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Sample records for plasmid partition complex

  1. Concerted action of plasmid maintenance functions: partition complexes create a requirement for dimer resolution.

    PubMed

    Bouet, Jean-Yves; Bouvier, Marie; Lane, David

    2006-12-01

    Partition of prokaryotic DNA requires formation of specific protein-centromere complexes, but an excess of the protein can disrupt segregation. The mechanisms underlying this destabilization are unknown. We have found that destabilization by the F plasmid partition protein, SopB, of plasmids carrying the F centromere, sopC, results from the capacity of the SopB-sopC partition complex to stimulate plasmid multimerization. Mutant SopBs unable to destabilize failed to increase multimerization. Stability of wild-type mini-F, whose ResD/rfsF site-specific recombination system enables it to resolve multimers to monomers, was barely affected by excess SopB. Destabilization of plasmids lacking the rfsF site was suppressed by recF, recO and recR, but not by recB, mutant alleles, indicating that multimerization is initiated from single-strand gaps. SopB did not alter the amounts or distribution of replication intermediates, implying that SopB-DNA complexes do not create single-strand gaps by blocking replication forks. Rather, the results are consistent with SopB-DNA complexes channelling gapped molecules into the RecFOR recombination pathway. We suggest that extended SopB-DNA complexes increase the likelihood of recombination between sibling plasmids by keeping them in close contact prior to SopA-mediated segregation. These results cast plasmid site-specific resolution in a new role - compensation for untoward consequences of partition complex formation. PMID:17059567

  2. ParB Partition Proteins: Complex Formation and Spreading at Bacterial and Plasmid Centromeres

    PubMed Central

    Funnell, Barbara E.

    2016-01-01

    In bacteria, active partition systems contribute to the faithful segregation of both chromosomes and low-copy-number plasmids. Each system depends on a site-specific DNA binding protein to recognize and assemble a partition complex at a centromere-like site, commonly called parS. Many plasmid, and all chromosomal centromere-binding proteins are dimeric helix-turn-helix DNA binding proteins, which are commonly named ParB. Although the overall sequence conservation among ParBs is not high, the proteins share similar domain and functional organization, and they assemble into similar higher-order complexes. In vivo, ParBs “spread,” that is, DNA binding extends away from the parS site into the surrounding non-specific DNA, a feature that reflects higher-order complex assembly. ParBs bridge and pair DNA at parS and non-specific DNA sites. ParB dimers interact with each other via flexible conformations of an N-terminal region. This review will focus on the properties of the HTH centromere-binding protein, in light of recent experimental evidence and models that are adding to our understanding of how these proteins assemble into large and dynamic partition complexes at and around their specific DNA sites. PMID:27622187

  3. ParB Partition Proteins: Complex Formation and Spreading at Bacterial and Plasmid Centromeres

    PubMed Central

    Funnell, Barbara E.

    2016-01-01

    In bacteria, active partition systems contribute to the faithful segregation of both chromosomes and low-copy-number plasmids. Each system depends on a site-specific DNA binding protein to recognize and assemble a partition complex at a centromere-like site, commonly called parS. Many plasmid, and all chromosomal centromere-binding proteins are dimeric helix-turn-helix DNA binding proteins, which are commonly named ParB. Although the overall sequence conservation among ParBs is not high, the proteins share similar domain and functional organization, and they assemble into similar higher-order complexes. In vivo, ParBs “spread,” that is, DNA binding extends away from the parS site into the surrounding non-specific DNA, a feature that reflects higher-order complex assembly. ParBs bridge and pair DNA at parS and non-specific DNA sites. ParB dimers interact with each other via flexible conformations of an N-terminal region. This review will focus on the properties of the HTH centromere-binding protein, in light of recent experimental evidence and models that are adding to our understanding of how these proteins assemble into large and dynamic partition complexes at and around their specific DNA sites.

  4. ParB Partition Proteins: Complex Formation and Spreading at Bacterial and Plasmid Centromeres.

    PubMed

    Funnell, Barbara E

    2016-01-01

    In bacteria, active partition systems contribute to the faithful segregation of both chromosomes and low-copy-number plasmids. Each system depends on a site-specific DNA binding protein to recognize and assemble a partition complex at a centromere-like site, commonly called parS. Many plasmid, and all chromosomal centromere-binding proteins are dimeric helix-turn-helix DNA binding proteins, which are commonly named ParB. Although the overall sequence conservation among ParBs is not high, the proteins share similar domain and functional organization, and they assemble into similar higher-order complexes. In vivo, ParBs "spread," that is, DNA binding extends away from the parS site into the surrounding non-specific DNA, a feature that reflects higher-order complex assembly. ParBs bridge and pair DNA at parS and non-specific DNA sites. ParB dimers interact with each other via flexible conformations of an N-terminal region. This review will focus on the properties of the HTH centromere-binding protein, in light of recent experimental evidence and models that are adding to our understanding of how these proteins assemble into large and dynamic partition complexes at and around their specific DNA sites. PMID:27622187

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

    PubMed

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

    2015-07-01

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

  6. A single 43-bp sopC repeat of plasmid mini-F is sufficient to allow assembly of a functional nucleoprotein partition complex.

    PubMed

    Biek, D P; Shi, J

    1994-08-16

    Stable maintenance of the low-copy-number mini-F plasmid in Escherichia coli is dependent on a functional partition system. The sop partition region encodes proteins SopA and SopB and a cis-acting element sopC, which contains multiple sites to which SopB binds. We have found that SopB protein acting at sopC in vivo is associated with a marked effect on plasmid DNA supercoiling, which may reflect the formation of a wrapped nucleoprotein complex. In this study, we demonstrate that a functional partition complex can form with a single 43-bp SopB binding site. Our experiments suggest that SopB bound at a single site nucleates the binding of additional SopB protein and wrapping of adjacent DNA sequences, such that approximately equal numbers of supercoils are restrained regardless of the number of tandem sopC repeats present. It is likely that this unusual nucleoprotein complex allows interaction of the plasmid with the partition apparatus.

  7. ParAB Partition Dynamics in Firmicutes: Nucleoid Bound ParA Captures and Tethers ParB-Plasmid Complexes

    PubMed Central

    Lioy, Virginia S.; Volante, Andrea; Soberón, Nora E.; Lurz, Rudi; Ayora, Silvia; Alonso, Juan C.

    2015-01-01

    In Firmicutes, small homodimeric ParA-like (δ2) and ParB-like (ω2) proteins, in concert with cis-acting plasmid-borne parS and the host chromosome, secure stable plasmid inheritance in a growing bacterial population. This study shows that (ω:YFP)2 binding to parS facilitates plasmid clustering in the cytosol. (δ:GFP)2 requires ATP binding but not hydrolysis to localize onto the cell’s nucleoid as a fluorescent cloud. The interaction of (δ:CFP)2 or δ2 bound to the nucleoid with (ω:YFP)2 foci facilitates plasmid capture, from a very broad distribution, towards the nucleoid and plasmid pairing. parS-bound ω2 promotes redistribution of (δ:GFP)2, leading to the dynamic release of (δ:GFP)2 from the nucleoid, in a process favored by ATP hydrolysis and protein-protein interaction. (δD60A:GFP)2, which binds but cannot hydrolyze ATP, also forms unstable complexes on the nucleoid. In the presence of ω2, (δD60A:GFP)2 accumulates foci or patched structures on the nucleoid. We propose that (δ:GFP)2 binding to different nucleoid regions and to ω2-parS might generate (δ:GFP)2 gradients that could direct plasmid movement. The iterative pairing and unpairing cycles may tether plasmids equidistantly on the nucleoid to ensure faithful plasmid segregation by a mechanism compatible with the diffusion-ratchet mechanism as proposed from in vitro reconstituted systems. PMID:26161642

  8. ParA-mediated plasmid partition driven by protein pattern self-organization

    PubMed Central

    Hwang, Ling Chin; Vecchiarelli, Anthony G; Han, Yong-Woon; Mizuuchi, Michiyo; Harada, Yoshie; Funnell, Barbara E; Mizuuchi, Kiyoshi

    2013-01-01

    DNA segregation ensures the stable inheritance of genetic material prior to cell division. Many bacterial chromosomes and low-copy plasmids, such as the plasmids P1 and F, employ a three-component system to partition replicated genomes: a partition site on the DNA target, typically called parS, a partition site binding protein, typically called ParB, and a Walker-type ATPase, typically called ParA, which also binds non-specific DNA. In vivo, the ParA family of ATPases forms dynamic patterns over the nucleoid, but how ATP-driven patterning is involved in partition is unknown. We reconstituted and visualized ParA-mediated plasmid partition inside a DNA-carpeted flowcell, which acts as an artificial nucleoid. ParA and ParB transiently bridged plasmid to the DNA carpet. ParB-stimulated ATP hydrolysis by ParA resulted in ParA disassembly from the bridging complex and from the surrounding DNA carpet, which led to plasmid detachment. Our results support a diffusion-ratchet model, where ParB on the plasmid chases and redistributes the ParA gradient on the nucleoid, which in turn mobilizes the plasmid. PMID:23443047

  9. Active stable maintenance functions in low copy-number plasmids of Gram-positive bacteria I. Partition systems.

    PubMed

    Dmowski, Michał; Jagura-Burdzy, Grazyna

    2013-01-01

    Low copy number plasmids cannot rely on the random segregation during bacterial cell division. To be stably maintained in the population they evolved two types of mechanisms (i) partition systems (PAR) that actively separate replicated plasmid molecules to the daughter cells and (ii) toxin-andidote systems (TA) that act after cell division to kill plasmid-less cells. Our knowledge of partition systems has been based mainly on analysis of plasmids from Gram-negative bacteria. Now, numerous partition systems of plasmids from Gram-positive bacteria have also been characterized and make significant contribution to our understanding of these mechanisms.

  10. Esc1, a Nuclear Periphery Protein Required for Sir4-Based Plasmid Anchoring and Partitioning

    PubMed Central

    Andrulis, Erik D.; Zappulla, David C.; Ansari, Athar; Perrod, Severine; Laiosa, Catherine V.; Gartenberg, Marc R.; Sternglanz, Rolf

    2002-01-01

    A targeted silencing screen was performed to identify yeast proteins that, when tethered to a telomere, suppress a telomeric silencing defect caused by truncation of Rap1. A previously uncharacterized protein, Esc1 (establishes silent chromatin), was recovered, in addition to well-characterized proteins Rap1, Sir1, and Rad7. Telomeric silencing was slightly decreased in Δesc1 mutants, but silencing of the HM loci was unaffected. On the other hand, targeted silencing by various tethered proteins was greatly weakened in Δesc1 mutants. Two-hybrid analysis revealed that Esc1 and Sir4 interact via a 34-amino-acid portion of Esc1 (residues 1440 to 1473) and a carboxyl-terminal domain of Sir4 known as PAD4 (residues 950 to 1262). When tethered to DNA, this Sir4 domain confers efficient partitioning to otherwise unstable plasmids and blocks the ability of bound DNA segments to rotate freely in vivo. Here, both phenomena were shown to require ESC1. Sir protein-mediated partitioning of a telomere-based plasmid also required ESC1. Fluorescence microscopy of cells expressing green fluorescent protein (GFP)-Esc1 showed that the protein localized to the nuclear periphery, a region of the nucleus known to be functionally important for silencing. GFP-Esc1 localization, however, was not entirely coincident with telomeres, the nucleolus, or nuclear pore complexes. Our data suggest that Esc1 is a component of a redundant pathway that functions to localize silencing complexes to the nuclear periphery. PMID:12417731

  11. Partition functions of mini-F affect plasmid DNA topology in Escherichia coli.

    PubMed

    Biek, D P; Strings, J

    1995-02-24

    Efficient segregation of the low copy number plasmid mini-F is dependent on partition functions encoded by the plasmid sopABC genes. The sop region encodes proteins SopA and SopB and a cis-acting element, sopC, which may function as a centromere analog. The SopC segment contains 12 imperfect 43 bp repeats to which the SopB protein binds. We have found that mutations in the sop genes affect superhelicity of isolated plasmid DNA. Plasmids with mutations in sopB or a deletion of the sopC segment were more highly negatively supercoiled than normal. In contrast, a mutation in the autoregulatory SopA protein resulted in plasmid DNA that was more relaxed. The SopAB proteins provided in trans to a pBR322 plasmid carrying sopC resulted in the relaxation of negative supercoils. We suggest that binding of SopB protein to the cis-acting sopC segment in vivo, alone or in conjunction with other proteins, produced a change in DNA topology in which positive superhelical turns were introduced locally. This higher-order nucleoprotein structure may allow interaction of plasmid mini-F with the partition apparatus.

  12. Partition locus-based classification of selected plasmids in Klebsiella pneumoniae, Escherichia coli and Salmonella enterica spp.: an additional tool.

    PubMed

    Bousquet, A; Henquet, S; Compain, F; Genel, N; Arlet, G; Decré, D

    2015-03-01

    The dissemination of antimicrobial resistance genes in Enterobacteriaceae has been largely attributed to plasmids, circular DNA molecules capable of autonomous replication. Whereas high-copy-number plasmids primarily rely on passive diffusion for plasmid maintenance, low-copy-number plasmids utilize so-called partition (par) systems. Plasmid partition relies on three structures, i.e. a centromere like DNA site, a centromere-binding protein and an ATPase or a GTPase motor protein for plasmid positioning. Identification and classification of plasmids is essential for tracing plasmids conferring drug resistance. PCR-based replicon typing is currently the standard method for plasmid identification but there are new classification schemes, especially the relaxase gene typing (PRaseT). Here we developed a multiplex PCR set targeting par loci found on the plasmids most frequently encountered in Enterobacteriaceae. This method, called "plasmid partition gene typing" (PAR-T), was validated with 136 transconjugants or transformants harboring various replicon types. The method was tested with 30 multidrug-resistant clinical isolates including Escherichia coli, Klebsiella pneumoniae and Salmonella enterica subsp. enterica carrying 1-4 replicons; all replicons were tested in parallel with PRaseT for comparison. Six multiplex PCRs and one simplex PCR including 18 pairs of primers recognized plasmids of groups IncA/C, FIA, FIB, FIC, FIIk, FII, HI1, HI2, I1, L/M, N, X. Our set of multiplex PCRs showed high specificity for the classification of resistance plasmids except for IncX replicons.

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed

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

    2008-05-01

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

  15. Incompatibility Protein IncC and Global Regulator KorB Interact in Active Partition of Promiscuous Plasmid RK2

    PubMed Central

    Rosche, Thomas M.; Siddique, Azeem; Larsen, Michelle H.; Figurski, David H.

    2000-01-01

    Replication of the broad-host-range, IncPα plasmid RK2 requires two plasmid loci: trfA, the replication initiator gene, and oriV, the origin of replication. While these determinants are sufficient for replication in a wide variety of bacteria, they do not confer the stable maintenance of parental RK2 observed in its hosts. The product of the incC gene has been proposed to function in the stable maintenance of RK2 because of its relatedness to the ParA family of ATPases, some of which are known to be involved in the active partition of plasmid and chromosomal DNA. Here we show that IncC has the properties expected of a component of an active partition system. The smaller polypeptide product of incC (IncC2) exhibits a strong, replicon-independent incompatibility phenotype with RK2. This incompatibility phenotype requires the global transcriptional repressor, KorB, and the target for incC-mediated incompatibility is a KorB-binding site (OB). We found that KorB and IncC interact in vivo by using the yeast two-hybrid system and in vitro by using partially purified proteins. Elevated expression of the incC and korB genes individually has no obvious effect on Escherichia coli cell growth, but their simultaneous overexpression is toxic, indicating a possible interaction of IncC-KorB complexes with a vital host target. A region of RK2 bearing incC, korB, and multiple KorB-binding sites is able to stabilize an unstable, heterologous plasmid in an incC-dependent manner. Finally, elevated levels of IncC2 cause RK2 to aggregate, indicating a possible role for IncC in plasmid pairing. These findings demonstrate that IncC, KorB, and at least one KorB-binding site are components of an active partition system for the promiscuous plasmid RK2. PMID:11029420

  16. Conjugative DNA Transfer Is Enhanced by Plasmid R1 Partitioning Proteins

    PubMed Central

    Gruber, Christian J.; Lang, Silvia; Rajendra, Vinod K. H.; Nuk, Monika; Raffl, Sandra; Schildbach, Joel F.; Zechner, Ellen L.

    2016-01-01

    Bacterial conjugation is a form of type IV secretion used to transport protein and DNA directly to recipient bacteria. The process is cell contact-dependent, yet the mechanisms enabling extracellular events to trigger plasmid transfer to begin inside the cell remain obscure. In this study of plasmid R1 we investigated the role of plasmid proteins in the initiation of gene transfer. We find that TraI, the central regulator of conjugative DNA processing, interacts physically, and functionally with the plasmid partitioning proteins ParM and ParR. These interactions stimulate TraI catalyzed relaxation of plasmid DNA in vivo and in vitro and increase ParM ATPase activity. ParM also binds the coupling protein TraD and VirB4-like channel ATPase TraC. Together, these protein-protein interactions probably act to co-localize the transfer components intracellularly and promote assembly of the conjugation machinery. Importantly these data also indicate that the continued association of ParM and ParR at the conjugative pore is necessary for plasmid transfer to start efficiently. Moreover, the conjugative pilus and underlying secretion machinery assembled in the absence of Par proteins mediate poor biofilm formation and are completely dysfunctional for pilus specific R17 bacteriophage uptake. Thus, functional integration of Par components at the interface of relaxosome, coupling protein, and channel ATPases appears important for an optimal conformation and effective activation of the transfer machinery. We conclude that low copy plasmid R1 has evolved an active segregation system that optimizes both its vertical and lateral modes of dissemination. PMID:27486582

  17. Conjugative DNA Transfer Is Enhanced by Plasmid R1 Partitioning Proteins.

    PubMed

    Gruber, Christian J; Lang, Silvia; Rajendra, Vinod K H; Nuk, Monika; Raffl, Sandra; Schildbach, Joel F; Zechner, Ellen L

    2016-01-01

    Bacterial conjugation is a form of type IV secretion used to transport protein and DNA directly to recipient bacteria. The process is cell contact-dependent, yet the mechanisms enabling extracellular events to trigger plasmid transfer to begin inside the cell remain obscure. In this study of plasmid R1 we investigated the role of plasmid proteins in the initiation of gene transfer. We find that TraI, the central regulator of conjugative DNA processing, interacts physically, and functionally with the plasmid partitioning proteins ParM and ParR. These interactions stimulate TraI catalyzed relaxation of plasmid DNA in vivo and in vitro and increase ParM ATPase activity. ParM also binds the coupling protein TraD and VirB4-like channel ATPase TraC. Together, these protein-protein interactions probably act to co-localize the transfer components intracellularly and promote assembly of the conjugation machinery. Importantly these data also indicate that the continued association of ParM and ParR at the conjugative pore is necessary for plasmid transfer to start efficiently. Moreover, the conjugative pilus and underlying secretion machinery assembled in the absence of Par proteins mediate poor biofilm formation and are completely dysfunctional for pilus specific R17 bacteriophage uptake. Thus, functional integration of Par components at the interface of relaxosome, coupling protein, and channel ATPases appears important for an optimal conformation and effective activation of the transfer machinery. We conclude that low copy plasmid R1 has evolved an active segregation system that optimizes both its vertical and lateral modes of dissemination. PMID:27486582

  18. High-copy bacterial plasmids diffuse in the nucleoid-free space, replicate stochastically and are randomly partitioned at cell division.

    PubMed

    Reyes-Lamothe, Rodrigo; Tran, Tung; Meas, Diane; Lee, Laura; Li, Alice M; Sherratt, David J; Tolmasky, Marcelo E

    2014-01-01

    Bacterial plasmids play important roles in the metabolism, pathogenesis and bacterial evolution and are highly versatile biotechnological tools. Stable inheritance of plasmids depends on their autonomous replication and efficient partition to daughter cells at cell division. Active partition systems have not been identified for high-copy number plasmids, and it has been generally believed that they are partitioned randomly at cell division. Nevertheless, direct evidence for the cellular location of replicating and nonreplicating plasmids, and the partition mechanism has been lacking. We used as model pJHCMW1, a plasmid isolated from Klebsiella pneumoniae that includes two β-lactamase and two aminoglycoside resistance genes. Here we report that individual ColE1-type plasmid molecules are mobile and tend to be excluded from the nucleoid, mainly localizing at the cell poles but occasionally moving between poles along the long axis of the cell. As a consequence, at the moment of cell division, most plasmid molecules are located at the poles, resulting in efficient random partition to the daughter cells. Complete replication of individual molecules occurred stochastically and independently in the nucleoid-free space throughout the cell cycle, with a constant probability of initiation per plasmid.

  19. Bacterial partition complexes segregate within the volume of the nucleoid

    PubMed Central

    Le Gall, Antoine; Cattoni, Diego I.; Guilhas, Baptiste; Mathieu-Demazière, Céline; Oudjedi, Laura; Fiche, Jean-Bernard; Rech, Jérôme; Abrahamsson, Sara; Murray, Heath; Bouet, Jean-Yves; Nollmann, Marcelo

    2016-01-01

    Precise and rapid DNA segregation is required for proper inheritance of genetic material. In most bacteria and archaea, this process is assured by a broadly conserved mitotic-like apparatus in which a NTPase (ParA) displaces the partition complex. Competing observations and models imply starkly different 3D localization patterns of the components of the partition machinery during segregation. Here we use super-resolution microscopies to localize in 3D each component of the segregation apparatus with respect to the bacterial chromosome. We show that Par proteins locate within the nucleoid volume and reveal that proper volumetric localization and segregation of partition complexes requires ATPase and DNA-binding activities of ParA. Finally, we find that the localization patterns of the different components of the partition system highly correlate with dense chromosomal regions. We propose a new mechanism in which the nucleoid provides a scaffold to guide the proper segregation of partition complexes. PMID:27377966

  20. Complexation Between Cationic Diblock Copolymers and Plasmid DNA

    NASA Astrophysics Data System (ADS)

    Jung, Seyoung; Reineke, Theresa; Lodge, Timothy

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

  1. The Active Partition Gene incC of IncP Plasmids Is Required for Stable Maintenance in a Broad Range of Hosts

    PubMed Central

    Siddique, Azeem; Figurski, David H.

    2002-01-01

    Plasmids of incompatibility group P (IncP) are capable of replication and stable inheritance in a wide variety of gram-negative bacteria. Three determinants of IncP plasmids are components of an active partition locus that is predicted to function in the segregation of plasmid copies to daughter cells. These determinants are incC, which codes for a member of the ParA family of partition ATPases; korB, which specifies a DNA-binding protein that also functions as a global transcriptional repressor; and OB, the DNA target for KorB, which occurs at multiple locations on IncP plasmids. To determine the importance and host range of the IncC/KorB partition system in the maintenance of IncP plasmids, we constructed an in-frame deletion of incC in the otherwise intact 60-kb IncPα plasmid R995. R995ΔincC was found to be highly unstable in Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Agrobacterium tumefaciens, and Acinetobacter calcoaceticus, whereas wild-type R995 is stable in all these hosts. In addition, R995ΔincC could not be established in Actinobacillus actinomycetemcomitans. trans-Complementation analysis showed that the coding region for IncC2 polypeptide, which is expressed from an internal translational start within the incC gene, was sufficient to restore stable maintenance to wild-type levels. The results show that the IncC/KorB active partition system of IncP plasmids is remarkably proficient for stable maintenance in diverse bacteria. PMID:11872733

  2. A highly selectable and highly transferable Ti plasmid to study conjugal host range and Ti plasmid dissemination in complex ecosystems.

    PubMed

    Teyssier-Cuvelle, S; Oger, P; Mougel, C; Groud, K; Farrand, S K; Nesme, X

    2004-07-01

    A conjugal donor system, ST2, was constructed to study the conjugal dissemination of a Ti plasmid to wild-type recipient bacteria in vitro and in situ. The system consisted of a polyauxotrophic derivative of C58 harboring a hyperconjugative and highly selectable Ti plasmid, pSTiEGK, which was constructed by inserting a multiple antibiotic resistance cassette in the traM- mcpA region of pTiC58Delta accR. ST2 transfers pSTiEGK constitutively at frequencies up to 10(-1) to plasmidless Agrobacterium recipients. The host range of pSTiEGK includes all the known genomic species of Agrobacterium, indigenous soil agrobacteria and some Rhizobium and Phyllobacterium spp. All transconjugants became pathogenic upon acquisition of the Ti plasmid and were also able to transfer pSTiEGK by conjugation. This host range was indistinguishable from that of its wild-type parent pTiC58, and therefore pSTiEGK constitute a valid proxy to study the dissemination of Ti plasmids directly in the environment. Transconjugants can be selected on a combination of four antibiotics, which efficiently prevents the growth of the indigenous microbiota present in complex environments. The transfer of pSTiEGK to members of the genus Agrobacterium was affected primarily by the plasmid content of the recipient strain (10(3)- to 10(5)-fold reduction), e.g., the presence of incompatible plasmids. As a consequence, a species should be considered permissive to Ti transfer whenever one permissive isolate is found. PMID:15164241

  3. Activity of site-specific endonucleases on complexes of plasmid DNA with multiwalled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Egorova, V. P.; Krylova, H. V.; Lipnevich, I. V.; Veligura, A. A.; Shulitsky, B. G.; Asayonok, A. A.; Vaskovtsev, E. V.

    2016-08-01

    We have synthesized and investigated structural and functional properties of plasmid DNA complexes with multi-walled carbon nanotubes (MWCNTs) for detection of changes in structural state of the plasmid DNA at its recognition by site-specific endonuclease. It has been also established that the site-specific endonuclease is functionally active on the surface of MWCNTs.

  4. Reconstructing the complex evolutionary history of mobile plasmids in red algal genomes

    PubMed Central

    Lee, JunMo; Kim, Kyeong Mi; Yang, Eun Chan; Miller, Kathy Ann; Boo, Sung Min; Bhattacharya, Debashish; Yoon, Hwan Su

    2016-01-01

    The integration of foreign DNA into algal and plant plastid genomes is a rare event, with only a few known examples of horizontal gene transfer (HGT). Plasmids, which are well-studied drivers of HGT in prokaryotes, have been reported previously in red algae (Rhodophyta). However, the distribution of these mobile DNA elements and their sites of integration into the plastid (ptDNA), mitochondrial (mtDNA), and nuclear genomes of Rhodophyta remain unknown. Here we reconstructed the complex evolutionary history of plasmid-derived DNAs in red algae. Comparative analysis of 21 rhodophyte ptDNAs, including new genome data for 5 species, turned up 22 plasmid-derived open reading frames (ORFs) that showed syntenic and copy number variation among species, but were conserved within different individuals in three lineages. Several plasmid-derived homologs were found not only in ptDNA but also in mtDNA and in the nuclear genome of green plants, stramenopiles, and rhizarians. Phylogenetic and plasmid-derived ORF analyses showed that the majority of plasmid DNAs originated within red algae, whereas others were derived from cyanobacteria, other bacteria, and viruses. Our results elucidate the evolution of plasmid DNAs in red algae and suggest that they spread as parasitic genetic elements. This hypothesis is consistent with their sporadic distribution within Rhodophyta. PMID:27030297

  5. A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during Streptomyces Conjugation

    PubMed Central

    Thoma, Lina; Dobrowinski, Hyazinth; Finger, Constanze; Guezguez, Jamil; Linke, Dirk; Sepulveda, Edgardo

    2015-01-01

    ABSTRACT Conjugative DNA transfer in mycelial Streptomyces is a unique process involving the transfer of a double-stranded plasmid from the donor into the recipient and the subsequent spreading of the transferred plasmid within the recipient mycelium. This process is associated with growth retardation of the recipient and manifested by the formation of circular inhibition zones, named pocks. To characterize the unique Streptomyces DNA transfer machinery, we replaced each gene of the conjugative 12.1-kbp Streptomyces venezuelae plasmid pSVH1, with the exception of the rep gene required for plasmid replication, with a hexanucleotide sequence. Only deletion of traB, encoding the FtsK-like DNA translocase, affected efficiency of the transfer dramatically and abolished pock formation. Deletion of spdB3, spd79, or spdB2 had a minor effect on transfer but prevented pock formation and intramycelial plasmid spreading. Biochemical characterization of the encoded proteins revealed that the GntR-type regulator TraR recognizes a specific sequence upstream of spdB3, while Orf108, SpdB2, and TraR bind to peptidoglycan. SpdB2 promoted spheroplast formation by T7 lysozyme and formed pores in artificial membranes. Bacterial two-hybrid analyses and chemical cross-linking revealed that most of the pSVH1-encoded proteins interacted with each other, suggesting a multiprotein DNA translocation complex of TraB and Spd proteins which directs intramycelial plasmid spreading. PMID:26015502

  6. Plasmid protein TubR uses a distinct mode of HTH-DNA binding and recruits the prokaryotic tubulin homolog TubZ to effect DNA partition.

    PubMed

    Ni, Lisheng; Xu, Weijun; Kumaraswami, Muthiah; Schumacher, Maria A

    2010-06-29

    The segregation of plasmid DNA typically requires three elements: a DNA centromere site, an NTPase, and a centromere-binding protein. Because of their simplicity, plasmid partition systems represent tractable models to study the molecular basis of DNA segregation. Unlike eukaryotes, which utilize the GTPase tubulin to segregate DNA, the most common plasmid-encoded NTPases contain Walker-box and actin-like folds. Recently, a plasmid stability cassette on Bacillus thuringiensis pBtoxis encoding a putative FtsZ/tubulin-like NTPase called TubZ and DNA-binding protein called TubR has been described. How these proteins collaborate to impart plasmid stability, however, is unknown. Here we show that the TubR structure consists of an intertwined dimer with a winged helix-turn-helix (HTH) motif. Strikingly, however, the TubR recognition helices mediate dimerization, making canonical HTH-DNA interactions impossible. Mutagenesis data indicate that a basic patch, encompassing the two wing regions and the N termini of the recognition helices, mediates DNA binding, which indicates an unusual HTH-DNA interaction mode in which the N termini of the recognition helices insert into a single DNA groove and the wings into adjacent DNA grooves. The TubZ structure shows that it is as similar structurally to eukaryotic tubulin as it is to bacterial FtsZ. TubZ forms polymers with guanine nucleotide-binding characteristics and polymer dynamics similar to tubulin. Finally, we show that the exposed TubZ C-terminal region interacts with TubR-DNA, linking the TubR-bound pBtoxis to TubZ polymerization. The combined data suggest a mechanism for TubZ-polymer powered plasmid movement. PMID:20534443

  7. Complex in vivo Ligation Using Homologous Recombination and High-efficiency Plasmid Rescue from Saccharomyces cerevisiae

    PubMed Central

    Finnigan, Gregory C.; Thorner, Jeremy

    2015-01-01

    The protocols presented here allow for the facile generation of a wide variety of complex multipart DNA constructs (tagged gene products, gene fusions, chimeric proteins, and other variants) using homologous recombination and in vivo ligation in budding yeast (Saccharomyces cerevisiae). This method is straightforward, efficient and cost-effective, and can be used both for vector creation and for subsequent one-step, high frequency integration into a chromosomal locus in yeast. The procedure utilizes PCR with extended oligonucleotide “tails” of homology between multiple fragments to allow for reassembly in yeast in a single transformation followed by a method for highly efficient plasmid extraction from yeast (for transformation into bacteria). The latter is an improvement on existing methods of yeast plasmid extraction, which, historically, has been a limiting step in recovery of desired constructs. We describe the utility and convenience of our techniques, and provide several examples. PMID:26523287

  8. Efficient plasmid DNA cleavage by a mononuclear copper(II) complex.

    PubMed

    Sissi, Claudia; Mancin, Fabrizio; Gatos, Maddalena; Palumbo, Manlio; Tecilla, Paolo; Tonellato, Umberto

    2005-04-01

    The Cu(II) complex of the ligand all-cis-2,4,6-triamino-1,3,5-trihydroxycyclohexane (TACI) is a very efficient catalyst of the cleavage of plasmid DNA in the absence of any added cofactor. The maximum rate of degradation of the supercoiled plasmid DNA form, obtained at pH 8.1 and 37 degrees C, in the presence of 48 microM TACI.Cu(II), is 2.3 x 10(-3) s(-1), corresponding to a half-life time of only 5 min for the cleavage of form I (supercoiled) to form II (relaxed circular). The dependence of the rate of plasmid DNA cleavage from the TACI.Cu(II) complex concentration follows an unusual and very narrow bell-like profile, which suggests an high DNA affinity of the complexes but also a great tendency to form unreactive dimers. The reactivity of the TACI.Cu(II) complexes is not affected by the presence of several scavengers for reactive oxygen species or when measured under anaerobic conditions. Moreover, no degradation of the radical reporter Rhodamine B is observed in the presence of such complexes. These results are consistent with the operation of a prevailing hydrolytic pathway under the normal conditions used, although the failure to obtain enzymatic religation of the linearized DNA does not allow one to rule out the occurrence of a nonhydrolytic oxygen-independent cleavage. A concurrent oxidative mechanism becomes competitive upon addition of reductants or in the presence of high levels of molecular oxygen: under such conditions, in fact, a remarkable increase in the rate of DNA cleavage is observed. PMID:15792466

  9. Preparation of plasmid DNA in transfection complexes for fluorescence and electron spectroscopic imaging.

    PubMed

    Malecki, M

    1996-01-01

    The aim of this project was to develop procedures necessary to study mechanisms of receptor mediated gene transfer by means of integrated microscopy. Plasmid DNA was incorporated into a transfection complex consisting of poly(L)lysine and transferrin to which the nuclear localization signal was conjugated. This complex was presented to cultured glioma cells. Preparation of the transfected DNA for imaging was pursued by two methods. In the first method tetramethylrhodamine, nanogold, and ferritin were linked through streptavidin to the biotinylated plasmid DNA. Trafficking of the fluorescent derivatives was studied in living cells with fluorescence microscopy. Then, selected cells were rapidly cryo-immobilized. Ultra-structural distribution of the transfected DNA was imaged with energy filtering transmission electron microscopy. In the second method, the unmodified transfected DNA was detected in cryo-immobilized cells by in situ polymerase chain reaction and in situ hybridization. For laser scanning fluorescence microscopy probes were labeled with tetramethylrhodamine. For ultrastructural analysis by electron spectroscopic imaging, probes containing incorporated digoxigenin were labeled with anti-digoxigenin boronated antibodies. Based upon the developed procedures, it has been demonstrated that the presence of the nuclear localization signal in the transfection complex resulted in rapid nuclear import of the transfected DNA. PMID:9601525

  10. Protein diversity confers specificity in plasmid segregation.

    PubMed

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

    2005-04-01

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

  11. Mapping environmental partitioning properties of nonpolar complex mixtures by use of GC × GC.

    PubMed

    Nabi, Deedar; Gros, Jonas; Dimitriou-Christidis, Petros; Arey, J Samuel

    2014-06-17

    Comprehensive two-dimensional gas chromatography (GC × GC) is effective for separating and quantifying nonpolar organic chemicals in complex mixtures. Here we present a model to estimate 11 environmental partitioning properties for nonpolar analytes based on GC × GC chromatogram retention time information. The considered partitioning properties span several phases including pure liquid, air, water, octanol, hexadecane, particle natural organic matter, dissolved organic matter, and organism lipids. The model training set and test sets are based on a literature compilation of 648 individual experimental partitioning property data. For a test set of 50 nonpolar environmental contaminants, predicted partition coefficients exhibit root-mean-squared errors ranging from 0.19 to 0.48 log unit, outperforming Abraham-type solvation models for the same chemical set. The approach is applicable to nonpolar organic chemicals containing C, H, F, Cl, Br, and I, having boiling points ≤402 °C. The presented model is calibrated, easy to apply, and requires the user only to identify a small set of known analytes that adapt the model to the GC × GC instrument program. The analyst can thus map partitioning property estimates onto GC × GC chromatograms of complex mixtures. For example, analyzed nonpolar chemicals can be screened for long-range transport potential, aquatic bioaccumulation potential, arctic contamination potential, and other characteristic partitioning behaviors. PMID:24901063

  12. Dinuclear Zinc (II) Complexes of Macrocyclic Polyamine Ligands Containing an Imidazolium Bridge: Synthesis, Characterization, and Their Interaction with Plasmid DNA

    PubMed Central

    Huang, Jun; Huang, Qing-Dong; Zhang, Ji; Zhou, Li-Hong; Li, Qiang-Lin; Li, Kun; Jiang, Ning; Lin, Hong-Hui; Wu, Jiang; Yu, Xiao-Qi

    2007-01-01

    Two novel macrocyclic polyamine ligands and their dinuclear zinc (II) complexes were synthesized and characterized. Their interaction with plasmid DNA was studied by gel electrophoresis and fluorescence quenching experiment. The result showed that these complexes could bind DNA efficiently under physiological conditions.

  13. Bacterial Genome Partitioning: N-Terminal Domain of IncC Protein Encoded by Broad-Host-Range Plasmid RK2 Modulates Oligomerisation and DNA Binding

    PubMed Central

    Batt, Sarah M.; Bingle, Lewis E.H.; Dafforn, Tim R.; Thomas, Christopher M.

    2009-01-01

    ParA Walker ATPases form part of the machinery that promotes better-than-random segregation of bacterial genomes. ParA proteins normally occur in one of two forms, differing by their N-terminal domain (NTD) of approximately 100 aa, which is generally associated with site-specific DNA binding. Unusually, and for as yet unknown reasons, parA (incC) of IncP-1 plasmids is translated from alternative start codons producing two forms, IncC1 (364 aa) and IncC2 (259 aa), whose ratio varies between hosts. IncC2 could be detected as an oligomeric form containing dimers, tetramers and octamers, but the N-terminal extension present in IncC1 favours nucleotide-stimulated dimerisation as well as high-affinity and ATP-dependent non-specific DNA binding. The IncC1 NTD does not dimerise or bind DNA alone, but it does bind IncC2 in the presence of nucleotides. Mixing IncC1 and IncC2 improved polymerisation and DNA binding. Thus, the NTD may modulate the polymerisation interface, facilitating polymerisation/depolymerisation and DNA binding, to promote the cycle that drives partitioning. PMID:19109978

  14. Chemical Partition of the Radiative Decay Rate of Luminescence of Europium Complexes

    PubMed Central

    Lima, Nathalia B. D.; Dutra, José Diogo L.; Gonçalves, Simone M. C.; Freire, Ricardo O.; Simas, Alfredo M.

    2016-01-01

    The spontaneous emission coefficient, Arad, a global molecular property, is one of the most important quantities related to the luminescence of complexes of lanthanide ions. In this work, by suitable algebraic transformations of the matrices involved, we introduce a partition that allows us to compute, for the first time, the individual effects of each ligand on Arad, a property of the molecule as a whole. Such a chemical partition thus opens possibilities for the comprehension of the role of each of the ligands and their interactions on the luminescence of europium coordination compounds. As an example, we applied the chemical partition to the case of repeating non-ionic ligand ternary complexes of europium(III) with DBM, TTA, and BTFA, showing that it allowed us to correctly order, in an a priori manner, the non-obvious pair combinations of non-ionic ligands that led to mixed-ligand compounds with larger values of Arad. PMID:26892900

  15. Chemical Partition of the Radiative Decay Rate of Luminescence of Europium Complexes

    NASA Astrophysics Data System (ADS)

    Lima, Nathalia B. D.; Dutra, José Diogo L.; Gonçalves, Simone M. C.; Freire, Ricardo O.; Simas, Alfredo M.

    2016-02-01

    The spontaneous emission coefficient, Arad, a global molecular property, is one of the most important quantities related to the luminescence of complexes of lanthanide ions. In this work, by suitable algebraic transformations of the matrices involved, we introduce a partition that allows us to compute, for the first time, the individual effects of each ligand on Arad, a property of the molecule as a whole. Such a chemical partition thus opens possibilities for the comprehension of the role of each of the ligands and their interactions on the luminescence of europium coordination compounds. As an example, we applied the chemical partition to the case of repeating non-ionic ligand ternary complexes of europium(III) with DBM, TTA, and BTFA, showing that it allowed us to correctly order, in an a priori manner, the non-obvious pair combinations of non-ionic ligands that led to mixed-ligand compounds with larger values of Arad.

  16. The evaluation of liposome-water partitioning of 8-hydroxyquinolines and their copper complexes.

    PubMed

    Kaiser, Sibylle M; Escher, Beate I

    2006-03-15

    Bioavailability and toxicity of mixtures are urgent research issues, but usually mixtures of exclusively organic chemicals or exclusively metals are investigated. In our study, we explored the role of combinations of hydrophobic ionogenic organic compounds (HIOCs) with copper (Cu2+)for uptake and bioavailability of metals and hydrophobic metal complexes in an in vitro membrane system. We investigated the influence of the interactions of copper and 8-hydroxyquinolines, both components used in formulations of pesticides, on their partitioning into liposomes, which are model systems for biological membranes and are composed of lipid bilayers made of phosphatidylcholine. The test set of compounds comprised the parent compound 8-hydroxyquinoline and 8-hydroxyquinolines with hydrophobic (e.g., 5-chloro-8-hydroxyquinoline, 5,7-dichloro-8-hydroxyquinoline, 5,7-dibromo-8-hydroxyquinoline) and with hydrophilic (e.g., 8-hydroxyquinoline-5-sulfonic acid) substituents. Hydrophobic 8-hydroxyquinolines facilitate the passive uptake of copper into phospholipid bilayers by complex formation. Not only the neutral species of the ligands and their neutral copper ligand complexes are significantly taken up into the membrane, but also the cationic and anionic species of the ligands and the cationic complexes. The neutral, anionic, and cationic species of 8-hydroxyquinoline and the hydrophobic substituted 8-hydroxyquinolines exhibit linear correlations between their logarithmic liposome-water partitioning coefficients (log Klipw) and the logarithmic octanol-water partitioning coefficients of their neutral species (log Kow, neutral). The neutral species show the strongest partitioning followed by the anionic and cationic species. The associated quantitative structure activity relationships describing the dependency of log Klipw of the various species from log Kow, neutral of the neutral ligand species have slopes between 0.9 and 1. In contrast, the partitioning of the neutral and cationic

  17. Delivery of rhBMP-2 Plasmid DNA Complexes via a PLLA/Collagen Electrospun Scaffold Induces Ectopic Bone Formation.

    PubMed

    Zhao, Xia; Komatsu, David E; Hadjiargyrou, Michael

    2016-06-01

    The development of effective strategies for gene delivery is a critical goal in DNA-based tissue engineering. Previously, our laboratory utilized the process of electrospinning to fabricate plasmid DNA-based polymeric scaffolds. Although there lease of DNA was robust, the in vitro transfection efficiency was low. In order to optimize these results, we recently modified our approach and utilized a strategy to adsorb plasmid DNA transfection complexes onto a PLLA/Collagen I electrospun scaffold for the delivery of recombinant human Bone Morphogenetic Protein-2 (rhBMP-2). BMP-2 was selected since it is currently clinically used to stimulate osteogenesis. Initially, we tested this approach using β-gal plasmid DNA complexes adsorbed onto PLLA/Collagen I scaffolds and obtained a transfection efficiency of 41% of that of the positive control (over 90%, DNA complexes in solution). Next, we utilized the same approach using the rhBMP-2 plasmid DNA complexes with the pre-osteoblastic. cell line, MC3T3, and detected robust (13-fold) expression of rhBMP-2 mRNA following transfection. Lastly, a mouse muscle pouch model was used to evaluate in vivo gene delivery efficacy and ectopic bone inducing capability of the scaffold adsorbed rhBMP-2 transfection complexes. Results showed that both rhBMP-2mRNA and protein were expressed and stimulated some ectopic bone formation. As such, adsorption of plasmid DNA complexes can be an effective strategy for tissue engineering in vivo, but further research is required to optimize our approach and obtain a clinically meaningful tissue response. PMID:27319221

  18. Degradative plasmids from sphingomonads.

    PubMed

    Stolz, Andreas

    2014-01-01

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

  19. Partitioning of aromatic constituents into water from gasoline and other complex solvent mixtures

    SciTech Connect

    Cline, P.V.; Delfino, J.J.; Rao, P.S.C. )

    1991-05-01

    Variations in gasoline composition (source variations) as well as complexity (nonideal behavior, cosolvent effects) contributing to variability in gasoline-water partitioning of aromatic hydrocarbon constituents were examined. Aromatic hydrocarbon concentrations in water extracts of 31 gasoline samples varied over 1 order of magnitude, reflecting the diversity in gasoline composition. However, the gasoline-water partition coefficients (K{sub fw}) varied by less than 30% among these samples. Partitioning between water and known mixtures of aromatic and aliphatic solvents was measured and used to estimate the upper and lower bounds of K{sub fw} values for more complex solvent mixtures such as gasoline and diesel fuel. Oxygenated additives, such as methanol and methyl tert-butyl ether (MTBE), were shown to have minimal cosolvent effects on hydrocarbon partitioning. The observed inverse, log-log linear dependence of K{sub fw} values on aqueous solubility could be well predicted by assuming gasoline to be an ideal solvent mixture (i.e., Raoult's law is valid).

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

    PubMed

    Million-Weaver, Samuel; Camps, Manel

    2014-09-01

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

  1. Synthesis, characterization, plasmid cleavage and cytotoxicity of cancer cells by a copper(II) complex of anthracenyl-terpyridine.

    PubMed

    Kumar, Amit; Chinta, Jugun Prakash; Ajay, Amrendra Kumar; Bhat, Manoj Kumar; Rao, Chebrolu P

    2011-11-01

    Metallo-organic compounds are interesting to study for their antitumor activity and related applications. This paper deals with the syntheses, characterization, structure determination of a copper complex of anthracenyl terpyridine (1) and its plasmid cleavage and cytotoxicity towards different cancer cell lines. The complex binds CT-DNA through partial intercalation mode. The plasmid cleavage studies carried out using pBR322 and pUC18 resulted in the formation of all the three forms of the plasmid DNA. Plasmid cleavage studies carried out with a non-redoxable Zn(2+) complex (2) supported the role of the redox activity of copper in 1. The complex 1 showed remarkable antiproliferative activity against cancer cell lines, viz., cervical (HeLa, SiHa, CaSki), breast (MCF-7), liver (HepG2) and lung (H1299). A considerable lowering was observed in the IC(50) values of HPV-infected (viz., HeLa, SiHa, CaSki) vs. non-HPV-infected cell lines (MCF-7, HepG2, H1299). Antiproliferative activity of 1 was found to be much higher than the carboplatin when treated with the same cell lines. Incubation of the cells with 1 results in granular structures only with the HPV-infected cells and not with others as studied by phase contrast and fluorescence microscopy. The lower IC(50) value observed in case of 1 with HPV-infected cell lines may be correlated with the involvement of HPV oncoprotein. The role of HPV has been further augmented by transfecting the MCF-7 cells (originally not possessing HPV copy) with e6 oncoprotein cDNA. To our knowledge this is the first copper complex that causes cell death by interacting with HPV oncoprotein followed by exhibition of remarkable antiproliferative activity.

  2. Photoinduced interactions of supramolecular ruthenium(II) complexes with plasmid DNA: synthesis and spectroscopic, electrochemical, and DNA photocleavage studies.

    PubMed

    Swavey, Shawn; DeBeer, Madeleine; Li, Kaiyu

    2015-04-01

    Two new bridging ligands have been synthesized by combining substituted benzaldehydes with phenanthrolinopyrrole (php), resulting in new polyazine bridging ligands. The ligands have been characterized by (1)H NMR, mass spectroscopy, and elemental analysis. These new ligands display π-π* transitions above 500 nm with modest molar absorptivities. Upon excitation at the ligand-centered charge-transfer transition, weak emission with a maximum wavelength of 612 nm is observed. When coordinated to two ruthenium(II) bis(bipyridyl) groups, the new bimetallic complexes generated give an overall 4+ charge. The electronic transitions of the bimetallic ruthenium(II) complexes display traditional π-π* transitions at 287 nm and metal-to-ligand charge-transfer transitions at 452 nm with molar absorptivities greater than 30000 M(-1) cm(-1). Oxidation of the ruthenium(II) metal centers to ruthenium(III) occurs at potentials above 1.4 V versus the Ag/AgCl reference electrode. Spectroscopic and electrochemical measurements indicate that the ruthenium(II) moieties behave independently. Both complexes are water-soluble and show the ability to photonick plasmid DNA when irradiated with low-energy light above 550 nm. In addition, one of the complexes, [Ru(bpy)2php]2Van(4+), shows the ability to linearize plasmid DNA and gives evidence, by gel electrophoresis, of photoinduced binding to plasmid DNA. PMID:25798576

  3. Efficient and sustained gene expression in primary T lymphocytes and primary and cultured tumor cells mediated by adeno-associated virus plasmid DNA complexed to cationic liposomes.

    PubMed

    Philip, R; Brunette, E; Kilinski, L; Murugesh, D; McNally, M A; Ucar, K; Rosenblatt, J; Okarma, T B; Lebkowski, J S

    1994-04-01

    We have used cationic liposomes to facilitate adeno-associated virus (AAV) plasmid transfections of primary and cultured cell types. AAV plasmid DNA complexed with liposomes showed levels of expression several fold higher than those of complexes with standard plasmids. In addition, long-term expression (> 30 days) of the gene, unlike the transient expression demonstrated by typical liposome-mediated transfection with standard plasmids, was observed. Southern analysis of chromosomal DNA further substantiated the hypothesis that the long-term expression was due to the presence of the transgene in the AAV plasmid-transfected group and not in the standard plasmid-transfected group. AAV plasmid-liposome complexes induced levels of transgene expression comparable to those obtained by recombinant AAV transduction. Primary breast, ovarian, and lung tumor cells were transfectable with the AAV plasmid DNA-liposome complexes. Transfected primary and cultured tumor cells were able to express transgene product even after lethal irradiation. High-level gene expression was also observed in freshly isolated CD3+, CD4+, and CD8+ T cells from normal human peripheral blood. Transfection efficiency ranged from 10 to 50% as assessed by intracellular interleukin-2 levels in interleukin-2-transfected cells. The ability to express transgenes in primary tumor and lymphoid cells may be applied toward tumor vaccine studies and protocols which may eventually permit highly specific modulation of the cellular immune response in cancer and AIDS.

  4. Efficient and sustained gene expression in primary T lymphocytes and primary and cultured tumor cells mediated by adeno-associated virus plasmid DNA complexed to cationic liposomes.

    PubMed Central

    Philip, R; Brunette, E; Kilinski, L; Murugesh, D; McNally, M A; Ucar, K; Rosenblatt, J; Okarma, T B; Lebkowski, J S

    1994-01-01

    We have used cationic liposomes to facilitate adeno-associated virus (AAV) plasmid transfections of primary and cultured cell types. AAV plasmid DNA complexed with liposomes showed levels of expression several fold higher than those of complexes with standard plasmids. In addition, long-term expression (> 30 days) of the gene, unlike the transient expression demonstrated by typical liposome-mediated transfection with standard plasmids, was observed. Southern analysis of chromosomal DNA further substantiated the hypothesis that the long-term expression was due to the presence of the transgene in the AAV plasmid-transfected group and not in the standard plasmid-transfected group. AAV plasmid-liposome complexes induced levels of transgene expression comparable to those obtained by recombinant AAV transduction. Primary breast, ovarian, and lung tumor cells were transfectable with the AAV plasmid DNA-liposome complexes. Transfected primary and cultured tumor cells were able to express transgene product even after lethal irradiation. High-level gene expression was also observed in freshly isolated CD3+, CD4+, and CD8+ T cells from normal human peripheral blood. Transfection efficiency ranged from 10 to 50% as assessed by intracellular interleukin-2 levels in interleukin-2-transfected cells. The ability to express transgenes in primary tumor and lymphoid cells may be applied toward tumor vaccine studies and protocols which may eventually permit highly specific modulation of the cellular immune response in cancer and AIDS. Images PMID:8139545

  5. Crystal Structure of pi Initiator Protein-iteron Complex of Plasmid R6K: Implications for Initiation of Plasmid DNA Replication

    SciTech Connect

    Swan,M.; Bastia, D.; Davies, C.

    2006-01-01

    We have determined the crystal structure of a monomeric biologically active form of the {pi} initiator protein of plasmid R6K as a complex with a single copy of its cognate DNA-binding site (iteron) at 3.1-{angstrom} resolution. The initiator belongs to the family of winged helix type of proteins. The structure reveals that the protein contacts the iteron DNA at two primary recognition helices, namely the C-terminal {alpha}4' and the N-terminal {alpha}4 helices, that recognize the 5' half and the 3' half of the 22-bp iteron, respectively. The base-amino acid contacts are all located in {alpha}4', whereas the {alpha}4 helix and its vicinity mainly contact the phosphate groups of the iteron. Mutational analyses show that the contacts of both recognition helices with DNA are necessary for iteron binding and replication initiation. Considerations of a large number of site-directed mutations reveal that two distinct regions, namely {alpha}2 and {alpha}5 and its vicinity, are required for DNA looping and initiator dimerization, respectively. Further analysis of mutant forms of {pi} revealed the possible domain that interacts with the DnaB helicase. Thus, the structure-function analysis presented illuminates aspects of initiation mechanism of R6K and its control.

  6. Relating the measured toxicity of complex hydrocarbon mixtures to their aqueous partitioning behavior

    SciTech Connect

    Peterson, D.R.

    1995-12-31

    Water extracts of petroleum products (complex mixtures of poorly water soluble hydrocarbons) are commonly used to assess the aquatic toxicity of such substances. When extracted into water, these substances give aqueous solutions whose soluble hydrocarbon composition depends upon both the individual partitioning behavior of each component and the initial hydrocarbon/water ratio. A standard aquatic toxicity testing procedure based upon varying the extraction ratios, dubbed lethal loading, is now being widely employed. The acute aquatic toxicity results (LL50s) from applying this procedure to hydrocarbon mixtures of reasonably well defined composition will be presented. The concentration of each individual hydrocarbon in solution at each extraction ratio may be calculated from its hydrocarbon/water partition coefficient, its original concentration in the mixture, and the hydrocarbon/water ratio. An additional factor to take into account is the partitioning of the more volatile hydrocarbons into air. The effect of this volatilization and of changes in headspace volume will be described. Taking these factors into account, aqueous concentrations of individual hydrocarbon components may be predicted, as will be demonstrated for a number of hydrocarbon mixtures. Furthermore, based upon the assumption of strict additivity of toxicity, the calculated water concentrations of components of complex hydrocarbons may be combined with QSAR (quantitative structure activity relationship) toxicity estimates in order to calculate a joint toxicity for components in the water extract. The relative contribution of various classes of hydrocarbon components to this toxicity, which is not widely appreciated, may be demonstrated using this QSAR approach. It will be shown that the results of these calculations agree well with the results o lethal loading toxicity tests on these same complex hydrocarbon mixtures.

  7. The Evolution of a Hierarchical Partitioning Algorithm for Large-Scale Scientific Data: Three Steps of Increasing Complexity

    SciTech Connect

    Baldwin, C; Eliassi-Rad, T; Abdulla, G; Critchlow, T

    2003-04-16

    As scientific data sets grow exponentially in size, the need for scalable algorithms that heuristically partition the data increases. In this paper, we describe the three-step evolution of a hierarchical partitioning algorithm for large-scale spatio-temporal scientific data sets generated by massive simulations. The first version of our algorithm uses a simple top-down partitioning technique, which divides the data by using a four-way bisection of the spatio-temporal space. The shortcomings of this algorithm lead to the second version of our partitioning algorithm, which uses a bottom-up approach. In this version, a partition hierarchy is constructed by systematically agglomerating the underlying Cartesian grid that is placed on the data. Finally, the third version of our algorithm utilizes the intrinsic topology of the data given in the original scientific problem to build the partition hierarchy in a bottom-up fashion. Specifically, the topology is used to heuristically agglomerate the data at each level of the partition hierarchy. Despite the growing complexity in our algorithms, the third version of our algorithm builds partition hierarchies in less time and is able to build trees for larger size data sets as compared to the previous two versions.

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

    PubMed

    Sengupta, Manjistha; Austin, Stuart

    2011-07-01

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

  9. Late Cretaceous to early Tertiary transtension and strain partitioning in the Chugach Accretionary Complex, SE Alaska

    USGS Publications Warehouse

    Davis, J.S.; Roeske, S.M.; Karl, S.M.

    1998-01-01

    Shear zones in the Late Cretaceous Sitka Graywacke of the Chugach accretionary complex in southeast Alaska record constrictional finite strains, with maximum principal s tretches plunging shallowly subparallel to strike of the shear zones. Macrostructural analysis indicates the finite strain formed during one deformation event. Microstructural analysis of the shear zones shows that this deformation is ductile, promoted mostly through deformation of low-strength lithic clasts and pressure solution. Kinematic indicators from some of the shear zones indicate dominantly dextral motion. Although multiple scenarios can explain constrictional finite strains in a shear zone, these dextral strike-slip shear zones must have experienced a component of extension across them in order to generate constrictional finite strains. Therefore, the shear zones are dextral transtensional shear zones, an uncommon tectinic regime in an accretionary complex. The transtensional shear zones reflect strike-slip motion related to partitioning of Late Cretaceous to Early Tertiary right-oblique convergence between North America and the Farallon plate. The extensional component that was superposed on the strike-slip shear zones to generate transtension resulted from contemporaneous collapse of the forearc following thickening related to underplating.Shear zones in the Late Cretaceous Sitka Graywacke of the Chugach accretionary complex in southeast Alaska record constrictional finite strains, with maximum principal stretches plunging shallowy sub-parallel to strike of the shear zones. Macrostructural analysis indicates the finite strain formed during one deformation event. Microstructural analysis of the shear zones shows that this deformation is ductile, promoted mostly through deformation of low-strength lithic clasts and pressure solution. Kinematic indicators from some of the shear zones indicate dominantly dextral motion. Although multiple scenarios can explain constrictional finite strains

  10. Late Cretaceous to Early Tertiary transtension and strain partitioning in the Chugach accretionary complex, SE Alaska

    NASA Astrophysics Data System (ADS)

    Davis, J. Steven; Roeske, Sarah M.; Karl, Sue M.

