Evolution and dynamics of megaplasmids with genome sizes larger than 100 kb in the Bacillus cereus group.

PubMed

Zheng, Jinshui; Peng, Donghai; Ruan, Lifang; Sun, Ming

2013-12-02

Plasmids play a crucial role in the evolution of bacterial genomes by mediating horizontal gene transfer. However, the origin and evolution of most plasmids remains unclear, especially for megaplasmids. Strains of the Bacillus cereus group contain up to 13 plasmids with genome sizes ranging from 2 kb to 600 kb, and thus can be used to study plasmid dynamics and evolution. This work studied the origin and evolution of 31 B. cereus group megaplasmids (>100 kb) focusing on the most conserved regions on plasmids, minireplicons. Sixty-five putative minireplicons were identified and classified to six types on the basis of proteins that are essential for replication. Twenty-nine of the 31 megaplasmids contained two or more minireplicons. Phylogenetic analysis of the protein sequences showed that different minireplicons on the same megaplasmid have different evolutionary histories. Therefore, we speculated that these megaplasmids are the results of fusion of smaller plasmids. All plasmids of a bacterial strain must be compatible. In megaplasmids of the B. cereus group, individual minireplicons of different megaplasmids in the same strain belong to different types or subtypes. Thus, the subtypes of each minireplicon they contain may determine the incompatibilities of megaplasmids. A broader analysis of all 1285 bacterial plasmids with putative known minireplicons whose complete genome sequences were available from GenBank revealed that 34% (443 plasmids) of the plasmids have two or more minireplicons. This indicates that plasmid fusion events are general among bacterial plasmids. Megaplasmids of B. cereus group are fusion of smaller plasmids, and the fusion of plasmids likely occurs frequently in the B. cereus group and in other bacterial taxa. Plasmid fusion may be one of the major mechanisms for formation of novel megaplasmids in the evolution of bacteria.

Enterococcus faecalis Ebp pili are important for cell-cell aggregation and intraspecies gene transfer

PubMed Central

Leanti La Rosa, Sabina; Camila Montealegre, Maria; Singh, Kavindra V.

2016-01-01

Enterococcus faecalis is an opportunistic pathogen that ranks among the leading causes of biofilm-associated infections. We previously demonstrated that the endocarditis- and biofilm-associated pili (Ebp) of E. faecalis play a major role in biofilm formation, adherence to abiotic surfaces and experimental infections. In this study, derivatives of E. faecalis strain OG1 were engineered to further characterize functions of Ebp pili. Loss of pili resulted in a 36-fold decrease in the number of closely associated cells when OG1RFΔebpABC was mixed with OG1SSpΔebpABC, compared with mixing the Ebp+ parental strains. In addition, using the Ebp+ parental strains as donor and recipient, we found a statistically significant increase (280–360 %, P < 0.05) in the frequency of plasmid transfer versus using Ebp−  mutants in the conjugation experiments. These results demonstrate a previously unrecognized role of Ebp pili, namely, as important contributors to microscale cell aggregation and horizontal spread of genetic material. PMID:26967674

Mining new crystal protein genes from Bacillus thuringiensis on the basis of mixed plasmid-enriched genome sequencing and a computational pipeline.

PubMed

Ye, Weixing; Zhu, Lei; Liu, Yingying; Crickmore, Neil; Peng, Donghai; Ruan, Lifang; Sun, Ming

2012-07-01

We have designed a high-throughput system for the identification of novel crystal protein genes (cry) from Bacillus thuringiensis strains. The system was developed with two goals: (i) to acquire the mixed plasmid-enriched genomic sequence of B. thuringiensis using next-generation sequencing biotechnology, and (ii) to identify cry genes with a computational pipeline (using BtToxin_scanner). In our pipeline method, we employed three different kinds of well-developed prediction methods, BLAST, hidden Markov model (HMM), and support vector machine (SVM), to predict the presence of Cry toxin genes. The pipeline proved to be fast (average speed, 1.02 Mb/min for proteins and open reading frames [ORFs] and 1.80 Mb/min for nucleotide sequences), sensitive (it detected 40% more protein toxin genes than a keyword extraction method using genomic sequences downloaded from GenBank), and highly specific. Twenty-one strains from our laboratory's collection were selected based on their plasmid pattern and/or crystal morphology. The plasmid-enriched genomic DNA was extracted from these strains and mixed for Illumina sequencing. The sequencing data were de novo assembled, and a total of 113 candidate cry sequences were identified using the computational pipeline. Twenty-seven candidate sequences were selected on the basis of their low level of sequence identity to known cry genes, and eight full-length genes were obtained with PCR. Finally, three new cry-type genes (primary ranks) and five cry holotypes, which were designated cry8Ac1, cry7Ha1, cry21Ca1, cry32Fa1, and cry21Da1 by the B. thuringiensis Toxin Nomenclature Committee, were identified. The system described here is both efficient and cost-effective and can greatly accelerate the discovery of novel cry genes.

Fast kinetic studies of plasmid DNA transfer in intact yeast cells mediated by electropulsation.

PubMed

Ganeva, V; Galutzov, B; Teissie, J

1995-09-25

Intact yeast cell Electrotransformation process was investigated. It is a two step process. The plasmid must be pre-mixed and present in contact with the cells during the pulse. During the millisecond field pulse, plasmid DNA is associated to the envelope. It therefore crosses the membrane by a process which lasts several seconds as shown by its sensitivity to a post pulse addition of DNase. Electrotransformation is not supported by an electrophoretic transfer due to the external field nor by a free diffusion across the electropermeabilized envelope. DNA is first bound during the field pulse and then is transferred by a still unknown active process due to cell metabolism.

Nitrogen-fixing nodules induced by Agrobacterium tumefaciens harboring Rhizobium phaseoli plasmids.

PubMed Central

Martínez, E; Palacios, R; Sánchez, F

1987-01-01

Rhizobium phaseoli CFN299 forms nitrogen-fixing nodules in Phaseolus vulgaris (bean) and in Leucaena esculenta. It has three plasmids of 185, 225, and 410 kilobases. The 410-kilobase plasmid contains the nitrogenase structural genes. We have transferred these plasmids to the plasmid-free strain Agrobacterium tumefaciens GMI9023. Transconjugants containing different combinations of the R. phaseoli plasmids were obtained, and they were exhaustively purified before nodulation was assayed. Only transconjugants harboring the 410-kilobase plasmid nodulate P. vulgaris and L. esculenta. Nodules formed by all such transconjugants are able to reduce acetylene. Transconjugants containing the whole set of plasmids from CFN299 nodulate better and fix more nitrogen than the transconjugants carrying only the Sym plasmid. Microscopic analysis of nodules induced by A. tumefaciens transconjugants reveals infected cells and vascular bundles. None of the A. tumefaciens transconjugants, not even the one with the whole set of plasmids from CFN299, behaves in symbiosis like the original R. phaseoli strain; the transconjugants produce fewer nodules and have lower acetylene reduction (25% as compared to the original R. phaseoli strain) and more amyloplasts per nodule. More than 2,000 bacterial isolates from nodules of P. vulgaris and L. esculenta formed by the transconjugants were analyzed by different criteria. Not a single rhizobium could be detected. Our results show that R. phaseoli plasmids may be expressed in the A. tumefaciens background and direct the formation of effective, differentiated nodules. Images PMID:3584072

Stability, Entrapment and Variant Formation of Salmonella Genomic Island 1

PubMed Central

Kiss, János; Nagy, Béla; Olasz, Ferenc

2012-01-01

Background The Salmonella genomic island 1 (SGI1) is a 42.4 kb integrative mobilizable element containing several antibiotic resistance determinants embedded in a complex integron segment In104. The numerous SGI1 variants identified so far, differ mainly in this segment and the explanations of their emergence were mostly based on comparative structure analyses. Here we provide experimental studies on the stability, entrapment and variant formation of this peculiar gene cluster originally found in S. Typhimurium. Methodology/Principal Findings Segregation and conjugation tests and various molecular techniques were used to detect the emerging SGI1 variants in Salmonella populations of 17 Salmonella enterica serovar Typhimurium DT104 isolates from Hungary. The SGI1s in these isolates proved to be fully competent in excision, conjugal transfer by the IncA/C helper plasmid R55, and integration into the E. coli chromosome. A trap vector has been constructed and successfully applied to capture the island on a plasmid. Monitoring of segregation of SGI1 indicated high stability of the island. SGI1-free segregants did not accumulate during long-term propagation, but several SGI1 variants could be obtained. Most of them appeared to be identical to SGI1-B and SGI1-C, but two new variants caused by deletions via a short-homology-dependent recombination process have also been detected. We have also noticed that the presence of the conjugation helper plasmid increased the formation of these deletion variants considerably. Conclusions/Significance Despite that excision of SGI1 from the chromosome was proven in SGI1+ Salmonella populations, its complete loss could not be observed. On the other hand, we demonstrated that several variants, among them two newly identified ones, arose with detectable frequencies in these populations in a short timescale and their formation was promoted by the helper plasmid. This reflects that IncA/C helper plasmids are not only involved in the horizontal spreading of SGI1, but may also contribute to its evolution. PMID:22384263

The ability of a collagen/calcium phosphate scaffold to act as its own vector for gene delivery and to promote bone formation via transfection with VEGF(165).

PubMed

Keeney, Michael; van den Beucken, Jeroen J J P; van der Kraan, Peter M; Jansen, John A; Pandit, Abhay

2010-04-01

Collagen/calcium phosphate scaffolds have been used for bone reconstruction due to their inherent similarities to the bone extracellular matrix. Calcium phosphate alone has also been used as a non-viral vector for gene delivery. The aim of this study was to determine the capability of a collagen/calcium phosphate scaffold to deliver naked plasmid DNA and mediate transfection in vivo. The second goal of the study was to deliver a plasmid encoding vascular endothelial growth factor(165) (pVEGF(165)) to promote angiogenesis, and hence bone formation, in a mouse intra-femoral model. The delivery of naked plasmid DNA resulted in a 7.6-fold increase in mRNA levels of beta-Galactosidase compared to the delivery of plasmid DNA complexed with a partially degraded PAMAM dendrimer (dPAMAM) in a subcutaneous murine model. When implanted in a muirne intra-femoral model, the delivery of pVEGF(165) resulted in a 2-fold increase in bone volume at the defect site relative to control scaffolds without pVEGF(165). It was concluded that a collagen/calcium phosphate scaffold can mediate transfection without the use of additional transfection vectors and can promote bone formation in a mouse model via the delivery of pVEGF(165). 2009 Elsevier Ltd. All rights reserved.

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

PubMed

Conlan, Sean; 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; Segre, Julia A

2016-06-28

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. In 2011, the NIH Clinical Center had a nosocomial outbreak involving 19 patients who became colonized or infected with blaKPC-positive Klebsiella pneumoniae Patients who have intestinal colonization with blaKPC-positive K. pneumoniae are at risk for developing infections that are difficult or nearly impossible to treat with existing antibiotic options. Two of those patients remained colonized with blaKPC-positive Klebsiella pneumoniae for over a year, leading to the initiation of a detailed genomic analysis exploring mixed colonization, plasmid recombination, and plasmid diversification. Whole-genome sequence analysis identified a variety of changes, both subtle and large, in the blaKPC-positive organisms. Long-term colonization of patients with blaKPC-positive Klebsiella pneumoniae creates new opportunities for horizontal gene transfer of plasmids encoding antibiotic resistance genes and poses complications for the delivery of health care. Copyright © 2016 Conlan et al.

Formation of Stable Cationic Lipid/DNA Complexes for Gene Transfer

NASA Astrophysics Data System (ADS)

Hofland, Hans E. J.; Shephard, Lee; Sullivan, Sean M.

1996-07-01

Stable cationic lipid/DNA complexes were formed by solubilizing cationic liposomes with 1% octylglucoside and complexing a DNA plasmid with the lipid in the presence of detergent. Removal of the detergent by dialysis yielded a lipid/DNA suspension that was able to transfect tissue culture cells up to 90 days after formation with no loss in activity. Similar levels of gene transfer were obtained by mixing the cationic lipid in a liposome form with DNA just prior to cell addition. However, expression was completely lost 24 hr after mixing. The transfection efficiency of the stable complex in 15% fetal calf serum was 30% of that obtained in the absence of serum, whereas the transient complex was completely inactivated with 2% fetal calf serum. A 90-day stability study comparing various storage conditions showed that the stable complex could be stored frozen or as a suspension at 4 degrees C with no loss in transfection efficiency. Centrifugation of the stable complex produced a pellet that contained approximately 90% of the DNA and 10% of the lipid. Transfection of cells with the resuspended pellet and the supernatant showed that the majority of the transfection activity was in the pellet and all the toxicity was in the supernatant. Formation of a stable cationic lipid/DNA complex has produced a transfection vehicle that can be stored indefinitely, can be concentrated with no loss in transfection efficiency, and the toxicity levels can be greatly reduced when the active complex is isolated from the uncomplexed lipid.

R-factor cointegrate formation in Salmonella typhimurium bacteriophage type 201 strains.

PubMed Central

Helmuth, R; Stephan, R; Bulling, E; van Leeuwen, W J; van Embden, J D; Guinée, P A; Portnoy, D; Falkow, S

1981-01-01

The genetic and molecular properties of the plasmids in Salmonella typhimurium phase type 201 isolated are described. Such strains are resistant to streptomycin, tetracycline, chloramphenicol, ampicillin, kanamycin, and several other antimicrobial drugs, and are highly pathogenic for calves. These strains have been encountered with increasing frequency since 1972 in West Germany and The Netherlands. We show that isolates of this phage type constitute a very homogeneous group with regard to their extrachromosomal elements. These bacteria carry three small plasmids: pRQ3, a 4.2-megadalton (Md) colicinogenic plasmid; pRQ4, 3.4-Md plasmid that interferes with the propagation of phages; and pRQ5, a 3.2-Md cryptic plasmid. Tetracycline resistance resides on a conjugative 120-MD plasmid pRQ1, belonging to the incompatibility class H2. Other antibiotic resistance determinants are encoded by a nonconjugative 108-Md plasmid pRQ2. Transfer of multiple-antibiotic resistance to appropriate recipient strains was associated with the appearance of a 230-Md plasmid, pRQ6. It appears that pRQ6 is a stable cointegrate of pRQ1 and pRQ2. This cointegrate plasmid was transferable with the same efficiency as pRQ1. Other conjugative plasmids could mobilize pRQ2, but stable cointegrates were not detected in the transconjugants. Phase type 201 strains carry a prophage, and we show that phage pattern 201 reflects the interference with propagation of typing phages effected by this prophage and plasmid pRQ4 in strains of phage type 201. Images PMID:7012128

Gene transfer device utilizing micron-spiked electrodes produced by the self-organization phenomenon of Fe-alloy.

PubMed

Miyano, Naoki; Inoue, Yuuki; Teramura, Yuji; Fujii, Keisuke; Tsumori, Fujio; Iwata, Hiroo; Kotera, Hidetoshi

2008-07-01

In the diffusional phase transformation of two-phase alloys, the new phase precipitates form the matrix phase at specific temperatures, followed by the formation of a mixed microstructure comprising the precipitate and the matrix. It has been found that by specific chemical-etching treatment, the precipitate in Fe-25Cr-6Ni alloy projects substantially and clusters at the surface. The configuration of the precipitate has an extremely high aspect ratio: it is several microns in width and several tens of microns in length (known as micron-spiked). This study targets the development of a gene transfer device with a micro-spike produced based on the self-organization phenomenon of the Fe-25Cr-6Ni alloy. With this spike-projected device, we tried to efficiently transfer plasmid DNA into adherent cells by electric pulse-triggered gene transfer using a plasmid-loaded electrode (electroporation-based reverse transfection). The spiked structure was applied to a substrate of the device to allow efficient gene transfer into adherent cells, although the general substrate was flat and had a smooth surface. The results suggest that this unique spike-projected device has potential applications in gene transfer devices for the analysis of the human genome in the post-genome period.

Fate of plasmids containing Mu DNA: chromosome association and mobilization.

PubMed

Bialy, H; Waggoner, B T; Pato, M L

1980-01-01

The fluorescent dye, diamidinophenylindole-dihydrochloride (DAPI) can be added to CsCl gradients to enhance the density resolution of DNA species, independent of their topological configurations. When Proteus mirabilis and Escherichia coli strains carrying an RP4::Mucts plasmid were examined with the use of such a technique, it was found that after thermal induction of the prophage essentially al of the plasmid DNA became associated with the chromosome. This quantitative association is detergent-RNase- and pronase-resistant and dependent on the expression of Mu genes. The association is temporally, and probably functionally, correlated with the onset of Mu DNA replication. Genetic studies with F'::mini Mu plasmids indicate that some of the association results in stable Hfr formation, and does not require the product of Mu gene B.

Large-scale production of lipoplexes with long shelf-life.

PubMed

Clement, Jule; Kiefer, Karin; Kimpfler, Andrea; Garidel, Patrick; Peschka-Süss, Regine

2005-01-01

The instability of lipoplex formulations is a major obstacle to overcome before their commercial application in gene therapy. In this study, a continuous mixing technique for the large-scale preparation of lipoplexes followed by lyophilisation for increased stability and shelf-life has been developed. Lipoplexes were analysed for transfection efficiency and cytotoxicity in human aorta smooth muscle cells (HASMC) and a rat smooth muscle cell line (A-10 SMC). Homogeneity of lipid/DNA-products was investigated by photon correlation spectroscopy (PCS) and cryotransmission electron microscopy (cryo-TEM). Studies have been undertaken with DAC-30, a composition of 3beta-[N-(N,N'-dimethylaminoethane)-carbamoyl]-cholesterol (DAC-Chol) and dioleylphosphatidylethanolamine (DOPE) and a green fluorescent protein (GFP) expressing marker plasmid. A continuous mixing technique was compared to the small-scale preparation of lipoplexes by pipetting. Individual steps of the continuous mixing process were evaluated in order to optimise the manufacturing technique: lipid/plasmid ratio, composition of transfection medium, pre-treatment of the lipid, size of the mixing device, mixing procedure and the influence of the lyophilisation process. It could be shown that the method developed for production of lipoplexes on a large scale under sterile conditions led to lipoplexes with good transfection efficiencies combined with low cytotoxicity, improved characteristics and long shelf-life.

Cloning of hydrogenase genes and fine structure analysis of an operon essential for H/sub 2/ metabolism in Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sankar, P.; Lee, J.H.; Shanmugam, K.T.

1985-04-01

Escherichia coli has two unlinked genes that code for hydrogenase synthesis and activity. The DNA fragments containing the two genes (hydA and hydB) were cloned into a plasmid vector, pBR322. The plasmids containing the hyd genes (pSE-290 and pSE-111 carrying the hydA and hydB genes, respectively) were used to genetically map a total of 51 mutant strains with defects in hydrogenase activity. A total of 37 mutants carried a mutation in the hydB gene, whereas the remaining 14 hyd were hydA. This complementation analysis also established the presence of two new genes, so far unidentified, one coding for formate dehydrogenase-2more » (fdv) and another producing an electron transport protein (fhl) coupling formate dehydrogenase-2 to hydrogenase. Three of the four genes, hydB, fhl, and fdv, may constitute a single operon, and all three genes are carried by a 5.6-kilobase-pair chromosomal DNA insert in plasmid pSE-128. Plasmids carrying a part of this 5.6-kilobase-pair DNA (pSE-130) or fragments derived from this DNA in different orientations (pSE-126 and pSE-129) inhibited the production of active formate hydrogenlyase. This inhibition occurred even in a prototrophic E. coli, strain K-10, but only during an early induction period. These results, based on complementation analysis with cloned DNA fragments, show that both hydA and hydB genes are essential for the production of active hydrogenase. For the expression of active formate hydrogenlyase, two other gene products, fhl and fdv are also needed. All four genes map between 58 and 59 min in the E. coli chromosome.« less

PEGylation enhances tumor targeting of plasmid DNA by an artificial cationized protein with repeated RGD sequences, Pronectin.

PubMed

Hosseinkhani, Hossein; Tabata, Yasuhiko

2004-05-31

The objective of this study is to investigate feasibility of a non-viral gene carrier with repeated RGD sequences (Pronectin F+) in tumor targeting for gene expression. The Pronectin F+ was cationized by introducing spermine (Sm) to the hydroxyl groups to allow to polyionically complex with plasmid DNA. The cationized Pronectin F+ prepared was additionally modified with poly(ethylene glycol) (PEG) molecules which have active ester and methoxy groups at the terminal, to form various PEG-introduced cationized Pronectin F+. The cationized Pronectin F+ with or without PEGylation at different extents was mixed with a plasmid DNA of LacZ to form respective cationized Pronectin F+-plasmid DNA complexes. The plasmid DNA was electrophoretically complexed with cationized Pronectin F+ and PEG-introduced cationized Pronectin F+, irrespective of the PEGylation extent, although the higher N/P ratio of complexes was needed for complexation with the latter Pronectin F+. The molecular size and zeta potential measurements revealed that the plasmid DNA was reduced in size to about 250 nm and the charge was changed to be positive by the complexation with cationized Pronectin F+. For the complexation with PEG-introduced cationized Pronectin F+, the charge of complex became neutral being almost 0 mV with the increasing PEGylation extents, while the molecular size was similar to that of cationized Pronectin F+. When cationized Pronectin F+-plasmid DNA complexes with or without PEGylation were intravenously injected to mice carrying a subcutaneous Meth-AR-1 fibrosarcoma mass, the PEG-introduced cationized Pronectin F+-plasmid DNA complex specifically enhanced the level of gene expression in the tumor, to a significantly high extent compared with the cationized Pronectin F+-plasmid DNA complexes and free plasmid DNA. The enhanced level of gene expression depended on the percentage of PEG introduced, the N/P ratio, and the plasmid DNA dose. A fluorescent microscopic study revealed that the localization of plasmid DNA in the tumor tissue was observed only for the PEG-introduced cationized Pronectin F+-plasmid DNA complex injected. We conclude that the PEGylation of cationized Pronectin F+ is a promising way to enable the plasmid DNA to target to the tumor for gene expression. Coyright 2004 Elsevier B.V.

A Transmissible Plasmid Controlling Camphor Oxidation in Pseudomonas putida

PubMed Central

Rheinwald, J. G.; Chakrabarty, A. M.; Gunsalus, I. C.

1973-01-01

Earlier papers demonstrated an extensive genetic exchange among fluorescent Pseudomonads; this one documents for genes specifying enzymes of peripheral dissimilation an extrachromosomal array, segregation, and frequent interstrain transfer. An hypothesis is presented of a general mechanism for the formation and maintenance of metabolic diversity. The example used, the path of oxidative cleavage of the carbocyclic rings of the bicyclic monoterpene D- and L-camphor, terminates in acetate release and isobutyrate chain debranching. By transduction, two gene linkage groups are shown for the reactions before and after isobutyrate. The group for reactions before isobutyrate is plasmid borne, contransferable by conjugation, mitomycin curable, and shows a higher segregation rate from cells that are multiplasmid rather than carrying a single plasmid. The genes that code for isobutyrate and essential anaplerotic and amphibolic metabolism are chromosomal. By conjugation plasmid-borne genes are transferred at a higher frequency than are chromosomal, and are transferred in homologous crosses more frequently than between heterologous species. Most isobutyrate-positive fluorescent pseudomonad strains will accept and express the camphor plasmid. PMID:4351810

Construction of plasmid-free Escherichia coli for the production of arabitol-free xylitol from corncob hemicellulosic hydrolysate.

PubMed

Su, Buli; Zhang, Zhe; Wu, Mianbin; Lin, Jianping; Yang, Lirong

2016-05-26

High costs and low production efficiency are a serious constraint to bio-based xylitol production. For industrial-scale production of xylitol, a plasmid-free Escherichia coli for arabitol-free xylitol production from corncob hemicellulosic hydrolysate has been constructed. Instead of being plasmid and inducer dependent, this strain relied on multiple-copy integration of xylose reductase (XR) genes into the chromosome, where their expression was controlled by the constitutive promoter P43. In addition, to minimize the flux from L-arabinose to arabitol, two strategies including low XR total activity and high selectivity of XR has been adopted. Arabitol was significantly decreased using plasmid-free strain which had lower XR total activity and an eight point-mutations of XR with a 27-fold lower enzyme activity toward L-arabinose was achieved. The plasmid-free strain in conjunction with this mutant XR can completely eliminate arabitol formation in xylitol production. In fed-batch fermentation, this plasmid-free strain produced 143.8 g L(-1) xylitol at 1.84 g L(-1) h(-1) from corncob hemicellulosic hydrolysate. From these results, we conclude that this route by plasmid-free E. coli has potential to become a commercially viable process for xylitol production.

Construction of plasmid-free Escherichia coli for the production of arabitol-free xylitol from corncob hemicellulosic hydrolysate

PubMed Central

Su, Buli; Zhang, Zhe; Wu, Mianbin; Lin, Jianping; Yang, Lirong

2016-01-01

High costs and low production efficiency are a serious constraint to bio-based xylitol production. For industrial-scale production of xylitol, a plasmid-free Escherichia coli for arabitol-free xylitol production from corncob hemicellulosic hydrolysate has been constructed. Instead of being plasmid and inducer dependent, this strain relied on multiple-copy integration of xylose reductase (XR) genes into the chromosome, where their expression was controlled by the constitutive promoter P43. In addition, to minimize the flux from L-arabinose to arabitol, two strategies including low XR total activity and high selectivity of XR has been adopted. Arabitol was significantly decreased using plasmid-free strain which had lower XR total activity and an eight point-mutations of XR with a 27-fold lower enzyme activity toward L-arabinose was achieved. The plasmid-free strain in conjunction with this mutant XR can completely eliminate arabitol formation in xylitol production. In fed-batch fermentation, this plasmid-free strain produced 143.8 g L−1 xylitol at 1.84 g L−1 h−1 from corncob hemicellulosic hydrolysate. From these results, we conclude that this route by plasmid-free E. coli has potential to become a commercially viable process for xylitol production. PMID:27225023

Liposomal lipid and plasmid DNA delivery to B16/BL6 tumors after intraperitoneal administration of cationic liposome DNA aggregates.

PubMed

Reimer, D L; Kong, S; Monck, M; Wyles, J; Tam, P; Wasan, E K; Bally, M B

1999-05-01

The transfer of plasmid expression vectors to cells is essential for transfection after administration of lipid-based DNA formulations (lipoplexes). A murine i.p. B16/BL6 tumor model was used to characterize DNA delivery, liposomal lipid delivery, and gene transfer after regional (i.p.) administration of free plasmid DNA and DNA lipoplexes. DNA lipoplexes were prepared using cationic dioleoyldimethylammonium chloride/dioleoylphosphatidylethanolamine (50:50 mol ratio) liposomes mixed with plasmid DNA (1 microgram DNA/10 nmol lipid). The plasmid used contained the chloramphenicol acetyltransferase gene and chloramphenicol acetyltransferase expression (mU/g tumor) was measured to estimate transfection efficiency. Tumor-associated DNA and liposomal lipid levels were measured to estimate the efficiency of lipid-mediated DNA delivery to tumors. Plasmid DNA delivery was estimated using [3H]-labeled plasmid as a tracer, dot blot analysis, and/or Southern analysis. Liposomal lipid delivery was estimated using [14C]-dioleoylphosphatidylethanolamine as a liposomal lipid marker. Gene expression in the B16/BL6 tumors was highly variable, with values ranging from greater than 2,000 mU/g tumor to less than 100 mU/g tumor. There was a tendency to observe enhanced transfection in small (<250 mg) tumors. Approximately 18% of the injected dose of DNA was associated with these small tumors 2 h after i.p. administration. Southern analysis of extracted tumor DNA indicated that plasmid DNA associated with tumors was intact 24 h after administration. DNA and associated liposomal lipid are efficiently bound to tumors after regional administration; however, it is unclear whether delivery is sufficient to abet internalization and appropriate subcellular localization of the expression vector.

Formation of AAV Single Stranded DNA Genome from a Circular Plasmid in Saccharomyces cerevisiae

PubMed Central

Cervelli, Tiziana; Backovic, Ana; Galli, Alvaro

2011-01-01

Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3+ clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway. PMID:21853137

Formation of AAV single stranded DNA genome from a circular plasmid in Saccharomyces cerevisiae.

PubMed

Cervelli, Tiziana; Backovic, Ana; Galli, Alvaro

2011-01-01

Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3(+) clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway.

Garvicin A, a Novel Class IId Bacteriocin from Lactococcus garvieae That Inhibits Septum Formation in L. garvieae Strains

PubMed Central

Cárdenas, Nivia; Martínez, Beatriz; Ruiz-Barba, José Luis; Fernández-Garayzábal, José F.; Rodríguez, Juan M.; Gibello, Alicia

2013-01-01

Lactococcus garvieae 21881, isolated in a human clinical case, produces a novel class IId bacteriocin, garvicin A (GarA), which is specifically active against other L. garvieae strains, including fish- and bovine-pathogenic isolates. Purification from active supernatants, sequence analyses, and plasmid-curing experiments identified pGL5, one of the five plasmids found in L. garvieae [M. Aguado-Urda et al., PLoS One 7(6):e40119, 2012], as the coding plasmid for the structural gene of GarA (lgnA), its putative immunity protein (lgnI), and the ABC transporter and its accessory protein (lgnC and lgnD). Interestingly, pGL5-cured strains were still resistant to GarA. Other putative bacteriocins encoded by the remaining plasmids were not detected during purification, pointing to GarA as the main inhibitor secreted by L. garvieae 21881. Mode-of-action studies revealed a potent bactericidal activity of GarA. Moreover, transmission microscopy showed that GarA seems to act by inhibiting septum formation in L. garvieae cells. This potent and species-specific inhibition by GarA holds promise for applications in the prevention or treatment of infections caused by pathogenic strains of L. garvieae in both veterinary and clinical settings. PMID:23666326

Structures of actin-like ParM filaments show architecture of plasmid-segregating spindles.

PubMed

Bharat, Tanmay A M; Murshudov, Garib N; Sachse, Carsten; Löwe, Jan

2015-07-02

Active segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments. Growing ParMRC spindles push sister plasmids to the cell poles. Here, using modern electron cryomicroscopy methods, we investigate the structures and arrangements of ParM filaments in vitro and in cells, revealing at near-atomic resolution how subunits and filaments come together to produce the simplest known mitotic machinery. To understand the mechanism of dynamic instability, we determine structures of ParM filaments in different nucleotide states. The structure of filaments bound to the ATP analogue AMPPNP is determined at 4.3 Å resolution and refined. The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions. Also using electron cryomicroscopy, we reconstruct ParM doublets forming antiparallel spindles. Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.

Biofilm Formation Potential of Heat-Resistant Escherichia coli Dairy Isolates and the Complete Genome of Multidrug-Resistant, Heat-Resistant Strain FAM21845

PubMed Central

Schmid, Michael; Kulli, Sandra; Schneeberger, Kerstin; Naskova, Javorka; Knøchel, Susanne; Ahrens, Christian H.

2017-01-01

ABSTRACT We tested the biofilm formation potential of 30 heat-resistant and 6 heat-sensitive Escherichia coli dairy isolates. Production of curli and cellulose, static biofilm formation on polystyrene (PS) and stainless steel surfaces, biofilm formation under dynamic conditions (Bioflux), and initial adhesion rates (IAR) were evaluated. Biofilm formation varied greatly between strains, media, and assays. Our results highlight the importance of the experimental setup in determining biofilm formation under conditions of interest, as correlation between different assays was often not a given. The heat-resistant, multidrug-resistant (MDR) strain FAM21845 showed the strongest biofilm formation on PS and the highest IAR and was the only strain that formed significant biofilms on stainless steel under conditions relevant to the dairy industry, and it was therefore fully sequenced. Its chromosome is 4.9 Mb long, and it harbors a total of five plasmids (147.2, 54.2, 5.8, 2.5, and 1.9 kb). The strain carries a broad range of genes relevant to antimicrobial resistance and biofilm formation, including some on its two large conjugative plasmids, as demonstrated in plate mating assays. IMPORTANCE In biofilms, cells are embedded in an extracellular matrix that protects them from stresses, such as UV radiation, osmotic shock, desiccation, antibiotics, and predation. Biofilm formation is a major bacterial persistence factor of great concern in the clinic and the food industry. Many tested strains formed strong biofilms, and especially strains such as the heat-resistant, MDR strain FAM21845 may pose a serious issue for food production. Strong biofilm formation combined with diverse resistances (some encoded on conjugative plasmids) may allow for increased persistence, coselection, and possible transfer of these resistance factors. Horizontal gene transfer may conceivably occur in the food production setting or the gastrointestinal tract after consumption. PMID:28550056

Biofilm Formation Potential of Heat-Resistant Escherichia coli Dairy Isolates and the Complete Genome of Multidrug-Resistant, Heat-Resistant Strain FAM21845.

PubMed

Marti, Roger; Schmid, Michael; Kulli, Sandra; Schneeberger, Kerstin; Naskova, Javorka; Knøchel, Susanne; Ahrens, Christian H; Hummerjohann, Jörg

2017-08-01

We tested the biofilm formation potential of 30 heat-resistant and 6 heat-sensitive Escherichia coli dairy isolates. Production of curli and cellulose, static biofilm formation on polystyrene (PS) and stainless steel surfaces, biofilm formation under dynamic conditions (Bioflux), and initial adhesion rates (IAR) were evaluated. Biofilm formation varied greatly between strains, media, and assays. Our results highlight the importance of the experimental setup in determining biofilm formation under conditions of interest, as correlation between different assays was often not a given. The heat-resistant, multidrug-resistant (MDR) strain FAM21845 showed the strongest biofilm formation on PS and the highest IAR and was the only strain that formed significant biofilms on stainless steel under conditions relevant to the dairy industry, and it was therefore fully sequenced. Its chromosome is 4.9 Mb long, and it harbors a total of five plasmids (147.2, 54.2, 5.8, 2.5, and 1.9 kb). The strain carries a broad range of genes relevant to antimicrobial resistance and biofilm formation, including some on its two large conjugative plasmids, as demonstrated in plate mating assays. IMPORTANCE In biofilms, cells are embedded in an extracellular matrix that protects them from stresses, such as UV radiation, osmotic shock, desiccation, antibiotics, and predation. Biofilm formation is a major bacterial persistence factor of great concern in the clinic and the food industry. Many tested strains formed strong biofilms, and especially strains such as the heat-resistant, MDR strain FAM21845 may pose a serious issue for food production. Strong biofilm formation combined with diverse resistances (some encoded on conjugative plasmids) may allow for increased persistence, coselection, and possible transfer of these resistance factors. Horizontal gene transfer may conceivably occur in the food production setting or the gastrointestinal tract after consumption. Copyright © 2017 Marti et al.

Gene Electrotransfer of Plasmid with Tissue Specific Promoter Encoding shRNA against Endoglin Exerts Antitumor Efficacy against Murine TS/A Tumors by Vascular Targeted Effects.

PubMed

Stimac, Monika; Dolinsek, Tanja; Lampreht, Ursa; Cemazar, Maja; Sersa, Gregor

2015-01-01

Vascular targeted therapies, targeting specific endothelial cell markers, are promising approaches for the treatment of cancer. One of the targets is endoglin, transforming growth factor-β (TGF-β) co-receptor, which mediates proliferation, differentiation and migration of endothelial cells forming neovasculature. However, its specific, safe and long-lasting targeting remains the challenge. Therefore, in our study we evaluated the transfection efficacy, vascular targeted effects and therapeutic potential of the plasmid silencing endoglin with the tissue specific promoter, specific for endothelial cells marker endothelin-1 (ET) (TS plasmid), in comparison to the plasmid with constitutive promoter (CON plasmid), in vitro and in vivo. Tissue specificity of TS plasmid was demonstrated in vitro on several cell lines, and its antiangiogenic efficacy was demonstrated by reducing tube formation of 2H11 endothelial cells. In vivo, on a murine mammary TS/A tumor model, we demonstrated good antitumor effect of gene electrotransfer (GET) of either of both plasmids in treatment of smaller tumors still in avascular phase of growth, as well as on bigger tumors, already well vascularized. In support to the observations on predominantly vascular targeted effects of endoglin, histological analysis has demonstrated an increase in necrosis and a decrease in the number of blood vessels in therapeutic groups. A significant antitumor effect was observed in tumors in avascular and vascular phase of growth, possibly due to both, the antiangiogenic and the vascular disrupting effect. Furthermore, the study indicates on the potential use of TS plasmid in cancer gene therapy since the same efficacy as of CON plasmid was determined.

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

Identification of an amino acid residue on influenza C virus M1 protein responsible for formation of the cord-like structures of the virus.

PubMed

Muraki, Yasushi; Washioka, Hiroshi; Sugawara, Kanetsu; Matsuzaki, Yoko; Takashita, Emi; Hongo, Seiji

2004-07-01

Influenza C virus-like particles (VLPs) have been generated from cloned cDNAs. A cDNA of the green fluorescent protein (GFP) gene in antisense orientation was flanked by the 5' and 3' non-coding regions of RNA segment 5 of the influenza C virus. The cDNA cassette was inserted between an RNA polymerase I promoter and terminator of the Pol I vector. This plasmid DNA was transfected into 293T cells together with plasmids encoding virus proteins of C/Ann Arbor/1/50 or C/Yamagata/1/88. Transfer of the supernatants of the transfected 293T cells to HMV-II cells resulted in GFP expression in the HMV-II cells. The quantification of the GFP-positive HMV-II cells indicated the presence of approximately 10(6) VLPs (ml supernatant)(-1). Cords 50-300 microm in length were observed on transfected 293T cells, although the cords were not observed when the plasmid for M1 protein of C/Ann Arbor/1/50 was replaced with that of C/Taylor/1233/47. A series of transfection experiments with plasmids encoding M1 mutants of C/Ann Arbor/1/50 or C/Taylor/1233/47 showed that an amino acid at residue 24 of the M1 protein is responsible for cord formation. This finding provides direct evidence for a previous hypothesis that M1 protein is involved in the formation of cord-like structures protruding from the C/Yamagata/1/88-infected cells. Evidence was obtained by electron microscopy that transfected cells bearing cords produced filamentous VLPs, suggesting the potential role of the M1 protein in determining the filamentous/spherical morphology of influenza C virus.

Cryo-EM reconstruction of AlfA from Bacillus subtilis reveals the structure of a simplified actin-like filament at 3.4-Å resolution.

PubMed

Szewczak-Harris, Andrzej; Löwe, Jan

2018-03-27

Low copy-number plasmid pLS32 of Bacillus subtilis subsp. natto contains a partitioning system that ensures segregation of plasmid copies during cell division. The partitioning locus comprises actin-like protein AlfA, adaptor protein AlfB, and the centromeric sequence parN Similar to the ParMRC partitioning system from Escherichia coli plasmid R1, AlfA filaments form actin-like double helical filaments that arrange into an antiparallel bipolar spindle, which attaches its growing ends to sister plasmids through interactions with AlfB and parN Because, compared with ParM and other actin-like proteins, AlfA is highly diverged in sequence, we determined the atomic structure of nonbundling AlfA filaments to 3.4-Å resolution by cryo-EM. The structure reveals how the deletion of subdomain IIB of the canonical actin fold has been accommodated by unique longitudinal and lateral contacts, while still enabling formation of left-handed, double helical, polar and staggered filaments that are architecturally similar to ParM. Through cryo-EM reconstruction of bundling AlfA filaments, we obtained a pseudoatomic model of AlfA doublets: the assembly of two filaments. The filaments are antiparallel, as required by the segregation mechanism, and exactly antiphasic with near eightfold helical symmetry, to enable efficient doublet formation. The structure of AlfA filaments and doublets shows, in atomic detail, how deletion of an entire domain of the actin fold is compensated by changes to all interfaces so that the required properties of polymerization, nucleotide hydrolysis, and antiparallel doublet formation are retained to fulfill the system's biological raison d'être.

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

PubMed Central

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

1995-01-01

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

Inhibition of gamma-radiation induced DNA damage in plasmid pBR322 by TMG, a water-soluble derivative of vitamin E.

PubMed

Rajagopalan, Rema; Wani, Khalida; Huilgol, Nagaraj G; Kagiya, Tsutomu V; Nair, Cherupally K Krishnan

2002-06-01

Alpha-tocopherol monoglucoside (TMG), a water-soluble derivative of alpha-tocopherol, has been examined for its ability to protect DNA against radiation-induced strand breaks. Gamma radiation, up to a dose of 6 Gy (dose rate, 0.7 Gy/minute), induced a dose-dependent increase in single strand breaks (SSBs) in plasmid pBR322 DNA. TMG inhibited the formation of gamma-radiation induced DNA single strand breaks (SSBs) in a concentration-dependent manner; 500 microM of TMG protected the single strand breaks completely. It also protected thymine glycol formation induced by gamma-radiation in a dose-dependent manner, based on an estimation of thymine glycol by HPLC.

Characterization of replication and conjugation of plasmid pWTY27 from a widely distributed Streptomyces species

PubMed Central

2012-01-01

Background Streptomyces species are widely distributed in natural habitats, such as soils, lakes, plants and some extreme environments. Replication loci of several Streptomyces theta-type plasmids have been reported, but are not characterized in details. Conjugation loci of some Streptomyces rolling-circle-type plasmids are identified and mechanism of conjugal transferring are described. Results We report the detection of a widely distributed Streptomyces strain Y27 and its indigenous plasmid pWTY27 from fourteen plants and four soil samples cross China by both culturing and nonculturing methods. The complete nucleotide sequence of pWTY27 consisted of 14,288 bp. A basic locus for plasmid replication comprised repAB genes and an adjacent iteron sequence, to a long inverted-repeat (ca. 105 bp) of which the RepA protein bound specifically in vitro, suggesting that RepA may recognize a second structure (e.g. a long stem-loop) of the iteron DNA. A plasmid containing the locus propagated in linear mode when the telomeres of a linear plasmid were attached, indicating a bi-directional replication mode for pWTY27. As for rolling-circle plasmids, a single traA gene and a clt sequence (covering 16 bp within traA and its adjacent 159 bp) on pWTY27 were required for plasmid transfer. TraA recognized and bound specifically to the two regions of the clt sequence, one containing all the four DC1 of 7 bp (TGACACC) and one DC2 (CCCGCCC) and most of IC1, and another covering two DC2 and part of IC1, suggesting formation of a high-ordered DNA-protein complex. Conclusions This work (i) isolates a widespread Streptomyces strain Y27 and sequences its indigenous theta-type plasmid pWTY27; (ii) identifies the replication and conjugation loci of pWTY27 and; (iii) characterizes the binding sequences of the RepA and TraA proteins. PMID:23134842

Peptides containing antigenic and cationic domains have enhanced, multivalent immunogenicity when bound to DNA vaccines.

PubMed

Riedl, Petra; Reimann, Jörg; Schirmbeck, Reinhold

2004-02-01

We explored strategies to codeliver DNA- and peptide-based vaccines in a way that enhances the immunogenicity of both components of the combination vaccine for T cells. Specific CD8(+) T cell responses to an antigenic peptide are primed when the peptide is fused to a cationic peptide domain that is bound to plasmid DNA or oligonucleotides (ODN; with or without CpG motifs). Plasmid DNA mixed with antigenic/cationic peptides or histones forms large complexes with different biological properties depending on the molar ratios of peptide/protein and polynucleotide. Complexes containing high (but not low) molar ratios of cationic peptide to DNA facilitate transfection (DNA uptake and expression of the plasmid-encoded product) of cells. In contrast, complexes containing low (but not high) molar ratios of cationic peptide to DNA prime potent multispecific T cell responses after a single intramuscular injection of the complexes. The general validity of this observation was confirmed mixing different antigenic/cationic peptides with different DNA vaccines. In these vaccine formulations, multispecific CD8(+) T cell responses specific for epitopes of the peptide- as well as the DNA-based vaccine were efficiently coprimed, together with humoral antibody responses to conformational determinants of large viral antigens encoded by the DNA vaccine. The data indicate that mixtures of DNA vaccines with antigenic, cationic peptides are immunogenic vaccine formulations particularly suited for the induction of multispecific T cell responses.

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

Rapid detection of biothreat agents based on cellular machinery.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lane, Todd W.; Gantt, Richard W.

This research addresses rapid and sensitive identification of biological agents in a complex background. We attempted to devise a method by which the specificity of the cellular transcriptional machinery could be used to detect and identify bacterial bio-terror agents in a background of other organisms. Bacterial cells contain RNA polymerases and transcription factors that transcribe genes into mRNA for translation into proteins. RNA polymerases in conjunction with transcription factors recognize regulatory elements (promoters) upstream of the gene. These promoters are, in many cases, recognized by the polymerase and transcription factor combinations of one species only. We have engineered a plasmid,more » for Escherichia coli, containing the virA promoter from the target species Shigella flexneri. This promoter was fused to a reporter gene Green Fluorescent Protein (GFP). In theory the indicator strain (carrying the plasmid) is mixed with the target strain and the two are lysed. The cellular machinery from both cells mixes and the GFP is produced. This report details the results of testing this system.« less

Design of antisense RNA constructs for downregulation of the acetone formation pathway of Clostridium acetobutylicum.

PubMed

Tummala, Seshu B; Welker, Neil E; Papoutsakis, Eleftherios T

2003-03-01

We investigated the effect of antisense RNA (asRNA) structural properties on the downregulation efficacy of enzymes in the acetone-formation pathway (acetoacetate decarboxylase [AADC] and coenzyme A-transferase [CoAT]) of Clostridium acetobutylicum strain ATCC 824. First, we generated three strains, C. acetobutylicum ATCC 824 (pADC38AS), 824(pADC68AS), and 824(pADC100AS), which contain plasmids that produce asRNAs of various lengths against the AADC (adc) transcript. Western analysis showed that all three strains exhibit low levels of AADC compared to the plasmid control [ATCC 824(pSOS95del)]. By using computational algorithms, the three different asRNAs directed toward AADC, along with previously reported clostridial asRNAs, were examined for structural features (free nucleotides and components). When the normalized metrics of these structural features were plotted against percent downregulation, only the component/nucleotide ratio correlated well with in vivo asRNA effectiveness. Despite the significant downregulation of AADC in these strains, there were no concomitant effects on acetone formation. These findings suggest that AADC does not limit acetone formation and, thus, we targeted next the CoAT. Using the component/nucleotide ratio as a selection parameter, we developed three strains [ATCC 824 (pCTFA2AS), 824(pCTFB1AS), and 824(pCOAT11AS)] which express asRNAs to downregulate either or both of the CoAT subunits. Compared to the plasmid control strain, these strains produced substantially low levels of acetone and butanol and Western blot analyses showed significantly low levels of both CoAT subunits. These results show that CoAT is the rate-limiting enzyme in acetone formation and strengthen the hypothesis that the component/nucleotide ratio is a predictive indicator of asRNA effectiveness.

Design of Antisense RNA Constructs for Downregulation of the Acetone Formation Pathway of Clostridium acetobutylicum

PubMed Central

Tummala, Seshu B.; Welker, Neil E.; Papoutsakis, Eleftherios T.

2003-01-01

We investigated the effect of antisense RNA (asRNA) structural properties on the downregulation efficacy of enzymes in the acetone-formation pathway (acetoacetate decarboxylase [AADC] and coenzyme A-transferase [CoAT]) of Clostridium acetobutylicum strain ATCC 824. First, we generated three strains, C. acetobutylicum ATCC 824 (pADC38AS), 824(pADC68AS), and 824(pADC100AS), which contain plasmids that produce asRNAs of various lengths against the AADC (adc) transcript. Western analysis showed that all three strains exhibit low levels of AADC compared to the plasmid control [ATCC 824(pSOS95del)]. By using computational algorithms, the three different asRNAs directed toward AADC, along with previously reported clostridial asRNAs, were examined for structural features (free nucleotides and components). When the normalized metrics of these structural features were plotted against percent downregulation, only the component/nucleotide ratio correlated well with in vivo asRNA effectiveness. Despite the significant downregulation of AADC in these strains, there were no concomitant effects on acetone formation. These findings suggest that AADC does not limit acetone formation and, thus, we targeted next the CoAT. Using the component/nucleotide ratio as a selection parameter, we developed three strains [ATCC 824 (pCTFA2AS), 824(pCTFB1AS), and 824(pCOAT11AS)] which express asRNAs to downregulate either or both of the CoAT subunits. Compared to the plasmid control strain, these strains produced substantially low levels of acetone and butanol and Western blot analyses showed significantly low levels of both CoAT subunits. These results show that CoAT is the rate-limiting enzyme in acetone formation and strengthen the hypothesis that the component/nucleotide ratio is a predictive indicator of asRNA effectiveness. PMID:12618456

Instability of plasmid DNA sequences: macro and micro evolution of the antibiotic resistance plasmid R6-5.

PubMed

Timmis, K N; Cabello, F; Andrés, I; Nordheim, A; Burkhardt, H J; Cohen, S N

1978-11-16

Detailed examination of the structure of cloned DNA fragments of the R6-5 antibiotic resistance plasmid has revealed a substantial degree of polynucleotide sequence heterogeneity and indicates that sequence rearrangements in plasmids and possible other replicons occur more frequently than has hitherto been appreciated. The sequences changes in cloned R6-5 fragments were shown in some instances to have occurred prior to cloning, i.e. existing in the original population of R6-5 molecules that was obtained from a single bacterial clone and by several different criteria judged to be homogeneous, and in others to have occurred either during the cloning procedure or during subsequent propagation of hybrid molecules. The molecular changes that are described involved insertion/deletion of the previously characterized IS2 insertion element, formation of a new inverted repeat structure probably by duplication of a preexisting R6-5 DNA sequence, sequence inversion, and loss and gain of restriction endonuclease cleavage sites.

A three-dimensional ParF meshwork assembles through the nucleoid to mediate plasmid segregation

PubMed Central

McLeod, Brett N.; Allison-Gamble, Gina E.; Barge, Madhuri T.; Tonthat, Nam K.; Schumacher, Maria A.; Hayes, Finbarr

2017-01-01

Abstract Genome segregation is a fundamental step in the life cycle of every cell. Most bacteria rely on dedicated DNA partition proteins to actively segregate chromosomes and low copy-number plasmids. Here, by employing super resolution microscopy, we establish that the ParF DNA partition protein of the ParA family assembles into a three-dimensional meshwork that uses the nucleoid as a scaffold and periodically shuttles between its poles. Whereas ParF specifies the territory for plasmid trafficking, the ParG partner protein dictates the tempo of ParF assembly cycles and plasmid segregation events by stimulating ParF adenosine triphosphate hydrolysis. Mutants in which this ParG temporal regulation is ablated show partition deficient phenotypes as a result of either altered ParF structure or dynamics and indicate that ParF nucleoid localization and dynamic relocation, although necessary, are not sufficient per se to ensure plasmid segregation. We propose a Venus flytrap model that merges the concepts of ParA polymerization and gradient formation and speculate that a transient, dynamic network of intersecting polymers that branches into the nucleoid interior is a widespread mechanism to distribute sizeable cargos within prokaryotic cells. PMID:28034957

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

PubMed

Rogers, Elizabeth E; Stenger, Drake C

2012-01-01

A ≈ 38kB plasmid (pXF-RIV5) was present in the Riv5 strain of Xylella fastidiosa subsp. multiplex isolated from ornamental plum in southern California. The complete nucleotide sequence of pXF-RIV5 is almost identical to that of pXFAS01 from X. fastidiosa subsp. fastidiosa strain M23; the two plasmids vary at only 6 nucleotide positions. BLAST searches and phylogenetic analyses indicate pXF-RIV5 and pXFAS01 share some similarity to chromosomal and plasmid (pXF51) sequences of X. fastidiosa subsp. pauca strain 9a5c and more distant similarity to plasmids from a wide variety of bacteria. Both pXF-RIV5 and pXFAS01 encode homologues of a complete Type IV secretion system involved in conjugation and DNA transfer among bacteria. Mating pair formation proteins (Trb) from Yersinia pseudotuberculosis IP31758 are the mostly closely related non-X. fastidiosa proteins to most of the Trb proteins encoded by pXF-RIV5 and pXFAS01. Unlike many bacterial conjugative plasmids, pXF-RIV5 and pXFAS01 do not carry homologues of known accessory modules that confer selective advantage on host bacteria. However, both plasmids encode seven hypothetical proteins of unknown function and possess a small transposon-associated region encoding a putative transposase and associated factor. Vegetative replication of pXF-RIV5 and pXFAS01 appears to be under control of RepA protein and both plasmids have an origin of DNA replication (oriV) similar to that of pRP4 and pR751 from Escherichia coli. In contrast, conjugative plasmids commonly encode TrfA and have an oriV similar to those found in IncP-1 incompatibility group plasmids. The presence of nearly identical plasmids in single strains from two distinct subspecies of X. fastidiosa is indicative of recent horizontal transfer, probably subsequent to the introduction of subspecies fastidiosa to the United States in the late 19(th) century.

