb) from wild-type Rhodococcus strains was confirmed. PMID:8292332

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  2. The mosaicism of plasmids revealed by atypical genes detection and analysis

    PubMed Central

    2011-01-01

    Background From an evolutionary viewpoint, prokaryotic genomes are extremely plastic and dynamic, since large amounts of genetic material are continuously added and/or lost through promiscuous gene exchange. In this picture, plasmids play a key role, since they can be transferred between different cells and, through genetic rearrangement(s), undergo gene(s) load, leading, in turn, to the appearance of important metabolic innovations that might be relevant for cell life. Despite their central position in bacterial evolution, a massive analysis of newly acquired functional blocks [likely the result of horizontal gene transfer (HGT) events] residing on plasmids is still missing. Results We have developed a computational, composition-based, pipeline to scan almost 2000 plasmids for genes that differ significantly from their hosting molecule. Plasmids atypical genes (PAGs) were about 6% of the total plasmids ORFs and, on average, each plasmid possessed 4.4 atypical genes. Nevertheless, conjugative plasmids were shown to possess an amount of atypical genes than that found in not mobilizable plasmids, providing strong support for the central role suggested for conjugative plasmids in the context of HGT. Part of the retrieved PAGs are organized into (mainly short) clusters and are involved in important biological processes (detoxification, antibiotic resistance, virulence), revealing the importance of HGT in the spreading of metabolic pathways within the whole microbial community. Lastly, our analysis revealed that PAGs mainly derive from other plasmid (rather than coming from phages and/or chromosomes), suggesting that plasmid-plasmid DNA exchange might be the primary source of metabolic innovations in this class of mobile genetic elements. Conclusions In this work we have performed the first large scale analysis of atypical genes that reside on plasmid molecules to date. Our findings on PAGs function, organization, distribution and spreading reveal the importance of

  3. The broad-host-range plasmid pSFA231 isolated from petroleum-contaminated sediment represents a new member of the PromA plasmid family

    PubMed Central

    Li, Xiaobin; Top, Eva M.; Wang, Yafei; Brown, Celeste J.; Yao, Fei; Yang, Shan; Jiang, Yong; Li, Hui

    2015-01-01

    A self-transmissible broad-host-range (BHR) plasmid pSFA231 was isolated from petroleum-contaminated sediment in Shen-fu wastewater irrigation zone, China, using the triparental mating exogenous plasmid capture method. Based on its complete sequence the plasmid has a size of 41.5 kb and codes for 50 putative open reading frames (orfs), 29 of which represent genes involved in replication, partitioning and transfer functions of the plasmid. Phylogenetic analysis grouped pSFA231 into the newly defined PromA plasmid family, which currently includes five members. Further comparative genomic analysis shows that pSFA231 shares the common backbone regions with the other PromA plasmids, i.e., genes involved in replication, maintenance and control, and conjugative transfer. Nevertheless, phylogenetic divergence was found in specific gene products. We propose to divide the PromA group into two subgroups, PromA-α (pMRAD02, pSB102) and PromA-β (pMOL98, pIPO2T, pSFA231, pTer331), based on the splits network analysis of the RepA protein. Interestingly, a cluster of hypothetical orfs located between parA and traA of pSFA231 shows high similarity with the corresponding regions on pMOL98, pIPO2T, and pTer331, suggesting these hypothetical orfs may represent “essential” plasmid backbone genes for the PromA-β subgroup. Alternatively, they may also be accessory genes that were first acquired and then stayed as the plasmid diverged. Our study increases the available collection of complete genome sequences of BHR plasmids, and since pSFA231 is the only characterized PromA plasmid from China, our findings also enhance our understanding of the genetic diversity of this plasmid group in different parts of the world. PMID:25628616

  4. Involvement of plasmids in total degradation of chlorinated biphenyls

    SciTech Connect

    Furukawa, K.; Chakrabarty, A.M.

    1982-09-01

    Acinetobacter sp. stain P6 has previously been reported to utilize biphenyl (BP) and chlorinated BPs, with accumulation of corresponding chlorbenzoic acids. Arthrobacter sp. strain M5 was isolated as a contaminant in the culture of Acinetobacter sp. strain P6 growing on 4-chlorobiphenyl and showed properties similar to P6 in the degradation of chlorinated BPs. Both strains harbored an identical plasmid of 53.7 megadaltons. These strains spontaneously lost the ability to utilize BP and 4-chlorobiphenyl with high frequency (4 to 8%) after overnight growth in nutrient broth. The BP/sup -/ derivatives could not regain the BP-assimilating ability (reversion frequency, <10/sup -9/ per cell per generation) but retained the plasmid with small, detectable deletions. BP/sup +/ P6 cells grown on BP or benzoate oxidized BP and 2,3-dihydroxybiphenyl and produced meta cleavage compounds from the latter compound (lambda/sub max/, 434 nm) and also from catechol (lambda/sub max/, 375 nm) through the meta pathway. On the other hand, benzoate-grown BP/sup -/ segregants totally lost the BP-metabolizig activities and oxidized catechol through the ortho pathway. A combined culture of the chlorinated BP-dissimilating P6 or M5 strain (harboring the putative 53.7-megadalton plasmid specifying conversion of chlorobiphenyl to chlorobenzoic acids) and genetically constructed mono- or dichlorobenzoate-utilizing pseudomonads (harboring plasmids encoding complete utilization of mono- or dichlorobenzoates) allowed greater than 98% utilization of mono- and dichlorobiphenyls, with the liberation of equivalent amounts of chloride ions.

  5. Characterization of a multiple antibiotic resistance plasmid from Haemophilus ducreyi.

    PubMed Central

    Willson, P J; Albritton, W L; Slaney, L; Setlow, J K

    1989-01-01

    Plasmid pLS88 from a clinical isolate of Haemophilus ducreyi encoded resistance determinants for sulfonamides and streptomycin related to those of RSF1010 and for kanamycin related to Tn903 but lacked the inverted repeats of the transposon. Its host range included Haemophilus influenzae, Actinobacillus pleuropneumoniae, and Escherichia coli; and it was compatible with pDM2 and RSF1010. Images PMID:2684012

  6. Spaceflight Effects on Genetics and Plasmids of Streptomycetes

    NASA Astrophysics Data System (ADS)

    Voeikova, T. A.; Emelyanova, L. K.; Tyaglov, B. V.; Novikova, L. M.; Goins, T. L.; Pyle, B. H.

    2008-06-01

    In 2007, experiments with streptomycetes were conducted during a 12-day flight of the Russian Foton-M3 spacecraft. The flight (F), synchronous control (SC) and laboratory control (LC) specimens were kept at 30°C. The objective of the experiments was to study spaceflight effects on the streptomycetes growth, differentiation, pigmentation, enzyme formation, genetic stability of plasmid and crossing between strains. It was found that the frequency of strain Streptomyces lividans segregation, the enzyme synthesis, pigmentation, and the level of sporulation were higher in F than in SC organisms. The study of pIJ702 plasmid inheritance in S. lividans showed that the frequency of plasmid loss in F and LC was similar and lower than that in SC specimens. The study of melanin synthesis in S. lividans (pIJ702) strain demonstrated decreased melanin specific yield and increased biomass accumulation in F microorganisms. HPTLC analysis of melanin showed that the number, molecular mass and the percentage of fractions were similar in SC and LC but different in F organisms. The study of spaceflight effects on genetic recombination in crosses between Streptomyces coelicolor A3(2) auxotrophic mutants showed that the frequency of various recombinant classes in F specimens differed from that in SC and LC. The frequency of a distal donor marker entry to the recipient in F was higher than in SC and LC.

  7. An insight of traditional plasmid curing in Vibrio species.

    PubMed

    Letchumanan, Vengadesh; Chan, Kok-Gan; Lee, Learn-Han

    2015-01-01

    As the causative agent of foodborne related illness, Vibrio species causes a huge impact on the public health and management. Vibrio species is often associated with seafood as the latter plays a role as a vehicle to transmit bacterial infections. Hence, antibiotics are used not to promote growth but rather to prevent and treat bacterial infections. The extensive use of antibiotics in the aquaculture industry and environment has led to the emerging of antibiotic resistant strains. This phenomenon has triggered an alarming public health concern due to the increase number of pathogenic Vibrio strains that are resistant to clinically used antibiotics and is found in the environment. Antibiotic resistance and the genes location in the strains can be detected through plasmid curing assay. The results derived from plasmid curing assay is fast, cost effective, sufficient in providing insights, and influence the antibiotic management policies in the aquaculture industry. This presentation aims in discussing and providing insights on various curing agents in Vibrio species. To our best of knowledge, this is a first review written discussing on plasmid curing in Vibrio species. PMID:26347714

  8. An insight of traditional plasmid curing in Vibrio species

    PubMed Central

    Letchumanan, Vengadesh; Chan, Kok-Gan; Lee, Learn-Han

    2015-01-01

    As the causative agent of foodborne related illness, Vibrio species causes a huge impact on the public health and management. Vibrio species is often associated with seafood as the latter plays a role as a vehicle to transmit bacterial infections. Hence, antibiotics are used not to promote growth but rather to prevent and treat bacterial infections. The extensive use of antibiotics in the aquaculture industry and environment has led to the emerging of antibiotic resistant strains. This phenomenon has triggered an alarming public health concern due to the increase number of pathogenic Vibrio strains that are resistant to clinically used antibiotics and is found in the environment. Antibiotic resistance and the genes location in the strains can be detected through plasmid curing assay. The results derived from plasmid curing assay is fast, cost effective, sufficient in providing insights, and influence the antibiotic management policies in the aquaculture industry. This presentation aims in discussing and providing insights on various curing agents in Vibrio species. To our best of knowledge, this is a first review written discussing on plasmid curing in Vibrio species. PMID:26347714

  9. Chlamydial Lytic Exit from Host Cells Is Plasmid Regulated

    PubMed Central

    Yang, Chunfu; Starr, Tregei; Song, Lihua; Carlson, John H.; Sturdevant, Gail L.; Beare, Paul A.; Whitmire, William M.

    2015-01-01

    ABSTRACT Chlamydia trachomatis is an obligate intracellular bacterium that is a globally important human pathogen. The chlamydial plasmid is an attenuating virulence factor, but the molecular basis for attenuation is not understood. Chlamydiae replicate within a membrane-bound vacuole termed an inclusion, where they undergo a biphasic developmental growth cycle and differentiate from noninfectious into infectious organisms. Late in the developmental cycle, the fragile chlamydia-laden inclusion retains its integrity by surrounding itself with scaffolds of host cytoskeletal proteins. The ability of chlamydiae to developmentally free themselves from this cytoskeleton network is a fundamental virulence trait of the pathogen. Here, we show that plasmidless chlamydiae are incapable of disrupting their cytoskeletal entrapment and remain intracellular as stable mature inclusions that support high numbers of infectious organisms. By using deletion mutants of the eight plasmid-carried genes (Δpgp1 to Δpgp8), we show that Pgp4, a transcriptional regulator of multiple chromosomal genes, is required for exit. Exit of chlamydiae is dependent on protein synthesis and is inhibited by the compound C1, an inhibitor of the type III secretion system (T3S). Exit of plasmid-free and Δpgp4 organisms, which failed to lyse infected cells, was rescued by latrunculin B, an inhibitor of actin polymerization. Our findings describe a genetic mechanism of chlamydial exit from host cells that is dependent on an unknown pgp4-regulated chromosomal T3S effector gene. PMID:26556273

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  11. Effect of plasmid copy number and lac operator sequence on antibiotic-free plasmid selection by operator-repressor titration in Escherichia coli.

    PubMed

    Cranenburgh, Rocky M; Lewis, Kathryn S; Hanak, Julian A J

    2004-01-01

    The Escherichia coli strain DH1lacdapD enables plasmid selection and maintenance that is free from antibiotics and selectable marker genes. This is achieved by using only the lac operator sequence as a selectable element. This strain is currently used to generate high copy number plasmids with no antibiotic resistance genes for use as DNA vaccines and for expression of recombinant proteins. Until now these have been limited to pUC-based plasmids containing a high copy number pMB1-derived origin of replication, and the principle lacO(1) and auxiliary lacO(3) operators. In this study we have shown that this system can also be used to select and maintain pBR322-based plasmids with the lower copy number pMB1 origin of replication, and that lacO(1) alone or a palindromic version of lacO(1) can provide a sufficient level of repressor titration for plasmid selection. This is advantageous for recombinant protein production, where low copy number plasmids are often used and plasmid maintenance is important. The degree of repressor titration due to these plasmids was measured using the natural lactose operon in E. coli DH1 as a model. PMID:15383717

  12. Mulberry strains of Xylella fastidiosa contain a 25 kilobase pair plasmid with extensive sequence identity to a plasmid from Verminephrobacter eiseniae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A 25 kbp plasmid was present in each of four Californian strains of Xylella fastidiosa from mulberry affected with leaf scorch disease. Fragments of each plasmid were cloned into E. coli, sequenced, and assembled into circular contigs of 25,105 bp (pXF-RIV11 and pXF-RIV16) or 24,372 bp (pXF-RIV19 an...

  13. Xylella fastidiosa isolates from mulberry harbor a 25 kilobase pair plasmid with extensive sequence identity to a plasmid from Verminephrobacter eiseniae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A 25 kbp plasmid was present in each of four Californian isolates of Xylella fastidiosa from mulberry affected with leaf scorch disease. Fragments of each plasmid were cloned into E. coli, sequenced, and assembled into circular contigs of 25,105 bp (pXF-RIV11 and pXF-RIV16) or 24,372 bp (pXF-RIV19 a...

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

    PubMed

    Bi, Dexi; Xie, Yingzhou; Tai, Cui; Jiang, Xiaofei; Zhang, Jie; Harrison, Ewan M; Jia, Shiru; Deng, Zixin; Rajakumar, Kumar; Ou, Hong-Yu

    2016-01-01

    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. PMID:26841043

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Large multidrug resistance plasmids of the A/C incompatibility complex (IncA/C) have been found in a diverse group of Gram-negative commensal and pathogenic bacteria. We present three completed sequences from IncA/C plasmids that originated from Escherichia coli (cattle) and Salmonella enterica sero...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed Central

    Tai, Cui; Jiang, Xiaofei; Zhang, Jie; Harrison, Ewan M.; Jia, Shiru; Deng, Zixin; Rajakumar, Kumar; Ou, Hong-Yu

    2016-01-01

    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. PMID:26841043

  18. Genetic Transformation of a Clinical (Genital Tract), Plasmid-Free Isolate of Chlamydia trachomatis: Engineering the Plasmid as a Cloning Vector

    PubMed Central

    Wang, Yibing; Kahane, Simona; Cutcliffe, Lesley T.; Skilton, Rachel J.; Lambden, Paul R.; Persson, Kenneth; Bjartling, Carina; Clarke, Ian N.

    2013-01-01

    Our study had three objectives: to extend the plasmid-based transformation protocol to a clinical isolate of C. trachomatis belonging to the trachoma biovar, to provide “proof of principle” that it is possible to “knock out” selected plasmid genes (retaining a replication competent plasmid) and to investigate the plasticity of the plasmid. A recently developed, plasmid-based transformation protocol for LGV isolates of C. trachomatis was modified and a plasmid-free, genital tract C. trachomatis isolate from Sweden (SWFP-) was genetically transformed. Transformation of this non-LGV C. trachomatis host required a centrifugation step, but the absence of the natural plasmid removed the need for plaque purification of transformants. Transformants expressed GFP, were penicillin resistant and iodine stain positive for accumulated glycogen. The transforming plasmid did not recombine with the host chromosome. A derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene was engineered. CDS5 encodes pgp3, a protein secreted from the inclusion into the cell cytoplasm. This plasmid (pCDS5KO) was used to transform C. trachomatis SWFP-, and established that pgp3 is dispensable for plasmid function. The work shows it is possible to selectively delete segments of the chlamydial plasmid, and this is the first step towards a detailed molecular dissection of the role of the plasmid. The 3.6 kb β-galactosidase cassette was inserted into the deletion site of CDS5 to produce plasmid placZ-CDS5KO. Transformants were penicillin resistant, expressed GFP and stained for glycogen. In addition, they expressed β-galactosidase showing that the lacZ cassette was functional in C. trachomatis. An assay was developed that allowed the visualisation of individual inclusions by X-gal staining. The ability to express active β-galactosidase within chlamydial inclusions is an important advance as it allows simple, rapid assays to measure directly chlamydial infectivity without the need

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

    PubMed

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

    2015-09-01

    During the past 2 decades, Bacillus megaterium has been systematically developed for the gram-per-liter scale production of recombinant proteins. The plasmid-based expression systems employed use a xylose-controlled promoter. Protein production analyses at the single-cell level using green fluorescent protein as a model product revealed cell culture heterogeneity characterized by a significant proportion of less productive bacteria. Due to the enormous size of B. megaterium, such bistable behavior seen in subpopulations was readily analyzed by time lapse microscopy and flow cytometry. Cell culture heterogeneity was not caused simply by plasmid loss: instead, an asymmetric distribution of plasmids during cell division was detected during the exponential-growth phase. Multicopy plasmids are generally randomly distributed between daughter cells. However, in vivo and in vitro experiments demonstrated that under conditions of strong protein production, plasmids are retained at one of the cell poles. Furthermore, it was found that cells with accumulated plasmids and high protein production ceased cell division. As a consequence, the overall protein production of the culture was achieved mainly by the subpopulation with a sufficient plasmid copy number. Based on our experimental data, we propose a model whereby the distribution of multicopy plasmids is controlled by polar fixation under protein production conditions. Thereby, cell lines with fluctuating plasmid abundance arise, which results in population heterogeneity. Our results provide initial insights into the mechanism of cellular heterogeneity during plasmid-based recombinant protein production in a Bacillus species. PMID:26116677

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

    PubMed Central

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

    2015-01-01

    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. PMID:26116677

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

    PubMed

    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. PMID:26441947

  2. Insights into Dynamics of Mobile Genetic Elements in Hyperthermophilic Environments from Five New Thermococcus Plasmids

    PubMed Central

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

    2013-01-01

    Mobilome of hyperthermophilic archaea dwelling in deep-sea hydrothermal vents is poorly characterized. To gain insight into genetic diversity and dynamics of mobile genetic elements in these environments we have sequenced five new plasmids from different Thermococcus strains that have been isolated from geographically remote hydrothermal vents. The plasmids were ascribed to two subfamilies, pTN2-like and pEXT9a-like. Gene content and phylogenetic analyses illuminated a robust connection between pTN2-like plasmids and Pyrococcus abyssi virus 1 (PAV1), with roughly half of the viral genome being composed of genes that have homologues in plasmids. Unexpectedly, pEXT9a-like plasmids were found to be closely related to the previously sequenced plasmid pMETVU01 from Methanocaldococcus vulcanius M7. Our data suggests that the latter observation is most compatible with an unprecedented horizontal transfer of a pEXT9a-like plasmid from Thermococcales to Methanococcales. Gene content analysis revealed that thermococcal plasmids encode Hfq-like proteins and toxin-antitoxin (TA) systems of two different families, VapBC and RelBE. Notably, although abundant in archaeal genomes, to our knowledge, TA and hfq-like genes have not been previously found in archaeal plasmids or viruses. Finally, the plasmids described here might prove to be useful in developing new genetic tools for hyperthermophiles. PMID:23326305

  3. Comparative Genomics Provides Insight into the Diversity of the Attaching and Effacing Escherichia coli Virulence Plasmids

    PubMed Central

    Hazen, Tracy H.; Kaper, James B.; Nataro, James P.

    2015-01-01

    Attaching and effacing Escherichia coli (AEEC) strains are a genomically diverse group of diarrheagenic E. coli strains that are characterized by the presence of the locus of enterocyte effacement (LEE) genomic island, which encodes a type III secretion system that is essential to virulence. AEEC strains can be further classified as either enterohemorrhagic E. coli (EHEC), typical enteropathogenic E. coli (EPEC), or atypical EPEC, depending on the presence or absence of the Shiga toxin genes or bundle-forming pilus (BFP) genes. Recent AEEC genomic studies have focused on the diversity of the core genome, and less is known regarding the genetic diversity and relatedness of AEEC plasmids. Comparative genomic analyses in this study demonstrated genetic similarity among AEEC plasmid genes involved in plasmid replication conjugative transfer and maintenance, while the remainder of the plasmids had sequence variability. Investigation of the EPEC adherence factor (EAF) plasmids, which carry the BFP genes, demonstrated significant plasmid diversity even among isolates within the same phylogenomic lineage, suggesting that these EAF-like plasmids have undergone genetic modifications or have been lost and acquired multiple times. Global transcriptional analyses of the EPEC prototype isolate E2348/69 and two EAF plasmid mutants of this isolate demonstrated that the plasmid genes influence the expression of a number of chromosomal genes in addition to the LEE. This suggests that the genetic diversity of the EAF plasmids could contribute to differences in the global virulence regulons of EPEC isolates. PMID:26238712

  4. A polymerization-based method to construct a plasmid containing clustered DNA damage and a mismatch.

    PubMed

    Takahashi, Momoko; Akamatsu, Ken; Shikazono, Naoya

    2016-10-01

    Exposure of biological materials to ionizing radiation often induces clustered DNA damage. The mutagenicity of clustered DNA damage can be analyzed with plasmids carrying a clustered DNA damage site, in which the strand bias of a replicating plasmid (i.e., the degree to which each of the two strands of the plasmid are used as the template for replication of the plasmid) can help to clarify how clustered DNA damage enhances the mutagenic potential of comprising lesions. Placement of a mismatch near a clustered DNA damage site can help to determine the strand bias, but present plasmid-based methods do not allow insertion of a mismatch at a given site in the plasmid. Here, we describe a polymerization-based method for constructing a plasmid containing clustered DNA lesions and a mismatch. The presence of a DNA lesion and a mismatch in the plasmid was verified by enzymatic treatment and by determining the relative abundance of the progeny plasmids derived from each of the two strands of the plasmid. PMID:27449134

  5. The genetic basis of plasmid tropism between Chlamydia trachomatis and Chlamydia muridarum.

    PubMed

    Wang, Yibing; Cutcliffe, Lesley T; Skilton, Rachel J; Ramsey, Kyle H; Thomson, Nicholas R; Clarke, Ian N

    2014-10-01

    The development of genetic transformation technology for Chlamydia trachomatis using its endogenous plasmid has recently been described. Chlamydia muridarum cannot be transformed by the C. trachomatis plasmid, indicating a barrier between chlamydial species. To determine which regions of the plasmid conferred the species specificity, we used the novel approach of transforming wild-type C. muridarum carrying the endogenous plasmid pNigg and forced recombination with the C. trachomatis vector pGFP::SW2 which carries the complete C. trachomatis plasmid (pSW2). Penicillin and chloramphenicol-resistant transformants expressing the green fluorescent protein were selected. Recovery of plasmids from these transformants showed they were recombinants. The differences between the pSW2 and pNigg allowed identification of the recombination breakpoints and showed that pGFP::SW2 had exchanged a ~ 1 kbp region with pNigg covering CDS 2. The recombinant plasmid (pSW2NiggCDS2) is maintained under antibiotic selection when transformed into plasmid-cured C. muridarum. The ability to select for recombinants in C. muridarum shows that the barrier is not at transformation, but at the level of plasmid replication or maintenance. Our studies show that CDS 2, together with adjoining sequences, is the main determinant of plasmid tropism. PMID:24700815

  6. The genetic basis of plasmid tropism between Chlamydia trachomatis and Chlamydia muridarum

    PubMed Central

    Wang, Yibing; Cutcliffe, Lesley T; Skilton, Rachel J; Ramsey, Kyle H; Thomson, Nicholas R; Clarke, Ian N

    2014-01-01

    The development of genetic transformation technology for Chlamydia trachomatis using its endogenous plasmid has recently been described. Chlamydia muridarum cannot be transformed by the C. trachomatis plasmid, indicating a barrier between chlamydial species. To determine which regions of the plasmid conferred the species specificity, we used the novel approach of transforming wild-type C. muridarum carrying the endogenous plasmid pNigg and forced recombination with the C. trachomatis vector pGFP::SW2 which carries the complete C. trachomatis plasmid (pSW2). Penicillin and chloramphenicol-resistant transformants expressing the green fluorescent protein were selected. Recovery of plasmids from these transformants showed they were recombinants. The differences between the pSW2 and pNigg allowed identification of the recombination breakpoints and showed that pGFP::SW2 had exchanged a ∼ 1 kbp region with pNigg covering CDS 2. The recombinant plasmid (pSW2NiggCDS2) is maintained under antibiotic selection when transformed into plasmid-cured C. muridarum. The ability to select for recombinants in C. muridarum shows that the barrier is not at transformation, but at the level of plasmid replication or maintenance. Our studies show that CDS 2, together with adjoining sequences, is the main determinant of plasmid tropism. PMID:24700815

  7. Plasmid CDS5 influences infectivity and virulence in a mouse model of Chlamydia trachomatis urogenital infection.

    PubMed

    Ramsey, K H; Schripsema, J H; Smith, B J; Wang, Y; Jham, B C; O'Hagan, K P; Thomson, N R; Murthy, A K; Skilton, R J; Chu, P; Clarke, I N

    2014-08-01

    The native plasmid of both Chlamydia muridarum and Chlamydia trachomatis has been shown to control virulence and infectivity in mice and in lower primates. We recently described the development of a plasmid-based genetic transformation protocol for Chlamydia trachomatis that for the first time provides a platform for the molecular dissection of the function of the chlamydial plasmid and its individual genes or coding sequences (CDS). In the present study, we transformed a plasmid-free lymphogranuloma venereum isolate of C. trachomatis, serovar L2, with either the original shuttle vector (pGFP::SW2) or a derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene (pCDS5KO). Female mice were inoculated with these strains either intravaginally or transcervically. We found that transformation of the plasmid-free isolate with the intact pGFP::SW2 vector significantly enhanced infectivity and induction of host inflammatory responses compared to the plasmid-free parental isolate. Transformation with pCDS5KO resulted in infection courses and inflammatory responses not significantly different from those observed in mice infected with the plasmid-free isolate. These results indicate a critical role of plasmid CDS5 in in vivo fitness and in induction of inflammatory responses. To our knowledge, these are the first in vivo observations ascribing infectivity and virulence to a specific plasmid gene. PMID:24866804

  8. Plasmid CDS5 Influences Infectivity and Virulence in a Mouse Model of Chlamydia trachomatis Urogenital Infection

    PubMed Central

    Schripsema, J. H.; Smith, B. J.; Wang, Y.; Jham, B. C.; O'Hagan, K. P.; Thomson, N. R.; Murthy, A. K.; Skilton, R. J.; Chu, P.; Clarke, I. N.

    2014-01-01

    The native plasmid of both Chlamydia muridarum and Chlamydia trachomatis has been shown to control virulence and infectivity in mice and in lower primates. We recently described the development of a plasmid-based genetic transformation protocol for Chlamydia trachomatis that for the first time provides a platform for the molecular dissection of the function of the chlamydial plasmid and its individual genes or coding sequences (CDS). In the present study, we transformed a plasmid-free lymphogranuloma venereum isolate of C. trachomatis, serovar L2, with either the original shuttle vector (pGFP::SW2) or a derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene (pCDS5KO). Female mice were inoculated with these strains either intravaginally or transcervically. We found that transformation of the plasmid-free isolate with the intact pGFP::SW2 vector significantly enhanced infectivity and induction of host inflammatory responses compared to the plasmid-free parental isolate. Transformation with pCDS5KO resulted in infection courses and inflammatory responses not significantly different from those observed in mice infected with the plasmid-free isolate. These results indicate a critical role of plasmid CDS5 in in vivo fitness and in induction of inflammatory responses. To our knowledge, these are the first in vivo observations ascribing infectivity and virulence to a specific plasmid gene. PMID:24866804

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

    PubMed Central

    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. PMID:26441947

  10. PSI:Biology-Materials Repository: A Biologist’s Resource for Protein Expression Plasmids

    PubMed Central

    Cormier, Catherine Y.; Park, Jin G.; Fiacco, Michael; Steel, Jason; Hunter, Preston; Kramer, Jason; Singla, Rajeev; LaBaer, Joshua

    2011-01-01

    The Protein Structure Initiative:Biology-Materials Repository (PSI:Biology-MR; MR; http://psimr.asu.edu) sequence-verifies, annotates, stores, and distributes the protein expression plasmids and vectors created by the Protein Structure Initiative (PSI). The MR has developed an informatics and sample processing pipeline that manages this process for thousands of samples per month from nearly a dozen PSI centers. DNASU (http://dnasu.asu.edu), a freely searchable database, stores the plasmid annotations, which include the full-length sequence, vector information, and associated publications for over 130,000 plasmids created by our laboratory, by the PSI and other consortia, and by individual laboratories for distribution to researchers worldwide. Each plasmid links to external resources, including the PSI Structural Biology Knowledgebase (http://sbkb.org), which facilitates cross-referencing of a particular plasmid to additional protein annotations and experimental data. To expedite and simplify plasmid requests, the MR uses an expedited material transfer agreement (EP-MTA) network, where researchers from network institutions can order and receive PSI plasmids without institutional delays. Currently over 39,000 protein expression plasmids and 78 empty vectors from the PSI are available upon request from DNASU. Overall, the MR’s repository of expression-ready plasmids, its automated pipeline, and the rapid process for receiving and distributing these plasmids more effectively allows the research community to dissect the biological function of proteins whose structures have been studied by the PSI. PMID:21360289

  11. Evolutionary Paths That Expand Plasmid Host-Range: Implications for Spread of Antibiotic Resistance.

    PubMed

    Loftie-Eaton, Wesley; Yano, Hirokazu; Burleigh, Stephen; Simmons, Ryan S; Hughes, Julie M; Rogers, Linda M; Hunter, Samuel S; Settles, Matthew L; Forney, Larry J; Ponciano, José M; Top, Eva M

    2016-04-01

    The World Health Organization has declared the emergence of antibiotic resistance to be a global threat to human health. Broad-host-range plasmids have a key role in causing this health crisis because they transfer multiple resistance genes to a wide range of bacteria. To limit the spread of antibiotic resistance, we need to gain insight into the mechanisms by which the host range of plasmids evolves. Although initially unstable plasmids have been shown to improve their persistence through evolution of the plasmid, the host, or both, the means by which this occurs are poorly understood. Here, we sought to identify the underlying genetic basis of expanded plasmid host-range and increased persistence of an antibiotic resistance plasmid using a combined experimental-modeling approach that included whole-genome resequencing, molecular genetics and a plasmid population dynamics model. In nine of the ten previously evolved clones, changes in host and plasmid each slightly improved plasmid persistence, but their combination resulted in a much larger improvement, which indicated positive epistasis. The only genetic change in the plasmid was the acquisition of a transposable element from a plasmid native to the Pseudomonas host used in these studies. The analysis of genetic deletions showed that the critical genes on this transposon encode a putative toxin-antitoxin (TA) and a cointegrate resolution system. As evolved plasmids were able to persist longer in multiple naïve hosts, acquisition of this transposon also expanded the plasmid's host range, which has important implications for the spread of antibiotic resistance. PMID:26668183

  12. Curing the Megaplasmid pTT27 from Thermus thermophilus HB27 and Maintaining Exogenous Plasmids in the Plasmid-Free Strain

    PubMed Central

    Tomita, Masaru; Itaya, Mitsuhiro

    2015-01-01

    Stepwise deletions in the only plasmid in Thermus thermophilus HB27, megaplasmid pTT27, showed that two distantly located loci were important for maintenance of the plasmid. One is a minimum replicon including one gene, repT, coding a replication initiator, and the other encodes subunits of class I ribonucleotide reductase (RNR) for deoxynucleoside triphosphate (dNTP) synthesis. Since the initiator protein, RepT, bound to direct repeats downstream from its own gene, it was speculated that a more-downstream A+T-rich region, which was critical for replication ability, could be unwound for replication initiation. On the other hand, the class I RNR is not necessarily essential for cell growth, as evidenced by the generation of the plasmid-free strain by the loss of pTT27. However, the plasmid-free strain culture has fewer viable cells than the wild-type culture, probably due to a dNTP pool imbalance in the cell. This is because of the introduction of the class I RNR genes or the supplementation of 5′-deoxyadenosylcobalamin, which stimulated class II RNR encoded in the chromosome, resolved the decrease in the number of viable cells in the plasmid-free strain. Likewise, these treatments dramatically enhanced the efficiency of transformation by exogenous plasmids and the stability of the plasmids in the strain. Therefore, the class I RNR would enable the stable maintenance of plasmids, including pTT27, as a result of genome replication normalized by reversing the dNTP pool imbalance. The generation of this plasmid-free strain with great natural competence and its analysis in regard to exogenous plasmid maintenance will expand the availability of HB27 for thermophilic cell factories. PMID:26712540

  13. Conjugation is necessary for a bacterial plasmid to survive under protozoan predation.

    PubMed

    Cairns, Johannes; Jalasvuori, Matti; Ojala, Ville; Brockhurst, Michael; Hiltunen, Teppo

    2016-02-01

    Horizontal gene transfer by conjugative plasmids plays a critical role in the evolution of antibiotic resistance. Interactions between bacteria and other organisms can affect the persistence and spread of conjugative plasmids. Here we show that protozoan predation increased the persistence and spread of the antibiotic resistance plasmid RP4 in populations of the opportunist bacterial pathogen Serratia marcescens. A conjugation-defective mutant plasmid was unable to survive under predation, suggesting that conjugative transfer is required for plasmid persistence under the realistic condition of predation. These results indicate that multi-trophic interactions can affect the maintenance of conjugative plasmids with implications for bacterial evolution and the spread of antibiotic resistance genes. PMID:26843557

  14. Plasmid purification by phenol extraction from guanidinium thiocyanate solution: development of an automated protocol.