    1998-05-01

    Shear zones in the Late Cretaceous Sitka Graywacke of the Chugach accretionary complex in southeast Alaska record constrictional finite strains, with maximum principal stretches plunging shallowly subparallel to strike of the shear zones. Macrostructural analysis indicates the finite strain formed during one deformation event. Microstructural analysis of the shear zones shows that this deformation is ductile, promoted mostly through deformation of low-strength lithic clasts and pressure solution. Kinematic indicators from some of the shear zones indicate dominantly dextral motion. Although multiple scenarios can explain constrictional finite strains in a shear zone, these dextral strike-slip shear zones must have experienced a component of extension across them in order to generate constrictional finite strains. Therefore, the shear zones are dextral transtensional shear zones, an uncommon tectonic regime in an accretionary complex. The transtensional shear zones reflect strike-slip motion related to partitioning of Late Cretaceous to Early Tertiary right-oblique convergence between North America and the Farallon plate. The extensional component that was superposed on the strike-slip shear zones to generate transtension resulted from contemporaneous collapse of the forearc following thickening related to underplating.

  11. Crystal structure of the plasmid maintenance system ɛ/ζ: Functional mechanism of toxin ζ and inactivation by ɛ2ζ2 complex formation

    PubMed Central

    Meinhart, Anton; Alonso, Juan C.; Sträter, Norbert; Saenger, Wolfram

    2003-01-01

    Programmed cell death in prokaryotes is frequently found as postsegregational killing. It relies on antitoxin/toxin systems that secure stable inheritance of low and medium copy number plasmids during cell division and kill cells that have lost the plasmid. The broad-host-range, low-copy-number plasmid pSM19035 from Streptococcus pyogenes carries the genes encoding the antitoxin/toxin system ɛ/ζ and antibiotic resistance proteins, among others. The crystal structure of the biologically nontoxic ɛ2ζ2 protein complex at a 1.95-Å resolution and site-directed mutagenesis showed that free ζ acts as phosphotransferase by using ATP/GTP. In ɛ2ζ2, the toxin ζ is inactivated because the N-terminal helix of the antitoxin ɛ blocks the ATP/GTP-binding site. To our knowledge, this is the first prokaryotic postsegregational killing system that has been entirely structurally characterized. PMID:12571357

  12. Plasmid Biopharmaceuticals.

    PubMed

    Prazeres, Duarte Miguel F; Monteiro, Gabriel A

    2014-12-01

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

  13. Highly Effective Non-Viral Antitumor Gene Therapy System Comprised of Biocompatible Small Plasmid Complex Particles Consisting of pDNA, Anionic Polysaccharide, and Fully Deprotected Linear Polyethylenimine.

    PubMed

    Koyama, Yoshiyuki; Sugiura, Kikuya; Yoshihara, Chieko; Inaba, Toshio; Ito, Tomoko

    2015-07-23

    We have reported that ternary complexes of plasmid DNA with conventional linear polyethylenimine (l-PEI) and certain polyanions were very stably dispersed, and, with no cryoprotectant, they could be freeze-dried and re-hydrated without the loss of transfection ability. These properties enabled the preparation of a concentrated suspension of very small pDNA complex, by preparing the complexes at highly diluted conditions, followed by condensation via lyophilization-and-rehydration procedure. Recently, a high potency linear polyethylenimine having no residual protective groups, i.e., Polyethylenimine "Max" (PEI "Max"), is available, which has been reported to induce much higher gene expression than conventional l-PEI. We tried to prepare the small DNA/PEI "Max"/polyanion complexes by a similar freeze-drying method. Small complex particles could be obtained without apparent aggregation, but transfection activity of the rehydrated complexes was severely reduced. Complex-preparation conditions were investigated in details to achieve the freeze-dried DNA/PEI "Max"/polyanion small ternary complexes with high transfection efficiency. DNA/PEI "Max"/polyanion complexes containing cytokine-coding plasmids were then prepared, and their anti-tumor therapeutic efficacy was examined in tumor-bearing mice.

  14. Highly Effective Non-Viral Antitumor Gene Therapy System Comprised of Biocompatible Small Plasmid Complex Particles Consisting of pDNA, Anionic Polysaccharide, and Fully Deprotected Linear Polyethylenimine

    PubMed Central

    Koyama, Yoshiyuki; Sugiura, Kikuya; Yoshihara, Chieko; Inaba, Toshio; Ito, Tomoko

    2015-01-01

    We have reported that ternary complexes of plasmid DNA with conventional linear polyethylenimine (l-PEI) and certain polyanions were very stably dispersed, and, with no cryoprotectant, they could be freeze-dried and re-hydrated without the loss of transfection ability. These properties enabled the preparation of a concentrated suspension of very small pDNA complex, by preparing the complexes at highly diluted conditions, followed by condensation via lyophilization-and-rehydration procedure. Recently, a high potency linear polyethylenimine having no residual protective groups, i.e., Polyethylenimine “Max” (PEI “Max”), is available, which has been reported to induce much higher gene expression than conventional l-PEI. We tried to prepare the small DNA/PEI “Max”/polyanion complexes by a similar freeze-drying method. Small complex particles could be obtained without apparent aggregation, but transfection activity of the rehydrated complexes was severely reduced. Complex-preparation conditions were investigated in details to achieve the freeze-dried DNA/PEI “Max”/polyanion small ternary complexes with high transfection efficiency. DNA/PEI “Max”/polyanion complexes containing cytokine-coding plasmids were then prepared, and their anti-tumor therapeutic efficacy was examined in tumor-bearing mice. PMID:26213961

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

    PubMed

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

    2013-02-01

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

  16. Molecular docking between the RNA polymerase of the Moniliophthora perniciosa mitochondrial plasmid and Rifampicin produces a highly stable complex

    PubMed Central

    2013-01-01

    Background Moniliophthora perniciosa (Stahel) Aime & Phillips-Mora is the causal agent of witches’ broom disease (WBD) in cacao (Theobroma cacao). When the mitochondrial genome of this fungus had been completely sequenced, an integrated linear-type plasmid that encodes viral-like RNA polymerases was found. The structure of this polymerase was previously constructed using a homology modeling approach. Methods Using a virtual screening process, accessing the Kegg, PubChem and ZINC databases, we selected the eight most probable macrocyclic polymerase inhibitors to test against M. perniciosa RNA polymerase (RPO). AutoDock Vina was used to perform docking calculations for each molecule. This software returned affinity energy values for several ligand conformations. Subsequently, we used PyMOL 1.4 and Ligand Scout 3.1 to check the stereochemistry of chiral carbons, substructure, superstructure, number of rotatable bonds, number of rings, number of donor groups, and hydrogen bond receptors. Results On the basis of this evidence we selected Rifampicin, a bacterial RNA polymerase inhibitor, and then AMBER 12 was used to simulate the behavior of the RPO-Rifampicin complex after a set of 5000 ps and up to 300 K in water. This calculation returned a graph of potential energy against simulation time and showed that the ligand remained inside the active site after the simulation was complete, with an average energy of -15 x 102 Kcal/Mol. Conclusions The results indicate that Rifampicin could be a good inhibitor for testing in vitro and in vivo against M. perniciosa. PMID:23442217

  17. Partitioning of complex surfactant mixtures between oil/water/microemulsion phases at high surfactant concentrations

    SciTech Connect

    Graciaa, A.; Lachaise, J.; Sayous, J.G.; Grenier, P.; Yiv, S.

    1983-06-01

    A model describing the partitioning of surfactant molecules between excess and microemulsion phases which are in equilibrium is proposed. The important parameters characterizing the individual molecules comprising the mixture are the critical micelle concentrations in water and the partition coefficients between oil and water phases. The model considers the existence of a separate surfactant phase which is the palisade layer of a micelle and leads to predictions for both fractionation and phase concentrations of surfactant. Predictions based on this model have been compared to experimentally determined quantities and the agreement is good for all cases tested. The model leads to a relatively simple mathematical formulation which can be used to study the effect of varying the overall system surfactant concentration and of changing the system water-to-oil ratio. 21 references.

  18. Effect of DNA/liposome mixing ratio on the physicochemical characteristics, cellular uptake and intracellular trafficking of plasmid DNA/cationic liposome complexes and subsequent gene expression.

    PubMed

    Sakurai, F; Inoue, R; Nishino, Y; Okuda, A; Matsumoto, O; Taga, T; Yamashita, F; Takakura, Y; Hashida, M

    2000-05-15

    In order to identify the important factors involved in cationic liposome-mediated gene transfer, in vitro transfection efficiencies by plasmid DNA complexed with DOTMA/DOPE liposomes at different DNA/liposome mixing ratios were evaluated using four types of cultured cells with respect to their physicochemical properties. Significant changes were observed in the particle size and zeta potential of the complexes as well as in their structures, assessed by atomic force microscopy, which depended on the mixing ratio. In transfection experiments, except for RAW 264.7 cells (mouse macrophages), efficient gene expression was obtained in MBT-2 cells (mouse bladder tumor), NLH3T3 cells (mouse fibroblasts) and HUVEC (human umbilical vein endothelial cells) at an optimal ratio of 1:5, 1:7.5 or 1:5, respectively. On the other hand, cellular uptake of the [32P]DNA/liposome complexes increased in all cell types with an increase in the mixing ratio, which was not reflected by the transfection efficiency. The cellular damage determined by MTT assay was minimal even at the highest DNA/liposome ratio (1:10), indicating that the lower gene expression level at the higher ratio was not due to cytotoxicity induced by the complex. An ethidium bromide intercalation assay showed that the release of plasmid DNA from the complex, following the addition of negatively charged liposomes, was restricted as the mixing ratio increased. Furthermore, confocal microscopic studies using HUVEC showed that the 1:5 complexes exhibited a dispersed distribution in the cytoplasm whereas a punctuate intracellular distribution was observed for the 1:10 complexes. This suggests that there was a significant difference in intracellular trafficking, probably release from the endosomes or lysosomes, of the plasmid DNA/cationic liposome complexes between these mixing ratios. Taken together, these findings suggest that the DNA/liposome mixing ratio significantly affects the intracellular trafficking of plasmid DNA

  19. Heavy metal partitioning in soil profiles in the vicinity of an industrial complex and potential health implications

    NASA Astrophysics Data System (ADS)

    Martley, E.; Gulson, B. L.

    2003-05-01

    The industrial complex of Port Kembla, NSW, Australia comprises a Cu smelter, steelworks and associated industries. As part of an investigation into the regional extent in soils of a suite of metals, speciation in selected soil profiles was undertaken. A single aqua regia extraction and a six-step sequential extraction procedure were applied to soil samples at six different horizons to a depth of 50cm from: (i) a contaminated, disturbed site located about 70m from a major point source (C2); (ii) a contaminated undisturbed area situated 1.1km from the contamination source (CI); and (iii) an uncontaminated, undisturbed area located 22.1km from the complex (C3). In the uncontaminated soil profile, Pb was preferentially bound to hydrous Fe-Mn oxides and the less mobile fractions (crystalline Fe oxides, sulphides and organic matter). Lead partitioning was relatively constant with depth. In the contaminated undisturbed soil profile, the proportion of mobile Pb decreased with depth. Lead partitioning at the lower levels was similar to that of core C3. At the surface, Pb was preferentially bound to carbonates. In the contaminated disturbed soil profile, a large proportion of Pb was found in the labile forms to a depth of 40-50cm. The contaminated disturbed site represents potential health and environmental hazards.

  20. CONFORMATIONAL PROPERTIES AND ENTROPIC PARTITIONING OF TOPOLOGICALLY COMPLEX POLYMERS UNDER CONFINEMENT - Final Report

    SciTech Connect

    Escobedo, Fernando A.

    2005-03-24

    The effect of molecular topology (e.g., branch and loop structures) on the solution properties of polymers is subtle and not well characterized. Because the conformational entropy of a polymer depends on its topology, many properties are affected by it such as its size and shape, mobility, bulk-to-pore partitioning, adsorption strength on surfaces, and depletion-induced forces on colloidal surfaces. We have systematically studied the effect of molecular topology on the structure and entropic partitioning of linear, branched, hyper-branched, cyclic, and hyper-cyclic polymers in a bulk solution and in pores. Ours is the first simulation study aimed at characterizing the conformational properties of hyper-cyclic molecules. Key findings: Our results show how differences in molecular architecture can be used to partition polymers in a porous media e.g., a highly branched polymer tends to be depleted in narrow pores (smaller than the coil size) relative to a less branched chain of equal molecular weight, but this trend is reversed in wide pores. It was also found that intra-molecular crosslinking (associated with cyclic structures) is an effective way to tune the conformational entropy of a polymer; the more crosslinks a molecule has, the smaller its conformational entropy, and the easier it is to adsorb it onto attractive pore walls. Intra-crosslinked chains are thus more effective steric stabilizer of colloid particles than linear chains (which are better depleting agents). Simulations were also used to investigate the mechanism of entropic trapping for model linear and branched DNA molecules as they go from a deep channel to a shallow channel driven by an electric field. In such a system, a molecule whose radius of gyration is larger than the gap of the shallow channel tends to get temporarily trapped at its entrance. Our results show that at moderate and strong fields, longer chains escape faster than shorter ones because, in the absence of significant differences in

  1. Experimental partitioning of Zr, Ti, and Nb between silicate liquid and a complex noble metal alloy and the partitioning of Ti between perovskite and platinum metal

    NASA Technical Reports Server (NTRS)

    Jurewicz, Stephen R.; Jones, John H.

    1993-01-01

    El Goresy et al.'s observation of Nb, Zr, and Ta in refractory platinum metal nuggets (RPMN's) from Ca-Al-rich inclusions (CAI's) in the Allende meteorite led them to propose that these lithophile elements alloyed in the metallic state with noble metals in the early solar nebula. However, Grossman pointed out that the thermodynamic stability of Zr in the oxide phase is vastly greater than metallic Zr at estimated solar nebula conditions. Jones and Burnett suggested this discrepancy may be explained by the very non-ideal behavior of some lithophile transition elements in noble metal solutions and/or intermetallic compounds. Subsequently, Fegley and Kornacki used thermodynamic data taken from the literature to predict the stability of several of these intermetallic compounds at estimated solar nebula conditions. Palme and Schmitt and Treiman et al. conducted experiments to quantify the partitioning behavior of certain lithophile elements between silicate liquid and Pt-metal. Although their results were somewhat variable, they did suggest that Zr partition coefficients were too small to explain the observed 'percent' levels in some RPMN's. Palme and Schmitt also observed large partition coefficients for Nb and Ta. No intermetallic phases were identified. Following the work of Treiman et al., Jurewicz and Jones performed experiments to examine Zr, Nb, and Ti partitioning near solar nebula conditions. Their results showed that Zr, Nb, and Ti all have an affinity for the platinum metal, with Nb and Ti having a very strong preference for the metal. The intermetallic phases (Zr,Fe)Pt3, (Nb,Fe)Pt3, and (Ti,Fe)Pt3 were identified. Curiously, although both experiments and calculations indicate that Ti should partition strongly into Pt-metal (possibly as TiPt3), no Ti has ever been observed in any RPMN's. Fegley and Kornacki also noticed this discrepancy and hypothesized that the Ti was stabilized in perovskite which is a common phase in Allende CAI's.

  2. 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

  3. N15: the linear phage-plasmid.

    PubMed

    Ravin, Nikolai V

    2011-03-01

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

  4. A complex genetic switch involving overlapping divergent promoters and DNA looping regulates expression of conjugation genes of a gram-positive plasmid.

    PubMed

    Ramachandran, Gayetri; Singh, Praveen K; Luque-Ortega, Juan Roman; Yuste, Luis; Alfonso, Carlos; Rojo, Fernando; Wu, Ling J; Meijer, Wilfried J J

    2014-10-01

    Plasmid conjugation plays a significant role in the dissemination of antibiotic resistance and pathogenicity determinants. Understanding how conjugation is regulated is important to gain insights into these features. Little is known about regulation of conjugation systems present on plasmids from Gram-positive bacteria. pLS20 is a native conjugative plasmid from the Gram-positive bacterium Bacillus subtilis. Recently the key players that repress and activate pLS20 conjugation have been identified. Here we studied in detail the molecular mechanism regulating the pLS20 conjugation genes using both in vivo and in vitro approaches. Our results show that conjugation is subject to the control of a complex genetic switch where at least three levels of regulation are integrated. The first of the three layers involves overlapping divergent promoters of different strengths regulating expression of the conjugation genes and the key transcriptional regulator RcoLS20. The second layer involves a triple function of RcoLS20 being a repressor of the main conjugation promoter and an activator and repressor of its own promoter at low and high concentrations, respectively. The third level of regulation concerns formation of a DNA loop mediated by simultaneous binding of tetrameric RcoLS20 to two operators, one of which overlaps with the divergent promoters. The combination of these three layers of regulation in the same switch allows the main conjugation promoter to be tightly repressed during conditions unfavorable to conjugation while maintaining the sensitivity to accurately switch on the conjugation genes when appropriate conditions occur. The implications of the regulatory switch and comparison with other genetic switches involving DNA looping are discussed. PMID:25340403

  5. Comparison of the efficiency of complexes based on S4(13)-PV cell-penetrating peptides in plasmid DNA and siRNA delivery.

    PubMed

    Cardoso, Ana M; Trabulo, Sara; Cardoso, Ana L; Maia, Sílvia; Gomes, Paula; Jurado, Amália S; Pedroso de Lima, Maria C

    2013-07-01

    The successful application of gene therapy approaches is highly dependent on the efficient delivery of nucleic acids into target cells. In the present study, new peptide-based nonviral systems were developed to enhance plasmid DNA and siRNA delivery, aiming at generating appropriate gene delivery and gene silencing tools for preclinical and clinical application. For this purpose, a new cell-penetrating peptide derived from the wild-type S4(13)-PV peptide was synthesized through the addition of a five-histidine tail to its N-terminus (H5-S4(13)-PV), and its ability to mediate gene expression and gene silencing was evaluated and compared to that of the wild-type peptide. The histidine-enriched peptide, H5-S4(13)-PV, proved to be generally more efficient and less toxic than the wild-type peptide in the delivery of plasmid DNA. In addition, complexes of H5-S4(13)-PV with siRNAs, but not of S4(13)-PV, were efficiently internalized by cells and presented high knockdown activity (63%). Interestingly, systems containing the S4(13)-PV or the H5-S4(13)-PV peptide exhibited superior biological activity when compared to those containing the reverse NLS or scrambled peptides, suggesting that both the cell-penetrating sequence and the NLS of the S4(13)-PV peptide influence the competence of binary and ternary complexes to accomplish nucleic acid delivery. In order to unravel the cancer therapeutic potential of formulations with the histidine-enriched peptide, their efficiency to mediate silencing of the oncogenic protein survivin was evaluated. As opposed to complexes with the wild-type peptide, H5-S4(13)-PV complexes showed the ability to promote a high survivin knockdown at the level of both protein (44%) and mRNA (73%), in HT1080 cells.

  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. Spontaneous Partitioning of Californium from Curium: Curious Cases from the Crystallization of Curium Coordination Complexes

    SciTech Connect

    Cary, Samantha K.; Silver, Mark A.; Liu, Guokui; Wang, Jamie C.; Bogart, Justin A.; Stritzinger, Jared T.; Arico, Alexandra A.; Hanson, Kenneth; Schelter, Eric J.; Albrecht-Schmitt, Thomas E.

    2015-12-07

    The reaction of 248CmCl3 with excess 2,6-pyridinedicarboxylic acid (DPA) under mild solvothermal conditions results in crystallization of the tris-chelate complex Cm(HDPA)3·H2O. Approximately half of the curium remains in solution at the end of this process, and evaporation of the mother liquor results in crystallization of the bis-chelate complex [Cm(HDPA)- (H2DPA)(H2O)2Cl]Cl·2H2O. 248Cm is the daughter of the α decay of 252Cf and is extracted in high purity from this parent. However, trace amounts of 249,250,251Cf are still present in all samples of 248Cm. During the crystallization of Cm(HDPA)3·H2O and [Cm(HDPA)(H2DPA)(H2O)2Cl]Cl·2H2O, californium(III) spontaneously separates itself from the curium complexes and is found doped within crystals of DPA in the form of Cf(HDPA)3. These results add to the growing body of evidence that the chemistry of californium is fundamentally different from that of earlier actinides.

  8. Spontaneous Partitioning of Californium from Curium: Curious Cases from the Crystallization of Curium Coordination Complexes.

    PubMed

    Cary, Samantha K; Silver, Mark A; Liu, Guokui; Wang, Jamie C; Bogart, Justin A; Stritzinger, Jared T; Arico, Alexandra A; Hanson, Kenneth; Schelter, Eric J; Albrecht-Schmitt, Thomas E

    2015-12-01

    The reaction of (248)CmCl3 with excess 2,6-pyridinedicarboxylic acid (DPA) under mild solvothermal conditions results in crystallization of the tris-chelate complex Cm(HDPA)3 · H2O. Approximately half of the curium remains in solution at the end of this process, and evaporation of the mother liquor results in crystallization of the bis-chelate complex [Cm(HDPA)(H2DPA)(H2O)2Cl]Cl·2H2O. (248)Cm is the daughter of the α decay of (252)Cf and is extracted in high purity from this parent. However, trace amounts of (249,250,251)Cf are still present in all samples of (248)Cm. During the crystallization of Cm(HDPA)3 · H2O and [Cm(HDPA)(H2DPA)(H2O)2Cl]Cl · 2H2O, californium(III) spontaneously separates itself from the curium complexes and is found doped within crystals of DPA in the form of Cf(HDPA)3. These results add to the growing body of evidence that the chemistry of californium is fundamentally different from that of earlier actinides.

  9. Tn6026 and Tn6029 are found in complex resistance regions mobilised by diverse plasmids and chromosomal islands in multiple antibiotic resistant Enterobacteriaceae.

    PubMed

    Reid, Cameron J; Roy Chowdhury, Piklu; Djordjevic, Steven P

    2015-07-01

    Transposons flanked by direct copies of IS26 are important contributors to the evolution of multiple antibiotic resistance. Tn6029 and Tn6026 are examples of composite transposons that have become widely disseminated on small and large plasmids with different incompatibility markers in pathogenic and commensal Escherichia coli and various serovars of Salmonella enterica. Some of the plasmids that harbour these transposons also carry combinations of virulence genes. Recently, Tn6029 and Tn6026 and derivatives thereof have been found on chromosomal islands in both established and recently emerged pathogens. While Tn6029 and Tn6026 carry genes encoding resistance to older generation antibiotics, they also provide a scaffold for the introduction of genes encoding resistance to a wide variety of clinically relevant antibiotics that are mobilised by IS26. As a consequence, Tn6029 and Tn6026 or variants are likely to increasingly feature in complex resistance regions in multiple antibiotic resistant Enterobacteriaceae that threaten the health of humans and food production animals.

  10. The use of CpG-free plasmids to mediate persistent gene expression following repeated aerosol delivery of pDNA/PEI complexes.

    PubMed

    Davies, Lee A; Hyde, Stephen C; Nunez-Alonso, Graciela; Bazzani, Reto P; Harding-Smith, Rebekka; Pringle, Ian A; Lawton, Anna E; Abdullah, Syahril; Roberts, Thomas C; McCormick, Dominique; Sumner-Jones, Stephanie G; Gill, Deborah R

    2012-08-01

    Aerosol gene therapy offers great potential for treating acquired and inherited lung diseases. For treatment of chronic lung diseases such as cystic fibrosis, asthma and emphysema, non-viral gene therapy will likely require repeated administration to maintain transgene expression in slowly dividing, or terminally differentiated, lung epithelial cells. When complexed with plasmid DNA (pDNA), the synthetic polymer, 25 kDa branched Polyethylenimine (PEI), can be formulated for aerosol delivery to the lungs. We show that pDNA/PEI aerosol formulations can be repeatedly administered to airways of mice on at least 10 occasions with no detectable toxicity. Interestingly, peak reporter gene activity upon repeated delivery was significantly reduced by up to 75% compared with a single administration, despite similar pDNA lung deposition at each subsequent aerosol exposure. Although the precise mechanism of inhibition is unknown, it is independent of mouse strain, does not involve an immune response, and is mediated by PEI. Importantly, using a dosing interval of 56 days, delivery of a fourth-generation, CpG-free plasmid generated high-level, sustained transgene expression, which was further boosted at subsequent administrations. Together these data indicate that pDNA/PEI aerosol formulations offer a versatile platform for gene delivery to the lung resulting in sustained transgene expression suitable for treatment of chronic lung diseases. PMID:22575838

  11. Tri-partite complex for axonal transport drug delivery achieves pharmacological effect

    PubMed Central

    2010-01-01

    Background Targeted delivery of pharmaceutical agents into selected populations of CNS (Central Nervous System) neurons is an extremely compelling goal. Currently, systemic methods are generally used for delivery of pain medications, anti-virals for treatment of dermatomal infections, anti-spasmodics, and neuroprotectants. Systemic side effects or undesirable effects on parts of the CNS that are not involved in the pathology limit efficacy and limit clinical utility for many classes of pharmaceuticals. Axonal transport from the periphery offers a possible selective route, but there has been little progress towards design of agents that can accomplish targeted delivery via this intraneural route. To achieve this goal, we developed a tripartite molecular construction concept involving an axonal transport facilitator molecule, a polymer linker, and a large number of drug molecules conjugated to the linker, then sought to evaluate its neurobiology and pharmacological behavior. Results We developed chemical synthesis methodologies for assembling these tripartite complexes using a variety of axonal transport facilitators including nerve growth factor, wheat germ agglutinin, and synthetic facilitators derived from phage display work. Loading of up to 100 drug molecules per complex was achieved. Conjugation methods were used that allowed the drugs to be released in active form inside the cell body after transport. Intramuscular and intradermal injection proved effective for introducing pharmacologically effective doses into selected populations of CNS neurons. Pharmacological efficacy with gabapentin in a paw withdrawal latency model revealed a ten fold increase in half life and a 300 fold decrease in necessary dose relative to systemic administration for gabapentin when the drug was delivered by axonal transport using the tripartite vehicle. Conclusion Specific targeting of selected subpopulations of CNS neurons for drug delivery by axonal transport holds great promise

  12. Highly efficient in vivo gene transfection by plasmid/PEI complexes coated by anionic PEG derivatives bearing carboxyl groups and RGD peptide.

    PubMed

    Sakae, Mitsuko; Ito, Tomoko; Yoshihara, Chieko; Iida-Tanaka, Naoko; Yanagie, Hironobu; Eriguchi, Masazumi; Koyama, Yoshiyuki

    2008-09-01

    A new class of an anionic poly (ethylene glycol) derivative, PEG-Suc, bearing 17.7 pairs of carboxylic acid-side chains was synthesized. PEG-Suc deposited onto the DNA/polyethyleneimine complexes without destroying them even at high dose ratio. Coating of the DNA complexes by PEG-Suc recharged their surface to negative, and effectively protected them from the albumin-induced aggregation. Paired carboxyl groups in the side chains showed higher proton sponge effect. Negatively charged surface would diminish the electrostatic binding of the complexes to the cells, and the transfection efficiency on the cultured cells was not high. RGD peptide side chain as a ligand to malignant cell surfaces was then introduced to compensate the reduced electrical adhesion. RGD-PEG-Suc-coated plasmid/PEI complex brought about more than 3 times higher reporter protein activity on the cultured B16 cells. Those bio-compatible DNA complexes with ligand attained very high gene expression in tumor, lung, and liver after injection into mouse tail vein.

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

    PubMed Central

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

    2016-01-01

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

  14. Partitioning the non‑consumptive effects of predators on preywith complex life histories

    USGS Publications Warehouse

    Davenport, Jon M.; Hossack, Blake R.; Lowe, Winsor H.

    2014-01-01

    Non-consumptive effects (NCEs) of predators on prey can be as strong as consumptive effects (CEs) and may be driven by numerous mechanisms, including predator characteristics. Previous work has highlighted the importance of predator characteristics in predicting NCEs, but has not addressed how complex life histories of prey could mediate predator NCEs. We conducted a meta-analysis to compare the effects of predator gape limitation (gape limited or not) and hunting mode (active or sit-and-pursue) on the activity, larval period, and size at metamorphosis of larval aquatic amphibians and invertebrates. Larval prey tended to reduce their activity and require more time to reach metamorphosis in the presence of all predator functional groups, but the responses did not differ from zero. Prey metamorphosed at smaller size in response to non-gape-limited, active predators, but counter to expectations, prey metamorphosed larger when confronted by non-gape-limited, sit-and-pursue predators. These results indicate NCEs on larval prey life history can be strongly influenced by predator functional characteristics. More broadly, our results suggest that understanding predator NCEs would benefit from greater consideration of how prey life history attributes mediate population and community-level outcomes.

  15. Increasing Nitrogen Fixation and Seed Development in Soybean Requires Complex Adjustments of Nodule Nitrogen Metabolism and Partitioning Processes.

    PubMed

    Carter, Amanda M; Tegeder, Mechthild

    2016-08-01

    Legumes are able to access atmospheric di-nitrogen (N2) through a symbiotic relationship with rhizobia that reside within root nodules. In soybean, following N2 fixation by the bacteroids, ammonia is finally reduced in uninfected cells to allantoin and allantoic acid [1]. These ureides present the primary long-distance transport forms of nitrogen (N), and are exported from nodules via the xylem for shoot N supply. Transport of allantoin and allantoic acid out of nodules requires the function of ureide permeases (UPS1) located in cells adjacent to the vasculature [2, 3]. We expressed a common bean UPS1 transporter in cortex and endodermis cells of soybean nodules and found that delivery of N from nodules to shoot, as well as seed set, was significantly increased. In addition, the number of transgenic nodules was increased and symbiotic N2 fixation per nodule was elevated, indicating that transporter function in nodule N export is a limiting step in bacterial N acquisition. Further, the transgenic nodules showed considerable increases in nodule N assimilation, ureide synthesis, and metabolite levels. This suggests complex adjustments of nodule N metabolism and partitioning processes in support of symbiotic N2 fixation. We propose that the transgenic UPS1 plants display metabolic and allocation plasticity to overcome N2 fixation and seed yield limitations. Overall, it is demonstrated that transporter function in N export from nodules is a key step for enhancing atmospheric N2 fixation and nodule function and for improving shoot N nutrition and seed development in legumes.

  16. MSTor: A program for calculating partition functions, free energies, enthalpies, entropies, and heat capacities of complex molecules including torsional anharmonicity

    NASA Astrophysics Data System (ADS)

    Zheng, Jingjing; Mielke, Steven L.; Clarkson, Kenneth L.; Truhlar, Donald G.

    2012-08-01

    We present a Fortran program package, MSTor, which calculates partition functions and thermodynamic functions of complex molecules involving multiple torsional motions by the recently proposed MS-T method. This method interpolates between the local harmonic approximation in the low-temperature limit, and the limit of free internal rotation of all torsions at high temperature. The program can also carry out calculations in the multiple-structure local harmonic approximation. The program package also includes six utility codes that can be used as stand-alone programs to calculate reduced moment of inertia matrices by the method of Kilpatrick and Pitzer, to generate conformational structures, to calculate, either analytically or by Monte Carlo sampling, volumes for torsional subdomains defined by Voronoi tessellation of the conformational subspace, to generate template input files, and to calculate one-dimensional torsional partition functions using the torsional eigenvalue summation method. Catalogue identifier: AEMF_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEMF_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: Standard CPC licence, http://cpc.cs.qub.ac.uk/licence/licence.html No. of lines in distributed program, including test data, etc.: 77 434 No. of bytes in distributed program, including test data, etc.: 3 264 737 Distribution format: tar.gz Programming language: Fortran 90, C, and Perl Computer: Itasca (HP Linux cluster, each node has two-socket, quad-core 2.8 GHz Intel Xeon X5560 “Nehalem EP” processors), Calhoun (SGI Altix XE 1300 cluster, each node containing two quad-core 2.66 GHz Intel Xeon “Clovertown”-class processors sharing 16 GB of main memory), Koronis (Altix UV 1000 server with 190 6-core Intel Xeon X7542 “Westmere” processors at 2.66 GHz), Elmo (Sun Fire X4600 Linux cluster with AMD Opteron cores), and Mac Pro (two 2.8 GHz Quad-core Intel Xeon

  17. Plasmids from Euryarchaeota.

    PubMed

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

    2014-12-01

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

  18. Increasing Nitrogen Fixation and Seed Development in Soybean Requires Complex Adjustments of Nodule Nitrogen Metabolism and Partitioning Processes.

    PubMed

    Carter, Amanda M; Tegeder, Mechthild

    2016-08-01

    Legumes are able to access atmospheric di-nitrogen (N2) through a symbiotic relationship with rhizobia that reside within root nodules. In soybean, following N2 fixation by the bacteroids, ammonia is finally reduced in uninfected cells to allantoin and allantoic acid [1]. These ureides present the primary long-distance transport forms of nitrogen (N), and are exported from nodules via the xylem for shoot N supply. Transport of allantoin and allantoic acid out of nodules requires the function of ureide permeases (UPS1) located in cells adjacent to the vasculature [2, 3]. We expressed a common bean UPS1 transporter in cortex and endodermis cells of soybean nodules and found that delivery of N from nodules to shoot, as well as seed set, was significantly increased. In addition, the number of transgenic nodules was increased and symbiotic N2 fixation per nodule was elevated, indicating that transporter function in nodule N export is a limiting step in bacterial N acquisition. Further, the transgenic nodules showed considerable increases in nodule N assimilation, ureide synthesis, and metabolite levels. This suggests complex adjustments of nodule N metabolism and partitioning processes in support of symbiotic N2 fixation. We propose that the transgenic UPS1 plants display metabolic and allocation plasticity to overcome N2 fixation and seed yield limitations. Overall, it is demonstrated that transporter function in N export from nodules is a key step for enhancing atmospheric N2 fixation and nodule function and for improving shoot N nutrition and seed development in legumes. PMID:27451897

  19. Efficient Cellular Entry of (r-x-r)-Type Carbamate-Plasmid DNA Complexes and Its Implication for Noninvasive Topical DNA Delivery to Skin.

    PubMed

    Vij, Manika; Natarajan, Poornemaa; Yadav, Amit K; Patil, Kiran M; Pandey, Tanuja; Gupta, Nidhi; Santhiya, Deenan; Kumar, Vaijayanti A; Fernandes, Moneesha; Ganguli, Munia

    2016-06-01

    Arginine-rich cell penetrating peptides are powerful tools for in vitro as well as in vivo delivery of a wide plethora of biomolecules. However, presence of consecutive arginine residues leads to enhanced amenability for proteolytic degradation as well as steric hindrances for membrane interactions which compromise its bioavailability. In order to overcome these limitations we previously reported a safe and stable octaarginine based oligomer, i.e., (r-x-r)4-carbamate, where the backbone amide linkages were replaced by carbamate linkages and 6-aminohexanoic acid based spacer moieties were incorporated for better flexibility, hydrophobicity, optimal spacing of guanidinium groups, and protection against proteolytic cleavage; resulting in improved transfection efficiency over its amide counterpart. In the present work we have investigated the mechanism behind this enhanced transfection efficiency and, based on our observations, demonstrate how the synergistic effect of rationalized oligomer designing, complex characteristics, and cell type contributes to overall effective intracellular delivery. Our results indicate that the (r-x-r)4-carbamate-plasmid DNA complexes primarily utilize lipid raft dependent pathway of cellular entry more than other pathways, and this possibly facilitates their increased entry in the lipid raft rich milieu of skin cells. We also emphasize the utility of oligomer (r-x-r)4-carbamate as an efficient carrier for topical delivery of nucleic acids in skin tissue. This carrier can be utilized for safe, efficient, and noninvasive delivery of therapeutically relevant macromolecular hydrophilic cargo like nucleic acids to skin. PMID:27175623

  20. Efficient Cellular Entry of (r-x-r)-Type Carbamate-Plasmid DNA Complexes and Its Implication for Noninvasive Topical DNA Delivery to Skin.

    PubMed

    Vij, Manika; Natarajan, Poornemaa; Yadav, Amit K; Patil, Kiran M; Pandey, Tanuja; Gupta, Nidhi; Santhiya, Deenan; Kumar, Vaijayanti A; Fernandes, Moneesha; Ganguli, Munia

    2016-06-01

    Arginine-rich cell penetrating peptides are powerful tools for in vitro as well as in vivo delivery of a wide plethora of biomolecules. However, presence of consecutive arginine residues leads to enhanced amenability for proteolytic degradation as well as steric hindrances for membrane interactions which compromise its bioavailability. In order to overcome these limitations we previously reported a safe and stable octaarginine based oligomer, i.e., (r-x-r)4-carbamate, where the backbone amide linkages were replaced by carbamate linkages and 6-aminohexanoic acid based spacer moieties were incorporated for better flexibility, hydrophobicity, optimal spacing of guanidinium groups, and protection against proteolytic cleavage; resulting in improved transfection efficiency over its amide counterpart. In the present work we have investigated the mechanism behind this enhanced transfection efficiency and, based on our observations, demonstrate how the synergistic effect of rationalized oligomer designing, complex characteristics, and cell type contributes to overall effective intracellular delivery. Our results indicate that the (r-x-r)4-carbamate-plasmid DNA complexes primarily utilize lipid raft dependent pathway of cellular entry more than other pathways, and this possibly facilitates their increased entry in the lipid raft rich milieu of skin cells. We also emphasize the utility of oligomer (r-x-r)4-carbamate as an efficient carrier for topical delivery of nucleic acids in skin tissue. This carrier can be utilized for safe, efficient, and noninvasive delivery of therapeutically relevant macromolecular hydrophilic cargo like nucleic acids to skin.

  1. Cationic Lipid-Nucleic Acid Complexes for Gene Delivery And Silencing: Pathways And Mechanisms for Plasmid Dna And Sirna

    SciTech Connect

    Ewert, K.K.; Zidovska, A.; Ahmad, A.; Bouxsein, N.F.; Evans, H.M.; McAllister, C.S.; Samuel, C.E.; Safinya, C.R.; /SLAC

    2012-07-17

    Motivated by the promises of gene therapy, there is great interest in developing non-viral lipid-based vectors for therapeutic applications due to their low immunogenicity, low toxicity, ease of production, and the potential of transferring large pieces of DNA into cells. In fact, cationic liposome (CL) based vectors are among the prevalent synthetic carriers of nucleic acids (NAs) currently used in gene therapy clinical trials worldwide. These vectors are studied both for gene delivery with CL-DNA complexes and gene silencing with CL-siRNA (short interfering RNA) complexes. However, their transfection efficiencies and silencing efficiencies remain low compared to those of engineered viral vectors. This reflects the currently poor understanding of transfection-related mechanisms at the molecular and self-assembled levels, including a lack of knowledge about interactions between membranes and double stranded NAs and between CL-NA complexes and cellular components. In this review we describe our recent efforts to improve the mechanistic understanding of transfection by CL-NA complexes, which will help to design optimal lipid-based carriers of DNA and siRNA for therapeutic gene delivery and gene silencing.

  2. Partition search

    SciTech Connect

    Ginsberg, M.L.

    1996-12-31

    We introduce a new form of game search called partition search that incorporates dependency analysis, allowing substantial reductions in the portion of the tree that needs to be expanded. Both theoretical results and experimental data are presented. For the game of bridge, partition search provides approximately as much of an improvement over existing methods as {alpha}-{beta} pruning provides over minimax.

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

    PubMed

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

    2013-08-01

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

  4. Impact of next-generation sequencing error on analysis of barcoded plasmid libraries of known complexity and sequence

    PubMed Central

    Deakin, Claire T.; Deakin, Jeffrey J.; Ginn, Samantha L.; Young, Paul; Humphreys, David; Suter, Catherine M.; Alexander, Ian E.; Hallwirth, Claus V.

    2014-01-01

    Barcoded vectors are promising tools for investigating clonal diversity and dynamics in hematopoietic gene therapy. Analysis of clones marked with barcoded vectors requires accurate identification of potentially large numbers of individually rare barcodes, when the exact number, sequence identity and abundance are unknown. This is an inherently challenging application, and the feasibility of using contemporary next-generation sequencing technologies is unresolved. To explore this potential application empirically, without prior assumptions, we sequenced barcode libraries of known complexity. Libraries containing 1, 10 and 100 Sanger-sequenced barcodes were sequenced using an Illumina platform, with a 100-barcode library also sequenced using a SOLiD platform. Libraries containing 1 and 10 barcodes were distinguished from false barcodes generated by sequencing error by a several log-fold difference in abundance. In 100-barcode libraries, however, expected and false barcodes overlapped and could not be resolved by bioinformatic filtering and clustering strategies. In independent sequencing runs multiple false-positive barcodes appeared to be represented at higher abundance than known barcodes, despite their confirmed absence from the original library. Such errors, which potentially impact barcoding studies in an application-dependent manner, are consistent with the existence of both stochastic and systematic error, the mechanism of which is yet to be fully resolved. PMID:25013183

  5. Ability of plasmid DNA complexed with histidinylated lPEI and lPEI to cross in vitro lung and muscle vascular endothelial barriers.

    PubMed

    Gomez, Jean-Pierre; Pichon, Chantal; Midoux, Patrick

    2013-08-10

    DNA complexes made with cationic polymers (polyplexes) developed as nonviral vectors for gene therapy must be enabled to cross through vascular endothelium to transfect underlying tissues upon their administration in the blood circulation. Here, we evaluated the transendothelial passage (TEP) of DNA complexes made with histidinylated linear polyethylenimine (His-lPEI) or linear polyethylenimine (lPEI). In vitro studies were performed by using established transwell lung and skeletal muscle vascular endothelial barriers. The models were composed of a monolayer of human lung microvascular endothelial (HMVEC-L) cells and mouse cardiac endothelial (MCEC) cells formed on a PET insert and immortalized human tracheal epithelial (ΣCFTE29o-) cells and mouse myoblasts (C2C12) as target cells cultured in the lower chamber, respectively. When the vascular endothelium monolayer was established and characterized, the transfection efficiency of target (ΣCFTE29o- and C2C12) cells with plasmid DNA encoding luciferase was used to evaluate TEP of polyplexes. The luciferase activities with His-lPEI and lPEI polyplexes compared to those obtained in the absence of endothelial cell monolayer were 6.5% and 4.3% into ΣCFTE29o- cells, and 18.5% and 0.23% into C2C12 cells, respectively. The estimated rate for His-lPEI polyplexes was 0.135 μg/cm(2).h and 0.385 μg/cm(2).h through the HMVEC-L and MCEC monolayers, respectively. These results indicate that His-lPEI polyplexes can pass through the lung and skeletal muscle vascular endothelium and can transfect underlying cells. PMID:23562720

  6. In Vivo Expansion of Regulatory T cells With IL-2/IL-2 mAb Complexes Prevents Anti-factor VIII Immune Responses in Hemophilia A Mice Treated With Factor VIII Plasmid-mediated Gene Therapy

    PubMed Central

    Liu, Chao-Lien; Ye, Peiqing; Yen, Benjamin C; Miao, Carol H

    2011-01-01

    Generation of transgene-specific immune responses can constitute a major complication following gene therapy treatment. An in vivo approach to inducing selective expansion of Regulatory T (Treg) cells by injecting interleukin-2 (IL-2) mixed with a specific IL-2 monoclonal antibody (JES6-1) was adopted to modulate anti-factor VIII (anti-FVIII) immune responses. Three consecutive IL-2 complexes treatments combined with FVIII plasmid injection prevented anti-FVIII formation and achieved persistent, therapeutic-level of FVIII expression in hemophilia A (HemA) mice. The IL-2 complexes treatment expanded CD4+CD25+Foxp3+ Treg cells five- to sevenfold on peak day, and they gradually returned to normal levels within 7–14 days without changing other lymphocyte populations. The transiently expanded Treg cells are highly activated and display suppressive function in vitro. Adoptive transfer of the expanded Treg cells protected recipient mice from generation of high-titer antibodies following FVIII plasmid challenge. Repeated plasmid transfer is applicable in tolerized mice without eliciting immune responses. Mice treated with IL-2 complexes mounted immune responses against both T-dependent and T-independent neoantigens, indicating that IL-2 complexes did not hamper the immune system for long. These results demonstrate the important role of Treg cells in suppressing anti-FVIII immune responses and the potential of developing Treg cell expansion therapies that induce long-term tolerance to FVIII. PMID:21468007

  7. The F plasmid centromere, sopC, is required for full repression of the sopAB operon.

    PubMed

    Yates, P; Lane, D; Biek, D P

    1999-07-16

    The SopB protein of the F plasmid has a dual role in the partition of F plasmid copies to daughter cells prior to division. It binds to the sopC centromere site to form the partition complex needed for stabilizing the plasmid, and it interacts with SopA to repress transcription of the sopAB operon, thus preventing the destabilization that results from excess SopB. We have isolated sop mutants by screening for unstable inheritance of mutagenized mini-F DNA. Four of the mutants resulted from different missense mutations in sopB. All four were deficient, to varying degrees, in autoregulation of Sop protein synthesis. The mutant proteins showed diminished capacity for reducing the linking number of mini-F and for destabilizing a plasmid carrying sopC, indicating that reduced ability to form a normal complex with sopC might underlie the autoregulation defect. Repression of the transcription of a sop promoter- lacZ fusion by SopA and SopB was strongly enhanced in the presence of sopC, in cis or in trans, and the enhancement was reduced or nullified when wild-type sopB was replaced by the mutant sopB alleles. A single 43 bp unit of sopC was almost as effective as sopC itself in enhancing repression. The results show that sopC is necessary for full repression of the sop promoter. They thus indicate a previously unsuspected role for this centromere site, and suggest that autoregulation and partition might normally be coordinated processes.

  8. SPP1-mediated plasmid transduction.

    PubMed Central

    Canosi, U; Lüder, G; Trautner, T A

    1982-01-01

    The virulent Bacillus subtilis phage SPP1 transduces plasmid DNA. Plasmid-transducing phages contain only plasmid DNA. Such DNA represents a concatemer of monomeric plasmid molecules with the molecular weight of mature SPP1 DNA. Biological parameters of plasmid transduction are described. Images PMID:6292508

  9. Different Phenotypes of Walker-Like A Box Mutants of ParA Homolog IncC of Broad-Host-Range IncP Plasmids

    PubMed Central

    Siddique, Azeem; Figurski, David H.

    2012-01-01

    The promiscuous IncPα plasmids RK2 and R995 encode a broad-host-range partition system, whose essential components include the incC and korB genes and a DNA site (OB) to which the korB product binds. IncC2, the smaller of the two incC products, is sufficient for stabilization of R995ΔincC. It is a member of the type Ia ParA family of partition ATPases. To better understand the role of ATP in partition, we constructed three alanine-substitution mutants of IncC2. Each mutation changed a different residue of the Walker-like ATP-binding and hydrolysis motif, including a lysine (K10) conserved solely among members of the ParA and MinD families. All three IncC2 mutants were defective in plasmid partition, but they differed from one another in other respects. The IncC2 T16A mutant, predicted to be defective in Mg2+ coordination, was severely impaired in all activities tested. IncC2 K10A, predicted to be defective in ATP hydrolysis, mediated enhanced incompatibility with R995 derivatives. IncC2 K15A, predicted to be defective in ATP binding, exhibited two distinct incompatibility properties depending on the genotype of the target plasmid. When in trans to plasmids carrying a complementable incC deletion, IncC2 K15A caused dramatic plasmid loss, even at low levels of expression. In trans to wild-type R995 or to R995ΔincC carrying a functional P1 partition system, IncC2 K15A-mediated incompatibility was significantly less than that caused by wild-type IncC2. All three Walker-like A box mutants were also defective for the host toxicity that normally results from co-overexpression of incC and korB. The phenotypes of the mutants support a model in which nucleotide hydrolysis is required for separation of paired plasmid complexes and possible interaction with a host factor. PMID:22579980

  10. 3D Numerical Experiments of Lithospheric Transtension Reveal Complex Crustal-Scale Flow and Strain Partitioning in Transdomes

    NASA Astrophysics Data System (ADS)

    Rey, P. F.; Mondy, L. S.; Duclaux, G.; Teyssier, C. P.; Whitney, D. L.

    2015-12-01

    We have used Underworld to perform a series of numerical experiments involving a 256 x 256 x 128 km domain, at a grid resolution of 1.33 km. The kinematic boundary conditions simulate a lithospheric-scale pull-apart setting. We compare the structural and thermal evolution of a model involving a crust of thickness 40 km (TMoho=540ºC) with a model with a crust of thickness 60 km (TMoho=830ºC). We show that in the thick, hot crust model the flow in the pull-apart region is strongly partitioned between the strong upper crust and the weak lower crust. The weak, deep crust flows toward the pull-apart region to isostatically compensate the stretching and thinning of the upper crust. In contrast, the velocity field in the upper crust remains parallel to the imposed direction of extension. In the pull-apart region a transdome, made of two parallel foliation folds (or sub-domes), forms. In the dome, fabrics evolve from strong vertical flattening in between the two sub-domes, to shallow dipping constriction roughly parallel to the direction of extension in the upper part of the transdome.

  11. Forearc deformation and strain partitioning during growth of a continental magmatic arc: The northwestern margin of the Central Bohemian Plutonic Complex, Bohemian Massif

    NASA Astrophysics Data System (ADS)

    Žák, Jiří; Dragoun, František; Verner, Kryštof; Chlupáčová, Marta; Holub, František V.; Kachlík, Václav

    2009-04-01

    The Late Devonian subduction followed by the Early Carboniferous continental collision of the Saxothuringian and overriding upper-crustal Teplá-Barrandian units led to the growth of a large magmatic arc (the ˜ 354-337 Ma Central Bohemian Plutonic Complex) in the central part of the Bohemian Massif. Far-field tectonic forces resulting from the collision produced ˜WNW-ESE to ˜NW-SE regional shortening across the forearc upper crust above the subduction zone; the shortening was accommodated by predominantly top-to-the-ESE tectonic transport along the southeastern flank of the Teplá-Barrandian unit. Approaching the magmatic arc margin, the regional structural pattern changes and exhibits significant across- and along-strike variations interpreted as a result of strain partitioning, where the Saxothuringian/Teplá-Barrandian convergence interacted in different ways with the intruding magma pulses. Around the voluminous, northeasterly ˜ 354 Ma Sázava pluton the principal shortening was at high angle to the forearc-facing intrusive contact and the host rocks were significantly thermally softened. The regional top-to-the-ESE tectonic transport converted here into arc-parallel ductile flow within the structural aureole around and above the pluton. In contrast, a narrow to nonexistent ductile strain aureole is preserved in the host rocks around discordant sheet-like plutons (the southwesterly pre-354 (?) Ma Marginal granite and the Milín granodiorite of unknown radiometric age). Our AMS study of the Marginal granite and Milín granodiorite, and mapping of mesoscopic magmatic foliations and lineations in another neighboring sheet-like pluton (the ˜ 346 Ma Kozárovice granodiorite), reveals sigmoidal map-scale fabric patterns consistent with dextral transpression. We thus suggest that the thin sheet-like plutons were oriented obliquely to the principal shortening and were rheologically weaker than the host rocks prior to final crystallization, producing dextral

  12. Chlamydial plasmids and bacteriophages.

    PubMed

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

    2015-01-01

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

  13. 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

  14. The structural basis for partitioning of the XRCC1/DNA ligase III-[alpha] BRCT-mediated dimer complexes

    SciTech Connect

    Cuneo, Matthew J.; Gabel, Scott A.; Krahn, Joseph M.; Ricker, Melissa A.; London, Robert E.

    2011-11-17

    The ultimate step common to almost all DNA repair pathways is the ligation of the nicked intermediate to form contiguous double-stranded DNA. In the mammalian nucleotide and base excision repair pathways, the ligation step is carried out by ligase III-{alpha}. For efficient ligation, ligase III-{alpha} is constitutively bound to the scaffolding protein XRCC1 through interactions between the C-terminal BRCT domains of each protein. Although structural data for the individual domains has been available, no structure of the complex has been determined and several alternative proposals for this interaction have been advanced. Interpretation of the models is complicated by the formation of homodimers that, depending on the model, may either contribute to, or compete with heterodimer formation. We report here the structures of both homodimer complexes as well as the heterodimer complex. Structural characterization of the heterodimer formed from a longer XRCC1 BRCT domain construct, including residues comprising the interdomain linker region, revealed an expanded heterodimer interface with the ligase III-{alpha} BRCT domain. This enhanced linker-mediated binding interface plays a significant role in the determination of heterodimer/homodimer selectivity. These data provide fundamental insights into the structural basis of BRCT-mediated dimerization, and resolve questions related to the organization of this important repair complex.

  15. The Structural Basis for Partitioning of the XRCC1/DNA Ligase III-alpha BRCT-mediated Dimer Complexes

    SciTech Connect

    M Cuneo; S Gabel; J Krahn; M Ricker; R London

    2011-12-31

    The ultimate step common to almost all DNA repair pathways is the ligation of the nicked intermediate to form contiguous double-stranded DNA. In the mammalian nucleotide and base excision repair pathways, the ligation step is carried out by ligase III-{alpha}. For efficient ligation, ligase III-{alpha} is constitutively bound to the scaffolding protein XRCC1 through interactions between the C-terminal BRCT domains of each protein. Although structural data for the individual domains has been available, no structure of the complex has been determined and several alternative proposals for this interaction have been advanced. Interpretation of the models is complicated by the formation of homodimers that, depending on the model, may either contribute to, or compete with heterodimer formation. We report here the structures of both homodimer complexes as well as the heterodimer complex. Structural characterization of the heterodimer formed from a longer XRCC1 BRCT domain construct, including residues comprising the interdomain linker region, revealed an expanded heterodimer interface with the ligase III-{alpha} BRCT domain. This enhanced linker-mediated binding interface plays a significant role in the determination of heterodimer/homodimer selectivity. These data provide fundamental insights into the structural basis of BRCT-mediated dimerization, and resolve questions related to the organization of this important repair complex.

  16. Bioinformatics-Based Molecular Classification of Arthrobacter Plasmids.

    PubMed

    Mihăşan, Marius

    2015-12-01

    The omnipresence of Arthrobacter species in polluted and toxic soils indicates their great potential in environmental biotechnologies, but practical applications of these bacteria are scarce mainly due to the availability of useful genetic engineering tools. Although many fully sequenced Arthrobacter genomes have been deposited in GenBank, little is known about the biology of their plasmids, especially the core functions: replication and partition. In this study the available Arthrobacter plasmid sequences were analyzed in order to identify their putative replication origin. At least the oris from the cryptic plasmids pXZ10142, pCG1, and pBL1 appear to work in this genus. Based on ParA homolog sequences, the Arthrobacter specific plasmids were classified into 4 clades. Iteron-like sequences were identified on most of the plasmids, indicating the position of the putative Arthrobacter specific oris. Although attempts were made to identify the core gene set required for plasmid replication in this genus, it was not possible. The plasmid proteomes showed a rather low similarity.

  17. 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.

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

    PubMed

    Ravin, Nikolai V

    2015-02-01

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

  19. Low-Molecular Weight Polyethylenimine Modified with Pluronic 123 and RGD- or Chimeric RGD-NLS Peptide: Characteristics and Transfection Efficacy of Their Complexes with Plasmid DNA.

    PubMed

    Hu, Jing; Zhao, Wenfang; Liu, Kehai; Yu, Qian; Mao, Yuan; Lu, Zeyu; Zhang, Yaguang; Zhu, Manman

    2016-01-01

    To solve the problem of transfection efficiency vs. cytotoxicity and tumor-targeting ability when polyethylenimine (PEI) was used as a nonviral gene delivery vector, new degradable PEI polymers were synthesized via cross-linking low-molecular-weight PEI with Pluronic P123 and then further coupled with a targeting peptide R4 (RGD) and a bifunctional R11 (RGD-NLS), which were termed as P123-PEI-R4 and P123-PEI-R11, respectively. Agarose gel electrophoresis showed that both P123-PEI-R4 and P123-PEI-R11 efficaciously condense plasmid DNA at a polymer-to-pDNA w/w ratio of 3.0 and 0.4, respectively. The polyplexes were stable in the presence of serum and could protect plasmid DNA against DNaseI. They had uniform spherical nanoparticles with appropriate sizes around 100-280 nm and zeta-potentials about +40 mV. Furthermore, in vitro experiments showed that these polyplexes had lower cytotoxicity at any concentration compared with PEI 25 kDa, thus giving promise to high transfection efficiency as compared with another P123-PEI derivate conjugated with trifunctional peptide RGD-TAT-NLS (P123-PEI-R18). More importantly, compared with the other polymers, P123-PEI-R11 showed the highest transfection efficiency with relatively lower cytotoxicity at any concentration, indicating that the new synthetic polymer P123-PEI-R11 could be used as a safe and efficient gene deliver vector. PMID:27213305

  20. PCR-directed in vivo plasmid construction using homologous recombination in baker's yeast.

    PubMed

    Andersen, Erik C

    2011-01-01

    A variety of applications require the creation of custom-designed plasmids, including transgenic reporters, heterologous gene fusions, and phenotypic rescue plasmids. These plasmids are created traditionally using restriction digests and in vitro ligation reactions, but these techniques are dependent on available restriction sites and can be laborious given the size and number of fragments to be ligated. The baker's yeast Saccharomyces cerevisiae provides a powerful platform to create nearly any plasmid through PCR-directed yeast-mediated ligation. This technique can ligate complex plasmids of up to 50 kilobasepairs (kb) in vivo to produce plasmids with precisely defined sequences.

  1. High-resolution anion-exchange and partition thin-layer chromatography for complex mixtures of 32P-postlabeled DNA adducts.

    PubMed

    Spencer-Beach, G G; Beach, A C; Gupta, R C

    1996-03-01

    32P-Postlabeling has emerged as a major tool for detecting DNA adducts resulting from exposure to complex carcinogen mixtures. An integral component of this assay is multi-directional PEI-cellulose TLC in which lipophilic 32P-adducts are resolved in high-salt, high-urea solvents following removal of the bulk of non-adduct radioactivity. This TLC system is very effective for adducts formed following exposure to individual carcinogens; however, adducts resulting from exposure to complex mixtures (e.g. cigarette smoke) generally appear in the form of the so-called diagonal radioactive zones. By using mixtures of polycyclic aromatic hydrocarbon- and aromatic amine-DNA adducts as well as adducts in mouse skin treated with cigarette smoke condensate, we have demonstrated that a combination of 0.3-0.4 M NH4OH and isopropanol-4 M NH4OH (1-1.4:1) solvents can provide more sharply defined adduct spots than the commonly used urea solvents. The non-urea solvents also result in excellent resolution of many adducts which otherwise may remain buried in diagonal radioactive zones when using the urea solvents. In addition, the signal-to-noise ratio is increased 2- to 5-fold over the urea solvents enabling detection of discrete adducts at < or = 3 adducts per 10(10) nucleotides. These partition TLC solvents also involve fewer manipulations (e.g. no water washes to remove salt and urea), and are likely to be more informative with regards to the type of individual adducts detected in the biomonitoring of humans than has hitherto been possible. PMID:8704930

  2. In vitro DNA binding, pBR322 plasmid cleavage and molecular modeling study of chiral benzothiazole Schiff-base-valine Cu(II) and Zn(II) complexes to evaluate their enantiomeric biological disposition for molecular target DNA.