A Conjugative 38 kB Plasmid Is Present in Multiple Subspecies of Xylella fastidiosa

PubMed Central

Rogers, Elizabeth E.; Stenger, Drake C.

2012-01-01

A ∼38kB plasmid (pXF-RIV5) was present in the Riv5 strain of Xylella fastidiosa subsp. multiplex isolated from ornamental plum in southern California. The complete nucleotide sequence of pXF-RIV5 is almost identical to that of pXFAS01 from X. fastidiosa subsp. fastidiosa strain M23; the two plasmids vary at only 6 nucleotide positions. BLAST searches and phylogenetic analyses indicate pXF-RIV5 and pXFAS01 share some similarity to chromosomal and plasmid (pXF51) sequences of X. fastidiosa subsp. pauca strain 9a5c and more distant similarity to plasmids from a wide variety of bacteria. Both pXF-RIV5 and pXFAS01 encode homologues of a complete Type IV secretion system involved in conjugation and DNA transfer among bacteria. Mating pair formation proteins (Trb) from Yersinia pseudotuberculosis IP31758 are the mostly closely related non-X. fastidiosa proteins to most of the Trb proteins encoded by pXF-RIV5 and pXFAS01. Unlike many bacterial conjugative plasmids, pXF-RIV5 and pXFAS01 do not carry homologues of known accessory modules that confer selective advantage on host bacteria. However, both plasmids encode seven hypothetical proteins of unknown function and possess a small transposon-associated region encoding a putative transposase and associated factor. Vegetative replication of pXF-RIV5 and pXFAS01 appears to be under control of RepA protein and both plasmids have an origin of DNA replication (oriV) similar to that of pRP4 and pR751 from Escherichia coli. In contrast, conjugative plasmids commonly encode TrfA and have an oriV similar to those found in IncP-1 incompatibility group plasmids. The presence of nearly identical plasmids in single strains from two distinct subspecies of X. fastidiosa is indicative of recent horizontal transfer, probably subsequent to the introduction of subspecies fastidiosa to the United States in the late 19th century. PMID:23251694

Cholesterol-conjugated supramolecular assemblies of low generations polyamidoamine dendrimers for enhanced EGFP plasmid DNA transfection

NASA Astrophysics Data System (ADS)

Golkar, Nasim; Samani, Soliman Mohammadi; Tamaddon, Ali Mohammad

2016-05-01

Aimed to prepare an enhanced gene delivery system with low cytotoxicity and high transfection efficiency, various cholesterol-conjugated derivates of low generation polyamidoamine (PAMAM) dendrimers were prepared. The conjugates were characterized by TNBS assay, FTIR, and 1H-NMR spectroscopy. Self-assembly of the dendrimer conjugates (G1-Chol, G2-Chol, and G3-Chol) was investigated by pyrene assay. Following formation of the complexes between enhanced green fluorescence protein plasmid and the dendrimer conjugates at various N (primary amine)/P (phosphate) mole ratios, plasmid condensation, biologic stability, cytotoxicity, and protein expression were investigated. The conjugates self-assembled into micellar dispersions with the critical micelle concentration values (<50 µg/ml) depending on the dendrimer generation and cholesterol/amine mole ratio. Cholesterol conjugation resulted in higher resistance of the condensed plasmid DNA in a competition assay with heparin sulfate. Also, the transfection efficiency was determined higher for the cholesterol conjugates than unmodified dendrimers in HepG2 cells, showing the highest for G2-Chol at 40 % degree of cholesterol modification (G2-Chol40 %) among various dendrimer generations. Interestingly, such conjugate showed a complete protection of plasmid against serum nucleases. Our results confirmed that the cholesterol conjugation to PAMAM dendrimers of low generations bearing little cytotoxicity improves their several physicochemical and biological characteristics required for an enhanced delivery of plasmid DNA into cells.

Non-invasive determination of conjugative transfer of plasmids bearing antibiotic-resistance genes in biofilm-bound bacteria: effects of substrate loading and antibiotic selection

PubMed Central

Ma, Hongyan; Bryers, James D.

2012-01-01

Biofilms cause much of all human microbial infections. Attempts to eradicate biofilm-based infections rely on disinfectants and antibiotics. Unfortunately, biofilm bacteria are significantly less responsive to antibiotic stressors than their planktonic counterparts. Sublethal doses of antibiotics can actually enhance biofilm formation. Here, we have developed a non-invasive microscopic image analyses to quantify plasmid conjugation within a developing biofilm. Corroborating destructive samples were analyzed by a cultivation-independent flow cytometry analysis and a selective plate count method to cultivate transconjugants. Increases in substrate loading altered biofilm 3-D architecture and subsequently affected the frequency of plasmid conjugation (decreases at least two times) in the absence of any antibiotic selective pressure. More importantly, donor populations in biofilms exposed to a sublethal dose of kanamycin exhibited enhanced transfer efficiency of plasmids containing the kanamycin resistance gene, up to tenfold. However, when stressed with a different antibiotic, imipenem, transfer of plasmids containing the kanR+ gene was not enhanced. These preliminary results suggest biofilm bacteria “sense” antibiotics to which they are resistant, which enhances the spread of that resistance. Confocal scanning microscopy coupled with our non-invasive image analysis was able to estimate plasmid conjugative transfer efficiency either averaged over the entire biofilm landscape or locally with individual biofilm clusters. PMID:22669634

Peroxynitrite modified DNA presents better epitopes for anti-DNA autoantibodies in diabetes type 1 patients.

PubMed

Tripathi, Prashant; Moinuddin; Dixit, Kiran; Mir, Abdul Rouf; Habib, Safia; Alam, Khursheed; Ali, Asif

2014-07-01

Peroxynitrite (ONOO(-)), formed by the reaction between nitric oxide (NO) and superoxide (O2(-)), has been implicated in the etiology of numerous disease processes. Peroxynitrite interacts with DNA via direct oxidative reactions or via indirect radical-mediated mechanism. It can inflict both oxidative and nitrosative damages on DNA bases, generating abasic sites, resulting in the single strand breaks. Plasmid pUC 18 isolated from Escherichiacoli was modified with peroxynitrite, generated by quenched flow process. Modifications incurred in plasmid DNA were characterized by ultraviolet and fluorescence spectroscopy, circular dichroism, HPLC and melting temperature studies. Binding characteristics and specificity of antibodies from diabetes patients were analyzed by direct binding and inhibition ELISA. Peroxynitrite modification of pUC 18 plasmid resulted in the formation of strand breaks and base modification. The major compound formed when peroxynitrite reacted with DNA was 8-nitroguanine, a specific marker for peroxynitrite induced DNA damage in inflamed tissues. The concentration of 8-nitroguanine was found to be 3.8 μM. Sera from diabetes type 1 patients from different age groups were studied for their binding to native and peroxynitrite modified plasmid. Direct binding and competitive-inhibition ELISA results showed higher recognition of peroxynitrite modified plasmid, as compared to the native form, by auto-antibodies present in diabetes patients. The preferential recognition of modified plasmid by diabetes autoantibodies was further reiterated by gel shift assay. Experimentally induced anti-peroxynitrite-modified plasmid IgG was used as a probe to detect nitrosative lesions in the DNA isolated from diabetes patients. Copyright © 2014 Elsevier Inc. All rights reserved.

Comparison of 2,4-Dichlorophenoxyacetic Acid Degradation and Plasmid Transfer in Soil Resulting from Bioaugmentation with Two Different pJP4 Donors

PubMed Central

Newby, D. T.; Gentry, T. J.; Pepper, I. L.

2000-01-01

A pilot field study was conducted to assess the impact of bioaugmentation with two plasmid pJP4-bearing microorganisms: the natural host, Ralstonia eutropha JMP134, and a laboratory-generated strain amenable to donor counterselection, Escherichia coli D11. The R. eutropha strain contained chromosomal genes necessary for mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D), while the E. coli strain did not. The soil system was contaminated with 2,4-D alone or was cocontaminated with 2,4-D and Cd. Plasmid transfer to indigenous populations, plasmid persistence in soil, and degradation of 2,4-D were monitored over a 63-day period in the bioreactors. To assess the impact of contaminant reexposure, aliquots of bioreactor soil were reamended with additional 2,4-D. Both introduced donors remained culturable and transferred plasmid pJP4 to indigenous recipients, although to different extents. Isolated transconjugants were members of the Burkholderia and Ralstonia genera, suggesting multiple, if not successive, plasmid transfers. Upon a second exposure to 2,4-D, enhanced degradation was observed for all treatments, suggesting microbial adaptation to 2,4-D. Upon reexposure, degradation was most rapid for the E. coli D11-inoculated treatments. Cd did not significantly impact 2,4-D degradation or transconjugant formation. This study demonstrated that the choice of donor microorganism might be a key factor to consider for bioaugmentation efforts. In addition, the establishment of an array of stable indigenous plasmid hosts at sites with potential for reexposure or long-term contamination may be particularly useful. PMID:10919798

Different effects of enhanced and reduced expression of pub gene on the formation of embryoid bodies by cultured embryonic mouse stem cell.

PubMed

Novosadova, E V; Manuilova, E S; Arsen'eva, E L; Khaidarova, N V; Dolotov, O V; Inozemtseva, L S; Kozachenkov, K Yu; Tarantul, V Z; Grivennikov, I A

2005-07-01

The effects of pub gene on proliferation and initial stages of differentiation of embryonic mouse stem cells were studied in vitro. To this end we used enhanced expression of human pub gene (hpub) and suppression of expression of mouse endogenous pub gene with RNA-interference in embryonic stem cells. Proliferative activity of genetically modified polyclonal lines of the embryonic stem cells transfected with plasmids carrying expressing hpub gene or plasmids generating small interference RNA to this gene did not differ from that of the control cells. Inhibition of expression of endogenous pub gene in embryonic stem cells using small interference RNA 2-fold decreased the formation of embryoid bodies, at the same time additional expression of exogenous hpub gene almost 2-fold increased their number in comparison with the control. It was hypothesized that pub gene participates in early stages of differentiation of embryonic stem cells leading to the formation of embryoid bodies.

Combined VEGF and LMP-1 delivery enhances osteoprogenitor cell differentiation and ectopic bone formation.

PubMed

Wang, Xiuli; Cui, Fuai; Madhu, Vedavathi; Dighe, Abhijit S; Balian, Gary; Cui, Quanjun

2011-02-01

A novel strategy to enhance bone repair is to combine angiogenic factors and osteogenic factors. We combined vascular endothelial growth factor (VEGF) and LIM mineralization protein-1 (LMP-1) by using an internal ribosome entry site to link the genes within a single plasmid. We then evaluated the effects on osteoblastic differentiation in vitro and ectopic bone formation in vivo with a subcutaneously placed PLAGA scaffold loaded with a cloned mouse osteoprogenitor cell line, D1, transfected with plasmids containing VEGF and LMP-1 genes. The cells expressing both genes elevated mRNA expression of RunX2 and β-catenin and alkaline phosphatase activity compared to cells from other groups. In vivo, X-ray and micro-CT analysis of the retrieved implants revealed more ectopic bone formation at 2 and 3 weeks but not at 4 weeks compared to other groups. The results indicate that the combination of the therapeutic growth factors potentiates cell differentiation and may promote osteogenesis.

Complex structural behavior of oligopurine-oligopyrimidine sequence cloned within the supercoiled plasmid.

PubMed Central

Parniewski, P; Galazka, G; Wilk, A; Klysik, J

1989-01-01

Synthetic sequence GATCC(AG)7ATCG(AT)4CG(AG)7 was cloned into plasmid and its structural behavior under the influence of supercoiling was analysed by chemical modification at variety of experimental conditions. It was found that this sequence adopts at least two different non-B conformations depending on -delta and pH values. Moreover, 12 nucleotide long non-pur.pyr spacer region separating two identical (AG)7 blocks does not provide a significant energy barrier protecting against unusual structures formation. Images PMID:2644622

Osteogenic potential of the human bone morphogenetic protein 2 gene activated nanobone putty.

PubMed

Tian, Xiao-bin; Sun, Li; Yang, Shu-hua; Zhang, Yu-kun; Hu, Ru-yin; Fu, De-hao

2008-04-20

Nanobone putty is an injectable and bioresorbable bone substitute. The neutral-pH putty resembles hard bone tissue, does not contain polymers or plasticizers, and is self-setting and nearly isothermic, properties which are helpful for the adhesion, proliferation, and function of bone cells. The aim of this study was to investigate the osteogenic potential of human bone morphogenetic protein 2 (hBMP2) gene activated nanobone putty in inducing ectopic bone formation, and the effects of the hBMP2 gene activated nanobone putty on repairing bone defects. Twenty four Kunming mice were randomly divided into two groups. The nanobone putty + hBMP2 plasmid was injected into the right thigh muscle pouches of the mice (experiment side). The nanobone putty + blank plasmid or nanobone putty was injected into the left thigh muscle pouches of the group 1 (control side 1) or group 2 (control side 2), respectively. The effects of ectopic bone formation were evaluated by radiography, histology, and molecular biology analysis at 2 and 4 weeks after operation. Bilateral 15 mm radial defects were made in forty-eight rabbits. These rabbits were randomly divided into three groups: Group A, nanobone putty + hBMP2 plasmid; Group B, putty + blank plasmid; Group C, nanobone putty only. Six rabbits with left radial defects served as blank controls. The effect of bone repairing was evaluated by radiography, histology, molecular biology, and biomechanical analysis at 4, 8, and 12 weeks after operation. The tissue from the experimental side of the mice expressed hBMP2. Obvious cartilage and island-distributed immature bone formation in implants of the experiment side were observed at 2 weeks after operation, and massive mature bone observed at 4 weeks. No bone formation was observed in the control side of the mice. The ALP activity in the experiment side of the mice was higher than that in the control side. The tissue of Group A rabbits expressed hBMP2 protein and higher ALP level. The new bone formation rate and antibending strength of group A was significantly higher than those of group B and C. The defects in blank control were not healed. The hBMP2 gene activated nanobone putty exhibited osteoinductive ability, and had a better bone defect repair capability than that of nanobone putty only.

In vitro transfection of plasmid DNA by amine derivatives of gelatin accompanied with ultrasound irradiation.

PubMed

Hosseinkhani, Hossein; Aoyama, Ternyoshi; Yamamoto, Shingo; Ogawa, Osamu; Tabata, Yasuhiko

2002-10-01

The purpose of this study is to examine the ultrasound (US)-enhanced gene expression by the complexes of a plasmid DNA with gelatin derivatives of aminization. Gelatin derivatives with different introduced extents of ethylenediamine (Ed), spermidine (Sd), and spermine (Sm) were prepared with a water-soluble carbodiimide. The molecular size and zeta potential of the gelatin derivatives before and after complexation with the plasmid DNA were examined. After incubation with the complexes with or without US exposure, the DNA expression of rat gastric mucosal cells was measured to evaluate the effect of the type of gelatin derivatives on their gene expression. The cell uptake of the complexes, the cell viability, and the buffering effect of gelatin derivatives were examined. The apparent molecular size and zeta potential of gelatin derivatives became larger as their aminization extent increased although the Sm gelatin derivative of higher aminization showed a larger value than other corresponding derivatives. Irrespective of the type of gelatin derivatives, the apparent molecular size of plasmid DNA was reduced by increasing the gelatin-DNA mixing ratio to attain a saturated value of about 150 nm. The condensed gelatin-DNA complexes showed the zeta potential of 10-15 mV. The cells incubated with the complex exhibited significantly stronger luciferase activities than free plasmid DNA, and the activity was further enhanced by US irradiation. The enhancement was significant for the Sm derivative compared with the corresponding Ed and Sd derivatives. The amount of plasmid DNA internalized into the cells was significantly increased by the complexation with every gelatin derivative, whereas US irradiation did not significantly increase the DNA internalization. US irradiation had no effect on the viability of cells incubated with every gelatin derivative-plasmid DNA complex, although the viability was decreased by the complex incubation. The buffering capacity of Sm derivative was higher than that of Ed and Sd derivatives and comparable with that of polyethylene amine. Among amine derivatives of gelatin, the Sm derivative enabled the plasmid DNA to induce the US-enhanced gene expression of cells in vitro most effectively because of the superior buffering effect.

A three-dimensional ParF meshwork assembles through the nucleoid to mediate plasmid segregation.

PubMed

McLeod, Brett N; Allison-Gamble, Gina E; Barge, Madhuri T; Tonthat, Nam K; Schumacher, Maria A; Hayes, Finbarr; Barillà, Daniela

2017-04-07

Genome segregation is a fundamental step in the life cycle of every cell. Most bacteria rely on dedicated DNA partition proteins to actively segregate chromosomes and low copy-number plasmids. Here, by employing super resolution microscopy, we establish that the ParF DNA partition protein of the ParA family assembles into a three-dimensional meshwork that uses the nucleoid as a scaffold and periodically shuttles between its poles. Whereas ParF specifies the territory for plasmid trafficking, the ParG partner protein dictates the tempo of ParF assembly cycles and plasmid segregation events by stimulating ParF adenosine triphosphate hydrolysis. Mutants in which this ParG temporal regulation is ablated show partition deficient phenotypes as a result of either altered ParF structure or dynamics and indicate that ParF nucleoid localization and dynamic relocation, although necessary, are not sufficient per se to ensure plasmid segregation. We propose a Venus flytrap model that merges the concepts of ParA polymerization and gradient formation and speculate that a transient, dynamic network of intersecting polymers that branches into the nucleoid interior is a widespread mechanism to distribute sizeable cargos within prokaryotic cells. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Identification and characterization of an autolysin gene, atlg, from Streptococcus sobrinus.

PubMed

Yamada, Arisa; Tamura, Haruki; Kato, Hirohisa

2009-02-01

AtlA is a major cell-lytic enzyme called autolysin in Streptococcus mutans. In this study, we identified the atlg gene-encoding autolysin (Atlg), consisting of 863 residues from Streptococcus sobrinus 6715DP, and confirmed lytic activity of recombinant Atlg by zymography of S. sobrinus cells. An atlA-inactivated mutant was constructed in S. mutans Xc, and the atlg gene product was characterized by plasmid complementation. Microscopic analysis, saliva-induced aggregation assay and autolysis assay of static cultures in air revealed that the atlg gene product partially complemented the role of AtlA. Furthermore, the capability of biofilm formation of the atlA-deficient mutant cultivated in air was restored by plasmid comprising the atlg gene. These findings suggest that Atlg may be involved in cell separation and biofilm formation in S. sobrinus.

A double-pulse approach for electrotransfection.

PubMed

Pasquet, L; Bellard, E; Golzio, M; Rols, M P; Teissie, J

2014-12-01

Gene transfer and expression can be obtained by delivering calibrated electric pulses on cells in the presence of plasmids coding for the activity of interest. The electric treatment affects the plasma membrane and induces the formation of a transient complex between nucleic acids and the plasma membrane. It results in a delivery of the plasmid in the cytoplasm. Expression is only obtained if the plasmid is translocated inside the nucleus. This is a key limit in the process. We previously showed that delivery of a high-field short-duration electric pulse was inducing a structural alteration of the nuclear envelope. This study investigates if the double-pulse approach (first pulse to transfer the plasmid to the cytoplasm, and second pulse to induce the structural alteration of the envelope) was a way to enhance the protein expression using the green fluorescent protein as a reporter. We observed that not only the double-pulse approach induced the transfection of a lower number of cells but moreover, these transfected cells were less fluorescent than the cells treated only with the first pulse.

New Insights into the Mechanism Underlying the Synergistic Action of Ionizing Radiation With Platinum Chemotherapeutic Drugs: The Role of Low-Energy Electrons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rezaee, Mohammad, E-mail: Mohammad.Rezaee@USherbrooke.ca; Hunting, Darel John; Sanche, Léon

Purpose: To investigate the efficiencies of platinum chemotherapeutic drugs (Pt-drugs) in the sensitization of DNA to the direct effects of ionizing radiation and to determine the role of low-energy electrons (LEEs) in this process. Methods and Materials: Complexes of supercoiled plasmid DNA covalently bound to either cisplatin, carboplatin, or oxaliplatin were prepared in different molar ratios. Solid films of DNA and DNA modified by Pt-drugs were irradiated with either 10-KeV or 10-eV electrons. Damages to DNA were quantified by gel electrophoresis, and the yields for damage formation were obtained from exposure–response curves. Results: The presence of an average of 2more » Pt-drug–DNA adducts (Pt-adducts) in 3199-bp plasmid DNA increases the probability of a double-strand break by factors of 3.1, 2.5, and 2.4 for carboplatin, cisplatin, and oxaliplatin, respectively. Electrons with energies of 10 eV and 10 KeV interact with Pt-adducts to preferentially enhance the formation of cluster lesions. The maximum increase in radiosensitivity per Pt-adduct is found at ratios up to 3.1 × 10{sup −4} Pt-adducts per nucleotide, which is equivalent to an average of 2 adducts per plasmid. Carboplatin and oxaliplatin show higher efficiencies than cisplatin in the radiosensitization of DNA. Because carboplatin and cisplatin give rise to identical reactive species that attach to DNA, carboplatin must be considered as a better radiosensitizer for equal numbers of Pt-adducts. Conclusion: Platinum chemotherapeutic drugs preferentially enhance the formation of cluster damage to DNA induced by the direct effect of ionizing radiation, and LEEs are the main species responsible for such an enhancement via the formation of electron resonances.« less

New insights into the mechanism underlying the synergistic action of ionizing radiation with platinum chemotherapeutic drugs: the role of low-energy electrons.

PubMed

Rezaee, Mohammad; Hunting, Darel John; Sanche, Léon

2013-11-15

To investigate the efficiencies of platinum chemotherapeutic drugs (Pt-drugs) in the sensitization of DNA to the direct effects of ionizing radiation and to determine the role of low-energy electrons (LEEs) in this process. Complexes of supercoiled plasmid DNA covalently bound to either cisplatin, carboplatin, or oxaliplatin were prepared in different molar ratios. Solid films of DNA and DNA modified by Pt-drugs were irradiated with either 10-KeV or 10-eV electrons. Damages to DNA were quantified by gel electrophoresis, and the yields for damage formation were obtained from exposure-response curves. The presence of an average of 2 Pt-drug-DNA adducts (Pt-adducts) in 3199-bp plasmid DNA increases the probability of a double-strand break by factors of 3.1, 2.5, and 2.4 for carboplatin, cisplatin, and oxaliplatin, respectively. Electrons with energies of 10 eV and 10 KeV interact with Pt-adducts to preferentially enhance the formation of cluster lesions. The maximum increase in radiosensitivity per Pt-adduct is found at ratios up to 3.1×10(-4) Pt-adducts per nucleotide, which is equivalent to an average of 2 adducts per plasmid. Carboplatin and oxaliplatin show higher efficiencies than cisplatin in the radiosensitization of DNA. Because carboplatin and cisplatin give rise to identical reactive species that attach to DNA, carboplatin must be considered as a better radiosensitizer for equal numbers of Pt-adducts. Platinum chemotherapeutic drugs preferentially enhance the formation of cluster damage to DNA induced by the direct effect of ionizing radiation, and LEEs are the main species responsible for such an enhancement via the formation of electron resonances. Copyright © 2013 Elsevier Inc. All rights reserved.

New Insights into the Mechanism Underlying the Synergistic Action of Ionizing Radiation with Platinum Chemotherapeutic Drugs: The Role of Low-Energy Electrons

PubMed Central

Rezaee, Mohammad; Hunting, Darel John; Sanche, Léon

2013-01-01

Purpose To investigate the efficiencies of platinum chemotherapeutic drugs (Pt-drugs) in the sensitization of DNA to the direct effects of ionizing radiation and to determine the role of low-energy electrons (LEEs) in this process. Methods and Materials Complexes of supercoiled plasmid DNA covalently bound to either cisplatin, carboplatin or oxaliplatin were prepared in different molar ratios. Solid films of DNA and DNA modified by Pt-drugs were irradiated with either 10-KeV or 10-eV electrons. DNA damages were quantified by gel electrophoresis, and the yields for damage formation were obtained from exposure-response curves. Results The presence of an average of two Pt-adducts in 3199-bp plasmid DNA increases the probability of a double-strand break by factors of 3.1, 2.5 and 2.4 for carboplatin, cisplatin and oxaliplatin, respectively. Electrons with energies of 10-eV and 10-KeV interact with Pt-adducts to preferentially enhance the formation of cluster lesions. The maximum increase in radiosensitivity per Pt-adduct is found at ratios up to 3.1 × 10−4 Pt-adducts per nucleotide which is equivalent to an average of two adducts per plasmid. Carboplatin and oxaliplatin show higher efficiencies than cisplatin in the radiosensitization of DNA. Since carboplatin and cisplatin give rise to identical reactive species which attach to DNA, carboplatin must be considered as a better radiosensitizers for equal number of Pt-adducts. Conclusion Pt-drugs preferentially enhance the formation of cluster damage to DNA induced by the direct effect of ionizing radiation and LEEs are the main species responsible for such an enhancement via the formation of electron resonances. PMID:23910707

Polyplex formation between four-arm poly(ethylene oxide)-b-poly(2-(diethylamino)ethyl methacrylate) and plasmid DNA in gene delivery.

PubMed

He, E; Yue, C Y; Simeon, F; Zhou, L H; Too, H P; Tam, K C

2009-12-01

Amphiphilic polyelectrolytes comprising cationic and uncharged hydrophilic segments condensed negatively charged DNA to form a core-shell structure stabilized by a layer of hydrophilic corona chains. At physiological pH, four-arm star-shaped poly(ethylene oxide)-b-poly(2-(diethylamino)ethyl methacrylate) (four-arm PEO-b-PDEAEMA) block copolymer possessed positively charged amine groups that interacted with negatively charged plasmid DNA to form polymer/DNA complexes. The mechanism and physicochemical properties of the complex formation were investigated at varying molar ratio of amine groups on polymer chains and phosphate group on plasmid DNA segments (N/P ratio). The capability of the star block copolymer to condense DNA was demonstrated through gel electrophoresis and ethidium bromide exclusion assay. In the absence of salt, the hydrodynamic radius of polyplexes was about 94 nm at low polymer/DNA ratio, and it decreased to about 34 nm at large N/P ratios, forming a compact spherical structure with a weighted average molecular weight of 4.39 +/- 0.22 x 10(6) g/mol. Approximately 15 polymeric chains were required to condense a plasmid DNA. The addition of monovalent salt to the polyplexes significantly altered the size of the complexes, which would have an impact on cell transfection. Because of the electrostatic interaction induced by the diffusion of small ions, the polyplex increased in size to about 53 nm with a less compact structure. In vitro cytotoxicty of polymer and polymer/pDNA complexes were evaluated, and the polyplexes exhibited low toxicity at low N/P ratios. At N/P ratio of 4.5, the four-arm PEO-b-PDEAEMA showed the highest level of transfection in Neuro-2A cells. These observations showed that the star-shaped multi-arm polymers offers interesting properties in self-association and condensation ability for plasmid DNA and can serve as a nonviral DNA delivery system. Copyright 2008 Wiley Periodicals, Inc.

Capture of a catabolic plasmid that encodes only 2,4-dichlorophenoxyacetic acid:alpha-ketoglutaric acid dioxygenase (TfdA) by genetic complementation.

PubMed Central

Top, E M; Maltseva, O V; Forney, L J

1996-01-01

The modular pathway for the metabolism of 2,4-dichlorophenoxyacetic acid (2,4-D) encoded on plasmid pJP4 of Alcaligenes eutrophus JMP134 appears to be an example in which two genes, tfdA and tfdB, have been recruited during the evolution of a catabolic pathway. The products of these genes act to convert 2,4-D to a chloro-substituted catechol that can be further metabolized by enzymes of a modified ortho-cleavage pathway encoded by tfdCDEF. Given that modified ortho-cleavage pathways are comparatively common and widely distributed among bacteria, we sought to determine if microbial populations in soil carry tfdA on plasmid vectors that lack tfdCDEF or tfdB. To capture such plasmids from soil populations, we used a recipient strain of A. eutrophus that was rifampin resistant and carried a derivative of plasmid pJP4 (called pBH501aE) in which the tfdA had been deleted. Upon mating with mixed bacterial populations from soil treated with 2,4-D, transconjugants that were resistant to rifampin yet able to grow on 2,4-D were obtained. Among the transconjugants obtained were clones that contained a ca. 75-kb plasmid, pEMT8. Bacterial hosts that carried this plasmid in addition to pBH501aE metabolized 2,4-D, whereas strains with only pEMT8 did not. Southern hybridization showed that pEMT8 encoded a gene with a low level of similarity to the tfdA gene from plasmid pJP4. Using oligonucleotide primers based on known tfdA sequences, we amplified a 330-bp fragment of the gene and determined that it was 77% similar to the tfdA gene of plasmid pJP4 and 94% similar to tfdA from Burkholderia sp. strain RASC. Plasmid pEMT8 lacked genes that exhibited significant levels of homology to tfdB and tfdCDEF. Moreover, cell extracts from A. eutrophus(pEMT8) cultures did not exhibit TfdB, TfdC, TfdD, and TfdE activities, whereas cell extracts from A. eutrophus(pEMT8)(pBH501aE) cultures did. These data suggest that pEMT8 encodes only tfdA and that this gene can effectively complement the tfdA deletion mutation of pBH501aE. PMID:8779586

Properties of internalization factors contributing to the uptake of extracellular DNA into tumor-initiating stem cells of mouse Krebs-2 cell line.

PubMed

Dolgova, Evgeniya V; Potter, Ekaterina A; Proskurina, Anastasiya S; Minkevich, Alexandra M; Chernych, Elena R; Ostanin, Alexandr A; Efremov, Yaroslav R; Bayborodin, Sergey I; Nikolin, Valeriy P; Popova, Nelly A; Kolchanov, Nikolay A; Bogachev, Sergey S

2016-05-25

Previously, we demonstrated that poorly differentiated cells of various origins, including tumor-initiating stem cells present in the ascites form of mouse cancer cell line Krebs-2, are capable of naturally internalizing both linear double-stranded DNA and circular plasmid DNA. The method of co-incubating Krebs-2 cells with extracellular plasmid DNA (pUC19) or TAMRA-5'-dUTP-labeled polymerase chain reaction (PCR) product was used. It was found that internalized plasmid DNA isolated from Krebs-2 can be transformed into competent Escherichia coli cells. Thus, the internalization processes taking place in the Krebs-2 cell subpopulation have been analyzed and compared, as assayed by E. coli colony formation assay (plasmid DNA) and cytofluorescence (TAMRA-DNA). We showed that extracellular DNA both in the form of plasmid DNA and a PCR product is internalized by the same subpopulation of Krebs-2 cells. We found that the saturation threshold for Krebs-2 ascites cells is 0.5 μg DNA/10(6) cells. Supercoiled plasmid DNA, human high-molecular weight DNA, and 500 bp PCR fragments are internalized into the Krebs-2 tumor-initiating stem cells via distinct, non-competing internalization pathways. Under our experimental conditions, each cell may harbor 340-2600 copies of intact plasmid material, or up to 3.097 ± 0.044×10(6) plasmid copies (intact or not), as detected by quantitative PCR. The internalization dynamics of extracellular DNA, copy number of the plasmids taken up by the cells, and competition between different types of double-stranded DNA upon internalization into tumor-initiating stem cells of mouse ascites Krebs-2 have been comprehensively analyzed. Investigation of the extracellular DNA internalization into tumor-initiating stem cells is an important part of understanding their properties and possible destruction mechanisms. For example, a TAMRA-labeled DNA probe may serve as an instrument to develop a target for the therapy of cancer, aiming at elimination of tumor stem cells, as well as developing a straightforward test system for the quantification of poorly differentiated cells, including tumor-initiating stem cells, in the bulk tumor sample (biopsy or surgery specimen).

Sequence and Role in Virulence of the Three Plasmid Complement of the Model Tumor-Inducing Bacterium Pseudomonas savastanoi pv. savastanoi NCPPB 3335

PubMed Central

Bardaji, Leire; Pérez-Martínez, Isabel; Rodríguez-Moreno, Luis; Rodríguez-Palenzuela, Pablo; Sundin, George W.; Ramos, Cayo; Murillo, Jesús

2011-01-01

Pseudomonas savastanoi pv. savastanoi NCPPB 3335 is a model for the study of the molecular basis of disease production and tumor formation in woody hosts, and its draft genome sequence has been recently obtained. Here we closed the sequence of the plasmid complement of this strain, composed of three circular molecules of 78,357 nt (pPsv48A), 45,220 nt (pPsv48B), and 42,103 nt (pPsv48C), all belonging to the pPT23A-like family of plasmids widely distributed in the P. syringae complex. A total of 152 coding sequences were predicted in the plasmid complement, of which 38 are hypothetical proteins and seven correspond to putative virulence genes. Plasmid pPsv48A contains an incomplete Type IVB secretion system, the type III secretion system (T3SS) effector gene hopAF1, gene ptz, involved in cytokinin biosynthesis, and three copies of a gene highly conserved in plant-associated proteobacteria, which is preceded by a hrp box motif. A complete Type IVA secretion system, a well conserved origin of transfer (oriT), and a homolog of the T3SS effector gene hopAO1 are present in pPsv48B, while pPsv48C contains a gene with significant homology to isopentenyl-diphosphate delta-isomerase, type 1. Several potential mobile elements were found on the three plasmids, including three types of MITE, a derivative of IS801, and a new transposon effector, ISPsy30. Although the replication regions of these three plasmids are phylogenetically closely related, their structure is diverse, suggesting that the plasmid architecture results from an active exchange of sequences. Artificial inoculations of olive plants with mutants cured of plasmids pPsv48A and pPsv48B showed that pPsv48A is necessary for full virulence and for the development of mature xylem vessels within the knots; we were unable to obtain mutants cured of pPsv48C, which contains five putative toxin-antitoxin genes. PMID:22022435

Oncogenic collaboration of the cyclin D1 (PRAD1, bcl-1) gene with a mutated p53 and an activated ras oncogene in neoplastic transformation.

PubMed

Uchimaru, K; Endo, K; Fujinuma, H; Zukerberg, L; Arnold, A; Motokura, T

1996-05-01

Cyclin D1 is one of the key regulators in G1 progression in the cell cycle and is also a candidate oncogene (termed PRAD1 or bcl-1) in several types of human tumors. We report a collaboration of the cyclin D1 gene with ras and a mutated form of p53 (p53-mt) in neoplastic transformation. Transfection of cyclin D1 alone or in combination with ras or with p53-mt was not sufficient for focus formation of rat embryonic fibroblasts. However, focus formation induced by co-transfection of ras and p53-mt was enhanced in the presence of the cyclin D1-expression plasmid. Co-transfection of ras- and p53-mt-transformants with the cyclin D1-expression plasmid resulted in reduced serum dependency in vitro. Furthermore, the transformants expressing exogenous cyclin D1 grew faster than those without the cyclin D1 plasmid when injected into nude mice. These observations strengthen the significance of cyclin D1 overexpression through gene rearrangement or gene amplification observed in human tumors as a step in multistep oncogenesis; deregulated expression of cyclin D1 may reduce the requirement for growth factors and may stimulate in vivo growth.

Site-Specific Recombination at XerC/D Sites Mediates the Formation and Resolution of Plasmid Co-integrates Carrying a blaOXA-58- and TnaphA6-Resistance Module in Acinetobacter baumannii

PubMed Central

Cameranesi, María M.; Morán-Barrio, Jorgelina; Limansky, Adriana S.; Repizo, Guillermo D.; Viale, Alejandro M.

2018-01-01

Members of the genus Acinetobacter possess distinct plasmid types which provide effective platforms for the acquisition, evolution, and dissemination of antimicrobial resistance structures. Many plasmid-borne resistance structures are bordered by short DNA sequences providing potential recognition sites for the host XerC and XerD site-specific tyrosine recombinases (XerC/D-like sites). However, whether these sites are active in recombination and how they assist the mobilization of associated resistance structures is still poorly understood. Here we characterized the plasmids carried by Acinetobacter baumannii Ab242, a multidrug-resistant clinical strain belonging to the ST104 (Oxford scheme) which produces an OXA-58 carbapenem-hydrolyzing class-D β-lactamase (CHDL). Plasmid sequencing and characterization of replication, stability, and adaptive modules revealed the presence in Ab242 of three novel plasmids lacking self-transferability functions which were designated pAb242_9, pAb242_12, and pAb242_25, respectively. Among them, only pAb242_25 was found to carry an adaptive module encompassing an ISAba825-blaOXA-58 arrangement accompanied by a TnaphA6 transposon, the whole structure conferring simultaneous resistance to carbapenems and aminoglycosides. Ab242 plasmids harbor several XerC/D-like sites, with most sites found in pAb242_25 located in the vicinity or within the adaptive module described above. Electrotransformation of susceptible A. nosocomialis cells with Ab242 plasmids followed by imipenem selection indicated that the transforming plasmid form was a co-integrate resulting from the fusion of pAb242_25 and pAb242_12. Further characterization by cloning and sequencing studies indicated that a XerC/D site in pAb242_25 and another in pAb242_12 provided the active sister pair for the inter-molecular site-specific recombination reaction mediating the fusion of these two plasmids. Moreover, the resulting co-integrate was found also to undergo intra-molecular resolution at the new pair of XerC/D sites generated during fusion thus regenerating the original pAb242_25 and pAb242_12 plasmids. These observations provide the first evidence indicating that XerC/D-like sites in A. baumannii plasmids can provide active pairs for site-specific recombination mediating inter-molecular fusions and intra-molecular resolutions. The overall results shed light on the evolutionary dynamics of A. baumannii plasmids and the underlying mechanisms of dissemination of genetic structures responsible for carbapenem and other antibiotics resistance among the Acinetobacter clinical population. PMID:29434581

Advances in the Function and Regulation of Hydrogenase in the Cyanobacterium Synechocystis PCC6803

PubMed Central

Cassier-Chauvat, Corinne; Veaudor, Théo; Chauvat, Franck

2014-01-01

In order to use cyanobacteria for the biological production of hydrogen, it is important to thoroughly study the function and the regulation of the hydrogen-production machine in order to better understand its role in the global cell metabolism and identify bottlenecks limiting H2 production. Most of the recent advances in our understanding of the bidirectional [Ni-Fe] hydrogenase (Hox) came from investigations performed in the widely-used model cyanobacterium Synechocystis PCC6803 where Hox is the sole enzyme capable of combining electrons with protons to produce H2 under specific conditions. Recent findings suggested that the Hox enzyme can receive electrons from not only NAD(P)H as usually shown, but also, or even preferentially, from ferredoxin. Furthermore, plasmid-encoded functions and glutathionylation (the formation of a mixed-disulfide between the cysteines residues of a protein and the cysteine residue of glutathione) are proposed as possible new players in the function and regulation of hydrogen production. PMID:25365180

☆DNA assembly technique simplifies the construction of infectious clone of fowl adenovirus.

PubMed

Zou, Xiao-Hui; Bi, Zhi-Xiang; Guo, Xiao-Juan; Zhang, Zun; Zhao, Yang; Wang, Min; Zhu, Ya-Lu; Jie, Hong-Ying; Yu, Yang; Hung, Tao; Lu, Zhuo-Zhuang

2018-07-01

Plasmid bearing adenovirus genome is generally constructed with the method of homologous recombination in E. coli BJ5183 strain. Here, we utilized Gibson gene assembly technique to generate infectious clone of fowl adenovirus 4 (FAdV-4). Primers flanked with partial inverted terminal repeat (ITR) sequence of FAdV-4 were synthesized to amplify a plasmid backbone containing kanamycin-resistant gene and pBR322 origin (KAN-ORI). DNA assembly was carried out by combining the KAN-ORI fragment, virus genomic DNA and DNA assembly master mix. E. coli competent cells were transformed with the assembled product, and plasmids (pKFAV4) were extracted and confirmed to contain viral genome by restriction analysis and sequencing. Virus was successfully rescued from linear pKFAV4-transfected chicken LMH cells. This approach was further verified in cloning of human adenovirus 5 genome. Our results indicated that DNA assembly technique simplified the construction of infectious clone of adenovirus, suggesting its possible application in virus traditional or reverse genetics. Copyright © 2018 Elsevier B.V. All rights reserved.

GFP as a marker for transient gene transfer and expression in Mycoplasma hyorhinis.

PubMed

Ishag, Hassan Z A; Liu, Maojun; Yang, Ruosong; Xiong, Qiyan; Feng, Zhixin; Shao, Guoqing

2016-01-01

Mycoplasma hyorhinis (M. hyorhinis) is an opportunistic pathogen of pigs and has been shown to transform cell cultures, which has increased the interest of researchers. The green florescence proteins (GFP) gene of Aquorea victoria, proved to be a vital marker to identify transformed cells in mixed populations. Use of GFP to observe gene transfer and expression in M. hyorhinis (strain HUB-1) has not been described. We have constructed a pMD18-O/MHRgfp plasmid containing the p97 gene promoter, origin of replication, tetracycline resistance marker and GFP gene controlled by the p97 gene promoter. The plasmid transformed into M. hyorhinis with a frequency of ~4 × 10(-3) cfu/µg plasmid DNA and could be detected by PCR amplification of the GFP gene from the total DNA of the transformant mycoplasmas. Analysis of a single clone grown on KM2-Agar containing tetracycline, showed a green fluorescence color. Conclusively, this report suggests the usefulness of GFP to monitor transient gene transfer and expression in M. hyorhinis, eventually minimizing screening procedures for gene transfer and expression.

Lipofection of plasmid DNA into human mast cell lines using lipid nanoparticles generated by microfluidic mixing.

PubMed

Duguay, Brett A; Huang, Kate Wei-Chen; Kulka, Marianna

2018-04-18

Mast cells are important immune cells that have significant roles in mediating allergy and asthma. Therefore, studying the molecular mechanisms regulating these and other processes in mast cells is important to elucidate. Methods such as lipofection, transduction, and electroporation are often employed to dissect these mechanisms by disrupting gene expression in mast cell lines. However, as with other leukocytes, human mast cells (HMCs) are often refractory to the delivery of plasmids by lipofection. In this study, we investigated the utility of lipid nanoparticles (LNPs) containing the ionizable cationic lipids 1,2-dioleoyloxy-3-dimethylaminopropane, 1,2-dioleyloxy-3-dimethylaminopropane, or 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane for the delivery of plasmid DNA into HMC lines. Herein, we demonstrate for the first time the use of LNPs to achieve significant and reproducible levels of plasmid DNA transfection in HMC-1.2 and laboratory of allergic diseases 2 (LAD2) cells. These levels reached 53.2% and 16.0% in HMC-1.2 and LAD2 cells, respectively; and outperformed Lipofectamine 3000 in both cases. Moreover, cell viability in the transfected cells remained above 65% for all LNP conditions tested. Together, these observations illustrate the efficacy of this technique for mast cell researchers and further support the use of LNPs for nucleic acid delivery into leukocytes. ©2018 Society for Leukocyte Biology.

Type II Toxin–Antitoxin Systems in the Unicellular Cyanobacterium Synechocystis sp. PCC 6803

PubMed Central

Kopfmann, Stefan; Roesch, Stefanie K.; Hess, Wolfgang R.

2016-01-01

Bacterial toxin–antitoxin (TA) systems are genetic elements, which are encoded by plasmid as well as chromosomal loci. They mediate plasmid and genomic island maintenance through post-segregational killing mechanisms but may also have milder effects, acting as mobile stress response systems that help certain cells of a population in persisting adverse growth conditions. Very few cyanobacterial TA system have been characterized thus far. In this work, we focus on the cyanobacterium Synechocystis 6803, a widely used model organism. We expand the number of putative Type II TA systems from 36 to 69 plus seven stand-alone components. Forty-seven TA pairs are located on the chromosome and 22 are plasmid-located. Different types of toxins are associated with various antitoxins in a mix and match principle. According to protein domains and experimental data, 81% of all toxins in Synechocystis 6803 likely exhibit RNase activity, suggesting extensive potential for toxicity-related RNA degradation and toxin-mediated transcriptome remodeling. Of particular interest is the Ssr8013–Slr8014 system encoded on plasmid pSYSG, which is part of a larger defense island or the pSYSX system Slr6056–Slr6057, which is linked to a bacterial ubiquitin-like system. Consequently, Synechocystis 6803 is one of the most prolific sources of new information about these genetic elements. PMID:27455323

Design of different strategies of multivalent DNA-based vaccination against rabies and canine distemper in mice and dogs.

PubMed

Touihri, Leila; Ahmed, Sami Belhaj; Chtourou, Yacine; Daoud, Rahma; Bahloul, Chokri

2012-12-27

During the vaccination campaigns, puppies younger than 3 months old are not targeted and remain unvaccinated for at least the first year of their lives. Almost half of the reported rabid dogs are 6 months or younger. Hence, we should recommend the vaccination against rabies of young puppies. Unfortunately, owing to the exposure of puppies to infections with either canine parvovirus (CPV) or distemper virus (CDV) after the intervention of the vaccinators, owners are reluctant to vaccinate puppies against rabies. Therefore, it is necessary to include the CPV and CDV valences in the vaccine against rabies. Multivalent DNA-based vaccination in dogs, including rabies and distemper valences, could help in raising vaccine coverage. We have designed monovalent and multivalent DNA-based vaccine candidates for in vitro and in vivo assays. These plasmids encode to the rabies virus glycoprotein and/or the canine distemper virus hemagglutinin. The first strategy of multivalent DNA-based vaccination is by mixing plasmids encoding to a single antigen each. The second is by simply fusing the genes of the antigens together. The third is by adding the foot and mouth disease virus (FMDV) 2A oligopeptide gene into the antigen genes. The last strategy is by the design and use of a bicistronic plasmid with an "Internal Ribosome Entry Site" (IRES) domain. The monovalent construct against canine distemper was efficiently validated by inducing higher humoral immune responses compared to cell-culture-derived vaccine both in mice and dogs. All multivalent plasmids efficiently expressed both valences after in vitro transfection of BHK-21 cells. In BALB/c mice, the bicistronic IRES-dependant construct was the most efficient inducer of virus-neutralizing antibodies against both valences. It was able to induce better humoral immune responses compared to the administration of either cell-culture-derived vaccines or monovalent plasmids. The FMDV 2A was also efficient in the design of multivalent plasmids. In a single shot, the design of efficient multivalent plasmids will be very beneficial for DNA-based vaccination against numerous diseases.

The effects of DNA supercoiling on G-quadruplex formation.

PubMed

Sekibo, Doreen A T; Fox, Keith R

2017-12-01

Guanine-rich DNAs can fold into four-stranded structures that contain stacks of G-quartets. Bioinformatics studies have revealed that G-rich sequences with the potential to adopt these structures are unevenly distributed throughout genomes, and are especially found in gene promoter regions. With the exception of the single-stranded telomeric DNA, all genomic G-rich sequences will always be present along with their C-rich complements, and quadruplex formation will be in competition with the corresponding Watson-Crick duplex. Quadruplex formation must therefore first require local dissociation (melting) of the duplex strands. Since negative supercoiling is known to facilitate the formation of alternative DNA structures, we have investigated G-quadruplex formation within negatively supercoiled DNA plasmids. Plasmids containing multiple copies of (G3T)n and (G3T4)n repeats, were probed with dimethylsulphate, potassium permanganate and S1 nuclease. While dimethylsulphate footprinting revealed some evidence for G-quadruplex formation in (G3T)n sequences, this was not affected by supercoiling, and permanganate failed to detect exposed thymines in the loop regions. (G3T4)n sequences were not protected from DMS and showed no reaction with permanganate. Similarly, both S1 nuclease and 2D gel electrophoresis of DNA topoisomers did not detect any supercoil-dependent structural transitions. These results suggest that negative supercoiling alone is not sufficient to drive G-quadruplex formation. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Long-term increase in mVEGF164 in mouse hindlimb muscle mediated by phage phiC31 integrase after nonviral DNA delivery.

PubMed

Portlock, Joylette L; Keravala, Annahita; Bertoni, Carmen; Lee, Solomon; Rando, Thomas A; Calos, Michele P

2006-08-01

Peripheral vascular disease (PVD), characterized by insufficient blood supply to extremities, can be a devastating illness. Although many gene therapy strategies for PVD using vascular endothelial growth factor (VEGF) have resulted in increased blood vessel formation, the vessels are often impermanent and regress after therapy, probably because of the short-lived VEGF expression mediated by gene therapy vectors (14 days or less). phiC31 integrase is a recombinase originally isolated from a bacteriophage of Streptomyces. This integrase performs efficient chromosomal integration of plasmid DNA into mammalian genomes that results in long-term transgene expression. In this study, gene transfer was achieved by intramuscular injection of VEGF and integrase plasmid DNAs into the tibialis anterior muscle in the mouse hindlimb, followed by electroporation of the muscle with needle electrodes. We observed VEGF levels significantly above background 40 days after injection in animals that received phiC31 integrase and the VEGF plasmid. Site-specific integration of plasmid DNA in the chromosomes of muscle tissue was verified by polymerase chain reaction at a common integration site. These results suggest the possible utility of the phiC31 integrase system to treat ischemic disease.

Linear Streptomyces plasmids form superhelical circles through interactions between their terminal proteins

PubMed Central

Tsai, Hsiu-Hui; Huang, Chih-Hung; Tessmer, Ingrid; Erie, Dorothy A.; Chen, Carton W.

2011-01-01

Linear chromosomes and linear plasmids of Streptomyces possess covalently bound terminal proteins (TPs) at the 5′ ends of their telomeres. These TPs are proposed to act as primers for DNA synthesis that patches the single-stranded gaps at the 3′ ends during replication. Most (‘archetypal’) Streptomyces TPs (designated Tpg) are highly conserved in size and sequence. In addition, there are a number of atypical TPs with heterologous sequences and sizes, one of which is Tpc that caps SCP1 plasmid of Streptomyces coelicolor. Interactions between the TPs on the linear Streptomyces replicons have been suggested by electrophoretic behaviors of TP-capped DNA and circular genetic maps of Streptomyces chromosomes. Using chemical cross-linking, we demonstrated intramolecular and intermolecular interactions in vivo between Tpgs, between Tpcs and between Tpg and Tpc. Interactions between the chromosomal and plasmid telomeres were also detected in vivo. The intramolecular telomere interactions produced negative superhelicity in the linear DNA, which was relaxed by topoisomerase I. Such intramolecular association between the TPs poses a post-replicational complication in the formation of a pseudo-dimeric structure that requires resolution by exchanging TPs or DNA. PMID:21109537

The ClpXP protease is responsible for the degradation of the Epsilon antidote to the Zeta toxin of the streptococcal pSM19035 plasmid.

