Kbp Plasmid Research Articles

Emergence of CTX-M-12 extended-spectrum -lactamase-producing Escherichia coli in Korea

To characterize CTX-M-12 extended-spectrum beta-lactamase (ESBL) produced by clinical Escherichia coli isolates and to investigate its genetic environment. Antimicrobial susceptibilities were determined by disc diffusion and agar dilution methods, and the double-disc synergy test was carried out. Detection of genes encoding class A beta-lactamases was performed by PCR amplification, and the genetic environments of the bla(CTX-M-12) genes were investigated by PCR and sequencing of the regions surrounding the genes. Kinetic parameters were determined from purified CTX-M-12. Sequence data for the CTX-M-1 cluster from three clinical E. coli isolates indicated the presence of CTX-M-12. An ISEcp1 insertion sequence was located 49 bp upstream of bla(CTX-M-12) in all three E. coli isolates. CTX-M-12 had a more potent hydrolytic activity against cefotaxime than against ceftazidime and was encoded on a self-transferable approximately 18 kbp plasmid. This work shows that CTX-M-12, which confers high-level resistance to cefotaxime but not to ceftazidime, has emerged in Korea. The bla(CTX-M-12) gene was associated with an upstream ISEcp1 insertion sequence.

Plasmid size up to 20 kbp does not limit effective in vivo lung gene transfer using compacted DNA nanoparticles

Nanoparticles consisting of single molecules of DNA condensed with polyethylene glycol-substituted lysine 30-mers efficiently transfect lung epithelium following intrapulmonary administration. Nanoparticles formulated with lysine polymers having different counterions at the time of DNA mixing have distinct geometric shapes: trifluoroacetate or acetate counterions produce ellipsoids or rods, respectively. Based on intracytoplasmic microinjection studies, nanoparticle ellipsoids having a minimum diameter less than the 25 nm nuclear membrane pore efficiently transfect non-dividing cells. This 25 nm size restriction corresponds to a 5.8 kbp plasmid when compacted into spheroids, whereas the 8-11 nm diameter of rod-like particles is smaller than the nuclear pore diameter. In mice, up to 50% of lung cells are transfected after dosing with a rod-like compacted 6.9 kbp lacZ expression plasmid, and correction of the CFTR chloride channel was observed in humans following intranasal administration of a rod-like compacted 8.3 kbp plasmid. To further investigate the potential size and shape limitations of DNA nanoparticles for in vivo lung delivery, reporter gene activity of ellipsoidal and rod-like compacted luciferase plasmids ranging in size between 5.3 and 20.2 kbp was investigated. Equivalent molar reporter gene activities were observed for each formulation, indicating that microinjection size limitations do not apply to the in vivo gene transfer setting.

Replication regions of Sinorhizobium meliloti plasmids

The replication ( rep) regions of small plasmids from three Sinorhizobium meliloti strains were cloned by marker rescue. Two unique replication regions were identified, one of which was common to two different strains. Plasmid pBB83 carried a 7.2 kbp rep region from a 42 kbp plasmid, and pBB84 carried a 4.5 kbp rep region from a 36 kbp plasmid. The cloned rep regions were of different compatibility types, and were capable of displacing their parent plasmids from S. meliloti. Neither could function in a PolA − strain of Escherichia coli. The cloned replication regions were less stable in S. meliloti than their parent plasmids. The rep genes for each plasmid were localized to less than 2.5 kbp segments. Sequencing data revealed that the pBB83 Rep protein is uncommon, with partial identity to a protein encoded by a plasmid from S. meliloti GR4 [Mercado-Blanco, J., Olivares, J., 1994. The large nonsymbiotic plasmid pRmeGR4a of Rhizobium meliloti GR4 encodes a protein involved in replication that has homology with the RepC protein of Agrobacterium plasmids. Plasmid 32, 75–79]. However, the cloned DNA fragment also contains a truncated segment of the common repABC genes, suggesting that the parent plasmid contained two sets of replication genes. Other genes and an IS-element within the insert are most closely related to sequences derived from the Rhizobiaceae family, suggesting that the plasmid has a limited host range. In contrast, the pBB84 rep region contained genes similar to those associated with several broad host-range plasmids, and its Rep protein is related to that of a Pseudomonas aeruginosa broad host-range plasmid, pVS1 [Heeb, S., Itoh, Y., Nishijyo, T., Schnider, U., Keel, C., Wade, J., Walsh, U., O’Gara, F., Haas, D., 2000. Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plant-associated bacteria. Mol. Plant-Microbe Interact. 13, 232–237]. The pBB84 rep region also includes a probable origin of replication, consisting of DNA boxes flanking a series of direct repeats and an AT-rich sequence.