    PubMed

    Alizadeh, Rahman; Afzal, Mohd; Arjmand, Farukh

    2014-10-15

    Bicyclic heterocyclic compounds viz. benzothiazoles are key components of deoxyribonucleic acid (DNA) molecules and participate directly in the encoding of genetic information. Benzothiazoles, therefore, represent a potent and selective class of antitumor compounds. The design and synthesis of chiral antitumor chemotherapeutic agents of Cu(II) and Zn(II), L- and -D benzothiazole Schiff base-valine complexes 1a &b and 2a &b, respectively were carried out and thoroughly characterized by spectroscopic and analytical techniques. Interaction of 1a and b and 2a and b with CT DNA by employing UV-vis, florescence, circular dichroic methods and cleavage studies of 1a with pBR322 plasmid, molecular docking were done in order to demonstrate their enantiomeric disposition toward the molecular drug target DNA. Interestingly, these studies unambiguously demonstrated the greater potency of L-enantiomer in comparison to D-enantiomer.

  3. In vitro DNA binding, pBR322 plasmid cleavage and molecular modeling study of chiral benzothiazole Schiff-base-valine Cu(II) and Zn(II) complexes to evaluate their enantiomeric biological disposition for molecular target DNA

    NASA Astrophysics Data System (ADS)

    Alizadeh, Rahman; Afzal, Mohd; Arjmand, Farukh

    2014-10-01

    Bicyclic heterocyclic compounds viz. benzothiazoles are key components of deoxyribonucleic acid (DNA) molecules and participate directly in the encoding of genetic information. Benzothiazoles, therefore, represent a potent and selective class of antitumor compounds. The design and synthesis of chiral antitumor chemotherapeutic agents of Cu(II) and Zn(II), L- and -D benzothiazole Schiff base-valine complexes 1a &b and 2a &b, respectively were carried out and thoroughly characterized by spectroscopic and analytical techniques. Interaction of 1a and b and 2a and b with CT DNA by employing UV-vis, florescence, circular dichroic methods and cleavage studies of 1a with pBR322 plasmid, molecular docking were done in order to demonstrate their enantiomeric disposition toward the molecular drug target DNA. Interestingly, these studies unambiguously demonstrated the greater potency of L-enantiomer in comparison to D-enantiomer.

  4. Plasmid copy number noise in monoclonal populations of bacteria

    NASA Astrophysics Data System (ADS)

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

    2010-01-01

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

  5. Bacteriophages Limit the Existence Conditions for Conjugative Plasmids

    PubMed Central

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

    2015-01-01

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

  6. 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.

  7. Young Children's Partitioning Strategies.

    ERIC Educational Resources Information Center

    Charles, Kathy; Nason, Rod

    2000-01-01

    Studies knowledge of young children's partitioning strategies by setting out not only to identify new partitioning strategies, but also to develop taxonomy for classifying young children's partitioning strategies in terms of their abilities. Provides taxonomy utilizing children's informal partitioning strategies as the foundation upon which to…

  8. Peaceful coexistence amongst Borrelia plasmids: getting by with a little help from their friends?

    PubMed

    Chaconas, George; Norris, Steven J

    2013-09-01

    Borrelia species comprise a unique genus of bacterial pathogens. These organisms contain a segmented genome with up to two dozen plasmids ranging in size from 5 kb up to about 200 kb. The plasmids have also been referred to as mini-chromosomes or essential genetic elements, as some of them carry information important for infection of vertebrates or for survival in the tick vector. Most of the plasmids are linear with covalently closed hairpin telomeres and these linear plasmids are in a constant state of genetic rearrangement. The mechanisms of plasmid replication, maintenance and partitioning remain largely obscure and are complicated by a long doubling time, the requirement for expensive media and inefficient genetic manipulation. A set of five parologous protein families (PFs) are believed to confer the ability for autonomous replication and plasmid maintenance. The number of plasmids also complicates analyses because of the possibility that PFs from one plasmid may sometimes function in trans on other plasmids. Two papers in the last year have moved the field forward and their combined data suggest that trans complementation amongst Borrelia plasmids may sometimes occur.

  9. Intermediate partitioning kinetic isotope effects for the NIH shift of 4-hydroxyphenylpyruvate dioxygenase and the hydroxylation reaction of hydroxymandelate synthase reveal mechanistic complexity.

    PubMed

    Shah, Dhara D; Conrad, John A; Moran, Graham R

    2013-09-01

    4-Hydroxyphenylpyruvate dioxygenase (HPPD) and hydroxymandelate synthase (HMS) are similar enzymes that catalyze complex dioxygenation reactions using the substrates 4-hydroxyphenylpyruvate (HPP) and dioxygen. Both enzymes decarboxylate HPP and then hydroxylate the resulting hydroxyphenylacetate (HPA). The hydroxylation reaction catalyzed by HPPD displaces the aceto substituent of HPA in a 1,2-shift to form 2,5-dihydroxyphenylacetate (homogentisate, HG), whereas the hydroxylation reaction of HMS places a hydroxyl on the benzylic carbon forming 3'-hydroxyphenylacetate (S-hydroxymandelate, HMA) without ensuing chemistry. The wild-type form of HPPD and variants of both enzymes uncouple to form both native and non-native products. We have used intermediate partitioning to probe bifurcating steps that form these products by substituting deuteriums for protiums at the benzylic position of the HPP substrate. These substitutions result in altered ratios of products that can be used to calculate kinetic isotope effects (KIE) for the formation of a specific product. For HPPD, secondary normal KIEs indicate that cleavage of the bond in the displacement reaction prior to the shift occurs by a homolytic mechanism. NMR analysis of HG derived from HPPD reacting with enantiomerically pure R-3'-deutero-HPP indicates that no rotation about the bond to the radical occurs, suggesting that collapse of the biradical intermediate is rapid. The production of HMA was observed in HMS and HPPD variant reactions. HMS hydroxylates to form exclusively S-hydroxymandelate. When HMS is reacted with R-3'-deutero-HPP, the observed kinetic isotope effect represents geometry changes in the initial transition state for the nonabstracted proton. These data show evidence of sp(3) hybridization in a HPPD variant and sp(2) hybridization in HMS variants, suggesting that HMS stabilizes a more advanced transition state in order to catalyze H-atom abstraction.

  10. Inference of plasmid-copy-number mean and noise from single-cell gene expression data

    NASA Astrophysics Data System (ADS)

    Ghozzi, Stéphane; Wong Ng, Jérôme; Chatenay, Didier; Robert, Jérôme

    2010-11-01

    Plasmids are extrachromosomal DNA molecules which code for their own replication. We previously reported a setup using genes coding for fluorescent proteins of two colors that allowed us, using a simple model, to extract the plasmid-copy-number noise in a monoclonal population of bacteria [J. Wong Ng , Phys. Rev. E 81, 011909 (2010)10.1103/PhysRevE.81.011909]. Here we present a detailed calculation relating this noise to the measured levels of fluorescence, taking into account all sources of fluorescence fluctuations: not only the fluctuation of gene expression as in the simple model but also the growth and division of bacteria, the nonuniform distribution of their ages, the random partition of proteins at divisions, and the replication and partition of plasmids and chromosome. We show how to use the chromosome as a reference, which helps extracting the plasmid-copy-number noise in a self-consistent manner.

  11. Asymmetric partitioning of transfected DNA during mammalian cell division

    PubMed Central

    Wang, Xuan; Le, Nhung; Denoth-Lippuner, Annina; Barral, Yves; Kroschewski, Ruth

    2016-01-01

    Foreign DNA molecules and chromosomal fragments are generally eliminated from proliferating cells, but we know little about how mammalian cells prevent their propagation. Here, we show that dividing human and canine cells partition transfected plasmid DNA asymmetrically, preferentially into the daughter cell harboring the young centrosome. Independently of how they entered the cell, most plasmids clustered in the cytoplasm. Unlike polystyrene beads of similar size, these clusters remained relatively immobile and physically associated to endoplasmic reticulum-derived membranes, as revealed by live cell and electron microscopy imaging. At entry of mitosis, most clusters localized near the centrosomes. As the two centrosomes split to assemble the bipolar spindle, predominantly the old centrosome migrated away, biasing the partition of the plasmid cluster toward the young centrosome. Down-regulation of the centrosomal proteins Ninein and adenomatous polyposis coli abolished this bias. Thus, we suggest that DNA clustering, cluster immobilization through association to the endoplasmic reticulum membrane, initial proximity between the cluster and centrosomes, and subsequent differential behavior of the two centrosomes together bias the partition of plasmid DNA during mitosis. This process leads to their progressive elimination from the proliferating population and might apply to any kind of foreign DNA molecule in mammalian cells. Furthermore, the functional difference of the centrosomes might also promote the asymmetric partitioning of other cellular components in other mammalian and possibly stem cells. PMID:27298340

  12. Tubulin homolog TubZ in a phage-encoded partition system

    PubMed Central

    Oliva, María A.; Martin-Galiano, Antonio J.; Sakaguchi, Yoshihiko; Andreu, José M.

    2012-01-01

    Partition systems are responsible for the process whereby large and essential plasmids are accurately positioned to daughter cells during bacterial division. They are typically made of three components: a centromere-like DNA zone, an adaptor protein, and an assembling protein that is either a Walker-box ATPase (type I) or an actin-like ATPase (type II). A recently described type III segregation system has a tubulin/FtsZ-like protein, called TubZ, for plasmid movement. Here, we present the 2.3 Å structure and dynamic assembly of a TubZ tubulin homolog from a bacteriophage and unravel the Clostridium botulinum phage c-st type III partition system. Using biochemical and biophysical approaches, we prove that a gene upstream from tubZ encodes the partner TubR and localize the centromeric region (tubS), both of which are essential for anchoring phage DNA to the motile TubZ filaments. Finally, we describe a conserved fourth component, TubY, which modulates the TubZ-R-S complex interaction. PMID:22538818

  13. Characterization of epidemic IncI1-Iγ plasmids harboring ambler class A and C genes in Escherichia coli and Salmonella enterica from animals and humans.

    PubMed

    Smith, Hilde; Bossers, Alex; Harders, Frank; Wu, Guanghui; Woodford, Neil; Schwarz, Stefan; Guerra, Beatriz; Rodríguez, Irene; van Essen-Zandbergen, Alieda; Brouwer, Michael; Mevius, Dik

    2015-09-01

    The aim of the study was to identify the plasmid-encoded factors contributing to the emergence and spread of epidemic IncI1-Iγ plasmids obtained from Escherichia coli and Salmonella enterica isolates from animal and human reservoirs. For this, 251 IncI1-Iγ plasmids carrying various extended-spectrum β-lactamase (ESBL) or AmpC β-lactamase genes were compared using plasmid multilocus sequence typing (pMLST). Thirty-two of these plasmids belonging to different pMLST types were sequenced using Roche 454 and Illumina platforms. Epidemic IncI1-Iγ plasmids could be assigned to various dominant clades, whereas rarely detected plasmids clustered together as a distinct clade. Similar phylogenetic trees were obtained using only the plasmid backbone sequences, showing that the differences observed between the plasmids belonging to distinct clades resulted mainly from differences between their backbone sequences. Plasmids belonging to the various clades differed particularly in the presence/absence of genes encoding partitioning and addiction systems, which contribute to stable inheritance during cell division and plasmid maintenance. Despite this, plasmids belonging to the various phylogenetic clades also showed marked resistance gene associations, indicating the circulation of successful plasmid-gene combinations. The variation in traY and excA genes found in IncI1-Iγ plasmids is conserved within pMLST sequence types and plays a role in incompatibility, although functional study is needed to elucidate the role of these genes in plasmid epidemiology.

  14. Cationic Lipid–Nucleic Acid Complexes for Gene Delivery and Silencing: Pathways and Mechanisms for Plasmid DNA and siRNA

    PubMed Central

    Ewert, Kai K.; Zidovska, Alexandra; Ahmad, Ayesha; Bouxsein, Nathan F.; Evans, Heather M.; McAllister, Christopher S.; Samuel, Charles E.; Safinya, Cyrus R.

    2013-01-01

    Motivated by the promises of gene therapy, there is a large interest in developing non-viral lipid-based vectors for therapeutic applications due to their nonimmunogenicity, low toxicity, ease of production, and the potential of transferring large pieces of DNA into cells. In fact, cationic lipid (CL) based vectors are among the prevalent synthetic carriers of nucleic acids (NAs) currently used in human clinical gene therapy trials worldwide. These vectors are studied both for gene delivery with CL–DNA complexes and gene silencing with CL–siRNA (short-interfering RNA) complexes. However, their transfection efficiencies and silencing efficiencies remain low compared to those of engineered viral vectors. This reflects the currently poor understanding of transfection-related mechanisms at the molecular and self-assembled levels, including a lack of knowledge about interactions between membranes and double stranded NAs and between CL–NA complexes and cellular components. In this review, we describe our recent efforts to improve the mechanistic understanding of transfection by CL–NA complexes, which will help to design optimal lipid-based carriers of DNA and siRNA for therapeutic gene delivery and gene silencing. PMID:21504103

  15. 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

  16. Partitioning the Quaternary

    NASA Astrophysics Data System (ADS)

    Gibbard, Philip L.; Lewin, John

    2016-11-01

    We review the historical purposes and procedures for stratigraphical division and naming within the Quaternary, and summarize the current requirements for formal partitioning through the International Commission on Stratigraphy (ICS). A raft of new data and evidence has impacted traditional approaches: quasi-continuous records from ocean sediments and ice cores, new numerical dating techniques, and alternative macro-models, such as those provided through Sequence Stratigraphy and Earth-System Science. The practical usefulness of division remains, but there is now greater appreciation of complex Quaternary detail and the modelling of time continua, the latter also extending into the future. There are problems both of commission (what is done, but could be done better) and of omission (what gets left out) in partitioning the Quaternary. These include the challenge set by the use of unconformities as stage boundaries, how to deal with multiphase records in ocean and terrestrial sediments, what happened at the 'Early-Mid- (Middle) Pleistocene Transition', dealing with trends that cross phase boundaries, and the current controversial focus on how to subdivide the Holocene and formally define an 'Anthropocene'.

  17. 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

  18. Hepatocyte-targeted in vivo gene expression by intravenous injection of plasmid DNA complexed with synthetic multi-functional gene delivery system.

    PubMed

    Nishikawa, M; Yamauchi, M; Morimoto, K; Ishida, E; Takakura, Y; Hashida, M

    2000-04-01

    To achieve hepatocyte-targeted in vivo gene expression, a carrier that controls both the tissue and intracellular distribution of DNA was designed and synthesized. A cationic polymer, poly(L-ornithine) (pOrn), was modified first with galactose, then with a fusigenic peptide (mHA2) to obtain Gal-pOrn-mHA2. When applied with Gal-pOrn-mHA2 to asialoglycoprotein receptor-positive cells, fluorescein-labeled DNA showed a diffuse profile, suggesting the release of DNA from endosomes and/or lysosomes by the carrier. Then the biodistribution and gene expression after intravenous injection of DNA complexes (10 microg DNA per mouse) were examined. After injection of [32P]DNA/Gal-pOrn-mHA2, about 60% of the radioactivity was recovered in the liver, mostly in parenchymal cells. A large amount (81 ng/g tissue) of transgene product (luciferase) was detected in the liver of mice injected with DNA/Gal-pOm-mHA2, which was 280-fold greater than that obtained with DNA/DOTMA:Chol liposomes (50 microg DNA). Prior administration of galactosylated albumin reduced the gene expression to 1/100, indicating the asialoglycoprotein receptor-mediated gene transfer in liver parenchymal cells, ie hepatocytes. The luciferase activity in hepatocytes contributed more than 95% of the total activity in all the tissues examined. Thus, hepatocyte-targeted in vivo gene expression was achieved by the intravenous injection of DNA complex with the multifunctional gene carrier.

  19. Enhanced expressions and histological characteristics of intravenously administered plasmid DNA in rat lung.

    PubMed Central

    Rha, S. J.; Wang, Y. P.

    2001-01-01

    Cationic liposome-mediated gene transfection is a promising method for gene therapy. In this study, the transfection efficiency and histological patterns were evaluated in rat lung after intravenous administration via femoral vein of naked plasmid DNA, naked plasmid DNA with pretreatment of DOTAP, and DOTAP-cholesterol-plasmid DNA complex. Plasmid DNA encoding bacterial LacZ gene was used. For quantification of LacZ gene expression, beta-galactosidase assay was performed. For histologic examination, X-gal staining and immunohistochemical staining for transfected gene products were performed. Pretreatment of DOTAP prior to the infusion of naked plasmid DNA increased transfection efficiency up to a level comparable to DOTAP-cholesterol-plasmid DNA complex injection. Transfected genes were mainly expressed in type II pneumocytes and alveolar macrophages in all animals. We conclude that the high transfection efficiency is achievable by intravenous administration of naked plasmid DNA with pretreatment of DOTAP, to a level comparable to DOTAP-cholesterol-plasmid DNA complex. In this regard, naked plasmid DNA administration with pretreatment of DOTAP could be a more feasible option for intravenous gene transfer than DOTAP-cholesterol-plasmid DNA complex, in that the former is technically easier and more cost-effective than the latter with a comparable efficacy, in terms of intravenous gene delivery to the lung. PMID:11641524

  20. Using quantitative structural property relationships, chemical fate models, and the chemical partitioning space to investigate the potential for long range transport and bioaccumulation of complex halogenated chemical mixtures.

    PubMed

    Gawor, Anya; Wania, Frank

    2013-09-01

    Some substances are mixtures of very large number of constituents which vary widely in their properties, and thus also in terms of their environmental fate and the hazard that they may pose to humans and the environment. Examples of such substances include industrial chemicals such as the chlorinated paraffins, technical pesticides such as toxaphene, and unintended combustion side products, such as mixed halogenated dibenzo-p-dioxins and dibenzofurans. Here we describe a simple graphical superposition method that could precede a more detailed hazard assessment for such substances. First, partitioning and degradation properties for each individual constituent of a mixture are estimated with high-throughput quantitative structure-property relationships. Placed in a chemical partitioning space, i.e. a coordinate system defined by two partitioning coefficients, the mixtures appear as 'clouds'. When model-derived hazard assessment metrics, such as the potential for bioaccumulation and long range transport, are superimposed on these clouds, the resulting maps identify the constituents with the highest value for a particular parameter and thus potentially the greatest hazard. The maps also indicate transparently how the potential for long range transport and bioaccumulation is dependent on structural attributes, such as chain length, and the degree and type of halogenation. In contrast to previous approaches, in which the mixture is represented by a single set of properties or those of a few selected constituents, the whole range of environmental fate behaviors displayed by the constituents of a mixture are being considered. The approach is illustrated with three sets of chemical substances.

  1. 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.

  2. Plasmid mapping computer program.

    PubMed

    Nolan, G P; Maina, C V; Szalay, A A

    1984-01-11

    Three new computer algorithms are described which rapidly order the restriction fragments of a plasmid DNA which has been cleaved with two restriction endonucleases in single and double digestions. Two of the algorithms are contained within a single computer program (called MPCIRC). The Rule-Oriented algorithm, constructs all logical circular map solutions within sixty seconds (14 double-digestion fragments) when used in conjunction with the Permutation method. The program is written in Apple Pascal and runs on an Apple II Plus Microcomputer with 64K of memory. A third algorithm is described which rapidly maps double digests and uses the above two algorithms as adducts. Modifications of the algorithms for linear mapping are also presented. PMID:6320105

  3. A plasmid in Streptococcus pneumoniae.

    PubMed Central

    Smith, M D; Guild, W R

    1979-01-01

    Plasmid deoxyribonucleic acid has been detected in three related laboratory strains of Streptococcus pneumoniae. Strains D39S, R36, and R36NC each contain a minimum of two copies per cell of a 2.0-megadalton plasmid (pDP1). A plasmid twice as large as this smaller one is also present in much lower quantity in these strains, but neither plasmid is present in four strains related to these or in a drug-resistant clinical isolate from Paris. The plasmid yield was not amplified in the presence of chloramphenicol. No phenotype has been correlated with the presence of pDP1, which has existed in strains carried for many years in laboratory collections. Images PMID:33961

  4. Characterization of ESBL disseminating plasmids.

    PubMed

    Brolund, Alma; Sandegren, Linus

    2016-01-01

    Bacteria producing extended-spectrum β-lactamases (ESBLs) constitute a globally increasing problem that contributes to treatment complications and elevated death rates. The extremely successful dissemination by ESBL-producing Enterobacteriaceae during the latest decades is a result of the combination of mobilization, evolution and horizontal spread of β-lactamase genes on plasmids. In parallel, spread of these plasmids to particularly well-adapted bacterial clones (outbreak clones) has expanded. In this review we describe ESBL-producing bacteria and the genetic mechanisms for dissemination of ESBL resistance. We describe available methodology for studying plasmids and the importance of including plasmids in epidemiological typing as natural parts of the organisms. Plasmids play a fundamental role in how resistance arises and disseminates.

  5. [Evolutionary engineering in Salmonella: emergence of hybrid virulence-resistance plasmids in non-typhoid serotypes].

    PubMed

    Mendoza, María Del Carmen; Herrero, Ana; Rodicio, María Rosario

    2009-01-01

    An example of evolutive engineering in bacterial pathogens is the emergence of hybrid virulence-resistance (VR) plasmids in Salmonella enterica, resulting from an association between antimicrobial resistance determinants and specific virulence plasmids of the S. typhimurium and S. choleraesuis serotypes. VR plasmids all possess the spv (Salmonella plasmid virulence) operon, which is involved in systemic infection; however, they differ in the presence of other virulence determinants and in the resistance gene profile. VR plasmids of S. typhimurium have been found in Europe, and show resistance regions with different levels of complexity that can include class 1 integrons and various transposons. VR plasmids of S. choleraesuis, detected in strains isolated in Taiwan, only confer resistance to ampicillin and sulfonamides. Both serotypes are zoonotic and the presence of hybrid VR plasmids may confer an adaptive advantage under certain conditions, resulting in bacterial strains that are more difficult to treat and have a higher epidemic potential.

  6. 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.

  7. Stable persistence of the yeast plasmid by hitchhiking on chromosomes during vegetative and germ-line divisions of host cells

    PubMed Central

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

    2015-01-01

    The chromosome-like stability of the Saccharomyces cerevisiae plasmid 2 micron circle likely stems from its ability to tether to chromosomes and segregate by a hitchhiking mechanism. The plasmid partitioning system, responsible for chromosome-coupled segregation, is comprised of 2 plasmid coded proteins Rep1 and Rep2 and a partitioning locus STB. The evidence for the hitchhiking model for mitotic plasmid segregation, although compelling, is almost entirely circumstantial. Direct tests for plasmid-chromosome association are hampered by the limited resolving power of current cell biological tools for analyzing yeast chromosomes. Recent investigations, exploiting the improved resolution of yeast meiotic chromosomes, have revealed the plasmid's propensity to be present at or near chromosome tips. This localization is consistent with the rapid plasmid movements during meiosis I prophase, closely resembling telomere dynamics driven by a meiosis-specific nuclear envelope motor. Current evidence is consistent with the plasmid utilizing the motor as a platform for gaining access to telomeres. Episomes of viruses of the papilloma family and the gammaherpes subfamily persist in latently infected cells by tethering to chromosomes. Selfish genetic elements from fungi to mammals appear to have, by convergent evolution, arrived at the common strategy of chromosome association as a means for stable propagation. PMID:26442178

  8. Study of recombinant micro-organism populations characterized by their plasmid content per cell using a segregated model.

    PubMed

    Shene, C; Andrews, B A; Asenjo, J A

    2003-07-01

    Numerous observations from recombinant systems have shown that properties such as the specific cell growth rate and the plasmid-free cell formation rate are related, not only to the average plasmid content per cell, but also to the plasmid distribution within a population. The plasmid distribution in recombinant cultures can have an effect on the culture productivity that cannot be modelled using average values of the overall culture. The prediction of the behaviour of a plasmid content distribution and its causes and effects can only be studied using segregated models. A segregated model that describes populations of recombinant cells characterized by their plasmid content distribution has been developed. This model includes critical causes of recombinant culture instability such as the plasmid partition mechanism at cell division, plasmid replication kinetics and the effect of the plasmid content on the specific growth rate. The segregated model allows investigation of the effect of each of these causes and that of the plasmid content distribution on the observable behaviour of a recombinant culture. The effect of two partitioning mechanisms (Gaussian distribution and binomial distribution) on culture stability was investigated. The Gaussian distribution is slightly more stable. A small plasmid replication rate constant results in a very unstable culture even after short periods of time. This instability is dramatically improved for a larger value of this constant, hence improving protein synthesis. For a very narrow initial plasmid distribution, a given plasmid replication rate and partitioning mechanism can become broad even after a relatively short period of time. In contrast, a very "broad" initial distribution gave rise to a "Gamma-like" distribution profile. If we compare the results obtained in the simulations of the segregated model with those of the non-segregated one (average model), the latter model predicts much more stable behaviour, thus these

  9. Major Families of Multiresistant Plasmids from Geographically and Epidemiologically Diverse Staphylococci

    PubMed Central

    Shearer, Julia E. S.; Wireman, Joy; Hostetler, Jessica; Forberger, Heather; Borman, Jon; Gill, John; Sanchez, Susan; Mankin, Alexander; LaMarre, Jacqueline; Lindsay, Jodi A.; Bayles, Kenneth; Nicholson, Ainsley; O’Brien, Frances; Jensen, Slade O.; Firth, Neville; Skurray, Ronald A.; Summers, Anne O.

    2011-01-01

    Staphylococci are increasingly aggressive human pathogens suggesting that active evolution is spreading novel virulence and resistance phenotypes. Large staphylococcal plasmids commonly carry antibiotic resistances and virulence loci, but relatively few have been completely sequenced. We determined the plasmid content of 280 staphylococci isolated in diverse geographical regions from the 1940s to the 2000s and found that 79% of strains carried at least one large plasmid >20 kb and that 75% of these large plasmids were 20–30 kb. Using restriction fragment length polymorphism (RFLP) analysis, we grouped 43% of all large plasmids into three major families, showing remarkably conserved intercontinental spread of multiresistant staphylococcal plasmids over seven decades. In total, we sequenced 93 complete and 57 partial staphylococcal plasmids ranging in size from 1.3 kb to 64.9 kb, tripling the number of complete sequences for staphylococcal plasmids >20 kb in the NCBI RefSeq database. These plasmids typically carried multiple antimicrobial and metal resistances and virulence genes, transposases and recombinases. Remarkably, plasmids within each of the three main families were >98% identical, apart from insertions and deletions, despite being isolated from strains decades apart and on different continents. This suggests enormous selective pressure has optimized the content of certain plasmids despite their large size and complex organization. PMID:22384369

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

    PubMed

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

    2013-06-01

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

  11. 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

  12. A rational proposal for plasmid nomenclature.

    PubMed

    Campos, P; Martín Luengo, F

    1989-09-01

    We propose a more rational system for nomenclature of wild plasmids of bacteria. With this proposal for nomenclature of bacterial plasmids, it is established in an unambiguous way: 1) if a plasmid is wild or derivative, and 2) in which species and bacterial strain it was found (in the case of wild plasmids).

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

    PubMed Central

    2011-01-01

    plasmids-mediated HGT within the complex bacterial evolutionary network and in the dissemination of important biological traits. PMID:21824433

  14. Plasmid Stability in Pseudomonas fluorescens in the Rhizosphere

    PubMed Central

    van der Bij, A. J.; de Weger, L. A.; Tucker, W. T.; Lugtenberg, B.

    1996-01-01

    Plasmids belonging to various incompatibility (Inc) groups were introduced into the efficiently root-colonizing strain Pseudomonas fluorescens WCS365, and their stabilities in complex and minimal media and in the rhizospheres of tomato, wheat, and potato plants grown under gnotobiotic conditions without selection pressure were tested. The IncP plasmid was found to be highly unstable under all conditions tested, whereas the IncQ and IncW plasmids showed intermediate stabilities and the plasmids pVSP41 and pWTT2081, for which the Inc group is unknown, both containing the origin of replication (rep) and stability (sta) regions of the Pseudomonas aeruginosa pVS1 replicon, were stably maintained under all conditions tested. Growth experiments in which cells of strain WCS365 carrying the plasmid pWTT2081 were grown in the presence of WCS365 without the plasmid showed that the presence of pWTT2081 acts as a burden. We conclude that pVSP41 and pWTT2081 are valuable as stable vectors for the functional analysis of genes involved in root colonization, provided that control cells carry the empty vector. PMID:16535259

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

    PubMed

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

    2013-09-01

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

  16. Distribution of Genes Encoding Nucleoid-Associated Protein Homologs in Plasmids

    PubMed Central

    Takeda, Toshiharu; Yun, Choong-Soo; Shintani, Masaki; Yamane, Hisakazu; Nojiri, Hideaki

    2011-01-01

    Bacterial nucleoid-associated proteins (NAPs) form nucleoprotein complexes and influence the expression of genes. Recent studies have shown that some plasmids carry genes encoding NAP homologs, which play important roles in transcriptional regulation networks between plasmids and host chromosomes. In this study, we determined the distributions of the well-known NAPs Fis, H-NS, HU, IHF, and Lrp and the newly found NAPs MvaT and NdpA among the whole-sequenced 1382 plasmids found in Gram-negative bacteria. Comparisons between NAP distributions and plasmid features (size, G+C content, and putative transferability) were also performed. We found that larger plasmids frequently have NAP gene homologs. Plasmids with H-NS gene homologs had less G+C content. It should be noted that plasmids with the NAP gene homolog also carried the relaxase gene involved in the conjugative transfer of plasmids more frequently than did those without the NAP gene homolog, implying that plasmid-encoded NAP homologs positively contribute to transmissible plasmids. PMID:21350637

  17. The Benefits of Adaptive Partitioning for Parallel AMR Applications

    SciTech Connect

    Steensland, Johan

    2008-07-01

    Parallel adaptive mesh refinement methods potentially lead to realistic modeling of complex three-dimensional physical phenomena. However, the dynamics inherent in these methods present significant challenges in data partitioning and load balancing. Significant human resources, including time, effort, experience, and knowledge, are required for determining the optimal partitioning technique for each new simulation. In reality, scientists resort to using the on-board partitioner of the computational framework, or to using the partitioning industry standard, ParMetis. Adaptive partitioning refers to repeatedly selecting, configuring and invoking the optimal partitioning technique at run-time, based on the current state of the computer and application. In theory, adaptive partitioning automatically delivers superior performance and eliminates the need for repeatedly spending valuable human resources for determining the optimal static partitioning technique. In practice, however, enabling frameworks are non-existent due to the inherent significant inter-disciplinary research challenges. This paper presents a study of a simple implementation of adaptive partitioning and discusses implied potential benefits from the perspective of common groups of users within computational science. The study is based on a large set of data derived from experiments including six real-life, multi-time-step adaptive applications from various scientific domains, five complementing and fundamentally different partitioning techniques, a large set of parameters corresponding to a wide spectrum of computing environments, and a flexible cost function that considers the relative impact of multiple partitioning metrics and diverse partitioning objectives. The results show that even a simple implementation of adaptive partitioning can automatically generate results statistically equivalent to the best static partitioning. Thus, it is possible to effectively eliminate the problem of determining the

  18. Fuzzy Partition Models for Fitting a Set of Partitions.

    ERIC Educational Resources Information Center

    Gordon, A. D.; Vichi, M.

    2001-01-01

    Describes methods for fitting a fuzzy consensus partition to a set of partitions of the same set of objects. Describes and illustrates three models defining median partitions and compares these methods to an alternative approach to obtaining a consensus fuzzy partition. Discusses interesting differences in the results. (SLD)

  19. Carbon partitioning in photosynthesis.

    PubMed

    Melis, Anastasios

    2013-06-01

    The work seeks to raise awareness of a fundamental problem that impacts the renewable generation of fuels and chemicals via (photo)synthetic biology. At issue is regulation of the endogenous cellular carbon partitioning between different biosynthetic pathways, over which the living cell exerts stringent control. The regulation of carbon partitioning in photosynthesis is not understood. In plants, microalgae and cyanobacteria, methods need be devised to alter photosynthetic carbon partitioning between the sugar, terpenoid, and fatty acid biosynthetic pathways, to lower the prevalence of sugar biosynthesis and correspondingly upregulate terpenoid and fatty acid hydrocarbons production in the cell. Insight from unusual but naturally occurring carbon-partitioning processes can help in the design of blueprints for improved photosynthetic fuels and chemicals production.

  20. 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

  1. A comparison of ethanol partitioning in biological and model membranes: nonideal partitioning is enhanced in synaptosomal membranes.

    PubMed

    Sarasua, M M; Faught, K R; Steedman, S L; Gordin, M D; Washington, M K

    1989-10-01

    The partitioning of ethanol into mouse brain synaptosomes at 37 degrees C was characterized as a function of ethanol concentration. In addition, the partitioning of ethanol into multilamellar dipalmitoylphosphatidylcholine (DPPC) vesicles was characterized as a function of ethanol concentration and temperature. DPPC liposomes provided a model for ethanol partitioning into a phospholipid bilayer of defined composition allowing comparison to the more complex synaptosomal membrane. The values of the partition coefficients for ethanol depend on the convention used to express concentration in the partition coefficient ratio. We express these concentrations as mole fractions as ethanol in the membrane and aqueous phases. Ethanol partitioning is nonideal (ethanol membrane: buffer partition coefficients vary with total ethanol concentration). In synaptosomes, the partition coefficients vary markedly with concentration and asymptotically approach zero at higher concentrations. In the DPPC system, the variation of the partition coefficient is less pronounced, but significant. The ethanol: DPPC partition coefficients decrease by a factor of 2 at ethanol concentrations above 3.2 x 10(-3) M. This suggests a model involving at least two distinguishable types of interactions of ethanol with the membrane. Ethanol appears to undergo both bulk phase partitioning into the membrane bilayer core and nonspecific binding to the membrane surface. In pure DPPC, bulk phase hydrophobic partitioning predominates. In synaptosomes, nonspecific surface binding appears to be a major interaction. Temperature studies indicate ethanol partitioning into DPPC increases above the phospholipid gel to liquid crystalline phase transition temperature. This suggests a preferred partitioning of ethanol into fluid state lipid. However, significant membrane concentrations of ethanol are found in gel state DPPC.

  2. The Emergence of DP in the Partitive Structure

    ERIC Educational Resources Information Center

    Stickney, Helen

    2009-01-01

    This dissertation is a first look at English-speaking children's acquisition of the syntax of the partitive. It presents four experiments that contrast three types of structures and examines how they interact with adjectival modification: the partitive, the pseudopartitive and complex nouns with prepositional adjuncts. The experimentation…

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

    PubMed

    Klümper, Uli; Riber, Leise; Dechesne, Arnaud; Sannazzarro, Analia; Hansen, Lars H; Sørensen, Søren J; Smets, Barth F

    2015-03-17

    Conjugal plasmids can provide microbes with full complements of new genes and constitute potent vehicles for horizontal gene transfer. Conjugal plasmid transfer is deemed responsible for the rapid spread of antibiotic resistance among microbes. While broad host range plasmids are known to transfer to diverse hosts in pure culture, the extent of their ability to transfer in the complex bacterial communities present in most habitats has not been comprehensively studied. Here, we isolated and characterized transconjugants with a degree of sensitivity not previously realized to investigate the transfer range of IncP- and IncPromA-type broad host range plasmids from three proteobacterial donors to a soil bacterial community. We identified transfer to many different recipients belonging to 11 different bacterial phyla. The prevalence of transconjugants belonging to diverse Gram-positive Firmicutes and Actinobacteria suggests that inter-Gram plasmid transfer of IncP-1 and IncPromA-type plasmids is a frequent phenomenon. While the plasmid receiving fractions of the community were both plasmid- and donor- dependent, we identified a core super-permissive fraction that could take up different plasmids from diverse donor strains. This fraction, comprising 80% of the identified transconjugants, thus has the potential to dominate IncP- and IncPromA-type plasmid transfer in soil. Our results demonstrate that these broad host range plasmids have a hitherto unrecognized potential to transfer readily to very diverse bacteria and can, therefore, directly connect large proportions of the soil bacterial gene pool. This finding reinforces the evolutionary and medical significances of these plasmids.

  4. Building mosaics of therapeutic plasmid gene vectors.

    PubMed

    Tolmachov, Oleg E

    2011-12-01

    Plasmids are circular or linear DNA molecules propagated extra-chromosomally in bacteria. Evolution shaped plasmids are inherently mosaic structures with individual functional units represented by distinct segments in the plasmid genome. The patchwork of plasmid genetic modules is a convenient template and a model for the generation of artificial plasmids used as vehicles for gene delivery into human cells. Plasmid gene vectors are an important tool in gene therapy and in basic biomedical research, where these vectors offer efficient transgene expression in many settings in vitro and in vivo. Plasmid vectors can be attached to nuclear directing ligands or transferred by electroporation as naked DNA to deliver the payload genes to the nuclei of the target cells. Transgene expression silencing by plasmid sequences of bacterial origin and immune stimulation by bacterial unmethylated CpG motifs can be avoided by the generation of plasmid-based minimized DNA vectors, such as minicircles. Systems of efficient site-specific integration into human chromosomes and stable episomal maintenance in human cells are being developed for further reduction of the chances for transgene silencing. The successful generation of plasmid vectors is governed by a number of vector design rules, some of which are common to all gene vectors, while others are specific to plasmid vectors. This review is focused both on the guiding principles and on the technical know-how of plasmid gene vector design. PMID:22023476

  5. Partitioning structural VHDL circuits for parallel execution on hypercubes

    NASA Astrophysics Data System (ADS)

    Kapp, Kevin L.

    1993-12-01

    Distributing simulations among multiple processors is one approach to reducing VHDL simulation time for large VLSI circuit designs. However, parallel simulation introduces the problem of how to partition the logic gates and system behaviors among the available processors in order to obtain maximum speedup. This research investigates deliberate partitioning algorithms that account for the complex inter-dependency structure of the circuit behaviors. Once an initial partition has been obtained, a border annealing algorithm is used to iteratively improve the partition. In addition, methods of measuring the cost of a partition and relating it to the resulting simulation performance are investigated. Structural circuits ranging from one thousand to over four thousand behaviors are simulated. The deliberate partitions consistently provided superior speedup to a random distribution of the circuit behaviors.

  6. Monitoring plasmid replication in live mammalian cells over multiple generations by fluorescence microscopy.

    PubMed

    Norby, Kathryn; Chiu, Ya-Fang; Sugden, Bill

    2012-01-01

    Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies (1-3). These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres (4-8). Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell. Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest (9). The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed. This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to

  7. Partition density functional theory

    NASA Astrophysics Data System (ADS)

    Nafziger, Jonathan

    Partition density functional theory (PDFT) is a method for dividing a molecular electronic structure calculation into fragment calculations. The molecular density and energy corresponding to Kohn Sham density-functional theory (KS-DFT) may be exactly recovered from these fragments. Each fragment acts as an isolated system except for the influence of a global one-body 'partition' potential which deforms the fragment densities. In this work, the developments of PDFT are put into the context of other fragment-based density functional methods. We developed three numerical implementations of PDFT: One within the NWChem computational chemistry package using basis sets, and the other two developed from scratch using real-space grids. It is shown that all three of these programs can exactly reproduce a KS-DFT calculation via fragment calculations. The first of our in-house codes handles non-interacting electrons in arbitrary one-dimensional potentials with any number of fragments. This code is used to explore how the exact partition potential changes for different partitionings of the same system and also to study features which determine which systems yield non-integer PDFT occupations and which systems are locked into integer PDFT occupations. The second in-house code, CADMium, performs real-space calculations of diatomic molecules. Features of the exact partition potential are studied for a variety of cases and an analytical formula determining singularities in the partition potential is derived. We introduce an approximation for the non-additive kinetic energy and show how this quantity can be computed exactly. Finally a PDFT functional is developed to address the issues of static correlation and delocalization errors in approximations within DFT. The functional is applied to the dissociation of H2 + and H2.

  8. FNAS phase partitions

    NASA Technical Reports Server (NTRS)

    Vanalstine, James M.

    1993-01-01

    Project NAS8-36955 D.O. #100 initially involved the following tasks: (1) evaluation of various coatings' ability to control wall wetting and surface zeta potential expression; (2) testing various methods to mix and control the demixing of phase systems; and (3) videomicroscopic investigation of cell partition. Three complementary areas were identified for modification and extension of the original contract. They were: (1) identification of new supports for column cell partition; (2) electrokinetic detection of protein adsorption; and (3) emulsion studies related to bioseparations.

  9. Plasmids as stochastic model systems

    NASA Astrophysics Data System (ADS)

    Paulsson, Johan

    2003-05-01

    Plasmids are self-replicating gene clusters present in on average 2-100 copies per bacterial cell. To reduce random fluctuations and thereby avoid extinction, they ubiquitously autoregulate their own synthesis using negative feedback loops. Here I use van Kampen's Ω-expansion for a two-dimensional model of negative feedback including plasmids and ther replication inhibitors. This analytically summarizes the standard perspective on replication control -- including the effects of sensitivity amplification, exponential time-delays and noisy signaling. I further review the two most common molecular sensitivity mechanisms: multistep control and cooperativity. Finally, I discuss more controversial sensitivity schemes, such as noise-enhanced sensitivity, the exploitation of small-number combinatorics and double-layered feedback loops to suppress noise in disordered environments.

  10. Segrosome Complex Formation during DNA Trafficking in Bacterial Cell Division

    PubMed Central

    Oliva, María A.

    2016-01-01

    Bacterial extrachromosomal DNAs often contribute to virulence in pathogenic organisms or facilitate adaptation to particular environments. The transmission of genetic information from one generation to the next requires sufficient partitioning of DNA molecules to ensure that at least one copy reaches each side of the division plane and is inherited by the daughter cells. Segregation of the bacterial chromosome occurs during or after replication and probably involves a strategy in which several protein complexes participate to modify the folding pattern and distribution first of the origin domain and then of the rest of the chromosome. Low-copy number plasmids rely on specialized partitioning systems, which in some cases use a mechanism that show striking similarity to eukaryotic DNA segregation. Overall, there have been multiple systems implicated in the dynamic transport of DNA cargo to a new cellular position during the cell cycle but most seem to share a common initial DNA partitioning step, involving the formation of a nucleoprotein complex called the segrosome. The particular features and complex topologies of individual segrosomes depend on both the nature of the DNA binding protein involved and on the recognized centromeric DNA sequence, both of which vary across systems. The combination of in vivo and in vitro approaches, with structural biology has significantly furthered our understanding of the mechanisms underlying DNA trafficking in bacteria. Here, I discuss recent advances and the molecular details of the DNA segregation machinery, focusing on the formation of the segrosome complex. PMID:27668216

  11. Segrosome Complex Formation during DNA Trafficking in Bacterial Cell Division

    PubMed Central

    Oliva, María A.

    2016-01-01

    Bacterial extrachromosomal DNAs often contribute to virulence in pathogenic organisms or facilitate adaptation to particular environments. The transmission of genetic information from one generation to the next requires sufficient partitioning of DNA molecules to ensure that at least one copy reaches each side of the division plane and is inherited by the daughter cells. Segregation of the bacterial chromosome occurs during or after replication and probably involves a strategy in which several protein complexes participate to modify the folding pattern and distribution first of the origin domain and then of the rest of the chromosome. Low-copy number plasmids rely on specialized partitioning systems, which in some cases use a mechanism that show striking similarity to eukaryotic DNA segregation. Overall, there have been multiple systems implicated in the dynamic transport of DNA cargo to a new cellular position during the cell cycle but most seem to share a common initial DNA partitioning step, involving the formation of a nucleoprotein complex called the segrosome. The particular features and complex topologies of individual segrosomes depend on both the nature of the DNA binding protein involved and on the recognized centromeric DNA sequence, both of which vary across systems. The combination of in vivo and in vitro approaches, with structural biology has significantly furthered our understanding of the mechanisms underlying DNA trafficking in bacteria. Here, I discuss recent advances and the molecular details of the DNA segregation machinery, focusing on the formation of the segrosome complex.

  12. Segrosome Complex Formation during DNA Trafficking in Bacterial Cell Division.

    PubMed

    Oliva, María A

    2016-01-01

    Bacterial extrachromosomal DNAs often contribute to virulence in pathogenic organisms or facilitate adaptation to particular environments. The transmission of genetic information from one generation to the next requires sufficient partitioning of DNA molecules to ensure that at least one copy reaches each side of the division plane and is inherited by the daughter cells. Segregation of the bacterial chromosome occurs during or after replication and probably involves a strategy in which several protein complexes participate to modify the folding pattern and distribution first of the origin domain and then of the rest of the chromosome. Low-copy number plasmids rely on specialized partitioning systems, which in some cases use a mechanism that show striking similarity to eukaryotic DNA segregation. Overall, there have been multiple systems implicated in the dynamic transport of DNA cargo to a new cellular position during the cell cycle but most seem to share a common initial DNA partitioning step, involving the formation of a nucleoprotein complex called the segrosome. The particular features and complex topologies of individual segrosomes depend on both the nature of the DNA binding protein involved and on the recognized centromeric DNA sequence, both of which vary across systems. The combination of in vivo and in vitro approaches, with structural biology has significantly furthered our understanding of the mechanisms underlying DNA trafficking in bacteria. Here, I discuss recent advances and the molecular details of the DNA segregation machinery, focusing on the formation of the segrosome complex. PMID:27668216

  13. STRUCTURAL DYNAMICS OF METAL PARTITIONING TO MINERAL SURFACES

    EPA Science Inventory

    The conceptual understanding of surface complexation reactions that control trace element partitioning to mineral surfaces is limited by the assumption that the solid reactant possesses a finite, time-invariant population of surface functional groups. This assumption has limited...

  14. [The relationship of plasmids from environmental Yersinia isolates and the virulence plasmid of enteropathogenic Yersinia strains].

    PubMed

    Hoffmann, B; Strauch, E; Appel, B; Nattermann, H

    2002-01-01

    The human pathogenic strains of Yersinia harbour a conserved plasmid carrying the Yop virulon. The virulence plasmid of Yersinia enterocolitica strains belonging to the serogroups O:3 and O:9 were used as probes to detect homologous sequences in plasmids of "avirulent" Yersinia strains. "Avirulent" Yersinia strains (Y. enterocolitica biogroup 1A, Y. intermedia, Y. kristensenii and Y. frederiksenii) lack the virulence plasmid. They are widely distributed in the environment and can frequently be isolated from clinical samples. Hybridisation experiments revealed a number of common genetic elements of the virulence plasmid and the plasmids of "avirulent" Yersinia strains. These elements were identified as genes involved in plasmid replication, as an endonuclease gene and as mobile genetic elements. However, none of the plasmid encoded virulence genes was present in the plasmids of "avirulent" Yersinia strains. The frequent occurrence and the possible etiological relevance of "avirulent" isolates will be discussed.

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

    PubMed

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

    2014-12-01

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

  16. Biogenic SOA formation through gas-phase oxidation and gas-to-particle partitioning - comparison between process models of varying complexity

    NASA Astrophysics Data System (ADS)

    Hermansson, E.; Roldin, P.; Rusanen, A.; Mogensen, D.; Kivekäs, N.; Boy, M.; Swietlicki, E.

    2014-05-01

    Biogenic volatile organic compounds (BVOCs) emitted by the vegetation play an important role for the aerosol mass loadings since the oxidation products of these compounds can take part in the formation and growth of secondary organic aerosols (SOA). The concentrations and properties of BVOCs and their oxidation products in the atmosphere are poorly characterized, which leads to high uncertainties in modeled SOA mass and properties. In this study the formation of SOA has been modeled along an air mass trajectory over the northern European boreal forest using two aerosol dynamics box models where the prediction of the condensable organics from the gas-phase oxidation of BVOC is handled with schemes of varying complexity. The use of box model simulations along an air mass trajectory allows us to, under atmospheric relevant conditions, compare different model parameterizations and their effect on SOA formation. The result of the study shows that the modeled mass concentration of SOA is highly dependent on the organic oxidation scheme used to predict the oxidation products. A near-explicit treatment of organic gas-phase oxidation (Master Chemical Mechanism version 3.2) was compared to oxidation schemes that use the volatility basis set (VBS) approach. The resulting SOA mass modeled with different VBS-schemes varies by a factor of about 7 depending on how the first generation oxidation products are parameterized and how they subsequently age (e.g. how fast the gas-phase oxidation products react with the OH-radical, how they respond to temperature changes and if they are allowed to fragment during the aging process). Since the VBS approach is frequently used in regional and global climate models due to its relatively simple treatment of the oxidation products compared to near-explicit oxidation schemes; better understanding of the abovementioned processes are needed. Compared to the most commonly used VBS-schemes, the near-explicit method produces less - but more oxidized

  17. 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.

  18. Biogenic SOA formation through gas-phase oxidation and gas-to-particle partitioning - a comparison between process models of varying complexity

    NASA Astrophysics Data System (ADS)

    Hermansson, E.; Roldin, P.; Rusanen, A.; Mogensen, D.; Kivekäs, N.; Väänänen, R.; Boy, M.; Swietlicki, E.

    2014-11-01

    Biogenic volatile organic compounds (BVOCs) emitted by vegetation play an important role for aerosol mass loadings since the oxidation products of these compounds can take part in the formation and growth of secondary organic aerosols (SOA). The concentrations and properties of BVOCs and their oxidation products in the atmosphere are poorly characterized, which leads to high uncertainties in modeled SOA mass and properties. In this study, the formation of SOA has been modeled along an air-mass trajectory over northern European boreal forest using two aerosol dynamics box models where the prediction of the condensable organics from the gas-phase oxidation of BVOC is handled with schemes of varying complexity. The use of box model simulations along an air-mass trajectory allows us to compare, under atmospheric relevant conditions, different model parameterizations and their effect on SOA formation. The result of the study shows that the modeled mass concentration of SOA is highly dependent on the organic oxidation scheme used to predict oxidation products. A near-explicit treatment of organic gas-phase oxidation (Master Chemical Mechanism version 3.2) was compared to oxidation schemes that use the volatility basis set (VBS) approach. The resulting SOA mass modeled with different VBS schemes varies by a factor of about 7 depending on how the first-generation oxidation products are parameterized and how they subsequently age (e.g., how fast the gas-phase oxidation products react with the OH radical, how they respond to temperature changes, and if they are allowed to fragment during the aging process). Since the VBS approach is frequently used in regional and global climate models due to its relatively simple treatment of the oxidation products compared to near-explicit oxidation schemes, a better understanding of the above-mentioned processes is needed. Based on the results of this study, fragmentation should be included in order to obtain a realistic SOA formation

  19. Variable length adjacent partitioning for PTS based PAPR reduction of OFDM signal

    SciTech Connect

    Ibraheem, Zeyid T.; Rahman, Md. Mijanur; Yaakob, S. N.; Razalli, Mohammad Shahrazel; Kadhim, Rasim A.

    2015-05-15

    Peak-to-Average power ratio (PAPR) is a major drawback in OFDM communication. It leads the power amplifier into nonlinear region operation resulting into loss of data integrity. As such, there is a strong motivation to find techniques to reduce PAPR. Partial Transmit Sequence (PTS) is an attractive scheme for this purpose. Judicious partitioning the OFDM data frame into disjoint subsets is a pivotal component of any PTS scheme. Out of the existing partitioning techniques, adjacent partitioning is characterized by an attractive trade-off between cost and performance. With an aim of determining effects of length variability of adjacent partitions, we performed an investigation into the performances of a variable length adjacent partitioning (VL-AP) and fixed length adjacent partitioning in comparison with other partitioning schemes such as pseudorandom partitioning. Simulation results with different modulation and partitioning scenarios showed that fixed length adjacent partition had better performance compared to variable length adjacent partitioning. As expected, simulation results showed a slightly better performance of pseudorandom partitioning technique compared to fixed and variable adjacent partitioning schemes. However, as the pseudorandom technique incurs high computational complexities, adjacent partitioning schemes were still seen as favorable candidates for PAPR reduction.

  20. Positive epistasis between co-infecting plasmids promotes plasmid survival in bacterial populations.

    PubMed

    San Millan, Alvaro; Heilbron, Karl; MacLean, R Craig

    2014-03-01

    Plasmids have a key role in the horizontal transfer of genes among bacteria. Although plasmids are catalysts for bacterial evolution, it is challenging to understand how they can persist in bacterial populations over the long term because of the burden they impose on their hosts (the 'plasmid paradox'). This paradox is especially perplexing in the case of 'small' plasmids, which are unable to self-transfer by conjugation. Here, for the first time, we investigate how interactions between co-infecting plasmids influence plasmid persistence. Using an experimental model system based on interactions between a diverse assemblage of 'large' plasmids and a single small plasmid, pNI105, in the pathogenic bacterium Pseudomonas aeruginosa, we demonstrate that positive epistasis minimizes the cost associated with carrying multiple plasmids over the short term and increases the stability of the small plasmid over a longer time scale. In support of these experimental data, bioinformatic analysis showed that associations between small and large plasmids are more common than would be expected owing to chance alone across a range of families of bacteria; more generally, we find that co-infection with multiple plasmids is more common than would be expected owing to chance across a wide range of bacterial phyla. Collectively, these results suggest that positive epistasis promotes plasmid stability in bacterial populations. These findings pave the way for future mechanistic studies aimed at elucidating the molecular mechanisms of plasmid-plasmid interaction, and evolutionary studies aimed at understanding how the coevolution of plasmids drives the spread of plasmid-encoded traits.

  1. Effects of genes exerting growth inhibition and plasmid stability on plasmid maintenance.

    PubMed

    Boe, L; Gerdes, K; Molin, S

    1987-10-01

    Plasmid stabilization mediated by the parA+ and parB+ genes of the R1 plasmid and the ccd+ and sop+ genes of the F plasmid was tested on a mini-R1 plasmid and a pBR322 plasmid derivative. The mini-R1 plasmid is thought to be unstably inherited owing to a low copy number and to random segregation of the plasmid at cell division, whereas cells harboring the pBR322 derivative used in this work are lost through competition with plasmid-free cells, mainly as a result of the shorter generation time of cells without plasmids. The pBR322 derivative carries a fusion between part of the atp operon of Escherichia coli and the bacteriophage lambda pR promoter, and the cI857 repressor gene. The insertion of sop+ from the F plasmid or parB+ from the R1 plasmid reduced the loss frequency by a factor of 10(3) for the pBR322 derivative and by at least a factor of 10(2) for the mini-R1 plasmid. Insertion of parA+ from the R1 plasmid decreased the loss frequency of the pBR322 derivative by a factor of 10 and that of the mini-R1 plasmid by a factor of 50. When ccd+ from the F plasmid was inserted, the loss frequency of the pBR322 derivative was decreased by a factor of 10, but it had only a marginal effect on the stability of the mini-R1 plasmid. In no case was any significant structural instability of the plasmids observed.

  2. Mechanism of DNA Segregation in Prokaryotes: Replicon Pairing by parC of Plasmid R1

    NASA Astrophysics Data System (ADS)

    Jensen, Rasmus Bugge; Lurz, Rudi; Gerdes, Kenn

    1998-07-01

    Prokaryotic chromosomes and plasmids encode partitioning systems that are required for DNA segregation at cell division. The systems are thought to be functionally analogous to eukaryotic centromeres and to play a general role in DNA segregation. The parA system of plasmid R1 encodes two proteins ParM and ParR, and a cis-acting centromere-like site denoted parC. The ParR protein binds to parC in vivo and in vitro. The ParM protein is an ATPase that interacts with ParR specifically bound to parC. Using electron microscopy, we show here that parC mediates efficient pairing of plasmid molecules. The pairing requires binding of ParR to parC and is stimulated by the ParM ATPase. The ParM mediated stimulation of plasmid pairing is dependent on ATP hydrolysis by ParM. Using a ligation kinetics assay, we find that ParR stimulates ligation of parC-containing DNA fragments. The rate-of-ligation was increased by wild type ParM protein but not by mutant ParM protein deficient in the ATPase activity. Thus, two independent assays show that parC mediates pairing of plasmid molecules in vitro. These results are consistent with the proposal that replicon pairing is part of the mechanism of DNA segregation in prokaryotes.

  3. The Complete Sequence and Comparative Analysis of a Multidrug-Resistance and Virulence Multireplicon IncFII Plasmid pEC302/04 from an Extraintestinal Pathogenic Escherichia coli EC302/04 Indicate Extensive Diversity of IncFII Plasmids

    PubMed Central

    Ho, Wing Sze; Yap, Kien-Pong; Yeo, Chew Chieng; Rajasekaram, Ganeswrie; Thong, Kwai Lin

    2016-01-01

    Extraintestinal pathogenic Escherichia coli (ExPEC) that causes extraintestinal infections often harbor plasmids encoding fitness traits such as resistance and virulence determinants that are of clinical importance. We determined the complete nucleotide sequence of plasmid pEC302/04 from a multidrug-resistant E. coli EC302/04 which was isolated from the tracheal aspirate of a patient in Malaysia. In addition, we also performed comparative sequence analyses of 18 related IncFIIA plasmids to determine the phylogenetic relationship and diversity of these plasmids. The 140,232 bp pEC302/04 is a multireplicon plasmid that bears three replication systems (FII, FIA, and FIB) with subtype of F2:A1:B1. The plasmid is self-transmissible with a complete transfer region. pEC302/04 also carries antibiotic resistance genes such as blaTEM−1 and a class I integron containing sul1, cml and aadA resistance genes, conferring multidrug resistance (MDR) to its host, E. coli EC302/04. Besides, two iron acquisition systems (SitABCD and IutA-IucABCD) which are the conserved virulence determinants of ExPEC-colicin V or B and M (ColV/ColBM)-producing plasmids were identified in pEC302/04. Multiple toxin-antitoxin (TA)-based addiction systems (i.e., PemI/PemK, VagC/VagD, CcdA/CcdB, and Hok/Sok) and a plasmid partitioning system, ParAB, and PsiAB, which are important for plasmid maintenance were also found. Comparative plasmid analysis revealed only one conserved gene, the repA1 as the core genome, showing that there is an extensive diversity among the IncFIIA plasmids. The phylogenetic relationship of 18 IncF plasmids based on the core regions revealed that ColV/ColBM-plasmids and non-ColV/ColBM plasmids were separated into two distinct groups. These plasmids, which carry highly diverse genetic contents, are also mosaic in nature. The atypical combination of genetic materials, i.e., the MDR- and ColV/ColBM-plasmid-virulence encoding regions in a single ExPEC plasmid is rare but of clinical

  4. Multimedia partitioning of dioxin

    SciTech Connect

    Travis, C.C.; Hattemer-Frey, H.A.