PubMed

Brzozowska, Iwona; Zielenkiewicz, Urszula

2014-03-14

Most bacterial genomes contain different types of toxin-antitoxin (TA) systems. The ω-ε-ζ proteinaceous type II TA cassette from the streptococcal pSM19035 plasmid is a member of the ε/ζ family, which is commonly found in multiresistance plasmids and chromosomes of various human pathogens. Regulation of type II TA systems relies on the proteolysis of antitoxin proteins. Under normal conditions, the Epsilon antidote neutralizes the Zeta toxin through the formation of a tight complex. In this study, we show, using both in vivo and in vitro analyses, that the ClpXP protease is responsible for Epsilon antitoxin degradation. Using in vivo studies, we examined the stability of the plasmids with active or inactive ω-ε-ζ TA cassettes in B. subtilis mutants that were defective for different proteases. Using in vitro assays, the degradation of purified His6-Epsilon by the His6-LonBs, ClpPBs, and ClpXBs proteases from B. subtilis was analyzed. Additionally, we showed that purified Zeta toxin protects the Epsilon protein from rapid ClpXP-catalyzed degradation.

Growth on octane alters the membrane lipid fatty acids of Pseudomonas oleovorans due to the induction of alkB and synthesis of octanol.

PubMed Central

Chen, Q; Janssen, D B; Witholt, B

1995-01-01

Growth of Pseudomonas oleovorans GPo1, which contains the OCT plasmid, on octane results in changes in the membrane phospholipid fatty acid composition. These changes were not found for GPo12, an OCT-plasmid-cured variant of GPo1, during growth in the presence or absence of octane, implying the involvement of OCT-plasmid-encoded functions. When recombinant strain GPo12(pGEc47) carrying the alk genes from the OCT plasmid was grown on octane, the cells showed the same changes in fatty acid composition as those found for GPo1, indicating that such changes result from induction and expression of the alk genes. This finding was corroborated by inducing GPo12(pGEc47) with dicyclopropylketone (DCPK), a gratuitous inducer of the alk genes. Further experiments showed that the increase of the mean acyl chain length of fatty acids is related to the expression of alkB, which encodes a major integral membrane protein, while the formation of trans unsaturated fatty acids mainly results from the effects of 1-octanol, an octane oxidation product. PMID:7592483

Complete sequence of a plasmid from a bovine methicillin-resistant Staphylococcus aureus harbouring a novel ica-like gene cluster in addition to antimicrobial and heavy metal resistance genes.

PubMed

Feßler, Andrea T; Zhao, Qin; Schoenfelder, Sonja; Kadlec, Kristina; Brenner Michael, Geovana; Wang, Yang; Ziebuhr, Wilma; Shen, Jianzhong; Schwarz, Stefan

2017-02-01

The multiresistance plasmid pAFS11, obtained from a bovine methicillin-resistant Staphylococcus aureus (MRSA) isolate, was completely sequenced and analysed for its structure and organisation. Moreover, the susceptibility to the heavy metals cadmium and copper was determined by broth macrodilution. The 49,189-bp plasmid harboured the apramycin resistance gene apmA, two copies of the macrolide/lincosamide/streptogramin B resistance gene erm(B) (both located on remnants of a truncated transposon Tn917), the kanamycin/neomycin resistance gene aadD, the tetracycline resistance gene tet(L) and the trimethoprim resistance gene dfrK. The latter three genes were part of a 7,284-bp segment which was bracketed by two copies of IS431. In addition, the cadmium resistance operon cadDX as well as the copper resistance genes copA and mco were located on the plasmid and mediated a reduced susceptibility to cadmium and copper. Moreover, a complete novel ica-like gene cluster of so far unknown genetic origin was detected on this plasmid. The ica-like gene cluster comprised four different genes whose products showed 64.4-76.9% homology to the Ica proteins known to be involved in biofilm formation of the S. aureus strains Mu50, Mu3 and N315. However, 96.2-99.4% homology was seen to proteins from S. sciuri NS1 indicating an S. sciuri origin. The finding of five different antibiotic resistance genes co-located on a plasmid with heavy metal resistance genes and an ica-like gene cluster is alarming. With the acquisition of this plasmid, antimicrobial multiresistance, heavy metal resistances and potential virulence properties may be co-selected and spread via a single horizontal gene transfer event. Copyright © 2016 Elsevier B.V. All rights reserved.

The Origin of Mutants Under Selection: How Natural Selection Mimics Mutagenesis (Adaptive Mutation)

PubMed Central

Maisnier-Patin, Sophie; Roth, John R.

2015-01-01

Selection detects mutants but does not cause mutations. Contrary to this dictum, Cairns and Foster plated a leaky lac mutant of Escherichia coli on lactose medium and saw revertant (Lac+) colonies accumulate with time above a nongrowing lawn. This result suggested that bacteria might mutagenize their own genome when growth is blocked. However, this conclusion is suspect in the light of recent evidence that revertant colonies are initiated by preexisting cells with multiple copies the conjugative F′lac plasmid, which carries the lac mutation. Some plated cells have multiple copies of the simple F′lac plasmid. This provides sufficient LacZ activity to support plasmid replication but not cell division. In nongrowing cells, repeated plasmid replication increases the likelihood of a reversion event. Reversion to lac+ triggers exponential cell growth leading to a stable Lac+ revertant colony. In 10% of these plated cells, the high-copy plasmid includes an internal tandem lac duplication, which provides even more LacZ activity—sufficient to support slow growth and formation of an unstable Lac+ colony. Cells with multiple copies of the F′lac plasmid have an increased mutation rate, because the plasmid encodes the error-prone (mutagenic) DNA polymerase, DinB. Without DinB, unstable and stable Lac+ revertant types form in equal numbers and both types arise with no mutagenesis. Amplification and selection are central to behavior of the Cairns–Foster system, whereas mutagenesis is a system-specific side effect or artifact caused by coamplification of dinB with lac. Study of this system has revealed several broadly applicable principles. In all populations, gene duplications are frequent stable genetic polymorphisms, common near-neutral mutant alleles can gain a positive phenotype when amplified under selection, and natural selection can operate without cell division when variability is generated by overreplication of local genome subregions. PMID:26134316

One pyrimidine dimer inactivates expression of a transfected gene in xeroderma pigmentosum cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Protic-Sabljic, M.; Kraemer, K.H.

1985-10-01

The authors have developed a host cell reactivation assay of DNA repair utilizing UV-treated plasmid vectors. The assay primarily reflects cellular repair of transcriptional activity of damaged DNA measured indirectly as enzyme activity of the transfected genes. They studied three plasmids (pSV2cat, 5020 base pairs; pSV2catSVgpt, 7268 base pairs; and pRSVcat, 5027 base pairs) with different sizes and promoters carrying the bacterial cat gene (CAT, chloramphenicol acetyltransferase) in a construction that permits cat expression in human cells. All human simian virus 40-transformed cells studied expressed high levels of the transfected cat gene. UV treatment of the plasmids prior to transfectionmore » resulted in differential decrease in CAT activity in different cell lines. With pSV2catSVgpt, UV inactivation of CAT expression was greater in the xeroderma pigmentosum group A and D lines than in the other human cell lines tested. The D0 of the CAT inactivation curve was 50 J X m-2 for pSV2cat and for pRSVcat in the xeroderma pigmentosum group A cells. The similarity of the D0 data in the xeroderma pigmentosum group A cells for three plasmids of different size and promoters implies they all have similar UV-inactivation target size. UV-induced pyrimidine dimer formation in the plasmids was quantified by assay of the number of UV-induced T4 endonuclease V-sensitive sites. In the most sensitive xeroderma pigmentosum cells, with all three plasmids, one UV-induced pyrimidine dimer inactivates a target of about 2 kilobases, close to the size of the putative CAT mRNA.« less

Functional relationships between plasmids and their significance for metabolism and symbiotic performance of Rhizobium leguminosarum bv. trifolii.

PubMed

Stasiak, Grażyna; Mazur, Andrzej; Wielbo, Jerzy; Marczak, Małgorzata; Zebracki, Kamil; Koper, Piotr; Skorupska, Anna

2014-11-01

Rhizobium leguminosarum bv. trifolii TA1 (RtTA1) is a soil bacterium establishing a highly specific symbiotic relationship with clover, which is based on the exchange of molecular signals between the host plant and the microsymbiont. The RtTA1 genome is large and multipartite, composed of a chromosome and four plasmids, which comprise approximately 65 % and 35 % of the total genome, respectively. Extrachromosomal replicons were previously shown to confer significant metabolic versatility to bacteria, which is important for their adaptation in the soil and nodulation competitiveness. To investigate the contribution of individual RtTA1 plasmids to the overall cell phenotype, metabolic properties and symbiotic performance, a transposon-based elimination strategy was employed. RtTA1 derivatives cured of pRleTA1b or pRleTA1d and deleted in pRleTA1a were obtained. In contrast to the in silico predictions of pRleTA1b and pRleTA1d, which were described as chromid-like replicons, both appeared to be completely curable. On the other hand, for pRleTA1a (symbiotic plasmid) and pRleTA1c, which were proposed to be unessential for RtTA1 viability, it was not possible to eliminate them at all (pRleTA1c) or entirely (pRleTA1a). Analyses of the phenotypic traits of the RtTA1 derivatives obtained revealed the functional significance of individual plasmids and their indispensability for growth, certain metabolic pathways, production of surface polysaccharides, autoaggregation, biofilm formation, motility and symbiotic performance. Moreover, the results allow us to suggest broad functional cooperation among the plasmids in shaping the phenotypic properties and symbiotic capabilities of rhizobia.

Incision of trivalent chromium [Cr(III)]-induced DNA damage by Bacillus caldotenax UvrABC endonuclease.

PubMed

O'Brien, Travis J; Jiang, Guohui; Chun, Gina; Mandel, H George; Westphal, Craig S; Kahen, Kaveh; Montaser, Akbar; States, J Christopher; Patierno, Steven R

2006-11-07

Some hexavalent chromium [Cr(VI)]-containing compounds are lung carcinogens. Once within cells, Cr(VI) is reduced to trivalent chromium [Cr(III)] which displays an affinity for both DNA bases and the phosphate backbone. A diverse array of genetic lesions is produced by Cr including Cr-DNA monoadducts, DNA interstrand crosslinks (ICLs), DNA-Cr-protein crosslinks (DPCs), abasic sites, DNA strand breaks and oxidized bases. Despite the large amount of information available on the genotoxicity of Cr, little is known regarding the molecular mechanisms involved in the removal of these lesions from damaged DNA. Recent work indicates that nucleotide excision repair (NER) is involved in the processing of Cr-DNA adducts in human and rodent cells. In order to better understand this process at the molecular level and begin to identify the Cr-DNA adducts processed by NER, the incision of CrCl(3) [Cr(III)]-damaged plasmid DNA was studied using a thermal-resistant UvrABC NER endonuclease from Bacillus caldotenax (Bca). Treatment of plasmid DNA with Cr(III) (as CrCl(3)) increased DNA binding as a function of dose. For example, at a Cr(III) concentration of 1 microM we observed approximately 2 Cr(III)-DNA adducts per plasmid. At this same concentration of Cr(III) we found that approximately 17% of the plasmid DNA contained ICLs ( approximately 0.2 ICLs/plasmid). When plasmid DNA treated with Cr(III) (1 microM) was incubated with Bca UvrABC we observed approximately 0.8 incisions/plasmid. The formation of endonuclease IV-sensitive abasic lesions or Fpg-sensitive oxidized DNA bases was not detected suggesting that the incision of Cr(III)-damaged plasmid DNA by UvrABC was not related to the generation of oxidized DNA damage. Taken together, our data suggest that a sub-fraction of Cr(III)-DNA adducts is recognized and processed by the prokaryotic NER machinery and that ICLs are not necessarily the sole lesions generated by Cr(III) that are substrates for NER.

Strategies to optimize capsid protein expression and single-stranded DNA formation of adeno-associated virus in Saccharomyces cerevisiae.

PubMed

Galli, A; Della Latta, V; Bologna, C; Pucciarelli, D; Cipriani, F; Backovic, A; Cervelli, T

2017-08-01

Adeno-associated virus type 2 (AAV) is a nonpathogenic parvovirus that is a promising tool for gene therapy. We aimed to construct plasmids for optimal expression and assembly of capsid proteins and evaluate adenovirus (Ad) protein effect on AAV single-stranded DNA (ssDNA) formation in Saccharomyces cerevisiae. Yeast expression plasmids have been developed in which the transcription of AAV capsid proteins (VP1,2,3) is driven by the constitutive ADH1 promoter or galactose-inducible promoters. Optimal VP1,2,3 expression was obtained from GAL1/10 bidirectional promoter. Moreover, we demonstrated that AAP is expressed in yeast and virus-like particles (VLPs) assembled inside the cell. Finally, the expression of two Ad proteins, E4orf6 and E1b55k, had no effect on AAV ssDNA formation. This study confirms that yeast is able to form AAV VLPs; however, capsid assembly and ssDNA formation are less efficient in yeast than in human cells. Moreover, the expression of Ad proteins did not affect AAV ssDNA formation. New manufacturing strategies for AAV-based gene therapy vectors (rAAV) are needed to reduce costs and time of production. Our study explores the feasibility of yeast as alternative system for rAAV production. © 2017 The Society for Applied Microbiology.

Interactions of Kid-Kis toxin-antitoxin complexes with the parD operator-promoter region of plasmid R1 are piloted by the Kis antitoxin and tuned by the stoichiometry of Kid-Kis oligomers.

PubMed

Monti, Maria C; Hernández-Arriaga, Ana M; Kamphuis, Monique B; López-Villarejo, Juan; Heck, Albert J R; Boelens, Rolf; Díaz-Orejas, Ramón; van den Heuvel, Robert H H

2007-01-01

The parD operon of Escherichia coli plasmid R1 encodes a toxin-antitoxin system, which is involved in plasmid stabilization. The toxin Kid inhibits cell growth by RNA degradation and its action is neutralized by the formation of a tight complex with the antitoxin Kis. A fascinating but poorly understood aspect of the kid-kis system is its autoregulation at the transcriptional level. Using macromolecular (tandem) mass spectrometry and DNA binding assays, we here demonstrate that Kis pilots the interaction of the Kid-Kis complex in the parD regulatory region and that two discrete Kis-binding regions are present on parD. The data clearly show that only when the Kis concentration equals or exceeds the Kid concentration a strong cooperative effect exists between strong DNA binding and Kid2-Kis2-Kid2-Kis2 complex formation. We propose a model in which transcriptional repression of the parD operon is tuned by the relative molar ratio of the antitoxin and toxin proteins in solution. When the concentration of the toxin exceeds that of the antitoxin tight Kid2-Kis2-Kid2 complexes are formed, which only neutralize the lethal activity of Kid. Upon increasing the Kis concentration, (Kid2-Kis2)n complexes repress the kid-kis operon.

Earthquakes Promote Bacterial Genetic Exchange in Serpentinite Crevices

NASA Astrophysics Data System (ADS)

Naoto, Yoshida; Fujiura, Nori

2009-04-01

We report the results of our efforts to study the effects of seismic shaking on simulated biofilms within serpentinite fissures. A colloidal solution consisting of recipient bacterial cells (Pseudomonas sp. or Bacillus subtilis), donor plasmid DNA encoded for antibiotic resistance, and chrysotile (an acicular clay mineral that forms in crevices of serpentinite layers) were placed onto an elastic body made from gellan gum, which acted as the biofilm matrix. Silica beads, as rock analogues (i.e., chemically inert mechanical serpentinite), were placed on the gellan surface, which was coated with the colloidal solution. A rolling vibration similar to vibrations generated by earthquakes was applied, and the silica beads moved randomly across the surface of the gellan. This resulted in the recipient cells' acquiring plasmid DNA and thus becoming genetically transformed to demonstrate marked antibiotic resistance. Neither Pseudomonas sp. nor B. subtilis were transformed by plasmid DNA when chrysotile was substituted for by kaolinite or bentonite in the colloidal solution. Tough gellan (1.0%) promoted the introduction of plasmid DNA into Pseudomonas sp., but soft gellan (0.3%) had no such effect. Genetic transformation of bacteria on the surface of gellan by exposure to exogenous plasmid DNA required seismic shaking and exposure to the acicular clay mineral chrysotile. These experimental results suggest that bacterial genetic exchange readily occurs when biofilms that form in crevices of serpentinite are exposed to seismic shaking. Seismic activity may be a key factor in bacterial evolution along with the formation of biofilms within crevices of serpentinite.

Encapsulation of Nucleic Acids into Giant Unilamellar Vesicles by Freeze-Thaw: a Way Protocells May Form

NASA Astrophysics Data System (ADS)

Qiao, Hai; Hu, Na; Bai, Jin; Ren, Lili; Liu, Qing; Fang, Liaoqiong; Wang, Zhibiao

2017-12-01

Protocells are believed to consist of a lipid membrane and encapsulated nucleic acid. As the lipid membrane is impermeable to macromolecules like nucleic acids, the processes by which nucleic acids become encapsulated inside lipid membrane compartments are still unknown. In this paper, a freeze-thaw method was modified and applied to giant unilamellar vesicles (GUVs) and deoxyribonucleic acid (DNA) in mixed solution resulting in the efficient encapsulation of 6.4 kb plasmid DNA and similar length linear DNA into GUVs. The mechanism of encapsulation was followed by observing the effect of freeze-thaw temperatures on GUV morphological change, DNA encapsulation and ice crystal formation, and analyzing their correlation. Following ice crystal formation, the shape of spherical GUVs was altered and membrane integrity was damaged and this was found to be a necessary condition for encapsulation. Heating alone had no effects on DNA encapsulation, but was helpful for restoring the spherical shape and membrane integrity of GUVs damaged during freezing. These results suggested that freeze-thaw could promote the encapsulation of DNA into GUVs by a mechanism: the vesicle membrane was breached by ice crystal formation during freezing, DNA entered into damaged GUVs through these membrane gaps and was encapsulated after the membrane was resealed during the thawing process. The process described herein therefore describes a simple way for the encapsulation of nucleic acids and potentially other macromolecules into lipid vesicles, a process by which early protocells might have formed.

High-throughput assays for DNA gyrase and other topoisomerases

PubMed Central

Maxwell, Anthony; Burton, Nicolas P.; O'Hagan, Natasha

2006-01-01

We have developed high-throughput microtitre plate-based assays for DNA gyrase and other DNA topoisomerases. These assays exploit the fact that negatively supercoiled plasmids form intermolecular triplexes more efficiently than when they are relaxed. Two assays are presented, one using capture of a plasmid containing a single triplex-forming sequence by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by staining with a DNA-specific fluorescent dye. The other uses capture of a plasmid containing two triplex-forming sequences by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by a second oligonucleotide that is radiolabelled. The assays are shown to be appropriate for assaying DNA supercoiling by Escherichia coli DNA gyrase and DNA relaxation by eukaryotic topoisomerases I and II, and E.coli topoisomerase IV. The assays are readily adaptable to other enzymes that change DNA supercoiling (e.g. restriction enzymes) and are suitable for use in a high-throughput format. PMID:16936317

High-throughput assays for DNA gyrase and other topoisomerases.

PubMed

Maxwell, Anthony; Burton, Nicolas P; O'Hagan, Natasha

2006-01-01

We have developed high-throughput microtitre plate-based assays for DNA gyrase and other DNA topoisomerases. These assays exploit the fact that negatively supercoiled plasmids form intermolecular triplexes more efficiently than when they are relaxed. Two assays are presented, one using capture of a plasmid containing a single triplex-forming sequence by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by staining with a DNA-specific fluorescent dye. The other uses capture of a plasmid containing two triplex-forming sequences by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by a second oligonucleotide that is radiolabelled. The assays are shown to be appropriate for assaying DNA supercoiling by Escherichia coli DNA gyrase and DNA relaxation by eukaryotic topoisomerases I and II, and E.coli topoisomerase IV. The assays are readily adaptable to other enzymes that change DNA supercoiling (e.g. restriction enzymes) and are suitable for use in a high-throughput format.

Bioaugmentation of activated sludge towards 3-chloroaniline removal with a mixed bacterial population carrying a degradative plasmid.

PubMed

Bathe, Stephan; Schwarzenbeck, Norbert; Hausner, Martina

2009-06-01

A bioaugmentation approach combining several strategies was applied to achieve degradation of 3-chloroaniline (3CA) in semicontinuous activated sludge reactors. In a first step, a 3CA-degrading Comamonas testosteroni strain carrying the degradative plasmid pNB2 was added to a biofilm reactor, and complete 3CA degradation together with spread of the plasmid within the indigenous biofilm population was achieved. A second set of reactors was then bioaugmented with either a suspension of biofilm cells removed from the carrier material or with biofilm-containing carrier material. 3CA degradation was established rapidly in all bioaugmented reactors, followed by a slow adaptation of the non-bioaugmented control reactors. In response to variations in 3CA concentration, all reactors exhibited temporary performance breakdowns. Whereas duplicates of the control reactors deviated in their behaviour, the bioaugmented reactors appeared more reproducible in their performance and population dynamics. Finally, the carrier-bioaugmented reactors showed an improved performance in the presence of high 3CA influent concentrations over the suspension-bioaugmented reactors. In contrast, degradation in one control reactor failed completely, but was rapidly established in the remaining control reactor.

Plasmid-Encoded MCP Is Involved in Virulence, Motility, and Biofilm Formation of Cronobacter sakazakii ATCC 29544

PubMed Central

Choi, Younho; Kim, Seongok; Hwang, Hyelyeon; Kim, Kwang-Pyo; Kang, Dong-Hyun

2014-01-01

The aim of this study was to elucidate the function of the plasmid-borne mcp (methyl-accepting chemotaxis protein) gene, which plays pleiotropic roles in Cronobacter sakazakii ATCC 29544. By searching for virulence factors using a random transposon insertion mutant library, we identified and sequenced a new plasmid, pCSA2, in C. sakazakii ATCC 29544. An in silico analysis of pCSA2 revealed that it included six putative open reading frames, and one of them was mcp. The mcp mutant was defective for invasion into and adhesion to epithelial cells, and the virulence of the mcp mutant was attenuated in rat pups. In addition, we demonstrated that putative MCP regulates the motility of C. sakazakii, and the expression of the flagellar genes was enhanced in the absence of a functional mcp gene. Furthermore, a lack of the mcp gene also impaired the ability of C. sakazakii to form a biofilm. Our results demonstrate a regulatory role for MCP in diverse biological processes, including the virulence of C. sakazakii ATCC 29544. To the best of our knowledge, this study is the first to elucidate a potential function of a plasmid-encoded MCP homolog in the C. sakazakii sequence type 8 (ST8) lineage. PMID:25332122

Plasmid-encoded MCP is involved in virulence, motility, and biofilm formation of Cronobacter sakazakii ATCC 29544.

PubMed

Choi, Younho; Kim, Seongok; Hwang, Hyelyeon; Kim, Kwang-Pyo; Kang, Dong-Hyun; Ryu, Sangryeol

2015-01-01

The aim of this study was to elucidate the function of the plasmid-borne mcp (methyl-accepting chemotaxis protein) gene, which plays pleiotropic roles in Cronobacter sakazakii ATCC 29544. By searching for virulence factors using a random transposon insertion mutant library, we identified and sequenced a new plasmid, pCSA2, in C. sakazakii ATCC 29544. An in silico analysis of pCSA2 revealed that it included six putative open reading frames, and one of them was mcp. The mcp mutant was defective for invasion into and adhesion to epithelial cells, and the virulence of the mcp mutant was attenuated in rat pups. In addition, we demonstrated that putative MCP regulates the motility of C. sakazakii, and the expression of the flagellar genes was enhanced in the absence of a functional mcp gene. Furthermore, a lack of the mcp gene also impaired the ability of C. sakazakii to form a biofilm. Our results demonstrate a regulatory role for MCP in diverse biological processes, including the virulence of C. sakazakii ATCC 29544. To the best of our knowledge, this study is the first to elucidate a potential function of a plasmid-encoded MCP homolog in the C. sakazakii sequence type 8 (ST8) lineage. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Quorum Sensing and Quorum Quenching in Agrobacterium: A "Go/No Go System"?

PubMed

Dessaux, Yves; Faure, Denis

2018-04-16

The pathogen Agrobacterium induces gall formation on a wide range of dicotyledonous plants. In this bacteria, most pathogenicity determinants are borne on the tumour inducing (Ti) plasmid. The conjugative transfer of this plasmid between agrobacteria is regulated by quorum sensing (QS). However, processes involved in the disturbance of QS also occur in this bacteria under the molecular form of a protein, TraM, inhibiting the sensing of the QS signals, and two lactonases BlcC (AttM) and AiiB that degrade the acylhomoserine lactone (AHL) QS signal. In the model Agrobacterium fabrum strain C58, several data, once integrated, strongly suggest that the QS regulation may not be reacting only to cell concentration. Rather, these QS elements in association with the quorum quenching (QQ) activities may constitute an integrated and complex “go/no go system” that finely controls the biologically costly transfer of the Ti plasmid in response to multiple environmental cues. This decision mechanism permits the bacteria to sense whether it is in a gall or not, in a living or decaying tumor, in stressed plant tissues, etc. In this scheme, the role of the lactonases selected and maintained in the course of Ti plasmid and agrobacterial evolution appears to be pivotal.

The effect of dimethyl sulfoxide on the induction of DNA strand breaks in plasmid DNA and colony formation of PC Cl3 mammalian cells by alpha-, beta-, and Auger electron emitters (223)Ra, (188)Re, and (99m)Tc.

PubMed

Runge, Roswitha; Oehme, Liane; Kotzerke, Jörg; Freudenberg, Robert

2016-12-01

DNA damage occurs as a consequence of both direct and indirect effects of ionizing radiation. The severity of DNA damage depends on the physical characteristics of the radiation quality, e.g., the linear energy transfer (LET). There are still contrary findings regarding direct or indirect interactions of high-LET emitters with DNA. Our aim is to determine DNA damage and the effect on cellular survival induced by (223)Ra compared to (188)Re and (99m)Tc modulated by the radical scavenger dimethyl sulfoxide (DMSO). Radioactive solutions of (223)Ra, (188)Re, or (99m)Tc were added to either plasmid DNA or to PC Cl3 cells in the absence or presence of DMSO. Following irradiation, single strand breaks (SSB) and double strand breaks (DSB) in plasmid DNA were analyzed by gel electrophoresis. To determine the radiosensitivity of the rat thyroid cell line (PC Cl3), survival curves were performed using the colony formation assay. Exposure to 120 Gy of (223)Ra, (188)Re, or (99m)Tc leads to maximal yields of SSB (80 %) in plasmid DNA. Irradiation with 540 Gy (223)Ra and 500 Gy (188)Re or (99m)Tc induced 40, 28, and 64 % linear plasmid conformations, respectively. DMSO prevented the SSB and DSB in a similar way for all radionuclides. However, with the α-emitter (223)Ra, a low level of DSB could not be prevented by DMSO. Irradiation of PC Cl3 cells with (223)Ra, (188)Re, and (99m)Tc pre-incubated with DMSO revealed enhanced survival fractions (SF) in comparison to treatment without DMSO. Protection factors (PF) were calculated using the fitted survival curves. These factors are 1.23 ± 0.04, 1.20 ± 0.19, and 1.34 ± 0.05 for (223)Ra, (188)Re, and (99m)Tc, respectively. For (223)Ra, as well as for (188)Re and (99m)Tc, dose-dependent radiation effects were found applicable for plasmid DNA and PC Cl3 cells. The radioprotection by DMSO was in the same range for high- and low-LET emitter. Overall, the results indicate the contribution of mainly indirect radiation effects for each of the radionuclides regarding DNA damage and cell survival. In summary, our findings may contribute to fundamental knowledge about the α-particle induced DNA damage.

Electrostatic bio-manipulation for the modification of cellular functions

NASA Astrophysics Data System (ADS)

Washizu, Masao

2013-03-01

The use of electrostatic field effects, including field-induced reversible-breakdown of the membrane and dielectrophoresis (DEP), in microfabricated structures are investigated. With the use of field constriction created by a micro-orifice whose diameter is smaller than the cells, controlled magnitude of pulsed voltage can be applied across the cell membrane regardless of the cell size, shape or orientation. As a result, the breakdown occurs reproducibly and with minimal invasiveness. The breakdown is used for two purposes, electroporation by which foreign substances can be fed into cells, and electrofusion which creates genetic and/or cytoplasmic mixture among two cells. When GFP plasmid is fed into MSC cell, the gene expression started within 2 hours, and finally observed in more than 50% of cells. For cell fusion, several ten percent fusion yield is achieved for most cell types, with the colony formation in several percents. Timing-controlled feeding foreign substances or mixing cellular contents, with high-yield and low-invasiveness, is expected to bring about a new technology for both genetic and epigenetic modifications of cellular functions, in such field as regenerative medicine.

Interactions of Kid–Kis toxin–antitoxin complexes with the parD operator-promoter region of plasmid R1 are piloted by the Kis antitoxin and tuned by the stoichiometry of Kid–Kis oligomers

PubMed Central

Monti, Maria C.; Hernández-Arriaga, Ana M.; Kamphuis, Monique B.; López-Villarejo, Juan; Heck, Albert J. R.; Boelens, Rolf; Díaz-Orejas, Ramón; van den Heuvel, Robert H. H.

2007-01-01

The parD operon of Escherichia coli plasmid R1 encodes a toxin–antitoxin system, which is involved in plasmid stabilization. The toxin Kid inhibits cell growth by RNA degradation and its action is neutralized by the formation of a tight complex with the antitoxin Kis. A fascinating but poorly understood aspect of the kid–kis system is its autoregulation at the transcriptional level. Using macromolecular (tandem) mass spectrometry and DNA binding assays, we here demonstrate that Kis pilots the interaction of the Kid–Kis complex in the parD regulatory region and that two discrete Kis-binding regions are present on parD. The data clearly show that only when the Kis concentration equals or exceeds the Kid concentration a strong cooperative effect exists between strong DNA binding and Kid2–Kis2–Kid2–Kis2 complex formation. We propose a model in which transcriptional repression of the parD operon is tuned by the relative molar ratio of the antitoxin and toxin proteins in solution. When the concentration of the toxin exceeds that of the antitoxin tight Kid2–Kis2–Kid2 complexes are formed, which only neutralize the lethal activity of Kid. Upon increasing the Kis concentration, (Kid2–Kis2)n complexes repress the kid–kis operon. PMID:17317682

Virulence properties of multidrug resistant ocular isolates of Acinetobacter baumannii.

PubMed

Talreja, Deepa; Muraleedharan, Chithra; Gunathilaka, Gayathri; Zhang, Yifan; Kaye, Keith S; Walia, Satish K; Kumar, Ashok

2014-07-01

Acinetobacter (A.) baumannii is an opportunistic pathogen and has been reported as a causative agent of ocular infections. The aim of this study is to identify virulence properties (biofilm formation, adhesion, invasion and cytotoxicity) and antibiotic resistance among A. baumannii isolates recovered from the eye. The Microscan Walk-Away®, an automated bacterial identification and susceptibility testing system was used to determine antibiotic resistance. Clonal relatedness was assessed by Pulsed-field gel electrophoresis (PFGE) and plasmid profile analysis. Conjugation experiments were carried out to determine the transfer of antibiotic resistance genes and PCR was used to confirm gene transfer. Virulence properties of the isolates were determined by biofilm formation using crystal violet and immunofluorescence staining, adherence and internalization using cultured corneal epithelial cells, and cytotoxicity by TUNEL-staining and LDH release assays. All ocular isolates (n = 12) exhibited multidrug resistant (MDR) phenotype and one of the isolate (AB12) was resistant to 18 antibiotics (β-lactam, aminoglycosides, tetracycline, chloramphenicol and quinolones). The plasmid profile analysis showed the presence of multiple plasmids in each isolate and a total of 10 different profiles were observed. However, PFGE analysis was more discriminatory which revealed 12 distinct genotypes. Antibiotic resistance (tetracycline and quinolone) was transferable from the isolate AB12 to a recipient Escherichia coli J53. Ten isolates were strong biofilm producers and the remaining two (AB5 and AB7) were moderate producers. All isolates demonstrated adherence and invasive properties towards HCECs. A similar trend was observed in their ability to cause cell death and toxicity. Our results indicate that ocular isolates of A. baumannii are biofilm producers and adherent and invasive to corneal epithelium, a first step in the pathogenesis of ocular infection. In addition, they demonstrated plasmid-mediated transfer of MDR traits making them a reservoir of resistance genes at ocular surface.

The vector homology problem in diagnostic nucleic acid hybridization of clinical specimens.

PubMed Central

Ambinder, R F; Charache, P; Staal, S; Wright, P; Forman, M; Hayward, S D; Hayward, G S

1986-01-01

Nucleic acid hybridization techniques using cloned probes are finding application in assays of clinical specimens in research and diagnostic laboratories. The probes that we and others have used are recombinant plasmids composed of viral inserts and bacterial plasmid vectors such as pBR322. We suspected that there was material homologous to pBR322 present in many clinical samples. because hybridization occurred in samples which lacked evidence of virus by other techniques. If the presence of this vector-homologous material was unrecognized, hybridization in the test sample might erroneously be interpreted as indicating the presence of viral sequences. In this paper we demonstrate specific hybridization of labeled pBR322 DNA with DNA from various clinical samples. Evidence is presented that nonspecific probe trapping could not account for this phenomenon. In mixing experiments, it is shown that contamination of clinical samples with bacteria would explain such a result. Approaches tested to circumvent this problem included the use of isolated insert probes, alternate cloning vectors, and cold competitor pBR322 DNA in prehybridization and hybridization mixes. None proved entirely satisfactory. We therefore emphasize that it is essential that all hybridization detection systems use a control probe of the vector alone in order to demonstrate the absence of material with vector homology in the specimen tested. Images PMID:3013928

[Involvement of the global regulators GrrS, RpoS, and SplIR in formation of biofilms in Serratia plymuthica].

PubMed

Zaĭtseva, Iu V; Voloshina, P V; Liu, X; Ovadis, M I; Berg, G; Chernin, L S; Khmel', I A

2010-05-01

Most bacteria exist in the natural environment as biofilms, multicellular communities attached to hard surfaces. Biofilms have a characteristic architecture and are enclosed in the exopolymer matrix. Bacterial cells in biofilms are extremely resistant to antibacterial factors. It was shown in this work that the GrrA/GrrS system of global regulators of gene expression and the sigma S subunit of RNA polymerase (RpoS) play a significant role in positive regulation of biofilm formation in the rhizospheric bacterium Serratia plymuthica IC1270. Inactivation of grrS and rpoS genes resulted in an up to six-to-sevenfold and four-to-fivefold reduction in biofilm formation, respectively. Mutations in the grrS gene decreased the capacity of the bacterium for swarming motility. The splIR Quorum Sensing (QS) system was shown to negatively influence the biofilm formation. Transfer of the recombinant plasmid containing cloned genes splI/splR of S. plymuthica HRO-C48 into S. plymuthica IC1270 cells led to a twofold decrease of their ability to form biofilms. Inactivation of the splI gene coding for the synthase of N-acyl-homoserine lactones in S. plymuthica HRO-C48 resulted in a 2-2.5-fold increase in the level of biofilm formation, whereas the inclusion of plasmid carrying the cloned splI/splR genes into these mutant cells restored the biofilm formation to the normal level. The results obtained demonstrate that the formation of biofilms in S. plymuthica is positively regulated by the GrrA/GrrS and RpoS global regulators and is negatively regulated by the SplIR QS system.

Properties of an unusual DNA primase from an archaeal plasmid

PubMed Central

Beck, Kirsten; Lipps, Georg

2007-01-01

Primases are specialized DNA-dependent RNA polymerases that synthesize a short oligoribonucleotide complementary to single-stranded template DNA. In the context of cellular DNA replication, primases are indispensable since DNA polymerases are not able to start DNA polymerization de novo. The primase activity of the replication protein from the archaeal plasmid pRN1 synthesizes a rather unusual mixed primer consisting of a single ribonucleotide at the 5′ end followed by seven deoxynucleotides. Ribonucleotides and deoxynucleotides are strictly required at the respective positions within the primer. Furthermore, in contrast to other archaeo-eukaryotic primases, the primase activity is highly sequence-specific and requires the trinucleotide motif GTG in the template. Primer synthesis starts outside of the recognition motif, immediately 5′ to the recognition motif. The fidelity of the primase synthesis is high, as non-complementary bases are not incorporated into the primer. PMID:17709343

Effect of West Nile virus DNA-plasmid vaccination on response to live virus challenge in red-tailed hawks (Buteo jamaicensis).

PubMed

Redig, Patrick T; Tully, Thomas N; Ritchie, Branson W; Roy, Alma F; Baudena, M Alexandra; Chang, Gwong-Jen J

2011-08-01

To evaluate the safety and efficacy of an experimental adjuvanted DNA-plasmid vaccine against West Nile virus (WNV) in red-tailed hawks (Buteo jamaicensis). 19 permanently disabled but otherwise healthy red-tailed hawks of mixed ages and both sexes without detectable serum antibodies against WNV. Hawks were injected IM with an experimental WNV DNA-plasmid vaccine in an aluminum-phosphate adjuvant (n = 14) or with the adjuvant only (control group; 5). All birds received 2 injections at a 3-week interval. Blood samples for serologic evaluation were collected before the first injection and 4 weeks after the second injection (day 0). At day 0, hawks were injected SC with live WNV. Pre- and postchallenge blood samples were collected at intervals for 14 days for assessment of viremia and antibody determination; oropharyngeal and cloacal swabs were collected for assessment of viral shedding. Vaccination was not associated with morbidity or deaths. Three of the vaccinated birds seroconverted after the second vaccine injection; all other birds seroconverted following the live virus injection. Vaccinated birds had significantly less severe viremia and shorter and less-intense shedding periods, compared with the control birds. Use of the WNV DNA-plasmid vaccine in red-tailed hawks was safe, and vaccination attenuated but did not eliminate both the viremia and the intensity of postchallenge shedding following live virus exposure. Further research is warranted to conclusively determine the efficacy of this vaccine preparation for protection of red-tailed hawks and other avian species against WNV-induced disease.

Environmental emission of multiresistant Escherichia coli carrying the colistin resistance gene mcr-1 from German swine farms.

PubMed

Guenther, Sebastian; Falgenhauer, Linda; Semmler, Torsten; Imirzalioglu, Can; Chakraborty, Trinad; Roesler, Uwe; Roschanski, Nicole

2017-05-01

Pigs have been the focus of the worldwide spread of colistin resistance. However, there is little information on the transmission of mcr-1 -containing bacteria into the environment of pig farms. We therefore rescreened environmental Escherichia coli isolates from the surrounding farm areas of three previously mcr-1 -positive swine herds in Germany. Thirty-five mixed bacterial cultures obtained from boot swabs, flies, dog faeces and manure from three pig farms in Germany in 2011-12 were non-selectively recultivated and the presence of the mcr-1 gene was checked by real-time PCR. After separation, single E. coli colonies were subsequently isolated and the presence of mcr-1 was confirmed by PCR and sequencing. In addition, phenotypic antimicrobial resistance screening and WGS followed by phylogenetic analysis and resistance genotyping as well as plasmid typing were performed. Seven mcr-1 -positive E. coli strains originating from environmental boot swabs, dog faeces, stable flies and manure were found. The isolates belonged to five different STs (ST10, ST1011, ST1140, ST5281 and ST342) and harboured extensive additional resistance genes. Comparative plasmid analysis predominantly located mcr-1 on IncX4 plasmids, which are strongly related to a recently described plasmid of human clinical origin (pICBEC72Hmcr). WGS-based analysis of the environmental E. coli isolates of farm surroundings showed clear links to mcr-1 -harbouring E. coli recovered from pig production in Europe as well as from human clinical isolates worldwide, presenting another piece of the puzzle, which further complicates the rapidly evolving epidemiology of plasmid-mediated colistin-resistant E. coli strains. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Fine-tuning synthesis of Yersinia pestis LcrV from runaway-like replication balanced-lethal plasmid in a Salmonella enterica serovar typhimurium vaccine induces protection against a lethal Y. pestis challenge in mice.

PubMed

Torres-Escobar, Ascención; Juárez-Rodríguez, María Dolores; Gunn, Bronwyn M; Branger, Christine G; Tinge, Steven A; Curtiss, Roy

2010-06-01

A balanced-lethal plasmid expression system that switches from low-copy-number to runaway-like high-copy-number replication (pYA4534) was constructed for the regulated delayed in vivo synthesis of heterologous antigens by vaccine strains. This is an antibiotic resistance-free maintenance system containing the asdA gene (essential for peptidoglycan synthesis) as a selectable marker to complement the lethal chromosomal DeltaasdA allele in live recombinant attenuated Salmonella vaccines (RASVs) such as Salmonella enterica serovar Typhimurium strain chi9447. pYA4534 harbors two origins of replication, pSC101 and pUC (low and high copy numbers, respectively). The pUC replication origin is controlled by a genetic switch formed by the operator/promoter of the P22 cro gene (O/P(cro)) (P(R)), which is negatively regulated by an arabinose-inducible P22 c2 gene located on both the plasmid and the chromosome (araC P(BAD) c2). The absence of arabinose, which is unavailable in vivo, triggers replication to a high-copy-number plasmid state. To validate these vector attributes, the Yersinia pestis virulence antigen LcrV was used to develop a vaccine against plague. An lcrV sequence encoding amino acids 131 to 326 (LcrV196) was optimized for expression in Salmonella, flanked with nucleotide sequences encoding the signal peptide (SS) and the carboxy-terminal domain (CT) of beta-lactamase, and cloned into pYA4534 under the control of the P(trc) promoter to generate plasmid pYA4535. Our results indicate that the live Salmonella vaccine strain chi9447 harboring pYA4535 efficiently stimulated a mixed Th1/Th2 immune response that protected mice against lethal challenge with Y. pestis strain CO92 introduced through either the intranasal or subcutaneous route.

Fine-Tuning Synthesis of Yersinia pestis LcrV from Runaway-Like Replication Balanced-Lethal Plasmid in a Salmonella enterica Serovar Typhimurium Vaccine Induces Protection against a Lethal Y. pestis Challenge in Mice▿

PubMed Central

Torres-Escobar, Ascención; Juárez-Rodríguez, María Dolores; Gunn, Bronwyn M.; Branger, Christine G.; Tinge, Steven A.; Curtiss, Roy

2010-01-01

A balanced-lethal plasmid expression system that switches from low-copy-number to runaway-like high-copy-number replication (pYA4534) was constructed for the regulated delayed in vivo synthesis of heterologous antigens by vaccine strains. This is an antibiotic resistance-free maintenance system containing the asdA gene (essential for peptidoglycan synthesis) as a selectable marker to complement the lethal chromosomal ΔasdA allele in live recombinant attenuated Salmonella vaccines (RASVs) such as Salmonella enterica serovar Typhimurium strain χ9447. pYA4534 harbors two origins of replication, pSC101 and pUC (low and high copy numbers, respectively). The pUC replication origin is controlled by a genetic switch formed by the operator/promoter of the P22 cro gene (O/Pcro) (PR), which is negatively regulated by an arabinose-inducible P22 c2 gene located on both the plasmid and the chromosome (araC PBAD c2). The absence of arabinose, which is unavailable in vivo, triggers replication to a high-copy-number plasmid state. To validate these vector attributes, the Yersinia pestis virulence antigen LcrV was used to develop a vaccine against plague. An lcrV sequence encoding amino acids 131 to 326 (LcrV196) was optimized for expression in Salmonella, flanked with nucleotide sequences encoding the signal peptide (SS) and the carboxy-terminal domain (CT) of β-lactamase, and cloned into pYA4534 under the control of the Ptrc promoter to generate plasmid pYA4535. Our results indicate that the live Salmonella vaccine strain χ9447 harboring pYA4535 efficiently stimulated a mixed Th1/Th2 immune response that protected mice against lethal challenge with Y. pestis strain CO92 introduced through either the intranasal or subcutaneous route. PMID:20308296

Isolation and expression of a Bacillus cereus gene encoding benzil reductase.

PubMed

Maruyama, R; Nishizawa, M; Itoi, Y; Ito, S; Inoue, M

2001-12-20

Benzil was reduced stereospecifically to (S)-benzoin by Bacillus cereus strain Tim-r01. To isolate the gene responsible for asymmetric reduction, we constructed a library consisting of Escherichia coli clones that harbored plasmids expressing Bacillus cereus genes. The library was screened using the halo formation assay, and one clone showed benzil reduction to (S)-benzoin. Thus, this clone seemed to carry a plasmid encoding a Bacillus cereus benzil reductase. The deduced amino acid sequence had marked homologies to the Bacillus subtilis yueD protein (41% identity), the yeast open reading frame YIR036C protein (31%), and the mammalian sepiapterin reductases (28% to 30%), suggesting that benzil reductase is a novel short-chain de-hydrogenases/ reductase. Copyright 2001 John Wiley & Sons, Inc.

Recombinant DNA technology for melanoma immunotherapy: anti-Id DNA vaccines targeting high molecular weight melanoma-associated antigen.

PubMed

Barucca, A; Capitani, M; Cesca, M; Tomassoni, D; Kazmi, U; Concetti, F; Vincenzetti, L; Concetti, A; Venanzi, F M

2014-11-01

Anti-idiotypic MK2-23 monoclonal antibody (anti-Id MK2-23 mAb), which mimics the high molecular weight melanoma-associated antigen (HMW-MAA), has been used to implement active immunotherapy against melanoma. However, due to safety and standardization issues, this approach never entered extensive clinical trials. In the present study, we investigated the usage of DNA vaccines as an alternative to MK2-23 mAb immunization. MK2-23 DNA plasmids coding for single chain (scFv) MK2-23 antibody were constructed via the insertion of variable heavy (V H) and light (V L) chains of MK2-23 into the pVAC-1mcs plasmids. Two alternative MK2-23 plasmids format V H/V L, and V L/V H were assembled. We demonstrate that both polypeptides expressed by scFv plasmids in vitro retained the ability to mimic HMW-MAA antigen, and to elicit specific anti-HMW-MAA humoral and cellular immunoresponses in immunized mice. Notably, MK2-23 scFv DNA vaccines impaired the onset and growth of transplantable B16 melanoma cells not engineered to express HMW-MAA. This pilot study suggests that optimized MK2-23 scFv DNA vaccines could potentially provide a safer and cost-effective alternative to anti-Id antibody immunization, for melanoma immunotherapy.

Lactobacillus backii and Pediococcus damnosus isolated from 170-year-old beer recovered from a shipwreck lack the metabolic activities required to grow in modern lager beer.

PubMed

Kajala, Ilkka; Bergsveinson, Jordyn; Friesen, Vanessa; Redekop, Anna; Juvonen, Riikka; Storgårds, Erna; Ziola, Barry

2018-01-01

In 2010, bottles of beer containing viable bacteria of the common beer-spoilage species Lactobacillus backii and Pediococcus damnosus were recovered from a shipwreck near the Åland Islands, Finland. The 170-year quiescent state maintained by the shipwreck bacteria presented a unique opportunity to study lactic acid bacteria (LAB) evolution vis-a-vis growth and survival in the beer environment. Three shipwreck bacteria (one L. backii strain and two P. damnosus strains) and modern-day beer-spoilage isolates of the same two species were genome sequenced, characterized for hop iso-α-acid tolerance, and growth in degassed lager and wheat beer. In addition, plasmid variants of the modern-day P. damnosus strain were analyzed for the effect of plasmid-encoded genes on growth in lager beer. Coding content on two plasmids was identified as essential for LAB growth in modern lager beer. Three chromosomal regions containing genes related to sugar transport and cell wall polysaccharides were shared by pediococci able to grow in beer. Our results show that the three shipwreck bacteria lack the necessary plasmid-located genetic content to grow in modern lager beer, but carry additional genes related to acid tolerance and biofilm formation compared to their modern counterparts. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Fermentation of xylose to ethanol by genetically modified enteric bacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tolan, J.S.

1987-01-01

This thesis describes the fermentation of D-xylose by wild type and recombinant Klebsiella planticola ATCC 33531 and Erwinia chrysanthemi B374. The recombinant strains bear multi-copy plasmids containing the pdc gene inserted from Zymomonas mobilis. Expression of the gene in K. planticola markedly increased the yield of ethanol, up to 1.3 mole/mole xylose, or 25.1 g/L. Concurrently, there were significant decreases in the yields of formation acetate, lactate, and butanediol. Transconjugant Klebsiella grew almost as fast as the wild type and tolerated up to 4% ethanol. The plasmid was retained by the cells during at least one batch culture, even inmore » the absence of selective pressure by antibiotics to maintain the plasmid. The cells produced 31.6 g/L ethanol from 79.6 g/L of a D-glucose-D-xylose-L-arabinose mixture designed to simulate hydrolyzed hemicellulose. The physiology of the wild type K. planticola is described in more detail than in the original report of its isolation. E. chrysanthemi PDC transconjugants also produced ethanol in high yield (up to 1.45 mole/mole xylose). However, transconjugant E. chrysanthemi grew only 1/4 as rapidly as the wild type and tolerated only 2% ethanol. The plasmid PZM15 apparently exhibits pleiotropic effects when inserted into K. planticola and into E. chrysanthemi.« less

Treadmilling of a prokaryotic tubulin-like protein, TubZ, required for plasmid stability in Bacillus thuringiensis

PubMed Central

Larsen, Rachel A.; Cusumano, Christina; Fujioka, Akina; Lim-Fong, Grace; Patterson, Paula; Pogliano, Joe

2007-01-01

Prokaryotes rely on a distant tubulin homolog, FtsZ, for assembling the cytokinetic ring essential for cell division, but are otherwise generally thought to lack tubulin-like polymers that participate in processes such as DNA segregation. Here we characterize a protein (TubZ) from the Bacillus thuringiensis virulence plasmid pBtoxis, which is a member of the tubulin/FtsZ GTPase superfamily but is only distantly related to both FtsZ and tubulin. TubZ assembles dynamic, linear polymers that exhibit directional polymerization with plus and minus ends, movement by treadmilling, and a critical concentration for assembly. A point mutation (D269A) that alters a highly conserved catalytic residue within the T7 loop completely eliminates treadmilling and allows the formation of stable polymers at a much lower protein concentration than the wild-type protein. When expressed in trans, TubZ(D269A) coassembles with wild-type TubZ and significantly reduces the stability of pBtoxis, demonstrating a direct correlation between TubZ dynamics and plasmid maintenance. The tubZ gene is in an operon with tubR, which encodes a putative DNA-binding protein that regulates TubZ levels. Our results suggest that TubZ is representative of a novel class of prokaryotic cytoskeletal proteins important for plasmid stability that diverged long ago from the ancient tubulin/FtsZ ancestor. PMID:17510284

Spaceflight Effects on Genetics and Plasmids of Streptomycetes

NASA Astrophysics Data System (ADS)

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

2008-06-01

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

Plasmid linkage of the D-tagatose 6-phosphate pathway in Streptococcus lactis: effect on lactose and galactose metabolism.