Purification of plasmid DNA vectors by aqueous two-phase extraction and hydrophobic interaction chromatography

The current study explores the possibility of using a polyethyleneglycol(PEG)–ammonium sulphate aqueous two-phase system (ATPS) as an early step in a process for the purification of a model 6.1 kbp plasmid DNA (pDNA) vector. Neutralised alkaline lysates were fed directly to ATPS. Conditions were selected to direct pDNA towards the salt-rich bottom phase, so that this stream could be subsequently processed by hydrophobic interaction chromatography (HIC). Screening of the best conditions for ATPS extraction was performed using three PEG molecular weights (300, 400 and 600) and varying the tie-line length, phase volume ratio and lysate load. For a 20% (w/w) lysate load, the best results were obtained with PEG 600 using the shortest tie-line (38.16%, w/w). By further manipulating the system composition along this tie-line in order to obtain a top/bottom phase volume ratio of 9.3 (35%, w/w PEG 600, 6%, w/w NH 4) 2SO 4), it was possible to recover 100% of pDNA in the bottom phase with a three-fold increase in concentration. Further increase in the lysate load up to 40% (w/w) with this system resulted in a eight-fold increase in pDNA concentration, but with a yield loss of 15%. The ATPS extraction was integrated with HIC and the overall process compared with a previously defined process that uses sequential precipitations with iso-propanol and ammonium sulphate prior to HIC. Although the final yield is lower in the ATPS-based process the purity grade of the final pDNA product is higher. This shows that it is possible to substitute the time-consuming two-step precipitation procedure by a simple ATPS extraction.

Worldwide distribution of Pseudomonas aeruginosa clone C strains in the aquatic environment and cystic fibrosis patients

Highly successful bacterial clones have the ability to effectively colonize environmental niches and patients. However, the factors which determine the complex interplay between the colonization of environmental niches and patients are mainly unknown. In this study we show that Pseudomonas aeruginosa clone C strains are distributed worldwide and highly prone to infect cystic fibrosis (CF) patients in Canada, England, France and Germany. In Hanover, Germany and Vancouver, Canada, clone C strains are highly prevalent in the CF patient community, although the mechanisms of acquisition may have been different. All clone C strains showed highly related macrorestriction fragment pattern of the whole genome as visualized by pulsed-field gel electrophoresis and harboured the 102 kbp plasmid pKLC102. Comparison of three prevalent P. aeruginosa clones with different distribution between the environment and patients revealed that neither enhanced biofilm formation nor antibiotic resistance was responsible for the spread of clone C. Clone M, which was highly prevalent in the clinical environment such as sanitary facilities, lacked motility, which could explain its relatively low prevalence in CF patients. Elucidation of the mechanisms which lead to the prevalence of clone C strain in patients and the environment requires the investigation of additional phenotypes.

