Science.gov

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

    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

  5. 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

  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 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

  9. 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

  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 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

  9. 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

  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. 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

  11. 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

  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. 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)

  9. 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

  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-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

  6. 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

  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

    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

  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 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

  14. 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

  15. 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

  16. 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.

  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. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  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. 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

  13. 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...

  14. 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....

  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. 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

  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. 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...

  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. 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...

  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. 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

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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

  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. 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

  18. 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

  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. 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

  3. 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.

  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. 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

  6. 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

  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. 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

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  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. 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

  9. 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

  10. 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

  11. 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.

  12. 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

  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 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

  17. 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

  18. 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

  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. 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

  1. 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)

  2. 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...

  3. 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.

  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. [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

  6. 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

  7. 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.

  8. 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...

  9. 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

  10. 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

  11. [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

  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. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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...

  1. 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

  2. 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...

  3. 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

  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. 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.

  9. 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

  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. 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

  18. 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

  19. 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

  20. 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.

  1. 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.

  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. 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

  7. 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.

  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. 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

  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. 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

  12. 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...

  13. 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...

  14. 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

  15. 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...

  16. 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...

  17. 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

  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. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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