    PubMed

    Fisher, J A; Favreau, M B

    1991-05-01

    We have developed a novel plasmid isolation procedure and have adapted it for use on an automated nucleic acid extraction instrument. The protocol is based on the finding that phenol extraction of a 1 M guanidinium thiocyanate solution at pH 4.5 efficiently removes genomic DNA from the aqueous phase, while supercoiled plasmid DNA is retained in the aqueous phase. S1 nuclease digestion of the removed genomic DNA shows that it has been denatured, which presumably confers solubility in the organic phase. The complete automated protocol for plasmid isolation involves pretreatment of bacterial cells successively with lysozyme, RNase A, and proteinase K. Following these digestions, the solution is extracted twice with a phenol/chloroform/water mixture and once with chloroform. Purified plasmid is then collected by isopropanol precipitation. The purified plasmid is essentially free of genomic DNA, RNA, and protein and is a suitable substrate for DNA sequencing and other applications requiring highly pure supercoiled plasmid. PMID:1713749

  15. Strategies and approaches in plasmidome studies-uncovering plasmid diversity disregarding of linear elements?

    PubMed

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

    2015-01-01

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

  16. In vivo generation of linear plasmids with addition of telomeric sequences by Histoplasma capsulatum.

    PubMed

    Woods, J P; Goldman, W E

    1992-12-01

    Histoplasma capsulatum is a dimorphic pathogenic fungus that is a major cause of respiratory and systemic mycosis. We previously developed a transformation system for Histoplasma and demonstrated chromosomal integration of transforming plasmid sequences. In this study, we describe another Histoplasma mechanism for maintaining transforming DNA i.e. the generation of modified, multicopy linear plasmids carrying DNA from the transforming Escherichia coli plasmid. Under selective conditions, these linear plasmids were stable and capable of retransforming Histoplasma without further modification. In vivo modification of the transforming DNA included duplication of plasmid sequence and telomeric addition at the termini of linear DNA. Apparently Histoplasma telomerase, like that of other organisms such as humans and Tetrahymena, is able to act on non-telomeric substrates. The terminus of a Histoplasma linear plasmid was cloned and shown to contain multiple repeats of GGGTTA, the telomeric repeat unit also found in vertebrates, trypanosomes, and slime moulds. PMID:1474902

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

    PubMed Central

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

    2015-01-01

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

  18. Plasmid DNA damage caused by stibine and trimethylstibine.

    PubMed

    Andrewes, Paul; Kitchin, Kirk T; Wallace, Kathleen

    2004-01-01

    Antimony is classified as "possibly carcinogenic to humans" and there is also sufficient evidence for antimony carcinogenicity in experimental animals. Stibine is a volatile inorganic antimony compound to which humans can be exposed in occupational settings (e.g., lead-acid battery charging). Because it is highly toxic, stibine is considered a significant health risk; however, its genotoxicity has received little attention. For the work reported here, stibine was generated by sodium borohydride reduction of potassium antimony tartrate. Trimethylstibine is a volatile organometallic antimony compound found commonly in landfill and sewage fermentation gases at concentrations ranging between 0.1 and 100 microg/m3. Trimethylstibine is generally considered to pose little environmental or health risk. In the work reported here, trimethylstibine was generated by reduction of trimethylantimony dichloride using either sodium borohydride or the thiol compounds, dithioerythritol (DTE), L-cysteine, and glutathione. Here we report the evaluation of the in vitro genotoxicities of five antimony compounds-potassium antimony tartrate, stibine, potassium hexahydroxyantimonate, trimethylantimony dichloride, and trimethylstibine-using a plasmid DNA-nicking assay. Of these five antimony compounds, only stibine and trimethylstibine were genotoxic (significant nicking to pBR 322 plasmid DNA). We found stibine and trimethylstibine to be about equipotent with trimethylarsine using this plasmid DNA-nicking assay. Reaction of trimethylantimony dichloride with either glutathione or L-cysteine to produce DNA-damaging trimethylstibine was observed with a trimethylantimony dichloride concentration as low as 50 microM and L-cysteine or glutathione concentrations as low as 500 and 200 microM, respectively, for a 24 h incubation. PMID:14728978

  19. The Virulence Plasmid of Yersinia, an Antihost Genome

    PubMed Central

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

    1998-01-01

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

  20. Characterization of a Cryptic and Intriguing Low Molecular Weight Plasmid.

    PubMed

    Carneiro, Lilian C; Mendes, Paulo Vinicius C; Silva, Silvana P; Souza, Guilherme R L; Bataus, Luiz Artur M

    2016-03-01

    The complete nucleotide sequence of cryptic plasmid pVCM04 isolated from Salmonella enterica serovar Enteritidis was determined and analyzed. pVCM04 contains 3853 bp with 53.6 % GC content and has twelve ORFs with more than 50 amino acids. Five of these sequences showed homology with replication and mobilization proteins. ORF1 and ORF2 showed homology with replication proteins, while ORFs 3-5 showed homology with mobilization proteins. The pVCM04 possesses a region associated with the theta-type replication mechanism. BLASTn search analysis revealed unexpectedly no similarity with sequences deposited in GenBank. The nucleotide sequence of pVCM04 can be divided into two arms: the region between nucleotides 552-1774 (encoding RepA and RepB) and the region between nucleotides 1775-3853 (encoding MobA, MobB and MobC). Codon bias pattern is distinct between mobA and repA, so the program Modeltest was used to select the best evolutionary model to study these genes. The result of ModelTest (model GTR+G for mobA and model HKY+G for repA) suggests that these genes would be subject to different selective pressures. Considering the differences in the codon usage, the selection of two different evolutionary models, and the absence of plasmids with homology to pVCM04 in GenBank, we believe that pVCM04 is a chimeric molecule and represents a new plasmid lineage. PMID:26670037

  1. A stable luciferase reporter plasmid for in vivo imaging in murine models of Staphylococcus aureus infections.

    PubMed

    Bacconi, Marta; Haag, Andreas F; Torre, Antonina; Castagnetti, Andrea; Chiarot, Emiliano; Delany, Isabel; Bensi, Giuliano

    2016-04-01

    In vivo imaging of bioluminescent bacteria permits their visualization in infected mice, allowing spatial and temporal evaluation of infection progression. Most available bioluminescent strains were obtained by integration of the luciferase genes into the bacterial chromosome, a challenging and time-consuming approach. Recently, episomal plasmids were used, which were introduced in bacteria and expressed all genes required for bioluminescence emission. However, the plasmid was progressively lost in vitro and in vivo, if bacteria were not maintained under antibiotic selective pressure. Increased stability could be obtained inserting into the plasmid backbone sequences that assured plasmid partition between daughter bacterial cells, or caused death of bacteria that had lost the plasmid. So far, no detailed analysis was performed of either plasmid stability in vivo or contribution of different stabilizing sequence types. Here we report the construction of a plasmid, which includes the Photorhabdus luminescens lux cassette expressed under the control of a Staphylococcus aureus specific gene promoter, and toxin/antitoxin (T/A) and partition sequences (Par) conferring stability and transmissibility of the plasmid. Following infection of mice with S. aureus carrying this plasmid, we demonstrated that the promoter-lux fusion was functional in vivo, that the plasmid was retained by 70-100% of bacterial cells 7 days post-infection, and that both stabilizing sequence types were required to maximize plasmid retention. These data suggest that the plasmid can be a valuable tool to study gene expression and bacterial spread in small laboratory animals infected with S. aureus or possibly other Gram-positive human pathogens. PMID:26685857

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

    PubMed

    Kalinowski, Marcin; Grądzki, Zbigniew; Jarosz, Łukasz; Kato, Kiyoko; Hieda, Yu; Kakuda, Tsutomu; Takai, Shinji

    2016-01-01

    Rhodococcus equi is an important bacterial pathogen in foals up to 6 months old, widespread in horse farms all over the world. It was found that only virulent R. equi strains expressing 15-17 kDa virulence-associated protein (VapA) and having large virulence plasmid of 85-90 kb containing vapA gene are pathogenic for horses. To date, 12 plasmid types have been reported in VapA positive strains from horses. There are no data concerning plasmid types of Polish field R. equi strains isolated from horses and horse farm environment. The aim of the study is to determine plasmid profiles of virulent R. equi strains isolated in Poland from dead foals as well as from soil samples taken from horse breeding farms. Plasmid profiles of 10 clinical strains derived from 8 farms and 11 environmental strains from 3 farms, confirmed as virulent by PCR, were compared with 12 reference strains containing the known plasmid size and type. Plasmid DNAs were analysed by digestion with the restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII for detailed comparison and estimation of plasmid sizes. The results of RFLP analysis revealed that all except one isolates used in the study are classified as VapA 85 kb type I plasmid. One strain harboured VapA 87 kb type I plasmid. This is the first report of plasmid types of Polish field R. equi strains. The results of our preliminary investigations on horse farms located in central and eastern Poland indicate that the virulent R. equi strains thus far isolated from diseased foals and horse farms environment represent a highly uniform plasmid pattern. PMID:27074033

  3. Cloning in Streptococcus lactis of plasmid-mediated UV resistance and effect on prophage stability

    SciTech Connect

    Chopin, M.C.; Chopin, A.; Rouault, A.; Simon, D.

    1986-02-01

    Plasmid pIL7 (33 kilobases) from Streptococcus lactis enhances UV resistance and prophage stability. A 5.4-kilobase pIL7 fragment carrying genes coding for both characters was cloned into S. lactis, using plasmid pHV1301 as the cloning vector. The recombinant plasmid was subsequently transferred to three other S. lactis strains by transformation or protoplast fusion. Cloned genes were expressed in all tested strains.

  4. Native Plasmids of Fusobacterium nucleatum: Characterization and Use in Development of Genetic Systems†

    PubMed Central

    Haake, Susan Kinder; Yoder, Sean C.; Attarian, Gwynne; Podkaminer, Kara

    2000-01-01

    Three native plasmids of Fusobacterium nucleatum were characterized, including DNA sequence analysis of one plasmid, pFN1. A shuttle plasmid, pHS17, capable of transforming Escherichia coli and F. nucleatum ATCC 10953 was constructed with pFN1. pHS17 was stably maintained in the F. nucleatum transformants, and differences in the transformation efficiencies suggested the presence of a restriction-modification system in F. nucleatum. PMID:10648549

  5. Linear Plasmids and the Rate of Sequence Evolution in Plant Mitochondrial Genomes.

    PubMed

    Warren, Jessica M; Simmons, Mark P; Wu, Zhiqiang; Sloan, Daniel B

    2016-02-01

    The mitochondrial genomes of flowering plants experience frequent insertions of foreign sequences, including linear plasmids that also exist in standalone forms within mitochondria, but the history and phylogenetic distribution of plasmid insertions is not well known. Taking advantage of the increased availability of plant mitochondrial genome sequences, we performed phylogenetic analyses to reconstruct the evolutionary history of these plasmids and plasmid-derived insertions. Mitochondrial genomes from multiple land plant lineages (including liverworts, lycophytes, ferns, and gymnosperms) include fragmented remnants from ancient plasmid insertions. Such insertions are much more recent and widespread in angiosperms, in which approximately 75% of sequenced mitochondrial genomes contain identifiable plasmid insertions. Although conflicts between plasmid and angiosperm phylogenies provide clear evidence of repeated horizontal transfers, we were still able to detect significant phylogenetic concordance, indicating that mitochondrial plasmids have also experienced sustained periods of (effectively) vertical transmission in angiosperms. The observed levels of sequence divergence in plasmid-derived genes suggest that nucleotide substitution rates in these plasmids, which often encode their own viral-like DNA polymerases, are orders of magnitude higher than in mitochondrial chromosomes. Based on these results, we hypothesize that the periodic incorporation of mitochondrial genes into plasmids contributes to the remarkable heterogeneity in substitution rates among genes that has recently been discovered in some angiosperm mitochondrial genomes. In support of this hypothesis, we show that the recently acquired ψtrnP-trnW gene region in a maize linear plasmid is evolving significantly faster than homologous sequences that have been retained in the mitochondrial chromosome in closely related grasses. PMID:26759362

  6. Linear Plasmids and the Rate of Sequence Evolution in Plant Mitochondrial Genomes

    PubMed Central

    Warren, Jessica M.; Simmons, Mark P.; Wu, Zhiqiang; Sloan, Daniel B.

    2016-01-01

    The mitochondrial genomes of flowering plants experience frequent insertions of foreign sequences, including linear plasmids that also exist in standalone forms within mitochondria, but the history and phylogenetic distribution of plasmid insertions is not well known. Taking advantage of the increased availability of plant mitochondrial genome sequences, we performed phylogenetic analyses to reconstruct the evolutionary history of these plasmids and plasmid-derived insertions. Mitochondrial genomes from multiple land plant lineages (including liverworts, lycophytes, ferns, and gymnosperms) include fragmented remnants from ancient plasmid insertions. Such insertions are much more recent and widespread in angiosperms, in which approximately 75% of sequenced mitochondrial genomes contain identifiable plasmid insertions. Although conflicts between plasmid and angiosperm phylogenies provide clear evidence of repeated horizontal transfers, we were still able to detect significant phylogenetic concordance, indicating that mitochondrial plasmids have also experienced sustained periods of (effectively) vertical transmission in angiosperms. The observed levels of sequence divergence in plasmid-derived genes suggest that nucleotide substitution rates in these plasmids, which often encode their own viral-like DNA polymerases, are orders of magnitude higher than in mitochondrial chromosomes. Based on these results, we hypothesize that the periodic incorporation of mitochondrial genes into plasmids contributes to the remarkable heterogeneity in substitution rates among genes that has recently been discovered in some angiosperm mitochondrial genomes. In support of this hypothesis, we show that the recently acquired ψtrnP-trnW gene region in a maize linear plasmid is evolving significantly faster than homologous sequences that have been retained in the mitochondrial chromosome in closely related grasses. PMID:26759362

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

    PubMed Central

    Kalinowski, Marcin; Grądzki, Zbigniew; Jarosz, Łukasz; Kato, Kiyoko; Hieda, Yu; Kakuda, Tsutomu; Takai, Shinji

    2016-01-01

    Rhodococcus equi is an important bacterial pathogen in foals up to 6 months old, widespread in horse farms all over the world. It was found that only virulent R. equi strains expressing 15–17 kDa virulence-associated protein (VapA) and having large virulence plasmid of 85–90 kb containing vapA gene are pathogenic for horses. To date, 12 plasmid types have been reported in VapA positive strains from horses. There are no data concerning plasmid types of Polish field R. equi strains isolated from horses and horse farm environment. The aim of the study is to determine plasmid profiles of virulent R. equi strains isolated in Poland from dead foals as well as from soil samples taken from horse breeding farms. Plasmid profiles of 10 clinical strains derived from 8 farms and 11 environmental strains from 3 farms, confirmed as virulent by PCR, were compared with 12 reference strains containing the known plasmid size and type. Plasmid DNAs were analysed by digestion with the restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII for detailed comparison and estimation of plasmid sizes. The results of RFLP analysis revealed that all except one isolates used in the study are classified as VapA 85 kb type I plasmid. One strain harboured VapA 87 kb type I plasmid. This is the first report of plasmid types of Polish field R. equi strains. The results of our preliminary investigations on horse farms located in central and eastern Poland indicate that the virulent R. equi strains thus far isolated from diseased foals and horse farms environment represent a highly uniform plasmid pattern. PMID:27074033

  8. Cefotaxime Resistant Escherichia coli Collected from a Healthy Volunteer; Characterisation and the Effect of Plasmid Loss

    PubMed Central

    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 blaCTX-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 blaCTX-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. PMID:24386342

  9. Association of tellurium resistance and bacteriophage inhibition conferred by R plasmids.

    PubMed Central

    Taylor, D E; Summers, A O

    1979-01-01

    Concomitant resistance to tellurium compounds (Ter) and inhibition of coli-phage development (Phi) are properties mediated by many H2 incompatibility group R plasmids which have been isolated from diverse bacterial and geographic sources. Ter plasmids from tellurium-resistant bacteria that were isolated from sewage and industrial wastes also mediated phage inhibition. Of these Ter plasmids, three from Citrobacter freundii belonged to the H incompatibility group, whereas three from Klebsiella pneumoniae did not. Images PMID:374351

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

    PubMed

    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. PMID:8669391

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

    PubMed Central

    Bergsveinson, Jordyn; Baecker, Nina; Pittet, Vanessa

    2014-01-01

    Specific isolates of lactic acid bacteria (LAB) can grow in the harsh beer environment, thus posing a threat to brew quality and the economic success of breweries worldwide. Plasmid-localized genes, such as horA, horC, and hitA, have been suggested to confer hop tolerance, a trait required for LAB survival in beer. The presence and expression of these genes among LAB, however, do not universally correlate with the ability to grow in beer. Genome sequencing of the virulent beer spoilage organism Lactobacillus brevis BSO 464 revealed the presence of eight plasmids, with plasmids 1, 2, and 3 containing horA, horC, and hitA, respectively. To investigate the roles that these and the other five plasmids play in L. brevis BSO 464 growth in beer, plasmid curing with novobiocin was used to derive 10 plasmid variants. Multiplex PCRs were utilized to determine the presence or absence of each plasmid, and how plasmid loss affected hop tolerance and growth in degassed (noncarbonated) beer was assessed. Loss of three of the eight plasmids was found to affect hop tolerance and growth in beer. Loss of plasmid 2 (horC and 28 other genes) had the most dramatic effect, with loss of plasmid 4 (120 genes) and plasmid 8 (47 genes) having significant, but smaller, impacts. These results support the contention that genes on mobile genetic elements are essential for bacterial growth in beer and that beer spoilage ability is not dependent solely on the three previously described hop tolerance genes or on the chromosome of a beer spoilage LAB isolate. PMID:25501474

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

    PubMed

    Bergsveinson, Jordyn; Baecker, Nina; Pittet, Vanessa; Ziola, Barry

    2015-02-01

    Specific isolates of lactic acid bacteria (LAB) can grow in the harsh beer environment, thus posing a threat to brew quality and the economic success of breweries worldwide. Plasmid-localized genes, such as horA, horC, and hitA, have been suggested to confer hop tolerance, a trait required for LAB survival in beer. The presence and expression of these genes among LAB, however, do not universally correlate with the ability to grow in beer. Genome sequencing of the virulent beer spoilage organism Lactobacillus brevis BSO 464 revealed the presence of eight plasmids, with plasmids 1, 2, and 3 containing horA, horC, and hitA, respectively. To investigate the roles that these and the other five plasmids play in L. brevis BSO 464 growth in beer, plasmid curing with novobiocin was used to derive 10 plasmid variants. Multiplex PCRs were utilized to determine the presence or absence of each plasmid, and how plasmid loss affected hop tolerance and growth in degassed (noncarbonated) beer was assessed. Loss of three of the eight plasmids was found to affect hop tolerance and growth in beer. Loss of plasmid 2 (horC and 28 other genes) had the most dramatic effect, with loss of plasmid 4 (120 genes) and plasmid 8 (47 genes) having significant, but smaller, impacts. These results support the contention that genes on mobile genetic elements are essential for bacterial growth in beer and that beer spoilage ability is not dependent solely on the three previously described hop tolerance genes or on the chromosome of a beer spoilage LAB isolate. PMID:25501474

  13. In vitro footprinting of promoter regions within supercoiled plasmid DNA

    PubMed Central

    Sun, Daekyu

    2010-01-01

    Polypurine/polypyrimidine (pPU/pPY) tracts, which exist in the promoter regions of many growth-related genes, have been proposed to be very dynamic in their conformation. In this chapter, we describe a detailed protocol for DNase I and S1 nuclease footprinting experiments with supercoiled plasmid DNA containing such the promoter regions to probe whether there are conformational transitions to B-type DNA, melted DNA and G-quadruplex structures within this tract. This is demonstrated with the proximal promoter region of the human vascular endothelial growth factor (VEGF) gene, which also contains multiple binding sites for Sp1 and Egr-1 transcription factors. PMID:19997887

  14. [Significance of plasmid virulence factors for transmission of nosocomial infection].

    PubMed

    Lebek, G

    1990-02-01

    The genetical basis of germ change especially for the gain of the resistance, and virulence plasmids have been treated not only with regard to clinical consequences but also have been classified in their evolutionary importance. Mechanisms of virulence have been illustrated on example of origin of siderophors, the increase of virulence through cytotoxical damage, on the obtained ability of adhesion and colonisation as well as by enhancement of equipment with enzyms. The patient with reduced resistance has been put out as selection condition. PMID:2183500

  15. Cold atmospheric pressure plasma jet interactions with plasmid DNA

    SciTech Connect

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

    2011-01-24

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

  16. Replicon typing of virulence plasmids of enterotoxigenic Escherichia coli isolates from cattle.

    PubMed Central

    Mainil, J G; Bex, F; Dreze, P; Kaeckenbeeck, A; Couturier, M

    1992-01-01

    Plasmid DNA hybridization with probes for virulence factors used for basic replicons of plasmids was used to identify the virulence plasmids of a collection of enterotoxigenic Escherichia coli isolates from cattle. The virulence probes were derived from the genes coding for the heat-stable enterotoxin STaP and for the F5 (K99) and F41 fimbrial adhesins. The replicon probes were derived from 16 different basic replicons of plasmids (probes repFIA, repFIB, repFIC, repFIIA, repI1, repHI1, repHI2, repL/M, repN, repP, repQ, repT, repU, repW, repX, and repY). The virulence genes coding for the STaP enterotoxin and for the F5 adhesin were located on a single plasmid band in each isolate. The sizes of most of these virulence plasmids were from 65 to 95 MDa. The F41 probe failed to hybridize with any plasmid band. The virulence plasmids had multireplicon types typical of plasmids of the IncF groups. The most common basic replicon association was the triple RepFIA-RepFIB-RepFIC family association. Images PMID:1639505

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

    PubMed Central

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

    2014-01-01

    Public health officials have raised concerns that plasmid transfer between Enterobacteriaceae species may spread resistance to carbapenems, an antibiotic class of last resort, thereby rendering common healthcare-associated infections nearly impossible to treat. We performed comprehensive surveillance and genomic sequencing to identify carbapenem-resistant Enterobacteriaceae in the NIH Clinical Center patient population and hospital environment in order to to articulate the diversity of carbapenemase-encoding plasmids and survey the mobility of and assess the mobility of these plasmids between bacterial species. We isolated a repertoire of carbapenemase-encoding Enterobacteriaceae, including multiple strains of Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, Enterobacter cloacae, Citrobacter freundii, and Pantoea species. Long-read genome sequencing with full end-to-end assembly revealed that these organisms carry the carbapenem-resistance genes on a wide array of plasmids. Klebsiella pneumoniae and Enterobacter cloacae isolated simultaneously from a single patient harbored two different carbapenemase-encoding plasmids, overriding the epidemiological scenario of plasmid transfer between organisms within this patient. We did, however, find evidence supporting horizontal transfer of carbapenemase-encoding plasmids between Klebsiella pneumoniae, Enterobacter cloacae and Citrobacter freundii in the hospital environment. Our comprehensive sequence data, with full plasmid identification, challenges assumptions about horizontal gene transfer events within patients and identified wider possible connections between patients and the hospital environment. In addition, we identified a new carbapenemase-encoding plasmid of potentially high clinical impact carried by Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae and Pantoea species, from unrelated patients and the hospital environment. PMID:25232178

  18. Construction of pBR322-ara hybrid plasmids by in vivo recombination.

    PubMed

    Horwitz, A H; Heffernan, L; Cass, L; Miyada, C G; Wilcox, G

    1980-01-01

    In vivo recombination was used to clone deletions of the araBAD-araC genes of Escherichia coli onto a hybrid pBR322-ara plasmid. Genetic and physical analyses demonstrated that the desired deletions had been recombined onto the plasmid. In addition to permitting a detailed physical analysis of various ara deletions, this procedure has generated a series of plasmid cloning vehicles that can be used to clone, by in vivo recombination, any ara point mutation located within the region covered by the deletions. Hybrid plasmids containing the cloned point mutation can be distinguished from the original cloning vehicle by genetic complementation. The desired recombinant plasmid can be easily obtained because the frequency of recombination between the plasmid ara region and the chromosomal ara region is 0.025%--3%. A plasmid containing a deletion which removes the ara controlling site region and the araC gene was used to clone two types of araBAD promoter mutations and an araC mutation by in vivo recombination. Genetic and physical analysis of these plasmids established that the mutations in question had been recombined on to the ara deletion plasmid. The application of this procedure to the ara genes and to other genetic systems is discussed. PMID:6255287

  19. Conserved small RNAs govern replication and incompatibility of a diverse new plasmid family from marine bacteria

    PubMed Central

    Le Roux, Frédérique; Davis, Brigid M.; Waldor, Matthew K.

    2011-01-01

    Plasmids are autonomously replicating extrachromosomal DNA molecules that often impart key phenotypes to their bacterial hosts. Plasmids are abundant in marine bacteria, but there is scant knowledge of the mechanisms that control their replication in these hosts. Here, we identified and characterized the factors governing replication of a new family of plasmids from marine bacteria, typified by the virulence-linked plasmid pB1067 of Vibrio nigripulchritudo. Members of this family are prevalent among, yet restricted to, the Vibrionaceae. Unlike almost all plasmid families characterized to date, the ori regions of these plasmids do not encode a Rep protein to initiate DNA replication; instead, the ori regions encode two partially complementary RNAs. The smaller, termed RNA I, is ∼68-nt long and functions as a negative regulator and the key determinant of plasmid incompatibility. This Marine RNA-based (MRB) plasmid family is the first characterized family of replicons derived from marine bacteria. Only one other plasmid family (the ColE1 family) has previously been reported to rely on RNA-mediated replication initiation. However, since the sequences and structures of MRB RNA I transcripts are not related to those of ColE1 replicons, these two families of RNA-dependent replicons likely arose by convergent evolution. PMID:20923782

  20. Plasmid marker rescue transformation proceeds by breakage-reunion in Bacillus subtilis

    SciTech Connect

    Weinrauch, Y.; Dubnau, D.

    1987-03-01

    Bacillus subtilis carrying a plasmid which replicates with a copy number of about 1 was transformed with linearized homologous plasmid DNA labeled with the heavy isotopes /sup 2/H and /sup 15/N, in the presence of /sup 32/Pi and 6-(p-hydroxyphenylazo)-uracil to inhibit DNA replication. Plasmid DNA was isolated from the transformed culture and fractionated in cesium chloride density gradients. The distribution of total and donor plasmid DNA was examined, using specific hybridization probes. The synthesis of new DNA, associated with the integration of donor moiety, was also monitored. Donor-specific sequences were present at a density intermediate between that of light and hybrid DNA. This recombinant DNA represented 1.4% of total plasmid DNA. The latter value corresponded well with the transforming activity (1.7%) obtained for the donor marker. Newly synthesized material associated with plasmid DNA at the recombinant density amounted to a minor portion of the recombinant plasmid DNA. These data suggest that, like chromosomal transformation, plasmid marker rescue transformation does not require replication for the integration of donor markers and, also like chromosomal transformation, proceeds by a breakage-reunion mechanism. The extent of donor DNA replacement of recipient DNA per plasmid molecule of 54 kilobases (27 kilobase pairs) was estimated as 16 kilobases.

  1. Genomics of microbial plasmids: classification and identification based on replication and transfer systems and host taxonomy.

    PubMed

    Shintani, Masaki; Sanchez, Zoe K; Kimbara, Kazuhide

    2015-01-01

    Plasmids are important "vehicles" for the communication of genetic information between bacteria. The exchange of plasmids transmits pathogenically and environmentally relevant traits to the host bacteria, promoting their rapid evolution and adaptation to various environments. Over the past six decades, a large number of plasmids have been identified and isolated from different microbes. With the revolution of sequencing technology, more than 4600 complete sequences of plasmids found in bacteria, archaea, and eukaryotes have been determined. The classification of a wide variety of plasmids is not only important to understand their features, host ranges, and microbial evolution but is also necessary to effectively use them as genetic tools for microbial engineering. This review summarizes the current situation of the classification of fully sequenced plasmids based on their host taxonomy and their features of replication and conjugative transfer. The majority of the fully sequenced plasmids are found in bacteria in the Proteobacteria, Firmicutes, Spirochaetes, Actinobacteria, Cyanobacteria and Euryarcheota phyla, and key features of each phylum are included. Recent advances in the identification of novel types of plasmids and plasmid transfer by culture-independent methods using samples from natural environments are also discussed. PMID:25873913

  2. Plasmids with temperature-dependent copy number for amplification of cloned genes and their products.

    PubMed

    Uhlin, B E; Molin, S; Gustafsson, P; Nordström, K

    1979-06-01

    Miniplasmids (pKN402 and pKN410) were isolated from runaway-replication mutants of plasmid R1. At 30 degrees C these miniplasmids are present in 20--50 copies per cell of Escherichia coli, whereas at temperatures above 35 degrees C the plasmids replicate without copy number control during 2--3 h. At the end of this period plasmid DNA amounts to about 75% of the total DNA. During the gene amplification, growth and protein synthesis continue at normal rate leading to a drastic amplification of plasmid gene products. Plasmids pKN402 (4.6 Md) and pKN410 (10 Md) have single restriction sites for restriction endonucleases EcoRI and HindIII; in addition plamid pKN410 has a single BamHI site and carries ampicillin resistance. The plasmids can therefore be used as cloning vectors. Several genes were cloned into these vectors using the EcoRI sites; chromosomal as well as plasmid-coded beta-lactamase was found to be amplified up to 400-fold after thermal induction of the runaway replication. Vectors of this temperature-dependent class will be useful in the production of large quantities of genes and gene products. These plasmids have lost their mobilization capacity. Runaway replication is lethal to the host bacteria in rich media. These two properties contribute to the safe use of the plasmids as cloning vehicles. PMID:383579

  3. A procedure for large-scale plasmid isolation without using ultracentrifugation.

    PubMed

    Chakrabarti, A; Sitaric, S; Ohi, S

    1992-10-01

    An expedient procedure for large-scale plasmid isolation from Escherichia coli strains without using ultracentrifugation or special setups or reagents is described. The protocol, which utilizes a modified alkaline extraction procedure as well as differential precipitations by isopropanol and lithium chloride, is simple and rapid and yet produces plasmid DNA with a yield of about 2 mg/liter culture. The isolated plasmids consisted of mostly monomeric and dimeric covalently closed circular DNA. The plasmids could be digested by various restriction endonucleases and were compatible with gene cloning, transfection-gene expression, and viral production. PMID:1333773

  4. Conjugal transfer and characterization of bacteriocin plasmids in group N (lactic acid) streptococci.

    PubMed Central

    Neve, H; Geis, A; Teuber, M

    1984-01-01

    Thirteen bacteriocin-producing strains of group N (lactic acid) streptococci were screened for their potential to transfer this property by conjugation to Streptococcus lactis subsp. diacetylactis Bu2-60. Bacteriocin production in three strains was plasmid encoded as shown by conjugal transfer and by analysis of cured, bacteriocin-negative derivatives of the donor strains and the transconjugants. With Streptococcus cremoris strains 9B4 and 4G6 and S. lactis subsp. diacetylactis 6F7 as donors, bacteriocin-producing transconjugants were isolated with frequencies ranging from ca. 2 X 10(-2) to 2 X 10(-1) per recipient cell. Bacteriocin-producing transconjugants had acquired a 39.6-megadalton plasmid from the donor strains 9B4 and 4G6, and a 75-megadalton plasmid from the donor strain 6F7. As shown by restriction endonuclease analysis, the plasmids from strains 9B4 and 4G6 were almost identical. The plasmid from strain 6F7 yielded some additional fragments not present in the two other plasmids. In hybridization experiments any of the three plasmids strongly hybridized with each other and with some other bacteriocin but nontransmissible plasmids from other S. cremoris strains. Homology was also detected to a variety of cryptic plasmids in lactic acid streptococci. Images PMID:6321437

  5. Potential shuttle vectors based on the methanogen plasmid pME2001

    SciTech Connect

    Meile, L.; Reeve, J.N.

    1985-01-01

    Methane is produced by anaerobic archaebacteria known as methanogens. Currently the only available plasmid from a methanogen is pME2001. The authors incorporated pME2001 into plasmids which should be capable of replication in a range of microbial host species. Plasmid pET2411, a recombinant plasmid formed by joining pBR322 to pME2001, directs the synthesis of pME2001 encoded polypeptides in Escherichia coli but cannot replicate in E. coli in the absence of E. coli DNA polymerase I. 23 references, 3 figures, 1 table.

  6. Stable maintenance of plasmid in continuous culture of yeast under non-selective conditions.

    PubMed

    Gupta, J C; Mukherjee, K J

    2001-01-01

    A recombinant yeast plasmid containing the gene for beta-galactosidase was tested for stability in a host auxotrophic for leucine. Plasmid loss was studied at different dilution rates in continuous culture under selective as well as non-selective conditions. It was observed that the instability of the culture was higher at low dilution rates in selective medium, while the pattern was reversed when complex non-selective medium was used, with plasmid-containing cells competing effectively with plasmid-free cells at low dilution rates. This was attributed to a low residual yeast extract concentration in the medium at low dilution rates. Since yeast extract was the sole source of leucine, this limited the growth of plasmid-free cells, which were auxotrophic for leucine. Growth rate studies also indicated a competitive advantage of the plasmid-containing cells over the plasmid-free cells at low yeast extract concentrations in semi-defined medium. Using the above data, a modified continuous culture was run using non-selective medium at a low dilution rate of 0.05 h(-1). This resulted in stable coexistence of plasmid-containing and plasmid-free cells and hence sustained expression of beta-galactosidase at approximately 330 OD420l(-1) h(-1) throughout the period of cultivation (134 h). PMID:16233104

  7. The 2-micron plasmid as a nonselectable, stable, high copy number yeast vector

    NASA Technical Reports Server (NTRS)

    Ludwig, D. L.; Bruschi, C. V.

    1991-01-01

    The endogenous 2-microns plasmid of Saccharomyces cerevisiae has been used extensively for the construction of yeast cloning and expression plasmids because it is a native yeast plasmid that is able to be maintained stably in cells at high copy number. Almost invariably, these plasmid constructs, containing some or all 2-microns sequences, exhibit copy number levels lower than 2-microns and are maintained stably only under selective conditions. We were interested in determining if there was a means by which 2-microns could be utilized for vector construction, without forfeiting either copy number or nonselective stability. We identified sites in the 2-microns plasmid that could be used for the insertion of genetic sequences without disrupting 2-microns coding elements and then assessed subsequent plasmid constructs for stability and copy number in vivo. We demonstrate the utility of a previously described 2-microns recombination chimera, pBH-2L, for the manipulation and transformation of 2-microns as a pure yeast plasmid vector. We show that the HpaI site near the STB element in the 2-microns plasmid can be utilized to clone yeast DNA of at least 3.9 kb with no loss of plasmid stability. Additionally, the copy number of these constructs is as high as levels reported for the endogenous 2-microns.

  8. Mobilization functions of the bacteriocinogenic plasmid pRJ6 of Staphylococcus aureus.

    PubMed

    Varella Coelho, Marcus Livio; Ceotto, Hilana; Madureira, Danielle Jannuzzi; Nes, Ingolf F; Bastos, Maria do Carmo de Freire

    2009-06-01

    Plasmid pRJ6 is the first known bacteriocinogenic mobilizable (Mob) plasmid of Staphylococcus aureus. Its Mob region is composed of four mob genes (mobCDAB) arranged as an operon, a genetic organization uncommon among S. aureus Mob plasmids. oriT (pRJ6) was detected in a region of 431 bp, positioned immediately upstream of mobC. This region, when cloned into pCN37, was able to confer mobilization to the re-combinant plasmid only in the presence of pRJ6. The entire Mob region, including oriT (pRJ6), is much more similar to Mob regions from several coagulase-negative staphylococci plasmids, although some remarkable similarities with S. aureus Mob plasmids can also be noted. These similarities include the presence within oriT (pRJ6) of the three mcb (MobC binding sites), firstly described in pC221 and pC223, an identical nick site also found in these same plasmids, and a nearly identical sra(pC223) site (sequence recognized by MobA). pRJ6 was successfully transferred to S. epidermidis by conjugation in the presence of the conjugative plasmid pGOl. Altogether these findings suggest that pRJ6 might have been originally a coagulase-negative staphylococci plasmid that had been transferred successfully to S. aureus. PMID:19557350

  9. Use of locked nucleic acid oligonucleotides to add functionality to plasmid DNA

    PubMed Central

    Hertoghs, Kirsten M. L.; Ellis, Jonathan H.; Catchpole, Ian R.