PubMed Central

Crow, V L; Davey, G P; Pearce, L E; Thomas, T D

1983-01-01

The three enzymes of the D-tagatose 6-phosphate pathway (galactose 6-phosphate isomerase, D-tagatose 6-phosphate kinase, and tagatose 1,6-diphosphate aldolase) were absent in lactose-negative (Lac-) derivatives of Streptococcus lactis C10, H1, and 133 grown on galactose. The lactose phosphoenolpyruvate-dependent phosphotransferase system and phospho-beta-galactosidase activities were also absent in Lac- derivatives of strains H1 and 133 and were low (possibly absent) in C10 Lac-. In all three Lac- derivatives, low galactose phosphotransferase system activity was found. On galactose, Lac- derivatives grew more slowly (presumably using the Leloir pathway) than the wild-type strains and accumulated high intracellular concentrations of galactose 6-phosphate (up to 49 mM); no intracellular tagatose 1,6-diphosphate was detected. The data suggest that the Lac phenotype is plasmid linked in the three strains studied, with the evidence being more substantial for strain H1. A Lac- derivative of H1 contained a single plasmid (33 megadaltons) which was absent from the Lac- mutant. We suggest that the genes linked to the lactose plasmid in S. lactis are more numerous than previously envisaged, coding for all of the enzymes involved in lactose metabolism from initial transport to the formation of triose phosphates via the D-tagatose 6-phosphate pathway. Images PMID:6294064

Physical Characterization of Gemini Surfactant-Based Synthetic Vectors for the Delivery of Linear Covalently Closed (LCC) DNA Ministrings

PubMed Central

Sum, Chi Hong; Nafissi, Nafiseh; Slavcev, Roderick A.; Wettig, Shawn

2015-01-01

In combination with novel linear covalently closed (LCC) DNA minivectors, referred to as DNA ministrings, a gemini surfactant-based synthetic vector for gene delivery has been shown to exhibit enhanced delivery and bioavailability while offering a heightened safety profile. Due to topological differences from conventional circular covalently closed (CCC) plasmid DNA vectors, the linear topology of LCC DNA ministrings may present differences with regards to DNA interaction and the physicochemical properties influencing DNA-surfactant interactions in the formulation of lipoplexed particles. In this study, N,N-bis(dimethylhexadecyl)-α,ω-propanediammonium(16-3-16)gemini-based synthetic vectors, incorporating either CCC plasmid or LCC DNA ministrings, were characterized and compared with respect to particle size, zeta potential, DNA encapsulation, DNase sensitivity, and in vitro transgene delivery efficacy. Through comparative analysis, differences between CCC plasmid DNA and LCC DNA ministrings led to variations in the physical properties of the resulting lipoplexes after complexation with 16-3-16 gemini surfactants. Despite the size disparities between the plasmid DNA vectors (CCC) and DNA ministrings (LCC), differences in DNA topology resulted in the generation of lipoplexes of comparable particle sizes. The capacity for ministring (LCC) derived lipoplexes to undergo complete counterion release during lipoplex formation contributed to improved DNA encapsulation, protection from DNase degradation, and in vitro transgene delivery. PMID:26561857

The archaeo-eukaryotic primase of plasmid pRN1 requires a helix bundle domain for faithful primer synthesis

PubMed Central

Beck, Kirsten; Vannini, Alessandro; Cramer, Patrick; Lipps, Georg

2010-01-01

The plasmid pRN1 encodes for a multifunctional replication protein with primase, DNA polymerase and helicase activity. The minimal region required for primase activity encompasses amino-acid residues 40–370. While the N-terminal part of that minimal region (residues 47–247) folds into the prim/pol domain and bears the active site, the structure and function of the C-terminal part (residues 248–370) is unknown. Here we show that the C-terminal part of the minimal region folds into a compact domain with six helices and is stabilized by a disulfide bond. Three helices superimpose well with the C-terminal domain of the primase of the bacterial broad host range plasmid RSF1010. Structure-based site-directed mutagenesis shows that the C-terminal helix of the helix bundle domain is required for primase activity although it is distant to the active site in the crystallized conformation. Furthermore, we identified mutants of the C-terminal domain, which are defective in template binding, dinucleotide formation and conformation change prior to DNA extension. PMID:20511586

Characterization of protein-protein interaction domains within the baculovirus Autographa californica multiple nucleopolyhedrovirus late expression factor LEF-3.

PubMed

Downie, Kelsey; Adetola, Gbolagade; Carstens, Eric B

2013-11-01

Autographa californica nucleopolyhedrovirus late expression factor 3 (LEF-3) is required for late viral gene expression probably through its numerous functions related to DNA replication, including nuclear localization of the virus helicase P143 and binding to ssDNA. LEF-3 appears to interact with itself as a homo-oligomer, although the details of this oligomeric structure are not yet known. To examine LEF-3-LEF-3 interactions, a bimolecular fluorescent protein complementation assay was used. Pairs of recombinant plasmids expressing full-length LEF-3 fused to one of two complementary fragments (V1 or V2) of a variant of yellow fluorescent protein named 'Venus' were constructed. Plasmids expressing fusions with complementary fragments of Venus were co-transfected into Sf21 cells and analysed by fluorescence microscopy. Co-transfected plasmids expressing full-length V1-LEF-3 and V2-LEF-3 showed positive fluorescence, confirming the formation of homo-oligomers. A series of truncated V1/V2-LEF-3 fusions was constructed and used to investigate interactions with one another as well as with full-length LEF-3.

Genotoxic activity of 4,4',5'-trimethylazapsoralen on plasmid DNA.

PubMed

Lagatolla, C; Dolzani, L; Granzotto, M; Monti-Bragadin, C

1998-01-01

The genotoxic activities of 8-methoxypsoralen (8-MOP) and 4,4',5'-trimethylazapsoralen (4,4',5'-TMAP) on plasmid DNA have been compared. In a previous work, 4,4',5'-TMAP, a methyl derivative of a psoralen isoster, had shown potential photochemotherapeutic activity. The mutagenic activity of mono- and bifunctional lesions caused by these compounds was evaluated both after UVA irradiation, which causes the formation of both kinds of lesions, and after a two-step irradiation procedure of the psoralen-plasmid DNA complex, which allowed monoadducts and interstrand crosslinks to be studied separately. Furthermore, we used a procedure that allowed us to evaluate both the mutagenic and recombinogenic activity of the two compounds. Results indicate that the most important difference between 8-MOP and 4,4',5'-TMAP consists in their mode of photoreaction with DNA rather than in their mutagenic potential. In fact, in all of the experimental procedures, 4,4',5'-TMAP shows a lower ability than 8-MOP to generate interstrand crosslinks. However, when comparable toxicity levels are reached, the two compounds show the same mutagenic potentiality.

A toxicological study of inhalable particulates in an industrial region of Lanzhou City, northwestern China: Results from plasmid scission assay

NASA Astrophysics Data System (ADS)

Xiao, Zhenghui; Shao, Longyi; Zhang, Ning; Wang, Jing; Chuang, Hsiao-Chi; Deng, Zhenzhen; Wang, Zhen; BéruBé, Kelly

2014-09-01

The city of Lanzhou in northwestern China experiences serious air pollution episodes in the form of PM10 that is characterized by having high levels of heavy metals. The Xigu District represents the industrial core area of Lanzhou City and is denoted by having the largest petrochemical bases in western China. This study investigates heavy metal compositions and oxidative potential of airborne PM10 (particulate matter with aerodynamic diameter of 10 μm or less) collected in Xigu District in the summer and winter of 2010. An in vitro plasmid scission assay (PSA) was employed to study the oxidative potential of airborne PM10 and inductively coupled plasma-mass spectrometry (ICP-MS) was used to examine heavy metal compositions. Transmission electron microscopy coupled with energy-dispersive X-ray spectrometry (TEM/EDX) was used to investigate elemental compositions and mixing states of PM10. The average mass concentrations of PM10 collected in Xigu District were generally higher than the national standard for daily PM10 (150 μg/m3). Cr, Zn, Pb and Mn were the most abundant metals in the intact whole particles of PM10. Zn, Mn and As was the most abundant metal in the water-soluble fraction, while Cr, Pb, and V existed primarily in insoluble forms. TD20 values (i.e. toxic dosage of PM10 causing 20% of plasmid DNA damage) varied considerably in both winter and summer (from 19 μg/mL to >1000 μg/mL) but were typically higher in summer, suggesting that the winter PM10 exhibited greater bioreactivity. In addition, the PM10 collected during a dust storm episode had a highest TD20 value and thus the least oxidative damage to supercoiled plasmid DNA, while the particles collected on a hazy day had a lowest TD20 value and thus the highest oxidative damage to supercoiled plasmid DNA. The particles collected on the first day after snow fall and on a day of cold air intrusion exhibited minor oxidative potential (i.e. caused limited DNA damage). The water-soluble Zn, Mn, As, and Cu displayed a significant negative correlation with TD20 values, suggesting that these heavy metals were responsible for the increase of oxidative potential. The high mass concentration of PM10 and resulting high oxidative potential in Xigu District may be due to the constant low wind speed and high relative humidity, particularly in winter. Finally, TEM analysis suggested that the oxidative potential of PM10 may be associated with its degree of internal mixing, whereby the heterogeneous assortment of soot, mineral and metals created a highly reactive moiety.

The Complete Multipartite Genome Sequence of Cupriavidus necator JMP134, a Versatile Pollutant Degrader

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lykidis, Athanasios; Perez-Pantoja, Danilo; Ledger, Thomas

Cupriavidus necator JMP134 (formerly Ralstonia eutropha JMP134) is a Gram-negative {beta}-proteobacterium able to degrade a variety of chloroaromatic compounds and chemically-related pollutants. It was originally isolated based on its ability to use 2,4 dichlorophenoxyacetic acid (2,4-D) as a sole carbon and energy source [1]. In addition to 2,4-D, this strain can also grow on a variety of aromatic substrates, such as 4-chloro-2-methylphenoxyacetate (MCPA), 3-chlorobenzoic acid (3-CB) [2], 2,4,6-trichlorophenol [3], and 4-fluorobenzoate [4]. The genes necessary for 2,4-D utilization have been identified. They are located in two clusters on plasmid pPJ4: tfd{sub I} and tfd{sub II} [5,6,7,8]. The sequence and analysismore » of plasmid pJP4 was reported and a congruent model for bacterial adaptation to chloroaromatic pollutants was proposed [9]. According to this model, catabolic gene clusters assemble in a modular manner into broad-host-range plasmid backbones by means of repeated chromosomal capture events. Cupriavidus and related Burkholderia genomes are typically multipartite, composed of two large replicons (chromosomes) accompanied by classical plasmids. Previous work with Burkholderia xenovorans LB400 revealed a differential gene distribution with core functions preferentially encoded by the larger chromosome and secondary functions by the smaller [10]. It has been proposed that the secondary chromosomes in many bacteria originated from ancestral plasmids which, in turn, had been the recipient of genes transferred earlier from ancestral primary chromosomes [11]. The existence of multiple Cupriavidus and Burkholderia genomes provides the opportunity for comparative studies that will lead to a better understanding of the evolutionary mechanisms for the formation of multipartite genomes and the relation with biodegradation abilities.« less

Plasmids with a Chromosome-Like Role in Rhizobia ▿ †

PubMed Central

Landeta, Cristina; Dávalos, Araceli; Cevallos, Miguel Ángel; Geiger, Otto; Brom, Susana; Romero, David

2011-01-01

Replicon architecture in bacteria is commonly comprised of one indispensable chromosome and several dispensable plasmids. This view has been enriched by the discovery of additional chromosomes, identified mainly by localization of rRNA and/or tRNA genes, and also by experimental demonstration of their requirement for cell growth. The genome of Rhizobium etli CFN42 is constituted by one chromosome and six large plasmids, ranging in size from 184 to 642 kb. Five of the six plasmids are dispensable for cell viability, but plasmid p42e is unusually stable. One possibility to explain this stability would be that genes on p42e carry out essential functions, thus making it a candidate for a secondary chromosome. To ascertain this, we made an in-depth functional analysis of p42e, employing bioinformatic tools, insertional mutagenesis, and programmed deletions. Nearly 11% of the genes in p42e participate in primary metabolism, involving biosynthetic functions (cobalamin, cardiolipin, cytochrome o, NAD, and thiamine), degradation (asparagine and melibiose), and septum formation (minCDE). Synteny analysis and incompatibility studies revealed highly stable replicons equivalent to p42e in content and gene order in other Rhizobium species. A systematic deletion analysis of p42e allowed the identification of two genes (RHE_PE00001 and RHE_PE00024), encoding, respectively, a hypothetical protein with a probable winged helix-turn-helix motif and a probable two-component sensor histidine kinase/response regulator hybrid protein, which are essential for growth in rich medium. These data support the proposal that p42e and its homologous replicons (pA, pRL11, pRLG202, and pR132502) merit the status of secondary chromosomes. PMID:21217003

Cisplatin enhances the formation of DNA single- and double-strand breaks by hydrated electrons and hydroxyl radicals.

PubMed

Rezaee, Mohammad; Sanche, Léon; Hunting, Darel J

2013-03-01

The synergistic interaction of cisplatin with ionizing radiation is the clinical rationale for the treatment of several cancers including head and neck, cervical and lung cancer. The underlying molecular mechanism of the synergy has not yet been identified, although both DNA damage and repair processes are likely involved. Here, we investigate the indirect effect of γ rays on strand break formation in a supercoiled plasmid DNA (pGEM-3Zf-) covalently modified by cisplatin. The yields of single- and double-strand breaks were determined by irradiation of DNA and cisplatin/DNA samples with (60)Co γ rays under four different scavenging conditions to examine the involvement of hydrated electrons and hydroxyl radicals in inducing the DNA damage. At 5 mM tris in an N2 atmosphere, the presence of an average of two cisplatins per plasmid increased the yields of single- and double-strand breaks by factors of 1.9 and 2.2, respectively, relative to the irradiated unmodified DNA samples. Given that each plasmid of 3,200 base pairs contained an average of two cisplatins, this represents an increase in radiosensitivity of 3,200-fold on a per base pair basis. When hydrated electrons were scavenged by saturating the samples with N2O, these enhancement factors decreased to 1.5 and 1.2, respectively, for single- and double-strand breaks. When hydroxyl radicals were scavenged using 200 mM tris, the respective enhancement factors were 1.2 and 1.6 for single- and double-strand breaks, respectively. Furthermore, no enhancement in DNA damage by cisplatin was observed after scavenging both hydroxyl radicals and hydrated electrons. These findings show that hydrated electrons can induce both single- and double-strand breaks in the platinated DNA, but not in unmodified DNA. In addition, cisplatin modification is clearly an extremely efficient means of increasing the formation of both single- and double-strand breaks by the hydrated electrons and hydroxyl radicals created by ionizing radiation.

Involvement of a plasmid in growth on and dispersion of crude oil by Acinetobacter calcoaceticus RA57.

PubMed Central

Rusansky, S; Avigad, R; Michaeli, S; Gutnick, D L

1987-01-01

A crude-oil-degrading Acinetobacter species, Acinetobacter calcoaceticus RA57, was isolated by standard enrichment culture techniques on the basis of its ability to utilize the oily sludge found in the vicinity of a local gas station. Strain RA57 was found to contain four plasmids: pSR1 (5.1 kilobases [kb]), pSR2 (5.4 kb), pSR3 (10.5 kb), and pSR4 (20 kb). Both supercoiled and open circular forms of the first three plasmids were identified by two-dimensional gel electrophoresis. Restriction endonuclease analysis of pSR4 demonstrated that the plasmid contained a circular map. Colonies were isolated at random after growth in the presence of acridine orange and found to fall into two categories: (i) those which had lost the ability to grow on and disperse crude oil in liquid culture and concurrently were cured of pSR4 and (ii) those which retained the ability to both grow on and disperse crude oil and which contained pSR4. Strains from the first class continued to grow on hydrocarbon vapors, indicating that the defect associated with the curing of pSR4 was related to the physical interaction of the cells with the hydrocarbon substrate, rather than to its metabolism. No differences in either adherence to hydrocarbons or production of extracellular emulsifying activity were found between the two classes of mutants. In growth experiments on crude oil in mixed culture with strains which either contained or lacked pSR4, no sparing of the growth defect was observed. The results are consistent with the possibility that pSR4 encodes a factor(s) which is tightly associated with the cell surface. Images PMID:2821903

Increased B and T Cell Responses in M. bovis Bacille Calmette-Guérin Vaccinated Pigs Co-Immunized with Plasmid DNA Encoding a Prototype Tuberculosis Antigen

PubMed Central

Bruffaerts, Nicolas; Pedersen, Lasse E.; Vandermeulen, Gaëlle; Préat, Véronique; Stockhofe-Zurwieden, Norbert; Huygen, Kris; Romano, Marta

2015-01-01

The only tuberculosis vaccine currently available, bacille Calmette-Guérin (BCG) is a poor inducer of CD8+ T cells, which are particularly important for the control of latent tuberculosis and protection against reactivation. As the induction of strong CD8+ T cell responses is a hallmark of DNA vaccines, a combination of BCG with plasmid DNA encoding a prototype TB antigen (Ag85A) was tested. As an alternative animal model, pigs were primed with BCG mixed with empty vector or codon-optimized pAg85A by the intradermal route and boosted with plasmid delivered by intramuscular electroporation. Control pigs received unformulated BCG. The BCG-pAg85A combination stimulated robust and sustained Ag85A specific antibody, lymphoproliferative, IL-6, IL-10 and IFN-γ responses. IgG1/IgG2 antibody isotype ratio reflected the Th1 helper type biased response. T lymphocyte responses against purified protein derivative of tuberculin (PPD) were induced in all (BCG) vaccinated animals, but responses were much stronger in BCG-pAg85A vaccinated pigs. Finally, Ag85A-specific IFN-γ producing CD8+ T cells were detected by intracellular cytokine staining and a synthetic peptide, spanning Ag85A131-150 and encompassing two regions with strong predicted SLA-1*0401/SLA-1*0801 binding affinity, was promiscuously recognized by 6/6 animals vaccinated with the BCG-pAg85A combination. Our study provides a proof of concept in a large mammalian species, for a new Th1 and CD8+ targeting tuberculosis vaccine, based on BCG-plasmid DNA co-administration. PMID:26172261

Sequences of two related multiple antibiotic resistance virulence plasmids sharing a unique IS26-related molecular signature isolated from different Escherichia coli pathotypes from different hosts.

PubMed

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

2013-01-01

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

A Droplet Microfluidic Platform for Automating Genetic Engineering.

PubMed

Gach, Philip C; Shih, Steve C C; Sustarich, Jess; Keasling, Jay D; Hillson, Nathan J; Adams, Paul D; Singh, Anup K

2016-05-20

We present a water-in-oil droplet microfluidic platform for transformation, culture and expression of recombinant proteins in multiple host organisms including bacteria, yeast and fungi. The platform consists of a hybrid digital microfluidic/channel-based droplet chip with integrated temperature control to allow complete automation and integration of plasmid addition, heat-shock transformation, addition of selection medium, culture, and protein expression. The microfluidic format permitted significant reduction in consumption (100-fold) of expensive reagents such as DNA and enzymes compared to the benchtop method. The chip contains a channel to continuously replenish oil to the culture chamber to provide a fresh supply of oxygen to the cells for long-term (∼5 days) cell culture. The flow channel also replenished oil lost to evaporation and increased the number of droplets that could be processed and cultured. The platform was validated by transforming several plasmids into Escherichia coli including plasmids containing genes for fluorescent proteins GFP, BFP and RFP; plasmids with selectable markers for ampicillin or kanamycin resistance; and a Golden Gate DNA assembly reaction. We also demonstrate the applicability of this platform for transformation in widely used eukaryotic organisms such as Saccharomyces cerevisiae and Aspergillus niger. Duration and temperatures of the microfluidic heat-shock procedures were optimized to yield transformation efficiencies comparable to those obtained by benchtop methods with a throughput up to 6 droplets/min. The proposed platform offers potential for automation of molecular biology experiments significantly reducing cost, time and variability while improving throughput.

Large plasmids of Escherichia coli and Salmonella encode highly diverse arrays of accessory genes on common replicon families.

PubMed

Williams, Laura E; Wireman, Joy; Hilliard, Valda C; Summers, Anne O

2013-01-01

Plasmids are important in evolution and adaptation of host bacteria, yet we lack a comprehensive picture of their own natural variation. We used replicon typing and RFLP analysis to assess diversity and distribution of plasmids in the ECOR, SARA, SARB and SARC reference collections of Escherichia coli and Salmonella. Plasmids, especially large (≥30 kb) plasmids, are abundant in these collections. Host species and genotype clearly impact plasmid prevalence; plasmids are more abundant in ECOR than SAR, but, within ECOR, subgroup B2 strains have the fewest large plasmids. The majority of large plasmids have unique RFLP patterns, suggesting high variation, even within dominant replicon families IncF and IncI1. We found only four conserved plasmid types within ECOR, none of which are widely distributed. Within SAR, conserved plasmid types are primarily serovar-specific, including a pSLT-like plasmid in 13 Typhimurium strains. Conservation of pSLT contrasts with variability of other plasmids, suggesting evolution of serovar-specific virulence plasmids is distinct from that of most enterobacterial plasmids. We sequenced a conserved serovar Heidelberg plasmid but did not detect virulence or antibiotic resistance genes. Our data illustrate the high degree of natural variation in large plasmids of E. coli and Salmonella, even among plasmids sharing backbone genes. Copyright © 2012 Elsevier Inc. All rights reserved.

Formation and loss of large, unstable tandem arrays of the piggyBac transposable element in the yellow fever mosquito, Aedes aegypti.

PubMed

Adelman, Zach N; Jasinskiene, Nijole; Vally, K J M; Peek, Corrie; Travanty, Emily A; Olson, Ken E; Brown, Susan E; Stephens, Janice L; Knudson, Dennis L; Coates, Craig J; James, Anthony A

2004-10-01

The Class II transposable element, piggyBac, was used to transform the yellow fever mosquito, Aedes aegypti. In two transformed lines only 15-30% of progeny inherited the transgene, with these individuals displaying mosaic expression of the EGFP marker gene. Southern analyses, gene amplification of genomic DNA, and plasmid rescue experiments provided evidence that these lines contained a high copy number of piggyBac transformation constructs and that much of this DNA consisted of both donor and helper plasmids. A detailed analysis of one line showed that the majority of piggyBac sequences were unit-length donor or helper plasmids arranged in a large tandem array that could be lost en masse in a single generation. Despite the presence of a transposase source and many intact donor elements, no conservative (cut and paste) transposition of piggyBac was observed in these lines. These results reveal one possible outcome of uncontrolled and/or unexpected recombination in this mosquito, and support the conclusion that further investigation is necessary before transposable elements such as piggyBac can be used as genetic drive mechanisms to move pathogen-resistance genes into mosquito populations.

Multiple displacement amplification in combination with high-fidelity PCR improves detection of bacteria from single females or eggs of Metaseiulus occidentalis (Nesbitt) (Acari: Phytoseiidae).

PubMed

Jeyaprakash, Ayyamperumal; Hoy, Marjorie A

2004-07-01

Amplifying microbial DNA by the polymerase chain reaction (PCR) from single phytoseiid mites has been difficult, perhaps due to the low titer of bacteria and to interference by the relatively larger amounts of mite genomic DNA. In this paper we evaluate the efficiency of standard and high-fidelity PCR protocols subsequent to amplification of the whole genome by a multiple displacement amplification (MDA) procedure developed by Dean et al. DNA from the phytoseiid Phytoseiulus persimilis (Athias-Henriot) was tested because it lacks a Cytophaga-like organism (CLO) and we could add known amounts of a plasmid containing a cloned 16S rRNA gene fragment from a CLO from Metaseiulus occidentalis (Nesbitt). P. persimilis genomic DNA was mixed with the serially diluted plasmid and amplified using MDA followed by either standard or high-fidelity PCR. MDA followed by high-fidelity PCR was most efficient and successfully amplified an expected 1.5-kb band from as little as 0.01fg of the plasmid, which is equivalent to about 1 copy. MDA followed by high-fidelity PCR also consistently amplified Wolbachia- or CLO-specific products from naturally infected single females or eggs of M. occidentalis, which will allow detailed studies of infection frequency and transmission of several microorganisms associated with this predatory mite.

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.

Chemotherapy of Bacterial Plasmids

DTIC Science & Technology

1979-01-29

of drug resistance is interrupted 1960 Fukasawa. Watanabe by blender treatment of mixed cultures The term " R-factor" is introduced for the 1960 M ...bacterial . -.co DNA biosynthesis (rev. in [53]). At a concentration oI I!319- M tl:2 -,- O Oil of 6.25x 106 ,l1which did not inhibit the growth A of...sensitized 96 15;! .,\\litsuhdshi. S . I~ohe. S.. Inoue. M :bi, _"). 131 (1976, those bacteria to inhibitions by ampicillin and cepha- -. ~ J.. et al .J

Cas9 Nickase-Assisted RNA Repression Enables Stable and Efficient Manipulation of Essential Metabolic Genes in Clostridium cellulolyticum.

PubMed

Xu, Tao; Li, Yongchao; He, Zhili; Van Nostrand, Joy D; Zhou, Jizhong

2017-01-01

Essential gene functions remain largely underexplored in bacteria. Clostridium cellulolyticum is a promising candidate for consolidated bioprocessing; however, its genetic manipulation to reduce the formation of less-valuable acetate is technically challenging due to the essentiality of acetate-producing genes. Here we developed a Cas9 nickase-assisted chromosome-based RNA repression to stably manipulate essential genes in C. cellulolyticum . Our plasmid-based expression of antisense RNA (asRNA) molecules targeting the phosphotransacetylase ( pta ) gene successfully reduced the enzymatic activity by 35% in cellobiose-grown cells, metabolically decreased the acetate titer by 15 and 52% in wildtype transformants on cellulose and xylan, respectively. To control both acetate and lactate simultaneously, we transformed the repression plasmid into lactate production-deficient mutant and found the plasmid delivery reduced acetate titer by more than 33%, concomitant with negligible lactate formation. The strains with pta gene repression generally diverted more carbon into ethanol. However, further testing on chromosomal integrants that were created by double-crossover recombination exhibited only very weak repression because DNA integration dramatically lessened gene dosage. With the design of a tandem repetitive promoter-driven asRNA module and the use of a new Cas9 nickase genome editing tool, a chromosomal integrant (LM3P) was generated in a single step and successfully enhanced RNA repression, with a 27% decrease in acetate titer on cellulose in antibiotic-free medium. These results indicate the effectiveness of tandem promoter-driven RNA repression modules in promoting gene repression in chromosomal integrants. Our combinatorial method using a Cas9 nickase genome editing tool to integrate the gene repression module demonstrates easy-to-use and high-efficiency advantages, paving the way for stably manipulating genes, even essential ones, for functional characterization and microbial engineering.

Cas9 Nickase-Assisted RNA Repression Enables Stable and Efficient Manipulation of Essential Metabolic Genes in Clostridium cellulolyticum

PubMed Central

Xu, Tao; Li, Yongchao; He, Zhili; Van Nostrand, Joy D.; Zhou, Jizhong

2017-01-01

Essential gene functions remain largely underexplored in bacteria. Clostridium cellulolyticum is a promising candidate for consolidated bioprocessing; however, its genetic manipulation to reduce the formation of less-valuable acetate is technically challenging due to the essentiality of acetate-producing genes. Here we developed a Cas9 nickase-assisted chromosome-based RNA repression to stably manipulate essential genes in C. cellulolyticum. Our plasmid-based expression of antisense RNA (asRNA) molecules targeting the phosphotransacetylase (pta) gene successfully reduced the enzymatic activity by 35% in cellobiose-grown cells, metabolically decreased the acetate titer by 15 and 52% in wildtype transformants on cellulose and xylan, respectively. To control both acetate and lactate simultaneously, we transformed the repression plasmid into lactate production-deficient mutant and found the plasmid delivery reduced acetate titer by more than 33%, concomitant with negligible lactate formation. The strains with pta gene repression generally diverted more carbon into ethanol. However, further testing on chromosomal integrants that were created by double-crossover recombination exhibited only very weak repression because DNA integration dramatically lessened gene dosage. With the design of a tandem repetitive promoter-driven asRNA module and the use of a new Cas9 nickase genome editing tool, a chromosomal integrant (LM3P) was generated in a single step and successfully enhanced RNA repression, with a 27% decrease in acetate titer on cellulose in antibiotic-free medium. These results indicate the effectiveness of tandem promoter-driven RNA repression modules in promoting gene repression in chromosomal integrants. Our combinatorial method using a Cas9 nickase genome editing tool to integrate the gene repression module demonstrates easy-to-use and high-efficiency advantages, paving the way for stably manipulating genes, even essential ones, for functional characterization and microbial engineering. PMID:28936208

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. Copyright © 2013 Elsevier GmbH. All rights reserved.

Effects of local gene transfer of VEGF on neointima formation after balloon injury in hypercholesterolemic rabbits

PubMed Central

Dulak, Jozef; Schwarzacher, Severin P; Zwick, Ralf H; Alber, Hannes; Millonig, Gunda; Weiss, Caecilia; Hügel, Heike; Frick, Matthias; Jozkowicz, Alicja; Pachinger, Otmar; Weidinger, Franz

2006-01-01

Enhancement of the generation of nitric oxide (NO) and vascular endothelial growth factor (VEGF) are suggested to prevent restenosis after angioplasty. Accordingly, we tested whether the local delivery of l-arginine (l-Arg), a substrate for NO generation and the VEGF gene, alone or in combination, can influence neointima formation in hypercholesterolemic rabbits. Balloon injury of the iliac arteries was performed in 24 New Zealand White rabbits fed a 1% cholesterol diet for 3 weeks followed by a local infusion of: (1) pSG5VEGF165 plasmid alone (1000 μg); (2) pSG5VEGF165 (1000 μg) with l-Arg (800 mg); (3) l-Arg (800 mg) alone; and (4) l-Arg (800 mg) with naked pSVβ-gal plasmid (1000 μg). The animals were kept on the hypercholesterolemic diets for a further 28 days, when vessels were taken for morphometric analysis and immunocytochemistry. Endogenous rabbit VEGF concentration in the plasma increased significantly at 7 days after injury (17.06 ± 1.57 vs 23.01 ± 1.9 pg/ml; p < 0.02) and remained elevated for up to 28 days (28.46 ± 5.24; p < 0.01). Injured arteries exhibited strong immunocytochemical staining for rabbit VEGF. Rabbits that received a VEGF gene transfer revealed more prominent neointima formation, whereas treatment with l-Arg was associated with significantly less intimal thickness (p < 0.05). Local transfer of the VEGF gene does not inhibit neointima formation in hypercholesterolemic rabbits. Our results suggest that VEGF gene therapy applied locally in atherosclerotic arteries may not be beneficial. PMID:16444857

Plasmid diversity and phylogenetic consistency in the Lyme disease agent Borrelia burgdorferi.

PubMed

Casjens, Sherwood R; Gilcrease, Eddie B; Vujadinovic, Marija; Mongodin, Emmanuel F; Luft, Benjamin J; Schutzer, Steven E; Fraser, Claire M; Qiu, Wei-Gang

2017-02-15

Bacteria from the genus Borrelia are known to harbor numerous linear and circular plasmids. We report here a comparative analysis of the nucleotide sequences of 236 plasmids present in fourteen independent isolates of the Lyme disease agent B. burgdorferi. We have sequenced the genomes of 14 B. burgdorferi sensu stricto isolates that carry a total of 236 plasmids. These individual isolates carry between seven and 23 plasmids. Their chromosomes, the cp26 and cp32 circular plasmids, as well as the lp54 linear plasmid, are quite evolutionarily stable; however, the remaining plasmids have undergone numerous non-homologous and often duplicative recombination events. We identify 32 different putative plasmid compatibility types among the 236 plasmids, of which 15 are (usually) circular and 17 are linear. Because of past rearrangements, any given gene, even though it might be universally present in these isolates, is often found on different linear plasmid compatibility types in different isolates. For example, the arp gene and the vls cassette region are present on plasmids of four and five different compatibility types, respectively, in different isolates. A majority of the plasmid types have more than one organizationally different subtype, and the number of such variants ranges from one to eight among the 18 linear plasmid types. In spite of this substantial organizational diversity, the plasmids are not so variable that every isolate has a novel version of every plasmid (i.e., there appears to be a limited number of extant plasmid subtypes). Although there have been many past recombination events, both homologous and nonhomologous, among the plasmids, particular organizational variants of these plasmids correlate with particular chromosomal genotypes, suggesting that there has not been rapid horizontal transfer of whole linear plasmids among B. burgdorferi lineages. We argue that plasmid rearrangements are essentially non-revertable and are present at a frequency of only about 0.65% that of single nucleotide changes, making rearrangement-derived novel junctions (mosaic boundaries) ideal phylogenetic markers in the study of B. burgdorferi population structure and plasmid evolution and exchange.

Plasmids in Vibrio parahemolyticus strains isolated in Japan and Bangladesh with special reference to different distributions.

PubMed

Arai, T; Ando, T; Kusakabe, A; Ullah, M A

1983-01-01

We surveyed plasmids in naturally occurring Vibrio parahemolyticus strains isolated in Japan and Bangladesh. Among the strains isolated in Japan, about half of the strains isolated from stools of patients of domestic diarrhea outbreaks as well as of travelers returning from East Asia were found to have plasmids, but no strains from foods had plasmids. In contrast, among the strains isolated in Bangladesh, none of the four strains isolated from patients had plasmids, but two out of eight strains isolated from water had plasmids, suggesting that plasmids are common in strains from the water in Bangladesh. All plasmids so far reported in V. parahemolyticus were detected in strains isolated from stools of patients. Incidences of plasmids in this organism were not so high in either area. In Japan, all plasmids were detected in strains from human intestines at 37 C, but in Bangladesh, where the temperature is around 30-40 C, the plasmids were detected in strains from the natural environment. These results suggested the possibility that these plasmids can come from different bacteria under rather high temperatures and that incidences of plasmids are influenced by the incidences of plasmids in bacteria present in the vicinity of V. parahemolyticus strains. None of these plasmids were found to have any relation to the biological characters tested.

Population genomic analysis of strain variation in Leptospirillum group II bacteria involved in acid mine drainage formation.

PubMed

Simmons, Sheri L; Dibartolo, Genevieve; Denef, Vincent J; Goltsman, Daniela S Aliaga; Thelen, Michael P; Banfield, Jillian F

2008-07-22

Deeply sampled community genomic (metagenomic) datasets enable comprehensive analysis of heterogeneity in natural microbial populations. In this study, we used sequence data obtained from the dominant member of a low-diversity natural chemoautotrophic microbial community to determine how coexisting closely related individuals differ from each other in terms of gene sequence and gene content, and to uncover evidence of evolutionary processes that occur over short timescales. DNA sequence obtained from an acid mine drainage biofilm was reconstructed, taking into account the effects of strain variation, to generate a nearly complete genome tiling path for a Leptospirillum group II species closely related to L. ferriphilum (sampling depth approximately 20x). The population is dominated by one sequence type, yet we detected evidence for relatively abundant variants (>99.5% sequence identity to the dominant type) at multiple loci, and a few rare variants. Blocks of other Leptospirillum group II types ( approximately 94% sequence identity) have recombined into one or more variants. Variant blocks of both types are more numerous near the origin of replication. Heterogeneity in genetic potential within the population arises from localized variation in gene content, typically focused in integrated plasmid/phage-like regions. Some laterally transferred gene blocks encode physiologically important genes, including quorum-sensing genes of the LuxIR system. Overall, results suggest inter- and intrapopulation genetic exchange involving distinct parental genome types and implicate gain and loss of phage and plasmid genes in recent evolution of this Leptospirillum group II population. Population genetic analyses of single nucleotide polymorphisms indicate variation between closely related strains is not maintained by positive selection, suggesting that these regions do not represent adaptive differences between strains. Thus, the most likely explanation for the observed patterns of polymorphism is divergence of ancestral strains due to geographic isolation, followed by mixing and subsequent recombination.

Population Genomic Analysis of Strain Variation in Leptospirillum Group II Bacteria Involved in Acid Mine Drainage Formation

PubMed Central

Denef, Vincent J; Goltsman, Daniela S. Aliaga; Thelen, Michael P; Banfield, Jillian F

2008-01-01

Deeply sampled community genomic (metagenomic) datasets enable comprehensive analysis of heterogeneity in natural microbial populations. In this study, we used sequence data obtained from the dominant member of a low-diversity natural chemoautotrophic microbial community to determine how coexisting closely related individuals differ from each other in terms of gene sequence and gene content, and to uncover evidence of evolutionary processes that occur over short timescales. DNA sequence obtained from an acid mine drainage biofilm was reconstructed, taking into account the effects of strain variation, to generate a nearly complete genome tiling path for a Leptospirillum group II species closely related to L. ferriphilum (sampling depth ∼20×). The population is dominated by one sequence type, yet we detected evidence for relatively abundant variants (>99.5% sequence identity to the dominant type) at multiple loci, and a few rare variants. Blocks of other Leptospirillum group II types (∼94% sequence identity) have recombined into one or more variants. Variant blocks of both types are more numerous near the origin of replication. Heterogeneity in genetic potential within the population arises from localized variation in gene content, typically focused in integrated plasmid/phage-like regions. Some laterally transferred gene blocks encode physiologically important genes, including quorum-sensing genes of the LuxIR system. Overall, results suggest inter- and intrapopulation genetic exchange involving distinct parental genome types and implicate gain and loss of phage and plasmid genes in recent evolution of this Leptospirillum group II population. Population genetic analyses of single nucleotide polymorphisms indicate variation between closely related strains is not maintained by positive selection, suggesting that these regions do not represent adaptive differences between strains. Thus, the most likely explanation for the observed patterns of polymorphism is divergence of ancestral strains due to geographic isolation, followed by mixing and subsequent recombination. PMID:18651792

Conjugal properties of the Sinorhizobium meliloti plasmid mobilome.

PubMed

Pistorio, Mariano; Giusti, María A; Del Papa, María F; Draghi, Walter O; Lozano, Mauricio J; Tejerizo, Gonzalo Torres; Lagares, Antonio

2008-09-01

The biology and biochemistry of plasmid transfer in soil bacteria is currently under active investigation because of its central role in prokaryote adaptation and evolution. In this work, we examined the conjugal properties of the cryptic plasmids present in a collection of the N(2)-fixing legume-symbiont Sinorhizobium meliloti. The study was performed on 65 S. meliloti isolates recovered from 25 humic soils of Argentina, which were grouped into 22 plasmid-profile types [i.e. plasmid operational taxonomic units (OTUs)]. The cumulative Shannon index calculated for the observed plasmid profiles showed a clear saturation plateau, thus indicating an adequate representation of the S. meliloti plasmid-profile types in the isolates studied. The results show that isolates of nearly 14% of the plasmid OTUs hosted transmissible plasmids and that isolates of 29% of the plasmid OTUs were able to retransfer the previously characterized mobilizable-cryptic plasmid pSmeLPU88b to a third recipient strain. It is noteworthy that isolates belonging to 14% of the plasmid OTUs proved to be refractory to the entrance of the model plasmid pSmeLPU88b, suggesting either the presence of surface exclusion phenomena or the occurrence of restriction incompatibility with the incoming replicon. Incompatibility for replication between resident plasmids and plasmid pSmeLPU88b was observed in c. 20% of the OTUs. The results reported here reveal a widespread compatibility among the conjugal functions of the cryptic plasmids in S. meliloti, and this fact, together with the observed high proportion of existing donor genotypes, points to the extrachromosomal compartment of the species as being an extremely active plasmid mobilome.

A quantitative and high-throughput assay of human papillomavirus DNA replication.

PubMed

Gagnon, David; Fradet-Turcotte, Amélie; Archambault, Jacques

2015-01-01

Replication of the human papillomavirus (HPV) double-stranded DNA genome is accomplished by the two viral proteins E1 and E2 in concert with host DNA replication factors. HPV DNA replication is an established model of eukaryotic DNA replication and a potential target for antiviral therapy. Assays to measure the transient replication of HPV DNA in transfected cells have been developed, which rely on a plasmid carrying the viral origin of DNA replication (ori) together with expression vectors for E1 and E2. Replication of the ori-plasmid is typically measured by Southern blotting or PCR analysis of newly replicated DNA (i.e., DpnI digested DNA) several days post-transfection. Although extremely valuable, these assays have been difficult to perform in a high-throughput and quantitative manner. Here, we describe a modified version of the transient DNA replication assay that circumvents these limitations by incorporating a firefly luciferase expression cassette in cis of the ori. Replication of this ori-plasmid by E1 and E2 results in increased levels of firefly luciferase activity that can be accurately quantified and normalized to those of Renilla luciferase expressed from a control plasmid, thus obviating the need for DNA extraction, digestion, and analysis. We provide a detailed protocol for performing the HPV type 31 DNA replication assay in a 96-well plate format suitable for small-molecule screening and EC50 determinations. The quantitative and high-throughput nature of the assay should greatly facilitate the study of HPV DNA replication and the identification of inhibitors thereof.

Catabolite-mediated mutations in alternate toluene degradative pathways in Pseudomonas putida.

PubMed Central

Leddy, M B; Phipps, D W; Ridgway, H F

1995-01-01

Pseudomonas putida 54g grew on mineral salts with toluene and exhibited catechol-2,3-dioxygenase (C23O) activity, indicating a meta pathway. After 10 to 15 days on toluene, nondegrading (Tol-) variants approached nearly 10% of total CFU. Auxotrophs were not detected among variants, suggesting selective loss of catabolic function(s). Variant formation was substrate dependent, since Tol- cells were observed on neither ethylbenzene, glucose, nor peptone-based media nor when toluene catabolism was suppressed by glucose. Unlike wild-type cells, variants did not grow on gasoline, toluene, benzene, ethylbenzene, benzoate, or catechol, suggesting loss of meta pathway function. Catabolic and C23O activities were restored to variants via transfer of a 78-mDa TOL-like plasmid from a wild-type Tol+ donor. Tests for reversion of variants to Tol+ were uniformly negative, suggesting possible delection or excision of catabolic genes. Deletions were confirmed in some variants by failure to hybridize with a DNA probe specific for the xylE gene encoding C23O. Cells grown on benzoate remained Tol+ but were C23O- and contained a plasmid of reduced size or were plasmid free, suggesting an alternate chromosomal catabolic pathway, also defective in variants. Cells exposed to benzyl alcohol, the initial oxidation product of toluene, accumulated > 13% variants in 5 days, even when cell division was repressed by nitrogen deprivation to abrogate selection processes. No variants formed in identical ethylbenzene-exposed controls. The results suggest that benzyl alcohol mediates irreversible defects in both a plasmid-associated meta pathway and an alternate chromosomal pathway. PMID:7642499

NanoSMGT: transgene transmission into bovine embryos using halloysite clay nanotubes or nanopolymer to improve transfection efficiency.

PubMed

Campos, Vinicius Farias; de Leon, Priscila Marques Moura; Komninou, Eliza Rossi; Dellagostin, Odir Antônio; Deschamps, João Carlos; Seixas, Fabiana Kömmling; Collares, Tiago

2011-11-01

The objectives were to investigate whether: 1) nanotransfectants are more effective than other common transfection methods for SMGT; 2) NanoSMGT is able to transmit exogenous DNA molecules to bovine embryos; and 3) halloysite clay nanotubes (HCNs) can be used as a transfection reagent to improve transgene transmission. Four transfection systems were used: naked DNA (without transfectant), lipofection, nanopolymer, and halloysite clay nanotubes. Plasmid uptake by sperm and its transfer to embryos were quantified by conventional and real-time PCR, as well as EGFP expression by fluorescence microscopy. Furthermore, sperm motility and viability, and embryo development were investigated. Mean number of plasmids taken up was affected (P < 0.05) by transfection procedure, with the nanopolymer being the most effective transfectant (∼ 153 plasmids per spermatozoon). None of the treatments affected sperm motility or viability. The mean number of plasmids transmitted to four-cell stage embryos was higher (P < 0.05) in nanopolymer and HCNs than liposomes and naked DNA groups. The number of embryos carrying the transgene increased from 8-10% using naked DNA or liposomes to 40-45% using nanopolymer or HCN as transfectants (P < 0.05). There were no significant differences among transfection procedures regarding blastocyst formation rate of resulting embryos. However, no EGFP-expressing embryo was identified in any treatment. Therefore, nanotransfectants improved transgene transmission in bovine embryos without deleterious effects on embryo development. To our knowledge, this was the first time that bovine embryos carrying a transgene were produced by NanoSMGT. Copyright © 2011 Elsevier Inc. All rights reserved.

Characterization and Comparative Overview of Complete Sequences of the First Plasmids of Pandoraea across Clinical and Non-clinical Strains

PubMed Central

Yong, Delicia; Tee, Kok Keng; Yin, Wai-Fong; Chan, Kok-Gan

2016-01-01

To date, information on plasmid analysis in Pandoraea spp. is scarce. To address the gap of knowledge on this, the complete sequences of eight plasmids from Pandoraea spp. namely Pandoraea faecigallinarum DSM 23572T (pPF72-1, pPF72-2), Pandoraea oxalativorans DSM 23570T (pPO70-1, pPO70-2, pPO70-3, pPO70-4), Pandoraea vervacti NS15 (pPV15) and Pandoraea apista DSM 16535T (pPA35) were studied for the first time in this study. The information on plasmid sequences in Pandoraea spp. is useful as the sequences did not match any known plasmid sequence deposited in public databases. Replication genes were not identified in some plasmids, a situation that has led to the possibility of host interaction involvement. Some plasmids were also void of par genes and intriguingly, repA gene was also not discovered in these plasmids. This further leads to the hypothesis of host-plasmid interaction. Plasmid stabilization/stability protein-encoding genes were observed in some plasmids but were not established for participating in plasmid segregation. Toxin-antitoxin systems MazEF, VapBC, RelBE, YgiT-MqsR, HigBA, and ParDE were identified across the plasmids and their presence would improve plasmid maintenance. Conjugation genes were identified portraying the conjugation ability amongst Pandoraea plasmids. Additionally, we found a shared region amongst some of the plasmids that consists of conjugation genes. The identification of genes involved in replication, segregation, toxin-antitoxin systems and conjugation, would aid the design of drugs to prevent the survival or transmission of plasmids carrying pathogenic properties. Additionally, genes conferring virulence and antibiotic resistance were identified amongst the plasmids. The observed features in the plasmids shed light on the Pandoraea spp. as opportunistic pathogens. PMID:27790203

Flow cytometry and real-time quantitative PCR as tools for assessing plasmid persistence.

PubMed

Loftie-Eaton, Wesley; Tucker, Allison; Norton, Ann; Top, Eva M

2014-09-01

The maintenance of a plasmid in the absence of selection for plasmid-borne genes is not guaranteed. However, plasmid persistence can evolve under selective conditions. Studying the molecular mechanisms behind the evolution of plasmid persistence is key to understanding how plasmids are maintained under nonselective conditions. Given the current crisis of rapid antibiotic resistance spread by multidrug resistance plasmids, this insight is of high medical relevance. The conventional method for monitoring plasmid persistence (i.e., the fraction of plasmid-containing cells in a population over time) is based on cultivation and involves differentiating colonies of plasmid-containing and plasmid-free cells on agar plates. However, this technique is time-consuming and does not easily lend itself to high-throughput applications. Here, we present flow cytometry (FCM) and real-time quantitative PCR (qPCR) as alternative tools for monitoring plasmid persistence. For this, we measured the persistence of a model plasmid, pB10::gfp, in three Pseudomonas hosts and in known mixtures of plasmid-containing and -free cells. We also compared three performance criteria: dynamic range, resolution, and variance. Although not without exceptions, both techniques generated estimates of overall plasmid loss rates that were rather similar to those generated by the conventional plate count (PC) method. They also were able to resolve differences in loss rates between artificial plasmid persistence assays. Finally, we briefly discuss the advantages and disadvantages for each technique and conclude that, overall, both FCM and real-time qPCR are suitable alternatives to cultivation-based methods for routine measurement of plasmid persistence, thereby opening avenues for high-throughput analyses. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Molecular evolution of tetracycline-resistance plasmids carrying TetM found in Neisseria gonorrhoeae from different countries.

PubMed

Gascoyne, D M; Heritage, J; Hawkey, P M; Turner, A; van Klingeren, B

1991-08-01

High level tetracycline resistant strains of Neisseria gonorrhoeae (TRNG) have been shown to carry a 40.6 kb (25.2 MDa) conjugative plasmid with a Class M tetracycline resistance determinant. Restriction endonuclease analysis mapping showed that there were at least two different TRNG plasmid types which were found in geographically distinct locations. The physical maps of these two plasmids were compared to a gonococcal conjugative plasmid which did not encode tetracycline resistance. The plasmid type which is endemic in the Netherlands was found to be closely related to the gonococcal conjugative plasmid, which supports the established hypothesis that the 40.6 kb plasmid has evolved by transposition of the TetM determinant into the conjugative plasmid. The plasmid found in the United States has either evolved by substantial divergent evolution or it results from a different transposition event. In the UK there have been isolations of TRNGs carrying either of the two plasmid types reflecting a flow of people both across the Atlantic and in Europe. It is possible that further TetM-containing plasmids will be found in N. gonorrhoeae paralleling the family of TEM beta-lactamase encoding plasmids already described.

Plasmid-linked ampicillin resistance in haempohilus influenza type b.

PubMed

Elwell, L P; De Graaff, J; Seibert, D; Falkow, S

1975-08-01

Four ampicillin-resistant, beta-lactamase-producing strains of Haempohilus influenzae type b were examined for the presence of plasmid deoxyribonucleic acid (DNA). Three resistant strains contained a 30 x 10-6-dalton (30Mdal) plasmid and one resitant strain contained a 3-Mdal plasmid. The ampicillin-sensitive Haemophilus strains examined did not contain plasmid DNA. Transformation of a sensitive H. influenzae strain to ampicillin resistance with isolated plasmid DNA preparations revealed that the structural gene for beta-lactamase resided on both plasmid species. DNA-DNA hybridization studies showed that the 30-Mdal Haemophilus plasmid contained the ampicillin translocation DNA segment (TnA) found on some R-factors of enteric origin of the H. influenzae plasmids.

Engineering Pseudomonas putida KT2440 for simultaneous degradation of organophosphates and pyrethroids and its application in bioremediation of soil.

PubMed

Zuo, Zhenqiang; Gong, Ting; Che, You; Liu, Ruihua; Xu, Ping; Jiang, Hong; Qiao, Chuanling; Song, Cunjiang; Yang, Chao

2015-06-01

Agricultural soils are usually co-contaminated with organophosphate (OP) and pyrethroid pesticides. To develop a stable and marker-free Pseudomonas putida for co-expression of two pesticide-degrading enzymes, we constructed a suicide plasmid with expression cassettes containing a constitutive promoter J23119, an OP-degrading gene (mpd), a pyrethroid-hydrolyzing carboxylesterase gene (pytH) that utilizes the upp gene as a counter-selectable marker for upp-deficient P. putida. By introduction of suicide plasmid and two-step homologous recombination, both mpd and pytH genes were integrated into the chromosome of a robust soil bacterium P. putida KT2440 and no selection marker was left on chromosome. Functional expression of mpd and pytH in P. putida KT2440 was demonstrated by Western blot analysis and enzyme activity assays. Degradation experiments with liquid cultures showed that the mixed pesticides including methyl parathion, fenitrothion, chlorpyrifos, permethrin, fenpropathrin, and cypermethrin (0.2 mM each) were degraded completely within 48 h. The inoculation of engineered strain (10(6) cells/g) to soils treated with the above mixed pesticides resulted in a higher degradation rate than in noninoculated soils. All six pesticides could be degraded completely within 15 days in fumigated and nonfumigated soils with inoculation. Theses results highlight the potential of the engineered strain to be used for in situ bioremediation of soils co-contaminated with OP and pyrethroid pesticides.

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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Production and purification of recombinant human glucagon overexpressed as intein fusion protein in Escherichia coli.