The plasmids of Chlamydia trachomatis and Chlamydophila pneumoniae (N16): accurate determination of copy number and the paradoxical effect of plasmid-curing agents

A 7.5 kbp cryptic plasmid is found in almost all isolates of Chlamydia trachomatis. Real-time PCR assays, using TaqMan chemistry, were set up to quantify accurately both the chlamydial plasmid and the single copy, chromosomal omcB gene in the infectious, elementary bodies (EBs) of C. trachomatis L1 440. Plasmid copy number was also determined in the EBs of six other lymphogranuloma venereum (LGV) isolates (serovars L1-L3), ten trachoma isolates (serovars A-C) and nine urogenital isolates (serovars D-J). The results indicated an average plasmid copy number of 4.0+/-0.8 (mean+/-95 % confidence interval) plasmids per chromosome. During the chlamydial developmental cycle, up to 7.6 plasmids per chromosome were detected, indicating an increased plasmid copy number in the actively replicating reticulate bodies. Attempts to eliminate the plasmid from strain L1 440 using the plasmid-curing agents ethidium bromide, acridine orange or imipramine/novobiocin led to a paradoxical increase in plasmid copy number. It is speculated that the stress induced by chemical curing agents may stimulate the activity of plasmid-encoded replication (Rep) proteins. In contrast to C. trachomatis, only a single isolate of Chlamydophila pneumoniae bears a plasmid. C. pneumoniae strain N16 supports a 7.4 kbp plasmid in which ORF1, encoding one of the putative Rep proteins, is disrupted by a deletion and split into two smaller ORFs. Similar assay techniques revealed 1.3+/-0.2 plasmids per chromosome (mean+/-95 % confidence interval) in EBs of this strain. These findings are in agreement with the hypothesis that the ORF1-encoded protein is involved in, but not essential for, plasmid replication and control of copy number.

Characterization of Arthrobacter nicotinovorans HIM, an atrazine-degrading bacterium, from agricultural soil New Zealand

Arthrobacter nicotinovorans HIM was isolated directly from an agricultural sandy dune soil 6 months after a single application of atrazine. It grew in minimal medium with atrazine as sole nitrogen source but was unable to mineralize 14C-ring-labelled atrazine. Atrazine was degraded to cyanuric acid. In addition to atrazine the bacterium degraded simazine, terbuthylazine, propazine, cyanazine and prometryn but was unable to grow on terbumeton. When added to soil, A. nicotinovorans HIM did enhance mineralization of 14C-ring-labelled atrazine and simazine, in combination with naturally occurring cyanuric acid degrading microbes resident in the soil. Using PCR, the atrazine-degradation genes atzABC were identified in A. nicotinovorans HIM. Cloning of the atzABC genes revealed significant homology (>99%) with the atrazine degradation genes of Pseudomonas sp. strain ADP. The atrazine degradation genes were held on a 96 kbp plasmid.

Integrated process for purification of plasmid DNA using aqueous two-phase systems combined with membrane filtration and lid bead chromatography

An integrated process for purifying a 6.1 kilo base pair (kbp) plasmid from a clarified Escherichia coli cell lysate based on an ultra/diafiltration step combined with polymer/polymer aqueous two-phase system and a new type of chromatography is described. The process starts with a volume reduction (ultrafiltration) and buffer exchange (diafiltration) of the clarified lysate using a hollow fibre membrane system. The concentrated and desalted plasmid solution is then extracted in a thermoseparating aqueous two-phase system, where the contaminants (RNA and proteins) to a large extent are removed. While the buffer exchange (diafiltration) is necessary in order to extract the plasmid DNA exclusively to the top phase, experiments showed that the ultrafiltration step increased the productivity of the aqueous two-phase system by a factor of more than 10. The thermoseparated water phase was then subjected to a polishing step using lid bead chromatography. Lid beads are a new type of restricted access chromatography beads, here with a positively charged inner core that adsorbed the remaining RNA while its inert surface layer prevented adsorption of the plasmid DNA thus passing in the flow-through of the column. Differently-sized plasmid DNA in the range of 2.7–20.5 kbp were also partitioned in the aqueous two-phase system. Within this size range, all plasmid DNA was exclusively extracted to the top phase. The complete process is free of additives and easy scalable for use in large scale production of plasmid DNA. The overall process yield for plasmid DNA was 69%.

Differential large-scale chromatin compaction and intranuclear positioning of transcribed versus non-transcribed transgene arrays containing beta-globin regulatory sequences.

