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Leise Riber, Mette Burmølle, Martin Alm, Stefan M Milani, Peter Thomsen, Lars H Hansen, Søren J Sørensen
The spread of antimicrobial resistance, usually mediated by horizontal transfer of plasmids, limits the options of treating bacterial infections and thereby poses a crucial human health problem. The disturbance of plasmid stability within bacterial species in clinical environments serves as a novel strategy to reduce the development and dissemination of antibiotic resistance. We tested the ability of irgasan to destabilize plasmids from Escherichia coli K-12 cells when added directly into liquid growth medium at concentrations below levels of marked bacterial growth inhibition, or when released into liquid growth medium from irgasan-impregnated Interpenetrating Polymer Network (IPN) silicone hydrogel objects, a novel technology developed as drug-delivery platform...
October 12, 2016: Plasmid
Despoina A I Mavridou, Diego Gonzalez, Abigail Clements, Kevin R Foster
Fluorescent labeling has been an invaluable tool for the study of living organisms and bacterial species are no exception to this. Here we present and characterize the pUltra plasmids which express constitutively a fluorescent protein gene (GFP, RFP, YFP or CFP) from a strong synthetic promoter and are suitable for the fluorescent labeling of a broad range of Enterobacteria. The amount of expressed fluorophore from these genetic constructs is such, that the contours of the cells can be delineated on the basis of the fluorescent signal only...
September 29, 2016: Plasmid
L Thoma, G Muth
Conjugative DNA-transfer in the Gram-positive mycelial soil bacterium Streptomyces, well known for the production of numerous antibiotics, is a unique process involving the transfer of a double-stranded DNA molecule. Apparently it does not depend on a type IV secretion system but resembles the segregation of chromosomes during bacterial cell division. A single plasmid-encoded protein, TraB, directs the transfer from the plasmid-carrying donor to the recipient. TraB is a FtsK-like DNA-translocase, which recognizes a specific plasmid sequence, clt, via interaction with specific 8-bp repeats...
September 28, 2016: Plasmid
Christopher J Harmer, Mohammad Hamidian, Stephanie J Ambrose, Ruth M Hall
Both the Salmonella genomic islands (SGI) and the conjugative IncC plasmids are known to contribute substantially to the acquisition of resistance to multiple antibiotics, and plasmids in the A/C group are known to mobilize the Salmonella genomic island SGI1, which also carries multiple antibiotic resistance genes. Plasmid pRMH760 (IncC; A/C2) was shown to mobilize SGI1 variants SGI1-I, SGI1-F, SGI1-K and SGI2 from Salmonella enterica to Escherichia coli where it was integrated at the preferred location, at the end of the trmE (thdF) gene...
September 9, 2016: Plasmid
Qiang Yang, Mei Deng, Ling-Ling Zhang, Xiao-Wei Zhang, Le-Ning Wang, Hu Chen, Jian Ma, Peng-Fei Qi, Qian-Tao Jiang, Xiu-Jin Lan, Yu-Ming Wei, You-Liang Zheng
In this study, we designed and constructed a super twin T-DNA vector (pTRIDT313-g) containing two independent T-DNA cassettes-one for the selection gene Hyg and the other for the target gene Gus-to produce marker-free transgenic lines. The resulting vector was transformed into tobacco, and polymerase chain reaction (PCR) analysis showed four types of gene combinations in the T1 and T2 generations: Gus only, Hyg only, Gus+Hyg, and untransformed lines. The intermediate region from the T-DNA of the right border of Hyg to the left border of Gus in the Hyg and Gus lines was not amplified...
September 8, 2016: Plasmid
Mohammad Hamidian, Stephanie J Ambrose, Ruth M Hall
Acinetobacter baumannii is an important nosocomial pathogen that often complicates treatment because of its high level of resistance to antibiotics. Though plasmids can potentially introduce various genes into bacterial strains, compared to other Gram-negative bacteria, information about the unique A. baumannii plasmid repertoire is limited. Here, whole genome sequence data was used to determine the plasmid content of strain A297 (RUH875), the reference strain for the globally disseminated multiply resistant A...
September 3, 2016: Plasmid
Christopher J Harmer, Ruth M Hall
IncC (A/C2) plasmids are known to play an important role in the spread of multiple antibiotic resistance determinants, including extended-spectrum β-lactamases and carbapenamases, amongst Gram negative bacterial populations. The ability to identify and track these plasmids is valuable in epidemiological and clinical studies. A recent comparative analysis of the backbones of sequenced IncC plasmids identified two distinct lineages, type 1 and type 2, with different evolutionary histories. Here, a simple PCR method to rapidly assign plasmids to one of these lineages by detecting variable regions in the backbone was developed...