    1988-01-01

    The general population is continuously being exposed to trace amounts of dioxin as exemplified by the fact that virtually all human adipose tissue samples contain dioxin levels of three parts per trillion (ppT) or greater. The purpose of this study is to investigate how 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is partitioned in the environment and to identify the major pathways of human exposure. 61 refs., 6 tabs.

  5. 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.

  6. 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

  7. Characterization of plasmids in bacterial fish pathogen.

    PubMed Central

    Toranzo, A E; Barja, J L; Colwell, R R; Hetrick, F M

    1983-01-01

    Plasmid profiles of representative fish pathogens, Aeromonas salmonicida, Aeromonas hydrophila, Vibrio anguillarum, Pasteurella piscicida, Yersinia ruckeri, Edwardsiella tarda, and Renibacterium salmoninarum, were determined by agarose gel electrophoresis with four different plasmid detection methods. A combination of two methods was required to detect the plasmids present in these strains and to calculate precisely the molecular weights of the plasmids. Of 38 strains, 28 harbored one or more plasmids, with the majority of strains demonstrating multiplasmid banding. Similarity in plasmid banding between strains was noted and related to geographic source. Five strains of A. salmonicida possessed six plasmid bands having molecular weights of 8.6 X 10(6), 8.4 X 10(6), 8.1 X 10(6), 3.6 X 10(6), 3.5 X 10(6), and 3.4 X 10(6). Four P. piscicida isolates shared three plasmid bands having molecular weights of 37 X 10(6), 15 X 10(6), and 5 X 10(6), and five A. hydrophila strains harbored a common plasmid having a molecular weight of ca. 20 X 10(6) to 30 X 10(6). The highest-molecular-weight plasmids (145 X 10(6) and 130 X 10(6) were detected in V. anguillarum. From curing experiments, it was found that in A. hydrophila strain 79-62, a loss of resistance to tetracycline was associated with loss of plasmid content in all susceptible derivatives, suggesting plasmid-mediated tetracycline resistance. Cell surface characteristics and metabolic properties were also modified in cured derivatives of A. hydrophila strain 79-62. Images PMID:6822413

  8. Superporous agarose anion exchangers for plasmid isolation.

    PubMed

    Tiainen, Peter; Gustavsson, Per-Erik; Ljunglöf, Anders; Larsson, Per-Olof

    2007-01-01

    Superporous agarose beads have wide, connecting flow pores allowing large molecules such as plasmids to be transported into the interior of the beads by convective flow. The pore walls provide additional surface for plasmid binding thus increasing the binding capacity of the adsorbent. Novel superporous agarose anion exchangers have been prepared, differing with respect to bead diameter, superpore diameter and type of anion-exchange functional group (poly(ethyleneimine) and quaternary amine). The plasmid binding capacities were obtained from breakthrough curves and compared with the binding capacity of homogeneous agarose beads of the same particle size. Significantly, the smaller diameter superporous agarose beads were found to have four to five times higher plasmid binding capacity than the corresponding homogeneous agarose beads. The experimentally determined plasmid binding capacity was compared with the theoretically calculated surface area for each adsorbent and fair agreement was found. Confocal microscopy studies of beads with adsorbed, fluorescently labelled plasmids aided in the interpretation of the results. Superporous poly(ethyleneimine)-substituted beads with a high ion capacity (230 micromol/ml) showed a plasmid binding of 3-4 mg/ml adsorbent. Superporous quaternary amine-substituted beads had a lower ion capacity (81 micromol/ml) and showed a correspondingly lower plasmid binding capacity (1-2 mg/ml adsorbent). In spite of the lower capacity, the beads with quaternary amine ligand were preferred, due to their much better plasmid recovery (70-100% recovery). Interestingly, both capacity and recovery was improved when the plasmid adsorption step was carried out in the presence of a moderate salt concentration. The most suitable superporous bead type (45-75 microm diameter beads; 4 microm superpores; quaternary amine ligand) was chosen for the capture of plasmid DNA from a clarified alkaline lysate. Two strategies were evaluated, one with and one

  9. ParA2, a Vibrio cholerae chromosome partitioning protein, forms left-handed helical filaments on DNA.

    PubMed

    Hui, Monica P; Galkin, Vitold E; Yu, Xiong; Stasiak, Alicja Z; Stasiak, Andrzej; Waldor, Matthew K; Egelman, Edward H

    2010-03-01

    Most bacterial chromosomes contain homologs of plasmid partitioning (par) loci. These loci encode ATPases called ParA that are thought to contribute to the mechanical force required for chromosome and plasmid segregation. In Vibrio cholerae, the chromosome II (chrII) par locus is essential for chrII segregation. Here, we found that purified ParA2 had ATPase activities comparable to other ParA homologs, but, unlike many other ParA homologs, did not form high molecular weight complexes in the presence of ATP alone. Instead, formation of high molecular weight ParA2 polymers required DNA. Electron microscopy and three-dimensional reconstruction revealed that ParA2 formed bipolar helical filaments on double-stranded DNA in a sequence-independent manner. These filaments had a distinct change in pitch when ParA2 was polymerized in the presence of ATP versus in the absence of a nucleotide cofactor. Fitting a crystal structure of a ParA protein into our filament reconstruction showed how a dimer of ParA2 binds the DNA. The filaments formed with ATP are left-handed, but surprisingly these filaments exert no topological changes on the right-handed B-DNA to which they are bound. The stoichiometry of binding is one dimer for every eight base pairs, and this determines the geometry of the ParA2 filaments with 4.4 dimers per 120 A pitch left-handed turn. Our findings will be critical for understanding how ParA proteins function in plasmid and chromosome segregation.

  10. 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.

  11. 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.

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

    PubMed

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

    2014-01-01

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

  13. Expansive spread of IncI1 plasmids carrying blaCMY-2 amongst Escherichia coli.

    PubMed

    Sidjabat, Hanna E; Seah, Kwee Yong; Coleman, Lyndall; Sartor, Anna; Derrington, Petra; Heney, Claire; Faoagali, Joan; Nimmo, Graeme R; Paterson, David L

    2014-09-01

    Escherichia coli is a leading cause of urinary tract infections. One of the most common antibiotic classes used to treat such infections is the β-lactams, including cephalosporins. Resistance to the third-generation cephalosporins can be caused by production of extended-spectrum β-lactamases (ESBLs) or plasmid-mediated AmpC β-lactamases. The most commonly reported AmpC β-lactamase in E. coli is CMY-2. Plasmid-mediated CMY-2 has been frequently reported in E. coli and Salmonella sp. from food-producing animals. This study aimed to elucidate the molecular characteristics of clinical E. coli isolates carrying plasmids encoding CMY-2. A total of 67 CMY-2-producing E. coli were characterised by clonal analysis and phylogenetic typing. Characterisation of the plasmids carrying blaCMY-2 included replicon typing, plasmid profiling, plasmid transferability and sequencing of the blaCMY-2 genetic environment. As a result, E. coli producing CMY-2 was found to be highly polyclonal. The majority of CMY-2-producing E. coli belonged to phylogenetic group D. IncI1 plasmids were predominant among those carrying blaCMY-2 (96%). Restriction analysis revealed a single IncI1 plasmid carrying blaCMY-2 to be predominant and present in different clones of E. coli. IS1294-ISEcp1 complex or ISEcp1 that was truncated by IS1294 was the predominant insertion sequence upstream of blaCMY-2. The homogeneous genetic environment of blaCMY-2 observed among different strains of E. coli strongly suggests horizontal transfer of this IncI1, blaCMY-2-carrying plasmid. In summary, horizontal plasmid transfer plays a major role in the spread of blaCMY-2 in E. coli. PMID:25052868

  14. Selection of a Multidrug Resistance Plasmid by Sublethal Levels of Antibiotics and Heavy Metals

    PubMed Central

    Gullberg, Erik; Albrecht, Lisa M.; Karlsson, Christoffer; Sandegren, Linus

    2014-01-01

    ABSTRACT How sublethal levels of antibiotics and heavy metals select for clinically important multidrug resistance plasmids is largely unknown. Carriage of plasmids generally confers substantial fitness costs, implying that for the plasmid-carrying bacteria to be maintained in the population, the plasmid cost needs to be balanced by a selective pressure conferred by, for example, antibiotics or heavy metals. We studied the effects of low levels of antibiotics and heavy metals on the selective maintenance of a 220-kbp extended-spectrum β-lactamase (ESBL) plasmid identified in a hospital outbreak of Klebsiella pneumoniae and Escherichia coli. The concentrations of antibiotics and heavy metals required to maintain plasmid-carrying bacteria, the minimal selective concentrations (MSCs), were in all cases below (almost up to 140-fold) the MIC of the plasmid-free susceptible bacteria. This finding indicates that the very low antibiotic and heavy metal levels found in polluted environments and in treated humans and animals might be sufficiently high to maintain multiresistance plasmids. When resistance genes were moved from the plasmid to the chromosome, the MSC decreased, showing that MSC for a specific resistance conditionally depends on genetic context. This finding suggests that a cost-free resistance could be maintained in a population by an infinitesimally low concentration of antibiotic. By studying the effect of combinations of several compounds, it was observed that for certain combinations of drugs each new compound added lowered the minimal selective concentration of the others. This combination effect could be a significant factor in the selection of multidrug resistance plasmids/bacterial clones in complex multidrug environments. PMID:25293762

  15. Supercoiling, knotting and replication fork reversal in partially replicated plasmids

    PubMed Central

    Olavarrieta, L.; Martínez-Robles, M. L.; Sogo, J. M.; Stasiak, A.; Hernández, P.; Krimer, D. B.; Schvartzman, J. B.

    2002-01-01

    To study the structure of partially replicated plasmids, we cloned the Escherichia coli polar replication terminator TerE in its active orientation at different locations in the ColE1 vector pBR18. The resulting plasmids, pBR18-TerE@StyI and pBR18-TerE@EcoRI, were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis and electron microscopy. Replication forks stop at the Ter–TUS complex, leading to the accumulation of specific replication intermediates with a mass 1.26 times the mass of non-replicating plasmids for pBR18-TerE@StyI and 1.57 times for pBR18-TerE@EcoRI. The number of knotted bubbles detected after digestion with ScaI and the number and electrophoretic mobility of undigested partially replicated topoisomers reflect the changes in plasmid topology that occur in DNA molecules replicated to different extents. Exposure to increasing concentrations of chloroquine or ethidium bromide revealed that partially replicated topoisomers (CCCRIs) do not sustain positive supercoiling as efficiently as their non-replicating counterparts. It was suggested that this occurs because in partially replicated plasmids a positive ΔLk is absorbed by regression of the replication fork. Indeed, we showed by electron microscopy that, at least in the presence of chloroquine, some of the CCCRIs of pBR18-Ter@StyI formed Holliday-like junction structures characteristic of reversed forks. However, not all the positive supercoiling was absorbed by fork reversal in the presence of high concentrations of ethidium bromide. PMID:11809877

  16. Lipid metabolism and nutrient partitioning strategies.

    PubMed

    Morris, A M; Calsbeek, D J; Eckel, R H

    2004-10-01

    The increasing prevalence of overweight and obesity worldwide is daunting and requires prompt attention by the affected, health care profession, government and the pharmaceutical industry. Because overweight/obesity are defined as an excess of adipose tissue mass, all approaches in prevention and treatment must consider redirecting lipid storage in adipose tissue to oxidative metabolism. Lipid partitioning is a complex process that involves interaction between fat and other macronutrients, particularly carbohydrate. In an isocaloric environment, when fat is stored carbohydrate is oxidized and vice versa. Processes that influence fat partitioning in a manner in which weight is maintained must be modified by changes in organ-specific fat transport and metabolism. When therapy is considered, however, changes in lipid partitioning alone will be ineffective unless a negative energy balance is also achieved, i.e. energy expenditure exceeds energy intake. The intent of this review is to focus on molecules including hormones, enzymes, cytokines, membrane transport proteins, and transcription factors directly involved in fat trafficking and partitioning that could be potential drug targets. Some examples of favorably altering body composition by systemic and/or tissue specific modification of these molecules have already been provided with gene knockout and/or transgenic approaches in mice. The translation of this science to humans remains a challenging task. PMID:15544448

  17. Reduction-responsive cross-linked stearyl peptide for effective delivery of plasmid DNA.

    PubMed

    Yao, Chong; Tai, Zongguang; Wang, Xiaoyu; Liu, Jiyong; Zhu, Quangang; Wu, Xin; Zhang, Lijuan; Zhang, Wei; Tian, Jing; Gao, Yuan; Gao, Shen

    2015-01-01

    Low efficiency and significant toxicity are the main obstacles to successful gene delivery. We have developed a cationic reduction-responsive vector based on a disulfide cross-linked stearylated polyarginine peptide modified with histidine (C-SHR) for DNA delivery. The structure of the C-SHR was characterized, and the in vitro and in vivo transfection efficiency and cytotoxicity of C-SHR/plasmid DNA complexes were examined. Compared with non-cross-linked stearylated polyarginine peptide (SHR), C-SHR increased the intracellular uptake and dissociation behavior of the complexes. In addition, the gene transfection efficiency of C-SHR/plasmid DNA complexes in HEK293 and HeLa cells was improved and was comparable with that of bPEI-25K/plasmid DNA complexes, and the cytotoxicity of C-SHR was significantly less than that of bPEI-25K. Importantly, the in vivo gene transfection efficiency of C-SHR/plasmid DNA complexes was five fold higher than that of SHR/plasmid DNA complexes, suggesting that C-SHR is an efficient non-viral vector for DNA delivery.

  18. Chemical amplification based on fluid partitioning

    SciTech Connect

    Anderson, Brian L.; Colston, Jr., Billy W.; Elkin, Chris

    2006-05-09

    A system for nucleic acid amplification of a sample comprises partitioning the sample into partitioned sections and performing PCR on the partitioned sections of the sample. Another embodiment of the invention provides a system for nucleic acid amplification and detection of a sample comprising partitioning the sample into partitioned sections, performing PCR on the partitioned sections of the sample, and detecting and analyzing the partitioned sections of the sample.

  19. CpG-free plasmid expression cassettes for cystic fibrosis gene therapy.

    PubMed

    Pringle, Ian A; Hyde, Stephen C; Connolly, Mary M; Lawton, Anna E; Xu, Bihui; Nunez-Alonso, Graciela; Davies, Lee A; Sumner-Jones, Stephanie G; Gill, Deborah R

    2012-10-01

    Clinical studies are underway for the aerosol delivery of plasmid DNA complexed with Genzyme Lipid GL67A to the lungs of patients with cystic fibrosis (CF). Plasmid vectors contain several functional elements all of which play a role in determining the efficacy of the final clinical product. To optimise the final plasmid, variations of CpG-free 5' enhancer elements and 3'UTR regions were inserted into a common CpG-free, plasmid backbone containing Luciferase or CFTR transgenes. Plasmids were compared in immortalised cell culture, human airway liquid interface primary cell cultures, and mouse lung models to determine which design directed optimal transgene expression. Following aerosol delivery to mouse lung, plasmids containing the murine CMV enhancer showed higher peak Luciferase activity than the human CMV enhancer, but the human version resulted in persistent expression. In cell culture, the SV40 3'UTR and a novel BGH2 3'UTR exhibited up to 20-fold higher Luciferase activity than the commonly used BGH 3'UTR, but in mouse lung aerosol studies the activity and duration was greater for BGH 3'UTR. Systematic evaluation of each functional component of the plasmid has resulted in an improved design, exhibiting superior levels and duration of lung gene expression. PMID:22727465

  20. 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

  1. Fault block kinematics at a releasing stepover of the Eastern California shear zone: Partitioning of rotation style in and around the Coso geothermal area and nascent metamorphic core complex

    NASA Astrophysics Data System (ADS)

    Pluhar, Christopher J.; Coe, Robert S.; Lewis, Jonathan C.; Monastero, Francis C.; Glen, Jonathan M. G.

    2006-10-01

    , K. Richards-Dinger, The Coso geothermal field: a nascent metamorphic core complex, Geol. Soc. Amer. Bull. 117 (2005) 1534-1553.] characterize as a nascent metamorphic core complex. Consistent with upper plate disruption above a detachment, surface rocks (i.e. the upper plate of the detachment system) at the Coso geothermal area are tilted westward. However they appear to exhibit no detectable rotation. Thus, the style of block rotation may be partitioned: with clockwise vertical-axis rotation dominating in the Wild Horse Mesa and horizontal axis rotation (tilting) in the geothermal area.

  2. Thermosensitive antibiotic resistance plasmids in enterobacteria.

    PubMed

    Smith, H W; Parsell, Z; Green, P

    1978-11-01

    Of 775 conjugative plasmids found in enterobacteria mediating antibiotic resistance, 24 (3.1%) were thermosensitive (ts); they were most common in Klebsiella pneumoniae. Ts plasmids were also found in all the samples of sewage and river water examined. Over half of 73 ts plasmids from unrelated sources mediated resistance to chloramphenicol in addition to several other antibiotics. Many of them mediated resistance to mercury (53.4%), arsenite (38.4%) and tellurite (79.5%) but not to copper, cobalt and silver. Fifty-eight belonged to incompatibility group H2 and 12 belonged to the H1 group. Resistance to mercury, arsenite and tellurite was common in strains containing H2 plasmids but not in H1 plasmids. The 73 plasmids transferred at high rates at 22 and 28 degrees C and at lower rates at 15 degrees C; they transferred at very low rates or not at all at 37 degrees C. They could be divided into two sets according to whether they transferred at a high or at a low rate at 33 degrees C. Unlike the prototype plasmid, Rts 1, they were solely or mainly ts for transfer and not for replication and only one of them brought about a marked reduction in growth rate of its host organism at 42 degrees C. None of the 73 plasmids mediated colicin or haemolysin production. Three plasmids, all from K. pneumoniae, mediated utilization of lactose, two of sucrose and raffinose and three, all belonging to group H1, of citrate. None of the plasmids increased the pathogenicity of Salmonella typhimurium for chicks or Escherichia coli K12 for mice.

  3. Stabilization of the Virulence Plasmid pSLT of Salmonella Typhimurium by Three Maintenance Systems and Its Evaluation by Using a New Stability Test

    PubMed Central

    Lobato-Márquez, Damián; Molina-García, Laura; Moreno-Córdoba, Inma; García-del Portillo, Francisco; Díaz-Orejas, Ramón

    2016-01-01

    Certain Salmonella enterica serovars belonging to subspecies I carry low-copy-number virulence plasmids of variable size (50–90 kb). All of these plasmids share the spv operon, which is important for systemic infection. Virulence plasmids are present at low copy numbers. Few copies reduce metabolic burden but suppose a risk of plasmid loss during bacterial division. This drawback is counterbalanced by maintenance modules that ensure plasmid stability, including partition systems and toxin-antitoxin (TA) loci. The low-copy number virulence pSLT plasmid of Salmonella enterica serovar Typhimurium encodes three auxiliary maintenance systems: one partition system (parAB) and two TA systems (ccdABST and vapBC2ST). The TA module ccdABST has previously been shown to contribute to pSLT plasmid stability and vapBC2ST to bacterial virulence. Here we describe a novel assay to measure plasmid stability based on the selection of plasmid-free cells following elimination of plasmid-containing cells by ParE toxin, a DNA gyrase inhibitor. Using this new maintenance assay we confirmed a crucial role of parAB in pSLT maintenance. We also showed that vapBC2ST, in addition to contribute to bacterial virulence, is important for plasmid stability. We have previously shown that ccdABST encodes an inactive CcdBST toxin. Using our new stability assay we monitored the contribution to plasmid stability of a ccdABST variant containing a single mutation (R99W) that restores the toxicity of CcdBST. The “activation” of CcdBST (R99W) did not increase pSLT stability by ccdABST. In contrast, ccdABST behaves as a canonical type II TA system in terms of transcriptional regulation. Of interest, ccdABST was shown to control the expression of a polycistronic operon in the pSLT plasmid. Collectively, these results show that the contribution of the CcdBST toxin to pSLT plasmid stability may depend on its role as a co-repressor in coordination with CcdAST antitoxin more than on its toxic activity. PMID

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

    PubMed

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

    1996-10-14

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

  5. A predicted T4 secretion system and conserved DNA-repeats identified in a subset of related Arthrobacter plasmids.

    PubMed

    Mihăşan, Marius; Brandsch, Roderich

    2016-10-01

    BLAST analysis of pAO1 ORFs of Arthrobacter nicotinovorans revealed 12 ORFs, including the ORF of a transcriptional regulator, predicted to encode the components of a T4-secretion system involved in bacterial conjugation. These ORFs were conserved and showed synteny among 14 Arthrobacter plasmids. A DNA repeat of about 370 nucleotides was found to be present 5' to the pAO1 ORFs of DUF4192-, DprA- and ParB-like proteins. Similar repeats were present in identical positions on 12 additional Arthrobacter plasmids. The DNA repeats on a particular plasmid are highly identical duplications. The DNA repeats contain alternating GC and AT reach sequences, potential protein DNA-binding sites and purine reach stretches. The sequences end with 5'ATG.AAC3' which results in the amino terminal sequence methionine (M) and asparagine (N) for all predicted DprA, DUF4192 and ParB proteins. The presences of conserved ORFs of a T4-secretion system and of similar DNA repeats suggest that these Arthrobacter plasmids are related and evolved from a common ancestor. The functional significance of the DNA repeats in a coordinated common mechanism of regulation of expression of the dprA-(involved in natural competence), parB- (involved in plasmid partitioning) and duf4192- (unknown function in plasmid life cycle) genes remains to be established. PMID:27524651

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

    PubMed Central

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

    2010-01-01

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

  7. ParABS Systems of the Four Replicons of Burkholderia cenocepacia: New Chromosome Centromeres Confer Partition Specificity†

    PubMed Central

    Dubarry, Nelly; Pasta, Franck; Lane, David

    2006-01-01

    Most bacterial chromosomes carry an analogue of the parABS systems that govern plasmid partition, but their role in chromosome partition is ambiguous. parABS systems might be particularly important for orderly segregation of multipartite genomes, where their role may thus be easier to evaluate. We have characterized parABS systems in Burkholderia cenocepacia, whose genome comprises three chromosomes and one low-copy-number plasmid. A single parAB locus and a set of ParB-binding (parS) centromere sites are located near the origin of each replicon. ParA and ParB of the longest chromosome are phylogenetically similar to analogues in other multichromosome and monochromosome bacteria but are distinct from those of smaller chromosomes. The latter form subgroups that correspond to the taxa of their hosts, indicating evolution from plasmids. The parS sites on the smaller chromosomes and the plasmid are similar to the “universal” parS of the main chromosome but with a sequence specific to their replicon. In an Escherichia coli plasmid stabilization test, each parAB exhibits partition activity only with the parS of its own replicon. Hence, parABS function is based on the independent partition of individual chromosomes rather than on a single communal system or network of interacting systems. Stabilization by the smaller chromosome and plasmid systems was enhanced by mutation of parS sites and a promoter internal to their parAB operons, suggesting autoregulatory mechanisms. The small chromosome ParBs were found to silence transcription, a property relevant to autoregulation. PMID:16452432

  8. Replication and Control of Circular Bacterial Plasmids

    PubMed Central

    del Solar, Gloria; Giraldo, Rafael; Ruiz-Echevarría, María Jesús; Espinosa, Manuel; Díaz-Orejas, Ramón

    1998-01-01

    An essential feature of bacterial plasmids is their ability to replicate as autonomous genetic elements in a controlled way within the host. Therefore, they can be used to explore the mechanisms involved in DNA replication and to analyze the different strategies that couple DNA replication to other critical events in the cell cycle. In this review, we focus on replication and its control in circular plasmids. Plasmid replication can be conveniently divided into three stages: initiation, elongation, and termination. The inability of DNA polymerases to initiate de novo replication makes necessary the independent generation of a primer. This is solved, in circular plasmids, by two main strategies: (i) opening of the strands followed by RNA priming (theta and strand displacement replication) or (ii) cleavage of one of the DNA strands to generate a 3′-OH end (rolling-circle replication). Initiation is catalyzed most frequently by one or a few plasmid-encoded initiation proteins that recognize plasmid-specific DNA sequences and determine the point from which replication starts (the origin of replication). In some cases, these proteins also participate directly in the generation of the primer. These initiators can also play the role of pilot proteins that guide the assembly of the host replisome at the plasmid origin. Elongation of plasmid replication is carried out basically by DNA polymerase III holoenzyme (and, in some cases, by DNA polymerase I at an early stage), with the participation of other host proteins that form the replisome. Termination of replication has specific requirements and implications for reinitiation, studies of which have started. The initiation stage plays an additional role: it is the stage at which mechanisms controlling replication operate. The objective of this control is to maintain a fixed concentration of plasmid molecules in a growing bacterial population (duplication of the plasmid pool paced with duplication of the bacterial population

  9. Characterization of the stable maintenance properties of the par region of broad-host-range plasmid RK2.

    PubMed Central

    Sobecky, P A; Easter, C L; Bear, P D; Helinski, D R

    1996-01-01

    A 3.2-kb fragment encoding five genes, parCBA/DE, in two divergently transcribed operons promotes stable maintenance of the replicon of the broad-host-range plasmid RK2 in a vector-independent manner in Escherichia coli. The parDE operon has been shown to contribute to stabilization through the postsegregational killing of plasmid-free daughter cells, while the parCBA operon encodes a resolvase, ParA, that mediates the resolution of plasmid multimers through site-specific recombination. To date, evidence indicates that multimer resolution alone does not play a significant role in RK2 stable maintenance by the parCBA operon in E. coli. It has been proposed, instead, that the parCBA region encodes an additional stability mechanism, a partition system, that ensures that each daughter cell receives a plasmid copy at cell division. However, studies carried out to date have not directly determined the plasmid stabilization activity of the parCBA operon alone. An assessment was made of the relative contributions of postsegregational killing (parDE) and the putative partitioning system (parCBA) to the stabilization of mini-RK2 replicons in E. coli. Mini-RK2 replicons carrying either the entire 3.2-kb (parCBA/DE) fragment or the 2.3-kb parCBA region alone were found to be stably maintained in two E. coli strains tested. The stabilization found is not due to resolution of multimers. The stabilizing effectiveness of parCBA was substantially reduced when the plasmid copy number was lowered, as in the case of E. coli cells carrying a temperature-sensitive mini-RK2 replicon grown at a nonpermissive temperature. The presence of the entire 3.2-kb region effectively stabilized the replicon, however, under both low- and high-copy-number-conditions. In those instances of decreased plasmid copy number, the postsegregational killing activity, encoded by parDE, either as part of the 3.2-kb fragment or alone played the major role in the stabilization of mini-RK2 replicons within the

  10. Thermosensitive H1 plasmids determining citrate utilization.

    PubMed

    Smith, H W; Parsell, Z; Green, P

    1978-12-01

    Twelve thermosensitive H1 plasmids from strains of Salmonella typhi that had caused outbreaks of chloramphenicol-resistant typhoid fever in Vietnam, Thailand and India mediated citrate utilization (Cit+) in a prototrophic Escherichia coli K12 strain but not in the S. typhi strains from which they were derived. Four H1 plasmids from a similar outbreak in Mexico differed from the Far Eastern plasmids in not mediating citrate utlization but in mediating mercury resistance. H1 plasmids resembling the Far Eastern and the Mexican plasmids in regard to citrate utilization and mercury resistance were found in sewage in Britain. Citrate utilization was transferred to eight pathogenic strains of E. coli and to one strain each of Shigella flexneri and Shigella sonnei. Cultures of Cit+ bacteria grew more rapidly in citrate media at 28 degrees C than at 37 degrees C. Plasmid mutants that were more efficient at utilizing citrate were present in all such cultures--they grew equally well or better at 37 degrees C than at 28 degrees C. None of 222 strains of E. coli or Shigella that contained a variety of different plasmids were able to utilize citrate. This property was not transferred to the prototrophic E. coli K12 strain from Citrobacter (3 strains), Salmonella (39 strains), Proteus (44 strains), Klebsiella pneumoniae (33 strains) or Pseudomonas aeruginosa (44 strains).

  11. Rolling-circle replication of bacterial plasmids.

    PubMed Central

    Khan, S A

    1997-01-01

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

  12. Non-parametric partitioning of SAR images

    NASA Astrophysics Data System (ADS)

    Delyon, G.; Galland, F.; Réfrégier, Ph.

    2006-09-01

    We describe and analyse a generalization of a parametric segmentation technique adapted to Gamma distributed SAR images to a simple non parametric noise model. The partition is obtained by minimizing the stochastic complexity of a quantized version on Q levels of the SAR image and lead to a criterion without parameters to be tuned by the user. We analyse the reliability of the proposed approach on synthetic images. The quality of the obtained partition will be studied for different possible strategies. In particular, one will discuss the reliability of the proposed optimization procedure. Finally, we will precisely study the performance of the proposed approach in comparison with the statistical parametric technique adapted to Gamma noise. These studies will be led by analyzing the number of misclassified pixels, the standard Hausdorff distance and the number of estimated regions.

  13. Number Partitioning via Quantum Adiabatic Computation

    NASA Technical Reports Server (NTRS)

    Smelyanskiy, Vadim N.; Toussaint, Udo; Clancy, Daniel (Technical Monitor)

    2002-01-01

    We study both analytically and numerically the complexity of the adiabatic quantum evolution algorithm applied to random instances of combinatorial optimization problems. We use as an example the NP-complete set partition problem and obtain an asymptotic expression for the minimal gap separating the ground and exited states of a system during the execution of the algorithm. We show that for computationally hard problem instances the size of the minimal gap scales exponentially with the problem size. This result is in qualitative agreement with the direct numerical simulation of the algorithm for small instances of the set partition problem. We describe the statistical properties of the optimization problem that are responsible for the exponential behavior of the algorithm.

  14. Generalized transduction of small Yersinia enterocolitica plasmids.

    PubMed

    Hertwig, S; Popp, A; Freytag, B; Lurz, R; Appel, B

    1999-09-01

    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.

  15. 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

  16. [Sop proteins can cause transcriptional silencing of genes located close to the centromere sites of linear plasmid N15].

    PubMed

    Mardanov, A V; Lane, D; Ravin, N V

    2010-01-01

    Stable inheritance of bacterial chromosomes and low copy number plasmids is ensured by accurate partitioning of replicated molecules between the daughter cells at division. Partitioning of the prophage of the temperate bacteriophage N15, which exists as a linear plasmid molecule with covalently closed ends, depends on the sop locus, comprising genes sopA and sopB, as well as four centromere sites located in different regions of the N15 genome essential for replication and the control of lysogeny. We found that binding of SopB to the centromere can silence centromere-proximal promoters, presumably due to subsequent polymerizing of SopB along the DNA. Close to the IR4 centromere site we identified a promoter, P59, able to drive expression of phage late genes encoding the structural proteins of virion. We found that following binding to IR4 the N15 Sop proteins can cause repression of this promoter. The repression depends on SopB and became stronger in the presence of SopA. Sop-dependent silencing of centromere-proximal promoters control gene expression in phage N15, particularly preventing undesired expression of late genes in the N15 prophage. Thus, the phage N15 sop system not only ensures plasmid partitioning but is also involved in the genetic network controlling prophage replication and the maintenance of lysogeny.

  17. A study on a coincidence phenomenon in building partitions using Laser Doppler Vibrometer

    NASA Astrophysics Data System (ADS)

    Bolejko, Romuald

    2012-06-01

    In the paper results of vibrations measurements of the partitions with finished dimensions done in anechoic environment using scanning laser Doppler vibrometer are presented. It has allowed presentation a complex pattern of vibration and flow of vibration energy caused by bending waves in a partition at coincidence frequencies. A study was done for homogenous aluminum plates, aluminum plate with damping coatings and double-leaf partition composed by aluminum plates with different thickness. It was demonstrated that magnitude and pattern of vibration of each leafs of the double partition is correlated in specific way. The influence of structural modifications (stiffeners) and damping coating of a partition on its vibration and sound insulation was discussed. It was shown that such partition modification could decrease an effect of the coincidence on sound transmission through a partition.

  18. Partitioning tracers for in-situ detection and quantification of dense nonaqueous. 1997 annual progress report

    SciTech Connect

    Brusseau, M.L.

    1997-11-10

    'The overall goal of the proposed project is to explore the use of partitioning tracers to characterize dense nonaqueous phase liquids (DNAPLs) in subsurface systems. Bulk-phase partitioning tracers will be investigated to detect and determine DNAPL saturation, while interface partitioning tracers will be investigated to measure the area of the DNAPL-water interface. The specific objectives that will be addressed to accomplish this goal are: (1) investigate the use of partitioning tracers to detect and determine both the saturation and interfacial area of DNAPLs in saturated porous media; (2) investigate the effect of rate-limited mass transfer on the transport behavior of partitioning tracers; (3) investigate the effect of porous-media heterogeneity on the transport behavior of partitioning tracers; and (4) develop and evaluate mathematical models capable of simulating the transport of partitioning tracers in complex systems.'

  19. In-Situ Characterization of Dense Non-Aqueous Phase Liquids Using Partitioning Tracers

    SciTech Connect

    Gary A. Pope; Daene C. McKinney; Akhil Datta Gupta; Richard E. Jackson; Minquan Jin

    2000-03-20

    Majors advances have been made during the past three years in our research on interwell partitioning tracers tests (PITTs). These advances include (1) progress on the inverse problem of how to estimate the three-dimensional distribution of NAPL in aquifers from the tracer data, (2) the first ever partitioning tracer experiments in dual porosity media, (3) the first modeling of partitioning tracers in dual porosity media (4) experiments with complex NAPLs such as coal tar, (5) the development of an accurate and simple method to predict partition coefficients using the equivalent alkane carbon number approach, (6) partitioning tracer experiments in large model aquifers with permeability layers, (7) the first ever analysis of partitioning tracer data to estimate the change in composition of a NAPL before and after remediation (8) the first ever analysis of partitioning tracer data after a field demonstration of surfactant foam to remediate NAPL and (9) experiments at elevated temperatures .

  20. 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

  1. 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)

  2. Active stable maintenance functions in low copy-number plasmids of Gram-positive bacteria II. Post-segregational killing systems.

    PubMed

    Dmowski, Michał; Jagura-Burdzy, Grazyna

    2013-01-01

    Active support is needed for low copy-number plasmids to be stably maintained in bacterial cells. The mechanisms that fulfill this role are (i) partition systems (PAR) acting to separate plasmid molecules to daughter cells and (ii) toxin-andidote (TA) (post-segregational killing-PSK) systems which arrest cell growth until the plasmid reaches the correct copy-number or kill the cells that have not inherited the plasmid. Our knowledge of toxin-antidote systems comes mainly from studies on Gram-negative bacteria. However, some addiction systems of Gram-positive bacteria have been characterized in detail or recently identified. Altogether, they bring new interesting data on toxin-antidote functioning in bacteria.

  3. Comparison of 10 IncP plasmids: homology in the regions involved in plasmid replication.

    PubMed Central

    Chikami, G K; Guiney, D G; Schmidhauser, T J; Helinski, D R

    1985-01-01

    We have examined the DNA homology in the replication regions of 10 IncP plasmids independently isolated from several different countries. Two regions of RK2, the best-studied plasmid of this group, are required for vegetative DNA replication: the origin of replication (oriV) and the trfA region, which codes for a gene product necessary for replication. Six of nine IncP plasmids studied were identical to RK2 in the oriV and trfA regions as shown by Southern hybridization. Three P plasmids, R751, R772, and R906, showed weaker homology with the RK2 trfA, region and hybridized to different-sized HaeII fragments than the other six plasmids. R751, R772, and R906 hybridized to the region of the RK2 replication origin which expresses P incompatibility but differed markedly from RK2 and the other six plasmids in the GC-rich region of the origin required for replication. These data indicate that the P-group plasmids can be divided into two subgroups: IncP alpha, which includes the RK2-like plasmids, and IncP beta which includes the R751-like plasmids. Images PMID:2985542

  4. Plasmid-mediated mineralization of 4-chlorobiphenyl.

    PubMed Central

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

    1985-01-01

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

  5. Temporal stability of network partitions.

    PubMed

    Petri, Giovanni; Expert, Paul

    2014-08-01

    We present a method to find the best temporal partition at any time scale and rank the relevance of partitions found at different time scales. This method is based on random walkers coevolving with the network and as such constitutes a generalization of partition stability to the case of temporal networks. We show that, when applied to a toy model and real data sets, temporal stability uncovers structures that are persistent over meaningful time scales as well as important isolated events, making it an effective tool to study both abrupt changes and gradual evolution of a network mesoscopic structures.

  6. Quantum field theory of partitions

    SciTech Connect

    Bender, C.M.; Brody, D.C.; Meister, B.K.

    1999-07-01

    Given a sequence of numbers {l_brace}a{sub n}{r_brace}, it is always possible to find a set of Feynman rules that reproduce that sequence. For the special case of the partitions of the integers, the appropriate Feynman rules give rise to graphs that represent the partitions in a clear pictorial fashion. These Feynman rules can be used to generate the Bell numbers B(n) and the Stirling numbers S(n,k) that are associated with the partitions of the integers. {copyright} {ital 1999 American Institute of Physics.}

  7. Temporal stability of network partitions.

    PubMed

    Petri, Giovanni; Expert, Paul

    2014-08-01

    We present a method to find the best temporal partition at any time scale and rank the relevance of partitions found at different time scales. This method is based on random walkers coevolving with the network and as such constitutes a generalization of partition stability to the case of temporal networks. We show that, when applied to a toy model and real data sets, temporal stability uncovers structures that are persistent over meaningful time scales as well as important isolated events, making it an effective tool to study both abrupt changes and gradual evolution of a network mesoscopic structures. PMID:25215787

  8. Partitioning ecosystems for sustainability.

    PubMed

    Murray, Martyn G

    2016-03-01

    Decline in the abundance of renewable natural resources (RNRs) coupled with increasing demands of an expanding human population will greatly intensify competition for Earth's natural resources during this century, yet curiously, analytical approaches to the management of productive ecosystems (ecological theory of wildlife harvesting, tragedy of the commons, green economics, and bioeconomics) give only peripheral attention to the driving influence of competition on resource exploitation. Here, I apply resource competition theory (RCT) to the exploitation of RNRs and derive four general policies in support of their sustainable and equitable use: (1) regulate resource extraction technology to avoid damage to the resource base; (2) increase efficiency of resource use and reduce waste at every step in the resource supply chain and distribution network; (3) partition ecosystems with the harvesting niche as the basic organizing principle for sustainable management of natural resources by multiple users; and (4) increase negative feedback between consumer and resource to bring about long-term sustainable use. A simple policy framework demonstrates how RCT integrates with other elements of sustainability science to better manage productive ecosystems. Several problem areas of RNR management are discussed in the light of RCT, including tragedy of the commons, overharvesting, resource collapse, bycatch, single species quotas, and simplification of ecosystems.

  9. Partitioning ecosystems for sustainability.

    PubMed

    Murray, Martyn G

    2016-03-01

    Decline in the abundance of renewable natural resources (RNRs) coupled with increasing demands of an expanding human population will greatly intensify competition for Earth's natural resources during this century, yet curiously, analytical approaches to the management of productive ecosystems (ecological theory of wildlife harvesting, tragedy of the commons, green economics, and bioeconomics) give only peripheral attention to the driving influence of competition on resource exploitation. Here, I apply resource competition theory (RCT) to the exploitation of RNRs and derive four general policies in support of their sustainable and equitable use: (1) regulate resource extraction technology to avoid damage to the resource base; (2) increase efficiency of resource use and reduce waste at every step in the resource supply chain and distribution network; (3) partition ecosystems with the harvesting niche as the basic organizing principle for sustainable management of natural resources by multiple users; and (4) increase negative feedback between consumer and resource to bring about long-term sustainable use. A simple policy framework demonstrates how RCT integrates with other elements of sustainability science to better manage productive ecosystems. Several problem areas of RNR management are discussed in the light of RCT, including tragedy of the commons, overharvesting, resource collapse, bycatch, single species quotas, and simplification of ecosystems. PMID:27209800

  10. Clostridium perfringens type A-E toxin plasmids.

    PubMed

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

    2015-05-01

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

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

    PubMed

    Kado, Clarence I

    2014-10-01

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

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

    PubMed

    Kado, Clarence I

    2014-10-01

    This chapter revisits the historical development and outcome of studies focused on the transmissible, extrachromosomal genetic elements called plasmids. Early work on plasmids involved structural and genetic mapping of these molecules, followed by the development of an understanding of how plasmids replicate and segregate during cell division. The intriguing property of plasmid transmission between bacteria and between bacteria and higher cells has received considerable attention. The utilitarian aspects of plasmids are described, including examples of various plasmid vector systems. This chapter also discusses the functional attributes of plasmids needed for their persistence and survival in nature and in man-made environments. The term plasmid biology was first conceived at the Fallen Leaf Lake Conference on Promiscuous Plasmids, 1990, Lake Tahoe, California. The International Society for Plasmid Biology was established in 2004 (www.ISPB.org). PMID:26104369

  13. Biogeography of time partitioning in mammals

    PubMed Central

    Bennie, Jonathan J.; Duffy, James P.; Inger, Richard; Gaston, Kevin J.

    2014-01-01

    Many animals regulate their activity over a 24-h sleep–wake cycle, concentrating their peak periods of activity to coincide with the hours of daylight, darkness, or twilight, or using different periods of light and darkness in more complex ways. These behavioral differences, which are in themselves functional traits, are associated with suites of physiological and morphological adaptations with implications for the ecological roles of species. The biogeography of diel time partitioning is, however, poorly understood. Here, we document basic biogeographic patterns of time partitioning by mammals and ecologically relevant large-scale patterns of natural variation in “illuminated activity time” constrained by temperature, and we determine how well the first of these are predicted by the second. Although the majority of mammals are nocturnal, the distributions of diurnal and crepuscular species richness are strongly associated with the availability of biologically useful daylight and twilight, respectively. Cathemerality is associated with relatively long hours of daylight and twilight in the northern Holarctic region, whereas the proportion of nocturnal species is highest in arid regions and lowest at extreme high altitudes. Although thermal constraints on activity have been identified as key to the distributions of organisms, constraints due to functional adaptation to the light environment are less well studied. Global patterns in diversity are constrained by the availability of the temporal niche; disruption of these constraints by the spread of artificial lighting and anthropogenic climate change, and the potential effects on time partitioning, are likely to be critical influences on species’ future distributions. PMID:25225371

  14. Plasmid-like replicative intermediates of the Epstein-Barr virus lytic origin of DNA replication.

    PubMed Central

    Pfüller, R; Hammerschmidt, W

    1996-01-01

    During the lytic phase of herpesviruses, intermediates of viral DNA replication are found as large concatemeric molecules in the infected cells. It is not known, however, what the early events in viral DNA replication that yield these concatemers are. In an attempt to identify these early steps of DNA replication, replicative intermediates derived from the lytic origin of Epstein-Barr virus, oriLyt, were analyzed. As shown by density shift experiments with bromodeoxyuridine, oriLyt replicated semiconservatively soon after induction of the lytic cycle and oriLyt-containing DNA is amplified to yield monomeric plasmid progeny DNA (besides multimeric forms and high-molecular-weight DNA). A new class of plasmid progeny DNA which have far fewer negative supercoils than do plasmids extracted from uninduced cells is present only in cells undergoing the lytic cycle of Epstein-Barr virus. This finding is consistent with plasmid DNAs having fewer nucleosomes before extraction. The newly replicated plasmid DNAs are dependent on a functional oriLyt in cis and support an efficient marker transfer into Escherichia coli as monomeric plasmids. Multimeric forms of presumably circular progeny DNA of oriLyt, as well as detected recombination events, indicate that oriLyt-mediated DNA replication is biphasic: an early theta-like mode is followed by a complex pattern which could result from rolling-circle DNA replication. PMID:8648674

  15. EcoR124I: from Plasmid-Encoded Restriction-Modification System to Nanodevice

    PubMed Central

    Youell, James; Firman, Keith

    2008-01-01

    Plasmid R124 was first described in 1972 as being a new member of incompatibility group IncFIV, yet early physical investigations of plasmid DNA showed that this type of classification was more complex than first imagined. Throughout the history of the study of this plasmid, there have been many unexpected observations. Therefore, in this review, we describe the history of our understanding of this plasmid and the type I restriction-modification (R-M) system that it encodes, which will allow an opportunity to correct errors, or misunderstandings, that have arisen in the literature. We also describe the characterization of the R-M enzyme EcoR124I and describe the unusual properties of both type I R-M enzymes and EcoR124I in particular. As we approached the 21st century, we began to see the potential of the EcoR124I R-M enzyme as a useful molecular motor, and this leads to a description of recent work that has shown that the R-M enzyme can be used as a nanoactuator. Therefore, this is a history that takes us from a plasmid isolated from (presumably) an infected source to the potential use of the plasmid-encoded R-M enzyme in bionanotechnology. PMID:18535150

  16. Metal partitioning and toxicity in sewage sludge

    SciTech Connect

    Carlson-Ekvall, C.E.A.; Morrison, G.M.

    1995-12-31

    Over 20 years of research has failed to provide an unequivocal correlation between chemically extracted metals in sewage sludge applied to agricultural soil and either metal toxicity to soil organisms or crop uptake. Partitioning of metals between phases and species can provide a better estimation of mobility and potential bioavailability. Partition coefficients, K{sub D} for Cd, Cu, Pb and Zn in a sludge/water solution were determined considering the sludge/water solution as a three-phase system (particulate, colloidal and electrochemically available) over a range of pH values, ionic strengths, contact times and sludge/water ratios and compared with the KD values for sludge/water solution as a two-phase system (aqueous phase and particulate phase). Partitioning results were interpreted in terms of metal mobility from sludge to colloids and in terms of potential bioavailability from colloids to electrochemically available. The results show that both mobility and potential bioavailability are high for Zn, while Cu partitions into the mobile colloidal phase which is relatively non-bioavailable. Lead is almost completely bound to the solid phase, and is neither mobile nor bioavailable. A comparison between K, values and toxicity shows that Zn in sludge is more toxic than can be accounted for in the aqueous phase, which can be due to synergistic effects between sludge organics and Zn. Copper demonstrates clear synergism which can be attributed to the formation of lipid-soluble Cu complexes with known sludge components such as LAS, caffeine, myristic acid and nonylphenol.

  17. Graph Partitioning and Sequencing Software

    1995-09-19

    Graph partitioning is a fundemental problem in many scientific contexts. CHACO2.0 is a software package designed to partition and sequence graphs. CHACO2.0 allows for recursive application of several methods for finding small edge separators in weighted graphs. These methods include inertial, spectral, Kernighan Lin and multilevel methods in addition to several simpler strategies. Each of these approaches can be used to partition the graph into two, four, or eight pieces at each level of recursion.more » In addition, the Kernighan Lin method can be used to improve partitions generated by any of the other algorithms. CHACO2.0 can also be used to address various graph sequencing problems, with applications to scientific computing, database design, gene sequencing and other problems.« less

  18. Crystal-chemistry and partitioning of REE in whitlockite

    NASA Technical Reports Server (NTRS)

    Colson, R. O.; Jolliff, B. L.

    1993-01-01

    Partitioning of Rare Earth Elements (REE) in whitlockite is complicated by the fact that two or more charge-balancing substitutions are involved and by the fact that concentrations of REE in natural whitlockites are sufficiently high such that simple partition coefficients are not expected to be constant even if mixing in the system is completely ideal. The present study combines preexisting REE partitioning data in whitlockites with new experiments in the same compositional system and at the same temperature (approximately 1030 C) to place additional constraints on the complex variations of REE partition coefficients and to test theoretical models for how REE partitioning should vary with REE concentration and other compositional variables. With this data set, and by combining crystallographic and thermochemical constraints with a SAS simultaneous-equation best-fitting routine, it is possible to infer answers to the following questions: what is the speciation on the individual sites Ca(B), Mg, and Ca(IIA) (where the ideal structural formula is Ca(B)18 Mg2Ca(IIA)2P14O56); how are REE's charge-balanced in the crystal; and is mixing of REE in whitlockite ideal or non-ideal. This understanding is necessary in order to extrapolate derived partition coefficients to other compositional systems and provides a broadened understanding of the crystal chemistry of whitlockite.

  19. 25 CFR 152.33 - Partition.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 25 Indians 1 2011-04-01 2011-04-01 false Partition. 152.33 Section 152.33 Indians BUREAU OF INDIAN..., REMOVAL OF RESTRICTIONS, AND SALE OF CERTAIN INDIAN LANDS Partitions in Kind of Inherited Allotments § 152.33 Partition. (a) Partition without application. If the Secretary of the Interior shall find that...

  20. 25 CFR 152.33 - Partition.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 25 Indians 1 2010-04-01 2010-04-01 false Partition. 152.33 Section 152.33 Indians BUREAU OF INDIAN..., REMOVAL OF RESTRICTIONS, AND SALE OF CERTAIN INDIAN LANDS Partitions in Kind of Inherited Allotments § 152.33 Partition. (a) Partition without application. If the Secretary of the Interior shall find that...

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

    SciTech Connect

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

    2008-07-15

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

  2. blaNDM-5 carried by an IncX3 plasmid in Escherichia coli sequence type 167.

    PubMed

    Yang, Ping; Xie, Yi; Feng, Ping; Zong, Zhiyong

    2014-12-01

    bla(NDM-)5 was found in Escherichia coli strain 0215 from a Chinese patient without travel history. Genomic sequencing and conjugation experiments were performed. Strain 0215 belonged to sequence type 167 (ST167) and had other resistance determinants, including bla(TEM-135), bla(CTX-M-14), and aac(6')-Ib. bla(NDM-5) was carried by a 47-kb self-transmissible IncX3 plasmid and was in a complex genetic context similar to that of bla(NDM-1) on IncX3 plasmids. IncX3 plasmids might have emerged as a common vehicle mediating the spread of bla(NDM).

  3. Kinetic modeling of plasmid bioproduction in Escherichia coli DH5α cultures over different carbon-source compositions.

    PubMed

    Lopes, Marta B; Martins, Gabriel; Calado, Cecília R C

    2014-09-30

    The need for the development of economic high plasmid production in Escherichia coli cultures is emerging, as a result of the latest advances in DNA vaccination and gene therapy. In order to contribute to achieve that, a model describing the kinetics involved in the bioproduction of plasmid by recombinant E. coli DH5α is presented, as an attempt to understand the complex and non-linear metabolic relationships and the plasmid production occurring in dynamic batch culture environments, run under different media compositions of glucose and glycerol, that result in distinct maximum biomass growths (between 8.2 and 12.8 g DCW/L) and specific plasmid productions (between 1.1 and 7.4 mg/g DCW). The model based on mass balance equations for biomass, glucose, glycerol, acetate and plasmid accurately described different culture behaviors, using either glucose or glycerol as carbon source, or mixtures of both. From the 17 parameters obtained after model simplification, the following 10 parameters were found to be independent of the carbon source composition: the substrate affinity constants, the inhibitory constants of biomass growth on glycerol by glucose, of biomass growth on acetate by glycerol and the global biomass growth by acetate, and the yields of biomass on acetate, acetate on glucose and glycerol, and plasmid on glucose. The parameters that depend on the culture composition, and that might explain the differences found between cultures, were: maximum specific growth rates on glucose, glycerol and acetate; biomass yield on glucose and glycerol; and plasmid yield on glycerol and acetate. Moreover, a crucial role of acetate in the plasmid production was revealed by the model, with most of plasmid production being associated to the acetate consumption. The model provides meaningful insight on the E. coli dynamic cell behavior concerning the plasmid bioproduction, which might lead to important guidelines for culture optimization and process scale-up and control.

  4. Automatic partitioning of head CTA for enabling segmentation

    NASA Astrophysics Data System (ADS)

    Suryanarayanan, Srikanth; Mullick, Rakesh; Mallya, Yogish; Kamath, Vidya; Nagaraj, Nithin

    2004-05-01

    Radiologists perform a CT Angiography procedure to examine vascular structures and associated pathologies such as aneurysms. Volume rendering is used to exploit volumetric capabilities of CT that provides complete interactive 3-D visualization. However, bone forms an occluding structure and must be segmented out. The anatomical complexity of the head creates a major challenge in the segmentation of bone and vessel. An analysis of the head volume reveals varying spatial relationships between vessel and bone that can be separated into three sub-volumes: "proximal", "middle", and "distal". The "proximal" and "distal" sub-volumes contain good spatial separation between bone and vessel (carotid referenced here). Bone and vessel appear contiguous in the "middle" partition that remains the most challenging region for segmentation. The partition algorithm is used to automatically identify these partition locations so that different segmentation methods can be developed for each sub-volume. The partition locations are computed using bone, image entropy, and sinus profiles along with a rule-based method. The algorithm is validated on 21 cases (varying volume sizes, resolution, clinical sites, pathologies) using ground truth identified visually. The algorithm is also computationally efficient, processing a 500+ slice volume in 6 seconds (an impressive 0.01 seconds / slice) that makes it an attractive algorithm for pre-processing large volumes. The partition algorithm is integrated into the segmentation workflow. Fast and simple algorithms are implemented for processing the "proximal" and "distal" partitions. Complex methods are restricted to only the "middle" partition. The partitionenabled segmentation has been successfully tested and results are shown from multiple cases.

  5. Human clinical trials of plasmid DNA vaccines.

    PubMed

    Liu, Margaret A; Ulmer, Jeffrey B

    2005-01-01

    This article gives an overview of DNA vaccines with specific emphasis on the development of DNA vaccines for clinical trials and an overview of those trials. It describes the preclinical research that demonstrated the efficacy of DNA vaccines as well as an explication of the immunologic mechanisms of action. These include the induction of cognate immune responses, such as the generation of cytolytic T lymphocytes (CTL) as well as the effect of the plasmid DNA upon the innate immune system. Specific issues related to the development of DNA as a product candidate are then discussed, including the manufacture of plasmid, the qualification of the plasmid DNA product, and the safety testing necessary for initiating clinical trials. Various human clinical trials for infectious diseases and cancer have been initiated or completed, and an overview of these trials is given. Finally, because the early clinical trials have shown less than optimal immunogenicity, methods to increase the potency of the vaccines are described. PMID:16291211

  6. Preparation of Pichia pastoris expression plasmids.

    PubMed

    Logez, Christel; Alkhalfioui, Fatima; Byrne, Bernadette; Wagner, Renaud

    2012-01-01

    When planning any heterologous expression experiment, the very first critical step is related to the design of the overall strategy, hence to the selection of the most adapted expression vector. The very flexible Pichia pastoris system offers a broad range of possibilities for the production of secreted, endogenous or membrane proteins thanks to a combination of various plasmid backbones, selection markers, promoters and fusion sequences introduced into dedicated host strains. The present chapter provides some guidelines on the choice of expression vectors and expression strategies. It also brings the reader a complete toolbox from which plasmids and fusion sequences can be picked and assembled to set up appropriate expression vectors. Finally, it provides standard starting protocols for the preparation of the selected plasmids and their use for host strain transformation.

  7. Distribution of small native plasmids in Streptococcus pyogenes in India.

    PubMed

    Bergmann, René; Nerlich, Andreas; Chhatwal, Gursharan S; Nitsche-Schmitz, D Patric

    2014-05-01

    Complete characterization of a Streptococcus pyogenes population from a defined geographic region comprises information on the plasmids that circulate in these bacteria. Therefore, we determined the distribution of small plasmids (<5kb) in a collection of 279 S. pyogenes isolates from India, where diversity of strains and incidence rates of S. pyogenes infections are high. The collection comprised 77 emm-types. For plasmid detection and discrimination, we developed PCRs for different plasmid replication initiation protein genes, the putative repressor gene copG and bacteriocin genes dysA and scnM57. Plasmid distribution was limited to 13 emm-types. Co-detection analysis using aforementioned PCRs revealed four distinct plasmid sub-types, two of which were previously unknown. Representative plasmids pA852 and pA996 of the two uncharacterized plasmid sub-types were sequenced. These two plasmids could be assigned to the pMV158 and the pC194/pUB110 family of rolling-circle plasmids, respectively. The majority of small plasmids found in India belonged to the two newly characterized sub-types, with pA852- and pA996-like plasmids amounting to 42% and 22% of all detected plasmids, respectively. None of the detected plasmids coded for a known antibiotic resistance gene. Instead, all of the four plasmid sub-types carried known or potential bacteriocin genes. These genes may have influence on the evolutionary success of certain S. pyogenes genotypes. Notably, pA852-like plasmids were found in all isolates of the most prevalent emm-type 11.0. Together, a priori fitness of this genotype and increased fitness due to the acquired plasmids may have rendered type emm11.0 successful and caused the prevalence of pA852-like plasmids in India.

  8. 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.

  9. 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

  10. 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

  11. Plasmidal maintenance of composite DNA derived from polyoma related plasmid, L factor.

    PubMed Central

    Saito, H; Uehara, H; Kusano, T; Oishi, M

    1987-01-01

    Recently, we reported a multicopy mammalian plasmid with a structure related to polyoma. The plasmid, named L factor, was found at a high copy number (5,000 or more per cell) in a subclone derived from mouse L cells. We attempted to utilize L factor as a plasmid vector for mammalian cells. A series of composite DNA consisting of L factor and a foreign (herpes simplex virus tk) were constructed. These DNA could be established as plasmids after transfection to several mouse cell lines, although the copy number of the re-established plasmids was considerably less than that observed for the original subclone. The composite DNA maintained the structure of the original DNA after prolonged culture and the copy number remained constant even with no selective pressure. A composite DNA, with no DNA sequence corresponding to polyoma T antigen, could also be established as a plasmid in a mouse L cell line in which polyoma T antigen is expressed. The potential use of the plasmid is discussed. Images PMID:2825120

  12. 12. VIEW OF SPACE BETWEEN EAST FALSE PARTITION WALL IN ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    12. VIEW OF SPACE BETWEEN EAST FALSE PARTITION WALL IN CLEAN ROOM (102) AND EAST WALL OF VEHICLE SUPPORT BUILDING SHOWING PREFILTER NEAR SOUTH WALL - Vandenberg Air Force Base, Space Launch Complex 3, Vehicle Support Building, Napa & Alden Roads, Lompoc, Santa Barbara County, CA

  13. Plasmid introduction in metal-stressed, subsurface-derived microcosms: plasmid fate and community response.

    PubMed

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

    2003-07-01

    The nonconjugal IncQ plasmids pMOL187 and pMOL222, which contain the metal resistance-encoding genes czc and ncc, were introduced by using Escherichia coli as a transitory delivery strain into microcosms containing subsurface-derived parent materials. The microcosms were semicontinuously dosed with an artificial groundwater to set a low-carbon flux and a target metal stress (0, 10, 100, and 1,000 micro M CdCl(2)), permitting long-term community monitoring. The broad-host-range IncPalpha plasmid RP4 was also transitorily introduced into a subset of microcosms. No novel community phenotype was detected after plasmid delivery, due to the high background resistances to Cd and Ni. At fixed Cd doses, however, small but consistent increases in Cd(r) or Ni(r) density were measured due to the introduction of a single pMOL plasmid, and this effect was enhanced by the joint introduction of RP4; the effects were most significant at the highest Cd doses. The pMOL plasmids introduced could, however, be monitored via czc- and ncc-targeted infinite-dilution PCR (ID-PCR) methods, because these genes were absent from the indigenous community: long-term presence of czc (after 14 or 27 weeks) was contingent on the joint introduction of RP4, although RP4 cointroduction was not yet required to ensure retention of ncc after 8 weeks. Plasmids isolated from Ni(r) transconjugants further confirmed the presence and retention of a pMOL222-sized plasmid. ID-PCR targeting the RP4-specific trafA gene revealed retention of RP4 for at least 8 weeks. Our findings confirm plasmid transfer and long-term retention in low-carbon-flux, metal-stressed subsurface communities but indicate that the subsurface community examined has limited mobilization potential for the IncQ plasmids employed.