    2003-01-01

    The available reagents for the attachment of functional moieties to plasmid DNA are limiting. Most reagents bind plasmid DNA in a non-sequence- specific manner, with undefined stoichiometry, and affect DNA charge and delivery properties or involve chemical modifications that abolish gene expression. The design and ability of oligonucleotides (ODNs) containing locked nucleic acids (LNAs) to bind supercoiled, double-stranded plasmid DNA in a sequence-specific manner are described for the first time. The main mechanism for LNA ODNs binding plasmid DNA is demonstrated to be by strand displacement. LNA ODNs are more stably bound to plasmid DNA than similar peptide nucleic acid (PNA) ‘clamps’ for procedures such as particle-mediated DNA delivery (gene gun). It is shown that LNA ODNs remain associated with plasmid DNA after cationic lipid-mediated transfection into mammalian cells. LNA ODNs can bind to DNA in a sequence-specific manner so that binding does not interfere with plasmid conformation or gene expression. Attachment of CpG-based immune adjuvants to plasmid by ‘hybrid’ phosphorothioate–LNA ODNs induces tumour necrosis factor-α production in the macrophage cell line RAW264.7. This observation exemplifies an important new, controllable methodology for adding functionality to plasmids for gene delivery and DNA vaccination. PMID:14530430

  10. Transfer of plasmid RP4 to Myxococcus xanthus and evidence for its integration into the chromosome.

    PubMed Central

    Breton, A M; Jaoua, S; Guespin-Michel, J

    1985-01-01

    The broad-host-range plasmid RP4 and its derivative R68.45 were transferred to Myxococcus xanthus DK101 and DZ1; RP4 was maintained integrated in the chromosome. Loss of plasmid markers occurred during the growth of the transconjugants, which could be prevented by selective pressure with oxytetracycline. The integrated plasmid was transferred back to Escherichia coli often as RP4-prime plasmids carrying various segments of the M. xanthus chromosome. It also mediated chromosomal transfer between M. xanthus strains. Images PMID:3918015

  11. Selective Conditions for a Multidrug Resistance Plasmid Depend on the Sociality of Antibiotic Resistance

    PubMed Central

    Wood, A. Jamie; Brockhurst, Michael A.

    2016-01-01

    Multidrug resistance (MDR) plasmids frequently carry antibiotic resistance genes conferring qualitatively different mechanisms of resistance. We show here that the antibiotic concentrations selecting for the RK2 plasmid in Escherichia coli depend upon the sociality of the drug resistance: the selection for selfish drug resistance (efflux pump) occurred at very low drug concentrations, just 1.3% of the MIC of the plasmid-free antibiotic-sensitive strain, whereas selection for cooperative drug resistance (modifying enzyme) occurred at drug concentrations exceeding the MIC of the plasmid-free strain. PMID:26787694

  12. Genomics of microbial plasmids: classification and identification based on replication and transfer systems and host taxonomy

    PubMed Central

    Shintani, Masaki; Sanchez, Zoe K.; Kimbara, Kazuhide

    2015-01-01

    Plasmids are important “vehicles” for the communication of genetic information between bacteria. The exchange of plasmids transmits pathogenically and environmentally relevant traits to the host bacteria, promoting their rapid evolution and adaptation to various environments. Over the past six decades, a large number of plasmids have been identified and isolated from different microbes. With the revolution of sequencing technology, more than 4600 complete sequences of plasmids found in bacteria, archaea, and eukaryotes have been determined. The classification of a wide variety of plasmids is not only important to understand their features, host ranges, and microbial evolution but is also necessary to effectively use them as genetic tools for microbial engineering. This review summarizes the current situation of the classification of fully sequenced plasmids based on their host taxonomy and their features of replication and conjugative transfer. The majority of the fully sequenced plasmids are found in bacteria in the Proteobacteria, Firmicutes, Spirochaetes, Actinobacteria, Cyanobacteria and Euryarcheota phyla, and key features of each phylum are included. Recent advances in the identification of novel types of plasmids and plasmid transfer by culture-independent methods using samples from natural environments are also discussed. PMID:25873913

  13. Mechanism of action of tricyclic drugs on Escherichia coli and Yersinia enterocolitica plasmid maintenance and replication.

    PubMed

    Csiszar, K; Molnar, J

    1992-01-01

    Tricyclic medical compounds like many other non-antibiotics exhibit antimicrobial activities. Two chemically representative groups were tested in plasmid DNA transformation and replication to assign intracellular target sites responsible for the multiple effects in Escherichia coli and Yersinia enterocolitica cells. To analyse the mechanism of action at the molecular level, the effects of chlorpromazine, 7,8 dioxochlorpromazine, promethazine, methylene blue, imipramine, cannabidiolic acid and tetrahydrocannabidiolic acid were examined at several points in the course of transformation, in plasmid replication and on the topological state of plasmid DNA. Two possible target sites were identified, one of them involving membrane binding sites which participate in plasmid DNA replication. Drug binding at these sites interfered with the replicating plasmid DNA and membrane protein complex, preventing the proper processing of the replication that resulted in plasmid loss. The other in vivo and in vitro effect was observed on the topological state of plasmid DNA. Tricyclic drugs intefered with energy dependent gyrase activity and promoted the relaxation of plasmid DNA, causing disturbances in gene expression and in plasmid replication. The results give insight into the chemical structures connected with significant specific antimicrobial effects. PMID:1295474

  14. Use of plasmid profiles in epidemiologic surveillance of disease outbreaks and in tracing the transmission of antibiotic resistance.

    PubMed Central

    Mayer, L W

    1988-01-01

    Plasmids are circular deoxyribonucleic acid molecules that exist in bacteria, usually independent of the chromosome. The study of plasmids is important to medical microbiology because plasmids can encode genes for antibiotic resistance or virulence factors. Plasmids can also serve as markers of various bacterial strains when a typing system referred to as plasmid profiling, or plasmid fingerprinting is used. In these methods partially purified plasma deoxyribonucleic acid species are separated according to molecular size by agarose gel electrophoresis. In a second procedure, plasmid deoxyribonucleic acid which has been cleaved by restriction endonucleases can be separated by agarose gel electrophoresis and the resulting pattern of fragments can be used to verify the identity of bacterial isolates. Because many species of bacteria contain plasmids, plasmid profile typing has been used to investigate outbreaks of many bacterial diseases and to trace inter- and intra-species spread of antibiotic resistance. Images PMID:2852997

  15. Identification of Plasmid-Free Chlamydia muridarum Organisms Using a Pgp3 Detection-Based Immunofluorescence Assay.

    PubMed

    Chen, Chaoqun; Zhong, Guangming; Ren, Lin; Lu, Chunxue; Li, Zhonggyu; Wu, Yimou

    2015-10-01

    Chlamydia possesses a conserved 7.5 kb plasmid that is known to play an important role in chlamydial pathogenesis, since some chlamydial organisms lacking the plasmid are attenuated. The chlamydial transformation system developed recently required the use of plasmid-free organisms. Thus, the generation and identification of plasmid-free organisms represent a key step in understanding chlamydial pathogenic mechanisms. A tricolor immunofluorescence assay for simultaneously detecting the plasmid-encoded Pgp3 and whole organisms plus DNA staining was used to screen C. muridarum organisms selected with novobiocin. PCR was used to detect the plasmid genes. Next-generation sequencing was then used to sequence the genomes of plasmid-free C. muridarum candidates and the parental C. muridarum Nigg strain. We generated five independent clones of plasmid-free C. muridarum organisms by using a combination of novobiocin treatment and screening plaque-purified clones with anti-Pgp3 antibody. The clones were confirmed to lack plasmid genes by PCR analysis. No GlgA protein or glycogen accumulation was detected in cells infected with the plasmid-free clones. More importantly, whole-genome sequencing characterization of the plasmid-free C. muridarum organism and the parental C. muridarum Nigg strain revealed no additional mutations other than loss of the plasmid in the plasmid-free C. muridarum organism. Thus, the Pgp3-based immunofluorescence assay has allowed us to identify authentic plasmid-free organisms that are useful for further investigating chlamydial pathogenic mechanisms. PMID:26059520

  16. Dual control of Agrobacterium tumefaciens Ti plasmid virulence genes.

    PubMed Central

    Close, T J; Rogowsky, P M; Kado, C I; Winans, S C; Yanofsky, M F; Nester, E W

    1987-01-01

    The virulence genes of nopaline (pTiC58) and octopine (pTiA6NC) Ti plasmids are similarly affected by the Agrobacterium tumefaciens ros mutation. Of six vir region complementation groups (virA, virB, virG, virC, virD, and virE) examined by using fusions to reporter genes, the promoters of only two (virC and virD) responded to the ros mutation. For each promoter that was affected by ros, the level of expression of its associated genes was substantially elevated in the mutant. This increase was not influenced by Ti plasmid-encoded factors, and the mutation did not interfere with the induction of pTiC58 vir genes by phenolic compounds via the VirA/VirG regulatory control mechanism. The effects of the ros mutation and acetosyringone were cumulative for all vir promoters examined. The pleiotropic characteristics of the ros mutant include the complete absence of the major acidic capsular polysaccharide. Images PMID:3667525

  17. Long term stability of lyophilized plasmid DNA pDERMATT.

    PubMed

    van der Heijden, Iris; Beijnen, Jos H; Nuijen, Bastiaan

    2013-09-10

    In this short note we report on the shelf-life stability of pDERMATT (plasmid DNA encoding recombinant MART-1 and tetanus toxin fragment-c) 2mg lyophilized powder for reconstitution for intradermal administration, used in an in-house, investigator-initiated clinical phase I study. pDERMATT was stored at 25°C/60% relative humidity (6 months), 2-8°C (24 months), and -20°C (66 months) in the dark and analyzed at several timepoints during the conduct of the clinical study for appearance, identity, purity (plasmid topology), content and residual water content. pDERMATT appeared stable at all storage conditions for the periods tested which, although patient inclusion in the study was significantly delayed, ensured the clinical supply needs. This study shows that lyophilization is an useful tool to preserve the quality of the pDNA and can prevent the need for costly and time-consuming additional manufacture of drug product in case of study delays, not uncommon at the early stage of drug development. To our knowledge, this is the first study reporting shelf life stability of a pDNA formulation for more than 5 years. PMID:23792100

  18. Fusion-mediated transfer of plasmids into Spiroplasma floricola cells.

    PubMed

    Salman, M; Tarshis, M; Rottem, S

    1992-07-01

    We have developed and characterized a system for the transfer of plasmids encapsulated in large unilamellar vesicles (LUV) into Spiroplasma floricola BNR1 cells. The approach is based on the ability of S. floricola-derived LUV to fuse with S. floricola cells. The fusion was continuously monitored by an assay for lipid mixing based on the dequenching of the fluorescent probe octadecylrhodamine B (R18) that was incorporated into LUV at self-quenching concentrations. The fusion was also evaluated by fluorescence-activated cell sorter measurements and by sucrose density gradient analysis. LUV-cell fusion occurred only in the presence of low concentrations (5%) of polyethylene glycol (polyethylene glycol 8000) and depended on temperature, the LUV/cell ratio, and divalent cations in the incubation medium. Throughout the fusion process, spiroplasma cells remained intact and viable. Under optimal fusion conditions, the plasmid pACYC, encapsulated in LUV by reversed-phase evaporation, was transferred into live S. floricola cells and expressed chloramphenicol acetyltransferase activity. The expression was transient with maximal chloramphenicol acetyltransferase activity observed after 6 h of incubation of the transfected cells. PMID:1624433

  19. Description of a bacteriocinogenic plasmid in Beneckea harveyi.

    PubMed Central

    McCall, J O; Sizemore, R K

    1979-01-01

    A total of 795 strains of marine Vibrio species and Beneckea harveyi, a luminescent marine bacterium, were isolated from various sources in the area of Galveston Island, Tex., and screened for the production of bacteriocin-like substances. More than 8% of the Vibrio isolates produced low-molecular-weight (dialyzable) substances, which were lethal to a test strain of V. parahaemolyticus. Approximately 5% of the B. harveyi isolates produced higher-molecular-weight (nondialyzable) substances which were lethal to a test strain of B. harveyi. One of the B. harveyi strains (strain SY) produced a nondialyzable substance which was lethal to two of 39 strains of B. harveyi. The substance showed no activity toward 17 test strains drawn from the Vibrionaceae and Enterobacteriaceae. Strain SY showed no sensitivity to its own lethal agent and was shown by agarose gel electrophoresis and electron microscopy to harbor a single plasmid of 38 x 10(6) daltons. Variants of strain SY lacking the plasmid were produced by growth in the presence of the antibiotic novobiocin. These variants lacked both the ability to produce the lethal substance and the ability to survive in its presence. The lethal agent produced by strain SY is the first bacteriocin reported in marine bacteria. The term "harveyicin" is proposed to name this lethal substance. Images PMID:317423

  20. Germline Competent Pluripotent Mouse Stem Cells Generated by Plasmid Vectors.

    PubMed

    Chen, Chien-Hong; Su, Yu-Hsiu; Lee, Kun-Hsiung; Chuang, Chin-Kai

    2016-07-01

    We developed nonintegrated methods to reprogram mouse embryonic fibroblast (MEF) cells into induced pluripotent stem cells (iPSCs) using pig pOct4, pSox2, and pc-Myc as well as human hKLF4, hAID, and hTDG that were carried by plasmid vectors. The 4F method employed pOct4, pSox2, pc-Myc, and hKLF4 to derive iPSC clones with naive embryonic stem cell (ESC)-like morphology. These 4F clones expressed endogenous mouse Nanog protein and could generate chimeras. In addition to the four conventional reprogramming factors used in the 4F method, hAID and hTDG were utilized in a 6F method to increase the conversion efficiency of reprogramming by approximately five-fold. One of the 6F plasmid derived iPSC (piPSC) clones was shown to be germline transmission competent. PMID:26980563

  1. Mining Environmental Plasmids for Synthetic Biology Parts and Devices.

    PubMed

    Martínez-García, Esteban; Benedetti, Ilaria; Hueso, Angeles; De Lorenzo, Víctor

    2015-02-01

    The scientific and technical ambition of contemporary synthetic biology is the engineering of biological objects with a degree of predictability comparable to those made through electric and industrial manufacturing. To this end, biological parts with given specifications are sequence-edited, standardized, and combined into devices, which are assembled into complete systems. This goal, however, faces the customary context dependency of biological ingredients and their amenability to mutation. Biological orthogonality (i.e., the ability to run a function in a fashion minimally influenced by the host) is thus a desirable trait in any deeply engineered construct. Promiscuous conjugative plasmids found in environmental bacteria have evolved precisely to autonomously deploy their encoded activities in a variety of hosts, and thus they become excellent sources of basic building blocks for genetic and metabolic circuits. In this article we review a number of such reusable functions that originated in environmental plasmids and keep their properties and functional parameters in a variety of hosts. The properties encoded in the corresponding sequences include inter alia origins of replication, DNA transfer machineries, toxin-antitoxin systems, antibiotic selection markers, site-specific recombinases, effector-dependent transcriptional regulators (with their cognate promoters), and metabolic genes and operons. Several of these sequences have been standardized as BioBricks and/or as components of the SEVA (Standard European Vector Architecture) collection. Such formatting facilitates their physical composability, which is aimed at designing and deploying complex genetic constructs with new-to-nature properties. PMID:26104565

  2. Linearized oncolytic adenoviral plasmid DNA delivered by bioreducible polymers

    PubMed Central

    Kim, Jaesung; Kim, Pyung-Hwan; Nam, Hye Yeong; Lee, Jung-Sun; Yun, Chae-Ok; Kim, Sung Wan

    2011-01-01

    As an effort to overcome limits of adenovirus (Ad) as a systemic delivery vector for cancer therapy, we developed a novel system using oncolytic Ad plasmid DNA with two bioreducible polymers: arginine-grafted bioreducible poly(disulfide amine)polymer (ABP) and PEG5k-conjugated ABP (ABP5k) in expectation of oncolytic effect caused by progeny viral production followed by replication. The linearized Ad DNAs for active viral replication polyplexed with each polymer were able to replicate only in humancancer cells and produce progeny viruses. The non-immunogenic polymers delivering the DNAs markedly elicited to evade the innate and adaptive immune response. The biodistribution ratio of the polyplexes administered systemically was approximately 99% decreased in liver when compared with naked Ad. Moreover, tumor-to-liver ratio of the Ad DNA delivered by ABP or ABP5k was significantly elevated at 229- or 419-fold greater than that of naked Ad, respectively. The ABP5k improved the chance of the DNA to localize within tumor versus liver with 1.8-fold increased ratio. In conclusion, the innovative and simple system for delivering oncolytic Ad plasmid DNA with the bioreducible polymers, skipping time-consuming steps such as generation and characterization of oncolytic Ad vectors, can be utilized as an alternative approach for cancer therapy. PMID:22207073

  3. Origin of replication of colicin E1 plasmid DNA.

    PubMed Central

    Tomizawa, J I; Ohmori, H; Bird, R E

    1977-01-01

    Cleavage maps of colicin E1 plasmid DNA and its smaller derivative, pNT1 DNA, were constructed by using restriction endonucleases. The nucleotide sequence of a region that contains the orgin of replication was determined. The site of the nucleotide from which DNA replication is initiated was determined with 6S L-fragments, the DNA fragment first made on colicin E1 plasmid DNA. The fragments were labeled with [gamma-32P]ATP and polynucleotide 5'-hydroxyl-kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) at the 5'-OH groups which were uncovered by alkali treatment. The site is one of three consecutive nucleotides, dA, dA, and dC, located at a unique position. One or a few rA residues were found to be attached to some of the DNA molecules. The transition from the primer RNA to DNA occurs in a region consisting of a segment of five A residues. Both sides of this segment are rich in G and C. Images PMID:325558

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

    PubMed Central

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

    2016-01-01

    ABSTRACT Antimicrobial resistance in Staphylococcus aureus presents 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 the oriT region of pSK41, and here we establish that these contacts are essential for proper DNA cleavage and religation by the full 665-residue NES protein in vitro. Second, pSK156 and pCA347 are nonconjugative Staphylococcus aureus plasmids that contain sequences similar to the oriT region 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 the oriT sequences of these nonconjugative plasmids in vitro. Although pSK41 could mobilize a coresident plasmid harboring its cognate oriT, it was unable to mobilize plasmids containing the pSK156 and pCA347 variant oriT mimics, 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-like oriT. These data indicate that the conjugative relaxase in trans mechanism 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. IMPORTANCE Understanding the mechanism of antimicrobial resistance transfer in bacteria such as Staphylococcus aureus is an important step toward potentially slowing the spread of antimicrobial-resistant infections. This work establishes protein-DNA interactions essential for the transfer of the Staphylococcus aureus

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

    PubMed Central

    Luo, Wenting; Li, Li Li; Hansen, Lars Hestbjerg; Sørensen, Søren Johannes

    2014-01-01

    Two recently developed isolation methods have shown promise when recovering pure community plasmid DNA (metamobilomes/plasmidomes), which is useful in conducting culture-independent investigations into plasmid ecology. However, both methods employ multiple displacement amplification (MDA) to ensure suitable quantities of plasmid DNA for high-throughput sequencing. This study demonstrates that MDA greatly favors smaller circular DNA elements (<10 Kbp), which, in turn, leads to stark underrepresentation of upper size range plasmids (>10 Kbp). Throughout the study, we used two model plasmids, a 4.4 Kbp 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 the addition of an electroelution step that separates different plasmid size ranges prior to MDA in order to reduce size-dependent competition during incubation. Subsequent analyses of metamobilome data from wastewater spiked with the model plasmids showed in silica recovery of pKJK10 to be very poor with the established method and a 1,300-fold overrepresentation of pBR322. Conversely, complete recovery of pKJK10 was enabled with the new modified protocol although considerable care must be taken during electroelution to minimize cross-contamination between samples. For further validation, non-spiked wastewater metamobilomes were mapped to more than 2,500 known plasmid genomes. This displayed an overall recovery of plasmids well into the upper size range (median size: 30 kilobases) with the modified protocol. Analysis of de novo assembled metamobilome data also suggested distinctly better recovery of larger plasmids, as gene functions associated with these plasmids, such as conjugation, was exclusively encoded in the data output generated through the modified protocol. Thus

  6. Molecular and population analyses of a recombination event in the catabolic plasmid pJP4.

    PubMed

    Larraín-Linton, Juanita; De la Iglesia, Rodrigo; Melo, Francisco; González, Bernardo

    2006-10-01

    Cupriavidus necator JMP134(pJP4) harbors a catabolic plasmid, pJP4, which confers the ability to grow on chloroaromatic compounds. Repeated growth on 3-chlorobenzoate (3-CB) results in selection of a recombinant strain, which degrades 3-CB better but no longer grows on 2,4-dichlorophenoxyacetate (2,4-D). We have previously proposed that this phenotype is due to a double homologous recombination event between inverted repeats of the multicopies of this plasmid within the cell. One recombinant form of this plasmid (pJP4-F3) explains this phenotype, since it harbors two copies of the chlorocatechol degradation tfd gene clusters, which are essential to grow on 3-CB, but has lost the tfdA gene, encoding the first step in degradation of 2,4-D. The other recombinant plasmid (pJP4-FM) should harbor two copies of the tfdA gene but no copies of the tfd gene clusters. A molecular analysis using a multiplex PCR approach to distinguish the wild-type plasmid pJP4 from its two recombinant forms, was carried out. Expected PCR products confirming this recombination model were found and sequenced. Few recombinant plasmid forms in cultures grown in several carbon sources were detected. Kinetic studies indicated that cells containing the recombinant plasmid pJP4-FM were not selectable by sole carbon source growth pressure, whereas those cells harboring recombinant plasmid pJP4-F3 were selected upon growth on 3-CB. After 12 days of repeated growth on 3-CB, the complete plasmid population in C. necator JMP134 apparently corresponds to this form. However, wild-type plasmid forms could be recovered after growing this culture on 2,4-D, indicating that different plasmid forms can be found in C. necator JMP134 at the population level. PMID:16980481

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

    PubMed Central

    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

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

    PubMed Central

    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. PMID:24225319

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

    SciTech Connect

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

    1986-02-01

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

  10. Characterization of tet(Y)-carrying LowGC plasmids exogenously captured from cow manure at a conventional dairy farm.

    PubMed

    Kyselková, Martina; Chrudimský, Tomáš; Husník, Filip; Chroňáková, Alica; Heuer, Holger; Smalla, Kornelia; Elhottová, Dana

    2016-06-01

    Manure from dairy farms has been shown to contain diverse tetracycline resistance genes that are transferable to soil. Here, we focus on conjugative plasmids that may spread tetracycline resistance at a conventional dairy farm. We performed exogenous plasmid isolation from cattle feces using chlortetracycline for transconjugant selection. The transconjugants obtained harbored LowGC-type plasmids and tet(Y). A representative plasmid (pFK2-7) was fully sequenced and this was compared with previously described LowGC plasmids from piggery manure-treated soil and a GenBank record from Acinetobacter nosocomialis that we also identified as a LowGC plasmid. The pFK2-7 plasmid had the conservative backbone typical of LowGC plasmids, though this region was interrupted with an insert containing the tet(Y)-tet(R) tetracycline resistance genes and the strA-strB streptomycin resistance genes. Despite Acinetobacter populations being considered natural hosts of LowGC plasmids, these plasmids were not found in three Acinetobacter isolates from the study farm. The isolates harbored tet(Y)-tet(R) genes in identical genetic surroundings as pFK2-7, however, suggesting genetic exchange between Acinetobacter and LowGC plasmids. Abundance of LowGC plasmids and tet(Y) was correlated in manure and soil samples from the farm, indicating that LowGC plasmids may be involved in the spread of tet(Y) in the environment. PMID:27083193

  11. Replicon typing of plasmids encoding resistance to newer beta-lactams.

    PubMed

    Carattoli, Alessandra; Miriagou, Vivi; Bertini, Alessia; Loli, Alexandra; Colinon, Celine; Villa, Laura; Whichard, Jean M; Rossolini, Gian Maria

    2006-07-01

    Polymerase chain reaction-based replicon typing represents a novel method to describe the dissemination and follow the evolution of resistance plasmids. We used this approach to study 26 epidemiologically unrelated Enterobacteriaceae and demonstrate the dominance of incompatibility (Inc) A/C or Inc N-related plasmids carrying some emerging resistance determinants to extended-spectrum cephalosporins and carbapenems. PMID:16836838

  12. Replicon Typing of Plasmids Encoding Resistance to Newer β-Lactams

    PubMed Central

    Miriagou, Vivi; Bertini, Alessia; Loli, Alexandra; Colinon, Celine; Villa, Laura; Whichard, Jean M.; Rossolini, Gian Maria

    2006-01-01

    Polymerase chain reaction–based replicon typing represents a novel method to describe the dissemination and follow the evolution of resistance plasmids. We used this approach to study 26 epidemiologically unrelated Enterobacteriaceae and demonstrate the dominance of incompatibility (Inc) A/C or Inc N-related plasmids carrying some emerging resistance determinants to extended-spectrum cephalosporins and carbapenems. PMID:16836838

  13. Modified live Edwardsiella ictaluri vaccine, AQUAVAC-ESC, lacks multidrug resistance plasmids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plasmid mediated antibiotic resistance was first discovered in Edwardsiella ictaluri in the early 1990’s, and in 2007 an E. ictaluri isolate harboring an IncA/C plasmid was recovered from a moribund channel catfish infected with the bacterium. Due to the identification of multidrug resistance plasm...

  14. [IncP-7 plasmids' classification based on structural diversity of their basic replicons].

    PubMed

    Volkova, O V; Panov, A V; Kosheleva, I A; Boronin, A M

    2013-01-01

    The structural diversity of basic replicons and repB gene of the IncP-7 plasmids' collection was firstly assessed on the basis of PCR, restriction analysis and partial sequencing. It has been revealed that DNA fragment containing gene for UvrD-like helicase RepB is a part of all known P-7 replicons, but often serves as hot place for diverse IS-elements invasion. The first system of P-7 plasmids' classification has been worked out on the basis of determined repA-oriV-par WABC nucleotide divergency. Most degradation plasmids established to be belonging to large beta-subgroup, streptomycin resistance plasmid Rms148 (IncP-7 archetype)--to alpha-subgroup, carbazole degradation plasmid pCAR1 and NAH/SAL-plasmids from pY-line (Yamal oil deposits)--to gamma-subgroup and CAP-plasmid pBS270 with potentially reduced P-7 replicon--to delta-subgroup. It has been observed that the type of IncP-7 basic replicon molecular organization does not correlate with fixed phenotypic character in most cases, that is plasmids encoding different phenotypic markers could be members of the same P-7 subgroup. PMID:23808156

  15. Conjugative transfer of broad host range plasmids to an acidobacterial strain, Edaphobacter aggregans.

    PubMed

    Bouhajja, Emna; Efthymiopoulos, Theocharis; George, Isabelle F; Moreels, David; Van Houdt, Rob; Mergeay, Max; Agathos, Spiros N

    2016-03-10

    The Acidobacteria phylum is of high ecological interest. Its members are ubiquitous and particularly abundant in soils but many are recalcitrant to cultivation in the laboratory. Thus, the ability of Acidobacteria to capture and maintain plasmids remains largely unexplored. In this work we tested the transfer and the stability of (i) the PromA plasmid pMOL98 and (ii) the IncQ plasmid pKT230 to the acidobacterial strain Edaphobacter aggregans DSM 19364. To this end quantitative conjugation assays were performed and transconjugants were scored for plasmid-borne antibiotic selection markers. The tested plasmids were transferred and maintained in the new host. Plasmid pMOL98 was more stable than pKT230 in Ed. aggregans in the absence of positive selection. Thus, from an ecological point of view, we have extended the host range of PromA and IncQ plasmids for the first time to an acidobacterial strain. Furthermore, we have uncovered the potential of Acidobacteria to capture as-yet-unknown plasmids and to foster the development of new cloning and expression systems for the exploitation of biotechnologically valuable soil resources. PMID:26808872

  16. A conjugative 38kB plasmid is present in multiple subspecies of Xylella fastidiosa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A ~38kB plasmid was present in the Riv5 strain of Xylella fastidiosa subsp. multiplex isolated from ornamental plum in southern California. This plasmid, pXF-RIV5, encodes a complete type IV secretion system necessary for conjugation and DNA transfer. pXF-RIV5 is almost identical to pXFAS01 from X. ...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  19. Enhanced purification of plasmid DNA isoforms by exploiting ionic strength effects during ultrafiltration.

    PubMed

    Li, Ying; Currie, David; Zydney, Andrew L

    2016-04-01

    The solution structure of plasmid DNA is known to be a strong function of solution conditions due to intramolecular electrostatic interactions between the charged phosphate groups along the DNA backbone. The objective of this work was to determine whether it was possible to enhance the use of ultrafiltration for separation of different plasmid isoforms by proper selection of the solution ionic strength and ion type. Experiments were performed with a 3.0 kbp plasmid using composite regenerated cellulose ultrafiltration membranes. The transmission of the linear isoform was nearly independent of solution ionic strength, but increased significantly with increasing filtrate flux due to the elongation of the highly flexible plasmid in the converging flow field into the membrane pores. In contrast, the transmission of the open-circular and supercoiled plasmids both increased with increasing NaCl or MgCl2 concentration due to the change in plasmid size and conformational flexibility. The effect of ionic strength was greatest for the supercoiled plasmid, providing opportunities for enhanced purification of this therapeutically active isoform. This behavior was confirmed using experiments performed with binary mixtures of the different isoforms. These results clearly demonstrate the potential for enhancing the performance of membrane systems for plasmid DNA separations by proper selection of the ionic conditions. PMID:26370270

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

    SciTech Connect

    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.

  1. Complete DNA Sequence and Analysis of the Large Virulence Plasmid of Shigella flexneri

    PubMed Central

    Venkatesan, Malabi M.; Goldberg, Marcia B.; Rose, Debra J.; Grotbeck, Erik J.; Burland, Valerie; Blattner, Frederick R.

    2001-01-01

    The complete sequence analysis of the 210-kb Shigella flexneri 5a virulence plasmid was determined. Shigella spp. cause dysentery and diarrhea by invasion and spread through the colonic mucosa. Most of the known Shigella virulence determinants are encoded on a large plasmid that is unique to virulent strains of Shigella and enteroinvasive Escherichia coli; these known genes account for approximately 30 to 35% of the virulence plasmid. In the complete sequence of the virulence plasmid, 286 open reading frames (ORFs) were identified. An astonishing 153 (53%) of these were related to known and putative insertion sequence (IS) elements; no known bacterial plasmid has previously been described with such a high proportion of IS elements. Four new IS elements were identified. Fifty putative proteins show no significant homology to proteins of known function; of these, 18 have a G+C content of less than 40%, typical of known virulence genes on the plasmid. These 18 constitute potentially unknown virulence genes. Two alleles of shet2 and five alleles of ipaH were also identified on the plasmid. Thus, the plasmid sequence suggests a remarkable history of IS-mediated acquisition of DNA across bacterial species. The complete sequence will permit targeted characterization of potential new Shigella virulence determinants. PMID:11292750

  2. Microarray analysis of Inc A/C Plasmids in a population of Multidrug resistant Salmonella enterica

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteria plasmids are fragments of extra-chromosomal double stranded deoxyribonucleic acid (DNA) that can contain a variety of genes beneficial to the survival of the host bacteria. Classification and tracking of bacterial plasmids is valuable for the study of horizontal gene transfer of drug resis...

  3. Evaluating quantitative methods for measuring plasmid copy numbers in single cells

    PubMed Central

    Tal, Shay; Paulsson, Johan

    2013-01-01

    The life of plasmids is a constant battle against fluctuations: failing to correct copy number fluctuations can increase the plasmid loss rate by many orders of magnitude, as can a failure to more evenly divide the copies between daughters at cell division. Plasmids are therefore long-standing model systems for stochastic processes in cells, much thanks to the efforts of Kurt Nordström to whose memory this issue is dedicated. Here we analyze a range of experimental methods for measuring plasmid copy numbers in single cells, focusing on challenges, trade-offs and necessary experimental controls. In particular we analyze published and unpublished strategies to infer copy numbers from expression of plasmid-encoded reporters, direct labeling of plasmids with fluorescent probes or DNA binding proteins fused to fluorescent reporters, PCR based methods applied to single cell lysates, and plasmid-specific replication arrest. We conclude that no method currently exists to measure plasmid copy numbers in single cells, and that most methods instead inadvertently measure various types of experimental noise. We also discuss how accurate methods can be developed. PMID:22305922

  4. EFFECTS OF SEGREGATION AND SELECTION ON INSTABILITY OF PLASMID PACYC184 IN 'ESCHERICHIA COLI'B

    EPA Science Inventory

    The authors use a mathematical model to analyze the dynamics of loss of nonconjugative pACYC184 from populations of Escherichia coli B in glucose-limited continuous culture. The model incorporates both plasmid segregation and selection against plasmid carriage. It is concluded th...

  5. ESTIMATING THE RATE OF PLASMID TRANSFER: AN END-POINT METHOD

    EPA Science Inventory

    A method is described for determining rate parameter of conjugative plasmid transfer that is based on single estimates of donor, recipient and transconjugant densities, and the growth rate in exponential phase of the mating culture. he formula for estimating the plasmid transfer ...

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

    PubMed Central

    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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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% ...

  8. Quantification Bias Caused by Plasmid DNA Conformation in Quantitative Real-Time PCR Assay

    PubMed Central

    Lin, Chih-Hui; Chen, Yu-Chieh; Pan, Tzu-Ming

    2011-01-01

    Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification. PMID:22194997

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. Vector insert-targeted integrative antisense expression system for plasmid stabilization.

    PubMed

    Luke, Jeremy M; Carnes, Aaron E; Hodgson, Clague P; Williams, James A

    2011-01-01

    Some DNA vaccine and gene therapy vector-encoded transgenes are toxic to the E. coli plasmid production host resulting in poor production yields. For plasmid products undergoing clinical evaluation, sequence modification to eliminate toxicity is undesirable because an altered vector is a new chemical entity. We hypothesized that: (1) insert-encoded toxicity is mediated by unintended expression of a toxic insert-encoded protein from spurious bacterial promoters; and (2) that toxicity could be eliminated with antisense RNA-mediated translation inhibition. We developed the pINT PR PL vector, a chromosomally integrable RNA expression vector, and utilized it to express insert-complementary (anti-insert) RNA from a single defined site in the bacterial chromosome. Anti-insert RNA eliminated leaky fluorescent protein expression from a target plasmid. A toxic retroviral gag pol helper plasmid produced in a gag pol anti-insert strain had fourfold improved plasmid fermentation yields. Plasmid fermentation yields were also fourfold improved when a DNA vaccine plasmid containing a toxic Influenza serotype H1 hemagglutinin transgene was grown in an H1 sense strand anti-insert production strain, suggesting that in this case toxicity was mediated by an antisense alternative reading frame-encoded peptide. This anti-insert chromosomal RNA expression technology is a general approach to improve production yields with plasmid-based vectors that encode toxic transgenes, or toxic alternative frame peptides. PMID:20607625

  11. ColE1 plasmid incompatibility: localization and analysis of mutations affecting incompatibility.

    PubMed Central

    Hashimoto-Gotoh, T; Inselburg, J

    1979-01-01

    Deletion mutants of plasmid ColE1 that involve the replication origin and adjacent regions of the plasmid have been studied to determine the mechanism by which those mutations affect the expression of plasmid incompatibility. It was observed that (i) a region of ColE1 that is involved in the expression of plasmid incompatibility lies between base pairs -185 and -684; (ii) the integrity of at least part of the region of ColE1 DNA between base pairs -185 and -572 is essential for the expression of ColE1 incompatibility; (iii) the expression of incompatibility is independent of the ability of the ColE1 genome to replicate autonomously; (iv) plasmid incompatibility is affected by plasmid copy number; and (v) ColE1 plasmid-mediated DNA replication of the lambda phage-ColE1 chimera lambda imm434 Oam29 Pam3 ColE1 is inhibited by ColE1-incompatible but not by ColE1-compatible plasmids. Images PMID:378980

  12. Natural selection, infectious transfer and the existence conditions for bacterial plasmids.

    PubMed Central

    Bergstrom, C T; Lipsitch, M; Levin, B R

    2000-01-01

    Despite the near-ubiquity of plasmids in bacterial populations and the profound contribution of infectious gene transfer to the adaptation and evolution of bacteria, the mechanisms responsible for the maintenance of plasmids in bacterial populations are poorly understood. In this article, we address the question of how plasmids manage to persist over evolutionary time. Empirical studies suggest that plasmids are not infectiously transmitted at a rate high enough to be maintained as genetic parasites. In part i, we present a general mathematical proof that if this is the case, then plasmids will not be able to persist indefinitely solely by carrying genes that are beneficial or sometimes beneficial to their host bacteria. Instead, such genes should, in the long run, be incorporated into the bacterial chromosome. If the mobility of host-adaptive genes imposes a cost, that mobility will eventually be lost. In part ii, we illustrate a pair of mechanisms by which plasmids can be maintained indefinitely even when their rates of transmission are too low for them to be genetic parasites. First, plasmids may persist because they can transfer locally adapted genes to newly arriving strains bearing evolutionary innovations, and thereby preserve the local adaptations in the face of background selective sweeps. Second, plasmids may persist because of their ability to shuttle intermittently favored genes back and forth between various (noncompeting) bacterial strains, ecotypes, or even species. PMID:10924453

  13. Characterization of a plasmid-encoded urease gene cluster found in members of the family Enterobacteriaceae.

    PubMed

    D'Orazio, S E; Collins, C M

    1993-03-01

    Plasmid-encoded urease gene clusters found in uropathogenic isolates of Escherichia coli, Providencia stuartii, and Salmonella cubana demonstrated DNA homology, similar positions of restriction endonuclease cleavage sites, and manners of urease expression and therefore represent the same locus. DNA sequence analysis indicated that the plasmid-encoded urease genes are closely related to the Proteus mirabilis urease genes. PMID:8449894

  14. Genomic analysis and Next Generation Sequencing (NGS) of MDR plasmids in Salmonella enterica

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Food animals harboring Multi-Drug Resistant (MDR) Salmonella enterica are a possible source of zoonotic human infections and are a potential risk to human health. MDR genes can be transmitted in a number of ways including via plasmids. MDR plasmid prevalence and distribution was investigated by stud...