Previous work has demonstrated a more decondensed large-scale chromatin structure and a more internal nuclear position for gene-rich versus gene-poor chromosome regions. Here, we show that large-scale chromatin opening and changes in intranuclear positioning of chromosome regions can be induced by normal levels of endogenous transcription factors acting on mammalian regulatory sequences. We transfected mouse erythroleukemia cells with a 15 kbp plasmid containing a lac operator repeat plus beta-globin regulatory sequences driving a beta-galactosidase reporter gene. After green-fluorescent-protein/lac-repressor fusion-protein binding or after fluorescence in situ hybridization, the volume and location of the transgene array signal were measured. With both detection methods, we found that the volume was severalfold larger when transcription was on. While silent transgene arrays were located close to the nuclear membrane, we observed a significantly more internal position for the transcriptionally active state. Our results indicate that both large-scale chromatin decondensation and changes in nuclear positioning as observed for large, complex gene-rich chromosome regions can be reproduced by endogenous regulatory sequences acting within simple repetitive transgene arrays.

848. The Study on the VEGF Plasmids Purify by Magnetic Nanoparticles

In order to evaluate the usefulness of circular dichroism (CD) spectroscopy as a quality control assay in manufacturing DNA nanoparticles, well-characterized complexes consisting of single molecules of plasmid DNA condensed with lysine polymers were spectrally analyzed. Plasmids of various sizes encoding luciferase (pLuc: 5.3, 9.7, and 20.2 kbp) or green fluorescent protein (pGFP: 2.9, 5.1, and 28 kbp) were condensed at a final 2:1 charge ratio (+to-) using conjugates of 10 kDa polyethylene glycol (PEG)-substituted 30-mer lysine polymers having an N-terminal cysteine. At the time of DNA mixing, the polylysine counterions were either acetate or trifluoroacetate, which determine nanoparticle shape (rods or ellipsoids, respectively). The condensed plasmids were characterized by electron microscopy (DNA nanoparticle size and shape), turbidity and sedimentation assays (colloidal stability), serum stability tests (protection of DNA from nucleases), and gel analysis (DNA integrity). CD spectra for condensed DNA, free DNA, and free polylysine were recorded at 4 deg C over a wavelength range of 195-310 nm. Free polylysine had a random coil conformation with a peak at 215-219 nm. This conformation was apparently preserved after binding to DNA since the strong positive ellipticity peak at 200 nm, which is characteristic of α and βconformations, was not observed in the compacted DNA. The CD spectra for condensed pLuc 5.3 kbp plasmid in water, 0.15 M NaCl, or 0.3 M NaCl were almost identical and distinct from the spectra for free DNA. The native 280-nm peak of B-form DNA decreased and red-shifted, as did the 261-nm crossover point, and the troughs at ~200 nm and ~250 nm became more negative. Electron microscopy and sedimentation analyses of DNA nanoparticles in water, 0.15 M NaCl, or 0.3 M NaCl were identical, showing unaggregated unimolecularly compacted DNA. At 0.5 M NaCl, however, DNA nanoparticles demonstrated aggregates of partially condensed DNA. Interestingly, CD spectra of DNA nanoparticles at 0.5 M NaCl were typical of psi-form DNA, which has significantly enhanced peaks at 220 nm and 280 nm and a reduced trough at 250 nm. At 1 or 1.5 M NaCl, the CD spectra for condensed DNA were a sum of spectra for free polylysine and free DNA. This expected result is consistent with complete dissociation of those two components, as revealed by titration of free lysine residues with fluorescamine. Qualitatively, spectra of all non-aggregated condensed plasmids (irrespective of plasmid size, reporter gene, and polylysine counterion) showed unique and common features distinguishing them from free DNA, aggregates of partially neutralized DNA, and other aggregates reported in the literature as having psi-type spectra. The unique spectrum and reproducibility of this test makes CD a useful quality control assay in monitoring the production of unimolecularly-compacted DNA nanoparticles.