  14. First Report of Complete Sequence of a blaNDM-13-Harboring Plasmid from an Escherichia coli ST5138 Clinical Isolate

    PubMed Central

    Lv, Jingnan; Qi, Xiuqin; Zhang, Dan; Zheng, Zhou; Chen, Yuehui; Guo, Yinjuan; Wang, Shanshan; Chen, Liang; Kreiswirth, Barry N.; Tang, Yi-Wei; Chen, Zengqiang; Hu, Longhua; Wang, Liangxing; Yu, Fangyou

    2016-01-01

    Since the first report of blaNDM-1, 16 blaNDM variants have been identified among Gram-negative bacteria worldwide. Recently, a novel blaNDM variant, blaNDM-13, was identified in the chromosome of an ST101 Escherichia coli isolate from Nepal. Here we first reported plasmid-mediated blaNDM-13 in a carbapenem-resistant E. coli ST5138 clinical isolate associated with hospital-acquired urinary tract infection from China. blaNDM-13 and blaSHV-12 coexisted on the a ~54 Kb self-transferable plasmid. Compared with NDM-1, NDM-13, NDM-3, and NDM-4 had two amino acid substitutions (D95N and M154L), one amino acid substitution (D95N) and one amino acid substitutions (M154L), respectively. Complete plasmid sequencing showed that blaNDM-13-harboring plasmid (pNDM13-DC33) was highly similar to the blaNDM-1-harboring IncX3 plasmid pNDM-HN380, a common blaNDM-harboring vector circulating in China. In accordance with the structure of pNDM-HN380, pNDM13-DC33 consists of a 33-kb backbone encoding plasmid replication (repB), stability partitioning, and transfer (tra, trb, and pil) functions, and a 21-kb antimicrobial resistance region with high GC content between umuD and mpr genes. In conclusion, the present study is the first report of a plasmid-encoded blaNDM-13 and the complete sequence of a blaNDM-13-harboring plasmid (pNDM13-DC33). blaNDM-13 maybe originate from blaNDM-1 located on a pNDM-HN380-like plasmid by sequential mutations.

  15. A versatile low-copy-number cloning vector derived from plasmid F.

    PubMed

    Shi, J; Biek, D P

    1995-10-16

    We have constructed a cloning vector based on plasmid mini-F for use in Escherichia coli. Plasmid pZC320 consists of the ori-2 replication unit of F that confers very low copy number (lcn), and includes the sop partition functions to insure stable plasmid maintenance in the absence of selection. A multiple cloning site (MCS) containing 16 unique restriction sites is located within the 5' end of the lacZ alpha gene. Expression of lacZ alpha is under the control of the wild-type lactose operator/promoter (lacOP) region and is efficiently repressed by the lacI repressor. Clones containing inserts can be detected using the blue/white screen for beta-galactosidase (beta Gal). A T7 promoter allows transcription of cloned inserts in the presence of T7 RNA polymerase. We have demonstrated the use of this lcn vector for cloning the regulated tetracycline-resistance genes from Tn10, which confer only low-level resistance when present at high copy number.

  16. Molecular characterization and structural instability of the industrially important composite metabolic plasmid pLP712.

    PubMed

    Wegmann, Udo; Overweg, Karin; Jeanson, Sophie; Gasson, Mike; Shearman, Claire

    2012-12-01

    The widely used plasmid-free Lactococcus lactis strain MG1363 was derived from the industrial dairy starter strain NCDO712. This strain carries a 55.39 kb plasmid encoding genes for lactose catabolism and a serine proteinase involved in casein degradation. We report the DNA sequencing and annotation of pLP712, which revealed additional metabolic genes, including peptidase F, d-lactate dehydrogenase and α-keto acid dehydrogenase (E3 complex). Comparison of pLP712 with other large lactococcal lactose and/or proteinase plasmids from L. lactis subsp. cremoris SK11 (pSK11L, pSK11P) and the plant strain L. lactis NCDO1867 (pGdh442) revealed their close relationship. The plasmid appears to have evolved through a series of genetic events as a composite of pGdh442, pSK11L and pSK11P. We describe in detail a scenario by which the metabolic genes relevant to the growth of its host in a milk environment have been unified on one replicon, reflecting the evolution of L. lactis as it changed its biological niche from plants to dairy environments. The extensive structural instability of pLP712 allows easy isolation of derivative plasmids lacking genes for casein degradation and/or lactose catabolism. Plasmid pLP712 is transferable by transduction and conjugation, and both of these processes result in significant molecular rearrangements. We report the detailed molecular analysis of insertion sequence element-mediated genetic rearrangements within pLP712 and several different mechanisms, including homologous recombination and adjacent deletion. Analysis of the integration of the lactose operon into the chromosome highlights the fluidity of the MG1363 integration hotspot and the potential for frequent movement of genes between plasmids and chromosomes in Lactococcus. PMID:23023974

  17. Exometabolite niche partitioning among sympatric soil bacteria

    SciTech Connect

    Baran, Richard; Brodie, Eoin L.; Mayberry-Lewis, Jazmine; Hummel, Eric; Da Rocha, Ulisses Nunes; Chakraborty, Romy; Bowen, Benjamin P.; Karaoz, Ulas; Cadillo-Quiroz, Hinsby; Garcia-Pichel, Ferran; Northen, Trent R.

    2015-09-22

    Soils are arguably the most microbially diverse ecosystems. Physicochemical properties have been associated with the maintenance of this diversity. Yet, the role of microbial substrate specialization is largely unexplored since substrate utilization studies have focused on simple substrates, not the complex mixtures representative of the soil environment. Here we examine the exometabolite composition of desert biological soil crusts (biocrusts) and the substrate preferences of seven biocrust isolates. The biocrust's main primary producer releases a diverse array of metabolites, and isolates of physically associated taxa use unique subsets of the complex metabolite pool. Individual isolates use only 13-26% of available metabolites, with only 2 out of 470 used by all and 40% not used by any. An extension of this approach to a mesophilic soil environment also reveals high levels of microbial substrate specialization. In conclusion, these results suggest that exometabolite niche partitioning may be an important factor in the maintenance of microbial diversity.

  18. Exometabolite niche partitioning among sympatric soil bacteria

    PubMed Central

    Baran, Richard; Brodie, Eoin L.; Mayberry-Lewis, Jazmine; Hummel, Eric; Da Rocha, Ulisses Nunes; Chakraborty, Romy; Bowen, Benjamin P.; Karaoz, Ulas; Cadillo-Quiroz, Hinsby; Garcia-Pichel, Ferran; Northen, Trent R.

    2015-01-01

    Soils are arguably the most microbially diverse ecosystems. Physicochemical properties have been associated with the maintenance of this diversity. Yet, the role of microbial substrate specialization is largely unexplored since substrate utilization studies have focused on simple substrates, not the complex mixtures representative of the soil environment. Here we examine the exometabolite composition of desert biological soil crusts (biocrusts) and the substrate preferences of seven biocrust isolates. The biocrust's main primary producer releases a diverse array of metabolites, and isolates of physically associated taxa use unique subsets of the complex metabolite pool. Individual isolates use only 13−26% of available metabolites, with only 2 out of 470 used by all and 40% not used by any. An extension of this approach to a mesophilic soil environment also reveals high levels of microbial substrate specialization. These results suggest that exometabolite niche partitioning may be an important factor in the maintenance of microbial diversity. PMID:26392107

  19. Exometabolite niche partitioning among sympatric soil bacteria

    DOE PAGES

    Baran, Richard; Brodie, Eoin L.; Mayberry-Lewis, Jazmine; Hummel, Eric; Da Rocha, Ulisses Nunes; Chakraborty, Romy; Bowen, Benjamin P.; Karaoz, Ulas; Cadillo-Quiroz, Hinsby; Garcia-Pichel, Ferran; et al

    2015-09-22

    Soils are arguably the most microbially diverse ecosystems. Physicochemical properties have been associated with the maintenance of this diversity. Yet, the role of microbial substrate specialization is largely unexplored since substrate utilization studies have focused on simple substrates, not the complex mixtures representative of the soil environment. Here we examine the exometabolite composition of desert biological soil crusts (biocrusts) and the substrate preferences of seven biocrust isolates. The biocrust's main primary producer releases a diverse array of metabolites, and isolates of physically associated taxa use unique subsets of the complex metabolite pool. Individual isolates use only 13-26% of available metabolites,more » with only 2 out of 470 used by all and 40% not used by any. An extension of this approach to a mesophilic soil environment also reveals high levels of microbial substrate specialization. In conclusion, these results suggest that exometabolite niche partitioning may be an important factor in the maintenance of microbial diversity.« less

  20. Extended spectrum beta-lactamase and fluoroquinolone resistance genes and plasmids among Escherichia coli isolates from zoo animals, Czech Republic.

    PubMed

    Dobiasova, Hana; Dolejska, Monika; Jamborova, Ivana; Brhelova, Eva; Blazkova, Lucie; Papousek, Ivo; Kozlova, Marketa; Klimes, Jiri; Cizek, Alois; Literak, Ivan

    2013-09-01

    Commensal Escherichia coli isolates from healthy zoo animals kept in Ostrava Zoological Garden, Czech Republic, were investigated to evaluate the dissemination of extended-spectrum beta-lactamase (ESBL) and plasmid-mediated quinolone resistance (PMQR) genes. A total of 160 faecal samples of various animal species were inoculated onto MacConkey agar with cefotaxime (2 mg L(-1)) or ciprofloxacin (0.05 mg L(-1)) to obtain ESBL- or PMQR-positive E. coli isolates. Clonality of E. coli isolates was investigated by multilocus sequence typing and pulsed-field gel electrophoresis. Plasmids carrying ESBL or PMQR genes were typed by PCR-based replicon typing, plasmid multilocus sequence typing and restriction fragment length polymorphism. Forty-nine (71%, n = 69) cefotaxime-resistant and 15 (16%, n = 94) ciprofloxacin-resistant E. coli isolates harboured ESBL or PMQR genes. Isolates were assigned to 18 sequence types (ST) and 20 clusters according to their macrorestriction patterns by pulsed-field gel electrophoresis. The genes blaCTX -M-1 and qnrS1 were detected on highly related IncI1 plasmids assigned to clonal complex 3 (ST3, ST38) and on non-related IncN plasmids of ST1 and ST3, respectively. The gene qnrS1 was located on related IncX1 plasmids. Dissemination of antibiotic resistance is associated with spreading of particular E. coli clones and plasmids of specific incompatibility groups among various animal species.

  1. Ecological dynamics and complex interactions of Agrobacterium megaplasmids.

    PubMed

    Platt, Thomas G; Morton, Elise R; Barton, Ian S; Bever, James D; Fuqua, Clay

    2014-01-01

    As with many pathogenic bacteria, agrobacterial plant pathogens carry most of their virulence functions on a horizontally transmissible genetic element. The tumor-inducing (Ti) plasmid encodes the majority of virulence functions for the crown gall agent Agrobacterium tumefaciens. This includes the vir genes which drive genetic transformation of host cells and the catabolic genes needed to utilize the opines produced by infected plants. The Ti plasmid also encodes, an opine-dependent quorum sensing system that tightly regulates Ti plasmid copy number and its conjugal transfer to other agrobacteria. Many natural agrobacteria are avirulent, lacking the Ti plasmid. The burden of harboring the Ti plasmid depends on the environmental context. Away from diseased hosts, plasmid costs are low but the benefit of the plasmid is also absent. Consequently, plasmidless genotypes are favored. On infected plants the costs of the Ti plasmid can be very high, but balanced by the opine benefits, locally favoring plasmid bearing cells. Cheating derivatives which do not incur virulence costs but can benefit from opines are favored on infected plants and in most other environments, and these are frequently isolated from nature. Many agrobacteria also harbor an At plasmid which can stably coexist with a Ti plasmid. At plasmid genes are less well characterized but in general facilitate metabolic activities in the rhizosphere and bulk soil, such as the ability to breakdown plant exudates. Examination of A. tumefaciens C58, revealed that harboring its At plasmid is much more costly than harboring it's Ti plasmid, but conversely the At plasmid is extremely difficult to cure. The interactions between these co-resident plasmids are complex, and depend on environmental context. However, the presence of a Ti plasmid appears to mitigate At plasmid costs, consistent with the high frequency with which they are found together. PMID:25452760

  2. Ecological dynamics and complex interactions of Agrobacterium megaplasmids

    PubMed Central

    Platt, Thomas G.; Morton, Elise R.; Barton, Ian S.; Bever, James D.; Fuqua, Clay

    2014-01-01

    As with many pathogenic bacteria, agrobacterial plant pathogens carry most of their virulence functions on a horizontally transmissible genetic element. The tumor-inducing (Ti) plasmid encodes the majority of virulence functions for the crown gall agent Agrobacterium tumefaciens. This includes the vir genes which drive genetic transformation of host cells and the catabolic genes needed to utilize the opines produced by infected plants. The Ti plasmid also encodes, an opine-dependent quorum sensing system that tightly regulates Ti plasmid copy number and its conjugal transfer to other agrobacteria. Many natural agrobacteria are avirulent, lacking the Ti plasmid. The burden of harboring the Ti plasmid depends on the environmental context. Away from diseased hosts, plasmid costs are low but the benefit of the plasmid is also absent. Consequently, plasmidless genotypes are favored. On infected plants the costs of the Ti plasmid can be very high, but balanced by the opine benefits, locally favoring plasmid bearing cells. Cheating derivatives which do not incur virulence costs but can benefit from opines are favored on infected plants and in most other environments, and these are frequently isolated from nature. Many agrobacteria also harbor an At plasmid which can stably coexist with a Ti plasmid. At plasmid genes are less well characterized but in general facilitate metabolic activities in the rhizosphere and bulk soil, such as the ability to breakdown plant exudates. Examination of A. tumefaciens C58, revealed that harboring its At plasmid is much more costly than harboring it’s Ti plasmid, but conversely the At plasmid is extremely difficult to cure. The interactions between these co-resident plasmids are complex, and depend on environmental context. However, the presence of a Ti plasmid appears to mitigate At plasmid costs, consistent with the high frequency with which they are found together. PMID:25452760

  3. P1 plasmid replication requires methylated DNA.

    PubMed Central

    Abeles, A L; Austin, S J

    1987-01-01

    Plasmids driven by the plasmid replication origin of bacteriophage P1 cannot be established in Escherichia coli strains that are defective for the DNA adenine methylase (dam). Using a composite plasmid that has two origins, we show that the P1 origin cannot function even in a plasmid that is already established in a dam strain. An in vitro replication system for the P1 origin was developed that uses as a substrate M13 replicative-form DNA containing the minimal P1 origin. The reaction mixture contains a crude extract of E. coli and purified P1 RepA protein. In addition to being RepA dependent, synthesis was shown to be dependent on methylation of the dam methylase-sensitive sites of the substrate DNA. As the P1 origin contains five such sites in a small region known to be critical for origin function, it can be concluded that methylation of these sites is a requirement for initiation. This suggests that the postreplicational methylation of the origin may control reinitiation and contribute to the accuracy of the highly stringent copy-number control of the origin in vivo. PMID:2826133

  4. Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material

    PubMed Central

    Dong, Lianhua; Meng, Ying; Sui, Zhiwei; Wang, Jing; Wu, Liqing; Fu, Boqiang

    2015-01-01

    Digital polymerase chain reaction (dPCR) is a unique approach to measurement of the absolute copy number of target DNA without using external standards. However, the comparability of different dPCR platforms with respect to measurement of DNA copy number must be addressed before dPCR can be classified fundamentally as an absolute quantification technique. The comparability of four dPCR platforms with respect to accuracy and measurement uncertainty was investigated by using a certified plasmid reference material. Plasmid conformation was found to have a significant effect on droplet-based dPCR (QX100 and RainDrop) not shared with chip-based QuantStudio 12k or BioMark. The relative uncertainty of partition volume was determined to be 0.7%, 0.8%, 2.3% and 2.9% for BioMark, QX100, QuantStudio 12k and RainDrop, respectively. The measurements of the certified pNIM-001 plasmid made using the four dPCR platforms were corrected for partition volume and closely consistent with the certified value within the expended uncertainty. This demonstrated that the four dPCR platforms are of comparable effectiveness in quantifying DNA copy number. These findings provide an independent assessment of this method of determining DNA copy number when using different dPCR platforms and underline important factors that should be taken into consideration in the design of dPCR experiments. PMID:26302947

  5. Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material.

    PubMed

    Dong, Lianhua; Meng, Ying; Sui, Zhiwei; Wang, Jing; Wu, Liqing; Fu, Boqiang

    2015-01-01

    Digital polymerase chain reaction (dPCR) is a unique approach to measurement of the absolute copy number of target DNA without using external standards. However, the comparability of different dPCR platforms with respect to measurement of DNA copy number must be addressed before dPCR can be classified fundamentally as an absolute quantification technique. The comparability of four dPCR platforms with respect to accuracy and measurement uncertainty was investigated by using a certified plasmid reference material. Plasmid conformation was found to have a significant effect on droplet-based dPCR (QX100 and RainDrop) not shared with chip-based QuantStudio 12k or BioMark. The relative uncertainty of partition volume was determined to be 0.7%, 0.8%, 2.3% and 2.9% for BioMark, QX100, QuantStudio 12k and RainDrop, respectively. The measurements of the certified pNIM-001 plasmid made using the four dPCR platforms were corrected for partition volume and closely consistent with the certified value within the expended uncertainty. This demonstrated that the four dPCR platforms are of comparable effectiveness in quantifying DNA copy number. These findings provide an independent assessment of this method of determining DNA copy number when using different dPCR platforms and underline important factors that should be taken into consideration in the design of dPCR experiments.

  6. Simple method for identification of plasmid-coded proteins.

    PubMed

    Sancar, A; Hack, A M; Rupp, W D

    1979-01-01

    Proteins encoded by plasmid DNA are specifically labeled in UV-irradiated cells of Escherichia coli carrying recA and uvrA mutations because extensive degradation of the chromosome DNA occurs concurrently with amplification of plasmid DNA.

  7. Plasmids spread very fast in heterogeneous bacterial communities.

    PubMed Central

    Dionisio, Francisco; Matic, Ivan; Radman, Miroslav; Rodrigues, Olivia R; Taddei, François

    2002-01-01

    Conjugative plasmids can mediate gene transfer between bacterial taxa in diverse environments. The ability to donate the F-type conjugative plasmid R1 greatly varies among enteric bacteria due to the interaction of the system that represses sex-pili formations (products of finOP) of plasmids already harbored by a bacterial strain with those of the R1 plasmid. The presence of efficient donors in heterogeneous bacterial populations can accelerate plasmid transfer and can spread by several orders of magnitude. Such donors allow millions of other bacteria to acquire the plasmid in a matter of days whereas, in the absence of such strains, plasmid dissemination would take years. This "amplification effect" could have an impact on the evolution of bacterial pathogens that exist in heterogeneous bacterial communities because conjugative plasmids can carry virulence or antibiotic-resistance genes. PMID:12524329

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

    PubMed

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

    2011-07-01

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

  9. Rectilinear partitioning of irregular data parallel computations

    NASA Technical Reports Server (NTRS)

    Nicol, David M.

    1991-01-01

    New mapping algorithms for domain oriented data-parallel computations, where the workload is distributed irregularly throughout the domain, but exhibits localized communication patterns are described. Researchers consider the problem of partitioning the domain for parallel processing in such a way that the workload on the most heavily loaded processor is minimized, subject to the constraint that the partition be perfectly rectilinear. Rectilinear partitions are useful on architectures that have a fast local mesh network. Discussed here is an improved algorithm for finding the optimal partitioning in one dimension, new algorithms for partitioning in two dimensions, and optimal partitioning in three dimensions. The application of these algorithms to real problems are discussed.

  10. Evidence for plasmid-mediated salt tolerance in the human gut microbiome and potential mechanisms.

    PubMed

    Broaders, Eileen; O'Brien, Ciarán; Gahan, Cormac G M; Marchesi, Julian R

    2016-03-01

    The human gut microbiome is critical to health and wellbeing. It hosts a complex ecosystem comprising a multitude of bacterial species, which contributes functionality that would otherwise be absent from the host. Transient and commensal bacteria in the gut must withstand many stresses. The influence of mobile genetic elements such as plasmids in stress adaptation within the ecosystem is poorly understood. Using a mobilomic approach we found evidence for plasmid-mediated osmotolerance as a phenotype amongst the Proteobacteria in healthy faecal slurries. A transconjugant carrying multiple plasmids acquired from healthy faecal slurry demonstrated increased osmotolerance in the presence of metal salts, particularly potassium chloride, which was not evident in the recipient. Pyrosequencing and analysis of the total plasmid DNA demonstrated the presence of plasmid-borne osmotolerance systems (including KdpD and H-NS) which may be linked to the observed phenotype. This is the first report of a transferable osmotolerance phenotype in gut commensals and may have implications for the transfer of osmotolerance in other niches.

  11. The role of branch architecture in assimilate production and partitioning: the example of apple (Malus domestica)

    PubMed Central

    Fanwoua, Julienne; Bairam, Emna; Delaire, Mickael; Buck-Sorlin, Gerhard

    2014-01-01

    Understanding the role of branch architecture in carbon production and allocation is essential to gain more insight into the complex process of assimilate partitioning in fruit trees. This mini review reports on the current knowledge of the role of branch architecture in carbohydrate production and partitioning in apple. The first-order carrier branch of apple illustrates the complexity of branch structure emerging from bud activity events and encountered in many fruit trees. Branch architecture influences carbon production by determining leaf exposure to light and by affecting leaf internal characteristics related to leaf photosynthetic capacity. The dynamics of assimilate partitioning between branch organs depends on the stage of development of sources and sinks. The sink strength of various branch organs and their relative positioning on the branch also affect partitioning. Vascular connections between branch organs determine major pathways for branch assimilate transport. We propose directions for employing a modeling approach to further elucidate the role of branch architecture on assimilate partitioning. PMID:25071813

  12. Presence of a virulence-associated plasmid in Yersinia pseudotuberculosis.

    PubMed Central

    Gemski, P; Lazere, J R; Casey, T; Wohlhieter, J A

    1980-01-01

    We have shown that Yersinia pseudotuberculosis can possess plasmids which are similar in size and function to the previously described Vwa plasmids of Y. enterocolitica. These plasmids are associated with the production of V and W antigens (calcium dependency) and pathogenicity of the organism. Further investigation of these plasmids from Y. pseudotuberculosis and Y. enterocolitica with restriction endonucleases revealed significant differences in their fragmentation pattern. Images Fig. 1 Fig. 2 Fig. 3 PMID:6249747

  13. METAL PARTITIONING IN COMBUSTION PROCESSES

    EPA Science Inventory

    This article summarizes ongoing research efforts at the National Risk Management Research Laboratory of the U.S. Environmental Protection Agency examining [high temperature] metal behavior within combustion environments. The partitioning of non-volatile (Cr and Ni), semi-volatil...

  14. Demonstration of plasmid-mediated drug resistance in Mycobacterium abscessus.

    PubMed

    Matsumoto, Cristianne Kayoko; Bispo, Paulo José Martins; Santin, Katiane; Nogueira, Christiane Lourenço; Leão, Sylvia Cardoso

    2014-05-01

    Plasmid-mediated kanamycin resistance was detected in a strain of Mycobacterium abscessus subsp. bolletii responsible for a nationwide epidemic of surgical infections in Brazil. The plasmid did not influence susceptibility to tobramycin, streptomycin, trimethoprim-sulfamethoxazole, clarithromycin, or ciprofloxacin. Plasmid-mediated drug resistance has not been described so far in mycobacteria. PMID:24574286

  15. 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.

  16. pRMH760, a precursor of A/C₂ plasmids carrying blaCMY and blaNDM genes.

    PubMed

    Harmer, Christopher J; Hall, Ruth M

    2014-10-01

    To investigate the evolution of plasmids in the repA/C2 group carrying genes conferring resistance to cephalosporins (bla(CMY)) or to carbapenems (bla(NDM)) and cephalosporins (bla(CMY)), the sequence of plasmid pRMH760 that lacks the β-lactamase genes was determined and compared to all available A/C2 plasmid sequences. pRMH760 is 170.6 kb and carries several antibiotic resistance genes in a 45.1 kb complex transposon structure located upstream of the rhs gene. In plasmid pR148, the closest relative of pRMH760, the antibiotic resistance island is in the same position but the resistance genes differ. pRMH760 also contains a deletion in the rhs gene. Sequenced A/C2 plasmids containing bla(CMY) or bla(CMY) and bla(NDM) have backbones closely related to the pRMH760/pR148 backbone, and they include resistance islands in the same location, indicating that they arose from a plasmid related to pRMH760/pR148. However, the gene content of this resistance island differs in each case, and the island family was designated ARI-A. The bla(NDM) gene is within ARI-A. The ISEcp1-bla(CMY) fragment is located elsewhere and is always in the same location, consistent with a single acquisition event. Plasmids containing only bla(CMY) carry a second resistance island, designated ARI-B, which includes the sul2 gene and a variable set of further resistance genes. Nine A/C2 plasmids that were not of this type (type 1) were found to have a similar backbone that can be simply distinguished by the presence of two exchanged regions and two insertions. Antibiotic resistance islands in type 2 plasmids are in different locations and have different structures.

  17. Efficient replication of Epstein-Barr virus-derived plasmids requires tethering by EBNA1 to host chromosomes.

    PubMed

    Hodin, Theresa L; Najrana, Tanbir; Yates, John L

    2013-12-01

    The EBNA1 protein of Epstein-Barr virus enables plasmids carrying oriP both to duplicate and to segregate efficiently in proliferating cells. EBNA1 recruits the origin recognition complex (ORC) to establish a replication origin at one element of oriP, DS (dyad symmetry); at another element, FR (family of repeats), EBNA1 binds to an array of sites from which it tethers plasmids to host chromosomes for mitotic stability. We report experiments leading to the conclusion that tethering by EBNA1 to host chromosomes is also needed within interphase nuclei in order for plasmids to be replicated efficiently from oriP. The DNA-binding domain of EBNA1, which lacks chromosome-binding ability, was found to support weak, DS-specific replication in HEK293 cells after transient transfection, being 17% as active as wild-type EBNA1. The low efficiency of replication was not due to the failure of the DNA-binding domain to retain plasmids within nuclei, because plasmids were recovered in similar amounts and entirely from the nuclear fraction of these transiently transfected cells. A derivative of EBNA1 with its chromosome-tethering domains replaced by a 22-amino-acid nucleosome-binding domain was fully active in supporting oriP functions. The implication is that EBNA1's DNA-binding domain is able to recruit ORC to DS, but either this step or subsequent replication is only efficient if the plasmid is tethered to a host chromosome. Finally, with some cell lines, DS can hardly support even transient plasmid replication without FR. A loss of plasmids lacking FR from nuclei cannot account for this requirement, suggesting that the stronger tethering to chromosomes by FR is needed for plasmid replication within the nuclei of such cells.

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

    PubMed

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

    2015-02-01

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

  19. Some trees with partition dimension three

    NASA Astrophysics Data System (ADS)

    Fredlina, Ketut Queena; Baskoro, Edy Tri

    2016-02-01

    The concept of partition dimension of a graph was introduced by Chartrand, E. Salehi and P. Zhang (1998) [2]. Let G(V, E) be a connected graph. For S ⊆ V (G) and v ∈ V (G), define the distance d(v, S) from v to S is min{d(v, x)|x ∈ S}. Let Π be an ordered partition of V (G) and Π = {S1, S2, ..., Sk }. The representation r(v|Π) of vertex v with respect to Π is (d(v, S1), d(v, S2), ..., d(v, Sk)). If the representations of all vertices are distinct, then the partition Π is called a resolving partition of G. The partition dimension of G is the minimum k such that G has a resolving partition with k partition classes. In this paper, we characterize some classes of trees with partition dimension three, namely olive trees, weeds, and centipedes.

  20. Approximate algorithms for partitioning and assignment problems

    NASA Technical Reports Server (NTRS)

    Iqbal, M. A.

    1986-01-01

    The problem of optimally assigning the modules of a parallel/pipelined program over the processors of a multiple computer system under certain restrictions on the interconnection structure of the program as well as the multiple computer system was considered. For a variety of such programs it is possible to find linear time if a partition of the program exists in which the load on any processor is within a certain bound. This method, when combined with a binary search over a finite range, provides an approximate solution to the partitioning problem. The specific problems considered were: a chain structured parallel program over a chain-like computer system, multiple chain-like programs over a host-satellite system, and a tree structured parallel program over a host-satellite system. For a problem with m modules and n processors, the complexity of the algorithm is no worse than O(mnlog(W sub T/epsilon)), where W sub T is the cost of assigning all modules to one processor and epsilon the desired accuracy.

  1. Partitioning of Nanoparticles into Organic Phases and Model Cells

    SciTech Connect

    Posner, J.D.; Westerhoff, P.; Hou, W-C.

    2011-08-25

    There is a recognized need to understand and predict the fate, transport and bioavailability of engineered nanoparticles (ENPs) in aquatic and soil ecosystems. Recent research focuses on either collection of empirical data (e.g., removal of a specific NP through water or soil matrices under variable experimental conditions) or precise NP characterization (e.g. size, degree of aggregation, morphology, zeta potential, purity, surface chemistry, and stability). However, it is almost impossible to transition from these precise measurements to models suitable to assess the NP behavior in the environment with complex and heterogeneous matrices. For decades, the USEPA has developed and applies basic partitioning parameters (e.g., octanol-water partition coefficients) and models (e.g., EPI Suite, ECOSAR) to predict the environmental fate, bioavailability, and toxicity of organic pollutants (e.g., pesticides, hydrocarbons, etc.). In this project we have investigated the hypothesis that NP partition coefficients between water and organic phases (octanol or lipid bilayer) is highly dependent on their physiochemical properties, aggregation, and presence of natural constituents in aquatic environments (salts, natural organic matter), which may impact their partitioning into biological matrices (bioaccumulation) and human exposure (bioavailability) as well as the eventual usage in modeling the fate and bioavailability of ENPs. In this report, we use the terminology "partitioning" to operationally define the fraction of ENPs distributed among different phases. The mechanisms leading to this partitioning probably involve both chemical force interactions (hydrophobic association, hydrogen bonding, ligand exchange, etc.) and physical forces that bring the ENPs in close contact with the phase interfaces (diffusion, electrostatic interactions, mixing turbulence, etc.). Our work focuses on partitioning, but also provides insight into the relative behavior of ENPs as either "more like

  2. Terminology for trace-element partitioning

    SciTech Connect

    Beattie, P. ); Drake, M. ); Jones, J.; McKay, G. ); Leeman, W. ); Longhi, J. ); Nielsen, R. ); Palme, H. ); Shaw, D. ); Takahashi, E. ); Watson, B. )

    1993-04-01

    A self-consistent terminology for partitioning data is presented. Ratios of the concentration of a component in two phases are termed partition coefficients and given the symbol D. Ratios of partition coefficients are termed exchange coefficients and given the symbol K[sub D]. The prefix bulk implies that these coefficients are weighted according to the proportions of coexisting phases. Bulk partition and bulk exchange coefficients are denoted by [bar D] and [ovr K[sub D

  3. Analysis of pFQ31, a 8551-bp cryptic plasmid from the symbiotic nitrogen-fixing actinomycete Frankia.

    PubMed

    Lavire, C; Louis, D; Perrière, G; Briolay, J; Normand, P; Cournoyer, B

    2001-04-01

    The actinomycete Frankia has never been transformed genetically. To favour the development of Frankia cloning vectors, we have fully sequenced the Frankia alni pFQ31 cryptic plasmid and performed analyses to characterise its coding and non-coding regions. This plasmid is 8551 bp-long and contains 72% G+C. Computer-assisted analyses identified 18 open reading frames (ORFs). These ORFs show a synonymous codon usage different from the one of Frankia chromosomal genes, suggesting an evolutionary bias linked to the nature of the replicon or a horizontal transfer. Three ORFs were found to encode genes likely to be involved in plasmid replication and stability: parFA (partition protein), ptrFA (transcriptional repressor of the GntR family) and repFA (initiation of replication). DNA signatures of a replication origin were identified in the ptrFA-repFA intergenic region. These structural motifs are similar to those observed among origins of iteron-containing plasmids replicating via a θ mode. PMID:11287155

  4. Cloning of a Thiobacillus ferrooxidans plasmid in Escherichia coli

    SciTech Connect

    Holmes, D.S.; Lobos, J.H.; Bopp, L.H.; Welch, G.C.

    1984-01-01

    Three separate plasmids of 6, 7, 16, and >23 kilobases were purified from a single clone of Thiobacillus ferrooxidans ATCC 33020 grown in the presence of uranium. The 6.7-kilobase plasmid (pTfl) was cloned separately into the HindIII or BamHI site of Escherichia coli plasmid pBR322. Restriction maps of the recombinant plasmids, termed pTf100 and pTf110, respectively, were constructed, creating potential cloning vehicles for exchanging genetic information between E. coli and T. ferrooxidans. Evidence from restriction enzyme analysis and Southern blot DNA-DNA hybridization indicates that the three native plasmids share little sequence homology.

  5. 25 CFR 158.56 - Partition records.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 25 Indians 1 2011-04-01 2011-04-01 false Partition records. 158.56 Section 158.56 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR LAND AND WATER OSAGE LANDS § 158.56 Partition records. Upon completion of an action in partition, a copy of the judgment roll showing schedule of costs...

  6. 25 CFR 158.56 - Partition records.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 25 Indians 1 2010-04-01 2010-04-01 false Partition records. 158.56 Section 158.56 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR LAND AND WATER OSAGE LANDS § 158.56 Partition records. Upon completion of an action in partition, a copy of the judgment roll showing schedule of costs...

  7. 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

  8. Intrauterine Infection with Plasmid-Free Chlamydia muridarum Reveals a Critical Role of the Plasmid in Chlamydial Ascension and Establishes a Model for Evaluating Plasmid-Independent Pathogenicity

    PubMed Central

    Chen, Jianlin; Yang, Zhangsheng; Sun, Xin; Tang, Lingli; Ding, Yiling; Xue, Min; Zhou, Zhiguang; Baseman, Joel

    2015-01-01

    Intravaginal infection with plasmid-competent but not plasmid-free Chlamydia muridarum induces hydrosalpinx in mouse upper genital tract, indicating a critical role of the plasmid in chlamydial pathogenicity. To evaluate the contribution of the plasmid to chlamydial ascension and activation of tubal inflammation, we delivered plasmid-free C. muridarum directly into the endometrium by intrauterine inoculation. We found that three of the six mouse strains tested, including CBA/J, C3H/HeJ, and C57BL/6J, developed significant hydrosalpinges when 1 × 107 inclusion-forming units (IFU) of plasmid-free C. muridarum were intrauterinally inoculated. Even when the inoculum was reduced to 1 × 104 IFU, the CBA/J mice still developed robust hydrosalpinx. The hydrosalpinx development in CBA/J mice correlated with increased organism ascension to the oviduct following the intrauterine inoculation. The CBA/J mice intravaginally infected with the same plasmid-free C. muridarum strain displayed reduced ascending infection and failed to develop hydrosalpinx. These observations have demonstrated a critical role of the plasmid in chlamydial ascending infection. The intrauterine inoculation of the CBA/J mice with plasmid-free C. muridarum not only resulted in more infection in the oviduct but also stimulated more inflammatory infiltration and cytokine production in the oviduct than the intravaginal inoculation, suggesting that the oviduct inflammation can be induced by plasmid-independent factors, which makes the hydrosalpinx induction in CBA/J mice by intrauterine infection with plasmid-free C. muridarum a suitable model for investigating plasmid-independent pathogenic mechanisms. PMID:25870225

  9. 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

  10. 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.

  11. The Salmonella Genomic Island 1 Is Specifically Mobilized In Trans by the IncA/C Multidrug Resistance Plasmid Family

    PubMed Central

    Douard, Gregory; Praud, Karine; Cloeckaert, Axel; Doublet, Benoît

    2010-01-01

    Background The Salmonella genomic island 1 (SGI1) is a Salmonella enterica-derived integrative mobilizable element (IME) containing various complex multiple resistance integrons identified in several S. enterica serovars and in Proteus mirabilis. Previous studies have shown that SGI1 transfers horizontally by in trans mobilization in the presence of the IncA/C conjugative helper plasmid pR55. Methodology/Principal Findings Here, we report the ability of different prevalent multidrug resistance (MDR) plasmids including extended-spectrum β-lactamase (ESBL) gene-carrying plasmids to mobilize the multidrug resistance genomic island SGI1. Through conjugation experiments, none of the 24 conjugative plasmids tested of the IncFI, FII, HI2, I1, L/M, N, P incompatibility groups were able to mobilize SGI1 at a detectable level (transfer frequency <10−9). In our collection, ESBL gene-carrying plasmids were mainly from the IncHI2 and I1 groups and thus were unable to mobilize SGI1. However, the horizontal transfer of SGI1 was shown to be specifically mediated by conjugative helper plasmids of the broad-host-range IncA/C incompatibility group. Several conjugative IncA/C MDR plasmids as well as the sequenced IncA/C reference plasmid pRA1 of 143,963 bp were shown to mobilize in trans SGI1 from a S. enterica donor to the Escherichia coli recipient strain. Depending on the IncA/C plasmid used, the conjugative transfer of SGI1 occurred at frequencies ranging from 10−3 to 10−6 transconjugants per donor. Of particular concern, some large IncA/C MDR plasmids carrying the extended-spectrum cephalosporinase blaCMY-2 gene were shown to mobilize in trans SGI1. Conclusions/Significance The ability of the IncA/C MDR plasmid family to mobilize SGI1 could contribute to its spread by horizontal transfer among enteric pathogens. Moreover, the increasing prevalence of IncA/C plasmids in MDR S. enterica isolates worldwide has potential implications for the epidemic success of the antibiotic

  12. Chloramphenicol Resistance Plasmids in Escherichia coli Isolated from Diseased Piglets

    PubMed Central

    Jørgensen, Sigrid Tue

    1978-01-01

    The plasmids in 19 chloramphenicol-resistant Escherichia coli strains of three pig pathogenic antigen types were studied in conjugation and transduction experiments. The plasmids had identical resistance patterns: streptomycin, spectinomycin, sulfonamides, and chloramphenicol (Sm, Sp, Su, Cm) and belonged to IncFII. One plasmid carried ampicillin resistance in addition. Restriction enzyme analysis of the deoxyribonucleic acid from five of the plasmids originating from the same herd showed that their digestion patterns with EcoRI were indistinguishable. EcoRI cleaved the deoxyribonucleic acid of a sixth plasmid from the same herd and displayed nine of the ten bands of the other five plasmids plus an additional six. It appears that the five plasmids with identical restriction patterns have a common origin and may be copies of the same plasmid from which the sixth may have developed. Four strains carried two plasmids each. In two of these strains, a plasmid with a tetracycline marker (Tc), or possibly the tetracycline marker alone, recombined frequently with the Sm Sp Su Cm plasmid without destroying any known function of the latter. The possibility that Tc is carried on a translocation sequence is discussed. Images PMID:352263

  13. 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.

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

    PubMed

    Cook, Laura C C; Dunny, Gary M

    2014-10-01

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

  15. Construction of a stable plasmid vector for industrial production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by a recombinant Cupriavidus necator H16 strain.

    PubMed

    Sato, Shunsuke; Fujiki, Tetsuya; Matsumoto, Keiji

    2013-12-01

    A new stable plasmid vector (pCUP3) was developed for high and stable production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) using Cupriavidus necator H16 as the host strain. In pCUP3, it was found that the plasmid partition and replication region of the megaplasmid pMOL28 in the Cupriavidus metallidurans CH34 strain plays an important role in plasmid stability in C. necator H16. Moreover, the partition locus (comprising parA28 and parB28 and the parS28 region) is essential for plasmid maintenance under high-PHBH-accumulation. PHBH productivity by the C. necator H16/ds strain (phaC1 deactivated mutant strain) harboring a phaCAc NSDG within pCUP3 was identical to the productivity of poly(3-hydroxybutyrate) by the C. necator H16 strain when palm kernel oil was used as the sole carbon source without any antibiotics. This new vector is important for industrial mass production of polyhydroxyalkanoates using the C. necator H16 strain as the host, dispensing the necessity of the application of selective pressure such as antibiotics.

  16. Linear Time Vertex Partitioning on Massive Graphs

    PubMed Central

    Mell, Peter; Harang, Richard; Gueye, Assane

    2016-01-01

    The problem of optimally removing a set of vertices from a graph to minimize the size of the largest resultant component is known to be NP-complete. Prior work has provided near optimal heuristics with a high time complexity that function on up to hundreds of nodes and less optimal but faster techniques that function on up to thousands of nodes. In this work, we analyze how to perform vertex partitioning on massive graphs of tens of millions of nodes. We use a previously known and very simple heuristic technique: iteratively removing the node of largest degree and all of its edges. This approach has an apparent quadratic complexity since, upon removal of a node and adjoining set of edges, the node degree calculations must be updated prior to choosing the next node. However, we describe a linear time complexity solution using an array whose indices map to node degree and whose values are hash tables indicating the presence or absence of a node at that degree value. This approach also has a linear growth with respect to memory usage which is surprising since we lowered the time complexity from quadratic to linear. We empirically demonstrate linear scalability and linear memory usage on random graphs of up to 15000 nodes. We then demonstrate tractability on massive graphs through execution on a graph with 34 million nodes representing Internet wide router connectivity. PMID:27336059

  17. Pathway of plasmid transformation in pneumococcus

    SciTech Connect

    Guild, W.R.; Saunders, C.W.

    1981-01-01

    Plasmids transform Streptococcus pneumoniae by a process involving low efficiency assembly of replicons from fragments of single strands that have entered the cell separately. Transformation of preexisting replicons is much more efficient. We have cloned the erm gene of pIP501 into pMV158, which so far as we know is the first example of cloning in a pneumococcus host-vector system.

  18. Plasmid DNA production for therapeutic applications.

    PubMed

    Lara, Alvaro R; Ramírez, Octavio T; Wunderlich, Martin

    2012-01-01

    Plasmid DNA (pDNA) is the base for promising DNA vaccines and gene therapies against many infectious, acquired, and genetic diseases, including HIV-AIDS, Ebola, Malaria, and different types of cancer, enteric pathogens, and influenza. Compared to conventional vaccines, DNA vaccines have many advantages such as high stability, not being infectious, focusing the immune response to only those antigens desired for immunization and long-term persistence of the vaccine protection. Especially in developing countries, where conventional effective vaccines are often unavailable or too expensive, there is a need for both new and improved vaccines. Therefore the demand of pDNA is expected to rise significantly in the near future. Since the injection of pDNA usually only leads to a weak immune response, several milligrams of DNA vaccine are necessary for immunization protection. Hence, there is a special interest to raise the product yield in order to reduce manufacturing costs. In this chapter, the different stages of plasmid DNA production are reviewed, from the vector design to downstream operation options. In particular, recent advances on cell engineering for improving plasmid DNA production are discussed. PMID:22160904

  19. Functional dissection of the ParB homologue (KorB) from IncP-1 plasmid RK2

    PubMed Central

    Lukaszewicz, M.; Kostelidou, K.; Bartosik, A. A.; Cooke, G. D.; Thomas, C. M.; Jagura-Burdzy, G.

    2002-01-01

    Active partitioning of low-copy number plasmids requires two proteins belonging to the ParA and ParB families and a cis-acting site which ParB acts upon. Active separation of clusters of plasmid molecules to the defined locations in the cell before cell division ensures stable inheritance of the plasmids. The central control operon of IncP-1 plasmids codes for regulatory proteins involved in the global transcriptional control of operons for vegetative replication, stable maintenance and conjugative transfer. Two of these proteins, IncC and KorB, also play a role in active partitioning, as the ParA and ParB homologues, respectively. Here we describe mapping the regions in KorB responsible for four of its different functions: dimerisation, DNA binding, repression of transcription and interaction with IncC. For DNA binding, amino acids E151 to T218 are essential, while repression depends not only on DNA binding but, additionally, on the adjacent region amino acids T218 to R255. The C-terminus of KorB is the main dimerisation domain but a secondary oligomerisation region is located centrally in the region from amino acid I174 to T218. Using three different methods (potentiation of transcriptional repression, potentiation of DNA binding and activation in the yeast two-hybrid system) we identify this region as also responsible for interactions with IncC. This IncC–KorB contact differs in location from the ParA–ParB/SopA–SopB interactions in P1/F but is similar to these systems in lying close to a masked oligomerisation determinant. PMID:11842117

  20. Invasion of E. coli biofilms by antibiotic resistance plasmids.

    PubMed

    Król, Jaroslaw E; Wojtowicz, Andrzej J; Rogers, Linda M; Heuer, Holger; Smalla, Kornelia; Krone, Stephen M; Top, Eva M

    2013-07-01

    In spite of the contribution of plasmids to the spread of antibiotic resistance in human pathogens, little is known about the transferability of various drug resistance plasmids in bacterial biofilms. The goal of this study was to compare the efficiency of transfer of 19 multidrug resistance plasmids into Escherichia coli recipient biofilms and determine the effects of biofilm age, biofilm-donor exposure time, and donor-to-biofilm attachment on this process. An E. coli recipient biofilm was exposed separately to 19 E. coli donors, each with a different plasmid, and transconjugants were determined by plate counting. With few exceptions, plasmids that transferred well in a liquid environment also showed the highest transferability in biofilms. The difference in transfer frequency between the most and least transferable plasmid was almost a million-fold. The 'invasibility' of the biofilm by plasmids, or the proportion of biofilm cells that acquired plasmids within a few hours, depended not only on the type of plasmid, but also on the time of biofilm exposure to the donor and on the ability of the plasmid donor to attach to the biofilm, yet not on biofilm age. The efficiency of donor strain attachment to the biofilm was not affected by the presence of plasmids. The most invasive plasmid was pHH2-227, which based on genome sequence analysis is a hybrid between IncU-like and IncW plasmids. The wide range in transferability in an E. coli biofilm among plasmids needs to be taken into account in our fight against the spread of drug resistance.

  1. Plasmid Dynamics in KPC-Positive Klebsiella pneumoniae during Long-Term Patient Colonization

    PubMed Central

    Park, Morgan; Deming, Clayton; Thomas, Pamela J.; Young, Alice C.; Coleman, Holly; Sison, Christina; Weingarten, Rebecca A.; Lau, Anna F.; Dekker, John P.; Palmore, Tara N.; Frank, Karen M.

    2016-01-01

    ABSTRACT Carbapenem-resistant Klebsiella pneumoniae strains are formidable hospital pathogens that pose a serious threat to patients around the globe due to a rising incidence in health care facilities, high mortality rates associated with infection, and potential to spread antibiotic resistance to other bacterial species, such as Escherichia coli. Over 6 months in 2011, 17 patients at the National Institutes of Health (NIH) Clinical Center became colonized with a highly virulent, transmissible carbapenem-resistant strain of K. pneumoniae. Our real-time genomic sequencing tracked patient-to-patient routes of transmission and informed epidemiologists’ actions to monitor and control this outbreak. Two of these patients remained colonized with carbapenemase-producing organisms for at least 2 to 4 years, providing the opportunity to undertake a focused genomic study of long-term colonization with antibiotic-resistant bacteria. Whole-genome sequencing studies shed light on the underlying complex microbial colonization, including mixed or evolving bacterial populations and gain or loss of plasmids. Isolates from NIH patient 15 showed complex plasmid rearrangements, leaving the chromosome and the blaKPC-carrying plasmid intact but rearranging the two other plasmids of this outbreak strain. NIH patient 16 has shown continuous colonization with blaKPC-positive organisms across multiple time points spanning 2011 to 2015. Genomic studies defined a complex pattern of succession and plasmid transmission across two different K. pneumoniae sequence types and an E. coli isolate. These findings demonstrate the utility of genomic methods for understanding strain succession, genome plasticity, and long-term carriage of antibiotic-resistant organisms. PMID:27353756

  2. Twisted sectors from plane partitions

    NASA Astrophysics Data System (ADS)

    Datta, Shouvik; Gaberdiel, Matthias R.; Li, Wei; Peng, Cheng

    2016-09-01

    Twisted sectors arise naturally in the bosonic higher spin CFTs at their free points, as well as in the associated symmetric orbifolds. We identify the coset representations of the twisted sector states using the description of W_{∞} representations in terms of plane partitions. We confirm these proposals by a microscopic null-vector analysis, and by matching the excitation spectrum of these representations with the orbifold prediction.

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

    PubMed Central

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

    2011-01-01

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

  4. Antimicrobial resistance and the ecology of Escherichia coli plasmids.

    PubMed Central

    Platt, D. J.; Sommerville, J. S.; Kraft, C. A.; Timbury, M. C.

    1984-01-01

    Four hundred and seven clinical isolates of Escherichia coli were examined for the presence of plasmids. These isolates comprised 189 which were collected irrespective of antimicrobial resistance (VP) and 218 which were collected on the basis of high-level trimethoprim resistance (TPR). The VP isolates were divided into drug sensitive (VPS) and drug-resistant (VPR) subpopulations. Plasmids were detected in 88% of VP isolates (81% of VPS and 94% of VPR) and 98% of TPR isolates. The distribution of plasmids in both groups and subpopulations was very similar. However, there were small but statistically significant differences between the plasmid distributions. These showed that more isolates in the resistant groups harboured plasmids than in the sensitive subpopulation (VPS) and that the number of plasmids carried by resistant isolates was greater. Multiple drug resistance was significantly more common among TPR isolates than the VPR subpopulation and this was paralleled by increased numbers of plasmids. Fifty-eight per cent of VPR and 57% of TPR isolates transferred antimicrobial resistance and plasmids to E. coli K12. Of the R+ isolates, 60% carried small plasmids (MW less than 20Md) and 52% of these co-transferred with R-plasmids. These results are discussed. PMID:6389695

  5. TDAB-induced DNA plasmid condensation on the surface of a reconstructed boron doped silicon substrate

    NASA Astrophysics Data System (ADS)

    Mougin, Antoine; Babak, Valéry G.; Palmino, Frank; Bêche, Eric; Baros, Francis; Hunting, Darel J.; Sanche, Léon; Fromm, Michel

    Our study aims at a better control and understanding of the transfer of a complex [DNA supercoiled plasmid - dodecyltrimethylammonium surfactant] layer from a liquid-vapour water interface onto a silicon surface without any additional cross-linker. The production of the complexed layer and its transfer from the aqueous subphase to the substrate is achieved with a Langmuir-Blodgett device. The substrate consists of a reconstructed boron doped silicon substrate with a nanometer-scale roughness. Using X-ray photoelectron spectroscopy and atomic force microscopy measurements, it is shown that the DNA complexes are stretched in a disorderly manner throughout a 2-4 nm high net-like structure. This architecture is composed of tilted cationic surfactant molecules bound electrostatically to DNA, which exhibits a characteristic network arrangement with a measured average fiber diameter of about 45 ± 15 nm covering the entire surface. The mechanism of transfer of this layer onto the planar surface of the semi-conductor and the parameters of the process are analysed and illustrated by atomic force microscopy snapshots. The molecular layer exhibits the typical characteristics of a spinodal decomposition pattern or dewetting features. Plasmid molecules appear like long flattened fibers covering the surface, forming holes of various shapes and areas. The cluster-cluster aggregation of the complex structure gets very much denser on the substrate edge. The supercoiled DNA plasmids undergo conformational changes and a high degree of condensation and aggregation is observed. Perspectives and potential applications are considered.

  6. Exposing plasmids as the Achilles' heel of drug-resistant bacteria.

    PubMed

    Williams, Julia J; Hergenrother, Paul J

    2008-08-01

    Many multidrug-resistant bacterial pathogens harbor large plasmids that encode proteins conferring resistance to antibiotics. Although 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.

  7. Plasmid-borne prokaryotic gene expression: Sources of variability and quantitative system characterization

    NASA Astrophysics Data System (ADS)

    Bagh, Sangram; Mazumder, Mostafizur; Velauthapillai, Tharsan; Sardana, Vandit; Dong, Guang Qiang; Movva, Ashok B.; Lim, Len H.; McMillen, David R.

    2008-02-01

    One aim of synthetic biology is to exert systematic control over cellular behavior, either for medical purposes or to “program” microorganisms. An engineering approach to the design of biological controllers demands a quantitative understanding of the dynamics of both the system to be controlled and the controllers themselves. Here we focus on a widely used method of exerting control in bacterial cells: plasmid vectors bearing gene-promoter pairs. We study two variants of the simplest such element, an unregulated promoter constitutively expressing its gene, against the varying genomic background of four Escherichia coli cell strains. Absolute protein numbers and rates of expression vary with both cell strain and plasmid type, as does the variability of expression across the population. Total variability is most strongly coupled to the cell division process, and after cell size is scaled away, plasmid copy number regulation emerges as a significant effect. We present simple models that capture the main features of the system behavior. Our results confirm that complex interactions between plasmids and their hosts can have significant effects on both expression and variability, even in deliberately simplified systems.

  8. Program partitioning for NUMA multiprocessor computer systems. [Nonuniform memory access

    SciTech Connect

    Wolski, R.M.; Feo, J.T. )

    1993-11-01

    Program partitioning and scheduling are essential steps in programming non-shared-memory computer systems. Partitioning is the separation of program operations into sequential tasks, and scheduling is the assignment of tasks to processors. To be effective, automatic methods require an accurate representation of the model of computation and the target architecture. Current partitioning methods assume today's most prevalent models -- macro dataflow and a homogeneous/two-level multicomputer system. Based on communication channels, neither model represents well the emerging class of NUMA multiprocessor computer systems consisting of hierarchical read/write memories. Consequently, the partitions generated by extant methods do not execute well on these systems. In this paper, the authors extend the conventional graph representation of the macro-dataflow model to enable mapping heuristics to consider the complex communication options supported by NUMA architectures. They describe two such heuristics. Simulated execution times of program graphs show that the model and heuristics generate higher quality program mappings than current methods for NUMA architectures.

  9. Unsupervised segmentation of MRI knees using image partition forests

    NASA Astrophysics Data System (ADS)

    Marčan, Marija; Voiculescu, Irina

    2016-03-01

    Nowadays many people are affected by arthritis, a condition of the joints with limited prevention measures, but with various options of treatment the most radical of which is surgical. In order for surgery to be successful, it can make use of careful analysis of patient-based models generated from medical images, usually by manual segmentation. In this work we show how to automate the segmentation of a crucial and complex joint -- the knee. To achieve this goal we rely on our novel way of representing a 3D voxel volume as a hierarchical structure of partitions which we have named Image Partition Forest (IPF). The IPF contains several partition layers of increasing coarseness, with partitions nested across layers in the form of adjacency graphs. On the basis of a set of properties (size, mean intensity, coordinates) of each node in the IPF we classify nodes into different features. Values indicating whether or not any particular node belongs to the femur or tibia are assigned through node filtering and node-based region growing. So far we have evaluated our method on 15 MRI knee images. Our unsupervised segmentation compared against a hand-segmented gold standard has achieved an average Dice similarity coefficient of 0.95 for femur and 0.93 for tibia, and an average symmetric surface distance of 0.98 mm for femur and 0.73 mm for tibia. The paper also discusses ways to introduce stricter morphological and spatial conditioning in the bone labelling process.

  10. Intracellular Trafficking of Plasmids for Gene Therapy: Mechanisms of Cytoplasmic Movement and Nuclear Import

    PubMed Central

    Dean, David A.

    2015-01-01

    Under physiologically relevant conditions, the levels of non-viral gene transfer are low at best. The reason for this is that many barriers exist for the efficient transfer of genes to cells, even before any gene expression can occur. While many transfection strategies focus on DNA condensation and overcoming the plasma membrane, events associated with the intracellular trafficking of the DNA complexes have not been as extensively studied. Once internalized, plasmids must travel potentially long distances through the cytoplasm to reach their next barrier, the nuclear envelope. This review summarizes the current progress on the cytoplasmic trafficking and nuclear transport of plasmids used for gene therapy applications. Both of these processes utilize specific and defined mechanisms to facilitate movement of DNA complexes through the cell. The continued elucidation and exploitation of these mechanisms will lead to improved strategies for transfection and successful gene therapy. PMID:17168698

  11. 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

  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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  14. 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...

  15. On some trees having partition dimension four

    NASA Astrophysics Data System (ADS)

    Ida Bagus Kade Puja Arimbawa, K.; Baskoro, Edy Tri

    2016-02-01

    In 1998, G. Chartrand, E. Salehi and P. Zhang introduced the notion of partition dimension of a graph. Since then, the study of this graph parameter has received much attention. A number of results have been obtained to know the values of partition dimensions of various classes of graphs. However, for some particular classes of graphs, finding of their partition dimensions is still not completely solved, for instances a class of general tree. In this paper, we study the properties of trees having partition dimension 4. In particular, we show that, for olive trees O(n), its partition dimension is equal to 4 if and only if 8 ≤ n ≤ 17. We also characterize all centipede trees having partition dimension 4.