  15. Molecular epidemiology of plasmid patterns in Shigella flexneri types 1-6.

    PubMed Central

    Gebre-Yohannes, A.; Drasar, B. S.

    1991-01-01

    A total of 123 drug-resistant and drug-sensitive Shigella flexneri types 1-6, and their Escherichia coli K12 transconjugants were used for plasmid profile analysis by agarose gel electrophoresis. Resistance factors (R-factors) were further characterized by incompatibility testing. The overall distribution of small plasmids in S. flexneri showed that a cryptic plasmid of about 4.6 Kb was found in all serotypes, and a plasmid of about 4.2 Kb was found in serotypes 1-4. Shigella flexneri types 2, 4 and 6 showed a 6.5 Kb plasmid which correlated with SSu-resistance. All S. flexneri serotypes harboured large plasmids of about 217 Kb. Plasmid profile analysis of S. flexneri in Ethiopia showed a high degree of uniformity within individual serotypes. However, there was a limited variability which, at times, could be useful for epidemiological investigation. Shigella flexneri serotypes 1-6 harboured resistance plasmids with diverse molecular weights but mostly belonging to incompatibility groups N and X. PMID:1936154

  16. Microarray based analysis of Inc A/C Plasmids in Multidrug resistant Salmonella enterica

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteria plasmids are fragments of extra-chromosomal double stranded deoxyribonucleic acid (DNA) that can contain a variety of genes beneficial to the survival of the host bacteria. Classification and tracking of bacterial plasmids is valuable for the study of horizontal gene transfer of drug resis...

  17. Purification of transcriptionally active multimeric plasmid DNA using zwitterionic detergent and carbonate apatite nano-particles.

    PubMed

    Tee, Lau Khye; Ling, Chong Siew; Chua, Ming Jang; Abdullah, Syahril; Rosli, Rozita; Chowdhury, Ezharul Hoque

    2011-10-01

    Plasmid DNA is one of the indispensable components in molecular biology research and a potential biomaterial for gene therapy and DNA vaccination. Both quality and quantity of extracted plasmid DNA are of the great interests in cloning and subsequent expression of genes in vitro and in vivo for basic research and therapeutic interventions. Bacteria with extremely short generation times are the valuable source of plasmid DNA that can be isolated through a number of existing techniques. However, the current methods have some limitations in isolating high quality plasmid DNA since the multimeric plasmid which is believed to be more efficiently transcribed by RNA polymerase than the monomeric form, is almost lost during the extraction process. Recently, we developed a rapid isolation technique for multimeric plasmid based on generation of a 'protein aggregate' using a zwitterionic detergent and alkali. Here we have investigated the roles of different parameters in the whole extraction process to optimise the production of high quality multimeric plasmid DNA. Moreover, we have showed the advantageous effects of nanoparticles to effectively sediment the 'protein aggregate' for smooth elution of multimeric plasmid DNA from it. Finally, quality assessment study has revealed that the isolated multimeric DNA is at least 10 times more transcriptionally active than the monomeric form isolated by the commercially available Qiaget kit. PMID:21419794

  18. Homology of pCS1 Plasmid Sequences with Chromosomal DNA in Clavibacter michiganense subsp. sepedonicum: Evidence for the Presence of a Repeated Sequence and Plasmid Integration †

    PubMed Central

    Mogen, Bradley D.; Oleson, Arland E.

    1987-01-01

    Restriction fragments of pCS1, a 50.6-kilobase (kb) plasmid present in many strains of Clavibacter michiganense subsp. sepedonicum (“Corynebacterium sepedonicum”), have been cloned in an M13mp11 phage vector. Radiolabeled forms of these cloned fragments have been used as Southern hybridization probes for the presence of plasmid sequences in chromosomal DNA of this organism. These studies have shown that all tested strains lacking the covalently closed circular form of pCS1 contain the plasmid in integrated form. In each case the site of integration exists on a single plasmid restriction fragment with a size of 5.1 kb. Southern hybridizations with these probes have also revealed the existence of a major repeated sequence in C. michiganense subsp. sepedonicum. Hybridizations of chromosomal DNA with deletion subclones of a 2.9-kb plasmid fragment containing the repeated sequence indicate that the size of the repeated sequence is approximately 1.3 kb. One of the copies of the repeated sequence is on the plasmid fragment containing the site of integration. Images PMID:16347464

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

    PubMed Central

    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

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

    PubMed

    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

  1. UvrD-dependent replication of rolling-circle plasmids in Escherichia coli.

    PubMed

    Bruand, C; Ehrlich, S D

    2000-01-01

    The Escherichia coli UvrD helicase (or helicase II) is known for its involvement in DNA repair. We report that UvrD is required for DNA replication of several different rolling-circle plasmids in E. coli, whereas its homologue, the Rep helicase, is not. Lack of UvrD helicase does not impair the first step of plasmid replication, nicking of the double-stranded origin by the plasmid initiator protein. However, replication proceeds no further without UvrD. Indeed, the nicked plasmid molecules accumulate to a high level in uvrD mutants. We conclude that UvrD is the replicative helicase of various rolling-circle plasmids. This is the first description of a direct implication of UvrD in DNA replication in vivo. PMID:10632890

  2. Mutations in ARS1 increase the rate of simple loss of plasmids in Saccharomyces cerevisiae.

    PubMed

    Strich, R; Woontner, M; Scott, J F

    1986-09-01

    Autonomously replicating sequence (ARS) elements are DNA sequences that promote extrachromosomal maintenance of plasmids in yeast. Mutations generated in vitro in the ARS1 region were examined for their effect on plasmid maintenance in a yeast centromeric plasmid. Our data show that mutations in the regions surrounding the ARS1 consensus sequence cause increases in the frequency of simple loss (1:0) events without affecting the rate of nondisjunction (2:0). Removal of the consensus sequence itself causes a drastic increase in the rate of simple loss. Sequences sensitive to mutagenesis were identified in each flanking region and differ with respect to their location and importance to ARS function. These results suggest that the role ARS1 plays in plasmid maintenance deals with the replication and/or localization of the plasmid in yeast. PMID:3333306

  3. Narrow- and Broad-Host-Range Symbiotic Plasmids of Rhizobium spp. Strains That Nodulate Phaseolus vulgaris

    PubMed Central

    Brom, Susana; Martinez, Esperanza; Dávila, Guillermo; Palacios, Rafael

    1988-01-01

    Agrobacterium transconjugants containing symbiotic plasmids from different Rhizobium spp. strains that nodulate Phaseolus vulgaris were obtained. All transconjugants conserved the parental nodulation host range. Symbiotic (Sym) plasmids of Rhizobium strains isolated originally from P. vulgaris nodules, which had a broad nodulation host range, and single-copy nitrogenase genes conferred a Fix+ phenotype to the Agrobacterium transconjugants. A Fix− phenotype was obtained with Sym plasmids of strains isolated from P. vulgaris nodules that had a narrow host range and reiterated nif genes, as well as with Sym plasmids of strains isolated from other legumes that presented single nif genes and a broad nodulation host range. This indicates that different types of Sym plasmids can confer the ability to establish an effective symbiosis with P. vulgaris. Images PMID:16347637

  4. Effect of the atmospheric pressure nonequilibrium plasmas on the conformational changes of plasmid DNA

    SciTech Connect

    Yan Xu; He Guangyuan; Shi Mengjun; Gao Xuan; Li Yin; Ma Fengyun; Yu Men; Wang Changdong; Wang Yuesheng; Yang Guangxiao; Zou Fei; Lu Xinpei; Xiong Qing; Xiong Zilan

    2009-08-24

    The cold atmospheric pressure plasma, which has been widely used for biomedical applications, may potentially affect the conformation of DNA. In this letter, an atmospheric pressure plasma plume is used to investigate its effects on the conformational changes of DNA of plasmid pAHC25. It is found that the plasma plume could cause plasmid DNA topology alteration, resulting in the percentage of the supercoiled plasmid DNA form decreased while that of the open circular and linearized form of plasmid DNA increased as detected by agrose gel electrophoresis. On the other hand, further investigation by using polymerase chain reaction method shows that the atmospheric pressure plasma jet treatments under proper conditions does not affect the genes of the plasmid DNA, which may have potential application in increasing the transformation frequency by genetic engineering.

  5. Radioprotective effects produced by the condensation of plasmid DNA with avidin and biotinylated gold nanoparticles.

    PubMed

    Perry, Christopher C; Urata, Sarah M; Lee, Melissa; Aguilera, Joe A; Milligan, Jamie R

    2012-11-01

    The treatment of aqueous solutions of plasmid DNA with the protein avidin results in significant changes in physical, chemical, and biochemical properties. These effects include increased light scattering, formation of micron-sized particles containing both DNA and protein, and plasmid protection against thermal denaturation, radical attack, and nuclease digestion. All of these changes are consistent with condensation of the plasmid by avidin. Avidin can be displaced from the plasmid at higher ionic strengths. Avidin is not displaced from the plasmid by an excess of a tetra-arginine ligand, nor by the presence of biotin. Therefore, this system offers the opportunity to reversibly bind biotin-labeled species to a condensed DNA-protein complex. An example application is the use of biotinylated gold nanoparticles. This system offers the ability to examine in better detail the chemical mechanisms involved in important radiobiological effects. Examples include protein modulation of radiation damage to DNA, and radiosensitization by gold nanoparticles. PMID:22825766

  6. Rapid Tracing of Resistance Plasmids in a Nosocomial Outbreak Using Optical DNA Mapping.

    PubMed

    Müller, Vilhelm; Karami, Nahid; Nyberg, Lena K; Pichler, Christoffer; Torche Pedreschi, Paola C; Quaderi, Saair; Fritzsche, Joachim; Ambjörnsson, Tobias; Åhrén, Christina; Westerlund, Fredrik

    2016-05-13

    Resistance to life-saving antibiotics increases rapidly worldwide, and multiresistant bacteria have become a global threat to human health. Presently, the most serious threat is the increasing spread of Enterobacteriaceae carrying genes coding for extended spectrum β-lactamases (ESBL) and carbapenemases on highly mobile plasmids. We here demonstrate how optical DNA maps of single plasmids can be used as fingerprints to trace plasmids, for example, during resistance outbreaks. We use the assay to demonstrate a potential transmission route of an ESBL-carrying plasmid between bacterial strains/species and between patients, during a polyclonal outbreak at a neonatal ward at Sahlgrenska University Hospital (Gothenburg, Sweden). Our results demonstrate that optical DNA mapping is an easy and rapid method for detecting the spread of plasmids mediating resistance. With the increasing prevalence of multiresistant bacteria, diagnostic tools that can aid in solving ongoing routes of transmission, in particular in hospital settings, will be of paramount importance. PMID:27627201

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  8. Source–sink plasmid transfer dynamics maintain gene mobility in soil bacterial communities

    PubMed Central

    Wood, A. Jamie

    2016-01-01

    Horizontal gene transfer is a fundamental process in bacterial evolution that can accelerate adaptation via the sharing of genes between lineages. Conjugative plasmids are the principal genetic elements mediating the horizontal transfer of genes, both within and between bacterial species. In some species, plasmids are unstable and likely to be lost through purifying selection, but when alternative hosts are available, interspecific plasmid transfer could counteract this and maintain access to plasmid-borne genes. To investigate the evolutionary importance of alternative hosts to plasmid population dynamics in an ecologically relevant environment, we established simple soil microcosm communities comprising two species of common soil bacteria, Pseudomonas fluorescens and Pseudomonas putida, and a mercury resistance (HgR) plasmid, pQBR57, both with and without positive selection [i.e., addition of Hg(II)]. In single-species populations, plasmid stability varied between species: although pQBR57 survived both with and without positive selection in P. fluorescens, it was lost or replaced by nontransferable HgR captured to the chromosome in P. putida. A simple mathematical model suggests these differences were likely due to pQBR57’s lower intraspecific conjugation rate in P. putida. By contrast, in two-species communities, both models and experiments show that interspecific conjugation from P. fluorescens allowed pQBR57 to persist in P. putida via source–sink transfer dynamics. Moreover, the replacement of pQBR57 by nontransferable chromosomal HgR in P. putida was slowed in coculture. Interspecific transfer allows plasmid survival in host species unable to sustain the plasmid in monoculture, promoting community-wide access to the plasmid-borne accessory gene pool and thus potentiating future evolvability. PMID:27385827

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

    PubMed Central

    Casjens, Sherwood R.; Mongodin, Emmanuel F.; Qiu, Wei-Gang; Luft, Benjamin J.; Schutzer, Steven E.; Gilcrease, Eddie B.; Huang, Wai Mun; Vujadinovic, Marija; Aron, John K.; Vargas, Levy C.; Freeman, Sam; Radune, Diana; Weidman, Janice F.; Dimitrov, George I.; Khouri, Hoda M.; Sosa, Julia E.; Halpin, Rebecca A.; Dunn, John J.; Fraser, Claire M.

    2012-01-01

    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. PMID:22432010

  10. pA506, a conjugative plasmid of the plant epiphyte Pseudomonas fluorescens A506.

    PubMed

    Stockwell, Virginia O; Davis, Edward W; Carey, Alyssa; Shaffer, Brenda T; Mavrodi, Dmitri V; Hassan, Karl A; Hockett, Kevin; Thomashow, Linda S; Paulsen, Ian T; Loper, Joyce E

    2013-09-01

    Conjugative plasmids are known to facilitate the acquisition and dispersal of genes contributing to the fitness of Pseudomonas spp. Here, we report the characterization of pA506, the 57-kb conjugative plasmid of Pseudomonas fluorescens A506, a plant epiphyte used in the United States for the biological control of fire blight disease of pear and apple. Twenty-nine of the 67 open reading frames (ORFs) of pA506 have putative functions in conjugation, including a type IV secretion system related to that of MOBP6 family plasmids and a gene cluster for type IV pili. We demonstrate that pA506 is self-transmissible via conjugation between A506 and strains of Pseudomonas spp. or the Enterobacteriaceae. The origin of vegetative replication (oriV) of pA506 is typical of those in pPT23A family plasmids, which are present in many pathovars of Pseudomonas syringae, but pA506 lacks repA, a defining locus for pPT23A plasmids, and has a novel partitioning region. We selected a plasmid-cured derivative of A506 and compared it to the wild type to identify plasmid-encoded phenotypes. pA506 conferred UV resistance, presumably due to the plasmid-borne rulAB genes, but did not influence epiphytic fitness of A506 on pear or apple blossoms in the field. pA506 does not appear to confer resistance to antibiotics or other toxic elements. Based on the conjugative nature of pA506 and the large number of its genes that are shared with plasmids from diverse groups of environmental bacteria, the plasmid is likely to serve as a vehicle for genetic exchange between A506 and its coinhabitants on plant surfaces. PMID:23811504

  11. Source-sink plasmid transfer dynamics maintain gene mobility in soil bacterial communities.

    PubMed

    Hall, James P J; Wood, A Jamie; Harrison, Ellie; Brockhurst, Michael A

    2016-07-19

    Horizontal gene transfer is a fundamental process in bacterial evolution that can accelerate adaptation via the sharing of genes between lineages. Conjugative plasmids are the principal genetic elements mediating the horizontal transfer of genes, both within and between bacterial species. In some species, plasmids are unstable and likely to be lost through purifying selection, but when alternative hosts are available, interspecific plasmid transfer could counteract this and maintain access to plasmid-borne genes. To investigate the evolutionary importance of alternative hosts to plasmid population dynamics in an ecologically relevant environment, we established simple soil microcosm communities comprising two species of common soil bacteria, Pseudomonas fluorescens and Pseudomonas putida, and a mercury resistance (Hg(R)) plasmid, pQBR57, both with and without positive selection [i.e., addition of Hg(II)]. In single-species populations, plasmid stability varied between species: although pQBR57 survived both with and without positive selection in P. fluorescens, it was lost or replaced by nontransferable Hg(R) captured to the chromosome in P. putida A simple mathematical model suggests these differences were likely due to pQBR57's lower intraspecific conjugation rate in P. putida By contrast, in two-species communities, both models and experiments show that interspecific conjugation from P. fluorescens allowed pQBR57 to persist in P. putida via source-sink transfer dynamics. Moreover, the replacement of pQBR57 by nontransferable chromosomal Hg(R) in P. putida was slowed in coculture. Interspecific transfer allows plasmid survival in host species unable to sustain the plasmid in monoculture, promoting community-wide access to the plasmid-borne accessory gene pool and thus potentiating future evolvability. PMID:27385827

  12. Diversity of plasmids encoding histidine decarboxylase gene in Tetragenococcus spp. isolated from Japanese fish sauce.

    PubMed

    Satomi, Masataka; Furushita, Manabu; Oikawa, Hiroshi; Yano, Yutaka

    2011-07-15

    Nineteen isolates of histamine producing halophilic bacteria were isolated from four fish sauce mashes, each mash accumulating over 1000 ppm of histamine. The complete sequences of the plasmids encoding the pyruvoyl dependent histidine decarboxylase gene (hdcA), which is harbored in histamine producing bacteria, were determined. In conjunction, the sequence regions adjacent to hdcA were analyzed to provide information regarding its genetic origin. As reference strains, Tetragenococcus halophilus H and T. muriaticus JCM10006(T) were also studied. Phenotypic and 16S rRNA gene sequence analyses identified all isolates as T. halophilus, a predominant histamine producing bacteria present during fish sauce fermentation. Genetic analyses (PCR, Southern blot, and complete plasmid sequencing) of the histamine producing isolates confirmed that all the isolates harbored approximately 21-37 kbp plasmids encoding a single copy of the hdc cluster consisting of four genes related to histamine production. Analysis of hdc clusters, including spacer regions, indicated >99% sequence similarity among the isolates. All of the plasmids sequenced encoded traA, however genes related to plasmid conjugation, namely mob genes and oriT, were not identified. Two putative mobile genetic elements, ISLP1-like and IS200-like, respectively, were identified in the up- and downstream region of the hdc cluster of all plasmids. Most of the sequences, except hdc cluster and two adjacent IS elements, were diverse among plasmids, suggesting that each histamine producers harbored a different histamine-related plasmid. These results suggested that the hdc cluster was not spread by clonal dissemination depending on the specific plasmid and that the hdc cluster in tetragenococcal plasmid was likely encoded on transformable elements. PMID:21616548

  13. Selection of a Multidrug Resistance Plasmid by Sublethal Levels of Antibiotics and Heavy Metals

    PubMed Central

    Gullberg, Erik; Albrecht, Lisa M.; Karlsson, Christoffer; Sandegren, Linus

    2014-01-01

    ABSTRACT 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. PMID:25293762

  14. Distribution and diversity of mycoplasma plasmids: lessons from cryptic genetic elements

    PubMed Central

    2012-01-01

    Background The evolution of mycoplasmas from a common ancestor with Firmicutes has been characterized not only by genome down-sizing but also by horizontal gene transfer between mycoplasma species sharing a common host. The mechanisms of these gene transfers remain unclear because our knowledge of the mycoplasma mobile genetic elements is limited. In particular, only a few plasmids have been described within the Mycoplasma genus. Results We have shown that several species of ruminant mycoplasmas carry plasmids that are members of a large family of elements and replicate via a rolling-circle mechanism. All plasmids were isolated from species that either belonged or were closely related to the Mycoplasma mycoides cluster; none was from the Mycoplasma bovis-Mycoplasma agalactiae group. Twenty one plasmids were completely sequenced, named and compared with each other and with the five mycoplasma plasmids previously reported. All plasmids share similar size and genetic organization, and present a mosaic structure. A peculiar case is that of the plasmid pMyBK1 from M. yeatsii; it is larger in size and is predicted to be mobilizable. Its origin of replication and replication protein were identified. In addition, pMyBK1 derivatives were shown to replicate in various species of the M. mycoides cluster, and therefore hold considerable promise for developing gene vectors. The phylogenetic analysis of these plasmids confirms the uniqueness of pMyBK1 and indicates that the other mycoplasma plasmids cluster together, apart from the related replicons found in phytoplasmas and in species of the clade Firmicutes. Conclusions Our results unraveled a totally new picture of mycoplasma plasmids. Although they probably play a limited role in the gene exchanges that participate in mycoplasma evolution, they are abundant in some species. Evidence for the occurrence of frequent genetic recombination strongly suggests they are transmitted between species sharing a common host or niche. PMID

  15. Modified live Edwardsiella ictaluri vaccine, AQUAVAC-ESC, lacks multidrug resistance plasmids.

    PubMed

    Lafrentz, Benjamin R; Welch, Timothy J; Shoemaker, Craig A; Drennan, John D; Klesius, Phillip H

    2011-12-01

    Plasmid-mediated antibiotic resistance was first discovered in Edwardsiella ictaluri in the early 1990s, and in 2007 an E. ictaluri isolate harboring an IncA/C plasmid was recovered from a moribund channel catfish Ictalurus punctatus infected with the bacterium. Due to the identification of multidrug resistance plasmids in aquaculture and their potential clinical importance, we sought to determine whether the modified live E. ictaluri vaccine strain in AQUAVAC-ESC harbors such plasmids, so that the use of this vaccine will not directly contribute to the pool of bacteria carrying plasmid-borne resistance. Antimicrobial sensitivity testing of the E. ictaluri parent isolate and vaccine strain demonstrated that both were sensitive to 15 of the 16 antimicrobials tested. Total DNA from each isolate was analyzed by polymerase chain reaction (PCR) using a set of 13 primer pairs specific for conserved regions of the IncA/C plasmid backbone, and no specific products were obtained. PCR-based replicon typing of the parent isolate and vaccine strain demonstrated the absence of the 18 commonly occurring plasmid incompatibility groups. These results demonstrate that the vaccine strain does not carry resistance to commonly used antimicrobials and provide strong support for the absence of IncA/C and other commonly occurring plasmid incompatibility groups. Therefore, its use should not directly contribute to the pool of bacteria carrying plasmid-borne resistance. This work highlights the importance of thoroughly investigating potential vaccine strains for the presence of plasmids or other transmissible elements that may encode resistance to antibiotics. PMID:22372247

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

    SciTech Connect

    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. Comparison of electrically mediated and liposome-complexed plasmid DNA delivery to the skin

    PubMed Central

    Heller, Loree C; Jaroszeski, Mark J; Coppola, Domenico; Heller, Richard

    2008-01-01

    Background Electroporation is an established technique for enhancing plasmid delivery to many tissues in vivo, including the skin. We have previously demonstrated efficient delivery of plasmid DNA to the skin utilizing a custom-built four-plate electrode. The experiments described here further evaluate cutaneous plasmid delivery using in vivo electroporation. Plasmid expression levels are compared to those after liposome mediated delivery. Methods Enhanced electrically-mediated delivery, and less extensively, liposome complexed delivery, of a plasmid encoding the reporter luciferase was tested in rodent skin. Expression kinetics and tissue damage were explored as well as testing in a second rodent model. Results Experiments confirm that electroporation alone is more effective in enhancing reporter gene expression than plasmid injection alone, plasmid conjugation with liposomes followed by injection, or than the combination of liposomes and electroporation. However, with two time courses of multiple electrically-mediated plasmid deliveries, neither the levels nor duration of transgene expression are significantly increased. Tissue damage may increase following a second treatment, no further damage is observed after a third treatment. When electroporation conditions utilized in a mouse model are tested in thicker rat skin, only higher field strengths or longer pulses were as effective in plasmid delivery. Conclusion Electroporation enhances reporter plasmid delivery to the skin to a greater extent than the liposome conjugation method tested. Multiple deliveries do not necessarily result in higher or longer term expression. In addition, some impact on tissue integrity with respect to surface damage is observed. Pulsing conditions should be optimized for the model and for the expression profile desired. PMID:19055808

  18. Monoclonal antibodies produced by muscle after plasmid injection and electroporation.

    PubMed

    Tjelle, Torunn Elisabeth; Corthay, Alexandre; Lunde, Elin; Sandlie, Inger; Michaelsen, Terje E; Mathiesen, Iacob; Bogen, Bjarne

    2004-03-01

    Antibodies are useful for the treatment of a variety of diseases. We here demonstrate that mouse muscle produced monoclonal antibodies (mAb) after a single injection of immunoglobulin genes as plasmid DNA. In vivo electroporation of muscle greatly enhanced antibody production. For chimeric antibodies, levels of 50-200 ng mAb/ml serum were obtained but levels declined after 7-14 days due to an immune response against the xenogeneic parts of the antibody. By contrast, fully mouse antibodies persisted in serum for at least 7 months. mAb produced by the muscle had correct structure, specificity, and biological effector functions. The findings were extended to a larger animal, the sheep, in which mAb serum levels of 30-50 ng/ml were obtained. Sustained levels of serum mAb, induced by single injection of Ig genes and electroporation of muscle cells, may offer significant advantages in the treatment of human diseases. PMID:15006599

  19. Enhancing polyethylenimine's delivery of plasmid DNA into mammalian cells

    PubMed Central

    Thomas, Mini; Klibanov, Alexander M.

    2002-01-01

    The effect of various chemical modifications of nitrogen atoms on the efficiency of polyethylenimines (PEIs) as synthetic vectors for the delivery of plasmid DNA into monkey kidney cells in vitro has been systematically investigated. The resultant structure–activity relationship has both provided mechanistic insights and led to PEI derivatives with markedly enhanced performance. For example, N-acylation of PEI with the molecular mass of 25 kDa (PEI25, one of the most potent polycationic gene delivery vectors) with alanine nearly doubles its transfection efficiency in the presence of serum and also lowers its toxicity. Furthermore, dodecylation of primary amino groups of 2-kDa PEI yields a nontoxic polycation whose transfection efficiency in the presence of serum is 400 times higher than the parent's and which exceeds 5-fold even that of PEI25. PMID:12403826

  20. Single cell transfection using plasmid decorated AFM probes

    SciTech Connect

    Cuerrier, Charles M.; Lebel, Rejean; Grandbois, Michel . E-mail: michel.grandbois@usherbrooke.ca

    2007-04-13

    Eukaryotic cells were individually transfected using commercially available atomic force microscope tips decorated with plasmidic DNA encoding for the fluorescent protein EGFP. In a typical transfection attempt, the tip is forcibly incorporated into the cell thus allowing for the transfer of the genetic material through the cell membrane. A sharp discontinuity, corresponding to the passage of the tip through the cell membrane can be easily detected when monitoring the cellular deformation as a function of the applied force. In order for the transfection to be successful, the tip must reversibly penetrates the membrane without causing disturbance or damage to the cell. Transfection success rate (30%), cell survival, and growth are confirmed by epifluorescence microscopy. This technique provides an alternative tool to the transfection toolbox, allowing the transfection of specific individual cells with minimal disturbance.

  1. Distribution of Plasmids in Distinct Leptospira Pathogenic Species

    PubMed Central

    Wang, Yanzhuo; Zhuang, Xuran; Zhong, Yi; Zhang, Cuicai; Zhang, Yan; Zeng, Lingbing; Zhu, Yongzhang; He, Ping; Dong, Ke; Pal, Utpal; Guo, Xiaokui; Qin, Jinhong

    2015-01-01

    Leptospirosis, caused by pathogenic Leptospira, is a worldwide zoonotic infection. The genus Leptospira includes at least 21 species clustered into three groups—pathogens, non-pathogens, and intermediates—based on 16S rRNA phylogeny. Research on Leptospira is difficult due to slow growth and poor transformability of the pathogens. Recent identification of extrachromosomal elements besides the two chromosomes in L. interrogans has provided new insight into genome complexity of the genus Leptospira. The large size, low copy number, and high similarity of the sequence of these extrachromosomal elements with the chromosomes present challenges in isolating and detecting them without careful genome assembly. In this study, two extrachromosomal elements were identified in L. borgpetersenii serovar Ballum strain 56604 through whole genome assembly combined with S1 nuclease digestion following pulsed-field gel electrophoresis (S1-PFGE) analysis. Further, extrachromosomal elements in additional 15 Chinese epidemic strains of Leptospira, comprising L. borgpetersenii, L. weilii, and L. interrogans, were successfully separated and identified, independent of genome sequence data. Southern blot hybridization with extrachromosomal element-specific probes, designated as lcp1, lcp2 and lcp3-rep, further confirmed their occurrences as extrachromosomal elements. In total, 24 plasmids were detected in 13 out of 15 tested strains, among which 11 can hybridize with the lcp1-rep probe and 11 with the lcp2-rep probe, whereas two can hybridize with the lcp3-rep probe. None of them are likely to be species-specific. Blastp search of the lcp1, lcp2, and lcp3-rep genes with a nonredundant protein database of Leptospira species genomes showed that their homologous sequences are widely distributed among clades of pathogens but not non-pathogens or intermediates. These results suggest that the plasmids are widely distributed in Leptospira species, and further elucidation of their biological

  2. Distribution of Plasmids in Distinct Leptospira Pathogenic Species.

    PubMed

    Wang, Yanzhuo; Zhuang, Xuran; Zhong, Yi; Zhang, Cuicai; Zhang, Yan; Zeng, Lingbing; Zhu, Yongzhang; He, Ping; Dong, Ke; Pal, Utpal; Guo, Xiaokui; Qin, Jinhong

    2015-11-01

    Leptospirosis, caused by pathogenic Leptospira, is a worldwide zoonotic infection. The genus Leptospira includes at least 21 species clustered into three groups--pathogens, non-pathogens, and intermediates--based on 16S rRNA phylogeny. Research on Leptospira is difficult due to slow growth and poor transformability of the pathogens. Recent identification of extrachromosomal elements besides the two chromosomes in L. interrogans has provided new insight into genome complexity of the genus Leptospira. The large size, low copy number, and high similarity of the sequence of these extrachromosomal elements with the chromosomes present challenges in isolating and detecting them without careful genome assembly. In this study, two extrachromosomal elements were identified in L. borgpetersenii serovar Ballum strain 56604 through whole genome assembly combined with S1 nuclease digestion following pulsed-field gel electrophoresis (S1-PFGE) analysis. Further, extrachromosomal elements in additional 15 Chinese epidemic strains of Leptospira, comprising L. borgpetersenii, L. weilii, and L. interrogans, were successfully separated and identified, independent of genome sequence data. Southern blot hybridization with extrachromosomal element-specific probes, designated as lcp1, lcp2 and lcp3-rep, further confirmed their occurrences as extrachromosomal elements. In total, 24 plasmids were detected in 13 out of 15 tested strains, among which 11 can hybridize with the lcp1-rep probe and 11 with the lcp2-rep probe, whereas two can hybridize with the lcp3-rep probe. None of them are likely to be species-specific. Blastp search of the lcp1, lcp2, and lcp3-rep genes with a nonredundant protein database of Leptospira species genomes showed that their homologous sequences are widely distributed among clades of pathogens but not non-pathogens or intermediates. These results suggest that the plasmids are widely distributed in Leptospira species, and further elucidation of their biological

  3. Proposed model for the high rate of rearrangement and rapid migration observed in some IncA/C plasmid lineages

    Technology Transfer Automated Retrieval System (TEKTRAN)

    IncA/C plasmids are a class of plasmids from Enterobacteraciae that are relatively large (49 to >180 kbp), are readily transferred by conjugation, and carry multiple antimicrobial resistance genes. Reconstruction of the phylogeny of these plasmids has been difficult because of the high rate of remo...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    PubMed Central

    Cunningham, Drew S; Koepsel, Richard R; Ataai, Mohammad M; Domach, Michael M

    2009-01-01

    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 production; mutations that

  6. Persistence of antibiotic resistance and plasmid-associated genes in soil following application of sewage sludge and abundance on vegetables at harvest.

    PubMed

    Rahube, Teddie O; Marti, Romain; Scott, Andrew; Tien, Yuan-Ching; Murray, Roger; Sabourin, Lyne; Duenk, Peter; Lapen, David R; Topp, Edward

    2016-07-01

    Sewage sludge recovered from wastewater treatment plants contains antibiotic residues and is rich in antibiotic resistance genes, selected for and enriched in the digestive tracts of human using antibiotics. The use of sewage sludge as a crop fertilizer constitutes a potential route of human exposure to antibiotic resistance genes through consumption of contaminated crops. Several gene targets associated with antibiotic resistance (catA1, catB3, ereA, ereB, erm(B), str(A), str(B), qnrD, sul1, and mphA), mobile genetic elements (int1, mobA, IncW repA, IncP1 groups -α, -β, -δ, -γ, -ε), and bacterial 16S rRNA (rrnS) were quantified by qPCR from soil and vegetable samples obtained from unamended and sludge-amended plots at an experimental field in London, Ontario. The qPCR data reveals an increase in abundance of gene targets in the soil and vegetables samples, indicating that there is potential for additional crop exposure to antibiotic resistance genes carried within sewage sludge following field application. It is therefore advisable to allow an appropriate delay period before harvesting of vegetables for human consumption. PMID:27277701

  7. Mutations in a Partitioning Protein and Altered Chromatin Structure at the Partitioning Locus Prevent Cohesin Recruitment by the Saccharomyces cerevisiae Plasmid and Cause Plasmid Missegregation

    PubMed Central

    Yang, Xian-Mei; Mehta, Shwetal; Uzri, Dina; Jayaram, Makkuni; Velmurugan, Soundarapandian

    2004-01-01

    The 2μm circle is a highly persistent “selfish” DNA element resident in the Saccharomyces cerevisiae nucleus whose stability approaches that of the chromosomes. The plasmid partitioning system, consisting of two plasmid-encoded proteins, Rep1p and Rep2p, and a cis-acting locus, STB, apparently feeds into the chromosome segregation pathway. The Rep proteins assist the recruitment of the yeast cohesin complex to STB during the S phase, presumably to apportion the replicated plasmid molecules equally to daughter cells. The DNA-protein and protein-protein interactions of the partitioning system, as well as the chromatin organization at STB, are important for cohesin recruitment. Rep1p variants that are incompetent in binding to Rep2p, STB, or both fail to assist the assembly of the cohesin complex at STB and are nonfunctional in plasmid maintenance. Preventing the cohesin-STB association without impeding Rep1p-Rep2p-STB interactions also causes plasmid missegregation. During the yeast cell cycle, the Rep1p and Rep2p proteins are expelled from STB during a short interval between the late G1 and early S phases. This dissociation and reassociation event ensures that cohesin loading at STB is replication dependent and is coordinated with chromosomal cohesin recruitment. In an rsc2Δ yeast strain lacking a specific chromatin remodeling complex and exhibiting a high degree of plasmid loss, neither Rep1p nor the cohesin complex can be recruited to STB. The phenotypes of the Rep1p mutations and of the rsc2Δ mutant are consistent with the role of cohesin in plasmid partitioning being analogous to that in chromosome partitioning. PMID:15169893

  8. Partial Characterization of R-Plasmids from Pasteurella multocida Isolated from Turkeys

    PubMed Central

    Berman, Stephen M.; Hirsh, Dwight C.