PubMed

Esipov, Roman S; Stepanenko, Vasily N; Gurevich, Alexandr I; Chupova, Larisa A; Miroshnikov, Anatoly I

2006-01-01

Chemico-enzymatic synthesis and cloning in Esherichia coli of an artificial gene coding human glucagon was performed. Recombinant plasmid containing hybrid glucagons gene and intein Ssp dnaB from Synechocestis sp. was designed. Expression of the obtained hybrid gene in E. coli, properties of the formed hybrid protein, and conditions of its autocatalytic cleavage leading to glucagon formation were studied.

Dynamics in copy numbers of five plasmids of a dairy Lactococcus lactis in dairy-related conditions including near-zero growth rates.

PubMed

van Mastrigt, Oscar; Lommers, Marcel M A N; de Vries, Yorick C; Abee, Tjakko; Smid, Eddy J

2018-03-23

Lactic acid bacteria can carry multiple plasmids affecting their performance in dairy fermentations. The expression of plasmid-encoded genes and the activity of the corresponding proteins is severely affected by changes in the number of plasmid copies. We studied the impact of growth rate on dynamics of plasmid copy numbers at high growth rates in chemostat cultures and down to near-zero growth rates in retentostat cultures. Five plasmids of the dairy strain Lactococcus lactis FM03-V1 were selected which varied in size (3 to 39 kb), in replication mechanism (theta or rolling-circle) and in putative (dairy-associated) functions. Copy numbers ranged from 1.5 to 40.5 and the copy number of theta-type replicating plasmids were negatively correlated to the plasmid size. Despite the extremely wide range of growth rates (0.0003 h -1 to 0.6 h -1 ), copy numbers of the five plasmids were stable and only slightly increased at near-zero growth rates showing that the plasmid replication rate was strictly controlled. One low-copy number plasmid, carrying a large exopolysaccharide gene cluster, was segregationally unstable during retentostat cultivations reflected in complete loss of the plasmid in one of the retentostat cultures. The copy number of the five plasmids was also hardly affected by varying the pH value, nutrient limitation or presence of citrate (maximum 2.2-fold) signifying the stability in copy number of the plasmids. Importance Lactococcus lactis is extensively used in starter cultures for dairy fermentations. Important traits for growth and survival of L. lactis in dairy fermentations are encoded by genes located on plasmids, such as genes involved in lactose and citrate metabolism, protein degradation and oligopeptide uptake and bacteriophage resistance. Because the number of plasmid copies could affect the expression of plasmid-encoded genes, it is important to know the factors that influence the plasmid copy numbers. We monitored plasmid copy numbers of L. lactis at near-zero growth rates, characteristic for cheese ripening. Moreover, we analysed the effect of pH, nutrient limitation and presence of citrate. This showed that plasmid copy numbers were stable giving insight into plasmid copy number dynamics in dairy fermentations. Copyright © 2018 American Society for Microbiology.

Clostridium perfringens type A–E toxin plasmids

PubMed Central

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

2014-01-01

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

Novel RepA-MCM proteins encoded in plasmids pTAU4, pORA1 and pTIK4 from Sulfolobus neozealandicus

PubMed Central

Greve, Bo; Jensen, Susanne; Phan, Hoa; Brügger, Kim; Zillig, Wolfram; She, Qunxin; Garrett, Roger A.

2005-01-01

Three plasmids isolated from the crenarchaeal thermoacidophile Sulfolobus neozealandicus were characterized. Plasmids pTAU4 (7,192 bp), pORA1 (9,689 bp) and pTIK4 (13,638 bp) show unusual properties that distinguish them from previously characterized cryptic plasmids of the genus Sulfolobus. Plasmids pORA1 and pTIK4 encode RepA proteins, only the former of which carries the novel polymerase–primase domain of other known Sulfolobus plasmids. Plasmid pTAU4 encodes a mini-chromosome maintenance protein homolog and no RepA protein; the implications for DNA replication are considered. Plasmid pORA1 is the first Sulfolobus plasmid to be characterized that does not encode the otherwise highly conserved DNA-binding PlrA protein. Another encoded protein appears to be specific for the New Zealand plasmids. The three plasmids should provide useful model systems for functional studies of these important crenarchaeal proteins. PMID:15876565

Characterization of the complete sequences and stability of plasmids carrying the genes aac(6')-Ib-cr or qnrS in Shigella flexneri in the Hangzhou area of China.

PubMed

Pu, Xiao-Ying; Gu, Yaming; Li, Jun; Song, Shu-Juan; Lu, Zhe

2018-05-18

The aim of this study was to explore the fluoroquinolone resistance mechanism of aac (6')-Ib-cr and qnrS gene by comparing complete sequences and stability of the aac(6')-Ib-cr- and qnrS-positive plasmids from Shigella isolates in the Hangzhou area of China. The complete sequences of four newly acquired plasmids carrying aac(6')-Ib-cr or qnrS were compared with those of two plasmids obtained previously and two similar reference Escherichia coli plasmids. The results showed that the length, antibiotic resistance genes and genetic environment were different among the plasmids. Moreover, the plasmid stability of three wild-type isolates and five plasmid transformants carrying aac(6')-Ib-cr and/or qnrS was measured in vitro, and all eight isolates were found to have lost their aac(6')-Ib-cr- or qnrS-positive plasmids to a different extent at different stages. When the plasmids were electroporated into Shigella flexneri or they lost positive plasmids, the MICs of ciprofloxacin increased or decreased two- to eightfold for aac(6')-Ib-cr-positive plasmids and 16- to 32-fold for qnrS-positive plasmids. To our knowledge, this is the first report comparing the complete sequences and describing stability for the aac(6')-Ib-cr- and qnrS-positive plasmids from Shigella isolates.

Host range diversification within the IncP-1 plasmid group

PubMed Central

Yano, Hirokazu; Rogers, Linda M.; Knox, Molly G.; Heuer, Holger; Smalla, Kornelia; Brown, Celeste J.

2013-01-01

Broad-host-range plasmids play a critical role in the spread of antibiotic resistance and other traits. In spite of increasing information about the genomic diversity of closely related plasmids, the relationship between sequence divergence and host range remains unclear. IncP-1 plasmids are currently classified into six subgroups based on the genetic distance of backbone genes. We investigated whether plasmids from two subgroups exhibit a different host range, using two IncP-1γ plasmids, an IncP-1β plasmid and their minireplicons. Efficiencies of plasmid establishment and maintenance were compared using five species that belong to the Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria. The IncP-1β plasmid replicated and persisted in all five hosts in the absence of selection. Of the two IncP-1γ plasmids, both were unable to replicate in alphaproteobacterial host Sphingobium japonicum, and one established itself in Agrobacterium tumefaciens but was very unstable. In contrast, both IncP-1γ minireplicons, which produced higher levels of replication initiation protein than the wild-type plasmids, replicated in all strains, suggesting that poor establishment of the native plasmids is in part due to suboptimal replication initiation gene regulation. The findings suggest that host ranges of distinct IncP-1 plasmids only partially overlap, which may limit plasmid recombination and thus result in further genome divergence. PMID:24002747

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

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

2015-01-01

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 bla TEM-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 importance. Such phenomenon is bothersome when the plasmids are transmissible, facilitating the spread of virulence and resistance plasmids among pathogenic bacteria. Notably, certain TA systems are more commonly found in particular ExPEC plasmid types, indicating the possible relationships between certain TA systems and ExPEC pathogenesis.

Sequence Analysis of IncA/C and IncI1 Plasmids Isolated from Multidrug-Resistant Salmonella Newport Using Single-Molecule Real-Time Sequencing.

PubMed

Cao, Guojie; Allard, Marc; Hoffmann, Maria; Muruvanda, Tim; Luo, Yan; Payne, Justin; Meng, Kevin; Zhao, Shaohua; McDermott, Patrick; Brown, Eric; Meng, Jianghong

2018-06-01

Multidrug-resistant (MDR) plasmids play an important role in disseminating antimicrobial resistance genes. To elucidate the antimicrobial resistance gene compositions in A/C incompatibility complex (IncA/C) plasmids carried by animal-derived MDR Salmonella Newport, and to investigate the spread mechanism of IncA/C plasmids, this study characterizes the complete nucleotide sequences of IncA/C plasmids by comparative analysis. Complete nucleotide sequencing of plasmids and chromosomes of six MDR Salmonella Newport strains was performed using PacBio RSII. Open reading frames were assigned using prokaryotic genome annotation pipeline (PGAP). To understand genomic diversity and evolutionary relationships among Salmonella Newport IncA/C plasmids, we included three complete IncA/C plasmid sequences with similar backbones from Salmonella Newport and Escherichia coli: pSN254, pAM04528, and peH4H, and additional 200 draft chromosomes. With the exception of canine isolate CVM22462, which contained an additional IncI1 plasmid, each of the six MDR Salmonella Newport strains contained only the IncA/C plasmid. These IncA/C plasmids (including references) ranged in size from 80.1 (pCVM21538) to 176.5 kb (pSN254) and carried various resistance genes. Resistance genes floR, tetA, tetR, strA, strB, sul, and mer were identified in all IncA/C plasmids. Additionally, bla CMY-2 and sugE were present in all IncA/C plasmids, excepting pCVM21538. Plasmid pCVM22462 was capable of being transferred by conjugation. The IncI1 plasmid pCVM22462b in CVM22462 carried bla CMY-2 and sugE. Our data showed that MDR Salmonella Newport strains carrying similar IncA/C plasmids clustered together in the phylogenetic tree using chromosome sequences and the IncA/C plasmids from animal-derived Salmonella Newport contained diverse resistance genes. In the current study, we analyzed genomic diversities and phylogenetic relationships among MDR Salmonella Newport using complete plasmids and chromosome sequences and provided possible spread mechanism of IncA/C plasmids in Salmonella Newport Lineage II.

[Construction of plant expression plasmid of chimera SBR-CT delta A1].

PubMed

Mai, Sui; Ling, Junqi

2003-08-01

The purpose of this study is to construct plant expression plasmid containing the gene encoding chimera SBR-CT delta A1. The target gene fragment P2, including the gene-encoded chimera SBR-CT delta A1 (3,498-5,378 bp), was obtained by standard PCR amplification. The PCR products were ligated with pGEM-easy vector through TA clone to form plasmid pTSC. The plasmid pTSC and plasmid pPOKII were digested by restricted endonuclease BamHI and KpnI, and the digested products were extracted and purified for recombination. Then the purified P2 and plasmid pPOKII were recombined by T4 DNA ligase to form recombinant plasmid pROSC; inserting bar gene into the plasmid and form pROSB plasmid. The recombined plasmids were isolated and identified by restricted endonuclease cutting and Sanger dideoxy DNA sequencing. P2 gene was linked to pPOKII plasmid and formed recombinant plasmid pROSC. The DNA sequence and orientation were corrected. And bar gene was inserted into pPOSC and form recombinant plasmid pROSB. Plant expression vector pROSC and pROSB containing the gene encoding chimera SBR-CT delta A1, which may provide useful experiment foundation for further study on edible vaccine against caries have been successfully constructed.

Fast and efficient three-step target-specific curing of a virulence plasmid in Salmonella enterica.

PubMed

de Moraes, Marcos H; Teplitski, Max

2015-12-01

Virulence plasmids borne by serovars of Salmonella enterica carry genes involved in its pathogenicity, as well as other functions. Characterization of phenotypes associated with virulence plasmids requires a system for efficiently curing strains of their virulence plasmids. Here, we developed a 3-step protocol for targeted curing of virulence plasmids. The protocol involves insertion of an I-SecI restriction site linked to an antibiotic resistance gene into the target plasmid using λ-Red mutagenesis, followed by the transformation with a temperature-sensitive auxiliary plasmid which carries I-SecI nuclease expressed from a tetracycline-inducible promoter. Finally, the auxiliary plasmid is removed by incubation at 42 °C and the plasmid-less strains are verified on antibiotic-containing media. This method is fast and very efficient: over 90 % of recovered colonies lacked their virulence plasmid.

Radio- and photosensitization of DNA with compounds containing platinum and bromine atoms

NASA Astrophysics Data System (ADS)

Śmiałek, Małgorzata A.; Ptasińska, Sylwia; Gow, Jason; Vrønning Hoffmann, Søren; Mason, Nigel J.

2015-05-01

Irradiations of plasmid DNA by both X-rays and UV light in the presence and absence of compounds containing platinum and bromine atoms were performed in order to asses the sensitization potential of these compounds. Plasmid DNA pBR322 was incubated with platinum (II) bromide, hydrogen hexabromoplatinate (IV), hydrogen hexahydroxyplatinate (IV) and sodium hexahydroxyplatinate (IV). Incubation was followed by X-ray or UV irradiations. It was found that amongst the sensitizers tested, during irradiations carried out in the presence of platinum (II) bromide, the highest levels of double strand breaks formation upon X-ray treatment were recorded. In contrast much less damage was induced by UV light. Data presented here suggests that this compound may be a promising radiosensitizer for cancer treatment. Contribution to the Topical Issue "COST Action Nano-IBCT: Nano-scale Processes Behind Ion-Beam Cancer Therapy", edited by Andrey Solov'yov, Nigel Mason, Gustavo García, Eugene Surdutovich.

One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections

PubMed Central

Mingo, Janire; Erramuzpe, Asier; Luna, Sandra; Aurtenetxe, Olaia; Amo, Laura; Diez, Ibai; Schepens, Jan T. G.; Hendriks, Wiljan J. A. J.; Cortés, Jesús M.; Pulido, Rafael

2016-01-01

Site-directed mutagenesis (SDM) is a powerful tool to create defined collections of protein variants for experimental and clinical purposes, but effectiveness is compromised when a large number of mutations is required. We present here a one-tube-only standardized SDM approach that generates comprehensive collections of amino acid substitution variants, including scanning- and single site-multiple mutations. The approach combines unified mutagenic primer design with the mixing of multiple distinct primer pairs and/or plasmid templates to increase the yield of a single inverse-PCR mutagenesis reaction. Also, a user-friendly program for automatic design of standardized primers for Ala-scanning mutagenesis is made available. Experimental results were compared with a modeling approach together with stochastic simulation data. For single site-multiple mutagenesis purposes and for simultaneous mutagenesis in different plasmid backgrounds, combination of primer sets and/or plasmid templates in a single reaction tube yielded the distinct mutations in a stochastic fashion. For scanning mutagenesis, we found that a combination of overlapping primer sets in a single PCR reaction allowed the yield of different individual mutations, although this yield did not necessarily follow a stochastic trend. Double mutants were generated when the overlap of primer pairs was below 60%. Our results illustrate that one-tube-only SDM effectively reduces the number of reactions required in large-scale mutagenesis strategies, facilitating the generation of comprehensive collections of protein variants suitable for functional analysis. PMID:27548698

Genetic control of ColE1 plasmid stability that is independent of plasmid copy number regulation.

PubMed

Standley, Melissa S; Million-Weaver, Samuel; Alexander, David L; Hu, Shuai; Camps, Manel

2018-06-16

ColE1-like plasmid vectors are widely used for expression of recombinant genes in E. coli. For these vectors, segregation of individual plasmids into daughter cells during cell division appears to be random, making them susceptible to loss over time when no mechanisms ensuring their maintenance are present. Here we use the plasmid pGFPuv in a recA relA strain as a sensitized model to study factors affecting plasmid stability in the context of recombinant gene expression. We find that in this model, plasmid stability can be restored by two types of genetic modifications to the plasmid origin of replication (ori) sequence: point mutations and a novel 269 nt duplication at the 5' end of the plasmid ori, which we named DAS (duplicated anti-sense) ori. Combinations of these modifications produce a range of copy numbers and of levels of recombinant expression. In direct contradiction with the classic random distribution model, we find no correlation between increased plasmid copy number and increased plasmid stability. Increased stability cannot be explained by reduced levels of recombinant gene expression either. Our observations would be more compatible with a hybrid clustered and free-distribution model, which has been recently proposed based on detection of individual plasmids in vivo using super-resolution fluorescence microscopy. This work suggests a role for the plasmid ori in the control of segregation of ColE1 plasmids that is distinct from replication initiation, opening the door for the genetic regulation of plasmid stability as a strategy aimed at enhancing large-scale recombinant gene expression or bioremediation.

Sequence of Two Plasmids from Clostridium perfringens Chicken Necrotic Enteritis Isolates and Comparison with C. perfringens Conjugative Plasmids

PubMed Central

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

2012-01-01

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

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

PubMed

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

2012-01-01

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

Diverse Broad-Host-Range Plasmids from Freshwater Carry Few Accessory Genes

PubMed Central

Sen, Diya; Yano, Hirokazu; Bauer, Matthew L.; Rogers, Linda M.; Van der Auwera, Geraldine A.

2013-01-01

Broad-host-range self-transferable plasmids are known to facilitate bacterial adaptation by spreading genes between phylogenetically distinct hosts. These plasmids typically have a conserved backbone region and a variable accessory region that encodes host-beneficial traits. We do not know, however, how well plasmids that do not encode accessory functions can survive in nature. The goal of this study was to characterize the backbone and accessory gene content of plasmids that were captured from freshwater sources without selecting for a particular phenotype or cultivating their host. To do this, triparental matings were used such that the only required phenotype was the plasmid's ability to mobilize a nonconjugative plasmid. Based on complete genome sequences of 10 plasmids, only 5 carried identifiable accessory gene regions, and none carried antibiotic resistance genes. The plasmids belong to four known incompatibility groups (IncN, IncP-1, IncU, and IncW) and two potentially new groups. Eight of the plasmids were shown to have a broad host range, being able to transfer into alpha-, beta-, and gammaproteobacteria. Because of the absence of antibiotic resistance genes, we resampled one of the sites and compared the proportion of captured plasmids that conferred antibiotic resistance to their hosts with the proportion of such plasmids captured from the effluent of a local wastewater treatment plant. Few of the captured plasmids from either site encoded antibiotic resistance. A high diversity of plasmids that encode no or unknown accessory functions is thus readily found in freshwater habitats. The question remains how the plasmids persist in these microbial communities. PMID:24096417

In vivo transcription of R-plasmid deoxyribonucleic acid in Escherichia coli strains with altered antibiotic resistance levels and/or conjugal proficiency.

PubMed Central

Davis, R; Vapnek, D

1976-01-01

The amounts of plasmid deoxyribonucleic acid (DNA) and the levels of the in vivo transcription of the Escherichia coli plasmids R538-1 (repressed for conjugal transfer) and R538-1drd (derepressed for transfer) were determined by DNA-DNA hybridization and DNA-ribonucleic acid hybridization, respectively. The results demonstrate that the level of plasmid transcription is increased by two-fold in the strain carrying the derepressed plasmid, compared to an isogenic strain carrying the repressed plasmid, whereas the amount of plasmid DNA is approximately the same, suggesting that the transfer genes are under transcriptional control. Levels of plasmid DNA, plasmid DNA transcription, and chloramphenicol acetyltransferase activity were also compared in a mutant strain that carried the R538-1drd plasmid and was resistant to high levels of antibiotics. This strain produces about 13 copies of plasmid DNA per chromosome compared to five copies for the parent strain. The level of transcription of plasmid DNA was found to be twofold higher in the high-level resistant strain, whereas the level of chloramphenition, acetyltransferase activity was increased by 10-fold. In addition the levels of plasmid DNA transcription and chloramphenicol acetyltransferase activity in the high-level resistant strain were found to be further increased by the presence of high levels of chloramphenicol in the growth medium. The amount of plasmid DNA remained constant under these conditions, indicating that high levels of chloramphenicol can stimulate the expression of plasmid genes at the level of transcription in this strain. PMID:767321

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

PubMed

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

2016-06-01

Manure from dairy farms has been shown to contain diverse tetracycline resistance genes that are transferable to soil. Here, we focus on conjugative plasmids that may spread tetracycline resistance at a conventional dairy farm. We performed exogenous plasmid isolation from cattle feces using chlortetracycline for transconjugant selection. The transconjugants obtained harbored LowGC-type plasmids and tet(Y). A representative plasmid (pFK2-7) was fully sequenced and this was compared with previously described LowGC plasmids from piggery manure-treated soil and a GenBank record from Acinetobacter nosocomialis that we also identified as a LowGC plasmid. The pFK2-7 plasmid had the conservative backbone typical of LowGC plasmids, though this region was interrupted with an insert containing the tet(Y)-tet(R) tetracycline resistance genes and the strA-strB streptomycin resistance genes. Despite Acinetobacter populations being considered natural hosts of LowGC plasmids, these plasmids were not found in three Acinetobacter isolates from the study farm. The isolates harbored tet(Y)-tet(R) genes in identical genetic surroundings as pFK2-7, however, suggesting genetic exchange between Acinetobacter and LowGC plasmids. Abundance of LowGC plasmids and tet(Y) was correlated in manure and soil samples from the farm, indicating that LowGC plasmids may be involved in the spread of tet(Y) in the environment. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

How can macromolecular crowding inhibit biological reactions? The enhanced formation of DNA nanoparticles

PubMed Central

Hou, Sen; Trochimczyk, Piotr; Sun, Lili; Wisniewska, Agnieszka; Kalwarczyk, Tomasz; Zhang, Xuzhu; Wielgus-Kutrowska, Beata; Bzowska, Agnieszka; Holyst, Robert

2016-01-01

In contrast to the already known effect that macromolecular crowding usually promotes biological reactions, solutions of PEG 6k at high concentrations stop the cleavage of DNA by HindIII enzyme, due to the formation of DNA nanoparticles. We characterized the DNA nanoparticles and probed the prerequisites for their formation using multiple techniques such as fluorescence correlation spectroscopy, dynamic light scattering, fluorescence analytical ultracentrifugation etc. In >25% PEG 6k solution, macromolecular crowding promotes the formation of DNA nanoparticles with dimensions of several hundreds of nanometers. The formation of DNA nanoparticles is a fast and reversible process. Both plasmid DNA (2686 bp) and double-stranded/single-stranded DNA fragment (66bp/nt) can form nanoparticles. We attribute the enhanced nanoparticle formation to the depletion effect of macromolecular crowding. This study presents our idea to enhance the formation of DNA nanoparticles by macromolecular crowding, providing the first step towards a final solution to efficient gene therapy. PMID:26903405

How can macromolecular crowding inhibit biological reactions? The enhanced formation of DNA nanoparticles.

PubMed

Hou, Sen; Trochimczyk, Piotr; Sun, Lili; Wisniewska, Agnieszka; Kalwarczyk, Tomasz; Zhang, Xuzhu; Wielgus-Kutrowska, Beata; Bzowska, Agnieszka; Holyst, Robert

2016-02-23

In contrast to the already known effect that macromolecular crowding usually promotes biological reactions, solutions of PEG 6k at high concentrations stop the cleavage of DNA by HindIII enzyme, due to the formation of DNA nanoparticles. We characterized the DNA nanoparticles and probed the prerequisites for their formation using multiple techniques such as fluorescence correlation spectroscopy, dynamic light scattering, fluorescence analytical ultracentrifugation etc. In >25% PEG 6k solution, macromolecular crowding promotes the formation of DNA nanoparticles with dimensions of several hundreds of nanometers. The formation of DNA nanoparticles is a fast and reversible process. Both plasmid DNA (2686 bp) and double-stranded/single-stranded DNA fragment (66 bp/nt) can form nanoparticles. We attribute the enhanced nanoparticle formation to the depletion effect of macromolecular crowding. This study presents our idea to enhance the formation of DNA nanoparticles by macromolecular crowding, providing the first step towards a final solution to efficient gene therapy.

Dark proteins: effect of inclusion body formation on quantification of protein expression.

PubMed

Iafolla, Marco A J; Mazumder, Mostafizur; Sardana, Vandit; Velauthapillai, Tharsan; Pannu, Karanbir; McMillen, David R

2008-09-01

Plasmid-borne gene expression systems have found wide application in the emerging fields of systems biology and synthetic biology, where plasmids are used to implement simple network architectures, either to test systems biology hypotheses about issues such as gene expression noise or as a means of exerting artificial control over a cell's dynamics. In both these cases, fluorescent proteins are commonly applied as a means of monitoring the expression of genes in the living cell, and efforts have been made to quantify protein expression levels through fluorescence intensity calibration and by monitoring the partitioning of proteins among the two daughter cells after division; such quantification is important in formulating the predictive models desired in systems and synthetic biology research. A potential pitfall of using plasmid-based gene expression systems is that the high protein levels associated with expression from plasmids can lead to the formation of inclusion bodies, insoluble aggregates of misfolded, nonfunctional proteins that will not generate fluorescence output; proteins caught in these inclusion bodies are thus "dark" to fluorescence-based detection methods. If significant numbers of proteins are incorporated into inclusion bodies rather than becoming biologically active, quantitative results obtained by fluorescent measurements will be skewed; we investigate this phenomenon here. We have created two plasmid constructs with differing average copy numbers, both incorporating an unregulated promoter (P(LtetO-1) in the absence of TetR) expressing the GFP derivative enhanced green fluorescent protein (EGFP), and inserted them into Escherichia coli bacterial cells (a common model organism for work on the dynamics of prokaryotic gene expression). We extracted the inclusion bodies, denatured them, and refolded them to render them active, obtaining a measurement of the average number of EGFP per cell locked into these aggregates; at the same time, we used calibrated fluorescent intensity measurements to determine the average number of active EGFP present per cell. Both measurements were carried out as a function of cellular doubling time, over a range of 45-75 min. We found that the ratio of inclusion body EGFP to active EGFP varied strongly as a function of the cellular growth rate, and that the number of "dark" proteins in the aggregates could in fact be substantial, reaching ratios as high as approximately five proteins locked into inclusion bodies for every active protein (at the fastest growth rate), and dropping to ratios well below 1 (for the slowest growth rate). Our results suggest that efforts to compare computational models to protein numbers derived from fluorescence measurements should take inclusion body loss into account, especially when working with rapidly growing cells. 2008 Wiley-Liss, Inc.

Mutation of the Erwinia amylovora argD Gene Causes Arginine Auxotrophy, Nonpathogenicity in Apples, and Reduced Virulence in Pears

PubMed Central

Ramos, Laura S.; Lehman, Brian L.; Peter, Kari A.

2014-01-01

Fire blight is caused by Erwinia amylovora and is the most destructive bacterial disease of apples and pears worldwide. In this study, we found that E. amylovora argD(1000)::Tn5, an argD Tn5 transposon mutant that has the Tn5 transposon inserted after nucleotide 999 in the argD gene-coding region, was an arginine auxotroph that did not cause fire blight in apple and had reduced virulence in immature pear fruits. The E. amylovora argD gene encodes a predicted N-acetylornithine aminotransferase enzyme, which is involved in the production of the amino acid arginine. A plasmid-borne copy of the wild-type argD gene complemented both the nonpathogenic and the arginine auxotrophic phenotypes of the argD(1000)::Tn5 mutant. However, even when mixed with virulent E. amylovora cells and inoculated onto immature apple fruit, the argD(1000)::Tn5 mutant still failed to grow, while the virulent strain grew and caused disease. Furthermore, the pCR2.1-argD complementation plasmid was stably maintained in the argD(1000)::Tn5 mutant growing in host tissues without any antibiotic selection. Therefore, the pCR2.1-argD complementation plasmid could be useful for the expression of genes, markers, and reporters in E. amylovora growing in planta, without concern about losing the plasmid over time. The ArgD protein cannot be considered an E. amylovora virulence factor because the argD(1000)::Tn5 mutant was auxotrophic and had a primary metabolism defect. Nevertheless, these results are informative about the parasitic nature of the fire blight disease interaction, since they indicate that E. amylovora cannot obtain sufficient arginine from apple and pear fruit tissues or from apple vegetative tissues, either at the beginning of the infection process or after the infection has progressed to an advanced state. PMID:25172854

Characterization of Streptomycin Resistance in Isolates of Erwinia amylovora in California.

PubMed

Förster, Helga; McGhee, Gayle C; Sundin, George W; Adaskaveg, James E

2015-10-01

In surveys from 2006 to 2014, streptomycin resistance in Erwinia amylovora from pear-growing areas in California declined from very high incidence in 2006 and 2007 to very low incidence in 2013 and 2014. The majority of resistant strains were designated as moderately resistant-low (MR-L), and were almost exclusively found in Sacramento County, whereas highly resistant (HR) strains were only recovered in Sutter-Yuba and San Joaquin counties. Resistance of HR strains was associated with a mutation in codon 43 of the chromosomal rpsL gene that results in a change from lysine to arginine, the same mutation that was originally reported for resistant strains from California in the mid-1970s. MR-L strains were found to harbor the strA-strB streptomycin resistance genes on transposon Tn5393a. This transposon lacks insertion sequence IS1133 that provides a promoter for efficient expression of strA-strB, resulting in lower minimum inhibitory concentrations of MR-L strains compared with those from other locations that harbor strA-strB on Tn5393::IS1133. In contrast to previously described plasmid-mediated resistance where Tn5393 is inserted in pEa34, or pEA29, Tn5393a in MR-L strains was located on plasmid pEU30. This plasmid was first described in E. amylovora from the western United States but was not associated with streptomycin resistance determinants previously. We hypothesize that Tn5393a was introduced into an E. amylovora strain carrying pEU30 and transposed into that plasmid. This hypothesis was supported by clustered regularly interspaced short palindromic repeat (CRISPR) sequence analysis that showed that two MR-L strains share the same CRISPR1 pattern as a streptomycin-sensitive strain. With current low resistance levels in California growing regions, streptomycin could be successfully used again, but applications per season should be limited and the antibiotic should be mixed and rotated with different modes of action.

Mutation of the Erwinia amylovora argD gene causes arginine auxotrophy, nonpathogenicity in apples, and reduced virulence in pears.

PubMed

Ramos, Laura S; Lehman, Brian L; Peter, Kari A; McNellis, Timothy W

2014-11-01

Fire blight is caused by Erwinia amylovora and is the most destructive bacterial disease of apples and pears worldwide. In this study, we found that E. amylovora argD(1000)::Tn5, an argD Tn5 transposon mutant that has the Tn5 transposon inserted after nucleotide 999 in the argD gene-coding region, was an arginine auxotroph that did not cause fire blight in apple and had reduced virulence in immature pear fruits. The E. amylovora argD gene encodes a predicted N-acetylornithine aminotransferase enzyme, which is involved in the production of the amino acid arginine. A plasmid-borne copy of the wild-type argD gene complemented both the nonpathogenic and the arginine auxotrophic phenotypes of the argD(1000)::Tn5 mutant. However, even when mixed with virulent E. amylovora cells and inoculated onto immature apple fruit, the argD(1000)::Tn5 mutant still failed to grow, while the virulent strain grew and caused disease. Furthermore, the pCR2.1-argD complementation plasmid was stably maintained in the argD(1000)::Tn5 mutant growing in host tissues without any antibiotic selection. Therefore, the pCR2.1-argD complementation plasmid could be useful for the expression of genes, markers, and reporters in E. amylovora growing in planta, without concern about losing the plasmid over time. The ArgD protein cannot be considered an E. amylovora virulence factor because the argD(1000)::Tn5 mutant was auxotrophic and had a primary metabolism defect. Nevertheless, these results are informative about the parasitic nature of the fire blight disease interaction, since they indicate that E. amylovora cannot obtain sufficient arginine from apple and pear fruit tissues or from apple vegetative tissues, either at the beginning of the infection process or after the infection has progressed to an advanced state. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Hypochlorite-induced damage to DNA, RNA, and polynucleotides: formation of chloramines and nitrogen-centered radicals.

PubMed

Hawkins, Clare L; Davies, Michael J

2002-01-01

Stimulated monocytes and neutrophils generate hypochlorite (HOCl) via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl is a key bactericidal agent, but can also damage host tissue. As there is a strong link between chronic inflammation and some cancers, we have investigated HOCl damage to DNA, RNA, and polynucleotides. Reaction of HOCl with these materials is shown to yield multiple semistable chloramines (RNHCl/RR'NCl), which are the major initial products, and account for 50-95% of the added HOCl. These chloramines decay by thermal and metal-ion catalyzed processes, to give nucleoside-derived, nitrogen-centered, radicals. The latter have been characterized by EPR spin trapping. The propensity for radical formation with polynucleotides is cytidine > adenosine = guanosine > uridine = thymidine. The rates of decay, and yield of radicals formed, are dependent on the nature of the nucleobase on which they are formed, with chloramines formed from ring heterocyclic amine groups being less stable than those formed on exocyclic amines (RNH2 groups). Evidence is presented for chlorine transfer from the former, kinetically favored, sites to the more thermodynamically favored exocyclic amines. EPR experiments have also provided evidence for the rapid addition of pyrimidine-derived nitrogen-centered radicals to other nucleobases to give dimers and the oxidation of DNA by radicals derived from preformed nucleoside chloramines. Direct reaction of HOCl with plasmid DNA gives rise to single- and double-strand breaks via chloramine-mediated reactions. Preformed nucleoside chloramines also induce plasmid cleavage, though this only occurs to a significant extent with unstable thymidine- and uridine-derived chloramines, where radical formation is rapid. Overall the data rationalize the preferential formation of chlorinated 2'-deoxycytidine and 2'-deoxyadenosine in DNA and suggest that DNA damage induced by HOCl, and preformed chloramines, occurs at sequence-specific sites.

Ordering the mob: Insights into replicon and MOB typing schemes from analysis of a curated dataset of publicly available plasmids.

PubMed

Orlek, Alex; Phan, Hang; Sheppard, Anna E; Doumith, Michel; Ellington, Matthew; Peto, Tim; Crook, Derrick; Walker, A Sarah; Woodford, Neil; Anjum, Muna F; Stoesser, Nicole

2017-05-01

Plasmid typing can provide insights into the epidemiology and transmission of plasmid-mediated antibiotic resistance. The principal plasmid typing schemes are replicon typing and MOB typing, which utilize variation in replication loci and relaxase proteins respectively. Previous studies investigating the proportion of plasmids assigned a type by these schemes ('typeability') have yielded conflicting results; moreover, thousands of plasmid sequences have been added to NCBI in recent years, without consistent annotation to indicate which sequences represent complete plasmids. Here, a curated dataset of complete Enterobacteriaceae plasmids from NCBI was compiled, and used to assess the typeability and concordance of in silico replicon and MOB typing schemes. Concordance was assessed at hierarchical replicon type resolutions, from replicon family-level to plasmid multilocus sequence type (pMLST)-level, where available. We found that 85% and 65% of the curated plasmids could be replicon and MOB typed, respectively. Overall, plasmid size and the number of resistance genes were significant independent predictors of replicon and MOB typing success. We found some degree of non-concordance between replicon families and MOB types, which was only partly resolved when partitioning plasmids into finer-resolution groups (replicon and pMLST types). In some cases, non-concordance was attributed to ambiguous boundaries between MOBP and MOBQ types; in other cases, backbone mosaicism was considered a more plausible explanation. β-lactamase resistance genes tended not to show fidelity to a particular plasmid type, though some previously reported associations were supported. Overall, replicon and MOB typing schemes are likely to continue playing an important role in plasmid analysis, but their performance is constrained by the diverse and dynamic nature of plasmid genomes. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Isolation and characterization of two novel groups of kanamycin-resistance ColE1-like plasmids in Salmonella enterica serotypes from food animals.

PubMed

Chen, Chin-Yi; Strobaugh, Terence P; Nguyen, Ly-Huong T; Abley, Melanie; Lindsey, Rebecca L; Jackson, Charlene R

2018-01-01

While antimicrobial resistance in Salmonella enterica is mainly attributed to large plasmids, small plasmids may also harbor antimicrobial resistance genes. Previously, three major groups of ColE1-like plasmids conferring kanamycin-resistance (KanR) in various S. enterica serotypes from diagnostic samples of human or animals were reported. In this study, over 200 KanR S. enterica isolates from slaughter samples, collected in 2010 and 2011 as a part of the animal arm of the National Antimicrobial Resistance Monitoring System, were screened for the presence of ColE1-like plasmids. Twenty-three KanR ColE1-like plasmids were successfully isolated. Restriction fragment mapping revealed five major plasmid groups with subgroups, including two new groups, X (n = 3) and Y/Y2/Y3 (n = 4), in addition to the previously identified groups A (n = 7), B (n = 6), and C/C3 (n = 3). Nearly 75% of the plasmid-carrying isolates were from turkey and included all the isolates carrying X and Y plasmids. All group X plasmids were from serotype Hadar. Serotype Senftenberg carried all the group Y plasmids and one group B plasmid. All Typhimurium isolates (n = 4) carried group A plasmids, while Newport isolates (n = 3) each carried a different plasmid group (A, B, or C). The presence of the selection bias in the NARMS strain collection prevents interpretation of findings at the population level. However, this study demonstrated that KanR ColE1-like plasmids are widely distributed among different S. enterica serotypes in the NARMS isolates and may play a role in dissemination of antimicrobial resistance genes.

Isolation and characterization of two novel groups of kanamycin-resistance ColE1-like plasmids in Salmonella enterica serotypes from food animals

PubMed Central

Strobaugh, Terence P.; Nguyen, Ly-Huong T.; Abley, Melanie; Lindsey, Rebecca L.; Jackson, Charlene R.

2018-01-01

While antimicrobial resistance in Salmonella enterica is mainly attributed to large plasmids, small plasmids may also harbor antimicrobial resistance genes. Previously, three major groups of ColE1-like plasmids conferring kanamycin-resistance (KanR) in various S. enterica serotypes from diagnostic samples of human or animals were reported. In this study, over 200 KanR S. enterica isolates from slaughter samples, collected in 2010 and 2011 as a part of the animal arm of the National Antimicrobial Resistance Monitoring System, were screened for the presence of ColE1-like plasmids. Twenty-three KanR ColE1-like plasmids were successfully isolated. Restriction fragment mapping revealed five major plasmid groups with subgroups, including two new groups, X (n = 3) and Y/Y2/Y3 (n = 4), in addition to the previously identified groups A (n = 7), B (n = 6), and C/C3 (n = 3). Nearly 75% of the plasmid-carrying isolates were from turkey and included all the isolates carrying X and Y plasmids. All group X plasmids were from serotype Hadar. Serotype Senftenberg carried all the group Y plasmids and one group B plasmid. All Typhimurium isolates (n = 4) carried group A plasmids, while Newport isolates (n = 3) each carried a different plasmid group (A, B, or C). The presence of the selection bias in the NARMS strain collection prevents interpretation of findings at the population level. However, this study demonstrated that KanR ColE1-like plasmids are widely distributed among different S. enterica serotypes in the NARMS isolates and may play a role in dissemination of antimicrobial resistance genes. PMID:29513730

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

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.

Plasmid analyses in clinical isolates of Bacteroides fragilis and other Bacteroides species.

PubMed Central

Wallace, B L; Bradley, J E; Rogolsky, M

1981-01-01

Plasmid analyses were performed on Bacteroides strains isolated from clinical specimens. Of 32 Bacteroides strains, 8 were found to contain plasmids. Seven of these eight strains were B. fragilis, and the other one was B. distasonis. Three of these eight strains harbored only a 3.0-megadalton plasmid. Two strains had only a 2.0-megadalton plasmid, and one had 2.0-, 3.0-megadalton plasmid. Of the remaining two strains, one had 2.0-, 3.0-, and 5.0-megadalton plasmids, and the other had 3.0- and 5.0-megadalton plasmids. Beta-Lactamase was produced by 93% of the clinical isolates. Seven of the eight plasmid-carrying strains were cadmium resistant, five were zinc resistant, four were mercury resistant, and two expressed a brick-red fluorescence under ultraviolet light. None of these traits could be associated with a plasmid after performing either curing experiments or genetic transfer experiments by cell-to-cell contact. Images PMID:6974737

Hofmeister series salts enhance purification of plasmid DNA by non-ionic detergents

PubMed Central

Lezin, George; Kuehn, Michael R.; Brunelli, Luca

2011-01-01

Ion-exchange chromatography is the standard technique used for plasmid DNA purification, an essential molecular biology procedure. Non-ionic detergents (NIDs) have been used for plasmid DNA purification, but it is unclear whether Hofmeister series salts (HSS) change the solubility and phase separation properties of specific NIDs, enhancing plasmid DNA purification. After scaling-up NID-mediated plasmid DNA isolation, we established that NIDs in HSS solutions minimize plasmid DNA contamination with protein. In addition, large-scale NID/HSS solutions eliminated LPS contamination of plasmid DNA more effectively than Qiagen ion-exchange columns. Large-scale NID isolation/NID purification generated increased yields of high quality DNA compared to alkali isolation/column purification. This work characterizes how HSS enhance NID-mediated plasmid DNA purification, and demonstrates that NID phase transition is not necessary for LPS removal from plasmid DNA. Specific NIDs such as IGEPAL CA-520 can be utilized for rapid, inexpensive and efficient laboratory-based large-scale plasmid DNA purification, outperforming Qiagen-based column procedures. PMID:21351074

inhibitory effects of citral, cinnamaldehyde, and tea polyphenols on mixed biofilm formation by foodborne Staphylococcus aureus and Salmonella enteritidis.

PubMed

Zhang, Hongmei; Zhou, Wenyuan; Zhang, Wenyan; Yang, Anlin; Liu, Yanlan; Jiang, Yan; Huang, Shaosong; Su, Jianyu

2014-06-01

Biofilms are significant hazards in the food industry. In this study, we investigated the effects of food additive such as citral, cinnamaldehyde, and tea polyphenols on mixed biofilm formation by foodborne Staphylococcus aureus and Salmonella serotype Enteritidis. The adhesion rates of mixed strains in sub-MIC of additives were determined by a microtiter plate assay and bacterial communication signal autoinducer 2 (AI-2) production via a bioluminescence reporter Vibrio harveyi BB170. The structure of mixed biofilm was analyzed using scanning electron microscopy. The effect of the disinfectants hydrogen peroxide, sodium hypochlorite, and peracetic acid was tested on the mixed biofilm. Our results demonstrated that citral, cinnamaldehyde, and tea polyphenols were able to significantly inhibit mixed biofilm formation, while citral could reduce the synthesis of AI-2. Conversely, we observed a significant increase in AI-2 mediated by cinnamaldehyde. Tea polyphenols at lower concentrations induced AI-2 synthesis; however, AI-2 synthesis was significantly inhibited at higher concentrations (300 m g/ml). Food additives inhibited the adhesion of mixed bacteria on stainless steel chips and increased the sensitivity of the mixed biofilm to disinfectants. In conclusion, citral, cinnamaldehyde, and tea polyphenols had strong inhibitory effects on mixed biofilm formation and also enhanced the effect of disinfectant on mixed biofilm formation. This study provides a scientific basis for the application of natural food additives to control biofilm formation of foodborne bacteria.

Ecological and genetic determinants of plasmid distribution in Escherichia coli.

PubMed

Medaney, Frances; Ellis, Richard J; Raymond, Ben

2016-11-01

Bacterial plasmids are important carriers of virulence and antibiotic resistance genes. Nevertheless, little is known of the determinants of plasmid distribution in bacterial populations. Here the factors affecting the diversity and distribution of the large plasmids of Escherichia coli were explored in cattle grazing on semi-natural grassland, a set of populations with low frequencies of antibiotic resistance genes. Critically, the population genetic structure of bacterial hosts was chararacterized. This revealed structured E. coli populations with high diversity between sites and individuals but low diversity within cattle hosts. Plasmid profiles, however, varied considerably within the same E. coli genotype. Both ecological and genetic factors affected plasmid distribution: plasmid profiles were affected by site, E. coli diversity, E. coli genotype and the presence of other large plasmids. Notably 3/26 E. coli serotypes accounted for half the observed plasmid-free isolates indicating that within species variation can substantially affect carriage of the major conjugative plasmids. The observed population structure suggest that most of the opportunities for within species plasmid transfer occur between different individuals of the same genotype and support recent experimental work indicating that plasmid-host coevolution, and epistatic interactions on fitness costs are likely to be important in determining occupancy. © 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

Testing plasmid stability of Escherichia coli using the Continuously Operated Shaken BIOreactor System.

PubMed

Sieben, Michaela; Steinhorn, Gregor; Müller, Carsten; Fuchs, Simone; Ann Chin, Laura; Regestein, Lars; Büchs, Jochen

2016-11-01

Plasmids are common vectors to genetically manipulate Escherichia coli or other microorganisms. They are easy to use and considerable experience has accumulated on their application in heterologous protein production. However, plasmids can be lost during cell growth, if no selection pressure like, e.g., antibiotics is used, hampering the production of the desired protein and endangering the economic success of a biotechnological production process. Thus, in this study the Continuously Operated Shaken BIOreactor System (COSBIOS) is applied as a tool for fast parallel testing of strain stability and operation conditions and to evaluate measures to counter such plasmid loss. In specific, by applying various ampicillin concentrations, the lowest effective ampicillin dosage is investigated to secure plasmid stability while lowering adverse ecological effects. A significant difference was found in the growth rates of plasmid-bearing and plasmid-free cells. The undesired plasmid-free cells grew 30% faster than the desired plasmid-bearing cells. During the testing of plasmid stability without antibiotics, the population fraction of plasmid-bearing cells rapidly decreased in continuous culture to zero within the first 48 h. An initial single dosage of ampicillin did not prevent plasmid loss. By contrast, a continuous application of a low dosage of 10 µg/mL ampicillin in the feed medium maintained plasmid stability in the culture. Consequently, the COSBIOS is an apt reactor system for measuring plasmid stability and evaluating methods to enhance this stability. Hence, decreased production of heterologous protein can be prevented. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1418-1425, 2016. © 2016 American Institute of Chemical Engineers.

Shifts in Host Range of a Promiscuous Plasmid through Parallel Evolution of its Replication Initiation Protein

PubMed Central

Sota, Masahiro; Yano, Hirokazu; Hughes, Julie; Daughdrill, Gary W.; Abdo, Zaid; Forney, Larry J.; Top, Eva M.

2011-01-01

The ability of bacterial plasmids to adapt to novel hosts and thereby shift their host range is key to their long-term persistence in bacterial communities. Promiscuous plasmids of the IncP-1 group can colonize a wide range of hosts, but it is not known if and how they can contract, shift or further expand their host range. To understand the evolutionary mechanisms of host range shifts of IncP-1 plasmids, an IncP-1β mini-replicon was experimentally evolved in four hosts wherein it was initially unstable. After 1000 generations in serial batch cultures under antibiotic selection for plasmid maintenance (kanamycin resistance), the stability of the mini-plasmid had dramatically improved in all coevolved hosts. However, only plasmids evolved in Shewanella oneidensis showed improved stability in the ancestor, indicating that adaptive mutations had occurred in the plasmid itself. Complete genome sequence analysis of nine independently evolved plasmids showed seven unique plasmid genotypes that had various kinds of single mutations at one locus, namely the N-terminal region of the replication initiation protein TrfA. Such parallel evolution indicates that this region was under strong selection. In five of the seven evolved plasmids these trfA mutations resulted in a significantly higher plasmid copy number. Evolved plasmids were found to be stable in four other naïve hosts, but could no longer replicate in Pseudomonas aeruginosa. This study demonstrates that plasmids can specialize to a novel host through trade-offs between improved stability in the new host and the ability to replicate in a previously permissive host. PMID:20520653

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 partitioning and suggest underlying biological roles shared by such elements. PMID:26865697

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 plaquing, fluorescence or antibody staining.

Novel blaROB-1-Bearing Plasmid Conferring Resistance to β-Lactams in Haemophilus parasuis Isolates from Healthy Weaning Pigs

PubMed Central

Moleres, Javier; Santos-López, Alfonso; Lázaro, Isidro; Labairu, Javier; Prat, Cristina; Ardanuy, Carmen; González-Zorn, Bruno

2015-01-01

Haemophilus parasuis, the causative agent of Glässer's disease, is one of the early colonizers of the nasal mucosa of piglets. It is prevalent in swine herds, and lesions associated with disease are fibrinous polyserositis and bronchopneumonia. Antibiotics are commonly used in disease control, and resistance to several antibiotics has been described in H. parasuis. Prediction of H. parasuis virulence is currently limited by our scarce understanding of its pathogenicity. Some genes have been associated with H. parasuis virulence, such as lsgB and group 1 vtaA, while biofilm growth has been associated with nonvirulent strains. In this study, 86 H. parasuis nasal isolates from farms that had not had a case of disease for more than 10 years were obtained by sampling piglets at weaning. Isolates were studied by enterobacterial repetitive intergenic consensus PCR and determination of the presence of lsgB and group 1 vtaA, biofilm formation, inflammatory cell response, and resistance to antibiotics. As part of the diversity encountered, a novel 2,661-bp plasmid, named pJMA-1, bearing the blaROB-1 β-lactamase was detected in eight colonizing strains. pJMA-1 was shown to share a backbone with other small plasmids described in the Pasteurellaceae, to be 100% stable, and to have a lower biological cost than the previously described plasmid pB1000. pJMA-1 was also found in nine H. parasuis nasal strains from a separate collection, but it was not detected in isolates from the lesions of animals with Glässer's disease or in nontypeable Haemophilus influenzae isolates. Altogether, we show that commensal H. parasuis isolates represent a reservoir of β-lactam resistance genes which can be transferred to pathogens or other bacteria. PMID:25747001

[The Effect of Introduction of the Heterologous Gene Encoding the N-acyl-homoserine Lactonase (aiiA) on the Properties of Burkholderia cenocepacia 370].

PubMed

Plyuta, V A; Lipasova, V A; Koksharova, O A; Veselova, M A; Kuznetsov, A E; Khmel, I A

2015-08-01

To study the role of Quorum Sensing (QS) regulation in the control of the cellular processes of Burkholderia cenocepacia 370, plasmid pME6863 was transferred into its cells. The plasmid contains a heterologous gene encoding for AiiA N-acyl-homoserine lactonase, which degrades the signaling molecules of the QS system of N-acyl-homoserine lactones (AHL). An absence or reduction of AHL in the culture was revealed with the biosensors Chromobacterium violaceum CV026 and Agrobacterium tumifaciens NT1/pZLR4, respectively. The presence of the aiiA gene, which was cloned from Bacillus sp. A24 in the cells of B. cenocepacia 370, resulted in a lack of hemolytic activity, which reduced the extracellular proteolytic activity and decreased the cells' ability to migration in swarms on the surface of the agar medium. The introduction of the aiiA gene did not affect lipase activity, fatty acids synthesis, HCN synthesis, or biofilm formation. Hydrogen peroxide was shown to stimulate biofilm formation by B. cenocepacia 370 in concentrations that inhibited or weakly suppressed bacterial growth. The introduction of the aiiA gene into the cells did not eliminate this effect but it did reduce it.

Coupled transcription and processing of mouse ribosomal RNA in a cell-free system.

PubMed Central

Mishima, Y; Mitsuma, T; Ogata, K

1985-01-01

An in vitro processing system of mouse rRNA was achieved using an RNA polymerase I-specific transcription system, (S100) and recombinant plasmids consisting of mouse rRNA gene (rDNA) segments containing the transcription initiation and 5'-terminal region of 18S (or 41S) rRNA. Pulse-chase experiments showed that a specific processing occurred with transcripts of the plasmid DNAs when the direction of transcription was the correct orientation relative to the 18S rRNA coding sequence, but not with transcripts of the DNA templates in which this coding sequence was in the opposite orientation. From the S1 nuclease protection analyses, we concluded that there are several steps of endonucleolytic cleavage including one 105 nucleotides upstream from the 5' end of 18S rRNA. Intermediates cleaved at this site were identified in in vivo processing of rRNA. This result indicates that endonucleolytic cleavage takes place 105 nucleotides upstream from the 5' terminus of 18S rRNA prior to the formation of mature 18S rRNA. Trimming or cleavage of the 105 nucleotides may be involved in the formation of the 5' terminus of mature 18S rRNA. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:3004977

alpha,beta-poly(asparthylhydrazide)-glycidyltrimethylammonium chloride copolymers (PAHy-GTA): novel polymers with potential for DNA delivery.