  16. Transcriptome modulations due to A/C2 plasmid acquisition.

    PubMed

    Lang, Kevin S; Johnson, Timothy J

    2015-07-01

    Plasmids play an important role in driving the genetic diversity of bacteria. Horizontal gene transfer via plasmids is crucial for the dissemination of antimicrobial resistance genes. Many factors contribute to the persistence of plasmids within bacterial populations, and it has been suggested that epistatic interactions between the host chromosome and plasmid contribute to the fitness of a particular plasmid-host combination. However, such interactions have been shown to differ between bacterial hosts. In this study, RNA-Seq was performed in six different strains, spanning three species, to characterize the influence of host background on the A/C2 plasmid transcriptome. In five of these strains, chromosomal transcriptomes were compared in the presence and absence of A/C2 plasmid pAR060302. Host-specific effects on plasmid gene expression were identified, and acquisition of pAR060302 resulted in changes in the expression of chromosomal genes involved in metabolism and energy production. These results suggest that A/C2 plasmid fitness is, in part, dependent on host chromosome content, as well as environmental factors. PMID:26079188

  17. Plasmid genes required for microcin B17 production.

    PubMed Central

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

    1985-01-01

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

  18. 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.

  19. Plasmid Recombination in a Rad52 Mutant of Saccharomyces Cerevisiae

    PubMed Central

    Dornfeld, K. J.; Livingston, D. M.

    1992-01-01

    Using plasmids capable of undergoing intramolecular recombination, we have compared the rates and the molecular outcomes of recombination events in a wild-type and a rad52 strain of Saccharomyces cerevisiae. The plasmids contain his3 heteroalleles oriented in either an inverted or a direct repeat. Inverted repeat plasmids recombine approximately 20-fold less frequently in the mutant than in the wild-type strain. Most events from both cell types have continuous coconversion tracts extending along one of the homologous segments. Reciprocal exchange occurs in fewer than 30% of events. Direct repeat plasmids recombine at rates comparable to those of inverted repeat plasmids in wild-type cells. Direct repeat conversion tracts are similar to inverted repeat conversion tracts in their continuity and length. Inverted and direct repeat plasmid recombination differ in two respects. First, rad52 does not affect the rate of direct repeat recombination as drastically as the rate of inverted repeat recombination. Second, direct repeat plasmids undergo crossing over more frequently than inverted repeat plasmids. In addition, crossovers constitute a larger fraction of mutant than wild-type direct repeat events. Many crossover events from both cell types are unusual in that the crossover HIS3 allele is within a plasmid containing the parental his3 heteroalleles. PMID:1644271

  20. Plasmid genes required for microcin B17 production.

    PubMed

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

    1985-09-01

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

  1. Plasmids foster diversification and adaptation of bacterial populations in soil.

    PubMed

    Heuer, Holger; Smalla, Kornelia

    2012-11-01

    It is increasingly being recognized that the transfer of conjugative plasmids across species boundaries plays a vital role in the adaptability of bacterial populations in soil. There are specific driving forces and constraints of plasmid transfer within bacterial communities in soils. Plasmid-mediated genetic variation allows bacteria to respond rapidly with adaptive responses to challenges such as irregular antibiotic or metal concentrations, or opportunities such as the utilization of xenobiotic compounds. Cultivation-independent detection and capture of plasmids from soil bacteria, and complete sequencing have provided new insights into the role and ecology of plasmids. Broad host range plasmids such as those belonging to IncP-1 transfer a wealth of accessory functions which are carried by similar plasmid backbones. Plasmids with a narrower host range can be more specifically adapted to particular species and often transfer genes which complement chromosomally encoded functions. Plasmids seem to be an ancient and successful strategy to ensure survival of a soil population in spatial and temporal heterogeneous conditions with various environmental stresses or opportunities that occur irregularly or as a novel challenge in soil.

  2. Distribution of Plasmids in Distinct Leptospira Pathogenic Species.

    PubMed

    Wang, Yanzhuo; Zhuang, Xuran; Zhong, Yi; Zhang, Cuicai; Zhang, Yan; Zeng, Lingbing; Zhu, Yongzhang; He, Ping; Dong, Ke; Pal, Utpal; Guo, Xiaokui; Qin, Jinhong

    2015-11-01

    Leptospirosis, caused by pathogenic Leptospira, is a worldwide zoonotic infection. The genus Leptospira includes at least 21 species clustered into three groups--pathogens, non-pathogens, and intermediates--based on 16S rRNA phylogeny. Research on Leptospira is difficult due to slow growth and poor transformability of the pathogens. Recent identification of extrachromosomal elements besides the two chromosomes in L. interrogans has provided new insight into genome complexity of the genus Leptospira. The large size, low copy number, and high similarity of the sequence of these extrachromosomal elements with the chromosomes present challenges in isolating and detecting them without careful genome assembly. In this study, two extrachromosomal elements were identified in L. borgpetersenii serovar Ballum strain 56604 through whole genome assembly combined with S1 nuclease digestion following pulsed-field gel electrophoresis (S1-PFGE) analysis. Further, extrachromosomal elements in additional 15 Chinese epidemic strains of Leptospira, comprising L. borgpetersenii, L. weilii, and L. interrogans, were successfully separated and identified, independent of genome sequence data. Southern blot hybridization with extrachromosomal element-specific probes, designated as lcp1, lcp2 and lcp3-rep, further confirmed their occurrences as extrachromosomal elements. In total, 24 plasmids were detected in 13 out of 15 tested strains, among which 11 can hybridize with the lcp1-rep probe and 11 with the lcp2-rep probe, whereas two can hybridize with the lcp3-rep probe. None of them are likely to be species-specific. Blastp search of the lcp1, lcp2, and lcp3-rep genes with a nonredundant protein database of Leptospira species genomes showed that their homologous sequences are widely distributed among clades of pathogens but not non-pathogens or intermediates. These results suggest that the plasmids are widely distributed in Leptospira species, and further elucidation of their biological

  3. Distribution of Plasmids in Distinct Leptospira Pathogenic Species.

    PubMed

    Wang, Yanzhuo; Zhuang, Xuran; Zhong, Yi; Zhang, Cuicai; Zhang, Yan; Zeng, Lingbing; Zhu, Yongzhang; He, Ping; Dong, Ke; Pal, Utpal; Guo, Xiaokui; Qin, Jinhong

    2015-11-01

    Leptospirosis, caused by pathogenic Leptospira, is a worldwide zoonotic infection. The genus Leptospira includes at least 21 species clustered into three groups--pathogens, non-pathogens, and intermediates--based on 16S rRNA phylogeny. Research on Leptospira is difficult due to slow growth and poor transformability of the pathogens. Recent identification of extrachromosomal elements besides the two chromosomes in L. interrogans has provided new insight into genome complexity of the genus Leptospira. The large size, low copy number, and high similarity of the sequence of these extrachromosomal elements with the chromosomes present challenges in isolating and detecting them without careful genome assembly. In this study, two extrachromosomal elements were identified in L. borgpetersenii serovar Ballum strain 56604 through whole genome assembly combined with S1 nuclease digestion following pulsed-field gel electrophoresis (S1-PFGE) analysis. Further, extrachromosomal elements in additional 15 Chinese epidemic strains of Leptospira, comprising L. borgpetersenii, L. weilii, and L. interrogans, were successfully separated and identified, independent of genome sequence data. Southern blot hybridization with extrachromosomal element-specific probes, designated as lcp1, lcp2 and lcp3-rep, further confirmed their occurrences as extrachromosomal elements. In total, 24 plasmids were detected in 13 out of 15 tested strains, among which 11 can hybridize with the lcp1-rep probe and 11 with the lcp2-rep probe, whereas two can hybridize with the lcp3-rep probe. None of them are likely to be species-specific. Blastp search of the lcp1, lcp2, and lcp3-rep genes with a nonredundant protein database of Leptospira species genomes showed that their homologous sequences are widely distributed among clades of pathogens but not non-pathogens or intermediates. These results suggest that the plasmids are widely distributed in Leptospira species, and further elucidation of their biological

  4. Chemical amplification based on fluid partitioning in an immiscible liquid

    DOEpatents

    Anderson, Brian L.; Colston, Bill W.; Elkin, Christopher J.

    2010-09-28

    A system for nucleic acid amplification of a sample comprises partitioning the sample into partitioned sections and performing PCR on the partitioned sections of the sample. Another embodiment of the invention provides a system for nucleic acid amplification and detection of a sample comprising partitioning the sample into partitioned sections, performing PCR on the partitioned sections of the sample, and detecting and analyzing the partitioned sections of the sample.

  5. Mass partitioning effects in diffusion transport.

    PubMed

    Kojic, Milos; Milosevic, Miljan; Wu, Suhong; Blanco, Elvin; Ferrari, Mauro; Ziemys, Arturas

    2015-08-28

    Frequent mass exchange takes place in a heterogeneous environment among several phases, where mass partitioning may occur at the interface of phases. Analytical and computational methods for diffusion do not usually incorporate molecule partitioning masking the true picture of mass transport. Here we present a computational finite element methodology to calculate diffusion mass transport with a partitioning phenomenon included and the analysis of the effects of partitioning. Our numerical results showed that partitioning controls equilibrated mass distribution as expected from analytical solutions. The experimental validation of mass release from drug-loaded nanoparticles showed that partitioning might even dominate in some cases with respect to diffusion itself. The analysis of diffusion kinetics in the parameter space of partitioning and diffusivity showed that partitioning is an extremely important parameter in systems, where mass diffusivity is fast and that the concentration of nanoparticles can control payload retention inside nanoparticles. The computational and experimental results suggest that partitioning and physiochemical properties of phases play an important, if not crucial, role in diffusion transport and should be included in the studies of mass transport processes.

  6. Mass partitioning effects in diffusion transport.

    PubMed

    Kojic, Milos; Milosevic, Miljan; Wu, Suhong; Blanco, Elvin; Ferrari, Mauro; Ziemys, Arturas

    2015-08-28

    Frequent mass exchange takes place in a heterogeneous environment among several phases, where mass partitioning may occur at the interface of phases. Analytical and computational methods for diffusion do not usually incorporate molecule partitioning masking the true picture of mass transport. Here we present a computational finite element methodology to calculate diffusion mass transport with a partitioning phenomenon included and the analysis of the effects of partitioning. Our numerical results showed that partitioning controls equilibrated mass distribution as expected from analytical solutions. The experimental validation of mass release from drug-loaded nanoparticles showed that partitioning might even dominate in some cases with respect to diffusion itself. The analysis of diffusion kinetics in the parameter space of partitioning and diffusivity showed that partitioning is an extremely important parameter in systems, where mass diffusivity is fast and that the concentration of nanoparticles can control payload retention inside nanoparticles. The computational and experimental results suggest that partitioning and physiochemical properties of phases play an important, if not crucial, role in diffusion transport and should be included in the studies of mass transport processes. PMID:26204522

  7. Molecular characterisation of the Chlamydia pecorum plasmid from porcine, ovine, bovine, and koala strains indicates plasmid-strain co-evolution

    PubMed Central

    Jelocnik, Martina; Bachmann, Nathan L.; Seth-Smith, Helena; Thomson, Nicholas R.; Timms, Peter

    2016-01-01

    Background. Highly stable, evolutionarily conserved, small, non-integrative plasmids are commonly found in members of the Chlamydiaceae and, in some species, these plasmids have been strongly linked to virulence. To date, evidence for such a plasmid in Chlamydia pecorum has been ambiguous. In a recent comparative genomic study of porcine, ovine, bovine, and koala C. pecorum isolates, we identified plasmids (pCpec) in a pig and three koala strains, respectively. Screening of further porcine, ovine, bovine, and koala C. pecorum isolates for pCpec showed that pCpec is common, but not ubiquitous in C. pecorum from all of the infected hosts. Methods. We used a combination of (i) bioinformatic mining of previously sequenced C. pecorum genome data sets and (ii) pCpec PCR-amplicon sequencing to characterise a further 17 novel pCpecs in C. pecorum isolates obtained from livestock, including pigs, sheep, and cattle, as well as those from koala. Results and Discussion. This analysis revealed that pCpec is conserved with all eight coding domain sequences (CDSs) present in isolates from each of the hosts studied. Sequence alignments revealed that the 21 pCpecs show 99% nucleotide sequence identity, with 83 single nucleotide polymorphisms (SNPs) shown to differentiate all of the plasmids analysed in this study. SNPs were found to be mostly synonymous and were distributed evenly across all eight pCpec CDSs as well as in the intergenic regions. Although conserved, analyses of the 21 pCpec sequences resolved plasmids into 12 distinct genotypes, with five shared between pCpecs from different isolates, and the remaining seven genotypes being unique to a single pCpec. Phylogenetic analysis revealed congruency and co-evolution of pCpecs with their cognate chromosome, further supporting polyphyletic origin of the koala C. pecorum. This study provides further understanding of the complex epidemiology of this pathogen in livestock and koala hosts and paves the way for studies to evaluate

  8. Molecular characterisation of the Chlamydia pecorum plasmid from porcine, ovine, bovine, and koala strains indicates plasmid-strain co-evolution.

    PubMed

    Jelocnik, Martina; Bachmann, Nathan L; Seth-Smith, Helena; Thomson, Nicholas R; Timms, Peter; Polkinghorne, Adam M

    2016-01-01

    Background. Highly stable, evolutionarily conserved, small, non-integrative plasmids are commonly found in members of the Chlamydiaceae and, in some species, these plasmids have been strongly linked to virulence. To date, evidence for such a plasmid in Chlamydia pecorum has been ambiguous. In a recent comparative genomic study of porcine, ovine, bovine, and koala C. pecorum isolates, we identified plasmids (pCpec) in a pig and three koala strains, respectively. Screening of further porcine, ovine, bovine, and koala C. pecorum isolates for pCpec showed that pCpec is common, but not ubiquitous in C. pecorum from all of the infected hosts. Methods. We used a combination of (i) bioinformatic mining of previously sequenced C. pecorum genome data sets and (ii) pCpec PCR-amplicon sequencing to characterise a further 17 novel pCpecs in C. pecorum isolates obtained from livestock, including pigs, sheep, and cattle, as well as those from koala. Results and Discussion. This analysis revealed that pCpec is conserved with all eight coding domain sequences (CDSs) present in isolates from each of the hosts studied. Sequence alignments revealed that the 21 pCpecs show 99% nucleotide sequence identity, with 83 single nucleotide polymorphisms (SNPs) shown to differentiate all of the plasmids analysed in this study. SNPs were found to be mostly synonymous and were distributed evenly across all eight pCpec CDSs as well as in the intergenic regions. Although conserved, analyses of the 21 pCpec sequences resolved plasmids into 12 distinct genotypes, with five shared between pCpecs from different isolates, and the remaining seven genotypes being unique to a single pCpec. Phylogenetic analysis revealed congruency and co-evolution of pCpecs with their cognate chromosome, further supporting polyphyletic origin of the koala C. pecorum. This study provides further understanding of the complex epidemiology of this pathogen in livestock and koala hosts and paves the way for studies to evaluate

  9. Molecular characterisation of the Chlamydia pecorum plasmid from porcine, ovine, bovine, and koala strains indicates plasmid-strain co-evolution.

    PubMed

    Jelocnik, Martina; Bachmann, Nathan L; Seth-Smith, Helena; Thomson, Nicholas R; Timms, Peter; Polkinghorne, Adam M

    2016-01-01

    Background. Highly stable, evolutionarily conserved, small, non-integrative plasmids are commonly found in members of the Chlamydiaceae and, in some species, these plasmids have been strongly linked to virulence. To date, evidence for such a plasmid in Chlamydia pecorum has been ambiguous. In a recent comparative genomic study of porcine, ovine, bovine, and koala C. pecorum isolates, we identified plasmids (pCpec) in a pig and three koala strains, respectively. Screening of further porcine, ovine, bovine, and koala C. pecorum isolates for pCpec showed that pCpec is common, but not ubiquitous in C. pecorum from all of the infected hosts. Methods. We used a combination of (i) bioinformatic mining of previously sequenced C. pecorum genome data sets and (ii) pCpec PCR-amplicon sequencing to characterise a further 17 novel pCpecs in C. pecorum isolates obtained from livestock, including pigs, sheep, and cattle, as well as those from koala. Results and Discussion. This analysis revealed that pCpec is conserved with all eight coding domain sequences (CDSs) present in isolates from each of the hosts studied. Sequence alignments revealed that the 21 pCpecs show 99% nucleotide sequence identity, with 83 single nucleotide polymorphisms (SNPs) shown to differentiate all of the plasmids analysed in this study. SNPs were found to be mostly synonymous and were distributed evenly across all eight pCpec CDSs as well as in the intergenic regions. Although conserved, analyses of the 21 pCpec sequences resolved plasmids into 12 distinct genotypes, with five shared between pCpecs from different isolates, and the remaining seven genotypes being unique to a single pCpec. Phylogenetic analysis revealed congruency and co-evolution of pCpecs with their cognate chromosome, further supporting polyphyletic origin of the koala C. pecorum. This study provides further understanding of the complex epidemiology of this pathogen in livestock and koala hosts and paves the way for studies to evaluate

  10. Stabilized plasmid-lipid particles for regional gene therapy: formulation and transfection properties.

    PubMed

    Zhang, Y P; Sekirov, L; Saravolac, E G; Wheeler, J J; Tardi, P; Clow, K; Leng, E; Sun, R; Cullis, P R; Scherrer, P

    1999-08-01

    Previous work (Wheeler et al, Gene Therapy 1999; 6: 271-281) has shown that plasmid DNA can be entrapped in 'stabilized plasmid-lipid particles' (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating. The PEG moieties are attached to a ceramide anchor containing an arachidoyl acyl group (PEG-CerC20). These SPLP exhibit low transfection potencies in vitro, due in part to the long residence time of the PEG-CerC20 on the SPLP surface. In this work we employed SPLP stabilized by PEG attached to ceramide containing an octanoyl acyl group (PEG-CerC8), which is able to quickly exchange out of the SPLP, to develop systems that give rise to optimized in vitro and in vivo (regional) transfection. A particular objective was to achieve cationic lipid contents that give rise to maximum transfection levels. It is shown that by performing the dialysis procedure in the presence of increasing concentrations of citrate, SPLP containing up to 30 mol% of the cationic lipid dioleoydimethylammonium chloride (DODAC) could be generated. The SPLP produced could be isolated from empty vesicles by sucrose density gradient centrifugation, and exhibited a narrow size distribution (62 +/- 8 nm, as determined by freeze-fracture electron microscopy) and a high plasmid-to-lipid ratio of 65 microg/micromol (corresponding to one plasmid per particle) regardless of the DODAC content. It was found that isolated SPLP containing 20-24 mol% DODAC resulted in optimum transfection of COS-7 and HepG2 cells in vitro, with luciferase expression levels comparable to those achieved for plasmid DNA-cationic lipid complexes. In vivo studies employing an intraperitoneal B16 tumor model and intraperitoneal administration of SPLP also demonstrated maximum luciferase expression for DODAC contents of 20-24 mol% and significantly improved gene expression in tumor tissue as compared with complexes. We

  11. Characterization of a Novel Plasmid, pMAH135, from Mycobacterium avium Subsp. hominissuis

    PubMed Central

    Uchiya, Kei-ichi; Takahashi, Hiroyasu; Nakagawa, Taku; Yagi, Tetsuya; Moriyama, Makoto; Inagaki, Takayuki; Ichikawa, Kazuya; Nikai, Toshiaki; Ogawa, Kenji

    2015-01-01

    Mycobacterium avium complex (MAC) causes mainly two types of disease. The first is disseminated disease in immunocompromised hosts, such as individuals infected by human immunodeficiency virus (HIV). The second is pulmonary disease in individuals without systemic immunosuppression, and the incidence of this type is increasing worldwide. M. avium subsp. hominissuis, a component of MAC, causes infection in pigs as well as in humans. Many aspects of the different modes of M. avium infection and its host specificity remain unclear. Here, we report the characteristics and complete sequence of a novel plasmid, designated pMAH135, derived from M. avium strain TH135 in an HIV-negative patient with pulmonary MAC disease. The pMAH135 plasmid consists of 194,711 nucleotides with an average G + C content of 66.5% and encodes 164 coding sequences (CDSs). This plasmid was unique in terms of its homology to other mycobacterial plasmids. Interestingly, it contains CDSs with sequence homology to mycobactin biosynthesis proteins and type VII secretion system-related proteins, which are involved in the pathogenicity of mycobacteria. It also contains putative conserved domains of the multidrug efflux transporter. Screening of isolates from humans and pigs for genes located on pMAH135 revealed that the detection rate of these genes was higher in clinical isolates from pulmonary MAC disease patients than in those from HIV-positive patients, whereas the genes were almost entirely absent in isolates from pigs. Moreover, variable number tandem repeats typing analysis showed that isolates carrying pMAH135 genes are grouped in a specific cluster. Collectively, the pMAH135 plasmid contains genes associated with M. avium’s pathogenicity and resistance to antimicrobial agents. The results of this study suggest that pMAH135 influence not only the pathological manifestations of MAC disease, but also the host specificity of MAC infection. PMID:25671431

  12. Plasmids in antibiotic susceptible and antibiotic resistant commensal Escherichia coli from healthy Australian adults.

    PubMed

    Moran, Robert A; Anantham, Sashindran; Pinyon, Jeremy L; Hall, Ruth M

    2015-07-01

    A collection of 111 commensal Escherichia coli isolated from 84 faecal samples from healthy Australian adults were screened using PCR-based replicon typing. Each isolate represented a distinct strain found in a particular faecal sample. Fifty-one isolates were resistant to one or more of 12 antibiotics tested. FII and FIB replicons were most common and usually found together. The FII replicon was detected in 63 isolates (35 susceptible, 28 resistant), the FIB replicon was present in 65 (32 susceptible, 33 resistant) and 54 (30 susceptible, 24 resistant) included both. Other replicon types were found infrequently (A/C, I1, K, L/M, P, R, Y, FIA and FIC) or not at all (HI1, HI2, N, T, U, W, X). Only the B/O amplicon, found in 21 resistant but only 4 susceptible isolates, was associated with antibiotic resistance. Detailed analysis of this group revealed that the B/O PCR also detected Z plasmids of several distinguishable types. PCR assays were developed to detect the two repA genes (repABKI and repAZ) found in members of the I-complex (I, B/O, K and Z plasmids). These assays distinguished the B/O and Z plasmids detected by the original "B/O" PCR. One isolate carried repABKI and the remainder carried repAZ. These genes were also detected in further isolates in the collection. Conjugative transfer of resistance genes was detected for the B/O plasmid and two Z groups. Evidence for transfer of repAZ plasmids in the human colon in the absence of antibiotic selection was also obtained.

  13. The First Report of a Fully Sequenced Resistance Plasmid from Shigella boydii

    PubMed Central

    Wang, Li; Liu, Lei; Liu, Dong; Yin, Zhe; Feng, Jiao; Zhang, Defu; Fang, Haihong; Qiu, Yefeng; Chen, Weijun; Yang, Ruisheng; Wang, Jinglin; Fa, Yunzhi; Zhou, Dongsheng

    2016-01-01

    The purpose of this study was to characterize mechanisms of plasmid-mediated antimicrobial resistance in Shigella boydii. S. boydii strain 2246 with resistance to ciprofloxacin, ceftriaxone and azithromycin was isolated from a human case of watery diarrhea in a Chinese public hospital. Resistance in strain 2246 to ceftriaxone and azithromycin was attributable to the presence of blaCTX-M-14, and erm(B) and mph(A), respectively, which were co-located on a multidrug-resistant (MDR) plasmid p2246-CTXM. p2246-CTXM represented a novel IncFII-type MDR plasmid with a very complex chimera structure. Its master backbone was genetically closely related to the R100 plasmid, but p2246-CTXM had evolved to integrate additional R100-unrelated backbone regions as well as massive exogenous mobile elements that carried multiple resistance determinants. In p2246-CTXM, erm(B) together with its leading peptide gene erm(C), mph(A) together with its regulatory genes mrx and mphR(A), and blaCTX-M-14 were captured by three different mobile elements Tn6295, the IS26-mph(A)-mrx-mphR(A)-IS6100 unit, and a truncated ISEcp1-blaCTX-M-14-IS903D-iroN transposition unit, respectively, all of which were harbored in a large Tn3-family transposon Tn6285. p2246-CTXM still carried additional resistance determinants mer (mercury resistance), aacA4 (aminoglycoside resistance), cmlA1 (chloramphenicol resistance), and qacED1 (quaternary ammonium compound resistance). This is the first report of identifying a clinical S. boydii strain simultaneously resistant to ciprofloxacin, ceftriaxone, and azithromycin, and determining the complete sequence of a resistance plasmid from S. boydii. PMID:27766094

  14. Raster Data Partitioning for Supporting Distributed GIS Processing

    NASA Astrophysics Data System (ADS)

    Nguyen Thai, B.; Olasz, A.

    2015-08-01

    In the geospatial sector big data concept also has already impact. Several studies facing originally computer science techniques applied in GIS processing of huge amount of geospatial data. In other research studies geospatial data is considered as it were always been big data (Lee and Kang, 2015). Nevertheless, we can prove data acquisition methods have been improved substantially not only the amount, but the resolution of raw data in spectral, spatial and temporal aspects as well. A significant portion of big data is geospatial data, and the size of such data is growing rapidly at least by 20% every year (Dasgupta, 2013). The produced increasing volume of raw data, in different format, representation and purpose the wealth of information derived from this data sets represents only valuable results. However, the computing capability and processing speed rather tackle with limitations, even if semi-automatic or automatic procedures are aimed on complex geospatial data (Kristóf et al., 2014). In late times, distributed computing has reached many interdisciplinary areas of computer science inclusive of remote sensing and geographic information processing approaches. Cloud computing even more requires appropriate processing algorithms to be distributed and handle geospatial big data. Map-Reduce programming model and distributed file systems have proven their capabilities to process non GIS big data. But sometimes it's inconvenient or inefficient to rewrite existing algorithms to Map-Reduce programming model, also GIS data can not be partitioned as text-based data by line or by bytes. Hence, we would like to find an alternative solution for data partitioning, data distribution and execution of existing algorithms without rewriting or with only minor modifications. This paper focuses on technical overview of currently available distributed computing environments, as well as GIS data (raster data) partitioning, distribution and distributed processing of GIS algorithms

  15. Scalable recovery of plasmid DNA based on aqueous two-phase separation.

    PubMed

    Frerix, Andreas; Müller, Markus; Kula, Maria-Regina; Hubbuch, Jürgen

    2005-08-01

    Future developments in gene therapy and DNA vaccination depend on cost-effective large-scale production of pharmaceutical-grade pDNA (plasmid DNA). Given the large amount of impurities present in the feedstock, purification processes that have high specificity and capacity at a moderate cost are required. In the present study, we describe a non-chromatographic procedure based on aqueous two-phase extraction allowing a fast and simply scalable capture step. PEG [poly(ethylene glycol)] in combination with potassium citrate or potassium phosphate was tested as phase component for extraction. By increasing either PEG or salt concentration, the partitioning of nucleic acids changed from bottom to top phase. Phase systems with a composition of 15% PEG 800 and 20% potassium phosphate at pH 7.0 showed a strong partitioning of pDNA to the bottom phase, linked to a clear decrease in open circular pDNA, while proteins, genomic DNA and RNA remain at the top or at the interphase. A great advantage of the current process is that the complete procedure of lysis, precipitation, clarification and extraction can be performed in a single vessel. The number of denatured and sheared genomic DNAs in a spiking experiment was found to be depleted by more than 99%. PMID:15612880

  16. Evidence for plasmid DNA exchange after polyplex mixing.

    PubMed

    Pigeon, L; Gonçalves, C; Pichon, C; Midoux, P

    2016-08-17

    The self-assembly of a plasmid DNA (pDNA) with cationic polymers or cationic liposomes forms nanosized supramolecular structures called lipoplexes, polyplexes and lipopolyplexes. Here, we report that when two polyplex preparations made using the same polymer and the same pDNA but labelled with two different fluorophores are mixed together, pDNA molecules are exchanged. Indeed, when Flu-pDNA complexed with histidinylated lPEI (Flu-pDNA/His-lPEI) polyplexes are mixed with Cy5-pDNA complexed with histidinylated lPEI (Cy5-pDNA/His-lPEI) polyplexes, a high quantity of polyplexes emitting dual fluorescence is observed and FRET indicates that one single polyplex contains two kinds of fluorescent pDNA molecules. This phenomenon depends on the polymer-type and the strength of the pDNA/polymer interaction. No exchange is observed with polylysine polyplexes, caged His-lPEI polyplexes, lipoplexes, lipopolyplexes or when His-lPEI polyplexes are mixed with lipoplexes. Our results suggest that aggregation or collapse of polyplexes occurs after their interaction leading to their unpackaging followed by the formation of new polyplexes with the exchange of pDNA. PMID:27459887

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

    PubMed

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

    2014-12-01

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

  18. Rapid compensatory evolution promotes the survival of conjugative plasmids.

    PubMed

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

    2016-01-01

    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

  19. 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

  20. 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...

  1. High frequency generalized transduction by miniMu plasmid phage.

    PubMed

    Wang, B M; Liu, L; Groisman, E A; Casadaban, M J; Berg, C M

    1987-06-01

    Deletion derivatives of phage Mu which replicate as multicopy plasmids, and also transpose and package like Mu, have been developed for the in vivo cloning of bacterial genes. We show here that these miniMu plasmid phage are also efficient at generalized transduction and that both in vivo cloning and generalized transduction of a given gene can be accomplished in a single experiment.

  2. The master activator of IncA/C conjugative plasmids stimulates genomic islands and multidrug resistance dissemination.

    PubMed

    Carraro, Nicolas; Matteau, Dominick; Luo, Peng; Rodrigue, Sébastien; Burrus, Vincent

    2014-10-01

    Dissemination of antibiotic resistance genes occurs mostly by conjugation, which mediates DNA transfer between cells in direct contact. Conjugative plasmids of the IncA/C incompatibility group have become a substantial threat due to their broad host-range, the extended spectrum of antimicrobial resistance they confer, their prevalence in enteric bacteria and their very efficient spread by conjugation. However, their biology remains largely unexplored. Using the IncA/C conjugative plasmid pVCR94ΔX as a prototype, we have investigated the regulatory circuitry that governs IncA/C plasmids dissemination and found that the transcriptional activator complex AcaCD is essential for the expression of plasmid transfer genes. Using chromatin immunoprecipitation coupled with exonuclease digestion (ChIP-exo) and RNA sequencing (RNA-seq) approaches, we have identified the sequences recognized by AcaCD and characterized the AcaCD regulon. Data mining using the DNA motif recognized by AcaCD revealed potential AcaCD-binding sites upstream of genes involved in the intracellular mobility functions (recombination directionality factor and mobilization genes) in two widespread classes of genomic islands (GIs) phylogenetically unrelated to IncA/C plasmids. The first class, SGI1, confers and propagates multidrug resistance in Salmonella enterica and Proteus mirabilis, whereas MGIVmi1 in Vibrio mimicus belongs to a previously uncharacterized class of GIs. We have demonstrated that through expression of AcaCD, IncA/C plasmids specifically trigger the excision and mobilization of the GIs at high frequencies. This study provides new evidence of the considerable impact of IncA/C plasmids on bacterial genome plasticity through their own mobility and the mobilization of genomic islands.

  3. The Master Activator of IncA/C Conjugative Plasmids Stimulates Genomic Islands and Multidrug Resistance Dissemination

    PubMed Central

    Luo, Peng; Rodrigue, Sébastien; Burrus, Vincent

    2014-01-01

    Dissemination of antibiotic resistance genes occurs mostly by conjugation, which mediates DNA transfer between cells in direct contact. Conjugative plasmids of the IncA/C incompatibility group have become a substantial threat due to their broad host-range, the extended spectrum of antimicrobial resistance they confer, their prevalence in enteric bacteria and their very efficient spread by conjugation. However, their biology remains largely unexplored. Using the IncA/C conjugative plasmid pVCR94ΔX as a prototype, we have investigated the regulatory circuitry that governs IncA/C plasmids dissemination and found that the transcriptional activator complex AcaCD is essential for the expression of plasmid transfer genes. Using chromatin immunoprecipitation coupled with exonuclease digestion (ChIP-exo) and RNA sequencing (RNA-seq) approaches, we have identified the sequences recognized by AcaCD and characterized the AcaCD regulon. Data mining using the DNA motif recognized by AcaCD revealed potential AcaCD-binding sites upstream of genes involved in the intracellular mobility functions (recombination directionality factor and mobilization genes) in two widespread classes of genomic islands (GIs) phylogenetically unrelated to IncA/C plasmids. The first class, SGI1, confers and propagates multidrug resistance in Salmonella enterica and Proteus mirabilis, whereas MGIVmi1 in Vibrio mimicus belongs to a previously uncharacterized class of GIs. We have demonstrated that through expression of AcaCD, IncA/C plasmids specifically trigger the excision and mobilization of the GIs at high frequencies. This study provides new evidence of the considerable impact of IncA/C plasmids on bacterial genome plasticity through their own mobility and the mobilization of genomic islands. PMID:25340549

  4. Assimilate partitioning during reproductive growth

    SciTech Connect

    Finazzo, S.F.; Davenport, T.L.

    1987-04-01

    Leaves having various phyllotactic relationships to fruitlets were labeled for 1 hour with 10/sub r/Ci of /sup 14/CO/sub 2/. Fruitlets were also labeled. Fruitlets did fix /sup 14/CO/sub 2/. Translocation of radioactivity from the peel into the fruit occurred slowly and to a limited extent. No evidence of translocation out of the fruitlets was observed. Assimilate partitioning in avocado was strongly influenced by phyllotaxy. If a fruit and the labeled leaf had the same phyllotaxy then greater than 95% of the radiolabel was present in this fruit. When the fruit did not have the same phyllotaxy as the labeled leaf, the radiolabel distribution was skewed with 70% of the label going to a single adjacent position. Avocado fruitlets exhibit uniform labeling throughout a particular tissue. In avocado, assimilates preferentially move from leaves to fruits with the same phyllotaxy.

  5. HPAM: Hirshfeld partitioned atomic multipoles

    NASA Astrophysics Data System (ADS)

    Elking, Dennis M.; Perera, Lalith; Pedersen, Lee G.

    2012-02-01

    An implementation of the Hirshfeld (HD) and Hirshfeld-Iterated (HD-I) atomic charge density partitioning schemes is described. Atomic charges and atomic multipoles are calculated from the HD and HD-I atomic charge densities for arbitrary atomic multipole rank l on molecules of arbitrary shape and size. The HD and HD-I atomic charges/multipoles are tested by comparing molecular multipole moments and the electrostatic potential (ESP) surrounding a molecule with their reference ab initio values. In general, the HD-I atomic charges/multipoles are found to better reproduce ab initio electrostatic properties over HD atomic charges/multipoles. A systematic increase in precision for reproducing ab initio electrostatic properties is demonstrated by increasing the atomic multipole rank from l=0 (atomic charges) to l=4 (atomic hexadecapoles). Both HD and HD-I atomic multipoles up to rank l are shown to exactly reproduce ab initio molecular multipole moments of rank L for L⩽l. In addition, molecular dipole moments calculated by HD, HD-I, and ChelpG atomic charges only ( l=0) are compared with reference ab initio values. Significant errors in reproducing ab initio molecular dipole moments are found if only HD or HD-I atomic charges used. Program summaryProgram title: HPAM Catalogue identifier: AEKP_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEKP_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: GNU General Public License v2 No. of lines in distributed program, including test data, etc.: 500 809 No. of bytes in distributed program, including test data, etc.: 13 424 494 Distribution format: tar.gz Programming language: C Computer: Any Operating system: Linux RAM: Typically, a few hundred megabytes Classification: 16.13 External routines: The program requires 'formatted checkpoint' files obtained from the Gaussian 03 or Gaussian 09 quantum chemistry program. Nature of problem: An ab initio

  6. MULTIVARIATE KERNEL PARTITION PROCESS MIXTURES

    PubMed Central

    Dunson, David B.

    2013-01-01

    Mixtures provide a useful approach for relaxing parametric assumptions. Discrete mixture models induce clusters, typically with the same cluster allocation for each parameter in multivariate cases. As a more flexible approach that facilitates sparse nonparametric modeling of multivariate random effects distributions, this article proposes a kernel partition process (KPP) in which the cluster allocation varies for different parameters. The KPP is shown to be the driving measure for a multivariate ordered Chinese restaurant process that induces a highly-flexible dependence structure in local clustering. This structure allows the relative locations of the random effects to inform the clustering process, with spatially-proximal random effects likely to be assigned the same cluster index. An exact block Gibbs sampler is developed for posterior computation, avoiding truncation of the infinite measure. The methods are applied to hormone curve data, and a dependent KPP is proposed for classification from functional predictors. PMID:24478563

  7. Trace element partition coefficient in ionic crystals.

    PubMed

    Nagasawa, H

    1966-05-01

    Partition coefficient monovalent trace ions between liquids and either solid NaNO(2) or KCl were determined. The isotropic elastic model of ionic crystals was used for calculating the energy change caused by the ionic substitutions. The observed values of partition coefficients in KCl good agreement with calculate values.

  8. [On the partition of acupuncture academic schools].

    PubMed

    Yang, Pengyan; Luo, Xi; Xia, Youbing

    2016-05-01

    Nowadays extensive attention has been paid on the research of acupuncture academic schools, however, a widely accepted method of partition of acupuncture academic schools is still in need. In this paper, the methods of partition of acupuncture academic schools in the history have been arranged, and three typical methods of"partition of five schools" "partition of eighteen schools" and "two-stage based partition" are summarized. After adeep analysis on the disadvantages and advantages of these three methods, a new method of partition of acupuncture academic schools that is called "three-stage based partition" is proposed. In this method, after the overall acupuncture academic schools are divided into an ancient stage, a modern stage and a contemporary stage, each schoolis divided into its sub-school category. It is believed that this method of partition can remedy the weaknesses ofcurrent methods, but also explore a new model of inheritance and development under a different aspect through thedifferentiation and interaction of acupuncture academic schools at three stages.

  9. Building Ecology and Partition Design. Technical Bulletin.

    ERIC Educational Resources Information Center

    Maryland State Dept. of Education, Baltimore.

    This bulletin is intended as a resource for school system facility planners and architects who design schools. Ways in which decision makers can incorporate environmental concerns in the design of school buildings are detailed. Focus is on the design of interior partition systems. Partition systems in schools serve several purposes; they define…

  10. Isoperimetric graph partitioning for image segmentation.

    PubMed

    Grady, Leo; Schwartz, Eric L

    2006-03-01

    Spectral graph partitioning provides a powerful approach to image segmentation. We introduce an alternate idea that finds partitions with a small isoperimetric constant, requiring solution to a linear system rather than an eigenvector problem. This approach produces the high quality segmentations of spectral methods, but with improved speed and stability.

  11. Exact Abjm Partition Function from Tba

    NASA Astrophysics Data System (ADS)

    Putrov, Pavel; Yamazaki, Masahito

    2012-11-01

    We report on the exact computation of the S3 partition function of U(N)k × U(N)-k ABJM theory for k = 1, N = 1, …, 19. The result is a polynomial in π-1 with rational coefficients. As an application of our results, we numerically determine the coefficient of the membrane 1-instanton correction to the partition function.

  12. A mesh partitioning algorithm for preserving spatial locality in arbitrary geometries

    SciTech Connect

    Nivarti, Girish V. Salehi, M. Mahdi; Bushe, W. Kendal

    2015-01-15

    Highlights: •An algorithm for partitioning computational meshes is proposed. •The Morton order space-filling curve is modified to achieve improved locality. •A spatial locality metric is defined to compare results with existing approaches. •Results indicate improved performance of the algorithm in complex geometries. -- Abstract: A space-filling curve (SFC) is a proximity preserving linear mapping of any multi-dimensional space and is widely used as a clustering tool. Equi-sized partitioning of an SFC ignores the loss in clustering quality that occurs due to inaccuracies in the mapping. Often, this results in poor locality within partitions, especially for the conceptually simple, Morton order curves. We present a heuristic that improves partition locality in arbitrary geometries by slicing a Morton order curve at points where spatial locality is sacrificed. In addition, we develop algorithms that evenly distribute points to the extent possible while maintaining spatial locality. A metric is defined to estimate relative inter-partition contact as an indicator of communication in parallel computing architectures. Domain partitioning tests have been conducted on geometries relevant to turbulent reactive flow simulations. The results obtained highlight the performance of our method as an unsupervised and computationally inexpensive domain partitioning tool.

  13. Parallel hypergraph partitioning for scientific computing.

    SciTech Connect

    Heaphy, Robert; Devine, Karen Dragon; Catalyurek, Umit; Bisseling, Robert; Hendrickson, Bruce Alan; Boman, Erik Gunnar

    2005-07-01

    Graph partitioning is often used for load balancing in parallel computing, but it is known that hypergraph partitioning has several advantages. First, hypergraphs more accurately model communication volume, and second, they are more expressive and can better represent nonsymmetric problems. Hypergraph partitioning is particularly suited to parallel sparse matrix-vector multiplication, a common kernel in scientific computing. We present a parallel software package for hypergraph (and sparse matrix) partitioning developed at Sandia National Labs. The algorithm is a variation on multilevel partitioning. Our parallel implementation is novel in that it uses a two-dimensional data distribution among processors. We present empirical results that show our parallel implementation achieves good speedup on several large problems (up to 33 million nonzeros) with up to 64 processors on a Linux cluster.

  14. Cell partition in two phase polymer systems

    NASA Technical Reports Server (NTRS)

    Brooks, D. E.

    1979-01-01

    Aqueous phase-separated polymer solutions can be used as support media for the partition of biological macromolecules, organelles and cells. Cell separations using the technique have proven to be extremely sensitive to cell surface properties but application of the systems are limited to cells or aggregates which do not significantly while the phases are settling. Partition in zero g in principle removes this limitation but an external driving force must be applied to induce the phases to separate since their density difference disappears. We have recently shown that an applied electric field can supply the necessary driving force. We are proposing to utilize the NASA FES to study field-driven phase separation and cell partition on the ground and in zero g to help define the separation/partition process, with the ultimate goal being to develop partition as a zero g cell separation technique.

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

    PubMed

    Kuwahara, S

    1978-09-01

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

  16. Positive selection and compensatory adaptation interact to stabilize non-transmissible plasmids.

    PubMed

    San Millan, A; Peña-Miller, R; Toll-Riera, M; Halbert, Z V; McLean, A R; Cooper, B S; MacLean, R C

    2014-10-10

    Plasmids are important drivers of bacterial evolution, but it is challenging to understand how plasmids persist over the long term because plasmid carriage is costly. Classical models predict that horizontal transfer is necessary for plasmid persistence, but recent work shows that almost half of plasmids are non-transmissible. Here we use a combination of mathematical modelling and experimental evolution to investigate how a costly, non-transmissible plasmid, pNUK73, can be maintained in populations of Pseudomonas aeruginosa. Compensatory adaptation increases plasmid stability by eliminating the cost of plasmid carriage. However, positive selection for plasmid-encoded antibiotic resistance is required to maintain the plasmid by offsetting reductions in plasmid frequency due to segregational loss. Crucially, we show that compensatory adaptation and positive selection reinforce each other's effects. Our study provides a new understanding of how plasmids persist in bacterial populations, and it helps to explain why resistance can be maintained after antibiotic use is stopped.

  17. Positive selection and compensatory adaptation interact to stabilize non-transmissible plasmids

    PubMed Central

    Millan, A. San; Peña-Miller, R.; Toll-Riera, M.; Halbert, Z. V.; McLean, A. R.; Cooper, B. S.; MacLean, R. C.

    2014-01-01

    Plasmids are important drivers of bacterial evolution, but it is challenging to understand how plasmids persist over the long term because plasmid carriage is costly. Classical models predict that horizontal transfer is necessary for plasmid persistence, but recent work shows that almost half of plasmids are non-transmissible. Here we use a combination of mathematical modelling and experimental evolution to investigate how a costly, non-transmissible plasmid, pNUK73, can be maintained in populations of Pseudomonas aeruginosa. Compensatory adaptation increases plasmid stability by eliminating the cost of plasmid carriage. However, positive selection for plasmid-encoded antibiotic resistance is required to maintain the plasmid by offsetting reductions in plasmid frequency due to segregational loss. Crucially, we show that compensatory adaptation and positive selection reinforce each other’s effects. Our study provides a new understanding of how plasmids persist in bacterial populations, and it helps to explain why resistance can be maintained after antibiotic use is stopped. PMID:25302567

  18. Exogenous Isolation of Mobilizing Plasmids from Polluted Soils and Sludges

    PubMed Central

    Top, Eva; De Smet, Ingrid; Verstraete, Willy; Dijkmans, Roger; Mergeay, Max

    1994-01-01

    Exogenous plasmid isolation was used to assess the presence of mobilizing plasmids in several soils and activated sludges. Triparental matings were performed with Escherichia coli (a member of the γ subgroup of the Proteobacteria) as the donor of an IncQ plasmid (pMOL155, containing the heavy metal resistance genes czc: Cor, Znr, and Cdr), Alcaligenes eutrophus (a member of the β subgroup of the Proteobacteria) as the recipient, and indigenous microorganisms from soil and sludge samples as helper strains. We developed an assay to assess the plasmid mobilization potential of a soil ecosystem on the basis of the number of transconjugants obtained after exogenous isolations. After inoculation into soil of several concentrations of a helper strain (E. coli CM120 harboring IncP [IncP1] mobilizing plasmid RP4), the log numbers of transconjugants obtained from exogenous isolations with different soil samples were a linear function of the log numbers of helper strain CM120(RP4) present in the soils. Four soils were analyzed for the presence of mobilizing elements, and mobilizing plasmids were isolated from two of these soils. Several sludge samples from different wastewater treatment plants yielded much higher numbers of transconjugants than the soil samples, indicating that higher numbers of mobilizing strains were present. The mobilizing plasmids isolated from Gent-O sludge and one plasmid isolated from Eislingen soil hybridized to the repP probe, whereas the plasmids isolated from Essen soil did not hybridize to a large number of rep probes (repFIC, repHI1, repH12, repL/M, repN, repP, repT, repU, repW, repX). This indicates that in Essen soil, broad-host-range mobilizing plasmids belonging to other incompatibility groups may be present. Images PMID:16349216

  19. Dense Subgraph Partition of Positive Hypergraphs.

    PubMed

    Liu, Hairong; Latecki, Longin Jan; Yan, Shuicheng

    2015-03-01

    In this paper, we present a novel partition framework, called dense subgraph partition (DSP), to automatically, precisely and efficiently decompose a positive hypergraph into dense subgraphs. A positive hypergraph is a graph or hypergraph whose edges, except self-loops, have positive weights. We first define the concepts of core subgraph, conditional core subgraph, and disjoint partition of a conditional core subgraph, then define DSP based on them. The result of DSP is an ordered list of dense subgraphs with decreasing densities, which uncovers all underlying clusters, as well as outliers. A divide-and-conquer algorithm, called min-partition evolution, is proposed to efficiently compute the partition. DSP has many appealing properties. First, it is a nonparametric partition and it reveals all meaningful clusters in a bottom-up way. Second, it has an exact and efficient solution, called min-partition evolution algorithm. The min-partition evolution algorithm is a divide-and-conquer algorithm, thus time-efficient and memory-friendly, and suitable for parallel processing. Third, it is a unified partition framework for a broad range of graphs and hypergraphs. We also establish its relationship with the densest k-subgraph problem (DkS), an NP-hard but fundamental problem in graph theory, and prove that DSP gives precise solutions to DkS for all kin a graph-dependent set, called critical k-set. To our best knowledge, this is a strong result which has not been reported before. Moreover, as our experimental results show, for sparse graphs, especially web graphs, the size of critical k-set is close to the number of vertices in the graph. We test the proposed partition framework on various tasks, and the experimental results clearly illustrate its advantages.

  20. Competition between plasmid-bearing and plasmid-free organisms in a chemostat with nutrient recycling and an inhibitor.

    PubMed

    Yuan, Sanling; Xiao, Dongmei; Han, Maoan

    2006-07-01

    The asymptotic behavior of solutions of a model for competition between plasmid-bearing and plasmid-free organisms in the chemostat with two distributed delays and an external inhibitor is considered. The model presents a refinement of the one considered by Lu and Hadeler [Z. Lu, K.P. Hadeler, Model of plasmid-bearing plasmid-free competition in the chemostat with nutrient recycling and an inhibitor, Math. Biosci. 167 (2000) p. 177]. The delays model the fact that the nutrient is partially recycled after the death of the biomass by bacterial decomposition. Furthermore, it is assumed that there is inter-specific competition between the plasmid-bearing and plasmid-free organisms as well as intra-specific competition within each population. Conditions for boundedness of solutions and existence of non-negative equilibrium are given. Analysis of the extinction of the organisms, including plasmid-bearing and plasmid-free organisms, and the uniform persistence of the system are also carried out. By constructing appropriate Liapunov-like functionals, some sufficient conditions of global attractivity to the extinction equilibria are obtained and the combined effects of the delays and the inhibitor are studied.

  1. The worldwide distribution of genetically and phylogenetically diverse Bacillus cereus isolates harbouring Bacillus anthracis-like plasmids.

    PubMed

    Kaminska, Paulina Sylwia; Yernazarova, Aliya; Drewnowska, Justyna Malgorzata; Zambrowski, Grzegorz; Swiecicka, Izabela

    2015-10-01

    Bacillus cereus is a close relative of B. anthracis, the causative agent of anthrax whose pathogenic determinants are located on pXO1 and pXO2 plasmids. Bacillus anthracis-like plasmids have been also noted among B. cereus, however, genetic features of B. cereus harbouring these elements remain largely undescribed, especially from the global perspective. Herein, we present the genetic polymorphism, population structure and phylogeny of B. cereus with pXO1-/pXO2-like plasmids originating from Argentina, Kazakhstan, Kenya and Poland. The plasmids were found in about 17% of the isolates, but their frequencies and expression of replicons differed within and between populations. In the multi-locus sequence typing, the bacteria exhibited high genetic polymorphism reflected by 116 sequencing types, including 84 singletons and 10 clonal complexes, which mainly consisted of isolates of the same origin. The phylogenetic analysis of pXO1-/pXO2-like positive B. cereus isolates revealed six independent clades; in certain clades individual populations predominated. Generally, B. cereus with pXO1-/pXO2-like plasmids did not indicate the genetic relationship with B. anthracis, and cannot be classified into an evolutionary independent anthrax line within the B. cereus group. Our report is of a crucial importance for discovering the genetic specificity and evolution of B. cereus bacilli.

  2. Conjugative transfer of an IncA/C plasmid-borne blaCMY-2 gene through genetic re-arrangements with an IncX1 plasmid

    PubMed Central

    2013-01-01

    transfer of the YU39 blaCMY-2 gene harbored on a non- conjugative pA/C requires the machinery of a highly conjugative pX1 plasmid. Our experiments demonstrate the complex interactions a single strain can exploit to contend with the challenge of horizontal transfer and antibiotic selective pressure. PMID:24262067

  3. Binary partition trees for object detection.

    PubMed

    Vilaplana, Veronica; Marques, Ferran; Salembier, Philippe

    2008-11-01

    This paper discusses the use of Binary Partition Trees (BPTs) for object detection. BPTs are hierarchical region-based representations of images. They define a reduced set of regions that covers the image support and that spans various levels of resolution. They are attractive for object detection as they tremendously reduce the search space. In this paper, several issues related to the use of BPT for object detection are studied. Concerning the tree construction, we analyze the compromise between computational complexity reduction and accuracy. This will lead us to define two parts in the BPT: one providing accuracy and one representing the search space for the object detection task. Then we analyze and objectively compare various similarity measures for the tree construction. We conclude that different similarity criteria should be used for the part providing accuracy in the BPT and for the part defining the search space and specific criteria are proposed for each case. Then we discuss the object detection strategy based on BPT. The notion of node extension is proposed and discussed. Finally, several object detection examples illustrating the generality of the approach and its efficiency are reported.

  4. Disentangling event-scale hydrologic flow partitioning in mountains of the Korean Peninsula under extreme precipitation

    NASA Astrophysics Data System (ADS)

    Shope, Christopher L.

    2016-07-01

    Mountainous headwaters include a variety of spatial landscape units; however, the flow contribution from different hydrologic components is complex and often unclear. In addition to complex landscape controls, temporal meteorological drivers play an important role in the distribution between surface runoff and subsurface storage changes. This spatiotemporal variability in partitioning can influence catchment-wide flow accumulation and nutrient and sediment loading. We use a multi-year, multi-method analysis of stable isotopes, geochemical indicators, and discharge distributed throughout the Haean catchment in South Korea to identify temporal variability in hydrologic flow partitioning from surface runoff, springs, shallow interflow, and groundwater under monsoonal conditions. By combining a weighted, multi-method discharge approach, high frequency, synoptic, catchment-wide isotopic and geochemical sampling, and baseflow analysis, we characterize watershed-scale spatiotemporal hydrologic flow partitioning. Meteorological drivers are spatially variable throughout the catchment and temporally between individual events. Baseflow contributions in the high elevation, forested areas are up to 50%, while the majority of the catchment is approximately 20%. Our study builds on previously reported seasonality of isotopic signatures by quantifying trends in distributed event-based partitioning of isotopic tracers. We demonstrate that high frequency flow partitioning can accurately be determined in mountainous topography with high precipitation and that there is a need for multiple method characterizations. Our results further show the benefit of spatially distributed synoptic sampling for process understanding of hydrologic partitioning throughout the watersheds.

  5. Maximum-Likelihood Tree Estimation Using Codon Substitution Models with Multiple Partitions

    PubMed Central

    Zoller, Stefan; Boskova, Veronika; Anisimova, Maria

    2015-01-01

    Many protein sequences have distinct domains that evolve with different rates, different selective pressures, or may differ in codon bias. Instead of modeling these differences by more and more complex models of molecular evolution, we present a multipartition approach that allows maximum-likelihood phylogeny inference using different codon models at predefined partitions in the data. Partition models can, but do not have to, share free parameters in the estimation process. We test this approach with simulated data as well as in a phylogenetic study of the origin of the leucin-rich repeat regions in the type III effector proteins of the pythopathogenic bacteria Ralstonia solanacearum. Our study does not only show that a simple two-partition model resolves the phylogeny better than a one-partition model but also gives more evidence supporting the hypothesis of lateral gene transfer events between the bacterial pathogens and its eukaryotic hosts. PMID:25911229

  6. Domain Decomposition By the Advancing-Partition Method for Parallel Unstructured Grid Generation

    NASA Technical Reports Server (NTRS)

    Pirzadeh, Shahyar Z.; Zagaris, George

    2009-01-01

    A new method of domain decomposition has been developed for generating unstructured grids in subdomains either sequentially or using multiple computers in parallel. Domain decomposition is a crucial and challenging step for parallel grid generation. Prior methods are generally based on auxiliary, complex, and computationally intensive operations for defining partition interfaces and usually produce grids of lower quality than those generated in single domains. The new technique, referred to as "Advancing Partition," is based on the Advancing-Front method, which partitions a domain as part of the volume mesh generation in a consistent and "natural" way. The benefits of this approach are: 1) the process of domain decomposition is highly automated, 2) partitioning of domain does not compromise the quality of the generated grids, and 3) the computational overhead for domain decomposition is minimal. The new method has been implemented in NASA's unstructured grid generation code VGRID.

  7. 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

  8. 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

  9. Bacterial plasmids: autonomous replication and vehicles for gene cloning.

    PubMed

    Helinski, D R

    1979-11-01

    The use of recombinant DNA techniques in the analysis of the structure and replication of bacterial plasmids has provided much information on the properties of these genetic elements and has led to the construction of plasmid elements that are potentially very useful as gene cloning vehicles in Escherichia coli and other gram-negative bacteria. The genetic and molecular properties of plasmids mini-F, ColE1, and RK2 are described with particular emphasis on the origin and direction of replication and the identification of genetic regions essential for maintenance of these elements in the extra-chromosomal state. Low molecular weight derivatives of each of these plasmids have been obtained and a restriction enzyme map determined for these various derivatives. A hybrid DNA molecule consisting of a low molecular weight derivative of ColE1 joined to a segment of bacteriophage DNA has been constructed and shown to be capable of existing either as a plasmid element or packaged as an infectious viral particle. Finally, several of the low molecular weight derivatives of these plasmids described have certain advantages as vehicles for the cloning of DNA including derivatives of he broad host range plasmid RK2 that may be useful for gene cloning in gram-negative bacteria distantly related to E. coli.

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

    PubMed Central

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

    1993-01-01

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

  11. 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.

  12. Why There Are No Essential Genes on Plasmids.

    PubMed

    Tazzyman, Samuel J; Bonhoeffer, Sebastian

    2015-12-01

    Mobile genetic elements such as plasmids are important for the evolution of prokaryotes. It has been suggested that there are differences between functions coded for by mobile genes and those in the "core" genome and that these differences can be seen between plasmids and chromosomes. In particular, it has been suggested that essential genes, such as those involved in the formation of structural proteins or in basic metabolic functions, are rarely located on plasmids. We model competition between genotypically varying bacteria within a single population to investigate whether selection favors a chromosomal location for essential genes. We find that in general, chromosomal locations for essential genes are indeed favored. This is because the inheritance of chromosomes is more stable than that for plasmids. We define the "degradation" rate as the rate at which chance genetic processes, for example, mutation, deletion, or translocation, render essential genes nonfunctioning. The only way in which plasmids can be a location for functioning essential genes is if chromosomal genes degrade faster than plasmid genes. If the two degradation rates are equal, or if plasmid genes degrade faster than chromosomal genes, functioning essential genes will be found only on chromosomes.

  13. 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

  14. 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

  15. 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

  16. 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

  17. Introducing Meta-Partition, a Useful Methodology to Explore Factors That Influence Ecological Effect Sizes.

    PubMed

    Ortega, Zaida; Martín-Vallejo, Javier; Mencía, Abraham; Galindo-Villardón, Maria Purificación; Pérez-Mellado, Valentín

    2016-01-01

    The study of the heterogeneity of effect sizes is a key aspect of ecological meta-analyses. Here we propose a meta-analytic methodology to study the influence of moderators in effect sizes by splitting heterogeneity: meta-partition. To introduce this methodology, we performed a meta-partition of published data about the traits that influence species sensitivity to habitat loss, that have been previously analyzed through meta-regression. Thus, here we aim to introduce meta-partition and to make an initial comparison with meta-regression. Meta-partition algorithm consists of three steps. Step 1 is to study the heterogeneity of effect sizes under the assumption of fixed effect model. If heterogeneity is found, we perform step 2, that is, to partition the heterogeneity by the moderator that minimizes heterogeneity within a subset while maximizing heterogeneity between subsets. Then, if effect sizes of the subset are still heterogeneous, we repeat step 1 and 2 until we reach final subsets. Finally, step 3 is to integrate effect sizes of final subsets, with fixed effect model if there is homogeneity, and with random effects model if there is heterogeneity. Results show that meta-partition is valuable to assess the importance of moderators in explaining heterogeneity of effect sizes, as well as to assess the directions of these relations and to detect possible interactions between moderators. With meta-partition we have been able to evaluate the importance of moderators in a more objective way than with meta-regression, and to visualize the complex relations that may exist between them. As ecological issues are often influenced by several factors interacting in complex ways, ranking the importance of possible moderators and detecting possible interactions would make meta-partition a useful exploration tool for ecological meta-analyses.