505. Effective Transgene Expression in the Murine Lung Using Compacted DNA Nanoparticles Formulated with Plasmid DNAs of 5, 10, and 20 kbp

One concern of non-viral gene therapies is the potential limitation on the size of the DNA construct used in formulating an effective therapeutic. Some open reading frames, such as the coding sequence of the cystic fibrosis transmembrane regulator (4.4 kbp), are quite large and, when combined with other required regulatory elements, result in a therapeutic construct of significant size (> 8 kbp). Gene transfer optimization studies are usually performed using reporter gene constructs. Typical reporter genes have much smaller open reading frames (luciferase 1.7 kbp, green fluorescent protein 0.7 kbp) than many therapeutic genes. Therefore, we wanted to determine if our strategies for gene therapy of multiple inherited and acquired diseases will be limited in vivo by the required size of the plasmid DNA payload. To address this aim, we constructed three luciferase reporter plasmids of increasing size—~5, 10, and 20 kbp—using lambda DNA stuffer fragments inserted into the 5 kbp plasmid. These plasmids were compacted to DNA nanoparticles with a 10 kDa polyethylene glycol (PEG) substituted 30-mer lysine polymer containing either trifluoroacetate or acetate counterions at the time of DNA mixing. As evaluated by transmission electron microscopy, these formulations generate compacted DNA that appears as either ellipsoids or rods, respectively. These formulations were delivered to the lungs of Balb/C mice via the intranasal route, and the lungs were harvested and luciferase activity evaluated 2 days after gene transfer. All DNA nanoparticles tested—~5 kbp to 20 kbp plasmids formulated using PEG-lysine polymers with either counterion—had luciferase reporter activities of ~104 RLU/mg protein/pmol DNA above background. Within each formulation group, the reporter gene activity was equivalent (no statistically significant differences) among all three compacted plasmid DNAs. These results indicate that the size of the plasmid used, up to at least 20 kbp, should not be a limitation for lung gene transfer using this non-viral gene therapy platform.

Extraction of plasmid DNA from Escherichia coli cell lysate in a thermoseparating aqueous two-phase system

The primary purification of a 6.1 kilo base pair (kbp) plasmid from a desalted alkaline lysate has been accomplished by a thermoseparating aqueous two-phase system [(50% ethylene oxide–50% propylene oxide)–Dextran T 500]. The partitioning of the different nucleic acids (plasmid DNA, RNA, genomic DNA) in the thermoseparating aqueous two-phase system was followed both qualitatively by agarose gel electrophoresis and quantitatively by analytical chromatography (size exclusion- and anion-exchange mode) and PicoGreen fluorescence analysis. The experimental results showed a complete recovery of the plasmid DNA to the top phase, while 80% of total RNA and 58% of total protein was discarded to the bottom phase. Moreover, a 3.8-fold volume reduction of the plasmid DNA solution was achieved. By using a final thermoseparating step, the EO 50PO 50 polymer could be efficiently recycled, resulting in plasmid solution containing less than 1% polymer. The developed thermoseparating aqueous two-phase system shows great potential for the large-scale processing of plasmid DNA.

Reduced sulfhydryls maintain specific incision of BPDE–DNA adducts by recombinant thermoresistant Bacillus caldotenax UvrABC endonuclease

Prokaryotic DNA repair nucleases are useful reagents for detecting DNA lesions. Escherichia coli UvrABC endonuclease can incise DNA containing UV photoproducts and bulky chemical adducts. The limited stability of the E. coli UvrABC subunits leads to difficulty in estimating incision efficiency and quantitative adduct detection. To develop a more stable enzyme with greater utility for the detection of DNA adducts, thermoresistant UvrABC endonuclease was cloned from the eubacterium Bacillus caldotenax ( Bca) and individual recombinant protein subunits were overexpressed in and purified from E. coli. Here, we show that Bca UvrC that had lost activity or specificity could be restored by dialysis against buffer containing 500 mM KCl and 20 mM dithiothreitol. Our data indicate that UvrC solubility depended on high salt concentrations and UvrC nuclease activity and the specificity of incisions depended on the presence of reduced sulfhydryls. Optimal conditions for BCA UvrABC-specific cleavage of plasmid DNAs treated with [ 3H](+)-7 R,8 S-dihydroxy-9 S,10 R-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene (BPDE) (1–5 lesions/plasmid) were developed. Preincubation of substrates with UvrA and UvrB enhanced incision efficiency on damaged substrates and decreased non-specific nuclease activity on undamaged substrates. Under optimal conditions for damaged plasmid incision, ∼70% of adducts were incised in 1 nM plasmid DNA (2 BPDE adducts/5.4 kbp plasmid) with UvrA at 2.5 nM, UvrB at 62.5 nM, and UvrC at 25 nM. These results demonstrate the potential usefulness of the Bca UvrABC for monitoring the distribution of chemical carcinogen-induced lesions in DNA.