    1978-01-01

    Pasteurella multocida, isolated from turkeys during an outbreak of septicemic disease (“fowl cholera”), was found to be resistant to tetracycline, streptomycin, and sulfonamides. Agarose gel electrophoretic analysis of DNA from these isolates indicated the presence of extrachromosomal elements. Plasmid DNA was isolated by cesium chloride-ethidium bromide density centrifugation. Escherichia coli was transformed to antimicrobic resistance with this DNA. Two plasmids were isolated. One of these plasmids had a buoyant density of 1.7158 g/cm3 (56.9 mol% guanine plus cytosine) and a molecular weight of 4.4 × 106 and conferred resistance to tetracycline, streptomycin, and sulfonamides. The other, having a buoyant density of 1.7198 g/cm3 (61 mol% guanine plus cytosine) and a molecular weight of 3.44 × 106, conferred resistance to streptomycin and sulfonamides. Streptomycin resistance was mediated by streptomycin phosphotransferase. Compatibility group testing indicated that neither plasmid belonged to any of 13 compatibility groups (of conjugal plasmids). Both plasmids were also found to be compatible with three small, nonconjugative resistance plasmids. Images PMID:708012

  9. Reconstructing the complex evolutionary history of mobile plasmids in red algal genomes

    PubMed Central

    Lee, JunMo; Kim, Kyeong Mi; Yang, Eun Chan; Miller, Kathy Ann; Boo, Sung Min; Bhattacharya, Debashish; Yoon, Hwan Su

    2016-01-01

    The integration of foreign DNA into algal and plant plastid genomes is a rare event, with only a few known examples of horizontal gene transfer (HGT). Plasmids, which are well-studied drivers of HGT in prokaryotes, have been reported previously in red algae (Rhodophyta). However, the distribution of these mobile DNA elements and their sites of integration into the plastid (ptDNA), mitochondrial (mtDNA), and nuclear genomes of Rhodophyta remain unknown. Here we reconstructed the complex evolutionary history of plasmid-derived DNAs in red algae. Comparative analysis of 21 rhodophyte ptDNAs, including new genome data for 5 species, turned up 22 plasmid-derived open reading frames (ORFs) that showed syntenic and copy number variation among species, but were conserved within different individuals in three lineages. Several plasmid-derived homologs were found not only in ptDNA but also in mtDNA and in the nuclear genome of green plants, stramenopiles, and rhizarians. Phylogenetic and plasmid-derived ORF analyses showed that the majority of plasmid DNAs originated within red algae, whereas others were derived from cyanobacteria, other bacteria, and viruses. Our results elucidate the evolution of plasmid DNAs in red algae and suggest that they spread as parasitic genetic elements. This hypothesis is consistent with their sporadic distribution within Rhodophyta. PMID:27030297

  10. Fertility properties and regulation of antimicrobial substance production by plasmid SCP2 of Streptomyces coelicolor.

    PubMed Central

    Troost, T R; Danilenko, V N; Lomovskaya, N D

    1979-01-01

    Streptomyces coelicolor A3(2) possesses two plasmids (SCP1 and SCP2) that act as sex factors. The plasmid deoxyribonucleic acid isolated from S. coelicolor A3(2) SCP1- strains A617 and A585 had the same molecular weight and endonuclease cleavage pattern as the SCP2 plasmid. The plasmidless strain S18 SCP2- was isolated from the A617 X A585 cross. SCP2 plasmid-containing strains acted as donors of chromosomal markers, whereas the plasmidless strain acted as recipient. The transfer of SCP2+ donor strain markers into the SCP2- recipient occurred at high frequencies (approximately 75%), was unidirectional, was initiated from a fixed region of the chromosome, and had the SCP2 fertility factor transferred first. The introduction of the SCP2 plasmid into a recipient strain greatly reduced the recombination frequency. These fertility properties differed from those previously reported, thereby suggesting that the SCP2 plasmid examined in this investigation may be an additional variant to those described in the literature. The SCP2 plasmid also regulated production of three antibacterial substances and conveyed resistance for S. coelicolor A3(2) strains against growth inhibition by one of them. Images PMID:500559

  11. GenoLIB: a database of biological parts derived from a library of common plasmid features.

    PubMed

    Adames, Neil R; Wilson, Mandy L; Fang, Gang; Lux, Matthew W; Glick, Benjamin S; Peccoud, Jean

    2015-05-26

    Synthetic biologists rely on databases of biological parts to design genetic devices and systems. The sequences and descriptions of genetic parts are often derived from features of previously described plasmids using ad hoc, error-prone and time-consuming curation processes because existing databases of plasmids and features are loosely organized. These databases often lack consistency in the way they identify and describe sequences. Furthermore, legacy bioinformatics file formats like GenBank do not provide enough information about the purpose of features. We have analyzed the annotations of a library of ∼2000 widely used plasmids to build a non-redundant database of plasmid features. We looked at the variability of plasmid features, their usage statistics and their distributions by feature type. We segmented the plasmid features by expression hosts. We derived a library of biological parts from the database of plasmid features. The library was formatted using the Synthetic Biology Open Language, an emerging standard developed to better organize libraries of genetic parts to facilitate synthetic biology workflows. As proof, the library was converted into GenoCAD grammar files to allow users to import and customize the library based on the needs of their research projects. PMID:25925571

  12. The incC Sequence Is Required for R27 Plasmid Stability

    PubMed Central

    Tassinari, Eleonora; Aznar, Sonia; Urcola, Imanol; Prieto, Alejandro; Hüttener, Mário; Juárez, Antonio

    2016-01-01

    IncHI plasmids account for multiple antimicrobial resistance in Salmonella and other enterobacterial genera. These plasmids are generally very stable in their bacterial hosts. R27 is the archetype of IncHI1 plasmids. A high percentage of the R27-encoded open reading frames (ORFs) (66.7%) do not show similarity to any known ORFs. We performed a deletion analysis of all non-essential R27 DNA sequences to search for hitherto non-identified plasmid functions that might be required for plasmid stability. We report the identification of a short DNA sequence (incC) that is essential for R27 stability. That region contains several repeats (incC repeats), belongs to one of the three-plasmid replicons (R27 FIA-like) and is targeted by the R27 E protein. Deletion of the incC sequence drastically reduces R27 stability both in Escherichia coli and in Salmonella, the effect being more pronounced in this latter species. Interfering with incC–E protein interaction must lead to a reduced IncHI1 plasmid stability, and may represent a new approach to combat antimicrobial resistance. PMID:27199955

  13. The incC Sequence Is Required for R27 Plasmid Stability.

    PubMed

    Tassinari, Eleonora; Aznar, Sonia; Urcola, Imanol; Prieto, Alejandro; Hüttener, Mário; Juárez, Antonio

    2016-01-01

    IncHI plasmids account for multiple antimicrobial resistance in Salmonella and other enterobacterial genera. These plasmids are generally very stable in their bacterial hosts. R27 is the archetype of IncHI1 plasmids. A high percentage of the R27-encoded open reading frames (ORFs) (66.7%) do not show similarity to any known ORFs. We performed a deletion analysis of all non-essential R27 DNA sequences to search for hitherto non-identified plasmid functions that might be required for plasmid stability. We report the identification of a short DNA sequence (incC) that is essential for R27 stability. That region contains several repeats (incC repeats), belongs to one of the three-plasmid replicons (R27 FIA-like) and is targeted by the R27 E protein. Deletion of the incC sequence drastically reduces R27 stability both in Escherichia coli and in Salmonella, the effect being more pronounced in this latter species. Interfering with incC-E protein interaction must lead to a reduced IncHI1 plasmid stability, and may represent a new approach to combat antimicrobial resistance. PMID:27199955

  14. Characterization of Mobile Staphylococcus equorum Plasmids Isolated from Fermented Seafood That Confer Lincomycin Resistance.

    PubMed

    Lee, Jong-Hoon; Jeong, Do-Won

    2015-01-01

    The complete nucleotide sequences of lincomycin-resistance gene (lnuA)-containing plasmids in Staphylococcus equorum strains isolated from the high-salt-fermented seafood jeotgal were determined. These plasmids, designated pSELNU1-3, are 2638-bp long, have two polymorphic sites, and encode typical elements found in plasmids that replicate via a rolling-circle mechanism including the replication protein gene (rep), a double-stranded origin of replication, a single-stranded origin of replication, and counter-transcribed RNA sequence, as well as lnuA. Plasmid sequences exhibit over 83% identity to other Staphylococcus plasmids that harbor rep and lnuA genes. Further, three pairs of identified direct repeats may be involved in inter-plasmid recombination. One plasmid, pSELNU1, was successfully transferred to other Staphylococcus species, Enterococcus faecalis, and Tetragenococcus halophilus in vitro. Antibiotic susceptibility of the transconjugants was host-dependent, and transconjugants maintained a lincomycin resistance phenotype in the absence of selective pressure over 60 generations. PMID:26448648

  15. Characterization of Mobile Staphylococcus equorum Plasmids Isolated from Fermented Seafood That Confer Lincomycin Resistance

    PubMed Central

    Lee, Jong-Hoon; Jeong, Do-Won

    2015-01-01

    The complete nucleotide sequences of lincomycin-resistance gene (lnuA)-containing plasmids in Staphylococcus equorum strains isolated from the high-salt-fermented seafood jeotgal were determined. These plasmids, designated pSELNU1–3, are 2638-bp long, have two polymorphic sites, and encode typical elements found in plasmids that replicate via a rolling-circle mechanism including the replication protein gene (rep), a double-stranded origin of replication, a single-stranded origin of replication, and counter-transcribed RNA sequence, as well as lnuA. Plasmid sequences exhibit over 83% identity to other Staphylococcus plasmids that harbor rep and lnuA genes. Further, three pairs of identified direct repeats may be involved in inter-plasmid recombination. One plasmid, pSELNU1, was successfully transferred to other Staphylococcus species, Enterococcus faecalis, and Tetragenococcus halophilus in vitro. Antibiotic susceptibility of the transconjugants was host-dependent, and transconjugants maintained a lincomycin resistance phenotype in the absence of selective pressure over 60 generations. PMID:26448648

  16. Attempts to find phenotypic markers of the virulence plasmid of Rhodococcus equi.

    PubMed Central

    De La Peña-Moctezuma, A; Prescott, J F; Goodfellow, M

    1996-01-01

    Four isolates of Rhodococcus equi, from pneumonic foals, and containing the 85 kb virulence plasmid, a porcine isolate containing an 80 kb plasmid, and their plasmid cured derivatives, were examined for 239 phenotypic properties in an attempt to find characters other than the virulence-associated protein (VapA) which might be encoded by the virulence plasmid in organisms grown at 37 degrees C. Tests chosen included those which have previously given variable results for R. equi isolates, since such variability might be attributed to plasmid curing, and characteristics which have been described as properties of plasmids of Rhodococcus species other than R. equi. Tests included cadmium resistance, Congo red binding, resistance to 26 antibiotics, conventional clinical microbiological tests, utilization of 95 different carbon sources, enzymatic activities in API ZYM, fluorogenic assays for exo- and endopeptidase, glycosidase activities, and testosterone degradation. Apart from production of VapA by foal isolates, no phenotypic property was identified in the plasmid-positive isolates. Phenotypic characteristics of R. equi that have not been described before, and might be useful in identification were: metabolism of N-acetyl-beta D-glucopyranoside, alpha- and beta-hydroxybutyric, alpha-ketobutyric and N-acetyl-glutamic acids, of methylpyruvate, heptanoate, nonanoate and stearate esters; exopeptidase activity against alanine-alanine-tyrosine, alanine-phenylalanine-lysine, glycine-arginine, lysine-alanine, and valine-glycine-alanine; endopeptidase activity against arginine and methionine; and hydrolysis of bis-phosphate ester. PMID:8825990

  17. Activity of T-DNA borders in plant cell transformation by mini-T plasmids.

    PubMed

    Jen, G C; Chilton, M D

    1986-05-01

    By using a binary vector system, we examined the requirements for border sequences in T-DNA transformation of plant genomes. Mini-T plasmids consisting of small replicons with different extents of pTiT37 T-DNA were tested for plant tumor-inducing ability in Agrobacterium tumefaciens strain LBA4404 containing helper plasmid pAL4404 (which encodes virulence genes needed for T-DNA transfer). Assays of these bacteria on carrot disks, Kalanchoë leaves, and SR1 Nicotiana tabacum plantlets showed that mini-T plasmid containing full length T-DNA including left and right borders was highly virulent, as were mini-T plasmids containing all onc (oncogenicity) genes and only the right border. In contrast, mini-T plasmids containing all onc genes and only the left border induced tumors only rarely, and a mini-T plasmid containing all onc genes but no T-DNA borders was completely avirulent. Southern hybridization analyses of tumor DNA showed that T-DNA border sequences delimited the extent of the two-border mini-T plasmid transferred and integrated into the plant genome. When only one T-DNA border was present, it formed one end of the transferred DNA, and the other end mapped in the vector sequences. The implications of these results for the mechanism of T-DNA transfer and integration are discussed. PMID:3009403

  18. GenoLIB: a database of biological parts derived from a library of common plasmid features

    PubMed Central

    Adames, Neil R.; Wilson, Mandy L.; Fang, Gang; Lux, Matthew W.; Glick, Benjamin S.; Peccoud, Jean

    2015-01-01

    Synthetic biologists rely on databases of biological parts to design genetic devices and systems. The sequences and descriptions of genetic parts are often derived from features of previously described plasmids using ad hoc, error-prone and time-consuming curation processes because existing databases of plasmids and features are loosely organized. These databases often lack consistency in the way they identify and describe sequences. Furthermore, legacy bioinformatics file formats like GenBank do not provide enough information about the purpose of features. We have analyzed the annotations of a library of ∼2000 widely used plasmids to build a non-redundant database of plasmid features. We looked at the variability of plasmid features, their usage statistics and their distributions by feature type. We segmented the plasmid features by expression hosts. We derived a library of biological parts from the database of plasmid features. The library was formatted using the Synthetic Biology Open Language, an emerging standard developed to better organize libraries of genetic parts to facilitate synthetic biology workflows. As proof, the library was converted into GenoCAD grammar files to allow users to import and customize the library based on the needs of their research projects. PMID:25925571

  19. Reconstructing the complex evolutionary history of mobile plasmids in red algal genomes.

    PubMed

    Lee, JunMo; Kim, Kyeong Mi; Yang, Eun Chan; Miller, Kathy Ann; Boo, Sung Min; Bhattacharya, Debashish; Yoon, Hwan Su

    2016-01-01

    The integration of foreign DNA into algal and plant plastid genomes is a rare event, with only a few known examples of horizontal gene transfer (HGT). Plasmids, which are well-studied drivers of HGT in prokaryotes, have been reported previously in red algae (Rhodophyta). However, the distribution of these mobile DNA elements and their sites of integration into the plastid (ptDNA), mitochondrial (mtDNA), and nuclear genomes of Rhodophyta remain unknown. Here we reconstructed the complex evolutionary history of plasmid-derived DNAs in red algae. Comparative analysis of 21 rhodophyte ptDNAs, including new genome data for 5 species, turned up 22 plasmid-derived open reading frames (ORFs) that showed syntenic and copy number variation among species, but were conserved within different individuals in three lineages. Several plasmid-derived homologs were found not only in ptDNA but also in mtDNA and in the nuclear genome of green plants, stramenopiles, and rhizarians. Phylogenetic and plasmid-derived ORF analyses showed that the majority of plasmid DNAs originated within red algae, whereas others were derived from cyanobacteria, other bacteria, and viruses. Our results elucidate the evolution of plasmid DNAs in red algae and suggest that they spread as parasitic genetic elements. This hypothesis is consistent with their sporadic distribution within Rhodophyta. PMID:27030297

  20. Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture.

    PubMed

    Rozkov, Aleksei; Larsson, Bert; Gillström, Stefan; Björnestedt, Robert; Schmidt, Stefan R

    2008-02-15

    Transient expression of recombinant proteins in mammalian cell culture in a 100-L scale requires a large quantity of plasmid that is very labour intensive to achieve with shake flask cultures and commercially available plasmid purification kits. In this paper we describe a process for plasmid production in 100-mg scale. The fermentation is carried out in a 4-L fed-batch culture with a minimal medium. The detection of the end of batch and triggering the exponential (0.1 h(-1)) feed profile was unattended and controlled by Multi-fermenter Control System. A restricted specific growth rate in fed-batch culture increased the specific plasmid yield compared to batch cultures with minimal and rich media. This together with high biomass concentration (68-107 g L(-1) wet weight) achieves high volumetric yields of plasmid (95-277 mg L(-1) depending on the construct). The purification process consisted of alkaline lysis, lysate clarification and ultrafiltration, two-phase extraction with Triton X-114 for endotoxin removal, anion-exchange chromatography as a polishing step, ultrafiltration and sterile filtration. Both fermentation and purification processes were used without optimisation for production of four plasmids yielding from 39 to 163 mg of plasmids with endotoxin content of 2.5 EU mg(-1) or less. PMID:17680665

  1. Analysis of a Pool of Small Plasmids from Soil Heterotrophic Cultivable Bacterial Communities

    PubMed Central

    Papaleo, Maria Cristiana; Fondi, Marco; Maida, Isabel; Perrin, Elena; Bevivino, Annamaria; Dalmastri, Claudia; Fani, Renato

    2015-01-01

    In this work the analysis of the plasmid presence on soil aerobic cultivable heterotrophic bacterial communities was carried out checking a panel of 1,200 isolates, in order to establish the frequency of plasmid presence as well as the degree of plasmid flow between strains affiliated to the same or different taxon. Bacterial communities were isolated from two different sites of a 13-year experimental field with a clay-silt texture. Plasmid molecules were detected at low frequency (27 isolates, 2%) with a size ranging between 2 Kb and 40 Kb. The RAPD analysis performed on the plasmid-harboring isolates and the phylogenetic analysis of the whole community using the 16S rRNA gene sequences revealed the existence of transfer of the same plasmids between strains belonging to the same species and, in some cases, to different species of the same genus. As it might be expected, even though the viable cells title did not differ significantly between the two samplings, the overall data disclosed an uneven distribution of both species and plasmid-harboring strains. PMID:26464609

  2. CpG-free plasmid expression cassettes for cystic fibrosis gene therapy.

    PubMed

    Pringle, Ian A; Hyde, Stephen C; Connolly, Mary M; Lawton, Anna E; Xu, Bihui; Nunez-Alonso, Graciela; Davies, Lee A; Sumner-Jones, Stephanie G; Gill, Deborah R

    2012-10-01

    Clinical studies are underway for the aerosol delivery of plasmid DNA complexed with Genzyme Lipid GL67A to the lungs of patients with cystic fibrosis (CF). Plasmid vectors contain several functional elements all of which play a role in determining the efficacy of the final clinical product. To optimise the final plasmid, variations of CpG-free 5' enhancer elements and 3'UTR regions were inserted into a common CpG-free, plasmid backbone containing Luciferase or CFTR transgenes. Plasmids were compared in immortalised cell culture, human airway liquid interface primary cell cultures, and mouse lung models to determine which design directed optimal transgene expression. Following aerosol delivery to mouse lung, plasmids containing the murine CMV enhancer showed higher peak Luciferase activity than the human CMV enhancer, but the human version resulted in persistent expression. In cell culture, the SV40 3'UTR and a novel BGH2 3'UTR exhibited up to 20-fold higher Luciferase activity than the commonly used BGH 3'UTR, but in mouse lung aerosol studies the activity and duration was greater for BGH 3'UTR. Systematic evaluation of each functional component of the plasmid has resulted in an improved design, exhibiting superior levels and duration of lung gene expression. PMID:22727465

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

    PubMed

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

    2016-01-01

    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

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

    PubMed Central

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

    2016-01-01

    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

  5. Natural plasmid transformation in a high-frequency-of transformation marine Vibrio strain

    SciTech Connect

    Frischer, M.E.; Thurmond, J.M.; Paul, J.H. )

    1990-11-01

    The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 {times} 10{sup {minus}9} and 3.4 {times} 10{sup {minus}7} transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42,857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 {times} 10{sup {minus}8} to 1.3 {times} 10{sup {minus}4} transformants per recipient with plasmid DNA and at an average frequency of 8.3 {times} 10{sup {minus}5} transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound ({sup 3}H)bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations.

  6. The Coxiella burnetii Cryptic Plasmid Is Enriched in Genes Encoding Type IV Secretion System Substrates▿ †

    PubMed Central

    Voth, Daniel E.; Beare, Paul A.; Howe, Dale; Sharma, Uma M.; Samoilis, Georgios; Cockrell, Diane C.; Omsland, Anders; Heinzen, Robert A.

    2011-01-01

    The intracellular bacterial pathogen Coxiella burnetii directs biogenesis of a phagolysosome-like parasitophorous vacuole (PV), in which it replicates. The organism encodes a Dot/Icm type IV secretion system (T4SS) predicted to deliver to the host cytosol effector proteins that mediate PV formation and other cellular events. All C. burnetii isolates carry a large, autonomously replicating plasmid or have chromosomally integrated plasmid-like sequences (IPS), suggesting that plasmid and IPS genes are critical for infection. Bioinformatic analyses revealed two candidate Dot/Icm substrates with eukaryotic-like motifs uniquely encoded by the QpH1 plasmid from the Nine Mile reference isolate. CpeC, containing an F-box domain, and CpeD, possessing kinesin-related and coiled-coil regions, were secreted by the closely related Legionella pneumophila Dot/Icm T4SS. An additional QpH1-specific gene, cpeE, situated in a predicted operon with cpeD, also encoded a secreted effector. Further screening revealed that three hypothetical proteins (CpeA, CpeB, and CpeF) encoded by all C. burnetii plasmids and IPS are Dot/Icm substrates. By use of new genetic tools, secretion of plasmid effectors by C. burnetii during host cell infection was confirmed using β-lactamase and adenylate cyclase translocation assays, and a C-terminal secretion signal was identified. When ectopically expressed in HeLa cells, plasmid effectors trafficked to different subcellular sites, including autophagosomes (CpeB), ubiquitin-rich compartments (CpeC), and the endoplasmic reticulum (CpeD). Collectively, these results suggest that C. burnetii plasmid-encoded T4SS substrates play important roles in subversion of host cell functions, providing a plausible explanation for the absolute maintenance of plasmid genes by this pathogen. PMID:21216993

  7. Targeting relaxase genes for classification of the predominant plasmids in Enterobacteriaceae.

    PubMed

    Compain, Fabrice; Poisson, Agathe; Le Hello, Simon; Branger, Catherine; Weill, François-Xavier; Arlet, Guillaume; Decré, Dominique

    2014-05-01

    Plasmids are the main vectors of antimicrobial drug resistance and virulence genes, especially in Enterobacteriaceae. Identification and classification of plasmids is essential for analysis of their distribution. The most widely used typing method is PCR-based replicon typing (PBRT). A new classification scheme based on relaxase gene typing has been described recently. We propose a practical application of this method, with the development of a multiplex PCR set targeting relaxase genes found on plasmids most frequently encountered in Enterobacteriaceae. This method, here called "plasmid relaxase gene typing" (PRaseT), was validated with 60 transconjugants and transformants harboring various replicon types. The method was tested with 39 multidrug-resistant clinical isolates including Escherichia coli, Klebsiella pneumoniae and Salmonella enterica subsp. enterica carrying 1-7 replicons as well as with 17 plasmids non-typeable using PBRT; all replicons were tested in parallel with PBRT for comparison. Six multiplex PCRs and one simplex PCR, including 24 pairs of primers, recognized plasmids of groups A/C, B/O, colE, FIA, FIB, FIC, FV, FIIk, HI1, HI2, I1, K, L/M, N, P1α, Q1, U, W, X1, X2, X3 and X4. There was perfect correlation between PRaseT and PBRT results in 31/39 (79.5%) clinical isolates. Moreover, 11/17 (64.7%) plasmids non-typeable by PBRT could be typed by PRaseT. Our set of multiplex PCRs showed high sensitivity and specificity for the classification of resistance plasmids. It has proved complementary to the widely used PBRT and will improve the monitoring of plasmid distribution in every-day practice. PMID:24342269

  8. Identification of the pXO1 plasmid in attenuated Bacillus anthracis vaccine strains.

    PubMed

    Liang, Xudong; Zhang, Huijuan; Zhang, Enmin; Wei, Jianchun; Li, Wei; Wang, Bingxiang; Dong, Shulin; Zhu, Jin

    2016-07-01

    Anthrax toxins and capsule are the major virulence factors of Bacillus anthracis. They are encoded by genes located on the plasmids pXO1 and pXO2, respectively. The vaccine strain Pasteur II was produced from high temperature subcultures of B. anthracis, which resulted in virulence attenuation through the loss of the plasmid pXO1. However, it is unclear whether the high temperature culture completely abolishes the plasmid DNA or affects the replication of the plasmid pXO1. In this study, we tested 3 B. anthracis vaccine strains, including Pasteur II from France, Qiankefusiji II from Russia, and Rentian II from Japan, which were all generated from subcultures at high temperatures. Surprisingly, we detected the presence of pXO1 plasmid DNA using overlap PCR in all these vaccine strains. DNA sequencing analysis of overlap PCR products further confirmed the presence of pXO1. Moreover, the expression of the protective antigen (PA) encoded on pXO1 was determined by using SDS-PAGE and western blotting. In addition, we mimicked Pasteur's method and exposed the A16R vaccine strain, which lacks the pXO2 plasmid, to high temperature, and identified the pXO1 plasmid in the subcultures at high temperatures. This indicated that the high temperature treatment at 42.5°C was unable to eliminate pXO1 plasmid DNA from B. anthracis. Our results suggest that the attenuation of the Pasteur II vaccine strain is likely due to the impact of high temperature stress on plasmid replication, which in turn limits the copy number of pXO1. Our data provide new insights into the mechanisms of the remaining immunogenicity and toxicity of the vaccine strains. PMID:27029580

  9. Successful transfer of plasmid DNA into in vitro cells transfected with an inorganic plasmid-Mg/Al-LDH nanobiocomposite material as a vector for gene expression

    NASA Astrophysics Data System (ADS)

    Jaffri Masarudin, Mas; Yusoff, Khatijah; Rahim, Raha Abdul; Zobir Hussein, Mohd

    2009-01-01

    The delivery of a full plasmid, encoding the green fluorescent protein gene into African monkey kidney (Vero3) cells, was successfully achieved using nanobiocomposites based on layered double hydroxides. This demonstrated the potential of using the system as an alternative DNA delivery vector. Intercalation of the circular plasmid DNA, pEGFP-N2, into Mg/Al-NO3- layered double hydroxides (LDH) was accomplished through anion exchange routes to form the nanobiocomposite material. The host was previously synthesized at the Mg2+ to Al3+ molar ratio Ri = 2 and subsequently intercalated with plasmid DNA. Size expansion of the interlamellae host from 8.8 Å in LDH to 42 Å was observed in the resulting nanobiocomposite, indicating stable hybridization of the plasmid DNA. The powder x-ray diffraction (PXRD) results, supplemented with Fourier-transform infrared (FTIR) spectroscopy, compositional and electrophoresis studies confirmed the encapsulation episode of the biomaterial. In order to elucidate the use of this resulting nanobiocomposite as a delivery vector, an MTT assay was performed to determine any cytotoxic effects of the host towards cells. The intercalated pEGFP-N2 anion was later successfully recovered through acidification with HNO3 after treatment with DNA-degrading enzymes, thus also showing the ability of the LDH host to protect the intercalated biomaterial from degradation. Cell transfection studies on Vero3 cells were then performed, where cells transfected with the nanobiocomposite exhibited fluorescence as early as 12 h post-treatment compared to naked delivery of the plasmid itself.

  10. Influence of Different Functional Elements of Plasmid pGT232 on Maintenance of Recombinant Plasmids in Lactobacillus reuteri Populations In Vitro and In Vivo

    PubMed Central

    Heng, Nicholas C. K.; Bateup, Judith M.; Loach, Diane M.; Wu, Xiyang; Jenkinson, Howard F.; Morrison, Mark; Tannock, Gerald W.

    1999-01-01

    Plasmid pGT232 (5.1 kb), an indigenous plasmid of Lactobacillus reuteri 100-23, was determined, on the basis of nucleotide and deduced protein sequence data, to belong to the pC194-pUB110 family of plasmids that replicate via the rolling-circle mechanism. The minimal replicon of pGT232 was located on a 1.7-kb sequence consisting of a double-strand origin of replication and a gene encoding the replication initiation protein, repA. An erythromycin-selectable recombinant plasmid containing this minimal replicon was stably maintained (>97% erythromycin-resistant cells) without antibiotic selection in an L. reuteri population under laboratory growth conditions but was poorly maintained (<33% resistant cells) in the L. reuteri population inhabiting the murine gastrointestinal tract. Stable maintenance (>90% resistant cells) of pGT232-derived plasmids in the lactobacillus population in vivo required an additional 1.0-kb sequence which contained a putative single-strand replication origin (SSO). The SSO of pGT232 is believed to be novel and functions in an orientation-specific manner. PMID:10583992

  11. Plasmids in the driving seat: The regulatory RNA Rcd gives plasmid ColE1 control over division and growth of its E. coli host.

    PubMed

    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. PMID:25446541

  12. Susceptibility to antimicrobial agents and plasmid carrying in Aeromonas hydrophila isolated from two estuarine systems.

    PubMed

    Montoya, R; Dominguez, M; Gonzalez, C; Mondaca, M A; Zemelman, R

    1992-01-01

    Susceptibility to various antimicrobial agents and the presence of plasmids was investigated in eleven strains of Aeromonas hydrophila isolated from samples of sea water and these strains isolated from Aulacomya ater. Transference of resistance to Escherichia coli was attempted by conjugation and transformation experiments. The strains showed multiple resistance toward beta-lactam antibiotics and susceptibility to other antimicrobial agents. Five strains harboured plasmids with molecular weights below 5.7 MD. It was not possible to relate the resistance of the strains with the presence of their plasmids. PMID:1593967

  13. Plasmids of incompatibility group P code for the capacity to propagate bacteriophage IKe.

    PubMed Central

    Grant, R B; Whiteley, M H; Shapley, A J

    1978-01-01

    Seven of eight plasmids of incompatibility group P were found to code for the capacity to propagate bacteriophage IKe in Escherichia coli. Six of the seven plasmids allowed propagation of IKe by one bacterial host (RG172) but not by another (RG176); the other plasmid allowed IKe propagation by both hosts. IKe propagation by a number of E. coli K-12 strains was quite variable. IKeh, an extended host range mutant of IKe, was found to plaque specifically on N+ and P+ strains. PMID:361724

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

    NASA Astrophysics Data System (ADS)

    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.

  15. Effect of repeated insertions of curved sequences in DNA plasmids: a light-scattering study

    NASA Astrophysics Data System (ADS)

    Beretta, Stefano; Chirico, Giuseppe; Baldini, Giancarlo; Kapp, U.; Badaracco, G.

    1993-06-01

    The effect of the insertion of different amounts (from 0 to 6) of the curved sequence AluI in pUC18m plasmid (2686 base pairs, bp) is studied by dynamic light scattering. This sequence is a highly repeated 113 base pairs long sequence from Artemia Franciscana shrimp. A 30% compaction of the plasmids containing 2 and 6 adjacent AluI sequences compared to pUC8 plasmid (2717 bp) is observed. Furthermore the behavior of the translational diffusion coefficient Dt versus the number of adjacent AluI insertion is not monotonic.

  16. [Integration of plasmids into E. coli K12 chromosomes, caused by a Bordetella transposon].

    PubMed

    Sivov, I G; Beliavskiĭ, O A; karataev, G I

    2000-01-01

    Transposition of Bordetella pertussis transposon in E. coli chromosome has been studied on a model of exclusion of donor multicopy pKK3 plasmid with coumermicin. TnBP3 induced the formation of co-integrates between the plasmid and chromosome. The structure of co-integrate was determined. Facts of exclusion of integrated structure and transposon transposition within integrated plasmid into new sites on a recipient chromosome were detected. Relationship between these processes and activity of bacterial cell recombination system has been determined. PMID:10876766

  17. Separate F-Type Plasmids Have Shaped the Evolution of the H30 Subclone of Escherichia coli Sequence Type 131.

    PubMed

    Johnson, Timothy J; Danzeisen, Jessica L; Youmans, Bonnie; Case, Kyle; Llop, Katharine; Munoz-Aguayo, Jeannette; Flores-Figueroa, Cristian; Aziz, Maliha; Stoesser, Nicole; Sokurenko, Evgeni; Price, Lance B; Johnson, James R

    2016-01-01

    The extraintestinal pathogenic Escherichia coli (ExPEC) H30 subclone of sequence type 131 (ST131-H30) has emerged abruptly as a dominant lineage of ExPEC responsible for human disease. The ST131-H30 lineage has been well described phylogenetically, yet its plasmid complement is not fully understood. Here, single-molecule, real-time sequencing was used to generate the complete plasmid sequences of ST131-H30 isolates and those belonging to other ST131 clades. Comparative analyses revealed separate F-type plasmids that have shaped the evolution of the main fluoroquinolone-resistant ST131-H30 clades. Specifically, an F1:A2:B20 plasmid is strongly associated with the H30R/C1 clade, whereas an F2:A1:B- plasmid is associated with the H30Rx/C2 clade. A series of plasmid gene losses, gains, and rearrangements involving IS26 likely led to the current plasmid complements within each ST131-H30 sublineage, which contain several overlapping gene clusters with putative functions in virulence and fitness, suggesting plasmid-mediated convergent evolution. Evidence suggests that the H30Rx/C2-associated F2:A1:B- plasmid type was present in strains ancestral to the acquisition of fluoroquinolone resistance and prior to the introduction of a multidrug resistance-encoding gene cassette harboring bla CTX-M-15. In vitro experiments indicated a host strain-independent low frequency of plasmid transfer, differential levels of plasmid stability even between closely related ST131-H30 strains, and possible epistasis for carriage of these plasmids within the H30R/Rx lineages. IMPORTANCE A clonal lineage of Escherichia coli known as ST131 has emerged as a dominating strain type causing extraintestinal infections in humans. The evolutionary history of ST131 E. coli is now well understood. However, the role of plasmids in ST131's evolutionary history is poorly defined. This study utilized real-time, single-molecule sequencing to compare plasmids from various current and historical lineages of ST

  18. Transformation of microorganisms with the plasmid vector with the replicon from pAC1 from Acetobacter pasteurianus.

    PubMed

    Grones, J; Turna, J

    1995-01-26

    A number of gram-negative and gram-positive bacteria species was screened for the expression of the gram-negative plasmid pACK5 and pACT72 with replicon of pAC1 plasmid from Acetobacter pasteurianus. As was described previously, both plasmids were expressed in Escherichia coli, Acetobacter pasteurianus, Acetobacter aceti, Shigella spp. and Citrobacter spp. Expressions of plasmids were successful in twelve species tested, Comamonas terrigena, Salmonella typhimurium, Serratia marcescens, Bacillus cereus, Bacillus megatericum, Bacillus subtilis, Lactobacillus helveticus, Micrococcus luteus, Sarcina lutea, Staphylococcus aureus, Staphylococcus epidermidis, Streptoccocus feacalis, and the stability of plasmid DNA was tested after cultivation in non-selective conditions. PMID:7832808

  19. Separate F-Type Plasmids Have Shaped the Evolution of the H30 Subclone of Escherichia coli Sequence Type 131

    PubMed Central

    Danzeisen, Jessica L.; Youmans, Bonnie; Case, Kyle; Llop, Katharine; Munoz-Aguayo, Jeannette; Flores-Figueroa, Cristian; Aziz, Maliha; Sokurenko, Evgeni; Price, Lance B.; Johnson, James R.