August 30, 2016: Plasmid
Yuhua Wu, Jun Li, Yulei Wang, Xiaofei Li, Yunjing Li, Li Zhu, Jun Li, Gang Wu
The use of analytical controls is essential when performing GMO detection through screening tests. Additionally, the presence of taxon-specific sequences is analyzed mostly for quality control during GMO detection. In this study, 11 commonly used genetic elements involving three promoters (P-35S, P-FMV35S and P-NOS), four marker genes (Bar, NPTII, HPT and Pmi), and four terminators (T-NOS, T-35S, T-g7 and T-e9), together with the reference gene fragments from six major crops of maize, soybean, rapeseed, rice, cotton and wheat, were co-integrated into the same single plasmid to construct a general reference plasmid pBI121-Screening...
August 4, 2016: Plasmid
Kevin S Lang, Timothy J Johnson
Conjugation plays an important role in the horizontal movement of DNA between bacterial species and even genera. Large conjugative plasmids in Gram-negative bacteria are associated with multi-drug resistance and have been implicated in the spread of these phenotypes to pathogenic organisms. A/C plasmids often carry genes that confer resistance to multiple classes of antibiotics. Recently, transcription factors were characterized that regulate A/C conjugation. In this work, we expanded the regulon of the negative regulator Acr2...
August 1, 2016: Plasmid
Adam J Kolakowski, Jeffrey F Gardner
DOT Integrase (IntDOT) is a member of the tyrosine recombinase family. It catalyzes the integration and excision reactions of an integrative and conjugative element (ICE) called CTnDOT. Like other tyrosine recombinases, the integration reaction proceeds by two sets of strand exchanges between the attDOT site on CTnDOT and an attB site in the host chromosome. The strand exchanges occur seven bases apart and define an overlap region. After the first strand exchanges a Holliday Junction (HJ) intermediate is formed...
July 12, 2016: Plasmid
Elsi Pulkkinen, Saija Haapa-Paananen, Hilkka Turakainen, Harri Savilahti
Mu transposition-based cloning of DNA circles employs in vitro transposition reaction to deliver both the plasmid origin of replication and a selectable marker into the target DNA of interest. We report here the construction of a platform for the purpose that contains ten mini-Mu transposons with five different replication origins, enabling a variety of research approaches for the discovery and study of circular DNA. We also demonstrate that the simultaneous use of two transposons, one with the origin of replication and the other with selectable marker, is beneficial as it improves the cloning efficiency by reducing the fraction of autointegration-derived plasmid clones...
July 2016: Plasmid
Jennifer M Auchtung, Naira Aleksanyan, Artemisa Bulku, Melanie B Berkmen
Horizontal gene transfer plays a profound role in bacterial evolution by propelling the rapid transfer of genes and gene cassettes. Integrative and conjugative elements (ICEs) are one important mechanism driving horizontal gene transfer. ICEs, also known as conjugative transposons, reside on the host chromosome but can excise to form a conjugative DNA circle that is capable of transfer to other cells. Analysis of the large number of completed bacterial genome sequences has revealed many previously unrecognized ICEs, including ICEBs1, found in the Gram-positive model bacterium Bacillus subtilis...
July 2016: Plasmid
Christopher J Harmer, Sally R Partridge, Ruth M Hall
Most A/C plasmids sequenced to date were recovered in the last two decades. To gain insight into the evolution of this group, the IncC plasmid pDGO100, found in a multiply antibiotic-resistant Escherichia coli strain isolated in 1981, was sequenced. pDGO100 belongs to the type 1 lineage and carries an ARI-A antibiotic resistance island but not an ARI-B island. The A/C2 backbone of pDGO100 has a deletion in the rhs1 gene previously found in pRMH760 and differs by only six single base pair substitutions from pRMH760, recovered at the same hospital 16years later...
July 2016: Plasmid
M D Crespo, E Altermann, J Olson, W G Miller, K Chandrashekhar, S Kathariou
In Campylobacter spp., resistance to the antimicrobials kanamycin and tetracycline is frequently associated with plasmid-borne genes. However, relatively few plasmids of Campylobacter jejuni have been fully characterized to date. A novel plasmid (p11601MD; 44,095nt) harboring tet(O) was identified in C. jejuni strain 11601MD, which was isolated from the jejunum of a turkey produced conventionally in North Carolina. Analysis of the p11601MD sequence revealed the presence of a high-GC content cassette with four genes that included tet(O) and a putative aminoglycoside transferase gene (aphA-3) highly similar to kanamycin resistance determinants...