PubMed

Pedone, E; Cavallaro, G; Richardson, S C; Duncan, R; Giammona, G

2001-11-09

Hydrophilic polycations form complexes when mixed with plasmids. Following functionalisation with glycidyltrimethylammonium chloride (GTA) alpha,beta-poly(asparthylhydrazide) (PAHy), a water-soluble synthetic macromolecule, becomes polycationic and potentially useful for systemic gene delivery. Initially the biocompatibility of PAHy and PAHy-GTA derivatives with different degrees of positive charge substitution were studied and it was shown that PAHy-GTA was neither haemolytic nor cytotoxicity up to 1 mg/ml. After intravenous injection (125)I-labelled PAHy-GTA derivative containing 46 mol% (PAHy-GTA(b)) of trimethylammonium groups did not accumulate in the liver (4.1+/-0.9% of the recovered dose after 1 h) but was subjected to renal excretion (45+/-21% of the recovered dose was in the kidneys after 1 h). PAHy-GTA formed complexes with DNA (gel retardation) and they protected against degradation by DNase II. Finally the ability of the PAHy-GTA(b) derivative to mediate the transfection of HepG2 cells using the marker gene beta-galactosidase was studied. The optimum plasmid/polymer mass ratio was examined in comparison to LipofectACE, Lipofectin and polyethylenimine.

Characterization of a beta-lactamase-specifying plasmid isolated from Eikenella corrodens and its relationship to a commensal Neisseria plasmid.

PubMed Central

Rotger, R; García-Valdés, E; Trallero, E P

1986-01-01

A 9.4-kilobase plasmid encoding penicillin, streptomycin, and sulfonamide resistance was isolated from a beta-lactamase-producing Eikenella corrodens strain. This plasmid appears to be identical to a resistance plasmid common to saprophytic Neisseria strains. Images PMID:3535668

[Replication of Streptomyces plasmids: the DNA nucleotide sequence of plasmid pSB 24.2].

PubMed

Bolotin, A P; Sorokin, A V; Aleksandrov, N N; Danilenko, V N; Kozlov, Iu I

1985-11-01

The nucleotide sequence of DNA in plasmid pSB 24.2, a natural deletion derivative of plasmid pSB 24.1 isolated from S. cyanogenus was studied. The plasmid amounted by its size to 3706 nucleotide pairs. The G-C composition was equal to 73 per cent. The analysis of the DNA structure in plasmid pSB 24.2 revealed the protein-encoding sequence of DNA, the continuity of which was significant for replication of the plasmid containing more than 1300 nucleotide pairs. The analysis also revealed two A-T-rich areas of DNA, the G-C composition of which was less than 55 per cent and a DNA area with a branched pin structure. The results may be of value in investigation of plasmid replication in actinomycetes and experimental cloning of DNA with this plasmid as a vector.

IncX2 and IncX1-X2 Hybrid Plasmids Coexisting in a FosA6-Producing Escherichia coli Strain

PubMed Central

Su, Jiachun; McElheny, Christi Lee; Wang, Minggui

2017-01-01

ABSTRACT IncX plasmids are receiving much attention as vehicles of carbapenem and colistin resistance genes, such as blaNDM, blaKPC, and mcr-1. Among them, IncX2 subgroup plasmids remain rare. Here, we characterized IncX2 and IncX1-X2 hybrid plasmids coexisting in a FosA6-producing Escherichia coli strain that were possibly generated as a consequence of recombination events between an R6K-like IncX2 plasmid and a pLN126_33-like IncX1 plasmid. Variable multidrug resistance mosaic regions were observed in these plasmids, indicating their potential to serve as flexible carriers of resistance genes. The diversity of IncX group plasmid backbones and accessory genes and the evolution of hybrid IncX plasmids pose a challenge in detecting and classifying them. PMID:28438937

Coupling between the basic replicon and the Kis-Kid maintenance system of plasmid R1: modulation by Kis antitoxin levels and involvement in control of plasmid replication.

PubMed

López-Villarejo, Juan; Lobato-Márquez, Damián; Díaz-Orejas, Ramón

2015-02-05

kis-kid, the auxiliary maintenance system of plasmid R1 and copB, the auxiliary copy number control gene of this plasmid, contribute to increase plasmid replication efficiency in cells with lower than average copy number. It is thought that Kis antitoxin levels decrease in these cells and that this acts as the switch that activates the Kid toxin; activated Kid toxin reduces copB-mRNA levels and this increases RepA levels that increases plasmid copy number. In support of this model we now report that: (i) the Kis antitoxin levels do decrease in cells containing a mini-R1 plasmid carrying a repA mutation that reduces plasmid copy number; (ii) kid-dependent replication rescue is abolished in cells in which the Kis antitoxin levels or the CopB levels are increased. Unexpectedly we found that this coordination significantly increases both the copy number of the repA mutant and of the wt mini-R1 plasmid. This indicates that the coordination between plasmid replication functions and kis-kid system contributes significantly to control plasmid R1 replication.

Coupling between the Basic Replicon and the Kis-Kid Maintenance System of Plasmid R1: Modulation by Kis Antitoxin Levels and Involvement in Control of Plasmid Replication

PubMed Central

López-Villarejo, Juan; Lobato-Márquez, Damián; Díaz-Orejas, Ramón

2015-01-01

kis-kid, the auxiliary maintenance system of plasmid R1 and copB, the auxiliary copy number control gene of this plasmid, contribute to increase plasmid replication efficiency in cells with lower than average copy number. It is thought that Kis antitoxin levels decrease in these cells and that this acts as the switch that activates the Kid toxin; activated Kid toxin reduces copB-mRNA levels and this increases RepA levels that increases plasmid copy number. In support of this model we now report that: (i) the Kis antitoxin levels do decrease in cells containing a mini-R1 plasmid carrying a repA mutation that reduces plasmid copy number; (ii) kid-dependent replication rescue is abolished in cells in which the Kis antitoxin levels or the CopB levels are increased. Unexpectedly we found that this coordination significantly increases both the copy number of the repA mutant and of the wt mini-R1 plasmid. This indicates that the coordination between plasmid replication functions and kis-kid system contributes significantly to control plasmid R1 replication. PMID:25664511

Frequent conjugative transfer accelerates adaptation of a broad-host-range plasmid to an unfavorable Pseudomonas putida host.

PubMed

Heuer, Holger; Fox, Randal E; Top, Eva M

2007-03-01

IncP-1 plasmids are known to be promiscuous, but it is not understood if they are equally well adapted to various species within their host range. Moreover, little is known about their fate in bacterial communities. We determined if the IncP-1beta plasmid pB10 was unstable in some Proteobacteria, and whether plasmid stability was enhanced after long-term carriage in a single host and when regularly switched between isogenic hosts. Plasmid pB10 was found to be very unstable in Pseudomonas putida H2, and conferred a high cost (c. 20% decrease in fitness relative to the plasmid-free host). H2(pB10) was then evolved under conditions that selected for plasmid maintenance, with or without regular plasmid transfer (host-switching). When tested in the ancestral host, the evolved plasmids were more stable and their cost was significantly reduced (9% and 16% for plasmids from host-switched and nonswitched lineages, respectively). Our findings suggest that IncP-1 plasmids can rapidly adapt to an unfavorable host by improving their overall stability, and that regular conjugative transfer accelerates this process.

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

Mixed-Format Test Score Equating: Effect of Item-Type Multidimensionality, Length and Composition of Common-Item Set, and Group Ability Difference

ERIC Educational Resources Information Center

Wang, Wei

2013-01-01

Mixed-format tests containing both multiple-choice (MC) items and constructed-response (CR) items are now widely used in many testing programs. Mixed-format tests often are considered to be superior to tests containing only MC items although the use of multiple item formats leads to measurement challenges in the context of equating conducted under…

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

PubMed Central

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

1999-01-01

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

The partitioning and copy number control systems of the selfish yeast plasmid: an optimized molecular design for stable persistence in host cells

PubMed Central

Yen-Ting-Liu; Sau, Saumitra; Ma, Chien-Hui; Kachroo, Aashiq H; Rowley, Paul A; Chang, Keng-Ming; Fan, Hsiu-Fang; Jayaram, Makkuni

2014-01-01

Summary The multi-copy 2 micron plasmid of Saccharomyces cerevisiae, a resident of the nucleus, is remarkable for its high chromosome-like stability. The plasmid does not appear to contribute to the fitness of the host, nor does it impose a significant metabolic burden on the host at its steady state copy number. The plasmid may be viewed as a highly optimized selfish DNA element whose genome design is devoted entirely towards efficient replication, equal segregation and copy number maintenance. A partitioning system comprised of two plasmid coded proteins, Rep1 and Rep2, and a partitioning locus STB is responsible for equal or nearly equal segregation of plasmid molecules to mother and daughter cells. Current evidence supports a model in which the Rep-STB system promotes the physical association of the plasmid with chromosomes and thus plasmid segregation by a hitchhiking mechanism. The Flp site-specific recombination system housed by the plasmid plays a critical role in maintaining steady state plasmid copy number. A decrease in plasmid population due to rare missegregation events is rectified by plasmid amplification via a recombination induced rolling circle replication mechanism. Appropriate plasmid amplification, without runaway increase in copy number, is ensured by positive and negative regulation of FLP gene expression by plasmid coded proteins and by the control of Flp level/activity through host mediated post-translational modification(s) of Flp. The Flp system has been successfully utilized to understand mechanisms of site-specific recombination, to bring about directed genetic alterations for addressing fundamental problems in biology, and as a tool in biotechnological applications. PMID:25541598

The partitioning and copy number control systems of the selfish yeast plasmid: an optimized molecular design for stable persistence in host cells.

PubMed

Yen-Ting-Liu; Sau, Saumitra; Ma, Chien-Hui; Kachroo, Aashiq H; Rowley, Paul A; Chang, Keng-Ming; Fan, Hsiu-Fang; Jayaram, Makkuni

2014-10-01

The multi-copy 2 micron plasmid of Saccharomyces cerevisiae, a resident of the nucleus, is remarkable for its high chromosome-like stability. The plasmid does not appear to contribute to the fitness of the host, nor does it impose a significant metabolic burden on the host at its steady state copy number. The plasmid may be viewed as a highly optimized selfish DNA element whose genome design is devoted entirely towards efficient replication, equal segregation and copy number maintenance. A partitioning system comprised of two plasmid coded proteins, Rep1 and Rep2, and a partitioning locus STB is responsible for equal or nearly equal segregation of plasmid molecules to mother and daughter cells. Current evidence supports a model in which the Rep-STB system promotes the physical association of the plasmid with chromosomes and thus plasmid segregation by a hitchhiking mechanism. The Flp site-specific recombination system housed by the plasmid plays a critical role in maintaining steady state plasmid copy number. A decrease in plasmid population due to rare missegregation events is rectified by plasmid amplification via a recombination induced rolling circle replication mechanism. Appropriate plasmid amplification, without runaway increase in copy number, is ensured by positive and negative regulation of FLP gene expression by plasmid coded proteins and by the control of Flp level/activity through host mediated post-translational modification(s) of Flp. The Flp system has been successfully utilized to understand mechanisms of site-specific recombination, to bring about directed genetic alterations for addressing fundamental problems in biology, and as a tool in biotechnological applications.

Detection and Characterization of Conjugative Degradative Plasmids in Xenobiotic-Degrading Sphingomonas Strains

PubMed Central

Basta, Tamara; Keck, Andreas; Klein, Joachim; Stolz, Andreas

2004-01-01

A systematic survey for the presence of plasmids in 17 different xenobiotic-degrading Sphingomonas strains was performed. In almost all analyzed strains, two to five plasmids with sizes of about 50 to 500 kb were detected by using pulsed-field gel electrophoresis. A comparison of plasmid preparations untreated or treated with S1 nuclease suggested that, in general, Sphingomonas plasmids are circular. Hybridization experiments with labeled gene probes suggested that large plasmids are involved in the degradation of dibenzo-p-dioxin, dibenzofuran, and naphthalenesulfonates in S. wittichii RW1, Sphingomonas sp. HH69, and S. xenophaga BN6, respectively. The plasmids which are responsible for the degradation of naphthalene, biphenyl, and toluene by S. aromaticivorans F199 (pNL1) and of naphthalenesulfonates by S. xenophaga BN6 (pBN6) were site-specifically labeled with a kanamycin resistance cassette. The conjugative transfer of these labeled plasmids was attempted with various bacterial strains as putative recipient strains. Thus, a conjugative transfer of plasmid pBN6 from S. xenophaga BN6 to a cured mutant of strain BN6 and to Sphingomonas sp. SS3 was observed. The conjugation experiments with plasmid pNL1 suggested a broader host range of this plasmid, because it was transferred without any obvious structural changes to S. yanoikuyae B1, Sphingomonas sp. SS3, and S. herbicidovorans. In contrast, major plasmid rearrangements were observed in the transconjugants after the transfer of plasmid pNL1 to Sphingomonas sp. HH69 and of pBN6 to Sphingomonas sp. SS3. No indications for the transfer of a Sphingomonas plasmid to bacteria outside of the Sphingomonadaceae were obtained. PMID:15175300

Comparative symbiotic plasmid analysis indicates that symbiosis gene ancestor type affects plasmid genetic evolution.

PubMed

Wang, X; Zhao, L; Zhang, L; Wu, Y; Chou, M; Wei, G

2018-07-01

Rhizobial symbiotic plasmids play vital roles in mutualistic symbiosis with legume plants by executing the functions of nodulation and nitrogen fixation. To explore the gene composition and genetic constitution of rhizobial symbiotic plasmids, comparison analyses of 24 rhizobial symbiotic plasmids derived from four rhizobial genera was carried out. Results illustrated that rhizobial symbiotic plasmids had higher proportion of functional genes participating in amino acid transport and metabolism, replication; recombination and repair; carbohydrate transport and metabolism; energy production and conversion and transcription. Mesorhizobium amorphae CCNWGS0123 symbiotic plasmid - pM0123d had similar gene composition with pR899b and pSNGR234a. All symbiotic plasmids shared 13 orthologous genes, including five nod and eight nif/fix genes which participate in the rhizobia-legume symbiosis process. These plasmids contained nod genes from four ancestors and fix genes from six ancestors. The ancestral type of pM0123d nod genes was similar with that of Rhizobium etli plasmids, while the ancestral type of pM0123d fix genes was same as that of pM7653Rb. The phylogenetic trees constructed based on nodCIJ and fixABC displayed different topological structures mainly due to nodCIJ and fixABC ancestral type discordance. The study presents valuable insights into mosaic structures and the evolution of rhizobial symbiotic plasmids. This study compared 24 rhizobial symbiotic plasmids that included four genera and 11 species, illuminating the functional gene composition and symbiosis gene ancestor types of symbiotic plasmids from higher taxonomy. It provides valuable insights into mosaic structures and the evolution of symbiotic plasmids. © 2018 The Society for Applied Microbiology.

Plasmid Replicon Typing of Commensal and Pathogenic Escherichia coli Isolates▿

PubMed Central

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

2007-01-01

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

Cloning and Expression of a Ralstonia eutropha HF39 Gene Mediating Indigo Formation in Escherichia coli

PubMed Central

Drewlo, Sascha; Brämer, Christian O.; Madkour, Mohamed; Mayer, Frank; Steinbüchel, Alexander

2001-01-01

On complex medium Escherichia coli strains carrying hybrid plasmid pBEC/EE:11.0, pSKBEC/BE:9.0, pSKBEC/PP:3.3, or pSKBEC/PP:2.4 harboring genomic DNA of Ralstonia eutropha HF39 produced a blue pigment characterized as indigo by several chemical and spectroscopic methods. A 1,251-bp open reading frame (bec) was cloned and sequenced. The deduced amino acid sequence of bec showed only weak similarities to short-chain acyl-coenzyme A dehydrogenases, and the gene product catalyzed formation of indoxyl, a reactive preliminary stage for production of indigo. PMID:11282658

Kid cleaves specific mRNAs at UUACU sites to rescue the copy number of plasmid R1

PubMed Central

Pimentel, Belén; Madine, Mark A; de la Cueva-Méndez, Guillermo

2005-01-01

Stability and copy number of extra-chromosomal elements are tightly regulated in prokaryotes and eukaryotes. Toxin Kid and antitoxin Kis are the components of the parD stability system of prokaryotic plasmid R1 and they can also function in eukaryotes. In bacteria, Kid was thought to become active only in cells that lose plasmid R1 and to cleave exclusively host mRNAs at UA(A/C/U) trinucleotide sites to eliminate plasmid-free cells. Instead, we demonstrate here that Kid becomes active in plasmid-containing cells when plasmid copy number decreases, cleaving not only host- but also a specific plasmid-encoded mRNA at the longer and more specific target sequence UUACU. This specific cleavage by Kid inhibits bacterial growth and, at the same time, helps to restore the plasmid copy number. Kid targets a plasmid RNA that encodes a repressor of the synthesis of an R1 replication protein, resulting in increased plasmid DNA replication. This mechanism resembles that employed by some human herpesviruses to regulate viral amplification during infection. PMID:16163387

Facile Recovery of Individual High-Molecular-Weight, Low-Copy-Number Natural Plasmids for Genomic Sequencing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Williams, L.E.; Detter, C,; Barrie, K.

2006-06-01

Sequencing of the large (>50 kb), low-copy-number (<5 per cell) plasmids that mediate horizontal gene transfer has been hindered by the difficulty and expense of isolating DNA from individual plasmids of this class. We report here that a kit method previously devised for purification of bacterial artificial chromosomes (BACs) can be adapted for effective preparation of individual plasmids up to 220 kb from wild gram-negative and gram-positive bacteria. Individual plasmid DNA recovered from less than 10 ml of Escherichia coli, Staphylococcus, and Corynebacterium cultures was of sufficient quantity and quality for construction of highcoverage libraries, as shown by sequencing fivemore » native plasmids ranging in size from 30 kb to 94 kb. We also report recommendations for vector screening to optimize plasmid sequence assembly, preliminary annotation of novel plasmid genomes, and insights on mobile genetic element biology derived from these sequences. Adaptation of this BAC method for large plasmid isolation removes one major technical hurdle to expanding our knowledge of the natural plasmid gene pool.« less

A selfish DNA element engages a meiosis-specific motor and telomeres for germ-line propagation.

PubMed

Sau, Soumitra; Conrad, Michael N; Lee, Chih-Ying; Kaback, David B; Dresser, Michael E; Jayaram, Makkuni

2014-06-09

The chromosome-like mitotic stability of the yeast 2 micron plasmid is conferred by the plasmid proteins Rep1-Rep2 and the cis-acting locus STB, likely by promoting plasmid-chromosome association and segregation by hitchhiking. Our analysis reveals that stable plasmid segregation during meiosis requires the bouquet proteins Ndj1 and Csm4. Plasmid relocalization from the nuclear interior in mitotic cells to the periphery at or proximal to telomeres rises from early meiosis to pachytene. Analogous to chromosomes, the plasmid undergoes Csm4- and Ndj1-dependent rapid prophase movements with speeds comparable to those of telomeres. Lack of Ndj1 partially disrupts plasmid-telomere association without affecting plasmid colocalization with the telomere-binding protein Rap1. The plasmid appears to engage a meiosis-specific motor that orchestrates telomere-led chromosome movements for its telomere-associated segregation during meiosis I. This hitherto uncharacterized mode of germ-line transmission by a selfish genetic element signifies a mechanistic variation within the shared theme of chromosome-coupled plasmid segregation during mitosis and meiosis. © 2014 Sau et al.

[Mutants of bacterium Azospirillum brasilense Sp245 with Omegon insertion in mmsB or fabG genes of lipid metabolism are defective in motility and flagellation].

PubMed

Kovtunov, E A; Shelud'ko, A V; Chernyshova, M P; Petrova, L P; Katsy, E I

2013-11-01

Bacteria Azospirillum brasilense have mixed flagellation: in addition to the polar flagellum, numerous lateral flagella are formed in their cells on medium with increased density. Flagella determine the active swimming and swarming capacities of azospirilla. Using A. brasilense Sp245 as an example, we showed that the Omegon-Km artificial transposon insertion into the chromosomal gene for 3-hydroxyisobutyrate dehydrogenase (mmsB) was concurrent with the appearance of significant defects in the formation of polar flagella and with the paralysis of lateral flagella. The Sp245 mutant with the Omegon insertion into the plasmid AZOBR_p1-borne gene for 3-oxoacyl-[acyl-carrier protein]-reductase (fabG) showed the complete loss of flagella and the swarming capacity, as well as significant defects in polar flagellar assembly (though some cells are still motile in liquid medium). The viability of the A. brasilense Sp245 mutants with the Omegon insertion into the mmsB or fabG gene was not reduced. No considerable differences in the fatty acid composition of whole cell lipid extracts were found for the A. brasilense Sp245 strain and its mmsB and fabG mutants.

Efficient interrupting skills of amino acid metallointercalators with DNA at physiological pH: Evaluation of biological assays

NASA Astrophysics Data System (ADS)

Raman, Natarajan; Selvaganapathy, Muthusamy; Radhakrishnan, Srinivasan

2014-06-01

The 4-aminoantipyrine derivatives (sbnd NO2, sbnd OCH3) and their mixed-ligand complexes with amino acids have been synthesized and investigated for their binding with CT DNA using UV-visible spectroscopy, cyclic voltammetry, and viscosity measurements under physiological conditions of pH (stomach 4.7; blood 7.4). The results from all techniques i.e. binding constant (Kb), and free energy change (ΔG) were in good agreement and inferred spontaneous compound-DNA complexes formation via intercalation. Among all the compounds 1 and 4 showed comparatively greater binding at pH 7.4 as evident from its greater Kb values. All the complexes exhibit oxidative cleavage of supercoiled (SC) pBR322 plasmid DNA in the presence of H2O2 as an activator. It is remarkable that at 25 μM concentration 1 and 4 completely degrade SC DNA into undetectable minor fragments and thus they act as efficient chemical nucleases. Among the new complexes, complexes 1 and 4 have highest potential against all the microorganisms tested. The results of the above biological experiments also reveal that the choice of different metal ions has little influence on the DNA binding, DNA cleavage and antimicrobial assay.

Plasmid expression and maintenance during long-term starvation-survival of bacteria in well water.

PubMed Central

Caldwell, B A; Ye, C; Griffiths, R P; Moyer, C L; Morita, R Y

1989-01-01

Strains of enteric bacteria and pseudomonads containing plasmid R388::Tnl721 (Tpr, Tcr) or pRO101 (Hgr, Tcr) were starved for over 250 days in sterile well water to evaluate effects of starvation-survival on plasmid expression and maintenance. Viable populations dropped to between approximately 0.1 and 1% of the initial populations. Escherichia coli(pRO101) and Pseudomonas cepacia(pRO101) lost both viability and plasmid expression at a lower rate than strains containing R388::Tnl721. Three patterns of host-plasmid interaction were detected: (i) no apparent loss of plasmid expression, (ii) loss of plasmid expression on initial recovery with subsequent expression upon resuscitation, and (iii) loss of capability to produce functional plasmid resistance. PMID:2782868

Role of the GGDEF protein family in Salmonella cellulose biosynthesis and biofilm formation.

PubMed

García, Begoña; Latasa, Cristina; Solano, Cristina; García-del Portillo, Francisco; Gamazo, Carlos; Lasa, Iñigo

2004-10-01

Salmonella enterica serovar Typhimurium is capable of producing cellulose as the main exopolysaccharide compound of the biofilm matrix. It has been shown for Gluconacetobacter xylinum that cellulose biosynthesis is allosterically regulated by bis-(3',5') cyclic diguanylic acid, whose synthesis/degradation depends on diguanylate cyclase/phosphodiesterase enzymatic activities. A protein domain, named GGDEF, is present in all diguanylate cyclase/phosphodiesterase enzymes that have been studied to date. In this study, we analysed the molecular mechanisms responsible for the failure of Salmonella typhimurium strain SL1344 to form biofilms under different environmental conditions. Using a complementation assay, we were able to identify two genes, which can restore the biofilm defect of SL1344 when expressed from the plasmid pBR328. Based on the observation that one of the genes, STM1987, contains a GGDEF domain, and the other, mlrA, indirectly controls the expression of another GGDEF protein, AdrA, we proceeded on a mutational analysis of the additional GG[DE]EF motif containing proteins of S. typhimurium. Our results demonstrated that MlrA, and thus AdrA, is required for cellulose production and biofilm formation in LB complex medium whereas STM1987 (GGDEF domain containing protein A, gcpA) is critical for biofilm formation in the nutrient-deficient medium, ATM. Insertional inactivation of the other six members of the GGDEF family (gcpB-G) showed that only deletion of yciR (gcpE) affected cellulose production and biofilm formation. However, when provided on plasmid pBR328, most of the members of the GGDEF family showed a strong dominant phenotype able to bypass the need for AdrA and GcpA respectively. Altogether, these results indicate that most GGDEF proteins of S. typhimurium are functionally related, probably by controlling the levels of the same final product (cyclic di-GMP), which include among its regulatory targets the cellulose production and biofilm formation of S. typhimurium.

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

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

PubMed

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

2015-02-01

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

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

PubMed Central

Bergsveinson, Jordyn; Baecker, Nina; Pittet, Vanessa

2014-01-01

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

Origin-of-transfer sequences facilitate mobilisation of non-conjugative antimicrobial-resistance plasmids in Staphylococcus aureus

PubMed Central

O'Brien, Frances G.; Yui Eto, Karina; Murphy, Riley J. T.; Fairhurst, Heather M.; Coombs, Geoffrey W.; Grubb, Warren B.; Ramsay, Joshua P.

2015-01-01

Staphylococcus aureus is a common cause of hospital, community and livestock-associated infections and is increasingly resistant to multiple antimicrobials. A significant proportion of antimicrobial-resistance genes are plasmid-borne, but only a minority of S. aureus plasmids encode proteins required for conjugative transfer or Mob relaxase proteins required for mobilisation. The pWBG749 family of S. aureus conjugative plasmids can facilitate the horizontal transfer of diverse antimicrobial-resistance plasmids that lack Mob genes. Here we reveal that these mobilisable plasmids carry copies of the pWBG749 origin-of-transfer (oriT) sequence and that these oriT sequences facilitate mobilisation by pWBG749. Sequences resembling the pWBG749 oriT were identified on half of all sequenced S. aureus plasmids, including the most prevalent large antimicrobial-resistance/virulence-gene plasmids, pIB485, pMW2 and pUSA300HOUMR. oriT sequences formed five subfamilies with distinct inverted-repeat-2 (IR2) sequences. pWBG749-family plasmids encoding each IR2 were identified and pWBG749 mobilisation was found to be specific for plasmids carrying matching IR2 sequences. Specificity of mobilisation was conferred by a putative ribbon-helix-helix-protein gene smpO. Several plasmids carried 2–3 oriT variants and pWBG749-mediated recombination occurred between distinct oriT sites during mobilisation. These observations suggest this relaxase-in trans mechanism of mobilisation by pWBG749-family plasmids is a common mechanism of plasmid dissemination in S. aureus. PMID:26243776

Construction of Biologically Functional Bacterial Plasmids In Vitro

PubMed Central

Cohen, Stanley N.; Chang, Annie C. Y.; Boyer, Herbert W.; Helling, Robert B.

1973-01-01

The construction of new plasmid DNA species by in vitro joining of restriction endonuclease-generated fragments of separate plasmids is described. Newly constructed plasmids that are inserted into Escherichia coli by transformation are shown to be biologically functional replicons that possess genetic properties and nucleotide base sequences from both of the parent DNA molecules. Functional plasmids can be obtained by reassociation of endonuclease-generated fragments of larger replicons, as well as by joining of plasmid DNA molecules of entirely different origins. Images PMID:4594039

Effect of chromatographic conditions and plasmid DNA size on the dynamic binding capacity of a monolithic support.

PubMed

Bicho, Diana; Sousa, Ângela; Sousa, Fani; Queiroz, João; Tomaz, Cãndida

2014-09-01

DNA therapies are becoming recognized alternatives for the treatment and prevention of severe pathologies. Although most current trials have used plasmids <10 kbp, in the future larger plasmids would be required. The purpose of this work was to study the chromatographic behavior of nongrafted carbonyldiimidazole monolithic disks using plasmids with different sizes under hydrophobic conditions. Thereunto, the purification of several plasmids was performed. Higher size plasmids needed lower ammonium sulfate concentration, due to the greater number of interactions between the plasmids and monolith. The dynamic binding capacity experiments for the different plasmids revealed a lower capacity for bigger plasmids. It was also verified that the increase of salt concentration from 2.5 to 3 M of ammonium sulfate increased the capacity. At the highest salt concentration, a slight improvement in the capacity using lower flow rate was observed, possibly due to compaction of plasmid molecules and its better organization on the monolith channels. Finally, a low pH also had a positive effect on the capacity. So, this monolithic support proved to be appropriate to purify the supercoiled isoform of different plasmids with different sizes, providing a valuable instrument as a purification technique. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Plasmids in Gram negatives: molecular typing of resistance plasmids.

PubMed

Carattoli, Alessandra

2011-12-01

A plasmid is defined as a double stranded, circular DNA molecule capable of autonomous replication. By definition, plasmids do not carry genes essential for the growth of host cells under non-stressed conditions but they have systems which guarantee their autonomous replication also controlling the copy number and ensuring stable inheritance during cell division. Most of the plasmids confer positively selectable phenotypes by the presence of antimicrobial resistance genes. Plasmids evolve as an integral part of the bacterial genome, providing resistance genes that can be easily exchanged among bacteria of different origin and source by conjugation. A multidisciplinary approach is currently applied to study the acquisition and spread of antimicrobial resistance in clinically relevant bacterial pathogens and the established surveillance can be implemented by replicon typing of plasmids. Particular plasmid families are more frequently detected among Enterobacteriaceae and play a major role in the diffusion of specific resistance genes. For instance, IncFII, IncA/C, IncL/M, IncN and IncI1 plasmids carrying extended-spectrum beta-lactamase genes and acquired AmpC genes are currently considered to be "epidemic resistance plasmids", being worldwide detected in Enterobacteriaceae of different origin and sources. The recognition of successful plasmids is an essential first step to design intervention strategies preventing their spread. Copyright © 2011 Elsevier GmbH. All rights reserved.

Complete genome sequence and the expression pattern of plasmids of the model ethanologen Zymomonas mobilis ZM4 and its xylose-utilizing derivatives 8b and 2032.

PubMed

Yang, Shihui; Vera, Jessica M; Grass, Jeff; Savvakis, Giannis; Moskvin, Oleg V; Yang, Yongfu; McIlwain, Sean J; Lyu, Yucai; Zinonos, Irene; Hebert, Alexander S; Coon, Joshua J; Bates, Donna M; Sato, Trey K; Brown, Steven D; Himmel, Michael E; Zhang, Min; Landick, Robert; Pappas, Katherine M; Zhang, Yaoping

2018-01-01

Zymomonas mobilis is a natural ethanologen being developed and deployed as an industrial biofuel producer. To date, eight Z. mobilis strains have been completely sequenced and found to contain 2-8 native plasmids. However, systematic verification of predicted Z. mobilis plasmid genes and their contribution to cell fitness has not been hitherto addressed. Moreover, the precise number and identities of plasmids in Z. mobilis model strain ZM4 have been unclear. The lack of functional information about plasmid genes in ZM4 impedes ongoing studies for this model biofuel-producing strain. In this study, we determined the complete chromosome and plasmid sequences of ZM4 and its engineered xylose-utilizing derivatives 2032 and 8b. Compared to previously published and revised ZM4 chromosome sequences, the ZM4 chromosome sequence reported here contains 65 nucleotide sequence variations as well as a 2400-bp insertion. Four plasmids were identified in all three strains, with 150 plasmid genes predicted in strain ZM4 and 2032, and 153 plasmid genes predicted in strain 8b due to the insertion of heterologous DNA for expanded substrate utilization. Plasmid genes were then annotated using Blast2GO, InterProScan, and systems biology data analyses, and most genes were found to have apparent orthologs in other organisms or identifiable conserved domains. To verify plasmid gene prediction, RNA-Seq was used to map transcripts and also compare relative gene expression under various growth conditions, including anaerobic and aerobic conditions, or growth in different concentrations of biomass hydrolysates. Overall, plasmid genes were more responsive to varying hydrolysate concentrations than to oxygen availability. Additionally, our results indicated that although all plasmids were present in low copy number (about 1-2 per cell), the copy number of some plasmids varied under specific growth conditions or due to heterologous gene insertion. The complete genome of ZM4 and two xylose-utilizing derivatives is reported in this study, with an emphasis on identifying and characterizing plasmid genes. Plasmid gene annotation, validation, expression levels at growth conditions of interest, and contribution to host fitness are reported for the first time.

Comparison of the Deoxyribonucleic Acid Molecular Weights and Homologies of Plasmids Conferring Linked Resistance to Streptomycin and Sulfonamides

PubMed Central

Barth, Peter T.; Grinter, Nigel J.

1974-01-01

Bacterial strains showing linked resistance to streptomycin (Sm) and sulfonamides (Su) were chosen representing a wide taxonomic and geographical range. Their SmSu resistances were transferred to Escherichia coli K-12 and then plasmid deoxyribonucleic acid (DNA) was isolated by ethidium bromide CsCl centrifugation. The plasmid DNA was examined by electron microscopy and analyzed by sedimentation through 5 to 20% neutral sucrose gradients. Plasmid DNA from strains having transmissible SmSu resistance consisted of two or three molecular species, one of which had a molecular mass of about 5.7 Mdal (106 daltons), the others varying between 20 to 60 Mdal. By using transformation or F′ mobilization, we isolated the SmSu-resistance determinant from any fellow resident plasmids in each strain and again isolated the plasmid DNA. Cosedimentation of each of these with a differently labeled reference plasmid DNA (R300B) showed 9 out of 12 of the plasmids to have a molecular mass not significantly different from the reference (5.7 Mdal); two others were 6.3 and 9.2 Mdal, but PB165 consisted of three plasmids of 7.4, 14.7, and 21.4 Mdal. Three separate isolations of the SmSu determinant from PB165 gave the same three plasmids, which we conclude may be monomer, dimer, and trimer, respectively. DNA-DNA hybridizations at 75 C demonstrated 80 to 93% homology between reference R300B DNA and each isolated SmSu plasmid DNA, except for the 9.2-Mdal plasmid which had 45% homology and PB165 which had 35%. All the SmSu plasmids were present as multiple copies (about 10) per chromosome. The conjugative plasmid of R300 (present as 1.3 copies per chromosome) has been shown to have negligible effect on the number of copies of its accompanying SmSu plasmid R300B. We conclude that the SmSu plasmids are closely related and probably have a common evolutionary origin. Images PMID:4616941

Explanatory chapter: how plasmid preparation kits work.

PubMed

Koontz, Laura

2013-01-01

To isolate plasmid DNA from bacteria using a commercial plasmid miniprep kit (if interested, compare this protocol with Isolation of plasmid DNA from bacteria). Copyright © 2013 Elsevier Inc. All rights reserved.

Characterization of bla(CMY)-encoding plasmids among Salmonella isolated in the United States in 2007.

PubMed

Folster, Jason P; Pecic, Gary; McCullough, Andre; Rickert, Regan; Whichard, Jean M

2011-12-01

Salmonella enterica is one of the most common bacterial causes of foodborne illness, and nontyphoidal Salmonella is estimated to cause ∼1.2 million illnesses in the United States each year. Plasmids are mobile genetic elements that play a critical role in the dissemination of antimicrobial resistance determinants. AmpC-type CMY β-lactamases (bla(CMY)) confer resistance to extended-spectrum cephalosporins and β-lactam/β-lactamase inhibitor combinations and are commonly plasmid-encoded. A variety of plasmids have been shown to encode CMY β-lactamases and certain plasmids may be associated with particular Salmonella serotypes or environmental sources. In this study, we characterized bla(CMY) β-lactamase-encoding plasmids among Salmonella isolates. Isolates of Salmonella from specimens collected from humans in 2007 were submitted to the Centers for Disease Control and Prevention National Antimicrobial Resistance Monitoring System laboratory for susceptibility testing. Three percent (65/2161) of Salmonella isolates displayed resistance to ceftriaxone (minimum inhibitory concentration [MIC] ≥4 mg/L) and amoxicillin/clavulanic acid (MIC ≥32 mg/L), a combination associated with the presence of a bla(CMY) mechanism of resistance. Sixty-four (98.5%) isolates were polymerase chain reaction-positive for bla(CMY) genes. Transformation and conjugation studies showed that 95% (61/64) of the bla(CMY) genes were plasmid-encoded. Most of the bla(CMY)-positive isolates were serotype Typhimurium, Newport, Heidelberg, and Agona. Forty-three plasmids were replicon type IncA/C, 15 IncI1, 2 contained multiple replicon loci, and 1 was untypeable. IncI1 plasmids conferred only the bla(CMY)-associated resistance phenotype, whereas IncA/C plasmids conferred additional multi-drug resistance (MDR) phenotypes to drugs such as chloramphenicol, sulfisoxazole, and tetracycline. Most of the IncI1 plasmids (12/15) were sequence type 12 by plasmid multi-locus sequence typing. CMY β-lactamase-encoding plasmids among human isolates of Salmonella in the United States tended to be large MDR IncA/C plasmids or single resistance determinant IncI1 plasmids. In general, IncI1 plasmids were identified among serotypes commonly associated with poultry, whereas IncA/C plasmids were more likely to be identified among cattle/beef-associated serotypes.

Camphor Plasmid-Mediated Chromosomal Transfer in Pseudomonas putida

PubMed Central

Shaham, M.; Chakrabarty, A. M.; Gunsalus, I. C.

1973-01-01

Camphor-utilizing strains of Pseudomonas putida have been shown to carry the genetic information required for camphor degradation on a plasmid. The plasmid-carrying strains can serve as donors of both plasmid-borne and chromosomal genes. As recipients, plasmid-deleted strains are much superior to those carrying the camphor pathway genes. The transfer frequency of chromosomal, but not plasmid-borne, genes is markedly enhanced if the donor cells are irradiated with ultraviolet light followed by 3-h of growth on a rich medium in the dark. Recombinants selected for prototrophy are stable and most acquire the camphor (CAM) plasmid concomitantly; only a few of the Cam+ recombinants inherit the donor's ability to transfer chromosomal genes at a high frequency. Transfer-defective mutations occur on the CAM plasmid, affecting both CAM and chromosomal gene transfer. PMID:4745436

A 2,4-dichlorophenoxyacetic acid degradation plasmid pM7012 discloses distribution of an unclassified megaplasmid group across bacterial species.

PubMed

Sakai, Yoriko; Ogawa, Naoto; Shimomura, Yumi; Fujii, Takeshi

2014-03-01

Analysis of the complete nucleotide sequence of plasmid pM7012 from 2,4-dichlorophenoxyacetic-acid (2,4-D)-degrading bacterium Burkholderia sp. M701 revealed that the plasmid had 582 142 bp, with 541 putative protein-coding sequences and 39 putative tRNA genes for the transport of the standard 20 aa. pM7012 contains sequences homologous to the regions involved in conjugal transfer and plasmid maintenance found in plasmids byi_2p from Burkholderia sp. YI23 and pBVIE01 from Burkholderia sp. G4. No relaxase gene was found in any of these plasmids, although genes for a type IV secretion system and type IV coupling proteins were identified. Plasmids with no relaxase gene have been classified as non-mobile plasmids. However, nucleotide sequences with a high level of similarity to the genes for plasmid transfer, plasmid maintenance, 2,4-D degradation and arsenic resistance contained on pM7012 were also detected in eight other megaplasmids (~600 or 900 kb) found in seven Burkholderia strains and a strain of Cupriavidus, which were isolated as 2,4-D-degrading bacteria in Japan and the United States. These results suggested that the 2,4-D degradation megaplasmids related to pM7012 are mobile and distributed across various bacterial species worldwide, and that the plasmid group could be distinguished from known mobile plasmid groups.

Yersinia enterocolitica-Induced Interleukin-8 Secretion by Human Intestinal Epithelial Cells Depends on Cell Differentiation

PubMed Central

Schulte, Ralf; Autenrieth, Ingo B.

1998-01-01

In response to bacterial entry epithelial cells up-regulate expression and secretion of various proinflammatory cytokines, including interleukin-8 (IL-8). We studied Yersinia enterocolitica O:8-induced IL-8 secretion by intestinal epithelial cells as a function of cell differentiation. For this purpose, human T84 intestinal epithelial cells were grown on permeable supports, which led to the formation of tight monolayers of polarized intestinal epithelial cells. To analyze IL-8 secretion as a function of cell differentiation, T84 monolayers were infected from the apical or basolateral side at different stages of differentiation. Both virulent (plasmid-carrying) and nonvirulent (plasmid-cured) Y. enterocolitica strains invaded nondifferentiated T84 cells from the apical side. Yersinia invasion into T84 cells was followed by secretion of IL-8. After polarized differentiation of T84 cells Y. enterocolitica was no longer able to invade from the apical side or to induce IL-8 secretion by T84 cells. However, Y. enterocolitica invaded and induced IL-8 secretion by polarized T84 cells after infection from the basolateral side. Basolateral invasion required the presence of the Yersinia invasion locus, inv, suggesting β1 integrin-mediated cell invasion. After basolateral infection, Yersinia-induced IL-8 secretion was not strictly dependent on cell invasion. Thus, although the plasmid-carrying Y. enterocolitica strain did not significantly invade T84 cells, it induced significant IL-8 secretion. Taken together, these data show that Yersinia-triggered IL-8 secretion by intestinal epithelial cells depends on cell differentiation and might be induced by invasion as well as by basolateral adhesion, suggesting that invasion is not essential for triggering IL-8 production. Whether IL-8 secretion is involved in the pathogenesis of Yersinia-induced abscess formation in Peyer’s patch tissue remains to be shown. PMID:9488416

Comparison of antibiotic resistance, biofilm formation and conjugative transfer of Staphylococcus and Enterococcus isolates from International Space Station and Antarctic Research Station Concordia.

PubMed

Schiwon, Katarzyna; Arends, Karsten; Rogowski, Katja Marie; Fürch, Svea; Prescha, Katrin; Sakinc, Türkan; Van Houdt, Rob; Werner, Guido; Grohmann, Elisabeth

2013-04-01

The International Space Station (ISS) and the Antarctic Research Station Concordia are confined and isolated habitats in extreme and hostile environments. The human and habitat microflora can alter due to the special environmental conditions resulting in microbial contamination and health risk for the crew. In this study, 29 isolates from the ISS and 55 from the Antarctic Research Station Concordia belonging to the genera Staphylococcus and Enterococcus were investigated. Resistance to one or more antibiotics was detected in 75.8 % of the ISS and in 43.6 % of the Concordia strains. The corresponding resistance genes were identified by polymerase chain reaction in 86 % of the resistant ISS strains and in 18.2 % of the resistant Concordia strains. Plasmids are present in 86.2 % of the ISS and in 78.2 % of the Concordia strains. Eight Enterococcus faecalis strains (ISS) harbor plasmids of about 130 kb. Relaxase and/or transfer genes encoded on plasmids from gram-positive bacteria like pIP501, pRE25, pSK41, pGO1 and pT181 were detected in 86.2 % of the ISS and in 52.7 % of the Concordia strains. Most pSK41-homologous transfer genes were detected in ISS isolates belonging to coagulase-negative staphylococci. We demonstrated through mating experiments that Staphylococcus haemolyticus F2 (ISS) and the Concordia strain Staphylococcus hominis subsp. hominis G2 can transfer resistance genes to E. faecalis and Staphylococcus aureus, respectively. Biofilm formation was observed in 83 % of the ISS and in 92.7 % of the Concordia strains. In conclusion, the ISS isolates were shown to encode more resistance genes and possess a higher gene transfer capacity due to the presence of three vir signature genes, virB1, virB4 and virD4 than the Concordia isolates.

Escherichia coli O157:H7 strains isolated from High-Event Period beef contamination have strong biofilm-forming ability and low sanitizer susceptibility, which are associated with high pO157 plasmid copy number

USDA-ARS?s Scientific Manuscript database

In the meat industry, a “High Event Period” (HEP) is defined as a time period when beef processing establishments experience an increased occurrence of product contamination by E. coli O157:H7. Our previous studies suggested that bacterial biofilm formation and sanitizer resistance might contribute...

Formation of Orthopoxvirus Cytoplasmic A-Type Inclusion Bodies and Embedding of Virions Are Dynamic Processes Requiring Microtubules

PubMed Central

Howard, Amanda R.

2012-01-01

In cells infected with some orthopoxviruses, numerous mature virions (MVs) become embedded within large, cytoplasmic A-type inclusions (ATIs) that can protect infectivity after cell lysis. ATIs are composed of an abundant viral protein called ATIp, which is truncated in orthopoxviruses such as vaccinia virus (VACV) that do not form ATIs. To study ATI formation and occlusion of MVs within ATIs, we used recombinant VACVs that express the cowpox full-length ATIp or we transfected plasmids encoding ATIp into cells infected with VACV, enabling ATI formation. ATI enlargement and MV embedment required continued protein synthesis and an intact microtubular network. For live imaging of ATIs and MVs, plasmids expressing mCherry fluorescent protein fused to ATIp were transfected into cells infected with VACV expressing the viral core protein A4 fused to yellow fluorescent protein. ATIs appeared as dynamic, mobile bodies that enlarged by multiple coalescence events, which could be prevented by disrupting microtubules. Coalescence of ATIs was confirmed in cells infected with cowpox virus. MVs were predominantly at the periphery of ATIs early in infection. We determined that coalescence contributed to the distribution of MVs within ATIs and that microtubule-disrupting drugs abrogated coalescence-mediated MV embedment. In addition, MVs were shown to move from viral factories at speeds consistent with microtubular transport to the peripheries of ATIs, whereas disruption of microtubules prevented such trafficking. The data indicate an important role for microtubules in the coalescence of ATIs into larger structures, transport of MVs to ATIs, and embedment of MVs within the ATI matrix. PMID:22438543

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

USDA-ARS?s Scientific Manuscript database

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

[The plasmid profile of Neisseria meningitidis strains].

PubMed

Khetsuriani, K G; Namgaladze, M Z; Lomsadze, Kh V; Kakuberi, D R

1993-01-01

The distribution of plasmids in N. meningitidis strains according to their origin and serological groups has been studied. Plasmids have been discovered in N. meningitidis of all groups, plasmid-carrying strains constituting 55% of strains isolated from healthy carriers and 46.2% of strains isolated from patients. The molecular weight of N. meningitidis plasmid DNA varies from 2.9 MD to 95 MD.

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. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

PlasFlow: predicting plasmid sequences in metagenomic data using genome signatures

PubMed Central

Lipinski, Leszek; Dziembowski, Andrzej

2018-01-01

Abstract Plasmids are mobile genetics elements that play an important role in the environmental adaptation of microorganisms. Although plasmids are usually analyzed in cultured microorganisms, there is a need for methods that allow for the analysis of pools of plasmids (plasmidomes) in environmental samples. To that end, several molecular biology and bioinformatics methods have been developed; however, they are limited to environments with low diversity and cannot recover large plasmids. Here, we present PlasFlow, a novel tool based on genomic signatures that employs a neural network approach for identification of bacterial plasmid sequences in environmental samples. PlasFlow can recover plasmid sequences from assembled metagenomes without any prior knowledge of the taxonomical or functional composition of samples with an accuracy up to 96%. It can also recover sequences of both circular and linear plasmids and can perform initial taxonomical classification of sequences. Compared to other currently available tools, PlasFlow demonstrated significantly better performance on test datasets. Analysis of two samples from heavy metal-contaminated microbial mats revealed that plasmids may constitute an important fraction of their metagenomes and carry genes involved in heavy-metal homeostasis, proving the pivotal role of plasmids in microorganism adaptation to environmental conditions. PMID:29346586

Nucleotide sequence of the Varkud mitochondrial plasmid of Neurospora and synthesis of a hybrid transcript with a 5' leader derived from mitochondrial RNA.

PubMed

Akins, R A; Grant, D M; Stohl, L L; Bottorff, D A; Nargang, F E; Lambowitz, A M

1988-11-05

The Mauriceville and Varkud mitochondrial plasmids of Neurospora are closely related, closed circular DNAs (3.6 and 3.7 kb, respectively; 1 kb = 10(3) bases or base-pairs), whose characteristics suggest relationships to mitochondrial DNA introns and retrotransposons. Here, we characterized the structure of the Varkud plasmid, determined its complete nucleotide sequence and mapped its major transcripts. The Mauriceville and Varkud plasmids have more than 97% positional identity. Both plasmids contain a 710 amino acid open reading frame that encodes a reverse transcriptase-like protein. The amino acid sequence of this open reading frame is strongly conserved between the two plasmids (701/710 amino acids) as expected for a functionally important protein. Both plasmids have a 0.4 kb region that contains five PstI palindromes and a direct repeat of approximately 160 base-pairs. Comparison of sequences in this region suggests that the Varkud plasmid has diverged less from a common ancestor than has the Mauriceville plasmid. Two major transcripts of the Varkud plasmid were detected by Northern hybridization experiments: a full-length linear RNA of 3.7 kb and an additional prominent transcript of 4.9 kb, 1.2 kb longer than monomer plasmid. Remarkably, we find that the 4.9 kb transcript is a hybrid RNA consisting of the full-length 3.7 kb Varkud plasmid transcript plus a 5' leader of 1.2 kb that is derived from the 5' end of the mitochondrial small rRNA. This and other findings suggest that the Varkud plasmid, like certain RNA viruses, has a mechanism for joining heterologous RNAs to the 5' end of its major transcript, and that, under some circumstances, nucleotide sequences in mitochondria may be recombined at the RNA level.

The Use of a Combined Bioinformatics Approach to Locate Antibiotic Resistance Genes on Plasmids From Whole Genome Sequences of Salmonella enterica Serovars From Humans in Ghana.

PubMed

Kudirkiene, Egle; Andoh, Linda A; Ahmed, Shahana; Herrero-Fresno, Ana; Dalsgaard, Anders; Obiri-Danso, Kwasi; Olsen, John E

2018-01-01

In the current study, we identified plasmids carrying antimicrobial resistance genes in draft whole genome sequences of 16 selected Salmonella enterica isolates representing six different serovars from humans in Ghana. The plasmids and the location of resistance genes in the genomes were predicted using a combination of PlasmidFinder, ResFinder, plasmidSPAdes and BLAST genomic analysis tools. Subsequently, S1-PFGE was employed for analysis of plasmid profiles. Whole genome sequencing confirmed the presence of antimicrobial resistance genes in Salmonella isolates showing multidrug resistance phenotypically. ESBL, either bla TEM52-B or bla CTX-M15 were present in two cephalosporin resistant isolates of S . Virchow and S . Poona, respectively. The systematic genome analysis revealed the presence of different plasmids in different serovars, with or without insertion of antimicrobial resistance genes. In S . Enteritidis, resistance genes were carried predominantly on plasmids of IncN type, in S . Typhimurium on plasmids of IncFII(S)/IncFIB(S)/IncQ1 type. In S . Virchow and in S . Poona, resistance genes were detected on plasmids of IncX1 and TrfA/IncHI2/IncHI2A type, respectively. The latter two plasmids were described for the first time in these serovars. The combination of genomic analytical tools allowed nearly full mapping of the resistance plasmids in all Salmonella strains analyzed. The results suggest that the improved analytical approach used in the current study may be used to identify plasmids that are specifically associated with resistance phenotypes in whole genome sequences. Such knowledge would allow the development of rapid multidrug resistance tracking tools in Salmonella populations using WGS.