  18. Introducing Meta-Partition, a Useful Methodology to Explore Factors That Influence Ecological Effect Sizes

    PubMed Central

    Martín-Vallejo, Javier; Mencía, Abraham; Galindo-Villardón, Maria Purificación; Pérez-Mellado, Valentín

    2016-01-01

    The study of the heterogeneity of effect sizes is a key aspect of ecological meta-analyses. Here we propose a meta-analytic methodology to study the influence of moderators in effect sizes by splitting heterogeneity: meta-partition. To introduce this methodology, we performed a meta-partition of published data about the traits that influence species sensitivity to habitat loss, that have been previously analyzed through meta-regression. Thus, here we aim to introduce meta-partition and to make an initial comparison with meta-regression. Meta-partition algorithm consists of three steps. Step 1 is to study the heterogeneity of effect sizes under the assumption of fixed effect model. If heterogeneity is found, we perform step 2, that is, to partition the heterogeneity by the moderator that minimizes heterogeneity within a subset while maximizing heterogeneity between subsets. Then, if effect sizes of the subset are still heterogeneous, we repeat step 1 and 2 until we reach final subsets. Finally, step 3 is to integrate effect sizes of final subsets, with fixed effect model if there is homogeneity, and with random effects model if there is heterogeneity. Results show that meta-partition is valuable to assess the importance of moderators in explaining heterogeneity of effect sizes, as well as to assess the directions of these relations and to detect possible interactions between moderators. With meta-partition we have been able to evaluate the importance of moderators in a more objective way than with meta-regression, and to visualize the complex relations that may exist between them. As ecological issues are often influenced by several factors interacting in complex ways, ranking the importance of possible moderators and detecting possible interactions would make meta-partition a useful exploration tool for ecological meta-analyses. PMID:27409084

  19. 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.

  20. How pervasive is the Hirshfeld partitioning?

    SciTech Connect

    Heidar-Zadeh, Farnaz; Ayers, Paul W.

    2015-01-28

    One can partition the molecular density into its atomic contributions by minimizing the divergence of the atom-in-molecule densities from their corresponding reference pro-atomic densities, subject to the constraint that the sum of the atom-in-molecule densities is the total molecular density. We expose conditions on the divergence measure that are necessary, and sufficient, to recover the popular Hirshfeld partitioning. Specifically, among all local measures of the divergence between two probability distribution functions, the Hirshfeld partitioning is obtained only for f-divergences.

  1. Convex Regression with Interpretable Sharp Partitions

    PubMed Central

    Petersen, Ashley; Simon, Noah; Witten, Daniela

    2016-01-01

    We consider the problem of predicting an outcome variable on the basis of a small number of covariates, using an interpretable yet non-additive model. We propose convex regression with interpretable sharp partitions (CRISP) for this task. CRISP partitions the covariate space into blocks in a data-adaptive way, and fits a mean model within each block. Unlike other partitioning methods, CRISP is fit using a non-greedy approach by solving a convex optimization problem, resulting in low-variance fits. We explore the properties of CRISP, and evaluate its performance in a simulation study and on a housing price data set.

  2. Partitioning of regular computation on multiprocessor systems

    NASA Technical Reports Server (NTRS)

    Lee, Fung Fung

    1988-01-01

    Problem partitioning of regular computation over two dimensional meshes on multiprocessor systems is examined. The regular computation model considered involves repetitive evaluation of values at each mesh point with local communication. The computational workload and the communication pattern are the same at each mesh point. The regular computation model arises in numerical solutions of partial differential equations and simulations of cellular automata. Given a communication pattern, a systematic way to generate a family of partitions is presented. The influence of various partitioning schemes on performance is compared on the basis of computation to communication ratio.

  3. Convex Regression with Interpretable Sharp Partitions

    PubMed Central

    Petersen, Ashley; Simon, Noah; Witten, Daniela

    2016-01-01

    We consider the problem of predicting an outcome variable on the basis of a small number of covariates, using an interpretable yet non-additive model. We propose convex regression with interpretable sharp partitions (CRISP) for this task. CRISP partitions the covariate space into blocks in a data-adaptive way, and fits a mean model within each block. Unlike other partitioning methods, CRISP is fit using a non-greedy approach by solving a convex optimization problem, resulting in low-variance fits. We explore the properties of CRISP, and evaluate its performance in a simulation study and on a housing price data set. PMID:27635120

  4. Energy partitioning schemes: a dilemma.

    PubMed

    Mayer, I

    2007-01-01

    Two closely related energy partitioning schemes, in which the total energy is presented as a sum of atomic and diatomic contributions by using the "atomic decomposition of identity", are compared on the example of N,N-dimethylformamide, a simple but chemically rich molecule. Both schemes account for different intramolecular interactions, for instance they identify the weak C-H...O intramolecular interactions, but give completely different numbers. (The energy decomposition scheme based on the virial theorem is also considered.) The comparison of the two schemes resulted in a dilemma which is especially striking when these schemes are applied for molecules distorted from their equilibrium structures: one either gets numbers which are "on the chemical scale" and have quite appealing values at the equilibrium molecular geometries, but exhibiting a counter-intuitive distance dependence (the two-center energy components increase in absolute value with the increase of the interatomic distances)--or numbers with too large absolute values but "correct" distance behaviour. The problem is connected with the quick decay of the diatomic kinetic energy components.

  5. HPAM: Hirshfeld Partitioned Atomic Multipoles.

    PubMed

    Elking, Dennis M; Perera, Lalith; Pedersen, Lee G

    2012-02-01

    An implementation of the Hirshfeld (HD) and Hirshfeld-Iterated (HD-I) atomic charge density partitioning schemes is described. Atomic charges and atomic multipoles are calculated from the HD and HD-I atomic charge densities for arbitrary atomic multipole rank l(max) on molecules of arbitrary shape and size. The HD and HD-I atomic charges/multipoles are tested by comparing molecular multipole moments and the electrostatic potential (ESP) surrounding a molecule with their reference ab initio values. In general, the HD-I atomic charges/multipoles are found to better reproduce ab initio electrostatic properties over HD atomic charges/multipoles. A systematic increase in precision for reproducing ab initio electrostatic properties is demonstrated by increasing the atomic multipole rank from l(max) = 0 (atomic charges) to l(max) = 4 (atomic hexadecapoles). Both HD and HD-I atomic multipoles up to rank l(max) are shown to exactly reproduce ab initio molecular multipole moments of rank L for L ≤ l(max). In addition, molecular dipole moments calculated by HD, HD-I, and ChelpG atomic charges only (l(max) = 0) are compared with reference ab initio values. Significant errors in reproducing ab initio molecular dipole moments are found if only HD or HD-I atomic charges used.

  6. Energy partitioning schemes: a dilemma.

    PubMed

    Mayer, I

    2007-01-01

    Two closely related energy partitioning schemes, in which the total energy is presented as a sum of atomic and diatomic contributions by using the "atomic decomposition of identity", are compared on the example of N,N-dimethylformamide, a simple but chemically rich molecule. Both schemes account for different intramolecular interactions, for instance they identify the weak C-H...O intramolecular interactions, but give completely different numbers. (The energy decomposition scheme based on the virial theorem is also considered.) The comparison of the two schemes resulted in a dilemma which is especially striking when these schemes are applied for molecules distorted from their equilibrium structures: one either gets numbers which are "on the chemical scale" and have quite appealing values at the equilibrium molecular geometries, but exhibiting a counter-intuitive distance dependence (the two-center energy components increase in absolute value with the increase of the interatomic distances)--or numbers with too large absolute values but "correct" distance behaviour. The problem is connected with the quick decay of the diatomic kinetic energy components. PMID:17328441

  7. Partitioning technique for open systems

    NASA Astrophysics Data System (ADS)

    Brändas, Erkki J.

    2010-11-01

    The focus of the present contribution is essentially confined to three research areas carried out during the author's turns as visiting (assistant, associate and full) professor at the University of Florida's Quantum Theory Project, QTP. The first two topics relate to perturbation theory and spectral theory for self-adjoint operators in Hilbert space. The third subject concerns analytic extensions to non-self-adjoint problems, where particular consequences of the occurrence of continuous energy spectra are measured. In these studies general partitioning methods serve as general cover for perturbation-, variational- and general matrix theory. In addition we follow up associated inferences for the time dependent problem as well as recent results and conclusions of a rather general yet surprising character. Although the author spent most of his times at QTP during visits in the 1970s and 1980s, collaborations with department members and shorter stays continued through later decades. Nevertheless the impact must be somewhat fragmentary, yet it is hoped that the present account is sufficiently self-contained to be realistic and constructive.

  8. REE Partitioning in Lunar Minerals

    NASA Technical Reports Server (NTRS)

    Rapp, J. F.; Lapen, T. J.; Draper, D. S.

    2015-01-01

    Rare earth elements (REE) are an extremely useful tool in modeling lunar magmatic processes. Here we present the first experimentally derived plagioclase/melt partition coefficients in lunar compositions covering the entire suite of REE. Positive europium anomalies are ubiquitous in the plagioclase-rich rocks of the lunar highlands, and complementary negative Eu anomalies are found in most lunar basalts. These features are taken as evidence of a large-scale differentiation event, with crystallization of a global-scale lunar magma ocean (LMO) resulting in a plagioclase flotation crust and a mafic lunar interior from which mare basalts were subsequently derived. However, the extent of the Eu anomaly in lunar rocks is variable. Fagan and Neal [1] reported highly anorthitic plagioclase grains in lunar impact melt rock 60635,19 that displayed negative Eu anomalies as well as the more usual positive anomalies. Indeed some grains in the sample are reported to display both positive and negative anomalies. Judging from cathodoluminescence images, these anomalies do not appear to be associated with crystal overgrowths or zones.

  9. Spectral partitioning in diffraction tomography

    SciTech Connect

    Lehman, S K; Chambers, D H; Candy, J V

    1999-06-14

    The scattering mechanism of diffraction tomography is described by the integral form of the Helmholtz equation. The goal of diffraction tomography is to invert this equation in order to reconstruct the object function from the measured scattered fields. During the forward propagation process, the spatial spectrum of the object under investigation is ''smeared,'' by a convolution in the spectral domain, across the propagating and evanescent regions of the received field. Hence, care must be taken in performing the reconstruction, as the object's spectral information has been moved into regions where it may be considered to be noise rather than useful information. This will reduce the quality and resolution of the reconstruction. We show haw the object's spectrum can be partitioned into resolvable and non-resolvable parts based upon the cutoff between the propagating and evanescent fields. Operating under the Born approximation, we develop a beam-forming on transmit approach to direct the energy into either the propagating or evanescent parts of the spectrum. In this manner, we may individually interrogate the propagating and evanescent regions of the object spectrum.

  10. Complete Sequence of Four Multidrug-Resistant MOBQ1 Plasmids Harboring blaGES-5 Isolated from Escherichia coli and Serratia marcescens Persisting in a Hospital in Canada.

    PubMed

    Boyd, David; Taylor, Geoffrey; Fuller, Jeff; Bryce, Elizabeth; Embree, Joanne; Gravel, Denise; Katz, Kevin; Kibsey, Pamela; Kuhn, Magdalena; Langley, Joanne; Mataseje, Laura; Mitchell, Robyn; Roscoe, Diane; Simor, Andrew; Thomas, Eva; Turgeon, Nathalie; Mulvey, Michael

    2015-06-01

    The usefulness of carbapenems for gram-negative infections is becoming compromised by organisms harboring carbapenemases, enzymes which can hydrolyze the drug. Currently KPC (class A), NDM (class B), and OXA-48 types (class D) are the most globally widespread carbapenemases. However, among the GES-type class A extended-spectrum β-lactamases (ESBLs) there are variants that hydrolyze carbapenems, with blaGES-5 being the most common. Two Escherichia coli and two Serratia marcescens harboring blaGES-5 on plasmids were isolated by the Canadian Nosocomial Infection Surveillance Program (CNISP) from four different patients in a single hospital over a 2-year period. Complete sequencing of the blaGES-5 plasmids indicated that all four had nearly identical backbones consisting of genes for replication, partitioning, and stability, but contained variant accessory regions consisting of mobile elements and antimicrobial resistance genes. The plasmids were of a novel replicon type, but belonged to the MOBQ1 group based on relaxase sequences, and appeared to be mobilizable, but not self-transmissible. Considering the time periods of bacterial isolation, it would appear the blaGES-5 plasmid has persisted in an environmental niche for at least 2 years in the hospital. This has implications for infection control and clinical care when it is transferred to clinically relevant gram-negative organisms.

  11. [Isolation and determination of silver-resistant bacteria plasmids].

    PubMed

    Li, Junmin; Jin, Zexin

    2006-02-01

    Through the enrichment of the active mud obtained from three chemical plants and the domestication with different concentration Ag+ solution, thirty bacteria strains with silver (Ag+)-resistance were isolated, among which, the highest Ag+ -resistant concentration was 80 mg x ml(-1). The plasmids in these bacteria were extracted, with the detection rate of 76.67%. The elimination rate of the plasmid in HAg4 bacteria was 98.75% by 40 mmol x L(-1) sodium benzoate, and 77.78% by 350 microg x ml(-1) acridine orange. It was suggested that the Ag+ -resistance of bacteria was highly correlated with their plasmids.

  12. Pharmaceutical grade large-scale plasmid DNA manufacturing process.

    PubMed

    Schmeer, Marco; Schleef, Martin

    2014-01-01

    For pharmaceutical applications of plasmid DNA, either direct or indirect, certain quality standards are required. Whereas for direct gene transfer into human "Good Manufacturing Practice" (GMP) grade is mandatory, for GMP production of, e.g., viral vectors (AAV, etc.) the plasmid DNA used needs not necessarily be produced under GMP. Besides such regulatory aspects up-scaling of the plasmid DNA production process from research laboratory scale (up to a few milligrams) to industrial scales (milligram to gram scales) is an issue that is addressed here.

  13. Modern and simple construction of plasmid: saving time and cost.

    PubMed

    Nakayama, Hideki; Shimamoto, Nobuo

    2014-11-01

    Construction of plasmids has been occupying a significant fraction of laboratory work in most fields of experimental biology. Tremendous effort was made to improve the traditional method for constructing plasmids, in which DNA fragments digested with restriction enzymes were ligated. However, the traditional method remained to be a standard protocol more than 40 years. At last, several recent inventions are rapidly and completely replacing the traditional method, because they are far quicker with less cost, and requiring less material. We here introduce three such methods that cover up most of the cases. Moreover, they are complementary with each other. Our lab protocols are provided for "no strain, no pain" construction of plasmids.

  14. Equilibrium absorptive partitioning theory between multiple aerosol particle modes

    NASA Astrophysics Data System (ADS)

    Crooks, Matthew; Connolly, Paul; Topping, David; McFiggans, Gordon

    2016-10-01

    An existing equilibrium absorptive partitioning model for calculating the equilibrium gas and particle concentrations of multiple semi-volatile organics within a bulk aerosol is extended to allow for multiple involatile aerosol modes of different sizes and chemical compositions. In the bulk aerosol problem, the partitioning coefficient determines the fraction of the total concentration of semi-volatile material that is in the condensed phase of the aerosol. This work modifies this definition for multiple polydisperse aerosol modes to account for multiple condensed concentrations, one for each semi-volatile on each involatile aerosol mode. The pivotal assumption in this work is that each aerosol mode contains an involatile constituent, thus overcoming the potential problem of smaller particles evaporating completely and then condensing on the larger particles to create a monodisperse aerosol at equilibrium. A parameterisation is proposed in which the coupled non-linear system of equations is approximated by a simpler set of equations obtained by setting the organic mole fraction in the partitioning coefficient to be the same across all modes. By perturbing the condensed masses about this approximate solution a correction term is derived that accounts for many of the removed complexities. This method offers a greatly increased efficiency in calculating the solution without significant loss in accuracy, thus making it suitable for inclusion in large-scale models.

  15. Influenza Plasmid DNA Vaccines: Progress and Prospects.

    PubMed

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

    2015-01-01

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

  16. Paths and partitions: Combinatorial descriptions of the parafermionic states

    NASA Astrophysics Data System (ADS)

    Mathieu, Pierre

    2009-09-01

    The Zk parafermionic conformal field theories, despite the relative complexity of their modes algebra, offer the simplest context for the study of the bases of states and their different combinatorial representations. Three bases are known. The classic one is given by strings of the fundamental parafermionic operators whose sequences of modes are in correspondence with restricted partitions with parts at distance k -1 differing at least by 2. Another basis is expressed in terms of the ordered modes of the k -1 different parafermionic fields, which are in correspondence with the so-called multiple partitions. Both types of partitions have a natural (Bressoud) path representation. Finally, a third basis, formulated in terms of different paths, is inherited from the solution of the restricted solid-on-solid model of Andrews-Baxter-Forrester. The aim of this work is to review, in a unified and pedagogical exposition, these four different combinatorial representations of the states of the Zk parafermionic models. The first part of this article presents the different paths and partitions and their bijective relations; it is purely combinatorial, self-contained, and elementary; it can be read independently of the conformal-field-theory applications. The second part links this combinatorial analysis with the bases of states of the Zk parafermionic theories. With the prototypical example of the parafermionic models worked out in detail, this analysis contributes to fix some foundations for the combinatorial study of more complicated theories. Indeed, as we briefly indicate in ending, generalized versions of both the Bressoud and the Andrews-Baxter-Forrester paths emerge naturally in the description of the minimal models.

  17. Transposon-mediated mobilization of chromosomally located catabolic operons of the CAM plasmid by TOL plasmid transposon Tn4652 and CAM plasmid transposon Tn3614.

    PubMed

    Mäe, A A; Heinaru, A L

    1994-04-01

    The CAM (camphor degradation) plasmid is integrated into the chromosome of Pseudomonas putida PaW-line strains and is not self-transferable as a plasmid via conjugation. Our results show that the mobilization of chromosomally located CAM and the integration of cam-operons into the chromosome of the new Cam+ transconjugants is a recA-independent process mediated by transposons Tn4652 (17 kbp) and Tn3614 (7.2 kbp). Transposon Tn3614 is apparently identical to the left-hand and the right-hand sequences of the TOL plasmid pWW0 transposon Tn4654. The insertion of Tn401 inside the left-hand terminal IR of Tn4652 completely inhibited the mobilization of CAM. According to our data transposons Tn4652 and Tn3614 together with CAM plasmid catabolic operons are integrated into the chromosome. We propose that in pseudomonads the transposons Tn4652 and Tn3614 play a key role in the evolution and spread of new catabolic plasmids in nature. PMID:8012608

  18. Novel structures for optimal space partitions

    NASA Astrophysics Data System (ADS)

    Opsomer, E.; Vandewalle, N.

    2016-10-01

    Partitioning space into polyhedra with a minimum total surface area is a fundamental question in science and mathematics. In 1887, Lord Kelvin conjectured that the optimal partition of space is obtained with a 14-faced space-filling polyhedron, called tetrakaidecahedron. Kelvin’s conjecture resisted a century until Weaire and Phelan proposed in 1994 a new structure, made of eight polyhedra, obtained from numerical simulations. Herein, we propose a stochastic method for finding efficient polyhedral structures, maximizing the mean isoperimeter Q, instead of minimizing total area. We show that novel optimal structures emerge with non-equal cell volumes and uncurved facets. A partition made of 24 polyhedra, is found to surpass the previous known structures. Our work suggests that other structures with high isoperimeter values are still to be discovered in the pursuit of optimal space partitions.

  19. Connections between groundwater flow and transpiration partitioning.

    PubMed

    Maxwell, Reed M; Condon, Laura E

    2016-07-22

    Understanding freshwater fluxes at continental scales will help us better predict hydrologic response and manage our terrestrial water resources. The partitioning of evapotranspiration into bare soil evaporation and plant transpiration remains a key uncertainty in the terrestrial water balance. We used integrated hydrologic simulations that couple vegetation and land-energy processes with surface and subsurface hydrology to study transpiration partitioning at the continental scale. Both latent heat flux and partitioning are connected to water table depth, and including lateral groundwater flow in the model increases transpiration partitioning from 47 ± 13 to 62 ± 12%. This suggests that lateral groundwater flow, which is generally simplified or excluded in Earth system models, may provide a missing link for reconciling observations and global models of terrestrial water fluxes.

  20. Connections between groundwater flow and transpiration partitioning.

    PubMed

    Maxwell, Reed M; Condon, Laura E

    2016-07-22

    Understanding freshwater fluxes at continental scales will help us better predict hydrologic response and manage our terrestrial water resources. The partitioning of evapotranspiration into bare soil evaporation and plant transpiration remains a key uncertainty in the terrestrial water balance. We used integrated hydrologic simulations that couple vegetation and land-energy processes with surface and subsurface hydrology to study transpiration partitioning at the continental scale. Both latent heat flux and partitioning are connected to water table depth, and including lateral groundwater flow in the model increases transpiration partitioning from 47 ± 13 to 62 ± 12%. This suggests that lateral groundwater flow, which is generally simplified or excluded in Earth system models, may provide a missing link for reconciling observations and global models of terrestrial water fluxes. PMID:27463671

  1. Connections between groundwater flow and transpiration partitioning

    NASA Astrophysics Data System (ADS)

    Maxwell, Reed M.; Condon, Laura E.

    2016-07-01

    Understanding freshwater fluxes at continental scales will help us better predict hydrologic response and manage our terrestrial water resources. The partitioning of evapotranspiration into bare soil evaporation and plant transpiration remains a key uncertainty in the terrestrial water balance. We used integrated hydrologic simulations that couple vegetation and land-energy processes with surface and subsurface hydrology to study transpiration partitioning at the continental scale. Both latent heat flux and partitioning are connected to water table depth, and including lateral groundwater flow in the model increases transpiration partitioning from 47 ± 13 to 62 ± 12%. This suggests that lateral groundwater flow, which is generally simplified or excluded in Earth system models, may provide a missing link for reconciling observations and global models of terrestrial water fluxes.

  2. Reducing variance in batch partitioning measurements

    SciTech Connect

    Mariner, Paul E.

    2010-08-11

    The partitioning experiment is commonly performed with little or no attention to reducing measurement variance. Batch test procedures such as those used to measure K{sub d} values (e.g., ASTM D 4646 and EPA402 -R-99-004A) do not explain how to evaluate measurement uncertainty nor how to minimize measurement variance. In fact, ASTM D 4646 prescribes a sorbent:water ratio that prevents variance minimization. Consequently, the variance of a set of partitioning measurements can be extreme and even absurd. Such data sets, which are commonplace, hamper probabilistic modeling efforts. An error-savvy design requires adjustment of the solution:sorbent ratio so that approximately half of the sorbate partitions to the sorbent. Results of Monte Carlo simulations indicate that this simple step can markedly improve the precision and statistical characterization of partitioning uncertainty.

  3. Merging Groups to Maximize Object Partition Comparison.

    ERIC Educational Resources Information Center

    Klastorin, T. D.

    1980-01-01

    The problem of objectively comparing two independently determined partitions of N objects or variables is discussed. A similarity measure based on the simple matching coefficient is defined and related to previously suggested measures. (Author/JKS)

  4. Release of plasmid DNA from intravascular stents coated with ultrathin multilayered polyelectrolyte films.

    PubMed

    Jewell, Christopher M; Zhang, Jingtao; Fredin, Nathaniel J; Wolff, Matthew R; Hacker, Timothy A; Lynn, David M

    2006-09-01

    Materials that permit control over the release of DNA from the surfaces of topologically complex implantable devices, such as intravascular stents, could contribute to the development of new approaches to the localized delivery of DNA. We report the fabrication of ultrathin, multilayered polyelectrolyte films that permit both the immobilization and controlled release of plasmid DNA from the surfaces of stainless steel intravascular stents. Our approach makes use of an aqueous-based, layer-by-layer method for the assembly of nanostructured thin films consisting of alternating layers of plasmid DNA and a hydrolytically degradable polyamine. Characterization of coated stents using scanning electron microscopy (SEM) demonstrated that stents were coated uniformly with an ultrathin film ca. 120 nm thick that adhered conformally to the surfaces of stent struts. These ultrathin films did not crack, peel, or delaminate substantially from the surface after exposure to a range of mechanical challenges representative of those encountered during stent deployment (e.g., balloon expansion). Stents coated with eight bilayers of degradable polyamine and a plasmid encoding enhanced green fluorescent protein (EGFP) sustained the release of DNA into solution for up to four days when incubated in phosphate buffered saline at 37 degrees C, and coated stents were capable of mediating the expression of EGFP in a mammalian cell line without the aid of additional transfection agents. The approach reported here could, with further development, contribute to the development of localized gene-based approaches to the treatment of cardiovascular diseases or related conditions. PMID:16961308

  5. DNA Targeting Sequence Improves Magnetic Nanoparticle-Based Plasmid DNA Transfection Efficiency in Model Neurons

    PubMed Central

    Vernon, Matthew M.; Dean, David A.; Dobson, Jon

    2015-01-01

    Efficient non-viral plasmid DNA transfection of most stem cells, progenitor cells and primary cell lines currently presents an obstacle for many applications within gene therapy research. From a standpoint of efficiency and cell viability, magnetic nanoparticle-based DNA transfection is a promising gene vectoring technique because it has demonstrated rapid and improved transfection outcomes when compared to alternative non-viral methods. Recently, our research group introduced oscillating magnet arrays that resulted in further improvements to this novel plasmid DNA (pDNA) vectoring technology. Continued improvements to nanomagnetic transfection techniques have focused primarily on magnetic nanoparticle (MNP) functionalization and transfection parameter optimization: cell confluence, growth media, serum starvation, magnet oscillation parameters, etc. Noting that none of these parameters can assist in the nuclear translocation of delivered pDNA following MNP-pDNA complex dissociation in the cell’s cytoplasm, inclusion of a cassette feature for pDNA nuclear translocation is theoretically justified. In this study incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid improved transfection efficiency in model neurons, presumably from increased nuclear translocation. This observation became most apparent when comparing the response of the dividing SH-SY5Y precursor cell to the non-dividing and differentiated SH-SY5Y neuroblastoma cells. PMID:26287182

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

    PubMed

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

    2016-05-01

    Toxic gene therapy (or suicidal gene therapy) is gaining enormous interest, specifically for the treatment of cancer. The success of this therapy lies in several crucial factors, including the potency of gene products to kill the transfected tumor cells and the transfection ability of the transfection vehicles. To address the potency problem, in the present study, we engineered two separate mammalian transfection plasmids (pSAP and pGEL) containing genes encoding ribosome inactivating proteins (RIPs), gelonin and saporin. After the successful preparation and amplification of the plasmids, they were tested on various cancer cell lines (HeLa, U87, 9L, and MDA-MB-435) and a noncancerous cell line (293 HEK) using polyethyleneimine (PEI) as the transfection agent. Transfection studies performed under varying gene concentration, incubation time, and gene-to-PEI ratios revealed that, compared to the treatment of pGFP (GFP expression plasmid)/PEI, both pGEL/PEI and pSAP/PEI complexes could induce significantly augmented cytotoxic effects at only 2 μg/mL gene concentration. Importantly, these cytotoxic effects were observed universally in all tested cancer cell lines. Overall, this study demonstrated the potential of pGEL and pSAP as effective gene candidates for the toxic gene-based cancer therapy. PMID:27008027

  7. DNA Targeting Sequence Improves Magnetic Nanoparticle-Based Plasmid DNA Transfection Efficiency in Model Neurons.

    PubMed

    Vernon, Matthew M; Dean, David A; Dobson, Jon

    2015-01-01

    Efficient non-viral plasmid DNA transfection of most stem cells, progenitor cells and primary cell lines currently presents an obstacle for many applications within gene therapy research. From a standpoint of efficiency and cell viability, magnetic nanoparticle-based DNA transfection is a promising gene vectoring technique because it has demonstrated rapid and improved transfection outcomes when compared to alternative non-viral methods. Recently, our research group introduced oscillating magnet arrays that resulted in further improvements to this novel plasmid DNA (pDNA) vectoring technology. Continued improvements to nanomagnetic transfection techniques have focused primarily on magnetic nanoparticle (MNP) functionalization and transfection parameter optimization: cell confluence, growth media, serum starvation, magnet oscillation parameters, etc. Noting that none of these parameters can assist in the nuclear translocation of delivered pDNA following MNP-pDNA complex dissociation in the cell's cytoplasm, inclusion of a cassette feature for pDNA nuclear translocation is theoretically justified. In this study incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid improved transfection efficiency in model neurons, presumably from increased nuclear translocation. This observation became most apparent when comparing the response of the dividing SH-SY5Y precursor cell to the non-dividing and differentiated SH-SY5Y neuroblastoma cells. PMID:26287182

  8. Isorropia Partitioning and Load Balancing Package

    2006-09-01

    Isorropia is a partitioning and load balancing package which interfaces with the Zoltan library. Isorropia can accept input objects such as matrices and matrix-graphs, and repartition/redistribute them into a better data distribution on parallel computers. Isorropia is primarily an interface package, utilizing graph and hypergraph partitioning algorithms that are in the Zoltan library which is a third-party library to Tilinos.

  9. Deriving the Hirshfeld partitioning using distance metrics

    SciTech Connect

    Heidar-Zadeh, Farnaz; Ayers, Paul W.; Bultinck, Patrick

    2014-09-07

    The atoms in molecules associated with the Hirshfeld partitioning minimize the generalized Hellinger-Bhattacharya distance to the reference pro-atom densities. Moreover, the reference pro-atoms can be chosen by minimizing the distance between the pro-molecule density and the true molecular density. This provides an alternative to both the heuristic “stockholder” and the mathematical information-theoretic interpretations of the Hirshfeld partitioning. These results extend to any member of the family of f-divergences.

  10. Rapid targeting of plasmid DNA to zebrafish embryo nuclei by the nuclear localization signal of SV40 T antigen.

    PubMed

    Collas, P; Aleström, P

    1997-03-01

    Binding SV40 T antigen nuclear localization signals (NLSs) to plasmid DNA promotes transgene expression following injection of DNA-NLS complexes into the cytoplasm of zebrafish eggs. We now demonstrate that NLS peptides mediate import of DNA from the cytoplasm into embryo nuclei, under conditions in which naked DNA is not imported. Plasmid DNA was localized by polymerase chain reaction (PCR) in isolated nuclei, and relative amounts were quantified by densitometry. Binding DNA to NLSs, but not to nuclear-import-deficient peptides, promoted rapid targeting of DNA-NLS complexes to nuclei, and transport across the nuclear envelope. Import of DNA-NLS complexes was competed by co-injected albumin-NLS conjugates. NLS, but not reverse NLS, was detected on blots of nuclei probed with 32P-labeled DNA. The results suggest that NLS-mediated DNA transfer into nuclei may constitute a valuable tool for several gene transfer applications. PMID:9116870

  11. 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

  12. Plasmid maintenance systems suitable for GMO-based bacterial vaccines.

    PubMed

    Spreng, Simone; Viret, Jean-François

    2005-03-18

    Live carrier-based bacterial vaccines represent a vaccine strategy that offers exceptional flexibility. Commensal or attenuated strains of pathogenic bacteria can be used as live carriers to present foreign antigens from unrelated pathogens to the immune system, with the aim of eliciting protective immune responses. As for oral immunisation, such an approach obviates the usual loss of antigen integrity observed during gastrointestinal passage and allows the delivery of a sufficient antigen dose to the mucosal immune system. Antibiotic and antibiotic-resistance genes have traditionally been used for the maintenance of recombinant plasmid vectors in bacteria used for biotechnological purposes. However, their continued use may appear undesirable in the field of live carrier-based vaccine development. This review focuses on strategies to omit antibiotic resistance determinants in live bacterial vaccines and discusses several balanced lethal-plasmid stabilisation systems with respect to maintenance of plasmid inheritance and antigenicity of plasmid-encoded antigen in vivo.

  13. Augmented anti-tumor effect of dendritic cells genetically engineered by interleukin-12 plasmid DNA.

    PubMed

    Yoshida, Masataka; Jo, Jun-Ichiro; Tabata, Yasuhiko

    2010-01-01

    The objective of this study was to genetically engineer dendritic cells (DC) for biological activation and evaluate their anti-tumor activity in a tumor-bearing mouse model. Mouse DC were incubated on the surface of culture dishes which had been coated with the complexes of a cationized dextran and luciferase plasmid DNA complexes plus a cell adhesion protein, Pronectin, for gene transfection (reverse transfection). When compared with the conventional transfection where DC were transfected in the medium containing the complexes, the level of gene expression by the reverse method was significantly higher and the time period of gene expression was prolonged. Following the reverse transfection of DC by a plasmid DNA of mouse interleukin-12 (mIL-12) complexed with the cationized dextran, the mIL-12 protein was secreted at higher amounts for a longer time period. When injected intratumorally into mice carrying a mass of B16 tumor cells, the DC genetically activated showed significant anti-tumor activity. PMID:20338099

  14. Antibacterial effect of cationic porphyrazines and anionic phthalocyanine and their interaction with plasmid DNA

    NASA Astrophysics Data System (ADS)

    Hassani, Leila; Hakimian, Fatemeh; Safaei, Elham; Fazeli, Zahra

    2013-11-01

    Resistance to antibiotics is a public health issue and identification of new antibacterial agents is one of the most important goals of pharmacological research. Among the novel developed antibacterial agents, porphyrin complexes and their derivatives are ideal candidates for use in medical applications. Phthalocyanines differ from porphyrins by having nitrogen atoms link the individual pyrrol units. The aza analogues of the phthalocyanines (azaPcs) such as tetramethylmetalloporphyrazines are heterocyclic Pc analogues. In this investigation, interaction of an anionic phthalocyanine (Cu(PcTs)) and two cationic tetrapyridinoporphyrazines including [Cu(2,3-tmtppa)]4+ and [Cu(3,4-tmtppa)]4+ complexes with plasmid DNA was studied using spectroscopic and gel electrophoresis methods. In addition, antibacterial effect of the complexes against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria was investigated using dilution test method. The results indicated that both porphyrazines have significant antibacterial properties, but Cu(PcTs) has weak antibacterial effect. Compairing the binding of the phthalocyanine and the porphyrazines to DNA demonstrated that the interaction of cationic porphyrazines is stronger than the anionic phthalocyanine remarkably. The extent of hypochromicity and red shift of absorption spectra indicated preferential intercalation of the two porphyrazine into the base pairs of DNA helix. Gel electrophoresis result implied Cu(2,3-tmtppa) and Cu(3,4-tmtppa) are able to perform cleavage of the plasmid DNA. Consequently, DNA binding and cleavage might be one of the antibacterial mechanisms of the complexes.

  15. 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.

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

    PubMed

    Harmer, Christopher J; Hall, Ruth M

    2015-07-01

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

  17. β-Carotene production by Saccharomyces cerevisiae with regard to plasmid stability and culture media.

    PubMed

    Lange, Nicole; Steinbüchel, Alexander

    2011-09-01

    A recombinant Saccharomyces cerevisiae strain was used for the production of β-carotene. The episomal plasmid YEplac195YB/I/E was extended by a gene coding for the mevalonate kinase (mvaK1) from Staphylococcus aureus. The adh1 promoter was chosen for constitutive expression of mvaK1. The recombinant strain S. cerevisiae G175 (YEplac-CaroSA) synthesised β-carotene by expressing the carotenogenic genes of Xanthophyllomyces dendrorhous together with the mvaK1 gene. Cells of this strain were investigated for their carotenoid contents in YNB and YPD media. A corresponding mvaK1 transcript in the recombinant yeast host was verified. Growth experiments of a specific erg12 deletion mutant showed that the mevalonate kinase (MvaK1) was able to complement the function of the deleted native mevalonate kinase (Erg12) from S. cerevisiae in the MVA pathway under control of the constitutive adh1 promoter. Cells of S. cerevisiae G175 (YEplac-CaroSA) exhibited high plasmid stability under either selective or non-selective cultivation conditions. Time course experiments demonstrated high plasmid stability even over extended cultivation periods. Carotenoid production was therefore also stable in larger culture volumes. Due to the stability of the plasmid, cultivation of the cells in complex YPD medium was possible, and 14.3 mg β-carotene per litre and a cell density of 9 g cell dry matter (CDM) per litre were achieved. The highest amount of 3,897 μg β-carotene per gramme CDM at a cell density of 1 g CDM per litre was measured after cultivation of the cells in YNB medium with glucose as sole carbon source. PMID:21573686

  18. β-Carotene production by Saccharomyces cerevisiae with regard to plasmid stability and culture media.

    PubMed

    Lange, Nicole; Steinbüchel, Alexander

    2011-09-01

    A recombinant Saccharomyces cerevisiae strain was used for the production of β-carotene. The episomal plasmid YEplac195YB/I/E was extended by a gene coding for the mevalonate kinase (mvaK1) from Staphylococcus aureus. The adh1 promoter was chosen for constitutive expression of mvaK1. The recombinant strain S. cerevisiae G175 (YEplac-CaroSA) synthesised β-carotene by expressing the carotenogenic genes of Xanthophyllomyces dendrorhous together with the mvaK1 gene. Cells of this strain were investigated for their carotenoid contents in YNB and YPD media. A corresponding mvaK1 transcript in the recombinant yeast host was verified. Growth experiments of a specific erg12 deletion mutant showed that the mevalonate kinase (MvaK1) was able to complement the function of the deleted native mevalonate kinase (Erg12) from S. cerevisiae in the MVA pathway under control of the constitutive adh1 promoter. Cells of S. cerevisiae G175 (YEplac-CaroSA) exhibited high plasmid stability under either selective or non-selective cultivation conditions. Time course experiments demonstrated high plasmid stability even over extended cultivation periods. Carotenoid production was therefore also stable in larger culture volumes. Due to the stability of the plasmid, cultivation of the cells in complex YPD medium was possible, and 14.3 mg β-carotene per litre and a cell density of 9 g cell dry matter (CDM) per litre were achieved. The highest amount of 3,897 μg β-carotene per gramme CDM at a cell density of 1 g CDM per litre was measured after cultivation of the cells in YNB medium with glucose as sole carbon source.

  19. Large partition coefficients for trace elements in high-silica rhyolites

    USGS Publications Warehouse

    Mahood, G.; Hildreth, W.

    1983-01-01

    The partitioning of 25 trace elements between high-silica rhyolitic glass and unzoned phenocrysts of potassic and sodic sanidine, biotite, augite, ferrohedenbergite, hypersthene, fayalite, titanomagnetite, ilmenite, zircon, and allanite has been determined by INAA on suites of samples from the mildly peralkaline lavas and tuff of the Sierra La Primavera, Mexico, and the metaluminous, compo. sitionally zoned, Bishop Tuff, California. The partition coefficients are much larger than published values for less silicic compositions; the range of values among Primavera samples that differ only slightly in temperature or bulk composition approaches that previously reported from basalts to rhyodacites. Intrinsic temperature dependence of the crystal/liquid partitioning is apparently small. The high values of partition coefficients reflect principally the strongly polymerized nature of the alkali-aluminosilicate liquid, whereas the marked variability of values for partition coefficients is attributed to differences in the concentrations of complexing ligands and/or different degrees of melt polymerization. Great variation in the values of partition coefficients that are potentially applicable to early stages in the partial melting of crustal rocks complicates assessment of 1. (1) source regions for granitic melts and 2. (2) contributions by crustal-melt increments to andesites. ?? 1983.

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

    PubMed Central

    Kamens, Joanne

    2015-01-01

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

  1. Electrotransformation of Thiobacillus ferrooxidans with plasmids containing a mer determinant.

    PubMed Central

    Kusano, T; Sugawara, K; Inoue, C; Takeshima, T; Numata, M; Shiratori, T

    1992-01-01

    The mer operon from a strain of Thiobacillus ferrooxidans (C. Inoue, K. Sugawara, and T. Kusano, Mol. Microbiol. 5:2707-2718, 1991) consists of the regulatory gene merR and an operator-promoter region followed by merC and merA structural genes and differs from other known gram-negative mer operons. We have constructed four potential shuttle plasmids composed of a T. ferrooxidans-borne cryptic plasmid, a pUC18 plasmid, and the above-mentioned mer determinant as a selectable marker. Mercury ion-sensitive T. ferrooxidans strains were electroporated with constructed plasmids, and one strain, Y4-3 (of 30 independent strains tested), was found to have a transformation efficiency of 120 to 200 mercury-resistant colonies per microgram of plasmid DNA. This recipient strain was confirmed to be T. ferrooxidans by physiological, morphological, and chemotaxonomical data. The transformants carried a plasmid with no physical rearrangements through 25 passages under no selective pressure. Cell extracts showed mercury ion-dependent NADPH oxidation activity. Images PMID:1400213

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

    PubMed

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

    2015-10-01

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

  3. Transformation of Rhizobium meliloti 41 with plasmid DNA.

    PubMed Central

    Kiss, G B; Kálmán, Z

    1982-01-01

    Plasmid pGV1106, a derivative of the wide-host-range plasmid S-a of the W incompatibility group, was introduced into Rhizobium meliloti 41 by plasmid-mediated mobilization to overcome the restriction of foreign DNA. The mobilized plasmid pKK2 differed from the original pGV1106 by an extra piece of DNA of 1.3 kilobase pairs which supposedly originated from pJB3JI used for mobilization. If pKK2 was isolated from R. meliloti 41, it could be successfully reintroduced by transformation. The transformation frequency was low (10 to 54 colonies per micrograms of plasmid DNA) but reproducible, and several lines of evidence showed that it was the consequence of plasmid DNA uptake. The small size (10.3 kilobases) and elevated copy number (10 to 15 copies per cell) of pKK2 make it a potentially useful cloning vector for the study of symbiotic nitrogen fixation genes of R. meliloti 41. Images PMID:6279558

  4. Software Partitioning Schemes for Advanced Simulation Computer Systems. Final Report.

    ERIC Educational Resources Information Center

    Clymer, S. J.

    Conducted to design software partitioning techniques for use by the Air Force to partition a large flight simulator program for optimal execution on alternative configurations, this study resulted in a mathematical model which defines characteristics for an optimal partition, and a manually demonstrated partitioning algorithm design which…

  5. 47 CFR 101.1415 - Partitioning and disaggregation.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... GHz Band § 101.1415 Partitioning and disaggregation. (a) MVDDS licensees are permitted to partition...) MVDDS licensees may apply to the Commission to partition their licensed geographic service areas to eligible entities and are free to partition their licensed spectrum at any time following the grant of...

  6. Sex, prions, and plasmids in yeast.

    PubMed

    Kelly, Amy C; Shewmaker, Frank P; Kryndushkin, Dmitry; Wickner, Reed B

    2012-10-01

    Even deadly prions may be widespread in nature if they spread by infection faster than they kill off their hosts. The yeast prions [PSI+] and [URE3] (amyloids of Sup35p and Ure2p) were not found in 70 wild strains, while [PIN+] (amyloid of Rnq1p) was found in ∼16% of the same population. Yeast prion infection occurs only by mating, balancing the detrimental effects of carrying the prion. We estimated the frequency of outcross mating as about 1% of mitotic doublings from the known detriment of carrying the 2-μm DNA plasmid (∼1%) and its frequency in wild populations (38/70). We also estimated the fraction of total matings that are outcross matings (∼23-46%) from the fraction of heterozygosity at the highly polymorphic RNQ1 locus (∼46%). These results show that the detriment of carrying even the mildest forms of [PSI+], [URE3], or [PIN+] is greater than 1%. We find that Rnq1p polymorphisms in wild strains include several premature stop codon alleles that cannot propagate [PIN+] from the reference allele and others with several small deletions and point mutations which show a small transmission barrier. Wild strains carrying [PIN+] are far more likely to be heterozygous at RNQ1 and other loci than are [pin-] strains, probably reflecting its being a sexually transmitted disease. Because sequence differences are known to block prion propagation or ameliorate its pathogenic effects, we hypothesize that polymorphism of RNQ1 was selected to protect cells from detrimental effects of the [PIN+] prion.

  7. 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

  8. Molecular epidemiology of plasmid spread among extended broad-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates in a pediatric hospital.

    PubMed Central

    Bingen, E H; Desjardins, P; Arlet, G; Bourgeois, F; Mariani-Kurkdjian, P; Lambert-Zechovsky, N Y; Denamur, E; Philippon, A; Elion, J

    1993-01-01

    Over a 12-month period, 43 children in eight different wards of our hospital (Hôpital Robert Debré) were infected or colonized with Klebsiella pneumoniae strains producing extended broad-spectrum beta-lactamases. The epidemiology of the outbreak was studied by a molecular approach including the determination of the beta-lactamase physicochemical parameters and plasmid profiles, as well as analysis of the restriction fragment length polymorphisms of the rDNA regions (ribotyping). The last approach produced 12 and 5 different patterns with EcoRI and HindIII, respectively, thus identifying 15 different ribotypes among the 43 clinical K. pneumoniae strains. However, 60% of the strains in six wards belonged to only two ribotypes, whereas nine ribotypes were observed only once. Twelve isolates from different wards that were representative of the eight most common ribotypes showed four different beta-lactamase isoelectric focusing patterns and seven different plasmid profiles by direct analysis or after EcoRI digestion. Thus, at least two genetically unrelated strains in the same ward were found to have the same plasmid content. Our results show the complexity of the outbreak, which was associated with patient-to-patient cross-contamination with several epidemic strains with different plasmid contents, interspersed sporadic cases with nonepidemic strains, and the possible spread of a plasmid. The combination of plasmid profile analysis and ribotyping therefore seems to be powerful at deciphering the details of such outbreaks. Images PMID:8432800

  9. 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.

  10. Quantum algorithm for an additive approximation of Ising partition functions

    NASA Astrophysics Data System (ADS)

    Matsuo, Akira; Fujii, Keisuke; Imoto, Nobuyuki

    2014-08-01

    We investigate quantum-computational complexity of calculating partition functions of Ising models. We construct a quantum algorithm for an additive approximation of Ising partition functions on square lattices. To this end, we utilize the overlap mapping developed by M. Van den Nest, W. Dür, and H. J. Briegel [Phys. Rev. Lett. 98, 117207 (2007), 10.1103/PhysRevLett.98.117207] and its interpretation through measurement-based quantum computation (MBQC). We specify an algorithmic domain, on which the proposed algorithm works, and an approximation scale, which determines the accuracy of the approximation. We show that the proposed algorithm performs a nontrivial task, which would be intractable on any classical computer, by showing that the problem that is solvable by the proposed quantum algorithm is BQP-complete. In the construction of the BQP-complete problem coupling strengths and magnetic fields take complex values. However, the Ising models that are of central interest in statistical physics and computer science consist of real coupling strengths and magnetic fields. Thus we extend the algorithmic domain of the proposed algorithm to such a real physical parameter region and calculate the approximation scale explicitly. We found that the overlap mapping and its MBQC interpretation improve the approximation scale exponentially compared to a straightforward constant-depth quantum algorithm. On the other hand, the proposed quantum algorithm also provides partial evidence that there exist no efficient classical algorithm for a multiplicative approximation of the Ising partition functions even on the square lattice. This result supports the observation that the proposed quantum algorithm also performs a nontrivial task in the physical parameter region.

  11. Secure splenic delivery of plasmid DNA and its application to DNA vaccine.

    PubMed

    Kurosaki, Tomoaki; Kodama, Yukinobu; Muro, Takahiro; Higuchi, Norihide; Nakamura, Tadahiro; Kitahara, Takashi; Miyakoda, Mana; Yui, Katsuyuki; Sasaki, Hitoshi

    2013-01-01

    In this experiment, we developed a novel safe and effective gene delivery vector coated with γ-polyglutamic acid (γ-PGA-coated complexes). The γ-PGA-coated complex was composed of chiseled spherical nano-particles with anionic charges. The plasmid DNA/polyethyleneimine complex (non-coated complex) showed high transgene efficiency in the spleen and lung after intravenous administration in mice, with high liver toxicity and lethality. On the other hand, γ-PGA-coated complex selectively showed high transgene efficiency in the spleen without such toxicity. Furthermore, the γ-PGA-coated complex highly accumulated and showed high gene expression in the marginal zone of the spleen. Those results strongly indicated that γ-PGA-coated complex was suitable as a DNA vaccine vector. We therefore applied γ-PGA-coated complex to melanoma DNA vaccine, pUb-M. The γ-PGA-coated complex containing pUb-M significantly inhibited the growth and metastasis of a melanoma cell line, B16-F10 cells. In conclusion, we developed a splenic gene vector, γ-PGA-coated complex, as a novel technology for clinical vaccination. PMID:24189423

  12. Plasmid replication initiator interactions with origin 13-mers and polymerase subunits contribute to strand-specific replisome assembly

    PubMed Central

    Wawrzycka, Aleksandra; Gross, Marta; Wasaznik, Anna; Konieczny, Igor

    2015-01-01

    Although the molecular basis for replisome activity has been extensively investigated, it is not clear what the exact mechanism for de novo assembly of the replication complex at the replication origin is, or how the directionality of replication is determined. Here, using the plasmid RK2 replicon, we analyze the protein interactions required for Escherichia coli polymerase III (Pol III) holoenzyme association at the replication origin. Our investigations revealed that in E. coli, replisome formation at the plasmid origin involves interactions of the RK2 plasmid replication initiation protein (TrfA) with both the polymerase β- and α-subunits. In the presence of other replication proteins, including DnaA, helicase, primase and the clamp loader, TrfA interaction with the β-clamp contributes to the formation of the β-clamp nucleoprotein complex on origin DNA. By reconstituting in vitro the replication reaction on ssDNA templates, we demonstrate that TrfA interaction with the β-clamp and sequence-specific TrfA interaction with one strand of the plasmid origin DNA unwinding element (DUE) contribute to strand-specific replisome assembly. Wild-type TrfA, but not the TrfA QLSLF mutant (which does not interact with the β-clamp), in the presence of primase, helicase, Pol III core, clamp loader, and β-clamp initiates DNA synthesis on ssDNA template containing 13-mers of the bottom strand, but not the top strand, of DUE. Results presented in this work uncovered requirements for anchoring polymerase at the plasmid replication origin and bring insights of how the directionality of DNA replication is determined. PMID:26195759

  13. Sediment-water partitioning of inorganic mercury in estuaries.

    PubMed

    Turner, A; Millward, G E; Le Roux, S M

    2001-12-01

    The sediment-water partitioning and speciation of inorganic mercury have been studied under simulated estuarine conditions by monitoring the hydrophobicity and uptake of dissolved 203Hg(II) in samples from a variety of estuarine environments. A persistent increase in the distribution coefficientwith increasing salinity is inconsistent with inorganic speciation calculations, which predict an increase in the concentration of the soluble HgCl4(2-) complex (or reduction in sediment-water distribution coefficient) with increasing salinity. Partition data are, however, defined by an empirical equation relating to the salting out of nonelectrolytes via electrostriction and are characterized by salting constants between about 1.4 and 2.0 L mol(-1). Salting out of the neutral, covalent chloro-complex, HgCl2(0), is predicted but cannot account for the magnitude of salting out observed. Since Hg(II) strongly complexes with dissolved (and particulate) organic matter in natural environments, of more significance appears to be the salting out of Hg(II)-organic complexes. Operational measurements of the speciation of dissolved Hg(II) using Sep-Pak C18 columns indicate a reduction in the proportion of hydrophobic (C18-retained) dissolved Hg(II) complexes with increasing salinity, both in the presence and absence of suspended particles. Ratios of hydrophobic Hg(ll) before and after particle addition suggest a coupled salting out-sorption mechanism, with the precise nature of Hg(II) species salted out being determined bythe characteristics and concentrations of dissolved and sediment organic matter. PMID:11770766

  14. Extrachromosomal plasmids in the plant pathogenic fungus Rhizoctonia solani.

    PubMed

    Jabaji-Hare, S H; Burger, G; Forget, L; Lang, B F

    1994-05-01

    Extrachromosomal DNA elements were found in field isolates of Rhizoctonia solani belonging to anastomosis groups (AG) 1-5. An isolate of AG-5 (Rh41) contains a 3.6-kbp plasmid (pRS188) which has a similar A+T content to mitochondrial DNA. pRS188 is linear and has knob structures at its ends, as revealed by electron microscopy. Exonuclease digestions show that the linear ends of pRS188 are protected, and remain protected even after proteinase K digestion. pRS188 does not hybridise to nuclear or mitochondrial DNAs of its host isolate (Rh41), to total DNAs of other plasmid-less AG-5 isolates, or to total DNA of plasmid-harbouring isolates belonging to different AGs. Cellular-fractionation experiments suggest that pRS188 is associated with mitochondria, but it remains undecided whether this occurs inside or outside of the organelles. The nucleotide sequence of about 60% of the plasmid has been determined, revealing no open reading frame longer than 91 amino acids, and no known gene or genetic element is detected in the sequence contigs of 300-1572 bp length. Similar studies were performed with the plasmid pRS104 present in an isolate of AG-4 (Rh36), the sequence of which exhibits essentially the same features as pRS188 except that its A+T content resembles that of nuclear DNA. Pathogenicity tests reveal that the isolates Rh41 and R36 are as virulent as the plasmid-less isolates of AG-4 and -5, indicating that the plasmids do not play any role in pathogenicity.

  15. 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

  16. Design of amphiphilic oligopeptides as models for fine tuning peptide assembly with plasmid DNA.

    PubMed

    Goparaju, Geetha N; Gupta, Pardeep K

    2014-08-01

    We discuss the design of novel amphiphilic oligopeptides with hydrophobic and cationic amino acids to serve as models to understand peptide-DNA assembly. Biophysical and thermodynamic characterization of interaction of these amphiphilic peptides with plasmid DNA is presented. Peptides with at least +4 charges favor stable complex formation. Surface potential is dependent on the type of hydrophobic amino acid for a certain charge. Thermodynamically it is a spontaneous interaction between most of the peptides and plasmid DNA. Lys(7) and Tyr peptides with +4/+5 charges indicate cooperative binding with pDNA without saturation of interaction while Val(2)-Gly-Lys(4), Val-Gly-Lys(5), and Phe-Gly-Lys(5) lead to saturation of interaction indicating condensed pDNA within the range of N/Ps studied. We show that the biophysical properties of DNA-peptide complexes could be modulated by design and the peptides presented here could be used as building blocks for creating DNA-peptide complexes for various biomedical applications, mainly nucleic acid delivery.

  17. Screening of pesticides for environmental partitioning tendency.

    PubMed

    Gramatica, Paola; Di Guardo, Antonio

    2002-06-01

    The partitioning tendency of chemicals, in this study pesticides in particular, into different environmental compartments depends mainly on the concurrent relevance of the physico-chemical properties of the chemical itself. To rank the pesticides according to their distribution tendencies in the different environmental compartments we propose a multivariate approach: the combination, by principal component analysis, of those physico-chemical properties like organic carbon partition coefficient (Koc), n-octanol/water partition coefficient (Kow), water solubility (Sw), vapour pressure and Henry's law constant (H) that are more relevant to the determination of environmental partitioning. The resultant macrovariables, the PC1 and PC2 scores here named leaching index (LIN) and volatality index (VIN), are proposed as preliminary environmental partitioning indexes in different media. These two indexes are modeled by theoretical molecular descriptors with satisfactory predictive power. Such an approach allows a rapid pre-determination and screening of the environmental distribution of pesticides starting only from the molecular structure of the pesticide, without any a priori knowledge of the physico-chemical properties.

  18. Photosynthate Partitioning into Starch in Soybean Leaves

    PubMed Central

    Chatterton, N. Jerry; Silvius, John E.

    1979-01-01

    Photosynthesis, photosynthate partitioning into foliar starch, and translocation were investigated in soybean plants (Glycine max (L.) Merr. cv. Amsoy 71), grown under different photoperiods and photosynthetic periods to determine the controls of leaf starch accumulation. Starch accumulation rates in soybean leaves were inversely related to the length of the daily photosynthetic period under which the plants were grown. Photosynthetic period and not photoperiod per se appears to be the important factor. Plants grown in a 14-hour photosynthetic period partitioned approximately 60% of the daily foliar accumulation into starch whereas 7-hour plants partitioned about 90% of their daily foliar accumulation into starch. The difference in starch accumulation resulted from a change in photosynthate partitioning between starch and leaf residual dry weight. Residual dry weight is defined as leaf dry weight minus the weight of total nonstructural carbohydrates. Differences in photosynthate partitioning into starch were also associated with changes in photosynthetic and translocation rates, as well as with leaf and whole plant morphology. It is concluded that leaf starch accumulation is a programmed process and not simply the result of a limitation in translocation. PMID:16661047

  19. Multi-A Graph Patrolling and Partitioning

    NASA Astrophysics Data System (ADS)

    Elor, Y.; Bruckstein, A. M.

    2012-12-01

    We introduce a novel multi agent patrolling algorithm inspired by the behavior of gas filled balloons. Very low capability ant-like agents are considered with the task of patrolling an unknown area modeled as a graph. While executing the proposed algorithm, the agents dynamically partition the graph between them using simple local interactions, every agent assuming the responsibility for patrolling his subgraph. Balanced graph partition is an emergent behavior due to the local interactions between the agents in the swarm. Extensive simulations on various graphs (environments) showed that the average time to reach a balanced partition is linear with the graph size. The simulations yielded a convincing argument for conjecturing that if the graph being patrolled contains a balanced partition, the agents will find it. However, we could not prove this. Nevertheless, we have proved that if a balanced partition is reached, the maximum time lag between two successive visits to any vertex using the proposed strategy is at most twice the optimal so the patrol quality is at least half the optimal. In case of weighted graphs the patrol quality is at least (1)/(2){lmin}/{lmax} of the optimal where lmax (lmin) is the longest (shortest) edge in the graph.

  20. Computational prediction of solubilizers' effect on partitioning.

    PubMed

    Hoest, Jan; Christensen, Inge T; Jørgensen, Flemming S; Hovgaard, Lars; Frokjaer, Sven

    2007-02-01

    A computational model for the prediction of solubilizers' effect on drug partitioning has been developed. Membrane/water partitioning was evaluated by means of immobilized artificial membrane (IAM) chromatography. Four solubilizers were used to alter the partitioning in the IAM column. Two types of molecular descriptors were calculated: 2D descriptors using the MOE software and 3D descriptors using the Volsurf software. Structure-property relationships between each of the two types of descriptors and partitioning were established using partial least squares, projection to latent structures (PLS) statistics. Statistically significant relationships between the molecular descriptors and the IAM data were identified. Based on the 2D descriptors structure-property relationships R(2)Y=0. 99 and Q(2)=0.82-0.83 were obtained for some of the solubilizers. The most important descriptor was related to logP. For the Volsurf 3D descriptors models with R(2)Y=0.53-0.64 and Q(2)=0.40-0.54 were obtained using five descriptors. The present study showed that it is possible to predict partitioning of substances in an artificial phospholipid membrane, with or without the use of solubilizers.