Sequence and analysis of pBM02, a novel RCR cryptic plasmid from Lactococcus lactis subsp. cremoris P8-2-47

This paper reports the complete nucleotide sequence of the 3.85 kbp plasmid pBM02 from Lactococcus lactis subsp. cremoris P8-2-47. Analysis of the sequence predicted six ORFs larger than 25 amino acids. They all were transcribed from the same strand and organized in two functional cassettes: the replication region and a putative mobilization region. In the replication region, two ORFs specifying proteins homologous to others found in some classes of rolling circle-replicating plasmids were encountered ( copG and repB). In fact, single-stranded DNA was detected as a replication intermediate of pBM02. copG and repB, together with some upstream sequences, formed part of the minimal replication unit of the plasmid. Interestingly, pBM02 shared a 212 bp stretch with plasmids of the pWV01 type, in which the whole single-strand origin of replication is included. In the mobilization region, an ORF coding for a mobilization-like protein was present, preceded by a putative oriT sequence homologous to that of plasmid pMV158. The replicon of pBM02 is of the wide-host range type, and functions in both Gram-positive and Gram-negative bacteria, including Lactobacillus casei, Lactobacillus plantarum, Bacillus subtilis, and Escherichia coli.

Characterisation of silent and active genes for a variable large protein of Borrelia recurrentis.

BackgroundWe report the characterisation of the variable large protein (vlp) gene expressed by clinical isolate A1 of Borrelia recurrentis; the agent of the life-threatening disease louse-borne relapsing fever.MethodsThe major vlp protein of this isolate was characterised and a DNA probe created. Use of this together with standard molecular methods was used to determine the location of the vlp1B. recurrentis A1 gene in both this and other isolates.ResultsThis isolate was found to carry silent and expressed copies of the vlp1B. recurrentis A1 gene on plasmids of 54 kbp and 24 kbp respectively, whereas a different isolate, A17, had only the silent vlp1B. recurrentis A17 on a 54 kbp plasmid. Silent and expressed vlp1 have identical mature protein coding regions but have different 5' regions, both containing different potential lipoprotein leader sequences. Only one form of vlp1 is transcribed in the A1 isolate of B. recurrentis, yet both 5' upstream sequences of this vlp1 gene possess features of bacterial promoters.ConclusionTaken together these results suggest that antigenic variation in B. recurrentis may result from recombination of variable large and small protein genes at the junction between lipoprotein leader sequence and mature protein coding region. However, this hypothetical model needs to be validated by further identification of expressed and silent variant protein genes in other B. recurrentis isolates.

The application of fluorescence correlation spectroscopy in detecting DNA condensation