Isolation, Characterization, and Transfer of Cryptic Gene-Mobilizing Plasmids in the Wheat Rhizosphere

PubMed Central

van Elsas, Jan Dirk; McSpadden Gardener, Brian B.; Wolters, Anneke C.; Smit, Eric

1998-01-01

A set of self-transmissible plasmids with IncQ plasmid-mobilizing capacity was isolated by triparental exogenous isolation from the wheat rhizosphere with an Escherichia coli IncQ plasmid host and a Ralstonia eutropha recipient. Three plasmids of 38 to 45 kb, denoted pIPO1, pIPO2, and pIPO3, were selected for further study. No selectable traits (antibiotic or heavy-metal resistance) were identified in these plasmids. The plasmids were characterized by replicon typing via PCR and hybridization with replicon-specific probes and other hybridizations. pIPO1 and pIPO3 were similar to each other, whereas pIPO2 was different. None of these plasmids belonged to any known incompatibility group. pIPO2 was selected for further work, and a mini-Tn5-tet transposon was inserted to confer selectability. Plasmid pIPO2 had a broad IncQ plasmid mobilization and self-transfer range among the alpha, beta, and gamma subclasses of the Proteobacteria but did not show productive transfer to gram-positive bacteria. Plasmid pIPO2 mobilized IncQ plasmid pIE723 from Pseudomonas fluorescens to diverse indigenous proteobacteria in the rhizosphere of field-grown wheat. Transfer of pIE723 to indigenous bacteria was not observed in the absence of added pIPO2. A specific PCR primer system and a probe were developed for the detection of pIPO2-type plasmids in soil and rhizosphere. Analysis of soil DNA provided evidence for the presence of pIPO2 in inoculated wheat rhizosphere soil in the field study, as well as in the rhizosphere of uninoculated wheat plants growing in soil microcosms. The system failed to identify major reservoirs of pIPO2 in a variety of other soils. PMID:9501428

Characterization of a Large Antibiotic Resistance Plasmid Found in Enteropathogenic Escherichia coli Strain B171 and Its Relatedness to Plasmids of Diverse E. coli and Shigella Strains.

PubMed

Hazen, Tracy H; Michalski, Jane; Nagaraj, Sushma; Okeke, Iruka N; Rasko, David A

2017-09-01

Enteropathogenic Escherichia coli (EPEC) is a leading cause of severe infantile diarrhea in developing countries. Previous research has focused on the diversity of the EPEC virulence plasmid, whereas less is known regarding the genetic content and distribution of antibiotic resistance plasmids carried by EPEC. A previous study demonstrated that in addition to the virulence plasmid, reference EPEC strain B171 harbors a second, larger plasmid that confers antibiotic resistance. To further understand the genetic diversity and dissemination of antibiotic resistance plasmids among EPEC strains, we describe the complete sequence of an antibiotic resistance plasmid from EPEC strain B171. The resistance plasmid, pB171_90, has a completed sequence length of 90,229 bp, a GC content of 54.55%, and carries protein-encoding genes involved in conjugative transfer, resistance to tetracycline ( tetA ), sulfonamides ( sulI ), and mercury, as well as several virulence-associated genes, including the transcriptional regulator hha and the putative calcium sequestration inhibitor ( csi ). In silico detection of the pB171_90 genes among 4,798 publicly available E. coli genome assemblies indicates that the unique genes of pB171_90 ( csi and traI ) are primarily restricted to genomes identified as EPEC or enterotoxigenic E. coli However, conserved regions of the pB171_90 plasmid containing genes involved in replication, stability, and antibiotic resistance were identified among diverse E. coli pathotypes. Interestingly, pB171_90 also exhibited significant similarity with a sequenced plasmid from Shigella dysenteriae type I. Our findings demonstrate the mosaic nature of EPEC antibiotic resistance plasmids and highlight the need for additional sequence-based characterization of antibiotic resistance plasmids harbored by pathogenic E. coli . Copyright © 2017 American Society for Microbiology.

Prolonging the expression duration of ultrasound-mediated gene transfection using PEI nanoparticles.

PubMed

Lee, Jyun-Lin; Lo, Chia-Wen; Ka, Shuk-Man; Chen, Ann; Chen, Wen-Shiang

2012-05-30

Ultrasound (US) irradiation has been found to facilitate the inward transport of genetic materials across cell membranes (sonoporation). However, its transfection efficiency is generally low, and the expression duration of transfected gene is short. Polyethylenimine (PEI), a cationic polymer, has been shown to aggregate plasmid DNA and facilitate its internalization. The purpose of this study is to determine whether PEI can also prolong the expression duration after US-mediated transfection. A mixture of pCMViLUC and 22-kDa linear PEI was transfected to cultured cells or mouse muscle by exposure to 1-MHz pulsed US. The duration of expression was assessed periodically following US treatment. As expected, strong expression of luciferase could be found 30days after the treatment of DNA-PEI complex with US exposure, both in vitro and in vivo. However, without US, only very low transfection level could be obtained in vivo. The DNA/PEI complex showed protective effect against digestion of DNase I enzymes as compared with groups without PEI or to which PEI was added following the mixing of plasmid DNA with DNase I. PEI enhanced the US transfection efficiency by increasing both the intracellular uptake of plasmid DNA and the percentage of transfected cells. Most of the DNA uptake occurred at 3h after US exposure, suggesting that endocytosis took place. Moreover, the PEI-facilitated US gene transfection depended on the ratio of PEI and DNA (N/P ratio), which was different for in-vitro and in-vivo conditions. This system could be applied in future human gene therapies. Copyright © 2012 Elsevier B.V. All rights reserved.

Monitoring the colonization of sugarcane and rice plants by the endophytic diazotrophic bacterium Gluconacetobacter diazotrophicus marked with gfp and gusA reporter genes.

PubMed

Rouws, L F M; Meneses, C H S G; Guedes, H V; Vidal, M S; Baldani, J I; Schwab, S

2010-09-01

To evaluate the colonization process of sugarcane plantlets and hydroponically grown rice seedlings by Gluconacetobacter diazotrophicus strain PAL5 marked with the gusA and gfp reporter genes. Sugarcane plantlets inoculated in vitro with PAL5 carrying the gfp::gusA plasmid pHRGFPGUS did not present green fluorescence, but beta-glucuronidase (GUS)-stained bacteria could be observed inside sugarcane roots. To complement this existing inoculation methodology for micropropagated sugarcane with a more rapid colonization assay, we employed hydroponically grown gnotobiotic rice seedlings to study PAL5-plant interaction. PAL5 could be isolated from the root surface (10(8) CFU g(-1)) and from surface-disinfected root and stem tissues (10(4) CFU g(-1)) of inoculated plants, suggesting that PAL5 colonized the internal plant tissues. Light microscopy confirmed the presence of bacteria inside the root tissue. After inoculation of rice plantlets with PAL5 marked with the gfp plasmid pHRGFPTC, bright green fluorescent bacteria could be seen colonizing the rice root surface, mainly at the sites of lateral root emergence, at root caps and on root hairs. The plasmids pHRGFPGUS and pHRGFPTC are valid tools to mark PAL5 and monitor the colonization of micropropagated sugarcane and hydroponic rice seedlings. These tools are of use to: (i) study PAL5 mutants affected in bacteria-plant interactions, (ii) monitor plant colonization in real time and (iii) distinguish PAL5 from other bacteria during the study of mixed inoculants.

Mu-driven transposition of recombinant mini-Mu unit DNA in the Corynebacterium glutamicum chromosome.

PubMed

Gorshkova, Natalya V; Lobanova, Juliya S; Tokmakova, Irina L; Smirnov, Sergey V; Akhverdyan, Valerii Z; Krylov, Alexander A; Mashko, Sergey V

2018-03-01

A dual-component Mu-transposition system was modified for the integration/amplification of genes in Corynebacterium. The system consists of two types of plasmids: (i) a non-replicative integrative plasmid that contains the transposing mini-Mu(LR) unit bracketed by the L/R Mu ends or the mini-Mu(LER) unit, which additionally contains the enhancer element, E, and (ii) an integration helper plasmid that expresses the transposition factor genes for MuA and MuB. Efficient transposition in the C. glutamicum chromosome (≈ 2 × 10 -4 per cell) occurred mainly through the replicative pathway via cointegrate formation followed by possible resolution. Optimizing the E location in the mini-Mu unit significantly increased the efficiency of Mu-driven intramolecular transposition-amplification in C. glutamicum as well as in gram-negative bacteria. The new C. glutamicum genome modification strategy that was developed allows the consequent independent integration/amplification/fixation of target genes at high copy numbers. After integration/amplification of the first mini-Mu(LER) unit in the C. glutamicum chromosome, the E-element, which is bracketed by lox-like sites, is excised by Cre-mediated fashion, thereby fixing the truncated mini-Mu(LR) unit in its position for the subsequent integration/amplification of new mini-Mu(LER) units. This strategy was demonstrated using the genes for the citrine and green fluorescent proteins, yECitrine and yEGFP, respectively.

Use of lacticin 481 to facilitate delivery of the bacteriophage resistance plasmid, pCBG104 to cheese starters.

PubMed

Mills, S; Coffey, A; O'Sullivan, L; Stokes, D; Hill, C; Fitzgerald, G F; Ross, R P

2002-01-01

Use of lacticin 481 to facilitate the conjugal transfer of the bacteriophage resistance plasmid pCBG104 to various starter cultures. A raw milk isolate of Lactococcus was found to harbour determinants for lacticin 481 production and immunity and phage resistance on a plasmid designated pCBG104. The lacticin 481 was successfully used to mobilize the phage resistance determinant to a variety of cheese starters enabling the formation of highly phage resistant starters. In addition, it facilitated the stacking of a number of phage resistance genes, namely a type I restriction modification system, a phage abortive infection system and a phage adsorption blocking system in a single Lactococcus strain without the use of recombinant techniques. The transconjugants were all shown to produce lacticin 481 and to contain the entire 481 operon. Subsequently one transconjugant was selected and successfully used for large-scale cheddar cheese manufacture. Lacticin 481 could be used as a food-grade selectable marker to facilitate the introduction of advantageous traits to starter cultures for industrial food fermentations. Food-grade selectable markers greatly facilitate the introduction of various advantageous traits to starter cultures for industrial food fermentation. Indeed self-cloning which is becoming increasingly important for strain improvement has a requirement for the identification and demonstration of the utility of tools such as lacticin 481.

Design of an Escherichia coli system for whole cell mediated steroid synthesis and molecular evolution of steroid hydroxylases.

PubMed

Hannemann, Frank; Virus, Cornelia; Bernhardt, Rita

2006-06-25

The 15beta-hydroxylase (CYP106A2) from Bacillus megaterium, one of the few bacterial steroid hydroxylases, which has been isolated and characterized so far, catalyses the 15beta-hydroxylation of a variety of steroids. The enzyme can be supported in its activity with adrenodoxin (Adx) and adrenodoxin reductase (AdR) from bovine adrenals, supplying this enzyme with the reducing equivalents necessary for steroid hydroxylation activity. This three-component electron transfer chain was implemented in Escherichia coli by coexpression of the corresponding coding sequences from two plasmids, containing different selection markers and compatible origins of replication. The cDNAs of AdR and Adx on the first plasmid were separated by a ribosome binding sequence, with the reductase preceding the ferredoxin. The second plasmid for CYP106A2 expression was constructed with all features necessary for a molecular evolution approach. The transformed bacteria show the inducible ability to efficiently convert 11-deoxycorticosterone (DOC) to 15beta-DOC at an average rate of 1 mM/d in culture volumes of 300 ml. The steroid conversion system was downscaled to the microtiter plate format and a robot set-up was developed for a fluorescence-based conversion assay as well as a CO difference spectroscopy assay, which enables the screening for enzyme variants with higher activity and stability.

[Effects of ezrin silencing on pancreatic cancer cell line Panc-1].

PubMed

Meng, Yun-xiao; Yu, Shuang-ni; Lu, Zhao-hui; Chen, Jie

2012-12-01

To explore the effects of ezrin silencing on pancreatic cancer cell line Panc-1. Pancreatic cancer cell line Panc-1 was transfected with ezrin silencing plasmid. The proliferation and the cell cycle status were determined by CCK-8 assay and flow cytometry analysis, respectively. Cellular membrane protrusions/microvilli formation were visualized by scanning election microscopy. Colony formation assay was used to determine the cell anchor-independent growth ability in vitro. Trans-filter migration and invasion assays were performed with 8 µm pore inserts in a 24-well BioCoat chamber with/without Matrigel. Ezrin silencing decreased cellular protrusions/microvilli formation, anchorage-independent growth, cell migration and invasion, but had no effects on cell proliferation in vitro and cell cycle, in pancreatic cancer cell line Panc-1. Ezrin expression affects the cellular protrusions/microvilli formation, anchorage-independent growth, cell migration and invasion in pancreatic cancer cell line Panc-1.

pSK41-Like Plasmid Is Necessary for Inc18-Like vanA Plasmid Transfer from Enterococcus faecalis to Staphylococcus aureus In Vitro

PubMed Central

Clark, Nancye; Patel, Jean B.

2013-01-01

Vancomycin-resistant Staphylococcus aureus (VRSA) is thought to result from the in vivo conjugative transfer of a vanA plasmid from an Enterococcus sp. to S. aureus. We studied bacterial isolates from VRSA cases that occurred in the United States to identify microbiological factors which may contribute to this plasmid transfer. First, vancomycin-susceptible, methicillin-resistant S. aureus (MRSA) isolates from five VRSA cases were tested for their ability to accept foreign DNA by conjugation in mating experiments with Enterococcus faecalis JH2-2 containing pAM378, a pheromone-response conjugative plasmid. All of the MRSA isolates accepted the plasmid DNA with similar transfer efficiencies (∼10−7/donor CFU) except for one isolate, MRSA8, for which conjugation was not successful. The MRSA isolates were also tested as recipients in mating experiments between an E. faecalis isolate with an Inc18-like vanA plasmid that was isolated from a VRSA case patient. Conjugative transfer was successful for 3/5 MRSA isolates. Successful MRSA recipients carried a pSK41-like plasmid, a staphylococcal conjugative plasmid, whereas the two unsuccessful MRSA recipients did not carry pSK41. The transfer of a pSK41-like plasmid from a successful MRSA recipient to the two unsuccessful recipients resulted in conjugal transfer of the Inc18-like vanA plasmid from E. faecalis at a frequency of 10−7/recipient CFU. In addition, conjugal transfer could be achieved for pSK41-negative MRSA in the presence of a cell-free culture filtrate from S. aureus carrying a pSK41-like plasmid at a frequency of 10−8/recipient CFU. These results indicated that a pSK41-like plasmid can facilitate the transfer of an Inc18-like vanA plasmid from E. faecalis to S. aureus, possibly via an extracellular factor produced by pSK41-carrying isolates. PMID:23089754

Plasmid dynamics in Vibrio parahaemolyticus strains related to shrimp Acute Hepatopancreatic Necrosis Syndrome (AHPNS).

PubMed

Theethakaew, Chonchanok; Nakamura, Shota; Motooka, Daisuke; Matsuda, Shigeaki; Kodama, Toshio; Chonsin, Kaknokrat; Suthienkul, Orasa; Iida, Tetsuya

2017-07-01

Vibrio parahaemolyticus is a causative agent of acute hapatopancreatic necrosis syndrome (AHPNS) which causes early mortality in white shrimp. Emergence of AHPNS has caused tremendous economic loss for aquaculture industry particularly in Asia since 2010. Previous studies reported that strains causing AHPNS harbor a 69-kb plasmid with possession of virulence genes, pirA and pirB. However, genetic variation of the 69-kb plasmid among AHPNS related strains has not been investigated. This study aimed to analyze genetic composition and diversity of the 69-kb plasmid in strains isolated from shrimps affected by AHPNS. Plasmids recovered from V. parahaemolyticus strain VPE61 which represented typical AHPNS pathogenicity, strain VP2HP which did not represent AHPNS pathogenicity but was isolated from AHPNS affected shrimp and other AHPNS V. parahaemolyticus isolates in Genbank were investigated. Protein coding genes of the 69-kb plasmid from the strain VPE61 were identical to that of AHPNS strain from Vietnam except the inverted complement 3.4-kb transposon covering pirA and pirB. The strain VP2HP possessed remarkable large 183-kb plasmid which shared similar protein coding genes to those of the 69-kb plasmid from strain VPE61. However, the 3.4-kb transposon covering pirA and pirB was absent from the 183-kb plasmid in strain VP2HP. A number of protein coding genes from the 183-kb plasmid were also detected in other AHPNS strains. In summary, this study identified a novel 183-kb plasmid that is related to AHPNS causing strains. Homologous recombination of the 69-kb AHPNS plasmid and other naturally occurring plasmids together with loss and gain of AHPNS virulence genes in V. parahaemolyticus were observed. The outcome of this research enables understanding of plasmid dynamics that possibly affect variable degrees of AHPNS pathogenicity. Copyright © 2017 Elsevier B.V. All rights reserved.

Complete genetic analysis of plasmids carrying mcr-1 and other resistance genes in an Escherichia coli isolate of animal origin.

PubMed

Li, Ruichao; Xie, Miaomiao; Lv, Jingzhang; Wai-Chi Chan, Edward; Chen, Sheng

2017-03-01

To investigate the genetic features of three plasmids recovered from an MCR-1 and ESBL-producing Escherichia coli strain, HYEC7, and characterize the transmission mechanism of mcr-1 . The genetic profiles of three plasmids were determined by PCR, S1-PFGE, Southern hybridization and WGS analysis. The ability of the mcr-1 -bearing plasmid to undergo conjugation was also assessed. The mcr-1 -bearing transposon Tn 6330 was characterized by PCR and DNA sequencing. Complete sequences of three plasmids were obtained. A non-conjugative phage P7-like plasmid, pHYEC7- mcr1 , was found to harbour the mcr-1 -bearing transposon Tn 6330 , which could be excised from the plasmid by generating a circular intermediate harbouring mcr-1 and the IS Apl1 element. The insertion of the circular intermediate into another plasmid, pHYEC7-IncHI2, could form pHNSHP45-2, the original IncHI2-type mcr-1 -carrying plasmid that was reported. The third plasmid, pHYEC7-110, harboured two replicons, IncX1 and IncFIB, and comprised multiple antimicrobial resistance mobile elements, some of which were shared by pHYEC7-IncHI2. The Tn 6330 element located in the phage-like plasmid pHYEC7- mcr1 could be excised from the plasmid and formed a circular intermediate that could be integrated into plasmids containing the IS Apl1 element. This phenomenon indicated that Tn 6330 is a key element responsible for widespread dissemination of mcr-1 among various types of plasmids and bacterial chromosomes. The dissemination rate of such an element may be further enhanced upon translocation into phage-like vectors, which may also be transmitted via transduction events. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Development of a high-copy plasmid for enhanced production of recombinant proteins in Leuconostoc citreum.

PubMed

Son, Yeon Jeong; Ryu, Ae Jin; Li, Ling; Han, Nam Soo; Jeong, Ki Jun

2016-01-15

Leuconostoc is a hetero-fermentative lactic acid bacteria, and its importance is widely recognized in the dairy industry. However, due to limited genetic tools including plasmids for Leuconostoc, there has not been much extensive research on the genetics and engineering of Leuconostoc yet. Thus, there is a big demand for high-copy-number plasmids for useful gene manipulation and overproduction of recombinant proteins in Leuconostoc. Using an existing low-copy plasmid, the copy number of plasmid was increased by random mutagenesis followed by FACS-based high-throughput screening. First, a random library of plasmids was constructed by randomizing the region responsible for replication in Leuconostoc citreum; additionally, a superfolder green fluorescent protein (sfGFP) was used as a reporter protein. With a high-speed FACS sorter, highly fluorescent cells were enriched, and after two rounds of sorting, single clone exhibiting the highest level of sfGFP was isolated. The copy number of the isolated plasmid (pCB4270) was determined by quantitative PCR (qPCR). It was found that the isolated plasmid has approximately a 30-fold higher copy number (approx. 70 copies per cell) than that of the original plasmid. From the sequence analysis, a single mutation (C→T) at position 4690 was found, and we confirmed that this single mutation was responsible for the increased plasmid copy number. The effectiveness of the isolated high-copy-number plasmid for the overproduction of recombinant proteins was successfully demonstrated with two protein models Glutathione-S-transferase (GST) and α-amylase. The high-copy number plasmid was successfully isolated by FACS-based high-throughput screening of a plasmid library in L. citreum. The isolated plasmid could be a useful genetic tool for high-level gene expression in Leuconostoc, and for extending the applications of this useful bacteria to various areas in the dairy and pharmaceutical industries.

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

PubMed Central

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

2011-01-01

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

Replication-dependent and independent mechanisms for the chromosome-coupled persistence of a selfish genome.

PubMed

Liu, Yen-Ting; Chang, Keng-Ming; Ma, Chien-Hui; Jayaram, Makkuni

2016-09-30

The yeast 2-micron plasmid epitomizes the evolutionary optimization of selfish extra-chromosomal genomes for stable persistence without jeopardizing their hosts' fitness. Analyses of fluorescence-tagged single-copy reporter plasmids and/or the plasmid partitioning proteins in native and non-native hosts reveal chromosome-hitchhiking as the likely means for plasmid segregation. The contribution of the partitioning system to equal segregation is bipartite- replication-independent and replication-dependent. The former nearly eliminates 'mother bias' (preferential plasmid retention in the mother cell) according to binomial distribution, thus limiting equal segregation of a plasmid pair to 50%. The latter enhances equal segregation of plasmid sisters beyond this level, elevating the plasmid close to chromosome status. Host factors involved in plasmid partitioning can be functionally separated by their participation in the replication-independent and/or replication-dependent steps. In the hitchhiking model, random tethering of a pair of plasmids to chromosomes signifies the replication-independent component of segregation; the symmetric tethering of plasmid sisters to sister chromatids embodies the replication-dependent component. The 2-micron circle broadly resembles the episomes of certain mammalian viruses in its chromosome-associated propagation. This unifying feature among otherwise widely differing selfish genomes suggests their evolutionary convergence to the common logic of exploiting, albeit via distinct molecular mechanisms, host chromosome segregation machineries for self-preservation. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Replication-dependent and independent mechanisms for the chromosome-coupled persistence of a selfish genome

PubMed Central

Liu, Yen-Ting; Chang, Keng-Ming; Ma, Chien-Hui; Jayaram, Makkuni

2016-01-01

The yeast 2-micron plasmid epitomizes the evolutionary optimization of selfish extra-chromosomal genomes for stable persistence without jeopardizing their hosts’ fitness. Analyses of fluorescence-tagged single-copy reporter plasmids and/or the plasmid partitioning proteins in native and non-native hosts reveal chromosome-hitchhiking as the likely means for plasmid segregation. The contribution of the partitioning system to equal segregation is bipartite- replication-independent and replication-dependent. The former nearly eliminates ‘mother bias’ (preferential plasmid retention in the mother cell) according to binomial distribution, thus limiting equal segregation of a plasmid pair to 50%. The latter enhances equal segregation of plasmid sisters beyond this level, elevating the plasmid close to chromosome status. Host factors involved in plasmid partitioning can be functionally separated by their participation in the replication-independent and/or replication-dependent steps. In the hitchhiking model, random tethering of a pair of plasmids to chromosomes signifies the replication-independent component of segregation; the symmetric tethering of plasmid sisters to sister chromatids embodies the replication-dependent component. The 2-micron circle broadly resembles the episomes of certain mammalian viruses in its chromosome-associated propagation. This unifying feature among otherwise widely differing selfish genomes suggests their evolutionary convergence to the common logic of exploiting, albeit via distinct molecular mechanisms, host chromosome segregation machineries for self-preservation. PMID:27492289

The cryptic plasmid is more important for Chlamydia muridarum to colonize the mouse gastrointestinal tract than to infect the genital tract.

PubMed

Shao, Lili; Melero, Jose; Zhang, Nu; Arulanandam, Bernard; Baseman, Joel; Liu, Quanzhong; Zhong, Guangming

2017-01-01

Chlamydia has been detected in the gastrointestinal tracts of both animals and humans. However, the mechanism by which Chlamydia colonizes the gut remains unclear. Chlamydia muridarum is known to spread from the genital to the gastrointestinal tracts hematogenously. The C. muridarum plasmid is a key pathogenic determinant in the mouse upper genital tract although plasmid-deficient C. muridarum is still able to colonize the upper genital tract. We now report that plasmid-deficient C. muridarum exhibits significantly delayed/reduced spreading from the mouse genital to the gastrointestinal tracts. C. muridarum with or without plasmid maintained similar levels in the mouse circulatory system following intravenous inoculation but the hematogenous plasmid-deficient C. muridarum was significantly less efficient in colonizing the gastrointestinal tract. Consistently, plasmid-deficient C. muridarum failed to restore normal colonization in the gastrointestinal tract even after intragastric inoculation at a high dose. Thus, we have demonstrated a plasmid-dependent colonization of C. muridarum in the gastrointestinal tract, supporting the concept that C. muridarum may have acquired the plasmid for adaptation to the mouse gastrointestinal tract during oral-fecal transmission. Since the plasmid is more important for C. muridarum to colonize the gastrointestinal tract than to infect the genital tract, the current study has laid a foundation for further defining the host pathways targeted by the plasmid-encoded or -regulated chlamydial effectors.

The cryptic plasmid is more important for Chlamydia muridarum to colonize the mouse gastrointestinal tract than to infect the genital tract

PubMed Central

Shao, Lili; Melero, Jose; Zhang, Nu; Arulanandam, Bernard; Baseman, Joel; Liu, Quanzhong

2017-01-01

Chlamydia has been detected in the gastrointestinal tracts of both animals and humans. However, the mechanism by which Chlamydia colonizes the gut remains unclear. Chlamydia muridarum is known to spread from the genital to the gastrointestinal tracts hematogenously. The C. muridarum plasmid is a key pathogenic determinant in the mouse upper genital tract although plasmid-deficient C. muridarum is still able to colonize the upper genital tract. We now report that plasmid-deficient C. muridarum exhibits significantly delayed/reduced spreading from the mouse genital to the gastrointestinal tracts. C. muridarum with or without plasmid maintained similar levels in the mouse circulatory system following intravenous inoculation but the hematogenous plasmid-deficient C. muridarum was significantly less efficient in colonizing the gastrointestinal tract. Consistently, plasmid-deficient C. muridarum failed to restore normal colonization in the gastrointestinal tract even after intragastric inoculation at a high dose. Thus, we have demonstrated a plasmid-dependent colonization of C. muridarum in the gastrointestinal tract, supporting the concept that C. muridarum may have acquired the plasmid for adaptation to the mouse gastrointestinal tract during oral-fecal transmission. Since the plasmid is more important for C. muridarum to colonize the gastrointestinal tract than to infect the genital tract, the current study has laid a foundation for further defining the host pathways targeted by the plasmid-encoded or -regulated chlamydial effectors. PMID:28542376

Spatial Heterogeneity and Imperfect Mixing in Chemical Reactions: Visualization of Density-Driven Pattern Formation

DOE PAGES

Sobel, Sabrina G.; Hastings, Harold M.; Testa, Matthew

2009-01-01

Imore » mperfect mixing is a concern in industrial processes, everyday processes (mixing paint, bread machines), and in understanding salt water-fresh water mixing in ecosystems. The effects of imperfect mixing become evident in the unstirred ferroin-catalyzed Belousov-Zhabotinsky reaction, the prototype for chemical pattern formation. Over time, waves of oxidation (high ferriin concentration, blue) propagate into a background of low ferriin concentration (red); their structure reflects in part the history of mixing in the reaction vessel. However, it may be difficult to separate mixing effects from reaction effects. We describe a simpler model system for visualizing density-driven pattern formation in an essentially unmixed chemical system: the reaction of pale yellow Fe 3 + with colorless SCN − to form the blood-red Fe ( SCN ) 2 + complex ion in aqueous solution. Careful addition of one drop of Fe ( NO 3 ) 3 to KSCN yields striped patterns after several minutes. The patterns appear reminiscent of Rayleigh-Taylor instabilities and convection rolls, arguing that pattern formation is caused by density-driven mixing.« less

Spatial Heterogeneity and Imperfect Mixing in Chemical Reactions: Visualization of Density-Driven Pattern Formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sobel, Sabrina G.; Hastings, Harold M.; Testa, Matthew

Imore » mperfect mixing is a concern in industrial processes, everyday processes (mixing paint, bread machines), and in understanding salt water-fresh water mixing in ecosystems. The effects of imperfect mixing become evident in the unstirred ferroin-catalyzed Belousov-Zhabotinsky reaction, the prototype for chemical pattern formation. Over time, waves of oxidation (high ferriin concentration, blue) propagate into a background of low ferriin concentration (red); their structure reflects in part the history of mixing in the reaction vessel. However, it may be difficult to separate mixing effects from reaction effects. We describe a simpler model system for visualizing density-driven pattern formation in an essentially unmixed chemical system: the reaction of pale yellow Fe 3 + with colorless SCN − to form the blood-red Fe ( SCN ) 2 + complex ion in aqueous solution. Careful addition of one drop of Fe ( NO 3 ) 3 to KSCN yields striped patterns after several minutes. The patterns appear reminiscent of Rayleigh-Taylor instabilities and convection rolls, arguing that pattern formation is caused by density-driven mixing.« less

Fusion and Compatibility of Camphor and Octane Plasmids in Pseudomonas

PubMed Central

Chou, George I. N.; Katz, Dvorah; Gunsalus, I. C.

1974-01-01

The octane (OCT) plasmid in Pseudomonas putida derived from the ω-hydroxylase-carrying strain of Coon and coworkers is transferable to the camphor (CAM) plasmid-bearing strain by conjugation or by transduction. While the majority of the Cam +Oct+ exconjugants segregate Cam+ or Oct+ cells, exconjugants with stable Cam +Oct+ phenotype (CAM-OCT) can be detected at a low frequency. The transductants are all of the CAM-OCT phenotype. In the stable Cam +Oct+ strains, the OCT plasmid resembles the CAM plasmid with respect to curing by mitomycin C, transfer in conjugation, and reaction to ts (temperature-sensitive) mutation specifically affecting CAM plasmid replication. Therefore, it is suggested that certain regions of homology exist between the CAM and OCT plasmids that enable them to recombine to form a single plasmid, and to overcome the incompatibility barrier that prevents their coexisting. PMID:4527812

Characterization of two new plasmid DNAs found in mitochondria of wild-type Neurospora intermedia strains.

PubMed Central

Stohl, L L; Collins, R A; Cole, M D; Lambowitz, A M

1982-01-01

Mitochondria from two Neurospora intermedia strains (P4O5-Labelle and Fiji N6-6) were found to contain plasmid DNAs in addition to the standard mitochondrial DNA species. The plasmid DNAs consist of monomeric circles (4.1-4.3 kbp and 5.2-5.3 kbp for Labelle and Fiji, respectively) and oligomers in which monomers are organized as head-to-tail repeats. DNA-DNA hybridization experiments showed that the plasmids have no substantial sequence homology to mtDNA, to each other, or to a previously characterized mitochondrial plasmid from N. crassa strain Mauriceville-lc (Collins et al. Cell 24, 443-452, 1981). The intramitochondrial location of the plasmids was established by cell fractionation and nuclease protection experiments. In sexual crosses, the plasmids showed strict maternal inheritance, the same as Neurospora mitochondrial DNA. The plasmids may represent a novel class of mitochondrial genetic elements. Images PMID:6280144

Comparative Sequence Analysis of Multidrug-Resistant IncA/C Plasmids from Salmonella enterica.

PubMed

Hoffmann, Maria; Pettengill, James B; Gonzalez-Escalona, Narjol; Miller, John; Ayers, Sherry L; Zhao, Shaohua; Allard, Marc W; McDermott, Patrick F; Brown, Eric W; Monday, Steven R

2017-01-01

Determinants of multidrug resistance (MDR) are often encoded on mobile elements, such as plasmids, transposons, and integrons, which have the potential to transfer among foodborne pathogens, as well as to other virulent pathogens, increasing the threats these traits pose to human and veterinary health. Our understanding of MDR among Salmonella has been limited by the lack of closed plasmid genomes for comparisons across resistance phenotypes, due to difficulties in effectively separating the DNA of these high-molecular weight, low-copy-number plasmids from chromosomal DNA. To resolve this problem, we demonstrate an efficient protocol for isolating, sequencing and closing IncA/C plasmids from Salmonella sp. using single molecule real-time sequencing on a Pacific Biosciences (Pacbio) RS II Sequencer. We obtained six Salmonella enterica isolates from poultry, representing six different serovars, each exhibiting the MDR-Ampc resistance profile. Salmonella plasmids were obtained using a modified mini preparation and transformed with Escherichia coli DH10Br. A Qiagen Large-Construct kit™ was used to recover highly concentrated and purified plasmid DNA that was sequenced using PacBio technology. These six closed IncA/C plasmids ranged in size from 104 to 191 kb and shared a stable, conserved backbone containing 98 core genes, with only six differences among those core genes. The plasmids encoded a number of antimicrobial resistance genes, including those for quaternary ammonium compounds and mercury. We then compared our six IncA/C plasmid sequences: first with 14 IncA/C plasmids derived from S. enterica available at the National Center for Biotechnology Information (NCBI), and then with an additional 38 IncA/C plasmids derived from different taxa. These comparisons allowed us to build an evolutionary picture of how antimicrobial resistance may be mediated by this common plasmid backbone. Our project provides detailed genetic information about resistance genes in plasmids, advances in plasmid sequencing, and phylogenetic analyses, and important insights about how MDR evolution occurs across diverse serotypes from different animal sources, particularly in agricultural settings where antimicrobial drug use practices vary.

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

PubMed

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 ( ccdAB ST and vapBC2 ST ). The TA module ccdAB ST has previously been shown to contribute to pSLT plasmid stability and vapBC2 ST 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 vapBC2 ST , in addition to contribute to bacterial virulence, is important for plasmid stability. We have previously shown that ccdAB ST encodes an inactive CcdB ST toxin. Using our new stability assay we monitored the contribution to plasmid stability of a ccdAB ST variant containing a single mutation (R99W) that restores the toxicity of CcdB ST . The "activation" of CcdB ST (R99W) did not increase pSLT stability by ccdAB ST . In contrast, ccdAB ST behaves as a canonical type II TA system in terms of transcriptional regulation. Of interest, ccdAB ST was shown to control the expression of a polycistronic operon in the pSLT plasmid. Collectively, these results show that the contribution of the CcdB ST toxin to pSLT plasmid stability may depend on its role as a co-repressor in coordination with CcdA ST antitoxin more than on its toxic activity.

Mobile Insertion Cassette Elements Found in Small Non-Transmissible Plasmids in Proteeae May Explain qnrD Mobilization

PubMed Central

Guillard, Thomas; Grillon, Antoine; de Champs, Christophe; Cartier, Céline; Madoux, Janick; Berçot, Béatrice; Lebreil, Anne-Laure; Lozniewski, Alain; Riahi, Jacques; Vernet-Garnier, Véronique; Cambau, Emmanuelle

2014-01-01

qnrD is a plasmid mediated quinolone resistance gene from unknown origin, recently described in Enterobacteriaceae. It encodes a pentapeptide repeat protein 36–60% different from the other Qnr (A, B, C, S and VC). Since most qnrD-positive strains were described as strains belonging to Proteus or Providencia genera, we hypothesized that qnrD originated in Proteeae before disseminating to other enterobacterial species. We screened 317 strains of Proteeae for qnrD and its genetic support by PCR. For all the seven qnrD-positive strains (4 Proteus mirabilis, 1 Proteus vulgaris and 2 Providencia rettgeri) the gene was carried onto a small non-transmissible plasmid, contrarily to other qnr genes that are usually carried onto large multi-resistant plasmids. Nucleotide sequences of the qnrD-bearing plasmids were 96% identical. Plasmids contained 3 ORFs apart from qnrD and belonged to an undescribed incompatibility group. Only one plasmid, in P. vulgaris, was slightly different with a 1,568-bp insertion between qnrD and its promoter, leading to absence of quinolone resistance. We sought for similar plasmids in 15 reference strains of Proteeae, but which were tested negative for qnrD, and found a 48% identical plasmid (pVERM) in Providencia vermicola. In order to explain how qnrD could have been inserted into such native plasmid, we sought for gene mobilization structures. qnrD was found to be located within a mobile insertion cassette (mic) element which sequences are similar to one mic also found in pVERM. Our conclusions are that (i) the small non-transmissible qnrD-plasmids described here may result from the recombination between an as-yet-unknown progenitor of qnrD and pVERM, (ii) these plasmids are maintained in Proteeae being a qnrD reservoir (iii) the mic element may explain qnrD mobilization from non-transmissible plasmids to mobilizable or conjugative plasmids from other Enterobacteriaceae, (iv) they can recombined with larger multiresistant plasmids conjugated in Proteeae. PMID:24504382

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

PubMed Central

Seiler, Catherine Y.; Park, Jin G.; Sharma, Amit; Hunter, Preston; Surapaneni, Padmini; Sedillo, Casey; Field, James; Algar, Rhys; Price, Andrea; Steel, Jason; Throop, Andrea; Fiacco, Michael; LaBaer, Joshua

2014-01-01

The mission of the DNASU Plasmid Repository is to accelerate research by providing high-quality, annotated plasmid samples and online plasmid resources to the research community through the curated DNASU database, website and repository (http://dnasu.asu.edu or http://dnasu.org). The collection includes plasmids from grant-funded, high-throughput cloning projects performed in our laboratory, plasmids from external researchers, and large collections from consortia such as the ORFeome Collaboration and the NIGMS-funded Protein Structure Initiative: Biology (PSI:Biology). Through DNASU, researchers can search for and access detailed information about each plasmid such as the full length gene insert sequence, vector information, associated publications, and links to external resources that provide additional protein annotations and experimental protocols. Plasmids can be requested directly through the DNASU website. DNASU and the PSI:Biology-Materials Repositories were previously described in the 2010 NAR Database Issue (Cormier, C.Y., Mohr, S.E., Zuo, D., Hu, Y., Rolfs, A., Kramer, J., Taycher, E., Kelley, F., Fiacco, M., Turnbull, G. et al. (2010) Protein Structure Initiative Material Repository: an open shared public resource of structural genomics plasmids for the biological community. Nucleic Acids Res., 38, D743–D749.). In this update we will describe the plasmid collection and highlight the new features in the website redesign, including new browse/search options, plasmid annotations and a dynamic vector mapping feature that was developed in collaboration with LabGenius. Overall, these plasmid resources continue to enable research with the goal of elucidating the role of proteins in both normal biological processes and disease. PMID:24225319

Complete genome sequence and the expression pattern of plasmids of the model ethanologen Zymomonas mobilis ZM4 and its xylose-utilizing derivatives 8b and 2032

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Shihui; Vera, Jessica M.; Grass, Jeff

Zymomonas mobilis is a natural ethanologen being developed and deployed as an industrial biofuel producer. To date, eight Z. mobilis strains have been completely sequenced and found to contain 2-8 native plasmids. However, systematic verification of predicted Z. mobilis plasmid genes and their contribution to cell fitness has not been hitherto addressed. Moreover, the precise number and identities of plasmids in Z. mobilis model strain ZM4 have been unclear. The lack of functional information about plasmid genes in ZM4 impedes ongoing studies for this model biofuel-producing strain. In this study, we determined the complete chromosome and plasmid sequences of ZM4more » and its engineered xylose-utilizing derivatives 2032 and 8b. Compared to previously published and revised ZM4 chromosome sequences, the ZM4 chromosome sequence reported here contains 65 nucleotide sequence variations as well as a 2400-bp insertion. Four plasmids were identified in all three strains, with 150 plasmid genes predicted in strain ZM4 and 2032, and 153 plasmid genes predicted in strain 8b due to the insertion of heterologous DNA for expanded substrate utilization. Plasmid genes were then annotated using Blast2GO, InterProScan, and systems biology data analyses, and most genes were found to have apparent orthologs in other organisms or identifiable conserved domains. To verify plasmid gene prediction, RNA-Seq was used to map transcripts and also compare relative gene expression under various growth conditions, including anaerobic and aerobic conditions, or growth in different concentrations of biomass hydrolysates. Overall, plasmid genes were more responsive to varying hydrolysate concentrations than to oxygen availability. Additionally, our results indicated that although all plasmids were present in low copy number (about 1-2 per cell), the copy number of some plasmids varied under specific growth conditions or due to heterologous gene insertion. The complete genome of ZM4 and two xylose-utilizing derivatives is reported in this study, with an emphasis on identifying and characterizing plasmid genes. Furthermore, plasmid gene annotation, validation, expression levels at growth conditions of interest, and contribution to host fitness are reported for the first time.« less

Complete genome sequence and the expression pattern of plasmids of the model ethanologen Zymomonas mobilis ZM4 and its xylose-utilizing derivatives 8b and 2032

DOE PAGES

Yang, Shihui; Vera, Jessica M.; Grass, Jeff; ...

2018-05-02

Zymomonas mobilis is a natural ethanologen being developed and deployed as an industrial biofuel producer. To date, eight Z. mobilis strains have been completely sequenced and found to contain 2-8 native plasmids. However, systematic verification of predicted Z. mobilis plasmid genes and their contribution to cell fitness has not been hitherto addressed. Moreover, the precise number and identities of plasmids in Z. mobilis model strain ZM4 have been unclear. The lack of functional information about plasmid genes in ZM4 impedes ongoing studies for this model biofuel-producing strain. In this study, we determined the complete chromosome and plasmid sequences of ZM4more » and its engineered xylose-utilizing derivatives 2032 and 8b. Compared to previously published and revised ZM4 chromosome sequences, the ZM4 chromosome sequence reported here contains 65 nucleotide sequence variations as well as a 2400-bp insertion. Four plasmids were identified in all three strains, with 150 plasmid genes predicted in strain ZM4 and 2032, and 153 plasmid genes predicted in strain 8b due to the insertion of heterologous DNA for expanded substrate utilization. Plasmid genes were then annotated using Blast2GO, InterProScan, and systems biology data analyses, and most genes were found to have apparent orthologs in other organisms or identifiable conserved domains. To verify plasmid gene prediction, RNA-Seq was used to map transcripts and also compare relative gene expression under various growth conditions, including anaerobic and aerobic conditions, or growth in different concentrations of biomass hydrolysates. Overall, plasmid genes were more responsive to varying hydrolysate concentrations than to oxygen availability. Additionally, our results indicated that although all plasmids were present in low copy number (about 1-2 per cell), the copy number of some plasmids varied under specific growth conditions or due to heterologous gene insertion. The complete genome of ZM4 and two xylose-utilizing derivatives is reported in this study, with an emphasis on identifying and characterizing plasmid genes. Furthermore, plasmid gene annotation, validation, expression levels at growth conditions of interest, and contribution to host fitness are reported for the first time.« less

Using the CRISPR/Cas9 system to eliminate native plasmids of Zymomonas mobilis ZM4.

PubMed

Cao, Qing-Hua; Shao, Huan-Huan; Qiu, Hui; Li, Tao; Zhang, Yi-Zheng; Tan, Xue-Mei

2017-03-01

The CRISPR/Cas system can be used to simply and efficiently edit the genomes of various species, including animals, plants, and microbes. Zymomonas mobilis ZM4 is a highly efficient, ethanol-producing bacterium that contains five native plasmids. Here, we constructed the pSUZM2a-Cas9 plasmid and a single-guide RNA expression plasmid. The pSUZM2a-Cas9 plasmid was used to express the Cas9 gene cloned from Streptococcus pyogenes CICC 10464. The single-guide RNA expression plasmid pUC-T7sgRNA, with a T7 promoter, can be used for the in vitro synthesis of single-guide RNAs. This system was successfully employed to knockout the upp gene of Escherichia coli and the replicase genes of native Z. mobilis plasmids. This is the first study to apply the CRISPR/Cas9 system of S. pyogenes to eliminate native plasmids in Z. mobilis. It provides a new method for plasmid curing and paves the way for the genomic engineering of Z. mobilis.

The selfish yeast plasmid uses the nuclear motor Kip1p but not Cin8p for its localization and equal segregation.

PubMed

Cui, Hong; Ghosh, Santanu K; Jayaram, Makkuni

2009-04-20

The 2 micron plasmid of Saccharomyces cerevisiae uses the Kip1 motor, but not the functionally redundant Cin8 motor, for its precise nuclear localization and equal segregation. The timing and lifetime of Kip1p association with the plasmid partitioning locus STB are consistent with Kip1p being an authentic component of the plasmid partitioning complex. Kip1-STB association is not blocked by disassembling the mitotic spindle. Lack of Kip1p disrupts recruitment of the cohesin complex at STB and cohesion of replicated plasmid molecules. Colocalization of a 2 micron reporter plasmid with Kip1p in close proximity to the spindle pole body is reminiscent of that of a CEN reporter plasmid. Absence of Kip1p displaces the plasmid from this nuclear address, where it has the potential to tether to a chromosome or poach chromosome segregation factors. Exploiting Kip1p, which is subsidiary to Cin8p for chromosome segregation, to direct itself to a "partitioning center" represents yet another facet of the benign parasitism of the yeast plasmid.

A selfish DNA element engages a meiosis-specific motor and telomeres for germ-line propagation

PubMed Central

Sau, Soumitra; Conrad, Michael N.; Lee, Chih-Ying; Kaback, David B.; Dresser, Michael E.

2014-01-01

The chromosome-like mitotic stability of the yeast 2 micron plasmid is conferred by the plasmid proteins Rep1-Rep2 and the cis-acting locus STB, likely by promoting plasmid-chromosome association and segregation by hitchhiking. Our analysis reveals that stable plasmid segregation during meiosis requires the bouquet proteins Ndj1 and Csm4. Plasmid relocalization from the nuclear interior in mitotic cells to the periphery at or proximal to telomeres rises from early meiosis to pachytene. Analogous to chromosomes, the plasmid undergoes Csm4- and Ndj1-dependent rapid prophase movements with speeds comparable to those of telomeres. Lack of Ndj1 partially disrupts plasmid–telomere association without affecting plasmid colocalization with the telomere-binding protein Rap1. The plasmid appears to engage a meiosis-specific motor that orchestrates telomere-led chromosome movements for its telomere-associated segregation during meiosis I. This hitherto uncharacterized mode of germ-line transmission by a selfish genetic element signifies a mechanistic variation within the shared theme of chromosome-coupled plasmid segregation during mitosis and meiosis. PMID:24914236

Bioelectrochemical conversion of CO2 to value added product formate using engineered Methylobacterium extorquens.

PubMed

Jang, Jungho; Jeon, Byoung Wook; Kim, Yong Hwan

2018-05-08

The conversion of carbon dioxide to formate is a fundamental step for building C1 chemical platforms. Methylobacterium extorquens AM1 was reported to show remarkable activity converting carbon dioxide into formate. Formate dehydrogenase 1 from M. extorquens AM1 (MeFDH1) was verified as the key responsible enzyme for the conversion of carbon dioxide to formate in this study. Using a 2% methanol concentration for induction, microbial harboring the recombinant MeFDH1 expressing plasmid produced the highest concentration of formate (26.6 mM within 21 hours) in electrochemical reactor. 60 μM of sodium tungstate in the culture medium was optimal for the expression of recombinant MeFDH1 and production of formate (25.7 mM within 21 hours). The recombinant MeFDH1 expressing cells showed maximum formate productivity of 2.53 mM/g-wet cell/hr, which was 2.5 times greater than that of wild type. Thus, M. extorquens AM1 was successfully engineered by expressing MeFDH1 as recombinant enzyme to elevate the production of formate from CO 2 after elucidating key responsible enzyme for the conversion of CO 2 to formate.

Microneedle-mediated transcutaneous immunization with plasmid DNA coated on cationic PLGA nanoparticles

PubMed Central

Kumar, Amit; Wonganan, Piyanuch; Sandoval, Michael A.; Li, Xinran; Zhu, Saijie; Cui, Zhengrong

2012-01-01

Previously, it was shown that microneedle-mediated transcutaneous immunization with plasmid DNA can potentially induce a stronger immune response than intramuscular injection of the same plasmid DNA. In the present study, we showed that the immune responses induced by transcutaneous immunization by applying plasmid DNA onto a skin area pretreated with solid microneedles were significantly enhanced by coating the plasmid DNA on the surface of cationic nanoparticles. In addition, the net surface charge of the DNA-coated nanoparticles significantly affected their in vitro skin permeation and their ability to induce immune responses in vivo. Transcutaneous immunization with plasmid DNA-coated net positively charged anoparticles elicited a stronger immune response than with plasmid DNA-coated net negatively charged nanoparticles or by intramuscular immunization with plasmid DNA alone. Transcutaneous immunization with plasmid DNA-coated net positively charged nanoparticles induced comparable immune responses as intramuscular injection of them, but transcutaneous immunization was able to induce specific mucosal immunity and a more balanced T helper type 1 and type 2 response. The ability of the net positively charged DNA-coated nanoparticles to induce a strong immune response through microneedle-mediated transcutaneous immunization may be attributed to their ability to increase the expression of the antigen gene encoded by the plasmid and to more effectively stimulate the maturation of antigen-presenting cells. PMID:22921518

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

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

NASA Technical Reports Server (NTRS)

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

1991-01-01

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

Plasmid Frequency Fluctuations in Bacterial Populations from Chemically Stressed Soil Communities

PubMed Central

Wickham, Gene S.; Atlas, Ronald M.

1988-01-01

The frequency of plasmids in chemically stressed bacterial populations was investigated by individually adding various concentration of kanamycin, ampicillin, and mercuric chloride to soil samples. Viable bacterial populations were enumerated, soil respiration was monitored for up to 6 weeks as an indicator of physiological stress, and bacterial isolates from stressed and control soils were screened for the presence of plasmids. Low levels of the chemical stress factors did not for the most part significantly alter population viability, soil respiration, or plasmid frequency. Exposure to high stress levels of mercury and ampicillin, however, resulted in altered numbers of viable organisms, soil respiration, and plasmid frequency. Plasmid frequency increased in response to ampicillin exposure but was not significantly changed after exposure to kanamycin. In mercuric chloride-stressed soils, there was a decrease in plasmid frequency despite an increase in overall mercury resistance of the isolates, suggesting that mercury resistance in these populations is largely, if not completely, chromosome encoded. Chemical stress did not cause an increase in plasmid-mediated multiple resistance. A genetic response (change in plasmid frequency) was not found unless a physiological (phenotypic) response (change in viable cells and respiratory activity) was also observed. The results indicate that a change in plasmid frequency is dependent on both the amount and type of chemical stress. PMID:16347730

The Salmonella dublin virulence plasmid mediates systemic but not enteric phases of salmonellosis in cattle.

PubMed Central

Wallis, T S; Paulin, S M; Plested, J S; Watson, P R; Jones, P W

1995-01-01

Plasmid-bearing and plasmid-free isolates and a plasmid-cured strain of Salmonella dublin were compared for virulence in calves. The plasmid-bearing strains were highly virulent, causing severe enteric and systemic disease with high mortality. In contrast, the plasmid-free strains caused diarrhea but only low mortality. The infection kinetics of a wild-type and a derivative plasmid-cured strain were compared. Both strains were isolated in high numbers from intestinal sites at 3 and 6 days after oral challenge and were isolated at comparable frequencies from systemic sites at 3 days, but not at 6 days, when the wild-type strain was predominant. The strains were equally invasive in intestinal epithelia with and without Peyer's patch and elicited comparable secretory and inflammatory responses and intestinal pathology in ligated ileal loops. The effect of the virulence plasmid on growth kinetics and on the outer membrane protein profile was assessed in an in vivo growth chamber. The virulence plasmid did not influence either extracellular growth or the expression of major outer membrane proteins. These observations demonstrate that the virulence plasmid is not involved in either the enteric phase of infection or the systemic dissemination of S. dublin but probably mediates the persistence of S. dublin at systemic sites. PMID:7790094

Structural similarity and distribution of small cryptic plasmids of Lactobacillus curvatus and L. sake.