July 2016: Plasmid
Anna Karlowicz, Katarzyna Wegrzyn, Andrzej Dubiel, Malgorzata Ropelewska, Igor Konieczny
Plasmids, as extrachromosomal genetic elements, need to work out strategies that promote independent replication and stable maintenance in host bacterial cells. Their maintenance depends on constant formation and dissociation of nucleoprotein complexes formed on plasmid DNA. Plasmid replication initiation proteins (Rep) form specific complexes on direct repeats (iterons) localized within the plasmid replication origin. Formation of these complexes along with a strict control of Rep protein cellular concentration, quaternary structure, and activity, is essential for plasmid maintenance...
July 2016: Plasmid
C M VanDrisse, J C Escalante-Semerena
Galloway et al. recently described a method to alter vectors to include Type IIS restriction enzymes for high efficiency cloning. Utilizing this method, the multiple cloning sites of complementation and overexpression vectors commonly used in our laboratory were altered to contain recognition sequences of the Type IIS restriction enzyme, BspQI. Use of this enzyme increased the rate of cloning success to >97% efficiency. L(+)-Arabinose-inducible complementation vectors and overexpression vectors encoding N-terminal recombinant tobacco etch virus protease (rTEV)-cleavable H6-tags were altered to contain BspQI sites that allowed for cloning into all vectors using identical primer overhangs...
July 2016: Plasmid
Yao Zhai, Zilong He, Yu Kang, Haiying Yu, Jianfeng Wang, Pengcheng Du, Zhao Zhang, Songnian Hu, Zhancheng Gao
The complete 284,628bp sequence of pH11, an IncHI2 plasmid, was determined through single-molecule, real-time (SMRT) sequencing. Harbored by a clinical Klebsiella pneumoniae strain H11, and isolated in Beijing, this plasmid contains multiple antibiotic resistance genes, including catA2, aac(6')-Ib, strB, strA, dfrA19, blaTEM-1, blaSHV-12, sul1, qacE delta 1, ereA, arr2, and aac3. The aac(6')-Ib is carried by a class I integron. Plasmid pH11 also carries several genes associated with resistance to heavy metals, such as tellurium, mercury, cobalt, zinc, nickel, copper, lead and cadmium...
July 2016: Plasmid
Mohsen Teimouri, Azadeh Hashem Nia, Khalil Abnous, Hossein Eshghi, Mohammad Ramezani
Nanomedicine as the interface between nanotechnology and medical sciences is a new area that has attracted the attention of vast groups of researchers. Carbon nanomaterials are common platform for synthesis of nanoparticles for biomedical applications due to their low cytotoxicity and feasible internalization into mammalian cell lines (Yang et al., 2007; Arora et al., 2014; Oh and Park, 2014). Synthesis of vectors based on various cationic polymers polyethylenimine (PEI), polypropylenimine (PPI) and polyamidoamine (PAMAM) and their derivatives were considered as a strategy for transferring plasmid DNA and treatment of genetic diseases...
March 2016: Plasmid
Eigo Takahashi, Jae Man Lee, Hiroaki Mon, Yuuka Chieda, Chisa Yasunaga-Aoki, Takahiro Kusakabe, Kazuhiro Iiyama
Pseudomonas aeruginosa PAO1 organisms harbouring different plasmids were cultured in broths containing appropriate antibiotic(s). Extracellular proteins were more abundant in the presence of tetracycline or kanamycin than in the presence of other antibiotics. Zymography revealed that alkaline protease (AprA) production was interfered by these antibiotics. Extracellular proteins were not observed at the same level when AprA-deficient EG03 strains were cultured in the presence of different antibiotics. The extracellular protein levels were dependent on the antibiotics and plasmid derivative groups...
March 2016: Plasmid
Anton Kurakov, Sofia Mindlin, Alexey Beletsky, Natalya Shcherbatova, Andrey Rakitin, Aleksandra Ermakova, Andrey Mardanov, Mayya Petrova
The small mobilizable plasmid pALWED1.8 containing a novel variant of the streptomycin/spectinomycin resistance gene aadA27 was isolated from the permafrost strains of Acinetobacter lwoffii. The 4135bp plasmid carries mobА and mobC genes that mediate its mobilization by conjugative plasmids. The nucleotide sequences of mobА and mobC are similar to those of mobilization genes of the modern plasmid pRAY* and its variants, which contain aadB gene, and are widespread among the pathogenic strains of Acinetobacter baumannii...
March 2016: Plasmid
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