We report the application of fluorescence correlation spectroscopy (FCS) in characterizing conformational changes (condensation) of chemically well-defined DNA plasmids. The plasmids: pHβAPr-1-neo (10 kbp, contour length 3.4 μm) and pBluescript SKt (2.96 kbp, contour length 1.02 μm) were imaged by a confocal fluorescence microscope using two fluorescent probes: ethidium bromide (EtBr) and propidium iodide (PrIo). It became clear that the DNA molecule exhibits discrete conformational change between the coil and globule states with the addition of a small amount (the order of magnitude being 10 −5 M) of cationic surfactant, spermine and hexadecyltrimethyl ammonium bromide (HTAB). When the concentrations of both condensing agents are smaller than 6.0×10 −6 M and 2.0×10 −6 M for the 10 and 2.96 kbp, both plasmids are in the extended coil state with diffusion constants D 10 kbp=9.6×10 −13 m 2 s −1 and D 2.96 kbp=2.5×10 −12 m 2 s −1, respectively. When the condensing agent in a concentration higher than 1.10×10 −5 M is added to pHβAPr-1-neo (10 kbp), plasmids are in the condensed globular state and their diffusion constants are D 10 kbp=8.0×10 −12 m 2 s −1 (spermine) and D 10 kbp=5.5×10 −12 m 2 s −1 (HTAB). The globular state of the pBluescript SKt (2.96 kbp) plasmids is characterized by diffusion constants equal to D 2.96 kbp=9.2×10 −12 m 2 s −1 (spermine) and D 2.96 kbp=8.2×10 −12 m 2 s −1 (HTAB).

Flexprep: scale-flexible rapid plasmid preparation for analysis of recombinant clones.

A scale-flexible and cost-efective protocol for plasmid preparation is described to cover miniprep and midiprep scale work in a microcentriguge format for analysis of recombinant clones, this protocol relies on a modified alkaline lysis of Escherichia coli cells and subsequent purification of plasmid DNA with no organic extraction and alcohol precipitation. It can process up to 20 mL of E. coli cells carrying 3-10 kbp plasmid vectors in < 10 min. Flexprep delivers sufficient yield and purity of plasmid DNA for routine applications including restriction enzyme digestion and fluorescent automated sequencing.

Plasmid-mediated dimethoate degradation in Pseudomonas aeruginosa MCMB-427.

To investigate the genetics of dimethoate degradation in Pseudomonas aeruginosa MCMB-427. Pseudomonas aeruginosa MCMB-427 demonstrated the ability to degrade dimethoate, a synthetic organophosphate insecticide. Total DNA preparation of MCMB-427 revealed the presence of a 6.6 kbp plasmid (designated as pDMD427). Escherichia coli NovaBlue transformed with plasmid pDMD427 subsequently acquired the ability to degrade dimethoate. Curing of the plasmid by plumbagin or ethidium bromide resulted in the loss of ability of MCMB-427 to degrade dimethoate. Plasmid pDMD427 was stable in MCMB-427 over 20 passages without selection. Genes encoding resistance to norfloxacin and cobalt were also located on plasmid pDMD427. The ability of Ps. aeruginosa MCMB-427 to degrade dimethoate is plasmid-mediated and transferable to other strains. As far as is known, this is the first report of plasmid-mediated dimethoate biodegradation. This study contributes significantly towards an understanding of the genetics of bacterial dimethoate degradation.

A highly homologous 68 kbp plasmid found in Vibrio vulnificus strains virulent for eels

Vibrio vulnificus serovar E (biotype 2) strains are virulent for eels and have also been reported to cause illness in humans. Studies on the plasmid content of serovar E strains revealed the existence of a plasmid of approximately 70 kbp present in most of these strains. In this study we characterized the 70 kbp plasmids of seven biotype 2 strains isolated from seawater, diseased eels and wound infections in humans. We determined the exact size of the high molecular weight plasmids to be 68 kbp. A comparison of the plasmids of the seven strains by restriction length polymorphism and hybridization analysis showed them to be almost identical.

A highly homologous 68 kbp plasmid found inVibrio vulnificus strains virulent for eels

Vibrio vulnificus serovar E (biotype 2) strains are virulent for eels and have also been reported to cause illness in humans. Studies on the plasmid content of serovar E strains revealed the existence of a plasmid of approximately 70 kbp present in most of these strains. In this study we characterized the 70 kbp plasmids of seven biotype 2 strains isolated from seawater, diseased eels and wound infections in humans. We determined the exact size of the high molecular weight plasmids to be 68 kbp. A comparison of the plasmids of the seven strains by restriction length polymorphism and hybridization analysis showed them to be almost identical.