    2016-01-01

    ABSTRACT The extraintestinal pathogenic Escherichia coli (ExPEC) H30 subclone of sequence type 131 (ST131-H30) has emerged abruptly as a dominant lineage of ExPEC responsible for human disease. The ST131-H30 lineage has been well described phylogenetically, yet its plasmid complement is not fully understood. Here, single-molecule, real-time sequencing was used to generate the complete plasmid sequences of ST131-H30 isolates and those belonging to other ST131 clades. Comparative analyses revealed separate F-type plasmids that have shaped the evolution of the main fluoroquinolone-resistant ST131-H30 clades. Specifically, an F1:A2:B20 plasmid is strongly associated with the H30R/C1 clade, whereas an F2:A1:B− plasmid is associated with the H30Rx/C2 clade. A series of plasmid gene losses, gains, and rearrangements involving IS26 likely led to the current plasmid complements within each ST131-H30 sublineage, which contain several overlapping gene clusters with putative functions in virulence and fitness, suggesting plasmid-mediated convergent evolution. Evidence suggests that the H30Rx/C2-associated F2:A1:B− plasmid type was present in strains ancestral to the acquisition of fluoroquinolone resistance and prior to the introduction of a multidrug resistance-encoding gene cassette harboring blaCTX-M-15. In vitro experiments indicated a host strain-independent low frequency of plasmid transfer, differential levels of plasmid stability even between closely related ST131-H30 strains, and possible epistasis for carriage of these plasmids within the H30R/Rx lineages. IMPORTANCE A clonal lineage of Escherichia coli known as ST131 has emerged as a dominating strain type causing extraintestinal infections in humans. The evolutionary history of ST131 E. coli is now well understood. However, the role of plasmids in ST131’s evolutionary history is poorly defined. This study utilized real-time, single-molecule sequencing to compare plasmids from various current and historical

  20. Curing vector for IncI1 plasmids and its use to provide evidence for a metabolic burden of IncI1 CTX-M-1 plasmid pIFM3791 on Klebsiella pneumoniae.

    PubMed

    Freire Martín, Irene; Thomas, Christopher M; Laing, Emma; AbuOun, Manal; La Ragione, Roberto M; Woodward, Martin J

    2016-07-01

    Using a sequence-based approach we previously identified an IncI1 CTX-M-1 plasmid, pIFM3791, on a single pig farm in the UK that was harboured by Klebsiella pneumoniae, Escherichia coli and Salmonella enterica serotype 4,5,12:i:-. To test the hypothesis that the plasmid had spread rapidly into these differing host bacteria we wished to assess whether the plasmid conferred a fitness advantage. To do this an IncI1 curing vector was constructed and used to displace the IncI1 CTX-M-1 plasmids from K. pneumoniae strain B3791 and several other unrelated IncI1-harbouring strains indicating the potential wider application of the curing vector. The IncI1 CTX-M-1 plasmid was reintroduced by conjugation into the cured K. pneumoniae strain and also a naturally IncI1 plasmid free S. enterica serotype 4,5,12:i:-, S348/11. Original, cured and complemented strains were tested for metabolic competence using Biolog technology and in competitive growth, association to mammalian cells and biofilm formation experiments. The plasmid-cured K. pneumoniae strain grew more rapidly than either the original plasmid-carrying strain or plasmid-complemented strains in competition experiments. Additionally, the plasmid-cured strain was significantly better at respiring with l-sorbose as a carbon source and putrescine, γ-amino-n-butyric acid, l-alanine and l-proline as nitrogen sources. By contrast, no differences in phenotype were found when comparing plasmid-harbouring and plasmid-free S. enterica S348/11. In conclusion, the IncI1 curing vector successfully displaced multiple IncI plasmids. The IncI1 CTX-M1 plasmid conferred a growth disadvantage upon K. pneumoniae, possibly by imposing a metabolic burden, the mechanism of which remains to be determined. PMID:27166141

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

    PubMed Central

    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

  2. IncF plasmid diversity in multi-drug resistant Escherichia coli strains from animals in China

    PubMed Central

    Yang, Qiu-E.; Sun, Jian; Li, Liang; Deng, Hui; Liu, Bao-Tao; Fang, Liang-Xing; Liao, Xiao-Ping; Liu, Ya-Hong

    2015-01-01

    The purpose of this study was to characterize a collection of 103 multidrug resistance IncF plasmids recovered from Escherichia coli of food producing and companion animals between 2003 and 2012. A total of 103 incF plasmids were characterized using an established PCR-based IncF replicon sequence typing (RST) system to identify FII, FIA, and FIB (FAB) groups. Plasmids were also analyzed using-restriction fragment length polymorphism (RFLP). Antibiotic Resistance determinants blaCTX-M, plasmid-mediated quinolone resistance (PMQR) genes and rmtB and plasmid addiction systems (PAS) were identified by PCR screening. A total of 20 different RSTs from 103 IncF plasmids were identified. The groups F2 and F33 with the RST formulae A-: B- were the most frequently encountered types (63.1%). The antibiotic resistance genes (ARGs) blaCTX-M, rmtB, and oqxB were carried by 82, 37, and 34 IncF plasmids, respectively. Most of these plasmids carried more than one resistance gene (59.2%, 61/103). The IncF plasmids also had a high frequency of addiction systems (mean 2.54) and two antisense RNA-regulated systems (hok–sok and srnBC) and a protein antitoxin-regulated system (pemKI) were the most prevalent. Not surprisingly, RFLP profiles among the IncF plasmids were diverse even though some shared identical IncF-RSTs. This is the first extensive study of IncF plasmid-positive E. coli isolates from animals in China. Our results demonstrate that IncF is the most prevalent plasmid family in E. coli plasmids and they commonly carry multiple resistance determinants that render them resistant to different antibiotic classes simultaneously. IncF plasmids also harbor addiction systems, promoting their stability and maintenance in the bacterial host, under changing environmental conditions. PMID:26441898

  3. Evolution of Chromosomal Clostridium botulinum Type E Neurotoxin Gene Clusters: Evidence Provided by Their Rare Plasmid-Borne Counterparts.

    PubMed

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

    2016-03-01

    Analysis of more than 150 Clostridium botulinum Group II type E genomes identified a small fraction (6%) where neurotoxin-encoding genes were located on plasmids. Seven closely related (134-144 kb) neurotoxigenic plasmids of subtypes E1, E3, and E10 were characterized; all carried genes associated with plasmid mobility via conjugation. Each plasmid contained the same 24-kb neurotoxin cluster cassette (six neurotoxin cluster and six flanking genes) that had split a helicase gene, rather than the more common chromosomal rarA. The neurotoxin cluster cassettes had evolved as separate genetic units which had either exited their chromosomal rarA locus in a series of parallel events, inserting into the plasmid-borne helicase gene, or vice versa. A single intact version of the helicase gene was discovered on a nonneurotoxigenic form of this plasmid. The observed low frequency for the plasmid location may reflect one or more of the following: 1) Less efficient recombination mechanism for the helicase gene target, 2) lack of suitable target plasmids, and 3) loss of neurotoxigenic plasmids. Type E1 and E10 plasmids possessed a Clustered Regularly Interspaced Short Palindromic Repeats locus with spacers that recognized C. botulinum Group II plasmids, but not C. botulinum Group I plasmids, demonstrating their long-term separation. Clostridium botulinum Group II type E strains also carry nonneurotoxigenic plasmids closely related to C. botulinum Group II types B and F plasmids. Here, the absence of neurotoxin cassettes may be because recombination requires both a specific mechanism and specific target sequence, which are rarely found together. PMID:26936890

  4. Evolution of Chromosomal Clostridium botulinum Type E Neurotoxin Gene Clusters: Evidence Provided by Their Rare Plasmid-Borne Counterparts

    PubMed Central

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

    2016-01-01

    Analysis of more than 150 Clostridium botulinum Group II type E genomes identified a small fraction (6%) where neurotoxin-encoding genes were located on plasmids. Seven closely related (134–144 kb) neurotoxigenic plasmids of subtypes E1, E3, and E10 were characterized; all carried genes associated with plasmid mobility via conjugation. Each plasmid contained the same 24-kb neurotoxin cluster cassette (six neurotoxin cluster and six flanking genes) that had split a helicase gene, rather than the more common chromosomal rarA. The neurotoxin cluster cassettes had evolved as separate genetic units which had either exited their chromosomal rarA locus in a series of parallel events, inserting into the plasmid-borne helicase gene, or vice versa. A single intact version of the helicase gene was discovered on a nonneurotoxigenic form of this plasmid. The observed low frequency for the plasmid location may reflect one or more of the following: 1) Less efficient recombination mechanism for the helicase gene target, 2) lack of suitable target plasmids, and 3) loss of neurotoxigenic plasmids. Type E1 and E10 plasmids possessed a Clustered Regularly Interspaced Short Palindromic Repeats locus with spacers that recognized C. botulinum Group II plasmids, but not C. botulinum Group I plasmids, demonstrating their long-term separation. Clostridium botulinum Group II type E strains also carry nonneurotoxigenic plasmids closely related to C. botulinum Group II types B and F plasmids. Here, the absence of neurotoxin cassettes may be because recombination requires both a specific mechanism and specific target sequence, which are rarely found together. PMID:26936890

  5. Evidence for plasmid DNA exchange after polyplex mixing.

    PubMed

    Pigeon, L; Gonçalves, C; Pichon, C; Midoux, P

    2016-08-17

    The self-assembly of a plasmid DNA (pDNA) with cationic polymers or cationic liposomes forms nanosized supramolecular structures called lipoplexes, polyplexes and lipopolyplexes. Here, we report that when two polyplex preparations made using the same polymer and the same pDNA but labelled with two different fluorophores are mixed together, pDNA molecules are exchanged. Indeed, when Flu-pDNA complexed with histidinylated lPEI (Flu-pDNA/His-lPEI) polyplexes are mixed with Cy5-pDNA complexed with histidinylated lPEI (Cy5-pDNA/His-lPEI) polyplexes, a high quantity of polyplexes emitting dual fluorescence is observed and FRET indicates that one single polyplex contains two kinds of fluorescent pDNA molecules. This phenomenon depends on the polymer-type and the strength of the pDNA/polymer interaction. No exchange is observed with polylysine polyplexes, caged His-lPEI polyplexes, lipoplexes, lipopolyplexes or when His-lPEI polyplexes are mixed with lipoplexes. Our results suggest that aggregation or collapse of polyplexes occurs after their interaction leading to their unpackaging followed by the formation of new polyplexes with the exchange of pDNA. PMID:27459887

  6. Quantitative analysis of virus and plasmid trafficking in cells

    NASA Astrophysics Data System (ADS)

    Lagache, Thibault; Dauty, Emmanuel; Holcman, David

    2009-01-01

    Intracellular transport of DNA carriers is a fundamental step of gene delivery. By combining both theoretical and numerical approaches we study here single and several viruses and DNA particles trafficking in the cell cytoplasm to a small nuclear pore. We present a physical model to account for certain aspects of cellular organization, starting with the observation that a viral trajectory consists of epochs of pure diffusion and epochs of active transport along microtubules. We define a general degradation rate to describe the limitations of the delivery of plasmid or viral particles to a nuclear pore imposed by various types of direct and indirect hydrolysis activity inside the cytoplasm. By replacing the switching dynamics by a single steady state stochastic description, we obtain estimates for the probability and the mean time for the first one of many particles to go from the cell membrane to a small nuclear pore. Computational simulations confirm that our model can be used to analyze and interpret viral trajectories and estimate quantitatively the success of nuclear delivery.

  7. Complexation Between Cationic Diblock Copolymers and Plasmid DNA

    NASA Astrophysics Data System (ADS)

    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.

  8. In vitro replication of mitochondrial plasmid mp1 from the higher plant Chenopodium album (L.): a remnant of bacterial rolling circle and conjugative plasmids?

    PubMed

    Backert, S; Kunnimalaiyaan, M; Börner, T; Nielsen, B L

    1998-12-11

    According to the endosymbiotic theory, mitochondrial genomes evolved from the chromosome of an alpha-proteobacterium-like ancestor and developed during evolution an extraordinary variation in size, structure and replication. We studied in vitro DNA replication of the mitochondrial circular plasmid mp1 (1309 bp) from the higher plant Chenopodium album (L.) as a model system that replicates in a manner reminiscent of bacterial rolling circle plasmids. Several mp1 subclones were tested for their ability to support DNA replication using a newly developed in vitro system. Neutral/neutral two-dimensional gel electrophoresis of the in vitro products revealed typical simple Y patterns of intermediates consistent with a rolling circle type of replication. Replication activity was very high for a BamHI-restricted total plasmid DNA clone, a 464 bp BamHI/KpnI fragment and a 363 bp BamHI/SmaI fragment. Further subcloning of a 148 bp BamHI/EcoRI fragment resulted in the strongest in vitro DNA replication activity, while a 1161 bp-template outside of this region resulted in a substantial loss of activity. Electron microscopic studies of in vitro DNA replication products from the highly active clones also revealed sigma-shaped molecules. These results support our in vivo data for the presence of a predominant replication origin between positions 628 and 776 on the plasmid map. This sequence shares homology with double-stranded rolling circle origin (dso) or transfer origin (oriT) nicking motifs from bacterial plasmids. mp1 is the first described rolling circle plasmid in eukaryotes. PMID:9837722

  9. Development of simple and efficient protocol for isolation of plasmids from mycobacteria using zirconia beads.

    PubMed

    Madiraju, M V; Qin, M H; Rajagopalan, M

    2000-01-01

    A two-step protocol has been developed for isolation of plasmids from recombinant mycobacteria via Escherichia coli. First either mycobacterial primary transformants or propagated cultures were lysed in a mini-bead beater using zirconia beads and the lysate thus obtained was used to transform E. coli recA mutant cells. Secondly, plasmid DNA was isolated from recombinant E. coli cells and analysed. Bead beating times of 2 min for Mycobacterium smegmatis, a rapid grower, and 4 min for M. bovis BCG, a slow grower, were found to be optimal for recovery of plasmid DNA. This protocol was also amenable to other mycobacterial species such as M. avium, M. fortuitum and M. tuberculosis H37Ra. Plasmid recovery from the recombinant M. bovis BCG using this protocol is approximately 300-fold higher than that reported for the electroduction method. PMID:10728558

  10. Plasmid-Mediated Bioaugmentation of Wastewater Microbial Communities in a Laboratory-Scale Bioreactor

    NASA Astrophysics Data System (ADS)

    Bathe, Stephan; Hausner, Martina

    Xenobiotic degradation during biological wastewater treatment can be established or enhanced by bioaugmentation - the addition of biological agents carrying biodegradation genes required to perform the task. Whereas the addition of microbial cells carrying chromosomally encoded catabolic genes can be impaired by limited survival of the added microorganisms, the addition of donor organisms carrying a transmissible catabolic plasmid is a promising alternative. This plasmid can spread within the indigenous microbial community of the system, circumventing the need for extended survival of the introduced bacterial strain. Here we discuss how the catabolic plasmid pNB2 can be evaluated towards its potential to facilitate the degradation of a xenobiotic compound, 3-chloroaniline, and demonstrate the applicability of this plasmid to accomplish 3-chloroaniline degradation in a bioreactor setting after in situ transfer to suitable recipient strains.

  11. Derepression of conjugal transfer of the antibiotic resistance plasmid R100 by antisense RNA.

    PubMed Central

    Dempsey, W B

    1989-01-01

    Conjugal transfer of the normally repressed antibiotic resistance plasmid R100 was derepressed by fragments of R100 that carried the traJ promoter and the traJ leader but lacked the finP promoter. PMID:2468651

  12. Hydrocarbon mineralization in sediments and plasmid incidence in sediment bacteria from the Campeche bank

    SciTech Connect

    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 ({sup 14}C) hexadecane and ({sup 14}C)phenanthrene measured for sediments were negligible; <1% of the substrate was converted to CO{sub 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.

  13. Efficient non-enzymatic cleavage of Staphylococcus aureus plasmid DNAs mediated by neodymium ions.

    PubMed

    Zovčáková, Monika; Španová, Alena; Pantůček, Roman; Doškař, Jiří; Rittich, Bohuslav

    2016-08-15

    Staphylococcus aureus plasmids are the main factor in the spreading of antibacterial resistance among bacterial strains that has emerged on a worldwide scale. Plasmids recovered from 12 clinical and food isolates of S. aureus were treated with 10 mM free lanthanide Nd(3+) ions (non-enzymatic cleavage agent) in Hepes buffer (pH 7.5) at 70 °C. Topological forms of plasmids-closed circular (ccc), open circular (oc), and linear (lin)-produced by cleavage at different times were separated using pulsed-field agarose gel electrophoresis. The method is proposed to detect and differentiate several plasmids in the same bacterial strain according to their size. PMID:27237372

  14. A physical map of the 85 kb virulence plasmid of Rhodococcus equi 103.

    PubMed Central

    de la Peña-Moctezuma, A; Prescott, J F

    1995-01-01

    A physical map of the 85 kb virulence plasmid pOTS from Rhodococcus equi 103 was constructed. The restriction map contains 2 AsnI, 5 BglII, 9 EcoRI, 4 HindIII, and 3 XbaI sites. The positions of the EcoRI and HindIII of pOTS are identical to that of the 85 kb virulence plasmid of R. equi ATCC 33701 reported recently by others. EcoRI restriction fragment sizes were similar in the 85 kb plasmids isolated from 4 horse derived R. equi but, except apparently for the 28.3 and possibly 2.0 and 1.5 kb fragments, were different in an 80.1 kb plasmid isolated from a pig source R. equi. PMID:8521357

  15. Low-Cost Fabrication of Centimetre-Scale Periodic Arrays of Single Plasmid DNA Molecules

    PubMed Central

    Kirkland, Brett; Wang, Zhibin; Zhang, Peipei; Takebayashi, Shin-ichiro; Lenhert, Steven; Gilbert, David M.

    2013-01-01

    We report development of a low-cost method to generate a centimetre-scale periodic array of single plasmid DNA of 11 kilobase pairs. The arrayed DNA is amenable to enzymatic and physical manipulation. PMID:23824041

  16. Hydrocarbon Mineralization in Sediments and Plasmid Incidence in Sediment Bacteria from the Campeche Bank

    PubMed Central

    Leahy, Joseph G.; Somerville, Charles C.; Cunningham, Kelly A.; Adamantiades, Grammenos A.; Byrd, Jeffrey J.; Colwell, Rita R.

    1990-01-01

    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 [14C]hexadecane and [14C]phenanthrene measured for sediments were negligible; <1% of the substrate was converted to CO2 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. PMID:16348204

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  18. Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids.

    PubMed

    Bryksin, Anton V; Matsumura, Ichiro

    2010-06-01

    Here we describe a straightforward, efficient, and reliable way to clone an insert of choice into a plasmid of choice without restriction endonucleases or T4 DNA ligase. Chimeric primers containing plasmid sequence at the 5' ends and insert sequence at the 3' ends were used to PCR-amplify insertion sequences of various sizes, namely the genes for GFP (gfp), beta-d-glucuronidase (gusA), and beta-galactosidase (lacZ), as well as the entire luxABCDE operon. These inserts were employed as mega-primers in a second PCR with a circular plasmid template. The original plasmid templates were then destroyed in restriction digests with DpnI, and the overlap extension PCR products were used to transform competent Escherichia coli cells. Phusion DNA polymerase was used for the amplification and fusion reactions, so both reactions were easy to monitor and optimize. PMID:20569222

  19. EBV-based plasmid DNA rearrangements after transfection of eukaryotic cells.

    PubMed

    Morozova, O V; Maksimova, T G; Kostenko, E V

    2000-05-01

    The cDNA encoding influenza virus (A/Udorn/307/72 strain) M2 protein was subcloned into the EBV-based vector pREP9. Three continuous kidney cellular lines of different origin were transfected with recombinant plasmid pREP9-M2. One and 5 months after transfection plasmid DNA rearrangements were detected by means of restriction analysis of recovered plasmids and their hybridization with an influenza-virus-specific radioactive probe. Deletions were the most frequent type of pREP9-M2 mutations. PCR with primers corresponding to cellular genome and plasmid DNA followed by Southern blot analysis with the [(32)P]-labeled M2-fragment allowed host DNA rearrangements to be revealed in transfected cells. PMID:10783296

  20. Diversified mcr-1-Harbouring Plasmid Reservoirs Confer Resistance to Colistin in Human Gut Microbiota

    PubMed Central

    Ye, Huiyan; Li, Yihui; Li, Zhencui; Gao, Rongsui; Zhang, Han; Wen, Ronghui; Gao, George F.; Hu, Qinghua

    2016-01-01

    ABSTRACT Colistin is an ultimate line of refuge against multidrug-resistant Gram-negative pathogens. Very recently, the emergence of plasmid-mediated mcr-1 colistin resistance has become a great challenge to global public health, raising the possibility that dissemination of the mcr-1 gene is underestimated and diversified. Here, we report three cases of plasmid-carried MCR-1 colistin resistance in isolates from gut microbiota of diarrhea patients. Structural and functional analyses determined that the colistin resistance is conferred purely by the single mcr-1 gene. Genetic and sequence mapping revealed that mcr-1-harbouring plasmid reservoirs are present in diversity. Together, the data represent the first evidence of diversity in mcr-1-harbouring plasmid reservoirs of human gut microbiota. PMID:27048797

  1. Draft genome sequences of two Aeromonas salmonicida subsp. salmonicida isolates harboring plasmids conferring antibiotic resistance.

    PubMed

    Vincent, Antony T; Tanaka, Katherine H; Trudel, Melanie V; Frenette, Michel; Derome, Nicolas; Charette, Steve J

    2015-02-01

    The bacterium Aeromonas salmonicida is the etiological agent of furunculosis, a widespread fish disease causing important economic losses to the fish farming industry. Antibiotic treatments in fish farms may be challenging given the existence of multidrug-resistant isolates of this bacterium. Here, we report the draft genome sequences of the 2004-05MF26 and 2009-144K3 isolates, which harbor plasmids conferring antibiotic resistance. Both isolates also carry the large plasmid pAsa5, which is known to encode a type three secretion system (TTSS) and the pAsal1 plasmid which has the aopP gene producing a TTSS effector. These two isolates are good representatives of the plasmid diversity in A. salmonicida subsp. salmonicida. PMID:25724776

  2. PREDICTIVE MODEL OF CONJUGATIVE PLASMID TRANSFER IN THE RHIZOSPHERE AND PHYLLOSPHERE

    EPA Science Inventory

    A computer simulation model was used to predict the dynamics of survival and conjugation of Pseudomonas cepacia (carrying the transmissible recombinant plasmid R388:Tn1721) with a nonrecombinant recipient strain in simple rhizosphere and phyllosphere microcosms. lasmid transfer r...

  3. Plasmids of Staphylococcus cohnii isolated from the intensive-care unit.

    PubMed

    Szewczyk, E M; Rózalska, M; Cieślikowski, T; Nowak, T

    2004-01-01

    Numerous isolates of both subspecies of Staphylococcus cohnii were found in the environment of the intensive-care unit of a pediatric hospital. These isolates carried in their cells many plasmids, up to fourteen, of a wide range of sizes (< 2 to > 56 kb). Striking was the occurrence of large plasmids not very common in staphylococci. These were present in > 80% of S. cohnii isolates. Fifty-two different plasmid profiles were found in 79 investigated isolates belonging to S. cohnii ssp. cohnii and S. cohnii ssp. urealyticus. Isolates similar in plasmid profiles were grouped in antibiotic-resistance clusters established for 9 antibiotics (gentamicin, ciprofloxacin, clindamycin, erythromycin, tetracycline, chloramphenicol, mupirocin, trimethoprim-sulfamethoxazole, vancomycin) using the method of unweighted pair group mathematical averages (UPGMA). Many isolates were multiresistant to antibiotics and produced bacteriocins. PMID:15227782

  4. A bacteriocin gene cluster able to enhance plasmid maintenance in Lactococcus lactis

    PubMed Central

    2014-01-01

    Background Lactococcus lactis is widely used as a dairy starter and has been extensively studied. Based on the acquired knowledge on its physiology and metabolism, new applications have been envisaged and there is an increasing interest of using L. lactis as a cell factory. Plasmids constitute the main toolbox for L. lactis genetic engineering and most rely on antibiotic resistant markers for plasmid selection and maintenance. In this work, we have assessed the ability of the bacteriocin Lactococcin 972 (Lcn972) gene cluster to behave as a food-grade post-segregational killing system to stabilize recombinant plasmids in L. lactis in the absence of antibiotics. Lcn972 is a non-lantibiotic bacteriocin encoded by the 11-kbp plasmid pBL1 with a potent antimicrobial activity against Lactococcus. Results Attempts to clone the full lcn972 operon with its own promoter (P972), the structural gene lcn972 and the immunity genes orf2-orf3 in the unstable plasmid pIL252 failed and only plasmids with a mutated promoter were recovered. Alternatively, cloning under other constitutive promoters was approached and achieved, but bacteriocin production levels were lower than those provided by pBL1. Segregational stability studies revealed that the recombinant plasmids that yielded high bacteriocin titers were maintained for at least 200 generations without antibiotic selection. In the case of expression vectors such as pTRL1, the Lcn972 gene cluster also contributed to plasmid maintenance without compromising the production of the fluorescent mCherry protein. Furthermore, unstable Lcn972 recombinant plasmids became integrated into the chromosome through the activity of insertion sequences, supporting the notion that Lcn972 does apply a strong selective pressure against susceptible cells. Despite of it, the Lcn972 gene cluster was not enough to avoid the use of antibiotics to select plasmid-bearing cells right after transformation. Conclusions Inserting the Lcn972 cluster into

  5. Two Large, Related, Cryptic Plasmids from Geographically Distinct Isolates of Sulfobacillus thermotolerans▿†

    PubMed Central

    Deane, S. M.; Rawlings, D. E.

    2011-01-01

    Two large cryptic plasmids (59.2 and 65.9 kb) from isolates of Sulfobacillus thermotolerans from Yellowstone National Park (United States) and the Caribbean island of Montserrat were isolated and sequenced. This analysis revealed a common “backbone” region coding for a potential plasmid stability system plus a nonpheromone conjugation system containing homologues of both type IV and type II (tight adherence, or Tad-like) secretion systems. PMID:21926204

  6. A cell engineering strategy to enhance supercoiled plasmid DNA production for gene therapy.

    PubMed

    Hassan, Sally; Keshavarz-Moore, Eli; Ward, John

    2016-09-01

    With the recent revival of the promise of plasmid DNA vectors in gene therapy, a novel synthetic biology approach was used to enhance the quantity, (yield), and quality of the plasmid DNA. Quality was measured by percentage supercoiling and supercoiling density, as well as improving segregational stability in fermentation. We examined the hypothesis that adding a Strong Gyrase binding Site (SGS) would increase DNA gyrase-mediated plasmid supercoiling. SGS from three different replicons, (the Mu bacteriophage and two plasmids, pSC101 and pBR322) were inserted into the plasmid, pUC57. Different sizes of these variants were transformed into E. coli DH5α, and their supercoiling properties and segregational stability measured. A 36% increase in supercoiling density was found in pUC57-SGS, but only when SGS was derived from the Mu phage and was the larger sized version of this fragment. These results were also confirmed at fermentation scale. Total percentage supercoiled monomer was maintained to 85-90%. A twofold increase in plasmid yield was also observed for pUC57-SGS in comparison to pUC57. pUC57-SGS displayed greater segregational stability than pUC57-cer and pUC57, demonstrating a further potential advantage of the SGS site. These findings should augment the potential of plasmid DNA vectors in plasmid DNA manufacture. Biotechnol. Bioeng. 2016;113: 2064-2071. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:26928284

  7. Molecular Analysis of Antibiotic Resistance Determinants and Plasmids in Malaysian Isolates of Multidrug Resistant Klebsiella pneumoniae

    PubMed Central

    Al-Marzooq, Farah; Mohd Yusof, Mohd Yasim; Tay, Sun Tee

    2015-01-01

    Infections caused by multidrug resistant Klebsiella pneumoniae have been increasingly reported in many parts of the world. A total of 93 Malaysian multidrug resistant K. pneumoniae isolated from patients attending to University of Malaya Medical Center, Kuala Lumpur, Malaysia from 2010-2012 were investigated for antibiotic resistance determinants including extended-spectrum beta-lactamases (ESBLs), aminoglycoside and trimethoprim/sulfamethoxazole resistance genes and plasmid replicons. CTX-M-15 (91.3%) was the predominant ESBL gene detected in this study. aacC2 gene (67.7%) was the most common gene detected in aminoglycoside-resistant isolates. Trimethoprim/sulfamethoxazole resistance (90.3%) was attributed to the presence of sul1 (53.8%) and dfrA (59.1%) genes in the isolates. Multiple plasmid replicons (1-4) were detected in 95.7% of the isolates. FIIK was the dominant replicon detected together with 13 other types of plasmid replicons. Conjugative plasmids (1-3 plasmids of ~3-100 kb) were obtained from 27 of 43 K. pneumoniae isolates. An ESBL gene (either CTX-M-15, CTX-M-3 or SHV-12) was detected from each transconjugant. Co-detection with at least one of other antibiotic resistance determinants [sul1, dfrA, aacC2, aac(6ˊ)-Ib, aac(6ˊ)-Ib-cr and qnrB] was noted in most conjugative plasmids. The transconjugants were resistant to multiple antibiotics including β-lactams, gentamicin and cotrimoxazole, but not ciprofloxacin. This is the first study describing the characterization of plasmids circulating in Malaysian multidrug resistant K. pneumoniae isolates. The results of this study suggest the diffusion of highly diverse plasmids with multiple antibiotic resistance determinants among the Malaysian isolates. Effective infection control measures and antibiotic stewardship programs should be adopted to limit the spread of the multidrug resistant bacteria in healthcare settings. PMID:26203651

  8. Analysis of Plasmid Deletion Induced by Ionizing Radiation in Yeast Saccharomyces cerevisiae

    SciTech Connect

    Yatsevich, E.; Stepanova, A.; Koltovaya, N.; Sprincova, A.

    2007-11-26

    The article is dedicated to the research of plasmid system YCpL2 with help of quantitative analysis of deletion formation. The cells of yeast Saccharomyces cerevisiae were irradiated by {gamma}-ray with the flux of 0.7 Gy/min and energy of 1.3 MeV as well as heavy ion beam {sup 11}B with energy 32 MeV/n. The deletion of plasmid DNA has been analyzed by genetic and restriction analysis.

  9. Polyethylene glycol-facilitated transformation of Bacteroides fragilis with plasmid DNA

    SciTech Connect

    Smith, C.J.

    1985-10-01

    A method for the transformation of Bacteroides fragilis with plasmid DNA was developed by using the clindamycin resistance plasmid pBFTM10 as the source of transforming DNA. The method was technically simple to perform and resulted in an average of 4.2 x 10/sup 3/ tranformants per ..mu..g of pBFTM10 added. A method for the preparation of frozen competent cells is also described.

  10. Characterization of Plasmids in a Human Clinical Strain of Lactococcus garvieae

    PubMed Central

    Blanco, M. Mar; López-Campos, Guillermo H.; Cutuli, M. Teresa; Fernández-Garayzábal, José F.

    2012-01-01

    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. PMID:22768237

  11. Analysis of Plasmid Deletion Induced by Ionizing Radiation in Yeast Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Yatsevich, E.; Stepanova, A.; Sprincova, A.; Koltovaya, N.

    2007-11-01

    The article is dedicated to the research of plasmid system YCpL2 with help of quantitative analysis of deletion formation. The cells of yeast Saccharomyces cerevisiae were irradiated by γ-ray with the flux of 0.7 Gy/min and energy of 1.3 MeV as well as heavy ion beam 11B with energy 32 MeV/n. The deletion of plasmid DNA has been analyzed by genetic and restriction analysis.

  12. Development and Host Compatibility of Plasmids for Two Important Ruminant Pathogens, Mycoplasma bovis and Mycoplasma agalactiae

    PubMed Central

    Sharma, Shukriti; Citti, Chistine; Sagné, Eveline; Marenda, Marc S.

    2015-01-01

    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. PMID:25746296

  13. A cell engineering strategy to enhance supercoiled plasmid DNA production for gene therapy

    PubMed Central

    Hassan, Sally; Ward, John

    2016-01-01

    ABSTRACT With the recent revival of the promise of plasmid DNA vectors in gene therapy, a novel synthetic biology approach was used to enhance the quantity, (yield), and quality of the plasmid DNA. Quality was measured by percentage supercoiling and supercoiling density, as well as improving segregational stability in fermentation. We examined the hypothesis that adding a Strong Gyrase binding Site (SGS) would increase DNA gyrase‐mediated plasmid supercoiling. SGS from three different replicons, (the Mu bacteriophage and two plasmids, pSC101 and pBR322) were inserted into the plasmid, pUC57. Different sizes of these variants were transformed into E. coli DH5α, and their supercoiling properties and segregational stability measured. A 36% increase in supercoiling density was found in pUC57‐SGS, but only when SGS was derived from the Mu phage and was the larger sized version of this fragment. These results were also confirmed at fermentation scale. Total percentage supercoiled monomer was maintained to 85–90%. A twofold increase in plasmid yield was also observed for pUC57‐SGS in comparison to pUC57. pUC57‐SGS displayed greater segregational stability than pUC57‐cer and pUC57, demonstrating a further potential advantage of the SGS site. These findings should augment the potential of plasmid DNA vectors in plasmid DNA manufacture. Biotechnol. Bioeng. 2016;113: 2064–2071. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:26928284

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

    PubMed

    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. PMID:18533903

  15. Antibiotic resistance due to an unusual ColE1-type replicon plasmid in Aeromonas salmonicida.

    PubMed

    Vincent, Antony T; Emond-Rheault, Jean-Guillaume; Barbeau, Xavier; Attéré, Sabrina A; Frenette, Michel; Lagüe, Patrick; Charette, Steve J

    2016-06-01

    Aeromonas salmonicida subsp. salmonicida is a fish pathogen known to have a rich plasmidome. In the present study, we discovered an isolate of this bacterium bearing an additional unidentified small plasmid. After having sequenced the DNA of that isolate by next-generation sequencing, it appeared that the new small plasmid is a ColE1-type replicon plasmid, named here pAsa7. This plasmid bears a functional chloramphenicol-acetyltransferase-encoding gene (cat-pAsa7) previously unknown in A. salmonicida and responsible for resistance to chloramphenicol. A comparison of pAsa7 with pAsa2, the only known ColE1-type replicon plasmid usually found in A. salmonicida subsp. salmonicida, revealed that even if both plasmids share a high structural similarity, it is still unclear if pAsa7 is a derivative of pAsa2 since they showed several mutations at the nucleotide level. Transcriptomic analysis revealed that the cat-pAsa4 gene, another chloramphenicol-acetyltransferase-encoding gene, found on the large plasmid pAsa4, was significantly more transcribed than cat-pAsa7. This was correlated with a higher chloramphenicol resistance for isolates bearing pAsa4 compared with the one having pAsa7. Finally, a phylogenetic analysis showed that both CAT-pAsa4 and CAT-pAsa7 proteins were in different clusters. The clustering was supported by the identity of residues involved in the catalytic site. In addition, to give a better understanding of the large drug-resistance panel of A. salmonicida, this study reinforces the hypothesis that A. salmonicida subsp. salmonicida is a considerable reservoir for mobile genetic elements such as plasmids. PMID:27028891

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

    PubMed Central

    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. Images PMID:1653214

  17. Chromosome- and Plasmid-Encoded β-Lactamases in Capnocytophaga spp.

    PubMed Central

    Handal, Trude; Giraud-Morin, Chantal; Caugant, Dominique A.; Madinier, Isabelle; Olsen, Ingar; Fosse, Thierry

    2005-01-01

    Chromosome- and plasmid-encoded CfxA2 and CfxA3 β-lactamases were detected in Capnocytophaga spp. from oral sources in France, Norway, and the United States. Unidentified chromosome-encoded β-lactamases were present in Capnocytophaga sputigena. Nucleotide sequence analysis of the CfxA3-encoding plasmid from C. ochracea revealed an unreported insertion sequence (ISCoc1) upstream of the cfxA gene. PMID:16127077

  18. Identification of dfrA14 in two distinct plasmids conferring trimethoprim resistance in Actinobacillus pleuropneumoniae

    PubMed Central

    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.