  1. Identification of Three Noncontiguous Regions on Bacillus anthracis Plasmid pXO1 That Are Important for Its Maintenance

    PubMed Central

    Pomerantsev, Andrei P.; Chang, Zanetta; Rappole, Catherine

    2014-01-01

    Bacillus anthracis pXO1 minireplicon (MR) plasmid consisting of open reading frames (ORFs) GBAA_pXO1_0020 to GBAA_pXO1_0023 is not stably maintained in B. anthracis, whereas the full-size parent pXO1 plasmid (having 181,677 bp and 217 ORFs) is extremely stable under the same growth conditions. Two genetic tools developed for DNA manipulation in B. anthracis (Cre-loxP and Flp-FRT systems) were used to identify pXO1 regions important for plasmid stability. We localized a large segment of pXO1 that enables stable plasmid maintenance during vegetative growth. Further genetic analysis identified three genes that are necessary for pXO1 maintenance: amsP (GBAA_pXO1_0069), minP (GBAA_pXO1_0082), and sojP (GBAA_pXO1_0084). Analysis of conserved domains in the corresponding proteins indicated that only AmsP (activator of maintenance system of pXO1) is predicted to bind DNA, due to its strong helix-turn-helix domain. Two conserved domains were found in the MinP protein (Min protein from pXO1): an N-terminal domain having some similarity to the B. anthracis septum site-determining protein MinD and a C-terminal domain that resembles a baculovirus single-stranded-DNA-binding protein. The SojP protein (Soj from pXO1) contains putative Walker box motifs and belongs to the ParA family of ATPases. No sequences encoding other components of type I plasmid partition systems, namely, cis-acting centromere parS and its binding ParB protein, were identified within the pXO1 genome. A model describing the role of the MinP protein in pXO1 distribution between daughter cells is proposed. PMID:24914182

  2. Structural analysis of the 6 kb cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 and construction of Escherichia coli-Rhodococcus shuttle vectors.

    PubMed

    De Mot, R; Nagy, I; De Schrijver, A; Pattanapipitpaisal, P; Schoofs, G; Vanderleyden, J

    1997-10-01

    The complete nucleotide sequence of the 5936 bp cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 was determined. Based on the characteristics of its putative replication genes, repA and repB, pFAJ2600 was assigned to the family of pAL5000-related small replicons identified in Mycobacterium (pAL5000), Corynebacterium (pXZ10142), Brevibacterium (pRBL1), Bifidobacterium (pMB1) and Neisseria (pJD1). The replication systems of these plasmids show striking similarities to the ones used by the ColE2 family of plasmids from Enterobacteria with respect to both trans-acting factors and ori sequences. Two possible plasmid stabilization systems are encoded on pFAJ2600: a site-specific recombinase (PmrA) related to the Escherichia coli Xer proteins for plasmid multimer resolution and an ATPase (ParA) related to the A-type of proteins in sop/par partitioning systems. The proposed replication termination region of pFAJ2600 has features in common with the Ter loci of Bacillus subtilis. Chimeras composed of a pUC18-Cmr derivative inserted in the parA-repA intergenic region of vector pFAJ2600 produced vectors that could be shuttled between Escherichia coli and several Rhodococcus species (R. erythropolis, R. fascians, R. rhodochrous, R. ruber). The pFAJ2600-based shuttle vector pFAJ2574 was stably maintained in R. erythropolis and R. fascians growing under non-selective conditions.

  3. Peoples, Resources, and Lifestyles: The Hopi-Navajo Land Partition Act of 1974.

    ERIC Educational Resources Information Center

    Goodman, James M.

    The Hopi and Navajo tribes have been engaged in a long and complex land dispute within the 1882 Executive Order Area (Joint Use Area) of Arizona, an area recently redefined via the Partition Act of 1974 which calls for the relocation of 5 to 10,000 Navajos. This rearrangement of political domain threatens to influence the future management and…

  4. The extended regulatory networks of SXT/R391 integrative and conjugative elements and IncA/C conjugative plasmids

    PubMed Central

    Poulin-Laprade, Dominic; Carraro, Nicolas; Burrus, Vincent

    2015-01-01

    Nowadays, healthcare systems are challenged by a major worldwide drug resistance crisis caused by the massive and rapid dissemination of antibiotic resistance genes and associated emergence of multidrug resistant pathogenic bacteria, in both clinical and environmental settings. Conjugation is the main driving force of gene transfer among microorganisms. This mechanism of horizontal gene transfer mediates the translocation of large DNA fragments between two bacterial cells in direct contact. Integrative and conjugative elements (ICEs) of the SXT/R391 family (SRIs) and IncA/C conjugative plasmids (ACPs) are responsible for the dissemination of a broad spectrum of antibiotic resistance genes among diverse species of Enterobacteriaceae and Vibrionaceae. The biology, diversity, prevalence and distribution of these two families of conjugative elements have been the subject of extensive studies for the past 15 years. Recently, the transcriptional regulators that govern their dissemination through the expression of ICE- or plasmid-encoded transfer genes have been described. Unrelated repressors control the activation of conjugation by preventing the expression of two related master activator complexes in both types of elements, i.e., SetCD in SXT/R391 ICEs and AcaCD in IncA/C plasmids. Finally, in addition to activating ICE- or plasmid-borne genes, these master activators have been shown to specifically activate phylogenetically unrelated mobilizable genomic islands (MGIs) that also disseminate antibiotic resistance genes and other adaptive traits among a plethora of pathogens such as Vibrio cholerae and Salmonella enterica. PMID:26347724

  5. The extended regulatory networks of SXT/R391 integrative and conjugative elements and IncA/C conjugative plasmids.

    PubMed

    Poulin-Laprade, Dominic; Carraro, Nicolas; Burrus, Vincent

    2015-01-01

    Nowadays, healthcare systems are challenged by a major worldwide drug resistance crisis caused by the massive and rapid dissemination of antibiotic resistance genes and associated emergence of multidrug resistant pathogenic bacteria, in both clinical and environmental settings. Conjugation is the main driving force of gene transfer among microorganisms. This mechanism of horizontal gene transfer mediates the translocation of large DNA fragments between two bacterial cells in direct contact. Integrative and conjugative elements (ICEs) of the SXT/R391 family (SRIs) and IncA/C conjugative plasmids (ACPs) are responsible for the dissemination of a broad spectrum of antibiotic resistance genes among diverse species of Enterobacteriaceae and Vibrionaceae. The biology, diversity, prevalence and distribution of these two families of conjugative elements have been the subject of extensive studies for the past 15 years. Recently, the transcriptional regulators that govern their dissemination through the expression of ICE- or plasmid-encoded transfer genes have been described. Unrelated repressors control the activation of conjugation by preventing the expression of two related master activator complexes in both types of elements, i.e., SetCD in SXT/R391 ICEs and AcaCD in IncA/C plasmids. Finally, in addition to activating ICE- or plasmid-borne genes, these master activators have been shown to specifically activate phylogenetically unrelated mobilizable genomic islands (MGIs) that also disseminate antibiotic resistance genes and other adaptive traits among a plethora of pathogens such as Vibrio cholerae and Salmonella enterica. PMID:26347724

  6. Specific protein-DNA and protein-protein interaction in the hig gene system, a plasmid-borne proteic killer gene system of plasmid Rts1.

    PubMed

    Tian, Q B; Ohnishi, M; Murata, T; Nakayama, K; Terawaki, Y; Hayashi, T

    2001-03-01

    The hig (host inhibition of growth) gene system of plasmid Rts1 belongs to the plasmid-encoded proteic killer gene family. Among the proteic killer genes described so far, hig is unique in that the toxin gene (higB) exists upstream of the antidote gene (higA). There are two promoters in the hig locus, Phig and PhigA, and only the former, which expresses both higB and higA genes, is negatively controlled by HigA and HigB proteins. In this study, we purified HigA protein by means of GST fusion. The electrophoretic mobility shift assay using the purified protein revealed that HigA specifically bound to the Phig region, but not to PhigA. The HigA-binding sequence was determined by DNase I footprinting assay to be a 56-bp sequence that completely covered the -35 and -10 boxes of Phig. The presence of two inverted repeats in the binding sequence and the identification of a dimer form of HigA by cross-linking experiment suggested that the protein bound to the Phig region as a dimer. HigB was purified as a GST fusion protein as well, though it was achieved only in the presence of HigA. HigA and GST-HigB formed a highly stable complex where the two proteins were present in an equimolar ratio.

  7. New parallel SOR method by domain partitioning

    SciTech Connect

    Xie, D.; Adams, L.

    1999-07-01

    In this paper the authors propose and analyze a new parallel SOR method, the PSOR method, formulated by using domain partitioning and interprocessor data communication techniques. They prove that the PSOR method has the same asymptotic rate of convergence as the Red/Black (R/B) SOR method for the five-point stencil on both strip and block partitions, and as the four-color (R/B/G/O) SOR method for the nine-point stencil on strip partitions. They also demonstrate the parallel performance of the PSOR method on four different MIMD multiprocessors (a KSR1, an Intel Delta, a Paragon, and an IBM SP2). Finally, they compare the parallel performance of PSOR, R/B SOR, and R/B/G/O SOR. Numerical results on the Paragon indicate that PSOR is more efficient than R/B SOR and R/B/G/O SOR in both computation and interprocessor data communication.

  8. Parallel algorithms for dynamically partitioning unstructured grids

    SciTech Connect

    Diniz, P.; Plimpton, S.; Hendrickson, B.; Leland, R.

    1994-10-01

    Grid partitioning is the method of choice for decomposing a wide variety of computational problems into naturally parallel pieces. In problems where computational load on the grid or the grid itself changes as the simulation progresses, the ability to repartition dynamically and in parallel is attractive for achieving higher performance. We describe three algorithms suitable for parallel dynamic load-balancing which attempt to partition unstructured grids so that computational load is balanced and communication is minimized. The execution time of algorithms and the quality of the partitions they generate are compared to results from serial partitioners for two large grids. The integration of the algorithms into a parallel particle simulation is also briefly discussed.

  9. Topological analysis of plasmid DNA replication intermediates using two-dimensional agarose gels.

    PubMed

    Hyrien, Olivier

    2009-01-01

    A fundamental process in DNA replication is the disentangling of the two parental strands by DNA topoisomerases. In this chapter, I detail the topological analysis of plasmid replication intermediates using two-dimensional (2D) agarose gels. The method can resolve replication intermediates according to mass and topology, and can resolve unlinked monomeric circles from catenated dimers of varying topology. The method has been used, alone or in combination with a procedure for purifying covalent protein-DNA complexes, to analyse the effect oftopoisomerase inhibitors on the topology of replication intermediates, to map the location of drug-stabilized topoisomerase cleavage complexes with respect to replication forks and to detect the breakage and repair of replication forks following collision with cleavage complexes. Other applications include the detection of knots that form independently of, or concomitantly with, DNA replication.

  10. Complete sequence of a F2:A-:B- plasmid pHN3A11 carrying rmtB and qepA, and its dissemination in China.

    PubMed

    Chen, Xiaojie; He, Liangying; Li, Yugu; Zeng, Zhenling; Deng, Yuting; Liu, Yahong; Liu, Jian-Hua

    2014-11-01

    Previous studies have confirmed that the spread of rmtB and qepA was mainly mediated by similar F2:A-:B- plasmids. In this study, a representative rmtB and qepA-harbouring F2:A-:B- plasmid, pHN3A11, originating from an Escherichia coli strain of feline origin, was fully sequenced and compared with other IncFII plasmids. pHN3A11 is 76,626bp long with a backbone similar to that of the IncFII plasmids obtained from China (pHK23a, pFOS-HK151325, pXZ) and Canada (pC15-1a). It contains genes encoding addiction (pemI/pemK, hok/mok/sok) and partitioning (parM, parB, and stbB) systems that promote plasmid maintenance during vertical transmission. rmtB, qepA, blaTEM-1, and dfr were found in previously observed contexts, interspersed with different complete or truncated insertion sequences and transposons (ΔIS1, ΔTn2, ΔintI1, ISCR3, 3 IS26, Tn21). Further analyses confirmed that pHN3A11-like plasmids have disseminated in E. coli isolates from pets, food animals and farm environments in China. The successful dissemination of F2:A-:B- type multidrug resistant plasmid among animals may represent a public health risk, and may further worsen the clinical impact. PMID:25236985

  11. Isolation and characterization of linear deoxyribonucleic acid plasmids from Kluyveromyces lactis and the plasmid-associated killer character.

    PubMed Central

    Gunge, N; Tamaru, A; Ozawa, F; Sakaguchi, K

    1981-01-01

    Two linear deoxyribonucleic acid plasmids, designated pGK11 and pGK12, were isolated from the yeast Kluyveromyces lactis IFO 1267. pGK11 and pGK12 had molecular weights of 5.4 X 10(6) and 8.4 X 10(6), respectively. Both plasmids possessed the same density of 1.687 g/cm3, lighter than the densities of mitochondrial (1.692 g/cm3) and nuclear (1.699 g/cm3) deoxyribonucleic acids. A restriction map of pGK11 was constructed from digestions by EcoRI, HindIII, PstI, and BamHI. pGK12 was cleaved by EcoRI into seven fragments and by BamHI into two fragments K. lactis IFO 1267 killed Saccharomyces cerevisiae sensitive and killer strains and certain strains of Saccharomyces italicus, K. lactis, Kluyveromyces thermotolerans, and K. vanudenii. All K. lactis strains lacking the pGK1 plasmids were nonkillers. A hybrid was constructed between K. lactis IFO 1267 and a nonkiller K. lactis strain lacking the plasmids and subjected to tetrad analysis after sporulation. The killer character was extrachromosomally transmitted in all tetrads in association with the pGK1 plasmids. The double-stranded ribonucleic acid killer plasmid could not be detected in any K. lactis killer strains. It is thus highly probable that the killer character is mediated by the linear deoxyribonucleic acid plasmids. A single chromosomal gene was found which was responsible for the resistance to the K. lactis killer. Images PMID:6257636

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. Genetic transformation of Rhodopseudomonas sphaeroides by plasmid DNA.

    PubMed Central

    Fornari, C S; Kaplan, S

    1982-01-01

    A broad-host-range cloning vector, pUI81, was constructed in vitro from plasmids RSF1010 and pSL25 (a pBR322 derivative) and used to assay for transformation in Rhodopseudomonas sphaeroides. Washing cells with 500 mM Tris was an effective means of inducing competence for DNA uptake. Transformation frequencies as high as 10(-5) (transformants per viable cell) have been achieved by incubating Tris-treated cells with plasmid DNA, 100 mM CaCl2, and 20% polyethylene glycol 6000. Maximum frequencies were obtained when recipient cells were spread onto selective media after a 6.5-h outgrowth period in antibiotic-free medium. The structure (open circular versus closed, covalent circular), size, and concentration of plasmid DNA all significantly affected the transformation frequency. Four different plasmids, all small and suitable as cloning vectors, have been introduced by transformation into several different R. sphaeroides strains. Recombinant DNA carried on small, nonconjugative plasmids with broad host ranges can now be directly transferred to R. sphaeroides by this method. Images PMID:6981642

  20. Partition coefficients for calcic plagioclase - Implications for Archean anorthosites

    NASA Technical Reports Server (NTRS)

    Phinney, W. C.; Morrison, D. A.

    1990-01-01

    In most Archean cratons, cumulates of equant plagioclase megacrysts form anorthositic complexes, including those at Bad Vermilion Lake (Ontario). In this paper, partition coefficients (Ds) of REEs between natural high-Ca plagioclase megacrysts and their basaltic matrices were determined, using a multiple aliquot techique, and megacrystic plagioclases occurring in anorthosites were analyzed for the same components which, in conjunction with their Ds, were applied to calculations of melts in equilibrium with anorthosites. The REE's Ds were found to agree well with experimentally determined values and to predict equilibrium melts for Archean anorthosites that agree well with coeval basaltic flows and dikes. The Ds also appear to be valid for both the tholeiitic and alkali basalts over a wide range of mg numbers and REE concentrations. It is suggested that the moderately Fe-rich tholeiites that are hosts to plagioclase megacrysts in greenstone belts form the parental melts for megacrysts which make up the Bad Vermilion Lake Archean anorthositic complex.

  1. Chiral partition functions of quantum Hall droplets

    SciTech Connect

    Cappelli, Andrea Viola, Giovanni; Zemba, Guillermo R.

    2010-02-15

    Chiral partition functions of conformal field theory describe the edge excitations of isolated Hall droplets. They are characterized by an index specifying the quasiparticle sector and transform among themselves by a finite-dimensional representation of the modular group. The partition functions are derived and used to describe electron transitions leading to Coulomb blockade conductance peaks. We find the peak patterns for Abelian hierarchical states and non-Abelian Read-Rezayi states, and compare them. Experimental observation of these features can check the qualitative properties of the conformal field theory description, such as the decomposition of the Hilbert space into sectors, involving charged and neutral parts, and the fusion rules.

  2. Partitioning SAT Instances for Distributed Solving

    NASA Astrophysics Data System (ADS)

    Hyvärinen, Antti E. J.; Junttila, Tommi; Niemelä, Ilkka

    In this paper we study the problem of solving hard propositional satisfiability problem (SAT) instances in a computing grid or cloud, where run times and communication between parallel running computations are limited.We study analytically an approach where the instance is partitioned iteratively into a tree of subproblems and each node in the tree is solved in parallel.We present new methods for constructing partitions which combine clause learning and lookahead. The methods are incorporated into the iterative approach and its performance is demonstrated with an extensive comparison against the best sequential solvers in the SAT competition 2009 as well as against two efficient parallel solvers.

  3. DNA sequence and genetic characterization of plasmid pFQ11 from Frankia alni strain CpI1.

    PubMed

    Xu, Xudong; Kong, Renqiu; de Bruijn, Frans J; He, Sheng Yang; Murry, Marcia A; Newman, Thomas; Wolk, C Peter

    2002-01-22

    An 8551-bp plasmid, pFQ11, from Frankia alni strain CpI1 was sequenced. Its sequence was found to be very similar to that presented for pFQ31 from strain ArI3. Six potential protein-encoding open reading frames (ORFs) were identified, and transcriptional activity was shown within four of those regions of the plasmid by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. An earlier study reported that ORF E(F) of pFQ31, which is nearly identical to the 3' 45% of ORF1 of pFQ11, is significantly similar to RepF. We found no such similarity. ORF2 and ORF3 predict products that are similar to a repressor protein and a partition protein, respectively. We found inverted repeats within and covering the start codon of ORF3; palindromic sequences and direct repeats between ORF3 and ORF4; and 3' from ORF3, an AT-rich sequence that extensively overlaps the promoter region of a uvrB homolog in strain ArI3. PMID:11886759

  4. Experimental geochemistry of Pu and Sm and the thermodynamics of trace element partitioning

    NASA Technical Reports Server (NTRS)

    Jones, John H.; Burnett, Donald S.

    1987-01-01

    An experimental study of the partitioning of Pu and Sm between diopside/liquid and whitlockite/liquid supports the hypothesis that Pu behaves as a light rare earth element during igneous processes in reducing environments. D-Pu/D-Sm is found to be about 2 for both diopsidic pyroxene and whitlockite, and the amount of fractionation would be decreased further if Pu were compared to Ce or Nd. Data indicate that temperature, rather than melt composition, is the most important control on elemental partitioning, and that P2O5 in aluminosilicate melts serves as a complexing agent for the actinides and lanthanides.

  5. The effect of sulphur in silicate melt on partitioning of Ni and other trace elements

    NASA Astrophysics Data System (ADS)

    Wood, Bernard; Kiseeva, Ekaterina; Wohlers, Anke

    2016-04-01

    It has been suggested that variations in the sulphur contents of silicate melts affect the partitioning of trace chalcophile elements, particularly Ni, between silicate melt and crystalline phases such as olivine [1]. The general idea is that Ni (and other elements) complex with sulphur dissolved in the melt, thereby stabilising Ni in the melt and reducing the olivine-melt partition coefficient DNi. More recent experiments lead to the assertion that any sulphur effect, if present is small and can be ignored [2]. Experiments aimed at addressing this problem have, however, struggled with the difficulty that the maximum S contents of olivine- precipitating melts do not exceed ~0.5% even at sulphide saturation. Any effect is therefore difficult to establish unequivocally. Here we have taken advantage of the fact that experiments under strongly reducing conditions, where FeO activity in the silicate melt is very low lead to much higher concentrations of S than those associated with olivine precipitation. We have therefore investigated partitioning between sulphide melts and haplobasaltic silicate melt at concentrations of FeO between 0.3 and 10 weight% in order to investigate the "sulphur-effect" on partitioning. At the lowest FeO contents we are able to drive the S content of the melt to 10 weight% enabling the effects to be unequivocally established. We find that partitioning of strongly lithophile elements Nb, Ta, U, REE partition more strongly out of silicate melt as its S content increases. The effect is, surprisingly, predominantly due to the effect of S on the activity coefficient of FeO in the melt. In contrast strongly chalcophile Ni, Cu, Ag partition more strongly into the melt as its S content increases. This is due to a dramatic lowering of the activity coefficients of these elements in the silicate as S increases. Elements which show little effect of S include Pb, Co and In. The results enable us to predict the effects of sulphur on olivine-melt and

  6. Modern and simple construction of plasmid: saving time and cost.

    PubMed

    Nakayama, Hideki; Shimamoto, Nobuo

    2014-11-01

    Construction of plasmids has been occupying a significant fraction of laboratory work in most fields of experimental biology. Tremendous effort was made to improve the traditional method for constructing plasmids, in which DNA fragments digested with restriction enzymes were ligated. However, the traditional method remained to be a standard protocol more than 40 years. At last, several recent inventions are rapidly and completely replacing the traditional method, because they are far quicker with less cost, and requiring less material. We here introduce three such methods that cover up most of the cases. Moreover, they are complementary with each other. Our lab protocols are provided for "no strain, no pain" construction of plasmids. PMID:25359266

  7. Association between specific plasmids and relapse in typhoid fever.

    PubMed Central

    Gotuzzo, E; Morris, J G; Benavente, L; Wood, P K; Levine, O; Black, R E; Levine, M M

    1987-01-01

    We studied isolates from 73 patients hospitalized with typhoid fever in Lima, Peru. Of these 73 patients, 11 (15%) suffered a clinical relapse, with fever and positive blood cultures, within 3 months of their original illness. Using plasmids as epidemiologic markers, we found that three patients who subsequently relapsed were initially infected with more than one strain of Salmonella typhi. There was a highly significant association between relapse and isolation of a strain containing either a 24- or a 38-kilobase plasmid at the time of the original infection; however, we were unable to show any evidence of homology between these two plasmids. Our data indicate that infection with multiple strains is not uncommon in this endemic area and suggest that relapse may be partly strain dependent. Images PMID:2821064

  8. 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

  9. 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.

  10. Partition function zeros and finite size scaling for polymer adsorption

    SciTech Connect

    Taylor, Mark P.; Luettmer-Strathmann, Jutta

    2014-11-28

    The zeros of the canonical partition functions for a flexible polymer chain tethered to an attractive flat surface are computed for chains up to length N = 1536. We use a bond-fluctuation model for the polymer and obtain the density of states for the tethered chain by Wang-Landau sampling. The partition function zeros in the complex e{sup β}-plane are symmetric about the real axis and densest in a boundary region that has the shape of a nearly closed circle, centered at the origin, terminated by two flaring tails. This structure defines a root-free zone about the positive real axis and follows Yang-Lee theory. As the chain length increases, the base of each tail moves toward the real axis, converging on the phase-transition point in the thermodynamic limit. We apply finite-size scaling theory of partition-function zeros and show that the crossover exponent defined through the leading zero is identical to the standard polymer adsorption crossover exponent ϕ. Scaling analysis of the leading zeros locates the polymer adsorption transition in the thermodynamic (N → ∞) limit at reduced temperature T{sub c}{sup *}=1.027(3) [β{sub c}=1/T{sub c}{sup *}=0.974(3)] with crossover exponent ϕ = 0.515(25). Critical exponents for the order parameter and specific heat are determined to be β{sup ~}=0.97(5) and α = 0.03(4), respectively. A universal scaling function for the average number of surface contacts is also constructed.

  11. Partition function zeros and finite size scaling for polymer adsorption

    NASA Astrophysics Data System (ADS)

    Taylor, Mark P.; Luettmer-Strathmann, Jutta

    2014-11-01

    The zeros of the canonical partition functions for a flexible polymer chain tethered to an attractive flat surface are computed for chains up to length N = 1536. We use a bond-fluctuation model for the polymer and obtain the density of states for the tethered chain by Wang-Landau sampling. The partition function zeros in the complex eβ-plane are symmetric about the real axis and densest in a boundary region that has the shape of a nearly closed circle, centered at the origin, terminated by two flaring tails. This structure defines a root-free zone about the positive real axis and follows Yang-Lee theory. As the chain length increases, the base of each tail moves toward the real axis, converging on the phase-transition point in the thermodynamic limit. We apply finite-size scaling theory of partition-function zeros and show that the crossover exponent defined through the leading zero is identical to the standard polymer adsorption crossover exponent ϕ. Scaling analysis of the leading zeros locates the polymer adsorption transition in the thermodynamic (N → ∞) limit at reduced temperature T_c^*=1.027(3) [β _c=1/T_c^*=0.974(3)] with crossover exponent ϕ = 0.515(25). Critical exponents for the order parameter and specific heat are determined to be widetilde{β }=0.97(5) and α = 0.03(4), respectively. A universal scaling function for the average number of surface contacts is also constructed.

  12. Robust and efficient overset grid assembly for partitioned unstructured meshes

    SciTech Connect

    Roget, Beatrice Sitaraman, Jayanarayanan

    2014-03-01

    This paper presents a method to perform efficient and automated Overset Grid Assembly (OGA) on a system of overlapping unstructured meshes in a parallel computing environment where all meshes are partitioned into multiple mesh-blocks and processed on multiple cores. The main task of the overset grid assembler is to identify, in parallel, among all points in the overlapping mesh system, at which points the flow solution should be computed (field points), interpolated (receptor points), or ignored (hole points). Point containment search or donor search, an algorithm to efficiently determine the cell that contains a given point, is the core procedure necessary for accomplishing this task. Donor search is particularly challenging for partitioned unstructured meshes because of the complex irregular boundaries that are often created during partitioning. Another challenge arises because of the large variation in the type of mesh-block overlap and the resulting large load imbalance on multiple processors. Desirable traits for the grid assembly method are efficiency (requiring only a small fraction of the solver time), robustness (correct identification of all point types), and full automation (no user input required other than the mesh system). Additionally, the method should be scalable, which is an important challenge due to the inherent load imbalance. This paper describes a fully-automated grid assembly method, which can use two different donor search algorithms. One is based on the use of auxiliary grids and Exact Inverse Maps (EIM), and the other is based on the use of Alternating Digital Trees (ADT). The EIM method is demonstrated to be more efficient than the ADT method, while retaining robustness. An adaptive load re-balance algorithm is also designed and implemented, which considerably improves the scalability of the method.

  13. Conjugative plasmid transfer in gram-positive bacteria.

    PubMed

    Grohmann, Elisabeth; Muth, Günther; Espinosa, Manuel

    2003-06-01

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

  14. 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.

  15. 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

  16. 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.

  17. Characterization of the replicon from plasmid pAC1 from Acetobacter pasteurianus.

    PubMed

    Grones, J; Králová, A; Turna, J

    1993-02-26

    A panel of recombinant plasmids pACK5 and pACT7 was prepared by introducing kanamycin and tetracycline resistance into the partially split plasmid pAC1 which contained replicon isolated from Acetobacter pasteurianus. The replicon in plasmid pAC1 is compatible with the ColE1 replicon. Compared to pBR322, the plasmid had more than 30 copies per chromosome in Escherichia coli cells. Plasmids were transformed into E. coli DH1, Acetobacter pasteurianus 3614, Acetobacter aceti 3620, Shigella, Citrobacter, and Brevibacterium flavum cells, and the stability of plasmid DNA was tested after cultivation in nonselective conditions.

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

    PubMed

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

    2016-02-01

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

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

    SciTech Connect

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

    1990-05-01

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

  20. Construction of a bioluminescence reporter plasmid for Francisella tularensis.

    PubMed

    Bina, Xiaowen R; Miller, Mark A; Bina, James E

    2010-11-01

    A Francisella tularensis shuttle vector that constitutively expresses the Photorhabdus luminescens lux operon in type A and type B strains of F. tularensis was constructed. The bioluminescence reporter plasmid was introduced into the live vaccine strain of F. tularensis and used to follow F. tularensis growth in a murine intranasal challenge model in real-time by bioluminescence imaging. The results show that the new bioluminescence reporter plasmid represents a useful tool for tularemia research that is suitable for following F. tularensis growth in both in vitro and in vivo model systems. PMID:20620161

  1. First experimentally determined thermodynamic values of francium: hydration energy, energy of partitioning, and thermodynamic radius.

    PubMed

    Delmau, Lætitia H; Moine, Jérôme; Mirzadeh, Saed; Moyer, Bruce A

    2013-08-01

    The Gibbs energy of partitioning of Fr(+) ion between water and nitrobenzene has been determined to be 14.5 ± 0.6 kJ/mol at 25 °C, the first ever Gibbs energy of partitioning for francium in particular and the first ever solution thermodynamic quantity for francium in general. This value enabled the ionic radius and standard Gibbs energy of hydration for Fr(+) to be estimated as 173 pm and -251 kJ/mol, respectively, the former value being significantly smaller than previously thought. A new experimental method was established using a cesium dicarbollide as a cation-exchange agent, overcoming problems inherent to the trace-level concentrations of francium. The methodology opens the door to the study of the partitioning behavior of francium to other water-immiscible solvents and the determination of complexation constants for francium binding by receptor molecules. PMID:23848436

  2. Strainrange partitioning behavior of the nickel-base superalloys, Rene' 80 and in 100

    NASA Technical Reports Server (NTRS)

    Halford, G. R.; Nachtigall, A. J.

    1978-01-01

    A study was made to assess the ability of the method of Strainrange Partitioning (SRP) to both correlate and predict high-temperature, low cycle fatigue lives of nickel base superalloys for gas turbine applications. The partitioned strainrange versus life relationships for uncoated Rene' 80 and cast IN 100 were also determined from the ductility normalized-Strainrange Partitioning equations. These were used to predict the cyclic lives of the baseline tests. The life predictability of the method was verified for cast IN 100 by applying the baseline results to the cyclic life prediction of a series of complex strain cycling tests with multiple hold periods at constant strain. It was concluded that the method of SRP can correlate and predict the cyclic lives of laboratory specimens of the nickel base superalloys evaluated in this program.

  3. Partition, sorption and structure activity relation study of dialkoxybenzenes that modulate insect behavior.

    PubMed

    Ebrahimi, Parisa; Spooner, Jacob; Weinberg, Noham; Plettner, Erika

    2013-09-01

    Some dialkoxybenzenes are promising new insect control agents. These compounds mimic naturally occurring odorants that modulate insect behavior. Before applying these compounds, however, their persistence and biodegradability at the application site and in the environment should be understood. The fate of organic compounds in the environment is a complex phenomenon which is influenced by many processes such as sorption to soil components, sedimentation, volatilization, and uptake by plants, as well as biotic and abiotic chemical degradation. In this study, the octanol-water partition coefficient, volatility and sorption on soil components (sand, clay and organic matter) of selected dialkoxybenzenes as well as structure activity relationships with regard to partition, volatility and sorption were investigated. Additionally, calculations of partition, molar volume and molecular surface areas were done, to understand structure-activity relationships of the physical properties.

  4. UNCERTAINTY IN SOURCE PARTITIONING USING STABLE ISOTOPES

    EPA Science Inventory

    Stable isotope analyses are often used to quantify the contribution of multiple sources to a mixture, such as proportions of food sources in an animal's diet, C3 vs. C4 plant inputs to soil organic carbon, etc. Linear mixing models can be used to partition two sources with a sin...

  5. Partitioning of penoxsulam, a new sulfonamide herbicide.

    PubMed

    Jabusch, Thomas W; Tjeerdema, Ronald S

    2005-09-01

    Penoxsulam (trade name Granite) is a new acetolactate synthase (ALS) inhibitor herbicide for postemergence control of annual grasses, sedges, and broadleaf weeds in rice culture. This study was done to understand the equilibrium phase partitioning of penoxsulam to soil and air under conditions simulating California rice field conditions. Partitioning of penoxsulam was determined between soil and water (Kd) by the batch equilibrium method and between air and water (K(H)) by the gas-purge method. In four representative soils from the Sacramento Valley, the Kd values ranged from 0.14 to 5.05 and displayed a modest increase with soil pH. In soil amended with manure compost, soil sorption increased 4-fold with increasing soil organic matter content, but was still low with a Kd of 0.4 in samples with high organic carbon contents of 15%. Penoxsulam was confirmed to be extremely nonvolatile and did not partition into air at any measurable rate at 20 or 40 degrees C. K(H) (pH 7) was estimated at 4.6 x 10(-15) Pa x L x mol(-1) on the basis of available water solubility and vapor pressure data. The results imply that soil and air partitioning of penoxsulam do not significantly affect its potential for degradation or offsite movement in water.

  6. Hydrologic transport and partitioning of phosphorus fractions

    NASA Astrophysics Data System (ADS)

    Berretta, C.; Sansalone, J.

    2011-06-01

    SummaryPhosphorus (P) in rainfall-runoff partitions between dissolved and particulate matter (PM) bound phases. This study investigates the transport and partitioning of P to PM fractions in runoff from a landscaped and biogenically-loaded carpark in Gainesville, FL (GNV). Additionally, partitioning and concentration results are compared to a similarly-sized concrete-paved source area of a similar rainfall depth frequency distribution in Baton Rouge, LA (BTR), where in contrast vehicular traffic represents the main source of pollutants. Results illustrate that concentrations of P fractions (dissolved, suspended, settleable and sediment) for GNV are one to two orders of magnitude higher than BTR. Despite these differences the dissolved fraction ( f d) and partitioning coefficient ( K d) distributions are similar, illustrating that P is predominantly bound to PM fractions. Examining PM size fractions, specific capacity for P (PSC) indicates that the P concentration order is suspended > settleable > sediment for GNV, similarly to BTR. For GNV the dominant PM mass fraction is sediment (>75 μm), while the mass of P is distributed predominantly between sediment and suspended (<25 μm) fractions since these PM mass fractions dominated the settleable one. With respect to transport of PM and P fractions the predominance of events for both areas is mass-limited first-flush, although each fraction illustrated unique washoff parameters. However, while transport is predominantly mass-limited, the transport of each PM and P fraction is influenced by separate hydrologic parameters.

  7. hydrogen partitioning between postperovskite and bridgmanite

    NASA Astrophysics Data System (ADS)

    Townsend, J. P.; Jacobsen, S. D.; Bina, C. R.; Tsuchiya, J.

    2015-12-01

    We present new results from first-principles calculations of phonon spectra of lower mantle phases of MgSiO3 bridgmanite (brg) and postperovskite (ppv) including hydrous defects, and alumino-hydrous defects. We compute the partition coefficient of hydrogen between ppv and brg for hydrous and alumino-hydrous compositions at D" pressures and temperatures from first-principles lattice dynamics simulations and free energy calculations computed under the quasiharmonic approximation. We find that for aluminum free hydrous conditions the hydrogen partition coefficient between ppv and brg ranges from 0.2-0.8 within D". However, in the presence of aluminum the aluminum-hydrogen partition coefficient between ppv and brg is approximately 1.5. In general for a given pressure, lower temperature increases the partitioning of hydrogen into ppv for the aluminous models, but not for the aluminum free models. Because aluminum is is expected to occur in both natural slab and mantle compositions this suggests aluminous-hydrous ppv may be a host for water in D".

  8. Partitioning of selected antioxidants in mayonnaise.

    PubMed

    Jacobsen, C; Schwarz, K; Stöckmann, H; Meyer, A S; Adler-Nissen, J

    1999-09-01

    This study examined partitioning of alpha-, beta-, and gamma-tocopherol and six polar antioxidants (Trolox, ferulic acid, caffeic acid, propyl gallate, gallic acid, and catechin) in mayonnaise. Partitioning of antioxidants between different phases was determined after separation of mayonnaise by either (a) centrifugation + ultracentrifugation or (b) centrifugation + dialysis. Antioxidants partitioned in accordance with their chemical structure and polarity: Tocopherols were concentrated in the oil phase (93-96%), while the proportion of polar antioxidants in the oil phase ranged from 0% (gallic acid and catechin) to 83% (Trolox). Accordingly, proportions of 6% (Trolox) to 80% (gallic acid and catechin) were found in the aqueous phase. Similar trends were observed after dialysis. After ultracentrifugation, large proportions of polar antioxidants were found in the "emulsion phase" and the "precipitate" (7-34% and 2-7%, respectively). This indicated entrapment of antioxidants at the oil-water interface in mayonnaise. The results signify that antioxidants partitioning into different phases of real food emulsions may vary widely.

  9. Set Partitions and the Multiplication Principle

    ERIC Educational Resources Information Center

    Lockwood, Elise; Caughman, John S., IV

    2016-01-01

    To further understand student thinking in the context of combinatorial enumeration, we examine student work on a problem involving set partitions. In this context, we note some key features of the multiplication principle that were often not attended to by students. We also share a productive way of thinking that emerged for several students who…

  10. Measure-theoretic sensitivity via finite partitions

    NASA Astrophysics Data System (ADS)

    Li, Jian

    2016-07-01

    For every positive integer n≥slant 2 , we introduce the concept of measure-theoretic n-sensitivity for measure-theoretic dynamical systems via finite measurable partitions, and show that an ergodic system is measure-theoretically n-sensitive but not (n  +  1)-sensitive if and only if its maximal pattern entropy is log n .

  11. Application of partition technology to particle electrophoresis

    NASA Technical Reports Server (NTRS)

    Van Alstine, James M.; Harris, J. Milton; Karr, Laurel J.; Bamberger, Stephan; Matsos, Helen C.; Snyder, Robert S.

    1989-01-01

    The effects of polymer-ligand concentration on particle electrophoretic mobility and partition in aqueous polymer two-phase systems are investigated. Polymer coating chemistry and affinity ligand synthesis, purification, and analysis are conducted. It is observed that poly (ethylene glycol)-ligands are effective for controlling particle electrophoretic mobility.

  12. 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

  13. A physicochemical approach for predicting the effectiveness of peptide-based gene delivery systems for use in plasmid-based gene therapy.

    PubMed Central

    Duguid, J G; Li, C; Shi, M; Logan, M J; Alila, H; Rolland, A; Tomlinson, E; Sparrow, J T; Smith, L C

    1998-01-01

    Novel synthetic peptides, based on carrier peptide analogs (YKAKnWK) and an amphipathic peptide (GLFEALLELLESLWELLLEA), have been formulated with DNA plasmids to create peptide-based gene delivery systems. The carrier peptides are used to condense plasmids into nanoparticles with a hydrodynamic diameter (DH) ranging from 40 to 200 nm, which are sterically stable for over 100 h. Size and morphology of the carrier peptide/plasmid complex have been determined by photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM), respectively. The amphipathic peptide is used as a pH-sensitive lytic agent to facilitate release of the plasmid from endosomes after endocytosis of the peptide/plasmid complex. Hemolysis assays have shown that the amphipathic peptide destabilizes lipid bilayers at low pH, mimicking the properties of viral fusogenic peptides. However, circular dichroism studies show that unlike the viral fusion peptides, this amphipathic peptide loses some of its alpha-helical structure at low pH in the presence of liposomes. The peptide-based gene delivery systems were tested for transfection efficiency in a variety of cell lines, including 14-day C2C12 mouse myotubes, using gene expression systems containing the beta-galactosidase reporter gene. Transfection data demonstrate a correlation between in vitro transfection efficiency and the combination of several physical properties of the peptide/plasmid complexes, including 1) DNA dose, 2) the zeta potential of the particle, 3) the requirement of both lytic and carrier peptides, and 4) the number of lysine residues associated with the carrier peptide. Transfection data on 14-day C2C12 myotubes utilizing the therapeutic human growth hormone gene formulated in an optimal peptide gene delivery system show an increase in gene expression over time, with a maximum in protein levels at 96 h (approximately 18 ng/ml). PMID:9635734

  14. Density-based partitioning methods for ground-state molecular calculations.

    PubMed

    Nafziger, Jonathan; Wasserman, Adam

    2014-09-11

    With the growing complexity of systems that can be treated with modern electronic-structure methods, it is critical to develop accurate and efficient strategies to partition the systems into smaller, more tractable fragments. We review some of the various recent formalisms that have been proposed to achieve this goal using fragment (ground-state) electron densities as the main variables, with an emphasis on partition density-functional theory (PDFT), which the authors have been developing. To expose the subtle but important differences between alternative approaches and to highlight the challenges involved with density partitioning, we focus on the simplest possible systems where the various methods can be transparently compared. We provide benchmark PDFT calculations on homonuclear diatomic molecules and analyze the associated partition potentials. We derive a new exact condition determining the strength of the singularities of the partition potentials at the nuclei, establish the connection between charge-transfer and electronegativity equalization between fragments, test different ways of dealing with fractional fragment charges and spins, and finally outline a general strategy for overcoming delocalization and static-correlation errors in density-functional calculations.

  15. Partitioning, diffusion, and ligand binding of raft lipid analogs in model and cellular plasma membranes.

    PubMed

    Sezgin, Erdinc; Levental, Ilya; Grzybek, Michal; Schwarzmann, Günter; Mueller, Veronika; Honigmann, Alf; Belov, Vladimir N; Eggeling, Christian; Coskun, Unal; Simons, Kai; Schwille, Petra

    2012-07-01

    Several simplified membrane models featuring coexisting liquid disordered (Ld) and ordered (Lo) lipid phases have been developed to mimic the heterogeneous organization of cellular membranes, and thus, aid our understanding of the nature and functional role of ordered lipid-protein nanodomains, termed "rafts". In spite of their greatly reduced complexity, quantitative characterization of local lipid environments using model membranes is not trivial, and the parallels that can be drawn to cellular membranes are not always evident. Similarly, various fluorescently labeled lipid analogs have been used to study membrane organization and function in vitro, although the biological activity of these probes in relation to their native counterparts often remains uncharacterized. This is particularly true for raft-preferring lipids ("raft lipids", e.g. sphingolipids and sterols), whose domain preference is a strict function of their molecular architecture, and is thus susceptible to disruption by fluorescence labeling. Here, we analyze the phase partitioning of a multitude of fluorescent raft lipid analogs in synthetic Giant Unilamellar Vesicles (GUVs) and cell-derived Giant Plasma Membrane Vesicles (GPMVs). We observe complex partitioning behavior dependent on label size, polarity, charge and position, lipid headgroup, and membrane composition. Several of the raft lipid analogs partitioned into the ordered phase in GPMVs, in contrast to fully synthetic GUVs, in which most raft lipid analogs mis-partitioned to the disordered phase. This behavior correlates with the greatly enhanced order difference between coexisting phases in the synthetic system. In addition, not only partitioning, but also ligand binding of the lipids is perturbed upon labeling: while cholera toxin B binds unlabeled GM1 in the Lo phase, it binds fluorescently labeled GMI exclusively in the Ld phase. Fluorescence correlation spectroscopy (FCS) by stimulated emission depletion (STED) nanoscopy on intact

  16. Amelioration of the cost of conjugative plasmid carriage in Eschericha coli K12.

    PubMed Central

    Dahlberg, Cecilia; Chao, Lin

    2003-01-01

    Although plasmids can provide beneficial functions to their host bacteria, they might confer a physiological or energetic cost. This study examines how natural selection may reduce the cost of carrying conjugative plasmids with drug-resistance markers in the absence of antibiotic selection. We studied two plasmids, R1 and RP4, both of which carry multiple drug resistance genes and were shown to impose an initial fitness cost on Escherichia coli. To determine if and how the cost could be reduced, we subjected plasmid-containing bacteria to 1100 generations of evolution in batch cultures. Analysis of the evolved populations revealed that plasmid loss never occurred, but that the cost was reduced through genetic changes in both the plasmids and the bacteria. Changes in the plasmids were inferred by the demonstration that evolved plasmids no longer imposed a cost on their hosts when transferred to a plasmid-free clone of the ancestral E. coli. Changes in the bacteria were shown by the lowered cost when the ancestral plasmids were introduced into evolved bacteria that had been cured of their (evolved) plasmids. Additionally, changes in the bacteria were inferred because conjugative transfer rates of evolved R1 plasmids were lower in the evolved host than in the ancestral host. Our results suggest that once a conjugative bacterial plasmid has invaded a bacterial population it will remain even if the original selection is discontinued. PMID:14704155

  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.

  18. Open software tools for eddy covariance flux partitioning

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Agro-ecosystem management and assessment will benefit greatly from the development of reliable techniques for partitioning evapotranspiration (ET) into evaporation (E) and transpiration (T). Among other activities, flux partitioning can aid in evaluating consumptive vs. non-consumptive agricultural...

  19. Dynamics of Quantum Adiabatic Evolution Algorithm for Number Partitioning

    NASA Technical Reports Server (NTRS)

    Smelyanskiy, Vadius; vonToussaint, Udo V.; Timucin, Dogan A.; Clancy, Daniel (Technical Monitor)

    2002-01-01

    We have developed a general technique to study the dynamics of the quantum adiabatic evolution algorithm applied to random combinatorial optimization problems in the asymptotic limit of large problem size n. We use as an example the NP-complete Number Partitioning problem and map the algorithm dynamics to that of an auxiliary quantum spin glass system with the slowly varying Hamiltonian. We use a Green function method to obtain the adiabatic eigenstates and the minimum exitation gap, gmin = O(n2(sup -n/2)), corresponding to the exponential complexity of the algorithm for Number Partitioning. The key element of the analysis is the conditional energy distribution computed for the set of all spin configurations generated from a given (ancestor) configuration by simultaneous flipping of a fixed number of spins. For the problem in question this distribution is shown to depend on the ancestor spin configuration only via a certain parameter related to the energy of the configuration. As the result, the algorithm dynamics can be described in terms of one-dimensional quantum diffusion in the energy space. This effect provides a general limitation of a quantum adiabatic computation in random optimization problems. Analytical results are in agreement with the numerical simulation of the algorithm.

  20. Dynamics of Quantum Adiabatic Evolution Algorithm for Number Partitioning

    NASA Technical Reports Server (NTRS)

    Smelyanskiy, V. N.; Toussaint, U. V.; Timucin, D. A.

    2002-01-01

    We have developed a general technique to study the dynamics of the quantum adiabatic evolution algorithm applied to random combinatorial optimization problems in the asymptotic limit of large problem size n. We use as an example the NP-complete Number Partitioning problem and map the algorithm dynamics to that of an auxiliary quantum spin glass system with the slowly varying Hamiltonian. We use a Green function method to obtain the adiabatic eigenstates and the minimum excitation gap. g min, = O(n 2(exp -n/2), corresponding to the exponential complexity of the algorithm for Number Partitioning. The key element of the analysis is the conditional energy distribution computed for the set of all spin configurations generated from a given (ancestor) configuration by simultaneous flipping of a fixed number of spins. For the problem in question this distribution is shown to depend on the ancestor spin configuration only via a certain parameter related to 'the energy of the configuration. As the result, the algorithm dynamics can be described in terms of one-dimensional quantum diffusion in the energy space. This effect provides a general limitation of a quantum adiabatic computation in random optimization problems. Analytical results are in agreement with the numerical simulation of the algorithm.

  1. Threshold partitioning of sparse matrices and applications to Markov chains

    SciTech Connect

    Choi, Hwajeong; Szyld, D.B.

    1996-12-31

    It is well known that the order of the variables and equations of a large, sparse linear system influences the performance of classical iterative methods. In particular if, after a symmetric permutation, the blocks in the diagonal have more nonzeros, classical block methods have a faster asymptotic rate of convergence. In this paper, different ordering and partitioning algorithms for sparse matrices are presented. They are modifications of PABLO. In the new algorithms, in addition to the location of the nonzeros, the values of the entries are taken into account. The matrix resulting after the symmetric permutation has dense blocks along the diagonal, and small entries in the off-diagonal blocks. Parameters can be easily adjusted to obtain, for example, denser blocks, or blocks with elements of larger magnitude. In particular, when the matrices represent Markov chains, the permuted matrices are well suited for block iterative methods that find the corresponding probability distribution. Applications to three types of methods are explored: (1) Classical block methods, such as Block Gauss Seidel. (2) Preconditioned GMRES, where a block diagonal preconditioner is used. (3) Iterative aggregation method (also called aggregation/disaggregation) where the partition obtained from the ordering algorithm with certain parameters is used as an aggregation scheme. In all three cases, experiments are presented which illustrate the performance of the methods with the new orderings. The complexity of the new algorithms is linear in the number of nonzeros and the order of the matrix, and thus adding little computational effort to the overall solution.

  2. Space Partitioning for Privacy Enabled 3D City Models

    NASA Astrophysics Data System (ADS)

    Filippovska, Y.; Wichmann, A.; Kada, M.

    2016-10-01

    Due to recent technological progress, data capturing and processing of highly detailed (3D) data has become extensive. And despite all prospects of potential uses, data that includes personal living spaces and public buildings can also be considered as a serious intrusion into people's privacy and a threat to security. It becomes especially critical if data is visible by the general public. Thus, a compromise is needed between open access to data and privacy requirements which can be very different for each application. As privacy is a complex and versatile topic, the focus of this work particularly lies on the visualization of 3D urban data sets. For the purpose of privacy enabled visualizations of 3D city models, we propose to partition the (living) spaces into privacy regions, each featuring its own level of anonymity. Within each region, the depicted 2D and 3D geometry and imagery is anonymized with cartographic generalization techniques. The underlying spatial partitioning is realized as a 2D map generated as a straight skeleton of the open space between buildings. The resulting privacy cells are then merged according to the privacy requirements associated with each building to form larger regions, their borderlines smoothed, and transition zones established between privacy regions to have a harmonious visual appearance. It is exemplarily demonstrated how the proposed method generates privacy enabled 3D city models.

  3. A new plasmid-encoded proteic killer gene system: cloning, sequencing, and analyzing hig locus of plasmid Rts1.

    PubMed

    Tian, Q B; Ohnishi, M; Tabuchi, A; Terawaki, Y

    1996-03-18

    A new proteic killer gene system, hig, was identified on the plasmid Rts1. The hig locus consisting of a higA and higB is directly related to the temperature sensitive host cell growth conferred by Rts1. We proved that higB encoding presumably a 92-amino-acid polypeptide inhibited segregation of plasmid free cells, and higA encoding a 104-amino-acid polypeptide suppressed the higB function both in cis and in trans.

  4. Optimising query execution time in LHCb Bookkeeping System using partition pruning and Partition-Wise joins

    NASA Astrophysics Data System (ADS)

    Mathe, Zoltan; Charpentier, Philippe

    2014-06-01

    The LHCb experiment produces a huge amount of data which has associated metadata such as run number, data taking condition (detector status when the data was taken), simulation condition, etc. The data are stored in files, replicated on the Computing Grid around the world. The LHCb Bookkeeping System provides methods for retrieving datasets based on their metadata. The metadata is stored in a hybrid database model, which is a mixture of Relational and Hierarchical database models and is based on the Oracle Relational Database Management System (RDBMS). The database access has to be reliable and fast. In order to achieve a high timing performance, the tables are partitioned and the queries are executed in parallel. When we store large amounts of data the partition pruning is essential for database performance, because it reduces the amount of data retrieved from the disk and optimises the resource utilisation. This research presented here is focusing on the extended composite partitioning strategy such as range-hash partition, partition pruning and usage of the Partition-Wise joins. The system has to serve thousands of queries per minute, the performance and capability of the system is measured when the above performance optimization techniques are used.

  5. Cell separation by immunoaffinity partitioning with polyethylene glycol-modified Protein A in aqueous polymer two-phase systems

    NASA Technical Reports Server (NTRS)

    Karr, Laurel J.; Van Alstine, James M.; Snyder, Robert S.; Shafer, Steven G.; Harris, J. Milton

    1988-01-01

    Previous work has shown that polyethylene glycol (PEG)-bound antibodies can be used as affinity ligands in PEG-dextran two-phase systems to provide selective partitioning of cells to the PEG-rich phase. In the present work it is shown that immunoaffinity partitioning can be simplified by use of PEG-modified Protein A which complexes with unmodified antibody and cells and shifts their partitioning into the PEG-rich phase, thus eliminating the need to prepare a PEG-modified antibody for each cell type. In addition, the paper provides a more rigorous test of the original technique with PEG-bound antibodies by showing that it is effective at shifting the partitioning of either cell type of a mixture of two cell populations.

  6. 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

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

    PubMed

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

    2012-08-01

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

  8. Controlled plasmid gene transfer to murine renal carcinoma by hexadecylphosphocholine.

    PubMed

    Settelen, Nathalie; Roch, Olivier; Bock, David; Rooke, Ronald; Braun, Serge; Meyer, Olivier

    2004-01-01

    We report here that the anticancer drug hexadecylphosphocholine (HPC) can control plasmid DNA-mediated gene transfer to renal carcinoma following intratumoral administration. Significant improvement of gene expression levels could be achieved depending on HPC dose administered. Optimal concentration of HPC co-injected with plasmid DNA was found to be 0.2% (w/v) showing up to a 10-fold increase in reporter gene expression levels when compared to DNA administered alone. In vivo gene transfer activity of HPC was not affected by the nature of the diluent used, i.e. glucose-based or saline-based isotonic solutions. Although in vitro transfection activity of HPC formulations could not be evidenced, a liposome leakage assay revealed that HPC could significantly destabilize stable lipid membranes suggesting that a possible membrane permeation enhancer activity of HPC combined to the physical stress induced by the intratumor injection may facilitate plasmid DNA entry inside the cells resulting in increased gene expression. HPC/plasmid formulations represent new and attractive non-viral gene delivery systems with potential in cancer gene therapy and vaccination. PMID:14684287

  9. Tragedy of the commons among antibiotic resistance plasmids.

    PubMed

    Smith, Jeff

    2012-04-01

    As social interactions are increasingly recognized as important determinants of microbial fitness, sociobiology is being enlisted to better understand the evolution of clinically relevant microbes and, potentially, to influence their evolution to aid human health. Of special interest are situations in which there exists a "tragedy of the commons," where natural selection leads to a net reduction in fitness for all members of a population. Here, I demonstrate the existence of a tragedy of the commons among antibiotic resistance plasmids of bacteria. In serial transfer culture, plasmids evolved a greater ability to superinfect already-infected bacteria, increasing plasmid fitness when evolved genotypes were rare. Evolved plasmids, however, fell victim to their own success, reducing the density of their bacterial hosts when they became common and suffering reduced fitness through vertical transmission. Social interactions can thus be an important determinant of evolution for the molecular endosymbionts of bacteria. These results also identify an avenue of evolution that reduces proliferation of both antibiotic resistance genes and their bacterial hosts. PMID:22486703

  10. Tragedy of the commons among antibiotic resistance plasmids.

    PubMed

    Smith, Jeff

    2012-04-01

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

  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. 33. Elevation of Doors / Typical Cement Toilet Partitions / ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    33. Elevation of Doors / Typical Cement Toilet Partitions / Typical Cement Shower Bath Partitions / Typical Marble Shower Bath Partitions / Dispensary Cupboard Supply Room Cupboard Similar / Section / Kitchen Cupboard and Sink / Screened Porch Cupboard (drawing 10) - Whittier State School, Hospital & Receiving Building, 11850 East Whittier Boulevard, Whittier, Los Angeles County, CA

  13. 47 CFR 24.104 - Partitioning and disaggregation.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ..., regional, and MTA licensees may apply to partition their authorized geographic service area or disaggregate their authorized spectrum at any time following grant of their geographic area authorizations. (a... partitioned service area on a schedule to the application. The partitioned service area shall be defined by...

  14. 47 CFR 24.104 - Partitioning and disaggregation.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ..., regional, and MTA licensees may apply to partition their authorized geographic service area or disaggregate their authorized spectrum at any time following grant of their geographic area authorizations. (a... partitioned service area on a schedule to the application. The partitioned service area shall be defined by...

  15. 47 CFR 24.104 - Partitioning and disaggregation.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ..., regional, and MTA licensees may apply to partition their authorized geographic service area or disaggregate their authorized spectrum at any time following grant of their geographic area authorizations. (a... partitioned service area on a schedule to the application. The partitioned service area shall be defined by...

  16. 47 CFR 24.104 - Partitioning and disaggregation.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ..., regional, and MTA licensees may apply to partition their authorized geographic service area or disaggregate their authorized spectrum at any time following grant of their geographic area authorizations. (a... partitioned service area on a schedule to the application. The partitioned service area shall be defined by...

  17. 47 CFR 24.104 - Partitioning and disaggregation.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ..., regional, and MTA licensees may apply to partition their authorized geographic service area or disaggregate their authorized spectrum at any time following grant of their geographic area authorizations. (a... partitioned service area on a schedule to the application. The partitioned service area shall be defined by...

  18. 47 CFR 90.911 - Partitioned licenses and disaggregated spectrum.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 5 2014-10-01 2014-10-01 false Partitioned licenses and disaggregated spectrum... Specialized Mobile Radio Service § 90.911 Partitioned licenses and disaggregated spectrum. (a) Eligibility...) that constitute the partitioned area. (2) Disaggregation. Spectrum may be disaggregated in any...

  19. 47 CFR 95.823 - Geographic partitioning and spectrum disaggregation.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 5 2013-10-01 2013-10-01 false Geographic partitioning and spectrum... Geographic partitioning and spectrum disaggregation. (a) Eligibility. Parties seeking Commission approval of geographic partitioning or spectrum disaggregation of 218-219 MHz Service system licenses shall request...

  20. 47 CFR 95.823 - Geographic partitioning and spectrum disaggregation.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 5 2014-10-01 2014-10-01 false Geographic partitioning and spectrum... Geographic partitioning and spectrum disaggregation. (a) Eligibility. Parties seeking Commission approval of geographic partitioning or spectrum disaggregation of 218-219 MHz Service system licenses shall request...