    2015-01-01

    Objectives The objective of this study was to determine the distribution and genetic basis of trimethoprim resistance in Actinobacillus pleuropneumoniae isolates from pigs in England. Methods 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. Results 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. Conclusions 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. PMID:25957382

  19. Sequence characterization and comparative analysis of three plasmids isolated from environmental Vibrio spp.

    PubMed

    Hazen, Tracy H; Wu, Dongying; Eisen, Jonathan A; Sobecky, Patricia A

    2007-12-01

    The horizontal transfer of genes by mobile genetic elements such as plasmids and phages can accelerate genome diversification of Vibrio spp., affecting their physiology, pathogenicity, and ecological character. In this study, sequence analysis of three plasmids from Vibrio spp. previously isolated from salt marsh sediment revealed the remarkable diversity of these elements. Plasmids p0908 (81.4 kb), p23023 (52.5 kb), and p09022 (31.0 kb) had a predicted 99, 64, and 32 protein-coding sequences and G+C contents of 49.2%, 44.7%, and 42.4%, respectively. A phylogenetic tree based on concatenation of the host 16S rRNA and rpoA nucleotide sequences indicated p23023 and p09022 were isolated from strains most closely related to V. mediterranei and V. campbellii, respectively, while the host of p0908 forms a clade with V. fluvialis and V. furnissii. Many predicted proteins had amino acid identities to proteins of previously characterized phages and plasmids (24 to 94%). Predicted proteins with similarity to chromosomally encoded proteins included RecA, a nucleoid-associated protein (NdpA), a type IV helicase (UvrD), and multiple hypothetical proteins. Plasmid p0908 had striking similarity to enterobacteria phage P1, sharing genetic organization and amino acid identity for 23 predicted proteins. This study provides evidence of genetic exchange between Vibrio plasmids, phages, and chromosomes among diverse Vibrio spp. PMID:17921277

  20. High-throughput isolation of ultra-pure plasmid DNA by a robotic system

    PubMed Central

    Kachel, Volker; Sindelar, Georg; Grimm, Stefan

    2006-01-01

    Background With the availability of complete genomes, a systematic inventory of cellular processes becomes achievable. This requires assessing the function of all individual genes. Transfection of plasmid DNA into cell culture cells is an essential technique for this aim as it allows functional overexpression or downregulation of genes. While many robotic systems isolate plasmids for sequencing purposes, for more demanding applications such as transfections there is a shortage of robots for the high-throughput isolation of plasmid DNA. Results Here we describe a custom-made, automated device, which uses a special protocol to isolate plasmid DNAs with a purity sufficient for efficient transfections into mammalian cells. Approximately 1,600 ultra pure plasmids can be isolated in a 96-well plate format within 12 hours. As a unique feature the robot comprises the integration of a centrifuge instead of expensive columns, the use of a custom-made pipetting head with a movable gripper, especially designed shaking platforms and an acetone wash facility. Conclusion Using this robot we demonstrate how centrifugation steps with multiple precipitations, most notably through a precipitation step of SDS in isopropanol, lead to high purity plasmid DNA and make possible high-throughput transfections into mammalian cells for functional gene annotations. PMID:16483377

  1. A role for the tet(O) plasmid in maintaining Campylobacter plasticity.

    PubMed

    Friis, L M; Pin, C; Taylor, D E; Pearson, B M; Wells, J M

    2007-01-01

    Genomic sequencing projects are beginning to reveal regions of extensive DNA homology between bacterial genera. Public fears of the spread of genetically modified organisms into the food chain and the increasing prevalence of multi-drug resistant disease in humans highlight the implications of horizontal gene transfer. The striking DNA sequence similarity between the two uniquely identified tetracycline resistant (Tc(R)) Campylobacter plasmids, pCC31 and pTet, suggests their conserved acquisition and maintenance within Campylobacter [Batchelor, R.A., Pearson, B.M., Friis, L.M., Guerry, P., Wells, J.M. 2004. Nucleotide sequences and comparison of two large conjugative plasmids from different Campylobacter species. Microbiology 150, 3507-3517]. It is thus likely that these and other conjugative plasmids are highly prevalent and broadly distributed across several continents. Microarray technology is now enabling fast and extensive genomic comparisons to be made and allows us to investigate intra- and inter-genetic conservation and variability. This study details the development of a microarray specific for genes from Campylobacter plasmids pCC31, pTet and pVir and its application to the analysis of Campylobacter plasmid gene presence and preservation throughout environmental and clinical isolates. Application of the iterative algorithm GENCOM (freely available at ) is used as a rapid and effective way of comparing the content and conservation of plasmids in bacteria and provides details of the Campylobacter flexible gene pool and its contribution to genomic plasticity. PMID:16934869

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

    PubMed

    Jussila, Minna M; Zhao, Ji; Suominen, Leena; Lindström, Kristina

    2007-03-01

    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. PMID:17000041

  3. Virulence of an emerging pathogenic lineage of Vibrio nigripulchritudo is dependent on two plasmids.

    PubMed

    Le Roux, Frédérique; Labreuche, Yannick; Davis, Brigid M; Iqbal, Naeem; Mangenot, Sophie; Goarant, Cyrille; Mazel, Didier; Waldor, Matthew K

    2011-02-01

    Vibrioses are the predominant bacterial infections in marine shrimp farms. Vibrio nigripulchritudo is an emerging pathogen of the cultured shrimp Litopenaeus stylirostris in New Caledonia and other regions in the Indo-Pacific. The molecular determinants of V. nigripulchritudo pathogenicity are unknown; however, molecular epidemiological studies have revealed that recent pathogenic V. nigripulchritudo isolates from New Caledonia all cluster into a monophyletic clade and contain a small plasmid, pB1067. Here, we report that a large plasmid, pA1066 (247 kb), can also serve as a marker for virulent V. nigripulchritudo, and that an ancestral version of this plasmid was likely acquired prior to other virulence-linked markers. Additionally, we demonstrate that pA1066 is critical for the full virulence of V. nigripulchritudo in several newly developed experimental models of infection. Plasmid pB1067 also contributes to virulence; only strains containing both plasmids induced the highest level of shrimp mortality. Thus, it appears that these plasmids, which are absent from non-pathogenic isolates, may be driving forces, as well as markers, for the emergence of a pathogenic lineage of V. nigripulchritudo. PMID:20825454

  4. Virulence of an emerging pathogenic lineage of Vibrio nigripulchritudo is dependent on two plasmids

    PubMed Central

    Le Roux, Frédérique; Labreuche, Yannick; Davis, Brigid M; Iqbal, Naeem; Mangenot, Sophie; Goarant, Cyrille; Mazel, Didier; Waldor, Matthew K

    2011-01-01

    Summary Vibrioses are the predominant bacterial infections in marine shrimp farms. Vibrio nigripulchritudo is an emerging pathogen of the cultured shrimp Litopenaeus stylirostris in New Caledonia and other regions in the Indo-Pacific. The molecular determinants of V. nigripulchritudo pathogenicity are unknown; however, molecular epidemiological studies have revealed that recent pathogenic V. nigripulchritudo isolates from New Caledonia all cluster into a monophyletic clade and contain a small plasmid, pB1067. Here, we report that a large plasmid, pA1066 (247 kb), can also serve as a marker for virulent V. nigripulchritudo, and that an ancestral version of this plasmid was likely acquired prior to other virulence-linked markers. Additionally, we demonstrate that pA1066 is critical for the full virulence of V. nigripulchritudo in several newly developed experimental models of infection. Plasmid pB1067 also contributes to virulence; only strains containing both plasmids induced the highest level of shrimp mortality. Thus, it appears that these plasmids, which are absent from non-pathogenic isolates, may be driving forces, as well as markers, for the emergence of a pathogenic lineage of V. nigripulchritudo. PMID:20825454

  5. Rapid identification of intact bacterial resistance plasmids via optical mapping of single DNA molecules

    PubMed Central

    Nyberg, Lena K.; Quaderi, Saair; Emilsson, Gustav; Karami, Nahid; Lagerstedt, Erik; Müller, Vilhelm; Noble, Charleston; Hammarberg, Susanna; Nilsson, Adam N.; Sjöberg, Fei; Fritzsche, Joachim; Kristiansson, Erik; Sandegren, Linus; Ambjörnsson, Tobias; Westerlund, Fredrik

    2016-01-01

    The rapid spread of antibiotic resistance – currently one of the greatest threats to human health according to WHO – is to a large extent enabled by plasmid-mediated horizontal transfer of resistance genes. Rapid identification and characterization of plasmids is thus important both for individual clinical outcomes and for epidemiological monitoring of antibiotic resistance. Toward this aim, we have developed an optical DNA mapping procedure where individual intact plasmids are elongated within nanofluidic channels and visualized through fluorescence microscopy, yielding barcodes that reflect the underlying sequence. The assay rapidly identifies plasmids through statistical comparisons with barcodes based on publicly available sequence repositories and also enables detection of structural variations. Since the assay yields holistic sequence information for individual intact plasmids, it is an ideal complement to next generation sequencing efforts which involve reassembly of sequence reads from fragmented DNA molecules. The assay should be applicable in microbiology labs around the world in applications ranging from fundamental plasmid biology to clinical epidemiology and diagnostics. PMID:27460437

  6. Chromatin structure of simian virus 40-pBR322 recombinant plasmids in COS-1 cells

    SciTech Connect

    Innis, J.W.; Scott, W.A.

    1983-12-01

    To study the nucleoprotein structure formed by recombinant plasmid DNA in mammalian cells, nuclei were isolated from COS-1 cells after transfection with a recombinant (pJI1) containing pBR322 sequences and a segment of simian virus 40 containing information for a nuclease-sensitive chromatin structure. The nuclei were incubated with DNase I. DNA fragments which were the size of linear pJI1 DNA were isolated, redigested with restriction enzymes, fractionated by electrophoresis, and detected by hybridization with nick-translated segments prepared from the plasmid DNA. Two DNase I-sensitive sites were detected in the simian virus 40 portion of the plasmid at the same sites that were DNase I sensitive in simian virus 40 chromatin prepared late after infection of African green monkey kidney (BSC-1) cells. One site extended from the viral origin of replication to approximately nucleotide 40. The 21-base pair repeated sequences were relatively DNase I resistant. A second site occurred over the single copy of hte 72-base pair segment present in this plasmid. These results indicate that the nuclease-sensitive chromatin structure does not depend on the presence of viral structural proteins. In addition, late viral proteins added to PJI1-transfected COS-1 cells by superinfection with simian virus 40 caused no change in the distribution of DNase I-sensitive sites in plasmid chromatin. Analysis of transfected plasmid DNA may provide a general method applicable to the study of the chromatin structure of cloned segments of DNA.

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

    PubMed Central

    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.

    2016-01-01

    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. PMID:27321040

  8. [Curing effects of chlorination, ozone and UV treatments on plasmid DNAs].

    PubMed

    Nakamura, S

    1990-09-01

    Curing effects of chlorine, ozone and ultraviolet (UV) on plasmid DNAs were investigated as one of the measures of bio-hazards due to recombinant plasmids. For donor strains of plasmids, Pseudomonas aeruginosa RM 2046 harboring the self-transmissible plasmid R68 and RM 2021 harboring the cloning vector R1162, Escherichia coli C 600 ML 4903 and ML 1410 harboring the self-transmissible plasmid Rts-1 and RP4, and E. coli JM 109 harboring the cloning vector pBR 322 were used. The curing rate showed a tendency to increase with treatment time in a L-shaped curve for each plasmid DNA. The rates reached steady state after more than 5-10 minutes of the chlorination and ozone treatment, or 5-10 seconds of the UV treatment, independent of the treatment intensity. It became clear by use of replica technique that effective curing took place by chlorination for Rts-1, R1162 and pBR322, by ozone treatment for R68, and by UV treatment for PR4, respectively. Of the three disinfection treatments excellent curing effect occurred with chlorination, as more than 90% of the curing rate was obtained by that treatment for all donor strains. PMID:2132391

  9. Recombination between bacteriophage lambda and plasmid pBR322 in Escherichia coli.

    PubMed Central

    Pogue-Geile, K L; Dassarma, S; King, S R; Jaskunas, S R

    1980-01-01

    Recombinant lambda phages were isolated that resulted from recombination between the lambda genome and plasmid pBR322 in Escherichia coli, even though these deoxyribonucleic acids (DNAs) did not share extensive regions of homology. The characterization of these recombinant DNAs by heteroduplex analysis and restriction endonucleases is described. All but one of the recombinants appeared to have resulted from reciprocal recombination between a site on lambda DNA and a site on the plasmid. In general, there were two classes of recombinants. One class appeared to have resulted from recombination at the phage attachment site that probably resulted from lambda integration into secondary attachment sites on the plasmid. Seven different secondary attachment sites on pBR322 were found. The other class resulted from plasmid integration at other sites that were widely scattered on the lambda genome. For this second class of recombinants, more than one site on the plasmid could recombine with lambda DNA. Thus, the recombination did not appear to be site specific with respect to lambda or the plasmid. Possible mechanisms for generating these recombinants are discussed. Images PMID:6247334

  10. Rapid identification of intact bacterial resistance plasmids via optical mapping of single DNA molecules.

    PubMed

    Nyberg, Lena K; Quaderi, Saair; Emilsson, Gustav; Karami, Nahid; Lagerstedt, Erik; Müller, Vilhelm; Noble, Charleston; Hammarberg, Susanna; Nilsson, Adam N; Sjöberg, Fei; Fritzsche, Joachim; Kristiansson, Erik; Sandegren, Linus; Ambjörnsson, Tobias; Westerlund, Fredrik

    2016-01-01

    The rapid spread of antibiotic resistance - currently one of the greatest threats to human health according to WHO - is to a large extent enabled by plasmid-mediated horizontal transfer of resistance genes. Rapid identification and characterization of plasmids is thus important both for individual clinical outcomes and for epidemiological monitoring of antibiotic resistance. Toward this aim, we have developed an optical DNA mapping procedure where individual intact plasmids are elongated within nanofluidic channels and visualized through fluorescence microscopy, yielding barcodes that reflect the underlying sequence. The assay rapidly identifies plasmids through statistical comparisons with barcodes based on publicly available sequence repositories and also enables detection of structural variations. Since the assay yields holistic sequence information for individual intact plasmids, it is an ideal complement to next generation sequencing efforts which involve reassembly of sequence reads from fragmented DNA molecules. The assay should be applicable in microbiology labs around the world in applications ranging from fundamental plasmid biology to clinical epidemiology and diagnostics. PMID:27460437

  11. Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

    PubMed Central

    Chen, Yuan-Jyue; Rao, Sundipta D.; Seelig, Georg

    2015-01-01

    DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA. PMID:26649734

  12. Plasmid Specificity of The Origin of Transfer of Sex Factor F

    PubMed Central

    Reeves, Peter; Willetts, Neil

    1974-01-01

    The ability of F-like plasmids to promote transfer from the F origin of transfer was determined. Chromosome transfer was measured from plasmid derivatives of RecA− Hfr deletion strains which had lost all the F transfer genes but which in some cases retained, and in others had also lost, the origin sequence. ColV2 and ColVBtrp could initiate transfer from the F origin, but R100-1, R1-19, and R538-1 drd could not. These results can be correlated with the plasmid specificity of the traI components of the different plasmid transfer systems, supporting the hypothesis that the origin of transfer is the site of action of the traI product. Most F-like plasmids, including R1-19 and R538-1 drd, could transfer ColE1, consistent with previous findings that the (plasmid-specific) traI product is not necessary for ColE1 transfer by Flac; ColE1 transfer may be initiated by a ColE1-or host-determined product. R100-1 and R136fin− could not transfer ColE1 efficiently, apparently because of differences residing in their pilus-forming genes. PMID:4608577

  13. R plasmids in environmental Vibrio cholerae non-O1 strains.

    PubMed Central

    Amaro, C; Aznar, R; Garay, E; Alcaide, E

    1988-01-01

    The occurrence of drug resistance and its plasmid-mediated transferability was investigated in 140 environmental strains of Vibrio cholerae non-O1 and 6 strains of Vibrio cholerae, both O1 and non-O1, of clinical origin. Of the 146 strains tested, 93% were resistant to at least one drug and 74% were resistant to two or more antibiotics. The O1 strains were susceptible to all antibiotics used. A total of 26 of 28 selected resistant wild strains carried R plasmids that were transferable by intraspecific and intergeneric matings. The most common transmissible R factor determined resistance to ampicillin, amoxicillin, and sulfanilamide (30%), followed by resistance to ampicillin and amoxicillin (13%) and resistance to ampicillin, amoxicillin, phosphomycin, and sulfanilamide (9%). Comparison of the three methods of plasmid analysis showed that the method of Birnboim and Doly (Nucleic Acids Res. 7:1513-1523, 1979) without EDTA and lysozyme was optimal for isolation of both large and small plasmids in environmental V. cholerae strains. Most strains harbored more than one plasmid, and the molecular sizes ranged from 1.1 to 74.8 megadaltons. The plasmids of high molecular size (around 74 megadaltons) were responsible for the resistance pattern transferred and were maintained with high stability in the hosts. Images PMID:3214157

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

    NASA Astrophysics Data System (ADS)

    Chiang, Chen-Li; Sung, Ching-Shan

    2006-07-01

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

  15. Toxin–antitoxin systems are ubiquitous and plasmid-encoded in vancomycin-resistant enterococci

    PubMed Central

    Moritz, Elizabeth M.; Hergenrother, Paul J.

    2007-01-01

    Vancomycin-resistant enterococci (VRE) are common hospital pathogens that are resistant to most major classes of antibiotics. The incidence of VRE is increasing rapidly, to the point where over one-quarter of enterococcal infections in intensive care units are now resistant to vancomycin. The exact mechanism by which VRE maintains its plasmid-encoded resistance genes is ill-defined, and novel targets for the treatment of VRE are lacking. In an effort to identify novel protein targets for the treatment of VRE infections, we probed the plasmids obtained from 75 VRE isolates for the presence of toxin–antitoxin (TA) gene systems. Remarkably, genes for one particular TA pair, the mazEF system (originally identified on the Escherichia coli chromosome), were present on plasmids from 75/75 (100%) of the isolates. Furthermore, mazEF was on the same plasmid as vanA in the vast majority of cases (>90%). Plasmid stability tests and RT-PCR raise the possibility that this plasmid-encoded mazEF is indeed functional in enterococci. Given this ubiquity of mazEF in VRE and the deleterious activity of the MazF toxin, disruption of mazEF with pharmacological agents is an attractive strategy for tailored antimicrobial therapy. PMID:17190821

  16. A modified alkaline lysis method for the preparation of highly purified plasmid DNA from Escherichia coli.

    PubMed

    Feliciello, I; Chinali, G

    1993-08-01

    We have developed a very efficient and rapid method for the preparation on a small or large scale of highly purified plasmid DNA from Escherichia coli. The procedure consists of five steps: (1) cell lysis by NaOH-SDS, (2) precipitation of cell lysate with 2 M potassium acetate-1 M acetic acid, (3) precipitation of the resulting supernatant with isopropanol, (4) treatment of the precipitate with RNase, and (5) a second isopropanol precipitation. The new procedure yields a plasmid DNA that is more than 90% in the supercoiled form and virtually free from proteins, RNA, and chromosomal DNA. We have thoroughly tested the method in the preparation of several thousand samples of different plasmids from various E. coli strains. We found that it consistently produced samples of plasmid DNA suitable for all routine uses such as restriction analysis, sequencing, and preparation of DNA probes for cloning and hybridization experiments. Moreover, plasmids purified by this procedure could fully replace plasmids purified on CsCl gradients for more demanding tasks such as the in vitro synthesis of RNA probes by phage RNA polymerases, the generation of deletion mutants with exonuclease III, and the transfection of mammalian cells by the calcium phosphate coprecipitation method, as tested on human fibroblasts and on CV-1 cells. PMID:8214582

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

    PubMed Central

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

    1984-01-01

    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. Images PMID:6327644

  18. Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe.

    PubMed

    Kakui, Yasutaka; Sunaga, Tomonari; Arai, Kunio; Dodgson, James; Ji, Liang; Csikász-Nagy, Attila; Carazo-Salas, Rafael; Sato, Masamitsu

    2015-06-01

    Integration of an external gene into a fission yeast chromosome is useful to investigate the effect of the gene product. An easy way to knock-in a gene construct is use of an integration plasmid, which can be targeted and inserted to a chromosome through homologous recombination. Despite the advantage of integration, construction of integration plasmids is energy- and time-consuming, because there is no systematic library of integration plasmids with various promoters, fluorescent protein tags, terminators and selection markers; therefore, researchers are often forced to make appropriate ones through multiple rounds of cloning procedures. Here, we establish materials and methods to easily construct integration plasmids. We introduce a convenient cloning system based on Golden Gate DNA shuffling, which enables the connection of multiple DNA fragments at once: any kind of promoters and terminators, the gene of interest, in combination with any fluorescent protein tag genes and any selection markers. Each of those DNA fragments, called a 'module', can be tandemly ligated in the order we desire in a single reaction, which yields a circular plasmid in a one-step manner. The resulting plasmids can be integrated through standard methods for transformation. Thus, these materials and methods help easy construction of knock-in strains, and this will further increase the value of fission yeast as a model organism. PMID:26108218

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

    PubMed

    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

    2016-01-01

    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. PMID:27321040

  20. Rapid screening of endonuclease target site preference using a modified bacterial two-plasmid selection.

    PubMed

    Wolfs, Jason M; Kleinstiver, Benjamin P; Edgell, David R

    2014-01-01

    Homing endonucleases and other site-specific endonucleases have potential applications in genome editing, yet efficient targeting requires a thorough understanding of DNA-sequence specificity. Here, we describe a modified two-plasmid genetic selection in Escherichia coli that allows rapid profiling of nucleotide substitutions within a target site of given endonucleases. The selection utilizes a toxic plasmid (pTox) that encodes a DNA gyrase toxin in addition to the endonuclease target site. Cleavage of the toxic plasmid by an endonuclease expressed from a second plasmid (pEndo) facilitates growth under selective conditions. The modified protocol utilizes competent cells harboring the endonuclease expression plasmid into which target site plasmids are transformed. Replica plating on nonselective and selective media plates identifies cleavable and non-cleavable targets. Thus, a library of randomized target sites, or many individual target sites, can be analyzed using a single transformation. Both cleavable and non-cleavable targets can be analyzed by DNA sequencing to gain information about nucleotide preference in the endonuclease's target site. PMID:24510263

  1. [Possible control of capsule formation and intracellular synthesis of envelope antigen by each different plasmid].

    PubMed

    Tsukano, H

    1989-03-01

    Variants which lacked capsular envelopes on their cell surface were isolated from the culture of a highly virulent Yreka strain of Yersinia pestis grown in the presence of acridine orange, ethidium bromide or sodium dodecyl sulfate at incompletely growth-inhibitory concentrations. The variant could be divided into two types on the basis of the presence and the absence of intracellular envelope antigen. Both types of the variants lacked the 13 megadaltone (Md) plasmid. Thus, it may well be said that the 13 Md plasmid would play some decisive role in extracellular envelope formation and no concern with the synthesis of intracellular antigen. It was clarified that these characters were carried by each different gene. The intracellular envelope antigen synthesis could not be correlated with other plasmids isolated from Yreka strain, i.e., 7, 23, 44 and 59 Md plasmids. On the other hand, further treatment of the intracellular-positive variant with the above inhibitors resulted in the occurrence of the antigen-deficient type variant at a rate of 1-2%. The high frequency appearance of the variant by the plasmid-depleting agents might indicate possible presence of some yet unknown plasmid responsive to the intracellular synthesis of envelope antigen. PMID:2504836

  2. Genetic and physical evidence for plasmid control of Shigella sonnei form I cell surface antigen.

    PubMed Central

    Kopecko, D J; Washington, O; Formal, S B

    1980-01-01

    Virulent Shigella sonnei synthesize a surface antigen (form I) which appears to be one of several requirements needed for this host to invade epithelial cells. Upon restreaking on agar media, form I cells readily and irreversibly generate form II cells that lack the form I antigen. All form II cells are avirulent. Plasmid deoxyribonucleic acid of form I and II cells of four different S. sonnei isolates, obtained from different areas of the world, was analyzed by agarose gel electrophoresis. A large plasmid (approximately 120 megadaltons in three of the strains) that is present in form I cells was always absent from form II derivatives. Attempts to transfer conjugally only this large plasmid from form I to genetically marked form II cells were unsuccessful. However, a composite molecule, apparently formed by recombination between the large form I plasmid and a self-transmissible plasmid, was found to transfer the form I trait. Transconjugant S. sonnei strains acquiring the form I antigen could retransfer this trait to S. sonnei, Shigella flexneri, or Salmonella typhi. These preliminary findings demonstrate that S. sonnei form I antigen synthesis is mediated by a large plasmid which is lost spontaneously at a relatively high frequency from S. sonnei strains. Images Fig. 1 Fig. 2 PMID:6249756

  3. Propagation of pSC101 plasmids defective in binding of integration host factor.

    PubMed Central

    Biek, D P; Cohen, S N

    1992-01-01

    Integration host factor (IHF), a multifunctional protein of E. coli, normally is required for the replication of plasmid pSC101. T. T. Stenzel, P. Patel, and D. Bastia (Cell 49:709-717, 1987) have reported that IHF binds to a DNA locus near the pSC101 replication origin and enhances a static bend present in this region; mutation of the IHF binding site affects the plasmid's ability to replicate. We report here studies indicating that the requirement for IHF binding near the pSC101 replication origin is circumvented partially or completely by (i) mutation of the plasmid-encoded repA (replicase) gene or the chromosomally encoded topA gene, (ii) the presence on the plasmid of the pSC101 partition (par) locus, or (iii) replacement of the par locus by a strong transcriptional promoter. With the exception of the repA mutation, the factors that substitute for a functional origin region IHF binding site are known to alter plasmid topology by increasing negative DNA supercoiling, as does IHF itself. These results are consistent with the proposal that IHF binding near the pSC101 replication origin promotes plasmid replication by inducing a conformational change leading to formation of a repA-dependent DNA-protein complex. A variety of IHF-independent mechanisms can facilitate formation of the putative replication-initiation complex. PMID:1310092

  4. Noncanonical SMC protein in Mycobacterium smegmatis restricts maintenance of Mycobacterium fortuitum plasmids

    PubMed Central

    Panas, Michael W.; Jain, Paras; Yang, Hui; Mitra, Shimontini; Biswas, Debasis; Wattam, Alice Rebecca; Letvin, Norman L.; Jacobs, William R.

    2014-01-01

    Research on tuberculosis and leprosy was revolutionized by the development of a plasmid transformation system in the fast-growing surrogate, Mycobacterium smegmatis. This transformation system was made possible by the successful isolation of a M. smegmatis mutant strain mc2155, whose efficient plasmid transformation (ept) phenotype supported the replication of Mycobacterium fortuitum pAL5000 plasmids. In this report, we identified the EptC gene, the loss of which confers the ept phenotype. EptC shares significant amino acid sequence homology and domain structure with the MukB protein of Escherichia coli, a structural maintenance of chromosomes (SMC) protein. Surprisingly, M. smegmatis has three paralogs of SMC proteins: EptC and MSMEG_0370 both share homology with Gram-negative bacterial MukB; and MSMEG_2423 shares homology with Gram-positive bacterial SMCs, including the single SMC protein predicted for Mycobacterium tuberculosis and Mycobacterium leprae. Purified EptC was shown to bind ssDNA and stabilize negative supercoils in plasmid DNA. Moreover, an EptC–mCherry fusion protein was constructed and shown to bind to DNA in live mycobacteria, and to prevent segregation of plasmid DNA to daughter cells. To our knowledge, this is the first report of impaired plasmid maintenance caused by a SMC homolog, which has been canonically known to assist the segregation of genetic materials. PMID:25197070

  5. Transformation of Saccharomyces cerevisiae and Schizosaccharomyces pombe with linear plasmids containing 2 micron sequences.

    PubMed Central

    Guerrini, A M; Ascenzioni, F; Tribioli, C; Donini, P

    1985-01-01

    Linear plasmids were constructed by adding telomeres prepared from Tetrahymena pyriformis rDNA to a circular hybrid Escherichia coli-yeast vector and transforming Saccharomyces cerevisiae. The parental vector contained the entire 2 mu yeast circle and the LEU gene from S. cerevisiae. Three transformed clones were shown to contain linear plasmids which were characterized by restriction analysis and shown to be rearranged versions of the desired linear plasmids. The plasmids obtained were imperfect palindromes: part of the parental vector was present in duplicated form, part as unique sequences and part was absent. The sequences that had been lost included a large portion of the 2 mu circle. The telomeres were approximately 450 bp longer than those of T. pyriformis. DNA prepared from transformed S. cerevisiae clones was used to transform Schizosaccharomyces pombe. The transformed S. pombe clones contained linear plasmids identical in structure to their linear parents in S. cerevisiae. No structural re-arrangements or integration into S. pombe was observed. Little or no telomere growth had occurred after transfer from S. cerevisiae to S. pombe. A model is proposed to explain the genesis of the plasmids. Images Fig. 1. Fig. 2. Fig. 4. PMID:3896773

  6. Gene A protein cleavage of recombinant plasmids containing the phi X174 replication origin.

    PubMed Central

    Fluit, A C; Baas, P D; Van Boom, J H; Veeneman, G H; Jansz, H S

    1984-01-01

    Synthetic oligonucleotides, DNA ligase and DNA polymerase were used to construct double-stranded DNA fragments homologous to the first 25, 27 or 30 b.p. of the origin of replication of bacteriophage phi X174 (nucleotides 4299-4328 of the phi X174 DNA sequence). The double-stranded DNA fragments were cloned into the unique SmaI or HindIII restriction sites in the kanamycin-resistance gene of pACYC177 (AmpR, KmR). Recombinant plasmids were picked up by colony hybridization. DNA sequencing showed that not only recombinant plasmids with the expected insert were formed, but also recombinant plasmids with a shorter insert. Recombinant plasmids with an insert homologous to the first 24, 25, 26, 27, 28 or all 30 b.p. of the phi X174 origin region were thus obtained. Supercoiled plasmids containing a sequence homologous to the first 27, 28 or 30 b.p. of the phi X174 origin region are nicked by the phi X174 gene A protein. However, the other supercoiled plasmids are not nicked by the phi X174 gene A protein. These results show that the first 27 b.p. of the phi X174 origin region are sufficient as well as required for the initiation step in phi X174 RF DNA replication, i.e. the cleavage by gene A protein. Images PMID:6236428

  7. In vitro construction of deletion mutants of the bacteriocinogenic plasmid Clo DF13.

    PubMed Central

    Stuitje, A R; Veltkamp, E; van den Elzen, P J; Nijkamp, H J

    1978-01-01

    The isolation and characterization of deletion mutants of the bacteriocinogenic plasmid Clo DF13 is described. To construct these deletion mutants, DNA of Clo DF13::Tn901 and Clo DF13-rep3::Tn901 plasmids was digested with restriction endonucleases, ligated with T4 ligase and introduced by transformation into Escherichia coli. The presence of the ampicilline transposon Tn901 facilitated the selection of plasmids. The resulting Clo DF13::Tn901 deletion mutants were analyzed by digestion with restriction endonucleases and electron microscopy. From the properties of the various deletion mutants it was concluded that a Clo DF13 DNA region, extending from 5 to 11.5% on the physical map, is essential for the replication of Clo DF13. This region, comprising about 600 base pairs, contains in addition to an origin of replication, DNA sequences which are involved in the regulation of Clo DF13 DNA replication. Furthermore it was observed that in case of the Clo DF13 copy mutant, Clo DF13-rep3, deletion of the 43% to 63% part of the plasmid genome, resulted in the generation of multimeric plasmid structures, accompanied with an impaired segregation of the plasmids to daughter cells. Images PMID:353730

  8. A mitochondrial mutator plasmid that causes senescence under dietary restricted conditions

    PubMed Central

    Maas, Marc FPM; Hoekstra, Rolf F; Debets, Alfons JM

    2007-01-01

    Background Calorie or dietary restriction extends life span in a wide range of organisms including the filamentous fungus Podospora anserina. Under dietary restricted conditions, P. anserina isolates are several-fold longer lived. This is however not the case in isolates that carry one of the pAL2-1 homologous mitochondrial plasmids. Results We show that the pAL2-1 homologues act as 'insertional mutators' of the mitochondrial genome, which may explain their negative effect on life span extension. Sequencing revealed at least fourteen unique plasmid integration sites, of which twelve were located within the mitochondrial genome and two within copies of the plasmid itself. The plasmids were able to integrate in their entirety, via a non-homologous mode of recombination. Some of the integrated plasmid copies were truncated, which probably resulted from secondary, post-integrative, recombination processes. Integration sites were predominantly located within and surrounding the region containing the mitochondrial rDNA loci. Conclusion We propose a model for the mechanism of integration, based on innate modes of mtDNA recombination, and discuss its possible link with the plasmid's negative effect on dietary restriction mediated life span extension. PMID:17407571

  9. Improved heterologous erythromycin A production through expression plasmid re-design.

    PubMed

    Jiang, Ming; Fang, Lei; Pfeifer, Blaine A

    2013-01-01

    The production of complex compounds from technically convenient microorganisms is an emerging route to the chemical diversity found in the surrounding environment. In this study, the antibiotic compound erythromycin A is produced from Escherichia coli as an alternative to native production through the soil bacterium Saccharopolyspora erythraea. By doing so, there is an opportunity to apply and refine engineering strategies for the manipulation of the erythromycin biosynthetic pathway and for the overproduction of this and other complex natural compounds. Previously, E. coli-derived production was enabled by the introduction of the entire erythromycin pathway (20 genes total) using separately selectable expression plasmids which demonstrated negative effects on final biosynthesis through metabolic burden and plasmid instability. In this study, improvements to final production were made by altering the design of the expression plasmids needed for biosynthetic pathway introduction. Specifically, the total number of genes and plasmids was pruned to reduce both metabolic burden and plasmid instability. Further, a comparison was conducted between species-specific (E. coli vs. S. coelicolor) protein chaperonins. Results indicate improvements in growth and plasmid retention metrics. The newly designed expression platform also increased erythromycin A production levels 5-fold. In conclusion, the steps outlined in this report were designed to upgrade the E. coli erythromycin A production system, led to improved final compound titers, and suggest additional forms of pathway engineering to further improve results from heterologous production attempts. PMID:23804312

  10. Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe

    PubMed Central

    Kakui, Yasutaka; Sunaga, Tomonari; Arai, Kunio; Dodgson, James; Ji, Liang; Csikász-Nagy, Attila; Carazo-Salas, Rafael; Sato, Masamitsu

    2015-01-01

    Integration of an external gene into a fission yeast chromosome is useful to investigate the effect of the gene product. An easy way to knock-in a gene construct is use of an integration plasmid, which can be targeted and inserted to a chromosome through homologous recombination. Despite the advantage of integration, construction of integration plasmids is energy- and time-consuming, because there is no systematic library of integration plasmids with various promoters, fluorescent protein tags, terminators and selection markers; therefore, researchers are often forced to make appropriate ones through multiple rounds of cloning procedures. Here, we establish materials and methods to easily construct integration plasmids. We introduce a convenient cloning system based on Golden Gate DNA shuffling, which enables the connection of multiple DNA fragments at once: any kind of promoters and terminators, the gene of interest, in combination with any fluorescent protein tag genes and any selection markers. Each of those DNA fragments, called a ‘module’, can be tandemly ligated in the order we desire in a single reaction, which yields a circular plasmid in a one-step manner. The resulting plasmids can be integrated through standard methods for transformation. Thus, these materials and methods help easy construction of knock-in strains, and this will further increase the value of fission yeast as a model organism. PMID:26108218

  11. pIGWZ12--A cryptic plasmid with a modular structure.

    PubMed

    Zaleski, Piotr; Wawrzyniak, Paweł; Sobolewska, Agnieszka; Łukasiewicz, Natalia; Baran, Piotr; Romańczuk, Katarzyna; Daniszewska, Katarzyna; Kierył, Piotr; Płucienniczak, Grażyna; Płucienniczak, Andrzej

    2015-05-01

    We studied the detailed structure of the cryptic plasmid pIGWZ12, which was isolated from an Escherichia coli strain. pIGWZ12 is composed of two structural modules of distinct evolutionary origin. The REP module, which contains all the features necessary for replication and stable maintenance in the bacterial cell, was assigned by genotyping to the IncF family. The MOB module, which is responsible for plasmid mobilization, shows significant homology to MOBQ modules from broad-host-range plasmids belonging to the RSF1010/R1162 family. We showed that iterons located in the origin of replication are the target for specific binding by the replication initiator protein RepApIGWZ12. Furthermore, we proved that the promoter for the repA gene overlaps with the iterons, and that the latter are the sole determinant of incompatibility. We performed a mutagenesis analysis of the MOBpIGWZ12 module and characterized the roles played by all identified genes (mobA and mobC), as well as the role played by oriT in mobilization. Finally, we showed that it was possible to remove the MOB module from pIGWZ12 without any loss in plasmid replication and stability. Furthermore, the MOBpIGWZ12 module was fully functional after subcloning into another plasmid. Therefore, pIGWZ12 is yet another example of modular structure in small cryptic plasmids. PMID:25889268

  12. Environmentally co‐occurring mercury resistance plasmids are genetically and phenotypically diverse and confer variable context‐dependent fitness effects

    PubMed Central

    Harrison, Ellie; Lilley, Andrew K.; Paterson, Steve; Spiers, Andrew J.; Brockhurst, Michael A.

    2015-01-01

    Summary Plasmids are important mobile elements that can facilitate genetic exchange and local adaptation within microbial communities. We compared the sequences of four co‐occurring pQBR family environmental mercury resistance plasmids and measured their effects on competitive fitness of a P seudomonas fluorescens  SBW25 host, which was isolated at the same field site. Fitness effects of carriage differed between plasmids and were strongly context dependent, varying with medium, plasmid status of competitor and levels of environmental mercury. The plasmids also varied widely in their rates of conjugation and segregational loss. We found that few of the plasmid‐borne accessory genes could be ascribed functions, although we identified a putative chemotaxis operon, a type IV pilus‐encoding cluster and a region encoding putative arylsulfatase enzymes, which were conserved across geographically distant isolates. One plasmid, pQBR55, conferred the ability to catabolize sucrose. Transposons, including the mercury resistance Tn5042, appeared to have been acquired by different pQBR plasmids by recombination, indicating an important role for horizontal gene transfer in the recent evolution of pQBR plasmids. Our findings demonstrate extensive genetic and phenotypic diversity among co‐occurring members of a plasmid community and suggest a role for environmental heterogeneity in the maintenance of plasmid diversity. PMID:25969927

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

    PubMed Central

    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

  14. Monitoring plasmid replication in live mammalian cells over multiple generations by fluorescence microscopy.

    PubMed

    Norby, Kathryn; Chiu, Ya-Fang; Sugden, Bill

    2012-01-01

    Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies (1-3). These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres (4-8). Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell. Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest (9). The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed. This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to

  15. 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

    PubMed Central

    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.

    2015-01-01

    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 < 0.001) and increased movement of the 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. PMID:25769824

  16. Mobilization of Chimeric oriT Plasmids by F and R100-1: Role of Relaxosome Formation in Defining Plasmid Specificity

    PubMed Central

    Fekete, Richard A.; Frost, Laura S.

    2000-01-01

    Cleavage at the F plasmid nic site within the origin of transfer (oriT) requires the F-encoded proteins TraY and TraI and the host-encoded protein integration host factor in vitro. We confirm that F TraY, but not F TraM, is required for cleavage at nic in vivo. Chimeric plasmids were constructed which contained either the entire F or R100-1 oriT regions or various combinations of nic, TraY, and TraM binding sites, in addition to the traM gene. The efficiency of cleavage at nic and the frequency of mobilization were assayed in the presence of F or R100-1 plasmids. The ability of these chimeric plasmids to complement an F traM mutant or affect F transfer via negative dominance was also measured using transfer efficiency assays. In cases where cleavage at nic was detected, R100-1 TraI was not sensitive to the two-base difference in sequence immediately downstream of nic, while F TraI was specific for the F sequence. Plasmid transfer was detected only when TraM was able to bind to its cognate sites within oriT. High-affinity binding of TraY in cis to oriT allowed detection of cleavage at nic but was not required for efficient mobilization. Taken together, our results suggest that stable relaxosomes, consisting of TraI, -M, and -Y bound to oriT are preferentially targeted to the transfer apparatus (transferosome). PMID:10869081

  17. Plasmids of psychrophilic and psychrotolerant bacteria and their role in adaptation to cold environments.

    PubMed

    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

  18. Molecular characterization and structural instability of the industrially important composite metabolic plasmid pLP712.

    PubMed

    Wegmann, Udo; Overweg, Karin; Jeanson, Sophie; Gasson, Mike; Shearman, Claire

    2012-12-01

    The widely used plasmid-free Lactococcus lactis strain MG1363 was derived from the industrial dairy starter strain NCDO712. This strain carries a 55.39 kb plasmid encoding genes for lactose catabolism and a serine proteinase involved in casein degradation. We report the DNA sequencing and annotation of pLP712, which revealed additional metabolic genes, including peptidase F, d-lactate dehydrogenase and α-keto acid dehydrogenase (E3 complex). Comparison of pLP712 with other large lactococcal lactose and/or proteinase plasmids from L. lactis subsp. cremoris SK11 (pSK11L, pSK11P) and the plant strain L. lactis NCDO1867 (pGdh442) revealed their close relationship. The plasmid appears to have evolved through a series of genetic events as a composite of pGdh442, pSK11L and pSK11P. We describe in detail a scenario by which the metabolic genes relevant to the growth of its host in a milk environment have been unified on one replicon, reflecting the evolution of L. lactis as it changed its biological niche from plants to dairy environments. The extensive structural instability of pLP712 allows easy isolation of derivative plasmids lacking genes for casein degradation and/or lactose catabolism. Plasmid pLP712 is transferable by transduction and conjugation, and both of these processes result in significant molecular rearrangements. We report the detailed molecular analysis of insertion sequence element-mediated genetic rearrangements within pLP712 and several different mechanisms, including homologous recombination and adjacent deletion. Analysis of the integration of the lactose operon into the chromosome highlights the fluidity of the MG1363 integration hotspot and the potential for frequent movement of genes between plasmids and chromosomes in Lactococcus. PMID:23023974

  19. The writhe distribution in DNA plasmids as derived from the free energy of supercoiling

    NASA Astrophysics Data System (ADS)

    Tobias, Irwin

    2000-10-01

    In theoretical work on the molecule, DNA is often treated, approximately, as a naturally straight, inextensible, isotropic elastic rod of circular cross section. It is shown that, consistent with this level of approximation, there exists a general connection between the free energy of supercoiling of plasmids formed by the DNA, and the writhe distribution in plasmids having a given value of the linking number difference, ΔLk. In particular, the writhe distribution in a collection of torsionally relaxed (ΔLk=0), but non-nicked, plasmids is completely determined once the free energy of supercoiling as a function of ΔLk is known. The writhe distribution in the supercoiled plasmids characterized by any other value of ΔLk, we shall also show, is simply related to the distribution in the relaxed plasmid, and, therefore, it, too, is completely determined. These general results are illustrated for two cases: Large plasmids for which the measured free energy of supercoiling, a quadratic function of ΔLk, implies a normal writhe distribution, and miniplasmids for which a theoretical expression for the free energy of supercoiling involving the frequencies of the normal modes of vibration of a circular elastic ring has recently become available. In this latter case, the writhe distribution for supercoiled plasmids is not normal, but shows a skewness related to a property of elastic rings, namely, the loss of stability of the circular equilibrium configuration of the rings when they are twisted beyond a critical value. Such a skewed writhe distribution for miniplasmids is, according to the model, associated with a free energy of supercoiling which is not, as has been assumed, a rigorously quadratic function of ΔLk.

  20. Transferable Multiresistance Plasmids Carrying cfr in Enterococcus spp. from Swine and Farm Environment

    PubMed Central

    Liu, Yang; Wang, Yang; Schwarz, Stefan; Li, Yun; Shen, Zhangqi; Zhang, Qijing; Wu, Congming

    2013-01-01

    Seventy-seven porcine Enterococcus isolates with florfenicol MICs of ≥16 μg of were/ml screened for the presence of the multiresistance gene cfr, its location on plasmids, and its genetic environment. Three isolates—Enterococcus thailandicus 3-38 (from a porcine rectal swab collected at a pig farm), Enterococcus thailandicus W3, and Enterococcus faecalis W9-2 (the latter two from sewage at a different farm), carried the cfr gene. The SmaI pulsed-field gel electrophoresis patterns of the three isolates differed distinctly. In addition, E. faecalis W9-2 was assigned to a new multilocus sequence type ST469. Mating experiments and Southern blot analysis indicated that cfr is located on conjugative plasmids pW3 (∼75 kb) from E. thailandicus W3, p3-38 (∼72 kb) from E. thailandicus 3-38, and pW9-2 (∼55 kb) from E. faecalis W9-2; these plasmids differed in their sizes, additional resistance genes, and the analysis of the segments encompassing the cfr gene. Sequence analysis revealed that all plasmids harbored a 4,447-bp central region, in which cfr was bracketed by two copies of the novel insertion sequence ISEnfa4 located in the same orientation. The sequences flanking the central regions of these plasmids, including the partial tra gene regions and a ω-ε-ζ toxin-antitoxin module, exhibited >95% nucleotide sequence identity to the conjugative plasmid pAMβ1 from E. faecalis. Conjugative plasmids carrying cfr appear to play an important role in the dissemination and maintenance of the multiresistance gene cfr among enterococcal isolates and possibly other species of Gram-positive bacteria. PMID:23070165

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

    PubMed

    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. PMID:27114279

  2. Unique plasmid-like mitochondrial DNAs from indigenous maize races of Latin America

    PubMed Central

    Weissinger, A. K.; Timothy, D. H.; Levings, C. S.; Hu, W. W. L.; Goodman, M. M.

    1982-01-01

    Mitochondrial DNA from 81 races of Latin American maize were examined by agarose gel electrophoresis. Twelve South American races each contained two plasmid-like mtDNA molecules similar to those of the cytoplasmic male-sterile S type (cms-S). The plasmid-like elements from all 12 races, designated RU, appear to be identical. Both molecules appear in vitro as double-stranded linear DNAs terminated by repeated sequences arranged in reverse polarity (terminal inverted repeats). The larger molecule of the pair, R-1, contains about 7460 nucleotides. It shares considerable homology with the larger plasmid-like molecule of cms-S, S-1, but is about 1000 nucleotides longer than S-1, has a unique sequence of about 2576 nucleotides, and also contains a BamHI recognition site not present in S-1, R-2, the smaller plasmid-like element, consists of about 5450 nucleotides and appears to share complete homology with S-2, the smaller plasmid-like molecule of cms-S. Neither pollen sterility nor any other trait has been associated with the R-1 and R-2 plasmid-like mtDNAs. The BamHI restriction fragments of total mtDNA from the 12 RU cytoplasms display similar patterns, which differ only slightly but vividly from that of a normal maize standard, B73 × Mo17. BamHI restriction analysis of 22 additional races produced arrays similar to those of the RU cytoplasms, but which lacked plasmid-like mtDNAs. The taxonomic significance of this digestion pattern and of the RU cytoplasms is discussed. One Mexican race, Conico Norteño, has been shown to contain the cms-S cytoplasm. Images PMID:16593137

  3. Conjugative DNA Transfer Is Enhanced by Plasmid R1 Partitioning Proteins.

    PubMed

    Gruber, Christian J; Lang, Silvia; Rajendra, Vinod K H; Nuk, Monika; Raffl, Sandra; Schildbach, Joel F; Zechner, Ellen L

    2016-01-01

    Bacterial conjugation is a form of type IV secretion used to transport protein and DNA directly to recipient bacteria. The process is cell contact-dependent, yet the mechanisms enabling extracellular events to trigger plasmid transfer to begin inside the cell remain obscure. In this study of plasmid R1 we investigated the role of plasmid proteins in the initiation of gene transfer. We find that TraI, the central regulator of conjugative DNA processing, interacts physically, and functionally with the plasmid partitioning proteins ParM and ParR. These interactions stimulate TraI catalyzed relaxation of plasmid DNA in vivo and in vitro and increase ParM ATPase activity. ParM also binds the coupling protein TraD and VirB4-like channel ATPase TraC. Together, these protein-protein interactions probably act to co-localize the transfer components intracellularly and promote assembly of the conjugation machinery. Importantly these data also indicate that the continued association of ParM and ParR at the conjugative pore is necessary for plasmid transfer to start efficiently. Moreover, the conjugative pilus and underlying secretion machinery assembled in the absence of Par proteins mediate poor biofilm formation and are completely dysfunctional for pilus specific R17 bacteriophage uptake. Thus, functional integration of Par components at the interface of relaxosome, coupling protein, and channel ATPases appears important for an optimal conformation and effective activation of the transfer machinery. We conclude that low copy plasmid R1 has evolved an active segregation system that optimizes both its vertical and lateral modes of dissemination. PMID:27486582

  4. Nonselective Persistence of a Rickettsia conorii Extrachromosomal Plasmid during Mammalian Infection.

    PubMed

    Riley, Sean P; Fish, Abigail I; Garza, Daniel A; Banajee, Kaikhushroo H; Harris, Emma K; del Piero, Fabio; Martinez, Juan J

    2016-03-01

    Scientific analysis of the genus Rickettsia is undergoing a rapid period of change with the emergence of viable genetic tools. The development of these tools for the mutagenesis of pathogenic bacteria will permit forward genetic analysis of Rickettsia pathogenesis. Despite these advances, uncertainty still remains regarding the use of plasmids to study these bacteria in in vivo mammalian models of infection, namely, the potential for virulence changes associated with the presence of extrachromosomal DNA and nonselective persistence of plasmids in mammalian models of infection. Here, we describe the transformation of Rickettsia conorii Malish 7 with the plasmid pRam18dRGA[AmTrCh]. Transformed R. conorii stably maintains this plasmid in infected cell cultures, expresses the encoded fluorescent proteins, and exhibits growth kinetics in cell culture similar to those of nontransformed R. conorii. Using a well-established murine model of fatal Mediterranean spotted fever, we demonstrate that R. conorii(pRam18dRGA[AmTrCh]) elicits the same fatal outcomes in animals as its untransformed counterpart and, importantly, maintains the plasmid throughout infection in the absence of selective antibiotic pressure. Interestingly, plasmid-transformed R. conorii was readily observed both in endothelial cells and within circulating leukocytes. Together, our data demonstrate that the presence of an extrachromosomal DNA element in a pathogenic rickettsial species does not affect either in vitro proliferation or in vivo infectivity in models of disease and that plasmids such as pRam18dRGA[AmTrCh] are valuable tools for the further genetic manipulation of pathogenic rickettsiae. PMID:26755154

  5. Self-transmissible plasmids in staphylococci that encode resistance to aminoglycosides.

    PubMed Central

    Archer, G L; Johnston, J L

    1983-01-01

    High-level resistance to gentamicin, tobramycin, and kanamycin was transferred between staphylococci of the same and different species by filter mating. Resistance and transfer proficiency were mediated by plasmids ranging from 38 to 54 kilobases in size. All of the plasmids encoded intermediate resistance to amikacin and netilmicin and resistance to ethidium bromide; some encoded beta-lactamase production. None of these plasmids carried resistance to other antibiotics or heavy metals. Transfer of antibiotic resistance occurred by a mechanism similar to that of conjugation, because it was DNase resistant, required cell-to-cell contact, and did not appear to involve phage. The participation of phage in transfer appeared to be unlikely because mijtomicin C-induced lysates of donor isolates did not mediate transfer, filter mating transfer proceeded at high frequency between nonlysogenic donor and recipient cells, and transfer of the aminoglycoside resistance plasmid mobilized the transfer of as many as five additional plasmids. All 17 gentamicin-resistant Staphylococcus aureus and all 6 Staphylococcus epidermidis isolates obtained from an outbreak of staphylococcal infections in a newborn nursery contained conjugative plasmids, as did all 6 gentamicin-resistant S. aureus isolates from bacteremic adults. However, only 3 of 10 gentamicin-resistant S. epidermidis isolates from colonized cardiac surgery patients and 1 of 2 S. epidermidis isolates from patients with prosthetic valve endocarditis transferred gentamicin resistance by filter mating. The recent increase in nosocomial infections caused by gentamicin-resistant staphylococci may be partially explained by the evolution of self-transmissible plasmids in these isolates. Images PMID:6625557

  6. The complete plasmid sequences of Salmonella enterica serovar Typhimurium U288.

    PubMed

    Hooton, Steven P T; Timms, Andrew R; Cummings, Nicola J; Moreton, Joanna; Wilson, Ray; Connerton, Ian F

    2014-08-28

    Salmonella enterica Serovar Typhimurium U288 is an emerging pathogen of pigs. The strain contains three plasmids of diverse origin that encode traits that are of concern for food security and safety, these include antibiotic resistant determinants, an array of functions that can modify cell physiology and permit genetic mobility. At 148,711 bp, pSTU288-1 appears to be a hybrid plasmid containing a conglomerate of genes found in pSLT of S. Typhimurium LT2, coupled with a mosaic of horizontally-acquired elements. Class I integron containing gene cassettes conferring resistance against clinically important antibiotics and compounds are present in pSTU288-1. A curious feature of the plasmid involves the deletion of two genes encoded in the Salmonella plasmid virulence operon (spvR and spvA) following the insertion of a tnpA IS26-like element coupled to a blaTEM gene. The spv operon is considered to be a major plasmid-encoded Salmonella virulence factor that is essential for the intracellular lifecycle. The loss of the positive regulator SpvR may impact on the pathogenesis of S. Typhimurium U288. A second 11,067 bp plasmid designated pSTU288-2 contains further antibiotic resistance determinants, as well as replication and mobilization genes. Finally, a small 4675 bp plasmid pSTU288-3 was identified containing mobilization genes and a pleD-like G-G-D/E-E-F conserved domain protein that modulate intracellular levels of cyclic di-GMP, and are associated with motile to sessile transitions in growth. PMID:25175817

  7. [Group of derepressed pKMR plasmids found in wild strains of Shigella].

    PubMed

    Sidorov, I A

    1981-09-01

    A group of derepressed (drd) R plasmids was identified in 3 clinical isolates of Shigella, i. e. Sh. flexneri 1b, Sh. flexneri 3c and Sh. sonnei resistant to ampicillin (Ap), streptomycin (Sm), tetracycline (Tc), chloramphenicol (Cm), kanamycin (Km) and sulfathiazole (Su). The plasmids were designated as pKMR 202-2 (Sm, Tc, Cm, Km, Su), pKMR 203-3 (Ap, Tc, Cm, Su), pKMR 204--2 (Sm, Km, Su), pKMR 204-3 (Ap, Sm, Cm, Km, Su), pKMR 204-4 (Ap, Sm, Km, Su), pKMR 204-5 (Km, Su), pKMR 204-6 (Ap, Sm, Tc, Cm, Km, Su) and pKMR 204-7 (Sm, Tc, Cm, Km, Su). All of the plasmids were transferred with the R- -cells of E. coli in 5 minutes at a frequency of 2 . 10(-6) to 4 . 10(-5) and had the Fi+ phenotype. None of them except pKMR 203-2 transferred sensitivity to F- donor-specific phages (f2 and Q beta) to the E. coli cells. The plasmids had neither capacity for maintaining multiplication of phages Ike and PR4 possessing the donor-specific properties with respect to the Inc N-, Inc P- and Inc W-plasmids. Therefore, the pKMR plasmids do not belong to these incompatibility groups. It should be noted that several plasmid variants (2--6) were isolated from every of the Shigella strains studied. Since they were stable in the cells and could be transferred separately on conjugation it was concluded that each combination was presented by the R factors belonging to different Inc-groups. PMID:7027918

  8. Transforming DNA uptake gene orthologs do not mediate spontaneous plasmid transformation in Escherichia coli.

    PubMed

    Sun, Dongchang; Zhang, Xuewu; Wang, Lingyu; Prudhomme, Marc; Xie, Zhixiong; Martin, Bernard; Claverys, Jean-Pierre

    2009-02-01

    Spontaneous plasmid transformation of Escherichia coli occurs on nutrient-containing agar plates. E. coli has also been reported to use double-stranded DNA (dsDNA) as a carbon source. The mechanism(s) of entry of exogenous dsDNA that allows plasmid establishment or the use of DNA as a nutrient remain(s) unknown. To further characterize plasmid transformation, we first documented the stimulation of transformation by agar and agarose. We provide evidence that stimulation is not due to agar contributing a supplement of Ca(2+), Fe(2+), Mg(2+), Mn(2+), or Zn(2+). Second, we undertook to inactivate the E. coli orthologues of Haemophilus influenzae components of the transformation machine that allows the uptake of single-stranded DNA (ssDNA) from exogenous dsDNA. The putative outer membrane channel protein (HofQ), transformation pseudopilus component (PpdD), and transmembrane pore (YcaI) are not required for plasmid transformation. We conclude that plasmid DNA does not enter E. coli cells as ssDNA. The finding that purified plasmid monomers transform E. coli with single-hit kinetics supports this conclusion; it establishes that a unique monomer molecule is sufficient to give rise to a transformant, which is not consistent with the reconstitution of an intact replicon through annealing of partially overlapping complementary ssDNA, taken up from two independent monomers. We therefore propose that plasmid transformation involves internalization of intact dsDNA molecules. Our data together, with previous reports that HofQ is required for the use of dsDNA as a carbon source, suggest the existence of two routes for DNA entry, at least across the outer membrane of E. coli. PMID:19011021

  9. Conjugative DNA Transfer Is Enhanced by Plasmid R1 Partitioning Proteins

    PubMed Central

    Gruber, Christian J.; Lang, Silvia; Rajendra, Vinod K. H.; Nuk, Monika; Raffl, Sandra; Schildbach, Joel F.; Zechner, Ellen L.

    2016-01-01

    Bacterial conjugation is a form of type IV secretion used to transport protein and DNA directly to recipient bacteria. The process is cell contact-dependent, yet the mechanisms enabling extracellular events to trigger plasmid transfer to begin inside the cell remain obscure. In this study of plasmid R1 we investigated the role of plasmid proteins in the initiation of gene transfer. We find that TraI, the central regulator of conjugative DNA processing, interacts physically, and functionally with the plasmid partitioning proteins ParM and ParR. These interactions stimulate TraI catalyzed relaxation of plasmid DNA in vivo and in vitro and increase ParM ATPase activity. ParM also binds the coupling protein TraD and VirB4-like channel ATPase TraC. Together, these protein-protein interactions probably act to co-localize the transfer components intracellularly and promote assembly of the conjugation machinery. Importantly these data also indicate that the continued association of ParM and ParR at the conjugative pore is necessary for plasmid transfer to start efficiently. Moreover, the conjugative pilus and underlying secretion machinery assembled in the absence of Par proteins mediate poor biofilm formation and are completely dysfunctional for pilus specific R17 bacteriophage uptake. Thus, functional integration of Par components at the interface of relaxosome, coupling protein, and channel ATPases appears important for an optimal conformation and effective activation of the transfer machinery. We conclude that low copy plasmid R1 has evolved an active segregation system that optimizes both its vertical and lateral modes of dissemination. PMID:27486582

  10. Construction of an Escherichia coli-rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp

    SciTech Connect

    Singer, M.E.V.; Finnerty, W.R.

    1988-02-01

    A plasmid transformation system for Rhodococcus sp. strain H13-A was developed by using an Escherichia coli-Rhodococcus shuttle plasmid constructed in this study. Rhodococcus sp. strain H13-A contains three cryptic indigenous plasmids, designated pMVS100, pMVS200, and pMVS300, of 75, 19.5, and 13.4 kilobases (kb), respectively. A 3.8-kb restriction fragment of pMVS300 was cloned into pIJ30, a 6.3-kb pBR322 derivative, containing the E. coli origin of replication (ori) and ampicillin resistance determinant (bla), as well as a Streptomyces gene for thiostrepton resistance, tsr. The resulting 10.1-kb recombinant plasmid, designated pMVS301, was isolated from E. coli DH1 (pMVS301) and transformed into Rhodococccus sp. strain AS-50, a derivative of strain H13-A. The cloned 3.8-kb fragment of Rhodococcus DNA in pMVS301 contains a Rhodococcus origin of replication, since the hybrid plasmid was capable of replication in both genera. The plasmid was identical in E. coli and Rhodococcus transformants as determined by restriction analysis and was maintained as a stable, independent replicon in both organisms. A restriction map demonstrated 14 unique restriction sites in pMVS301, some of which are potentially useful for molecular cloning in Rhodococcus spp. and other actinomycetes. This is the first report of plasmid transformation and of heterologous gene expression in a Rhodococcus sp.

  11. A Degenerate Primer MOB Typing (DPMT) Method to Classify Gamma-Proteobacterial Plasmids in Clinical and Environmental Settings

    PubMed Central

    de la Cruz, Fernando

    2012-01-01

    Transmissible plasmids are responsible for the spread of genetic determinants, such as antibiotic resistance or virulence traits, causing a large ecological and epidemiological impact. Transmissible plasmids, either conjugative or mobilizable, have in common the presence of a relaxase gene. Relaxases were previously classified in six protein families according to their phylogeny. Degenerate primers hybridizing to coding sequences of conserved amino acid motifs were designed to amplify related relaxase genes from γ-Proteobacterial plasmids. Specificity and sensitivity of a selected set of 19 primer pairs were first tested using a collection of 33 reference relaxases, representing the diversity of γ-Proteobacterial plasmids. The validated set was then applied to the analysis of two plasmid collections obtained from clinical isolates. The relaxase screening method, which we call “Degenerate Primer MOB Typing” or DPMT, detected not only most known Inc/Rep groups, but also a plethora of plasmids not previously assigned to any Inc group or Rep-type. PMID:22792321

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

    PubMed

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

    2015-09-18

    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

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

    PubMed Central

    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. Extended-spectrum plasmid-mediated beta-lactamases.

    PubMed

    Sirot, D

    1995-07-01

    Extended-spectrum beta-lactamases (ESBLs) are mutant enzymes which derive from TEM or SHV (class A) enzymes. They confer variable levels of resistance to cefotaxime, ceftazidime and other broad-spectrum cephalosporins and to monobactams such as aztreonam but have no detectable activity against cephamycins and carbapenems. Recently, new plasmid-mediated ESBLs, not derived from TEM or SHV enzymes but related to cephalosporinases of Enterobacteriaceae (class C enzymes), that confer resistance to all cephalosporins including cephamycins, have been reported. However, to date there have been no reported outbreaks due to strains producing transferable cephalosporinases. Klebsiella pneumoniae is the species in which the ESBL enzymes have been most commonly reported around the world. Most of the clinical isolates that produce TEM- or SHV-derived ESBL, come from hospitalised patients and have frequently caused nosocomial outbreaks. Care should be taken in the selection of a beta-lactam for the treatment of infections because the presence of an ESBL does not prevent other mechanisms of resistance, such as decreased permeability, from emerging. Broad-spectrum cephalosporins including cefepime and cefpirome are hydrolysed by ESBL. However, low level resistance to cefotaxime, ceftriaxone, cefepime and aztreonam does occur in some strains producing certain TEM-derived ESBL. It remains to be seen, therefore, whether such isolates are clinically susceptible to these drugs. The combination of a third-generation cephalosporin and a beta-lactamase inhibitor such as sulbactam could be of interest against some strains producing certain ESBLs. Among the 7-alpha-methoxy cephalosporins, cefotetan and latamoxef are the most active. However, cephamycins should be used with caution to treat infections caused by ESBL-producing K. pneumoniae because of the relative ease with which clinical strains decrease the expression of outer membrane proteins. The most active beta-lactams are the

  15. Diversity and molecular variation among plasmids in Salmonella enterica serotype Dublin based on restriction enzyme fragmentation pattern analysis.

    PubMed Central

    Browning, L. M.; Wray, C.; Platt, D. J.

    1995-01-01

    Molecular variation within and between plasmids of Salmonella enterica serotype Dublin was analysed. Such variation has been demonstrated in the serotype-specific plasmids (SSP's) of Typhimurium and Enteritidis. The two aims of this study were to determine the plasmid diversity in a host-adapted serotype and also the incidence of molecular variation in the SSP among strains of Dublin using restriction endonuclease fragmentation pattern (REFP) analysis with Pst1, Sma1 and EcoRV. Sixty-five strains were examined from seven countries. Plasmid profile and REFP analysis showed that none of the strains was plasmid-free. Seventy-seven percent of the strains possessed the 72 kb SSP either alone or in combination with another plasmid; 23% harboured plasmids which were molecular variants of the SSP. Four of the variants were more closely related to each other than to the reference SSP and were harboured by Dublin isolated from both the USA and Europe. A further three were shown to be cointegrate plasmids and were similarly distributed. Thirty-two percent of strains possessed the SSP alone. None of the UK strains was resistant to any of the antimicrobial agents tested whereas 74% of the remaining strains were resistant to between one and five antimicrobial agents. This study corroborates previous findings concerning the high degree of stability of the SSP and confirmed the clonal nature of Dublin. Co-resident plasmids provided evidence of sub-clones within localized geographical areas. Images Fig. 2 PMID:7705487

  16. Facile Recovery of Individual High-Molecular-Weight, Low-Copy-Number Natural Plasmids for Genomic Sequencing

    SciTech Connect

    Williams, L.E.; Detter, C,; Barrie, K.; Lapidus, A.; Summers, A.O.

    2006-06-01

    Sequencing of the large (>50 kb), low-copy-number (<5 per cell) plasmids that mediate horizontal gene transfer has been hindered by the difficulty and expense of isolating DNA from individual plasmids of this class. We report here that a kit method previously devised for purification of bacterial artificial chromosomes (BACs) can be adapted for effective preparation of individual plasmids up to 220 kb from wild gram-negative and gram-positive bacteria. Individual plasmid DNA recovered from less than 10 ml of Escherichia coli, Staphylococcus, and Corynebacterium cultures was of sufficient quantity and quality for construction of highcoverage libraries, as shown by sequencing five native plasmids ranging in size from 30 kb to 94 kb. We also report recommendations for vector screening to optimize plasmid sequence assembly, preliminary annotation of novel plasmid genomes, and insights on mobile genetic element biology derived from these sequences. Adaptation of this BAC method for large plasmid isolation removes one major technical hurdle to expanding our knowledge of the natural plasmid gene pool.

  17. A high-salinity solution with calcium chloride enables RNase-free, easy plasmid isolation within 55 minutes.

    PubMed

    Sasagawa, Noboru; Koebis, Michinori; Yonemura, Yoji; Mitsuhashi, Hiroaki; Ishiura, Shoichi

    2013-12-01

    We dramatically improved a plasmid-isolation protocol based on the popular alkaline-sodium dodecyl sulfate plasmid isolation method. Our modified method provides significant time and cost savings. We used a modified solution during the neutralization step, which allowed us to skip several subsequent handling steps, saving a great amount of time. The plasmids purified by this method were of high quality, and the optical density ratio 260 and 280 was approximately 1.8. Plasmid DNA isolated by our method was of sufficient quality to perform subsequent restriction enzyme cuts and other downstream experiments, including budding yeast transformation, cultured cell transfection, and Caenorhabditis elegans injection experiments. PMID:24390365

  18. Complete Nucleotide Sequence of cfr-Carrying IncX4 Plasmid pSD11 from Escherichia coli

    PubMed Central

    Sun, Jian; Deng, Hui; Li, Liang; Chen, Mu-Ya; Fang, Liang-Xing; Yang, Qiu-E

    2014-01-01

    We report the complete nucleotide sequence of a plasmid carrying the multiresistance gene cfr. This plasmid was isolated from an Escherichia coli strain of swine origin in 2011. This 37,672-bp plasmid, pSD11, had an IncX4 backbone similar to those of the IncX4 plasmids obtained from the United States and Australia, in which the cfr gene was flanked by two copies of IS26 and a truncated Tn1331 was inserted. PMID:25403661

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

    PubMed Central

    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

  20. Construction of the recombinant broad-host-range plasmids providing their bacterial hosts arsenic resistance and arsenite oxidation ability.

    PubMed

    Drewniak, Lukasz; Ciezkowska, Martyna; Radlinska, Monika; Sklodowska, Aleksandra

    2015-02-20

    The plasmid pSinA of Sinorhizobium sp. M14 was used as a source of functional phenotypic modules, encoding proteins involved in arsenite oxidation and arsenic resistance, to obtain recombinant broad-host-range plasmids providing their bacterial hosts arsenic resistance and arsenite oxidative ability. An arsenite oxidation module was cloned into pBBR1MCS-2 vector yielding plasmid vector pAIO1, while an arsenic resistance module was cloned into pCM62 vector yielding plasmid pARS1. Both plasmid constructs were introduced (separately and together) into the cells of phylogenetically distant (representing Alpha-, Beta-, and Gammaproteobacteria) and physiologically diversified (unable to oxidize arsenite and susceptible/resistant to arsenite and arsenate) bacteria. Functional analysis of the modified strains showed that: (i) the plasmid pARS1 can be used for the construction of strains with an increased resistance to arsenite [up to 20mM of As(III), (ii) the presence of the plasmid pAIO1 in bacteria previously unable to oxidize As(III) to As(V), contributes to the acquisition of arsenite oxidation abilities by these cells, (iii) the highest arsenite utilization rate are observed in the culture of strains harbouring both the