PubMed

Vogel, R F; Lohmann, M; Weller, A N; Hugas, M; Hammes, W P

1991-11-15

Plasmid profiles of strains of Lactobacillus curvatus and L. sake isolated from meat or sauerkraut were analysed to investigate plasmid homology and distribution in relation to the ecology of these organisms in fermenting foods. A hybridisation probe was constructed by cloning of pLc2, a cryptic, 2.6-kbp plasmid from L. curvatus LTH683, into the Escherichia coli plasmid pRV50. In Southern hybridisations with the digoxygenine labeled pLc2 probe, pLc2-related small plasmids were frequently detected in meat-borne strains of L. casei subsp. pseudoplantarum, L. curvatus, L. sake, L. alimentarius, L. farciminis and L. halotolerans and in L. curvatus and L. sake isolated from sauerkraut. Among 27 Lactobacillus type strains originally isolated from habitats other than meat this type of homology was detected only with plasmids of L. buchneri and L. mali. Restriction-enzyme mapping of six small cryptic plasmids from L. curvatus and L. sake revealed strong structural homology but no similarity to previously characterized plasmids of lactobacilli. The presence of a variable region in addition to a conserved one and the occurrence of deletions during cloning of pLc2 suggest that vectors derived from these plasmids are likely to be structurally unstable.

Plasmid-Borne Antimicrobial Resistance of Staphylococcus aureus Isolated in a Hospital in Lisbon, Portugal.

PubMed

Costa, Sofia Santos; Palma, Cláudia; Kadlec, Kristina; Fessler, Andrea T; Viveiros, Miguel; Melo-Cristino, José; Schwarz, Stefan; Couto, Isabel

2016-12-01

Plasmids play a key role in the genetic plasticity and survival of Staphylococcus aureus in challenging environments. Although many S. aureus plasmids have been described, still few studies portray the plasmid content of a given S. aureus population. The aim of this work was to characterize the plasmids carried by a collection of 53 S. aureus isolates collected in a large hospital in Lisbon, Portugal, and investigate their role in conferring resistance to several antimicrobial agents. Plasmids were present in 44 out of the 53 isolates and were grouped into eleven AccI restriction profiles. Plasmid curing of representative strains and comparison of antimicrobial susceptibility profiles between pairs of isogenic strains proved to be a valuable guidance tool in the identification of plasmid-located resistance genes. The plasmids harbored several resistance genes, namely blaZ (resistance to β-lactams), erm(C) (resistance to macrolides, lincosamides, and streptogramin B), cadA (resistance to cadmium and zinc), cadD (resistance to cadmium), and qacA and smr (resistance to biocides and dyes). This study demonstrates the impact of plasmids on the resistance properties of S. aureus, highlighting their role in the dissemination of antibiotic, heavy metal, and biocide resistance genes, and survival of this major pathogen in the hospital environment.

Horizontal Dissemination of Antimicrobial Resistance Determinants in Multiple Salmonella Serotypes following Isolation from the Commercial Swine Operation Environment after Manure Application.

PubMed

Pornsukarom, Suchawan; Thakur, Siddhartha

2017-10-15

The aim of this study was to characterize the plasmids carrying antimicrobial resistance (AMR) determinants in multiple Salmonella serotypes recovered from the commercial swine farm environment after manure application on land. Manure and soil samples were collected on day 0 before and after manure application on six farms in North Carolina, and sequential soil samples were recollected on days 7, 14, and 21 from the same plots. All environmental samples were processed for Salmonella , and their plasmid contents were further characterized. A total of 14 isolates including Salmonella enterica serotypes Johannesburg ( n = 2), Ohio ( n = 2), Rissen ( n = 1), Typhimurium var5- ( n = 5), Worthington ( n = 3), and 4,12:i:- ( n = 1), representing different farms, were selected for plasmid analysis. Antimicrobial susceptibility testing was done by broth microdilution against a panel of 14 antimicrobials on the 14 confirmed transconjugants after conjugation assays. The plasmids were isolated by modified alkaline lysis, and PCRs were performed on purified plasmid DNA to identify the AMR determinants and the plasmid replicon types. The plasmids were sequenced for further analysis and to compare profiles and create phylogenetic trees. A class 1 integron with an ANT(2″)-Ia- aadA2 cassette was detected in the 50-kb IncN plasmids identified in S Worthington isolates. We identified 100-kb and 90-kb IncI1 plasmids in S Johannesburg and S Rissen isolates carrying the bla CMY-2 and tet (A) genes, respectively. An identical 95-kb IncF plasmid was widely disseminated among the different serotypes and across different farms. Our study provides evidence on the importance of horizontal dissemination of resistance determinants through plasmids of multiple Salmonella serotypes distributed across commercial swine farms after manure application. IMPORTANCE The horizontal gene transfer of antimicrobial resistance (AMR) determinants located on plasmids is considered to be the main reason for the rapid proliferation and spread of drug resistance. The deposition of manure generated in swine production systems into the environment is identified as a potential source of AMR dissemination. In this study, AMR gene-carrying plasmids were detected in multiple Salmonella serotypes across different commercial swine farms in North Carolina. The plasmid profiles were characterized based on Salmonella serotype donors and incompatibility (Inc) groups. We found that different Inc plasmids showed evidence of AMR gene transfer in multiple Salmonella serotypes. We detected an identical 95-kb plasmid that was widely distributed across swine farms in North Carolina. These conjugable resistance plasmids were able to persist on land after swine manure application. Our study provides strong evidence of AMR determinant dissemination present in plasmids of multiple Salmonella serotypes in the environment after manure application. Copyright © 2017 American Society for Microbiology.

Vaccination with plasmid DNA encoding TSA/LmSTI1 leishmanial fusion proteins confers protection against Leishmania major infection in susceptible BALB/c mice.

PubMed

Campos-Neto, A; Webb, J R; Greeson, K; Coler, R N; Skeiky, Y A W; Reed, S G

2002-06-01

We have recently shown that a cocktail containing two leishmanial recombinant antigens (LmSTI1 and TSA) and interleukin-12 (IL-12) as an adjuvant induces solid protection in both a murine and a nonhuman primate model of cutaneous leishmaniasis. However, because IL-12 is difficult to prepare, is expensive, and does not have the stability required for a vaccine product, we have investigated the possibility of using DNA as an alternative means of inducing protective immunity. Here, we present evidence that the antigens TSA and LmSTI1 delivered in a plasmid DNA format either as single genes or in a tandem digene construct induce equally solid protection against Leishmania major infection in susceptible BALB/c mice. Immunization of mice with either TSA DNA or LmSTI1 DNA induced specific CD4(+)-T-cell responses of the Th1 phenotype without a requirement for specific adjuvant. CD8 responses, as measured by cytotoxic-T-lymphocyte activity, were generated after immunization with TSA DNA but not LmSTI1 DNA. Interestingly, vaccination of mice with TSA DNA consistently induced protection to a much greater extent than LmSTI1 DNA, thus supporting the notion that CD8 responses might be an important accessory arm of the immune response for acquired resistance against leishmaniasis. Moreover, the protection induced by DNA immunization was specific for infection with Leishmania, i.e., the immunization had no effect on the course of infection of the mice challenged with an unrelated intracellular pathogen such as Mycobacterium tuberculosis. Conversely, immunization of BALB/c mice with a plasmid DNA that is protective against challenge with M. tuberculosis had no effect on the course of infection of these mice with L. major. Together, these results indicate that the protection observed with the leishmanial DNA is mediated by acquired specific immune response rather than by the activation of nonspecific innate immune mechanisms. In addition, a plasmid DNA containing a fusion construct of the two genes was also tested. Similarly to the plasmids encoding individual proteins, the fusion construct induced both specific immune responses to the individual antigens and protection against challenge with L. major. These results confirm previous observations about the possibility of DNA immunization against leishmaniasis and lend support to the idea of using a single polygenic plasmid DNA construct to achieve polyspecific immune responses to several distinct parasite antigens.

Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:H− Isolates from Czech Patients with Novel Plasmid Composition Not Previously Seen in German Isolates

PubMed Central

Bauwens, Andreas; Marejková, Monika; Middendorf-Bauchart, Barbara; Prager, Rita; Kossow, Annelene; Zhang, Wenlan; Karch, Helge

2017-01-01

ABSTRACT Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H− strains, first identified in Germany, have emerged as important pathogens throughout Europe. Besides chromosomally encoded Shiga toxin 2a (the major virulence factor), several putative virulence loci, including the hly, etp, and sfp operons, encoding EHEC hemolysin, type II secretion system proteins, and Sfp fimbriae, respectively, are located on the 121-kb plasmid pSFO157 in German strains. Here we report novel SF EHEC O157:H− strains isolated from patients in the Czech Republic. These strains share the core genomes and chromosomal virulence loci encoding toxins (stx2a and the cdtV-ABC operon) and adhesins (eae-γ, efa1, lpfAO157OI-141, and lpfAO157OI-154) with German strains but differ essentially in their plasmids. In contrast to all previously detected SF EHEC O157:H− strains, the Czech strains carry two plasmids, of 79 kb and 86 kb. The 79-kb plasmid harbors the sfp operon, but neither of the plasmids contains the hly and etp operons. Sequence analyses demonstrated that the 79-kb plasmid (pSFO157 258/98-1) evolved from pSFO157 of German strains by deletion of a 41,534-bp region via homologous recombination, resulting in loss of the hly and etp operons. The 86-kb plasmid (pSFO157 258/98-2) displays 98% sequence similarity to a 92.7-kb plasmid of an extraintestinal pathogenic E. coli bloodstream isolate. Our finding of this novel plasmid composition in SF EHEC O157:H− strains extends the evolutionary history of EHEC O157 plasmids. Moreover, the unique molecular plasmid characteristics permit the identification of such strains, thereby facilitating further investigations of their geographic distribution, clinical significance, and epidemiology. IMPORTANCE Since their first identification in Germany in 1989, sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H− (nonmotile) strains have emerged as important causes of the life-threatening disease hemolytic-uremic syndrome in Europe. They account for 10 to 20% of sporadic cases of this disease and have caused several large outbreaks. The strains isolated throughout Europe share conserved chromosomal and plasmid characteristics. Here we identified novel sorbitol-fermenting enterohemorrhagic E. coli O157:H− patient isolates in the Czech Republic which differ from all such strains reported previously by their unique plasmid characteristics, including plasmid number, composition of plasmid-carried virulence genes, and plasmid origins. Our findings contribute substantially to understanding the evolution of E. coli O157 strains and their plasmids. In practical terms, they enable the identification of strains with these novel plasmid characteristics in patient stool samples and thus the investigation of their roles as human pathogens in other geographic areas. PMID:28970221

Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:H- Isolates from Czech Patients with Novel Plasmid Composition Not Previously Seen in German Isolates.

PubMed

Bauwens, Andreas; Marejková, Monika; Middendorf-Bauchart, Barbara; Prager, Rita; Kossow, Annelene; Zhang, Wenlan; Karch, Helge; Mellmann, Alexander; Bielaszewska, Martina

2017-12-01

Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H - strains, first identified in Germany, have emerged as important pathogens throughout Europe. Besides chromosomally encoded Shiga toxin 2a (the major virulence factor), several putative virulence loci, including the hly , etp , and sfp operons, encoding EHEC hemolysin, type II secretion system proteins, and Sfp fimbriae, respectively, are located on the 121-kb plasmid pSFO157 in German strains. Here we report novel SF EHEC O157:H - strains isolated from patients in the Czech Republic. These strains share the core genomes and chromosomal virulence loci encoding toxins ( stx 2a and the cdtV -ABC operon) and adhesins ( eae -γ, efa1 , lpfA O157OI-141 , and lpfA O157OI-154 ) with German strains but differ essentially in their plasmids. In contrast to all previously detected SF EHEC O157:H - strains, the Czech strains carry two plasmids, of 79 kb and 86 kb. The 79-kb plasmid harbors the sfp operon, but neither of the plasmids contains the hly and etp operons. Sequence analyses demonstrated that the 79-kb plasmid (pSFO157 258/98-1) evolved from pSFO157 of German strains by deletion of a 41,534-bp region via homologous recombination, resulting in loss of the hly and etp operons. The 86-kb plasmid (pSFO157 258/98-2) displays 98% sequence similarity to a 92.7-kb plasmid of an extraintestinal pathogenic E. coli bloodstream isolate. Our finding of this novel plasmid composition in SF EHEC O157:H - strains extends the evolutionary history of EHEC O157 plasmids. Moreover, the unique molecular plasmid characteristics permit the identification of such strains, thereby facilitating further investigations of their geographic distribution, clinical significance, and epidemiology. IMPORTANCE Since their first identification in Germany in 1989, sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H - (nonmotile) strains have emerged as important causes of the life-threatening disease hemolytic-uremic syndrome in Europe. They account for 10 to 20% of sporadic cases of this disease and have caused several large outbreaks. The strains isolated throughout Europe share conserved chromosomal and plasmid characteristics. Here we identified novel sorbitol-fermenting enterohemorrhagic E. coli O157:H - patient isolates in the Czech Republic which differ from all such strains reported previously by their unique plasmid characteristics, including plasmid number, composition of plasmid-carried virulence genes, and plasmid origins. Our findings contribute substantially to understanding the evolution of E. coli O157 strains and their plasmids. In practical terms, they enable the identification of strains with these novel plasmid characteristics in patient stool samples and thus the investigation of their roles as human pathogens in other geographic areas. Copyright © 2017 American Society for Microbiology.

pTRA - A reporter system for monitoring the intracellular dynamics of gene expression.

PubMed

Wagner, Sabine G; Ziegler, Martin; Löwe, Hannes; Kremling, Andreas; Pflüger-Grau, Katharina

2018-01-01

The presence of standardised tools and methods to measure and represent accurately biological parts and functions is a prerequisite for successful metabolic engineering and crucial to understand and predict the behaviour of synthetic genetic circuits. Many synthetic gene networks are based on transcriptional circuits, thus information on transcriptional and translational activity is important for understanding and fine-tuning the synthetic function. To this end, we have developed a toolkit to analyse systematically the transcriptional and translational activity of a specific synthetic part in vivo. It is based on the plasmid pTRA and allows the assignment of specific transcriptional and translational outputs to the gene(s) of interest (GOI) and to compare different genetic setups. By this, the optimal combination of transcriptional strength and translational activity can be identified. The design is tested in a case study using the gene encoding the fluorescent mCherry protein as GOI. We show the intracellular dynamics of mRNA and protein formation and discuss the potential and shortcomings of the pTRA plasmid.

Introduction to the ultrasound targeted microbubble destruction technique.

PubMed

Walton, Chad B; Anderson, Cynthia D; Boulay, Rachel; Shohet, Ralph V

2011-06-12

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid microbubble contrast agents. In mice these vector-carrying microbubbles can be administered intravenously or directly to the left ventricle of the heart. In larger animals they can also be infused through an intracoronary catheter. The subsequent delivery from the circulation to a target organ occurs by acoustic cavitation at a resonant frequency of the microbubbles. It seems likely that the mechanical energy generated by the microbubble destruction results in transient pore formation in or between the endothelial cells of the microvasculature of the targeted region. As a result of this sonoporation effect, the transfection efficiency into and across the endothelial cells is enhanced, and transgene-encoding vectors are deposited into the surrounding tissue. Plasmid DNA remaining in the circulation is rapidly degraded by nucleases in the blood, which further reduces the likelihood of delivery to non-sonicated tissues and leads to highly specific target-organ transfection.

Isolation of a conjugative F-like plasmid from a multidrug-resistant Escherichia coli strain CM6 using tandem shock wave-mediated transformation.

PubMed

Soto-Alonso, G; Cruz-Medina, J A; Caballero-Pérez, J; Arvizu-Hernández, I; Ávalos-Esparza, L M; Cruz-Hernández, A; Romero-Gómez, S; Rodríguez, A L; Pastrana-Martínez, X; Fernández, F; Loske, A M; Campos-Guillén, J

2015-07-01

Genetic characterization of plasmids from bacterial strains provides insight about multidrug resistance. Ten wild type Escherichia coli (E. coli) strains isolated from cow fecal samples were characterized by their antibiotic resistance profile, plasmid patterns and three different identification methods. From one of the strains, a fertility factor-like plasmid was replicated using tandem shock wave-mediated transformation. Underwater shock waves with a positive pressure peak of up to approximately 40 MPa, followed by a pressure trough of approximately -19 MPa were generated using an experimental piezoelectric shock wave source. Three different shock wave energies and a fixed delay of 750 μs were used to study the relationship between energy and transformation efficiency (TE), as well as the influence of shock wave energy on the integrity of the plasmid. Our results showed that the mean shock wave-mediated TE and the integrity of the large plasmid (~70 kb) were reduced significantly at the energy levels tested. The sequencing analysis of the plasmid revealed a high identity to the pHK17a plasmid, including the replication system, which was similar to the plasmid incompatibility group FII. It also showed that it carried an extended spectrum beta-lactamase gene, ctx-m-14. Furthermore, diverse genes for the conjugative mechanism were identified. Our results may be helpful in improving methodologies for conjugative plasmid transfer and directly selecting the most interesting plasmids from environmental samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Prevalence of ColE1-like plasmids and kanamycin resistance genes in Salmonella enterica serovars.

PubMed

Chen, Chin-Yi; Lindsey, Rebecca L; Strobaugh, Terence P; Frye, Jonathan G; Meinersmann, Richard J

2010-10-01

Multi-antimicrobial-resistant Salmonella enterica strains frequently carry resistance genes on plasmids. Recent studies focus heavily on large conjugative plasmids, and the role that small plasmids play in resistance gene transfer is largely unknown. To expand our previous studies in assessing the prevalence of the isolates harboring ColE1-like plasmids carrying the aph gene responsible for kanamycin resistance (Kan(r)) phenotypes, 102 Kan(r) Salmonella isolates collected through the National Antimicrobial Resistance Monitoring System (NARMS) in 2005 were screened by PCR using ColE1 primer sets. Thirty isolates were found to be positive for ColE1-like replicon. Plasmids from 23 isolates were able to propagate in Escherichia coli and were subjected to further characterization. Restriction mapping revealed three major plasmid groups found in three or more isolates, with each group consisting of two to three subtypes. The aph genes from the Kan(r) Salmonella isolates were amplified by PCR, sequenced, and showed four different aph(3')-I genes. The distribution of the ColE1 plasmid groups in association with the aph gene, Salmonella serovar, and isolate source demonstrated a strong linkage of the plasmid with S. enterica serovar Typhimurium DT104. Due to their high copy number and mobility, the ColE1-like plasmids may play a critical role in transmission of antibiotic resistance genes among enteric pathogens, and these findings warrant a close monitoring of this plasmid incompatibility group.

Prevalence of ColE1-Like Plasmids and Kanamycin Resistance Genes in Salmonella enterica Serovars ▿

PubMed Central

Chen, Chin-Yi; Lindsey, Rebecca L.; Strobaugh, Terence P.; Frye, Jonathan G.; Meinersmann, Richard J.

2010-01-01

Multi-antimicrobial-resistant Salmonella enterica strains frequently carry resistance genes on plasmids. Recent studies focus heavily on large conjugative plasmids, and the role that small plasmids play in resistance gene transfer is largely unknown. To expand our previous studies in assessing the prevalence of the isolates harboring ColE1-like plasmids carrying the aph gene responsible for kanamycin resistance (Kanr) phenotypes, 102 Kanr Salmonella isolates collected through the National Antimicrobial Resistance Monitoring System (NARMS) in 2005 were screened by PCR using ColE1 primer sets. Thirty isolates were found to be positive for ColE1-like replicon. Plasmids from 23 isolates were able to propagate in Escherichia coli and were subjected to further characterization. Restriction mapping revealed three major plasmid groups found in three or more isolates, with each group consisting of two to three subtypes. The aph genes from the Kanr Salmonella isolates were amplified by PCR, sequenced, and showed four different aph(3′)-I genes. The distribution of the ColE1 plasmid groups in association with the aph gene, Salmonella serovar, and isolate source demonstrated a strong linkage of the plasmid with S. enterica serovar Typhimurium DT104. Due to their high copy number and mobility, the ColE1-like plasmids may play a critical role in transmission of antibiotic resistance genes among enteric pathogens, and these findings warrant a close monitoring of this plasmid incompatibility group. PMID:20693446

Strategy to approach stable production of recombinant nattokinase in Bacillus subtilis.

PubMed

Chen, Po Ting; Chiang, Chung-Jen; Chao, Yun-Peng

2007-01-01

Bacillus subtilis (B. subtilis) is widely accepted as an excellent host cell for the secretory production of recombinant proteins. In this study, a shuttle vector was constructed by fusion of Staphylococcus aureus (S. aureus) plasmid pUB110 with Escherichia coli (E. coli) plasmid pUC18 and used for the expression of nattokinase in B. subtilis. The pUB110/pUC-based plasmid was found to exhibit high structural instability with the identification of a DNA deletion between two repeated regions. An initial attempt was made to eliminate the homologous site in the plasmid, whereas the stability of the resulting plasmid was not improved. In an alternative way, the pUC18-derived region in this hybrid vector was replaced by the suicidal R6K plasmid origin of E. coli. As a consequence, the pUB110/R6K-based plasmid displayed full structural stability, leading to a high-level production of recombinant nattokinase in the culture broth. This was mirrored by the detection of a very low level of high molecular weight DNAs generated by the plasmid. Moreover, 2-fold higher nattokinase production was obtained by B. subtilis strain carrying the pUB110/R6K-based plasmid as compared to the cell with the pAMbeta1-derived vector, a plasmid known to have high structural stability. Overall, it indicates the feasibility of the approach by fusing two compatible plasmid origins for stable and efficient production of recombinant nattokinase in B. subtilis.

Transfer of Herb-Resistance Plasmid From Escherichia coli to Staphylococcus aureus Residing in the Human Urinary Tract

PubMed Central

Tong, Yan Qing; Xin, Bing; Zhu, Li

2014-01-01

Background: Plasmid transfer among bacteria provides a means for dissemination of resistance. Plasmid Analysis has made it possible to track plasmids that induce resistance in bacterial population. Objectives: To screen the presence of herb-resistance plasmid in Escherichia coli strains and determine the transferability of this resistance plasmid directly from E. coli to the Gram-positive, Staphylococcus aureus. Materials and Methods: The donor strain E. coli CP9 and recipient strain S. aureus RN450RF were isolated from UTI patients. E. coli CP9 was highly resistant to herbal concoction. Isolates of S. aureus RN450RF were fully susceptible. Total plasmid DNA was prepared and transferred into E. coli DH5α. Transconjugants were selected on agar plates containing serial dilutions of herbal concoction. Resistance plasmid was transferred to susceptible S. aureus RN450RFin triple replicas. The mating experiments were repeated twice. Results: The identified 45 kb herb-resistance plasmid could be transferred from E. coli CP9 isolates to E. coli DH5α. As a consequence E. coli DH5α transconjugant MIC increased from 0.0125 g/mL to 0.25 g/mL. The plasmid was easily transferred from E. coli CP9 strain to S. aureus RN450RF with a mean transfer rate of 1×10-2 transconjugants/recipient. The E. coli donor and the S. aureus RN450RF transconjugant contained a plasmid of the same size, which was absent in the recipient before mating. Susceptibility testing showed that the S. aureus RN450RF transconjugant was resistant to herbal concoction. Conclusions: E. coli herb-resistance plasmid can replicate and be expressed in S. aureus. PMID:25147679

Prevalence of plasmid-bearing and plasmid-free Chlamydia trachomatis infection among women who visited obstetrics and gynecology clinics in Malaysia.

PubMed

Yeow, Tee Cian; Wong, Won Fen; Sabet, Negar Shafiei; Sulaiman, Sofiah; Shahhosseini, Fatemeh; Tan, Grace Min Yi; Movahed, Elaheh; Looi, Chung Yeng; Shankar, Esaki M; Gupta, Rishien; Arulanandam, Bernard P; Hassan, Jamiyah; Abu Bakar, Sazaly

2016-03-18

The 7.5 kb cryptic plasmid of Chlamydia trachomatis has been shown to be a virulence factor in animal models, but its significance in humans still remains unknown. The aim of this study was to investigate the prevalence and potential involvement of the C. trachomatis cryptic plasmid in causing various clinical manifestations; including infertility, reproductive tract disintegrity, menstrual disorder, and polycystic ovarian syndrome (PCOS) among genital C. trachomatis-infected patients. A total of 180 female patients of child bearing age (mean 30.9 years old, IQR:27-35) with gynecological complications and subfertility issues, who visited Obstetrics and Gynecology clinics in Kuala Lumpur, Malaysia were recruited for the study. Prevalence of genital chlamydial infection among these patients was alarmingly high at 51.1% (92/180). Of the 92 chlamydia-infected patients, 93.5% (86/92) were infected with plasmid-bearing (+) C. trachomatis while the remaining 6.5% (6/92) were caused by the plasmid-free (-) variant. Our data showed that genital C. trachomatis infection was associated with infertility issues, inflammation in the reproductive tract (mucopurulent cervicitis or endometriosis), irregular menstrual cycles and polycystic ovarian syndrome (PCOS). However, no statistical significance was detected among patients with plasmid (+) versus plasmid (-) C. trachomatis infection. Interestingly, plasmid (+) C. trachomatis was detected in all patients with PCOS, and the plasmid copy numbers were significantly higher among PCOS patients, relative to non-PCOS patients. Our findings show a high incidence of C. trachomatis infection among women with infertility or gynecological problems in Malaysia. However, due to the low number of plasmid (-) C. trachomatis cases, a significant role of the plasmid in causing virulence in human requires further investigation of a larger cohort.

Changing plasmid types responsible for extended spectrum cephalosporin resistance in Escherichia coli O157:H7 in the United States, 1996–2009

PubMed Central

Folster, J. P.; Pecic, G.; Stroika, S.; Rickert, R.; Whichard, J.

2015-01-01

Escherichia coli O157 is a major cause of foodborne illness. Plasmids are genetic elements that mobilize antimicrobial resistance determinants including blaCMY β-lactamases that confer resistance to extended-spectrum cephalosporins (ESC). ESCs are important for treating a variety of infections. IncA/C plasmids are found among diverse sources, including cattle, the principal source of E. coli O157 infections in humans. IncI1 plasmids are common among E. coli and Salmonella from poultry and other avian sources. To broaden our understanding of reservoirs of blaCMY, we determined the types of plasmids carrying blaCMY among E. coli O157. From 1996 to 2009, 3742 E. coli O157 isolates were tested. Eleven (0.29%) were ceftriaxone resistant and had a blaCMY-2-containing plasmid. All four isolates submitted before 2001 and a single 2001 isolate had blaCMY encoded on IncA/C plasmids, while all five isolates submitted after 2001 and a single 2001 isolate had blaCMY carried on IncI1 plasmids. The IncI1 plasmids were ST2, ST20, and ST23. We conclude that cephalosporin resistance among E. coli O157:H7 is due to plasmid-encoded blaCMY genes and that plasmid types appear to have shifted from IncA/C to IncI1. This shift suggests either a change in plasmid type among animal reservoirs or that the organism has expanded into avian reservoirs. More analysis of human, retail meat, and food animal isolates is necessary to broaden our understanding of the antimicrobial resistance determinants of ESC resistance among E. coli O157. PMID:26478858

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

PubMed Central

Maj, Anna; Dziewit, Lukasz; Czarnecki, Jakub; Wlodarczyk, Miroslawa; Baj, Jadwiga; Skrzypczyk, Grazyna; Giersz, Dorota; Bartosik, Dariusz

2013-01-01

Plasmids are components of many bacterial genomes. They enable the spread of a large pool of genetic information via lateral gene transfer. Many bacterial strains contain mega-sized replicons and these are particularly common in Alphaproteobacteria. Considerably less is known about smaller alphaproteobacterial plasmids. We analyzed the genomes of 14 such plasmids residing in 4 multireplicon carotenoid-producing strains of the genus Paracoccus (Alphaproteobacteria): P. aestuarii DSM 19484, P. haeundaensis LG P-21903, P. marcusii DSM 11574 and P. marcusii OS22. Comparative analyses revealed mosaic structures of the plasmids and recombinational shuffling of diverse genetic modules involved in (i) plasmid replication, (ii) stabilization (including toxin-antitoxin systems of the relBE/parDE, tad-ata, higBA, mazEF and toxBA families) and (iii) mobilization for conjugal transfer (encoding relaxases of the MobQ, MobP or MobV families). A common feature of the majority of the plasmids is the presence of AT-rich sequence islets (located downstream of exc1-like genes) containing genes, whose homologs are conserved in the chromosomes of many bacteria (encoding e.g. RelA/SpoT, SMC-like proteins and a retron-type reverse transcriptase). The results of this study have provided insight into the diversity and plasticity of plasmids of Paracoccus spp., and of the entire Alphaproteobacteria. Some of the identified plasmids contain replication systems not described previously in this class of bacteria. The composition of the plasmid genomes revealed frequent transfer of chromosomal genes into plasmids, which significantly enriches the pool of mobile DNA that can participate in lateral transfer. Many strains of Paracoccus spp. have great biotechnological potential, and the plasmid vectors constructed in this study will facilitate genetic studies of these bacteria. PMID:24260361

Characterization of Plasmids in Extensively Drug-Resistant Acinetobacter Strains Isolated in India and Pakistan

PubMed Central

Carvalho, Maria J.; Toleman, Mark A.; White, P. Lewis; Connor, Thomas R.; Mushtaq, Ammara; Weeks, Janis L.; Kumarasamy, Karthikeyan K.; Raven, Katherine E.; Török, M. Estée; Peacock, Sharon J.; Howe, Robin A.; Walsh, Timothy R.

2014-01-01

The blaNDM-1 gene is associated with extensive drug resistance in Gram-negative bacteria. This probably spread to Enterobacteriaceae from Acinetobacter spp., and we characterized plasmids associated with blaNDM-1 in Acinetobacter spp. to gain insight into their role in this dissemination. Four clinical NDM-1-producing Acinetobacter species strains from India and Pakistan were investigated. A plasmid harboring blaNDM-1, pNDM-40-1, was characterized by whole-genome sequencing of Acinetobacter bereziniae CHI-40-1 and comparison with related plasmids. The presence of similar plasmids in strains from Pakistan was sought by PCR and sequencing of amplicons. Conjugation frequency was tested and stability of pNDM-40-1 investigated by real-time PCR of isolates passaged with and without antimicrobial selection pressure. A. bereziniae and Acinetobacter haemolyticus strains contained plasmids similar to the pNDM-BJ01-like plasmids identified in Acinetobacter spp. in China. The backbone of pNDM-40-1 was almost identical to that of pNDM-BJ01-like plasmids, but the transposon harboring blaNDM-1, Tn125, contained two short deletions. Escherichia coli and Acinetobacter pittii transconjugants were readily obtained. Transconjugants retained pNDM-40-1 after a 14-day passage experiment, although stability was greater with meropenem selection. Fragments of pNDM-BJ01-like plasmid backbones are found near blaNDM-1 in some genetic contexts from Enterobacteriaceae, suggesting that cross-genus transfer has occurred. pNDM-BJ01-like plasmids have been described in isolates originating from a wide geographical region in southern Asia. In vitro data on plasmid transfer and stability suggest that these plasmids could have contributed to the spread of blaNDM-1 into Enterobacteriaceae. PMID:25421466

Novel plasmids and resistance phenotypes in Yersinia pestis: unique plasmid inventory of strain Java 9 mediates high levels of arsenic resistance.

PubMed

Eppinger, Mark; Radnedge, Lyndsay; Andersen, Gary; Vietri, Nicholas; Severson, Grant; Mou, Sherry; Ravel, Jacques; Worsham, Patricia L

2012-01-01

Growing evidence suggests that the plasmid repertoire of Yersinia pestis is not restricted to the three classical virulence plasmids. The Java 9 strain of Y. pestis is a biovar Orientalis isolate obtained from a rat in Indonesia. Although it lacks the Y. pestis-specific plasmid pMT, which encodes the F1 capsule, it retains virulence in mouse and non-human primate animal models. While comparing diverse Y. pestis strains using subtractive hybridization, we identified sequences in Java 9 that were homologous to a Y. enterocolitica strain carrying the transposon Tn2502, which is known to encode arsenic resistance. Here we demonstrate that Java 9 exhibits high levels of arsenic and arsenite resistance mediated by a novel promiscuous class II transposon, named Tn2503. Arsenic resistance was self-transmissible from Java 9 to other Y. pestis strains via conjugation. Genomic analysis of the atypical plasmid inventory of Java 9 identified pCD and pPCP plasmids of atypical size and two previously uncharacterized cryptic plasmids. Unlike the Tn2502-mediated arsenic resistance encoded on the Y. enterocolitica virulence plasmid; the resistance loci in Java 9 are found on all four indigenous plasmids, including the two novel cryptic plasmids. This unique mobilome introduces more than 105 genes into the species gene pool. The majority of these are encoded by the two entirely novel self-transmissible plasmids, which show partial homology and synteny to other enterics. In contrast to the reductive evolution in Y. pestis, this study underlines the major impact of a dynamic mobilome and lateral acquisition in the genome evolution of the plague bacterium.

Novel Plasmids and Resistance Phenotypes in Yersinia pestis: Unique Plasmid Inventory of Strain Java 9 Mediates High Levels of Arsenic Resistance

PubMed Central

Eppinger, Mark; Radnedge, Lyndsay; Andersen, Gary; Vietri, Nicholas; Severson, Grant; Mou, Sherry; Ravel, Jacques; Worsham, Patricia L.

2012-01-01

Growing evidence suggests that the plasmid repertoire of Yersinia pestis is not restricted to the three classical virulence plasmids. The Java 9 strain of Y. pestis is a biovar Orientalis isolate obtained from a rat in Indonesia. Although it lacks the Y. pestis-specific plasmid pMT, which encodes the F1 capsule, it retains virulence in mouse and non-human primate animal models. While comparing diverse Y. pestis strains using subtractive hybridization, we identified sequences in Java 9 that were homologous to a Y. enterocolitica strain carrying the transposon Tn2502, which is known to encode arsenic resistance. Here we demonstrate that Java 9 exhibits high levels of arsenic and arsenite resistance mediated by a novel promiscuous class II transposon, named Tn2503. Arsenic resistance was self-transmissible from Java 9 to other Y. pestis strains via conjugation. Genomic analysis of the atypical plasmid inventory of Java 9 identified pCD and pPCP plasmids of atypical size and two previously uncharacterized cryptic plasmids. Unlike the Tn2502-mediated arsenic resistance encoded on the Y. enterocolitica virulence plasmid; the resistance loci in Java 9 are found on all four indigenous plasmids, including the two novel cryptic plasmids. This unique mobilome introduces more than 105 genes into the species gene pool. The majority of these are encoded by the two entirely novel self-transmissible plasmids, which show partial homology and synteny to other enterics. In contrast to the reductive evolution in Y. pestis, this study underlines the major impact of a dynamic mobilome and lateral acquisition in the genome evolution of the plague bacterium. PMID:22479347

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.

Molecular Diversity of Plasmids Bearing Genes That Encode Toluene and Xylene Metabolism in Pseudomonas Strains Isolated from Different Contaminated Sites in Belarus

PubMed Central

Sentchilo, Vladimir S.; Perebituk, Alexander N.; Zehnder, Alexander J. B.; van der Meer, Jan Roelof

2000-01-01

Twenty different Pseudomonas strains utilizing m-toluate were isolated from oil-contaminated soil samples near Minsk, Belarus. Seventeen of these isolates carried plasmids ranging in size from 78 to about 200 kb (assigned pSVS plasmids) and encoding the meta cleavage pathway for toluene metabolism. Most plasmids were conjugative but of unknown incompatibility groups, except for one, which belonged to the IncP9 group. The organization of the genes for toluene catabolism was determined by restriction analysis and hybridization with xyl gene probes of pWW0. The majority of the plasmids carried xyl-type genes highly homologous to those of pWW53 and organized in a similar manner (M. T. Gallegos, P. A. Williams, and J. L. Ramos, J. Bacteriol. 179:5024–5029, 1997), with two distinguishable meta pathway operons, one upper pathway operon, and three xylS-homologous regions. All of these plasmids also possessed large areas of homologous DNA outside the catabolic genes, suggesting a common ancestry. Two other pSVS plasmids carried only one meta pathway operon, one upper pathway operon, and one copy each of xylS and xylR. The backbones of these two plasmids differed greatly from those of the others. Whereas these parts of the plasmids, carrying the xyl genes, were mostly conserved between plasmids of each group, the noncatabolic parts had undergone intensive DNA rearrangements. DNA sequencing of specific regions near and within the xylTE and xylA genes of the pSVS plasmids confirmed the strong homologies to the xyl genes of pWW53 and pWW0. However, several recombinations were discovered within the upper pathway operons of the pSVS plasmids and pWW0. The main genetic mechanisms which are thought to have resulted in the present-day configuration of the xyl operons are discussed in light of the diversity analysis carried out on the pSVS plasmids. PMID:10877777

Identical plasmid AmpC beta-lactamase genes and plasmid types in E. coli isolates from patients and poultry meat in the Netherlands.

PubMed

Voets, Guido M; Fluit, Ad C; Scharringa, Jelle; Schapendonk, Claudia; van den Munckhof, Thijs; Leverstein-van Hall, Maurine A; Stuart, James Cohen

2013-11-01

The increasing prevalence of third-generation cephalosporin-resistant Enterobacteriaceae is a worldwide problem. Recent studies showed that poultry meat and humans share identical Extended-Spectrum Beta-Lactamase genes, plasmid types, and Escherichia coli strain types, suggesting that transmission from poultry meat to humans may occur. The aim of this study was to compare plasmid-encoded Ambler class C beta-lactamase (pAmpC) genes, their plasmids, and bacterial strain types between E. coli isolates from retail chicken meat and clinical isolates in the Netherlands. In total, 98 Dutch retail chicken meat samples and 479 third-generation cephalosporin non-susceptible human clinical E. coli isolates from the same period were screened for pAmpC production. Plasmid typing was performed using PCR-based replicon typing (PBRT). E coli strains were compared using Multi-Locus-Sequence-Typing (MLST). In 12 of 98 chicken meat samples (12%), pAmpC producing E. coli were detected (all blaCMY-2). Of the 479 human E. coli, 25 (5.2%) harboured pAmpC genes (blaCMY-2 n = 22, blaACT n = 2, blaMIR n = 1). PBRT showed that 91% of poultry meat isolates harboured blaCMY-2 on an IncK plasmid, and 9% on an IncI1 plasmid. Of the human blaCMY-2 producing isolates, 42% also harboured blaCMY-2 on an IncK plasmid, and 47% on an IncI1 plasmid. Thus, 68% of human pAmpC producing E. coli have the same AmpC gene (blaCMY-2) and plasmid type (IncI1 or IncK) as found in poultry meat. MLST showed one cluster containing one human isolate and three meat isolates, with an IncK plasmid. These findings imply that a foodborne transmission route of blaCMY-2 harbouring plasmids cannot be excluded and that further evaluation is required. © 2013.

Brownian Ratchet Mechanism for Faithful Segregation of Low-Copy-Number Plasmids.

PubMed

Hu, Longhua; Vecchiarelli, Anthony G; Mizuuchi, Kiyoshi; Neuman, Keir C; Liu, Jian

2017-04-11

Bacterial plasmids are extrachromosomal DNA that provides selective advantages for bacterial survival. Plasmid partitioning can be remarkably robust. For high-copy-number plasmids, diffusion ensures that both daughter cells inherit plasmids after cell division. In contrast, most low-copy-number plasmids need to be actively partitioned by a conserved tripartite ParA-type system. ParA is an ATPase that binds to chromosomal DNA; ParB is the stimulator of the ParA ATPase and specifically binds to the plasmid at a centromere-like site, parS. ParB stimulation of the ParA ATPase releases ParA from the bacterial chromosome, after which it takes a long time to reset its DNA-binding affinity. We previously demonstrated in vitro that the ParA system can exploit this biochemical asymmetry for directed cargo transport. Multiple ParA-ParB bonds can bridge a parS-coated cargo to a DNA carpet, and they can work collectively as a Brownian ratchet that directs persistent cargo movement with a ParA-depletion zone trailing behind. By extending this model, we suggest that a similar Brownian ratchet mechanism recapitulates the full range of actively segregated plasmid motilities observed in vivo. We demonstrate that plasmid motility is tuned as the replenishment rate of the ParA-depletion zone progressively increases relative to the cargo speed, evolving from diffusion to pole-to-pole oscillation, local excursions, and, finally, immobility. When the plasmid replicates, the daughters largely display motilities similar to that of their mother, except that when the single-focus progenitor is locally excursive, the daughter foci undergo directed segregation. We show that directed segregation maximizes the fidelity of plasmid partition. Given that local excursion and directed segregation are the most commonly observed modes of plasmid motility in vivo, we suggest that the operation of the ParA-type partition system has been shaped by evolution for high fidelity of plasmid segregation. Published by Elsevier Inc.

Complete sequences of a novel blaNDM-1-harbouring plasmid from Providencia rettgeri and an FII-type plasmid from Klebsiella pneumoniae identified in Canada.

PubMed

Mataseje, L F; Boyd, D A; Lefebvre, B; Bryce, E; Embree, J; Gravel, D; Katz, K; Kibsey, P; Kuhn, M; Langley, J; Mitchell, R; Roscoe, D; Simor, A; Taylor, G; Thomas, E; Turgeon, N; Mulvey, M R

2014-03-01

Emergence of plasmids harbouring bla(NDM-1) is a major public health concern due to their association with multidrug resistance and their potential mobility. PCR was used to detect bla(NDM-1) from clinical isolates of Providencia rettgeri (PR) and Klebsiella pneumoniae (KP). Antimicrobial susceptibilities were determined using Vitek 2. The complete DNA sequence of two bla(NDM-1) plasmids (pPrY2001 and pKp11-42) was obtained using a 454-Genome Sequencer FLX. Contig assembly and gap closures were confirmed by PCR-based sequencing. Comparative analysis was done using BLASTn and BLASTp algorithms. Both clinical isolates were resistant to all β-lactams, carbapenems, aminoglycosides, ciprofloxacin and trimethoprim/sulfamethoxazole, and susceptible to tigecycline. Plasmid pPrY2001 (113 295 bp) was isolated from PR. It did not show significant homology to any known plasmid backbone and contained a truncated repA and novel repB. Two bla(NDM-1)-harbouring plasmids from Acinetobacter lwoffii (JQ001791 and JQ060896) shared 100% similarity to a 15 kb region that contained bla(NDM-1). pPrY2001 also contained a type II toxin/antitoxin system. pKp11-42 (146 695 bp) was isolated from KP. It contained multiple repA genes. The plasmid backbone had the highest homology to the IncFIIk plasmid type (51% coverage, 100% nucleotide identity). The bla(NDM-1) region was unique in that it was flanked upstream by IS3000 and downstream by a novel transposon designated Tn6229. pKp11-42 also contained a number of mutagenesis and plasmid stability proteins. pPrY2001 differed from all known plasmids due to its novel backbone and repB. pKp11-42 was similar to IncFIIk plasmids and contained a number of genes that aid in plasmid persistence.

Asymmetry and Extent of In Vivo Transcripition of R-Plasmid Deoxyribonucleic Acid in Escherichia coli

PubMed Central

Vapnek, Daniel; Spingler, Elizabeth

1974-01-01

Deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization studies have been performed with R-plasmid DNA (R538-1drd) and in vivo-synthesized RNA. R-plasmid DNA was isolated from Escherichia coli K-12, and the complementary strands were separated in cesium chloride-polyuridylic acid-polyguanylic acid gradients. DNA-RNA hybridization was performed with the separated DNA strands and RNA purified from R-plasmid-carrying cells. The results demonstrated that an asymmetric transcription of the R-plasmid DNA occurs in vivo. Hybridization was only detected with the H strand (denser strand in cesium chloride-polyuridylic acid-polyguanylic acid). By determining the density of the RNA-DNA hybrid in CsCl gradients, it was estimated that greater than 60% of the nucleotide sequences in the R-plasmid DNA are transcribed in logarithmically growing E. coli cells. No R-plasmid-specific RNA was detected in E. coli cells that did not carry the plasmid. PMID:4612013

Characterization of new plasmids from methylotrophic bacteria.

PubMed

Brenner, V; Holubová, I; Benada, O; Hubácek, J

1991-07-01

Several tens of methanol-utilizing bacterial strains isolated from soil were screened for the presence of plasmids. From the obligate methylotroph Methylomonas sp. strain R103a plasmid pIH36 (36 kb) was isolated and its restriction map was constructed. In pink-pigmented facultative methylotrophs (PPFM), belonging to the genus Methylobacterium four plasmids were detected: plasmids pIB200 (200 kb) and pIB14 (14 kb) in the strain R15d and plasmids pWU14 (14 kb) and pWU7 (7.8 kb) in the strain M17. Because of the small size and the presence of several unique REN sites (HindIII, EcoRI, NcoI), plasmid pWU7 was chosen for the construction of a vector for cloning in methylotrophs. Cointegrates pKWU7A and pKWU7B were formed between pWU7 and the E. coli plasmid pK19 Kmr, which were checked for conjugative transfer from E. coli into the methylotrophic host.

Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR.

PubMed

Plotka, Magdalena; Wozniak, Mateusz; Kaczorowski, Tadeusz

2017-01-01

Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within Escherichia coli DH5α cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a straightforward and reliable method to quantify the plasmid copy number. Therefore we believe that the ddPCR designed in this study will be widely used for any plasmid copy number calculation in the future.

Wild corvid birds colonized with vancomycin-resistant Enterococcus faecium of human origin harbor epidemic vanA plasmids.

PubMed

Oravcová, Veronika; Peixe, Luísa; Coque, Teresa M; Novais, Carla; Francia, Maria V; Literák, Ivan; Freitas, Ana R

2018-06-02

The most prevalent type of acquired vancomycin resistance in Enterococcus faecium (VREfm) is encoded by the vanA transposon Tn1546, mainly located on transferable plasmids. vanA plasmids have been characterized in VREfm from a variety of sources but not wild birds. The aim of this study was to analyse the genetic context of VREfm strains recovered from wild corvid birds and to compare their plasmid and strain characteristics with human strains. To achieve that, 75 VREfm isolates, including strains from wild birds recovered during wide surveillance studies performed in Europe, Canada and the United States (2010-2013), and clinical and wastewater strains from Czech Republic, a region lacking data about vanA plasmids, were analysed. Their population structure, presence of major putative virulence markers and characterization of vanA transposons and plasmids were established. VREfm from wild birds were mainly associated with major human lineages (ST18 and ST78) circulating in hospitals worldwide and were enriched in putative virulence markers that are highly associated with clinical E. faecium from human infections. They also carried plasmids of the same families usually found in the clinical setting [RCR, small theta plasmids, RepA_N (pRUM/pLG1) and Inc18]. The clinically widespread IS1251-carrying Tn1546 type "F" was predominant and Tn1546-vanA was mainly located on pRUM/Axe-Txe (USA) and Inc18- or pLG1-like (Europe) plasmids. VREfm from hospitals and wastewaters carried Tn1546-vanA in different plasmid types including mosaic pRUM-Inc18 plasmids, not identified in wild birds. This is the first characterization of vanA plasmids obtained from wild birds. A similar plasmid pool seems to exist in different clonal E. faecium backgrounds of humans and wild birds. The isolation of VREfm strains from wild birds that belong to human E. faecium adapted lineages and carry virulence genes, Tn1546 and plasmid variants widespread in the clinical setting is of concern and highlight their role as potential drivers of the global dissemination of vancomycin resistance. Copyright © 2018 Elsevier Ltd. All rights reserved.

Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR

PubMed Central

Plotka, Magdalena; Wozniak, Mateusz; Kaczorowski, Tadeusz

2017-01-01

Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within Escherichia coli DH5α cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a straightforward and reliable method to quantify the plasmid copy number. Therefore we believe that the ddPCR designed in this study will be widely used for any plasmid copy number calculation in the future. PMID:28085908

pHTβ-promoted mobilization of non-conjugative resistance plasmids from Enterococcus faecium to Enterococcus faecalis.

PubMed

Di Sante, Laura; Morroni, Gianluca; Brenciani, Andrea; Vignaroli, Carla; Antonelli, Alberto; D'Andrea, Marco Maria; Di Cesare, Andrea; Giovanetti, Eleonora; Varaldo, Pietro E; Rossolini, Gian Maria; Biavasco, Francesca

2017-09-01

To analyse the recombination events associated with conjugal mobilization of two multiresistance plasmids, pRUM17i48 and pLAG (formerly named pDO1-like), from Enterococcus faecium 17i48 to Enterococcus faecalis JH2-2. The plasmids from two E. faecalis transconjugants (JH-4T, tetracycline resistant, and JH-8E, erythromycin resistant) and from the E. faecium donor (also carrying a pHTβ-like conjugative plasmid, named pHTβ17i48) were investigated by several methods, including PCR mapping and sequencing, S1-PFGE followed by Southern blotting and hybridization, and WGS. Two locations of repApHTβ were detected in both transconjugants, one on a ∼50 kb plasmid (as in the donor) and the other on plasmids of larger sizes. In JH-4T, WGS disclosed an 88.6 kb plasmid resulting from the recombination of pHTβ17i48 (∼50 kb) and a new plasmid, named pLAG (35.3 kb), carrying the tet(M), tet(L), lsa(E), lnu(B), spw and aadE resistance genes. In JH-8E, a 75 kb plasmid resulting from the recombination of pHTβ17i48 and pRUM17i48 was observed. In both cases, the cointegrates were apparently derived from replicative transposition of an IS1216 present in each of the multiresistance plasmids into pHTβ17i48. The cointegrates could resolve to yield the multiresistance plasmids and a pHTβ17i48 derivative carrying an IS1216 (unlike the pHTβ17i48 of the donor). Our results completed the characterization of the multiresistance plasmids carried by the E. faecium 17i48, confirming the role of pHT plasmids in the mobilization of non-conjugative antibiotic resistance elements among enterococci. Results also revealed that mobilization to E. faecalis was associated with the generation of cointegrate plasmids promoted by IS1216-mediated transposition. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Cloning of the citrate permease gene of Lactococcus lactis subsp. lactis biovar diacetylactis and expression in Escherichia coli.

PubMed Central

Sesma, F; Gardiol, D; de Ruiz Holgado, A P; de Mendoza, D

1990-01-01

The citrate plasmid (Cit+ plasmid) from Lactococcus lactis subsp. lactis biovar diacetylactis was cloned into the EcoRI site of plasmid pUC18. This recombinant plasmid enabled Escherichia coli K-12 to transport and utilize citrate as a source of energy, indicating expression of the citrate permease from L. lactis biovar diacetylactis. The citrate permease was under the control of the lac promoter of pUC18. Genetic expression of the Cit+ plasmid in maxicells revealed that the plasmid encoded two polypeptides of 47 and 32 kilodaltons, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Images PMID:2117878

Large-scale preparation of plasmid DNA.

PubMed

Heilig, J S; Elbing, K L; Brent, R

2001-05-01

Although the need for large quantities of plasmid DNA has diminished as techniques for manipulating small quantities of DNA have improved, occasionally large amounts of high-quality plasmid DNA are desired. This unit describes the preparation of milligram quantities of highly purified plasmid DNA. The first part of the unit describes three methods for preparing crude lysates enriched in plasmid DNA from bacterial cells grown in liquid culture: alkaline lysis, boiling, and Triton lysis. The second part describes four methods for purifying plasmid DNA in such lysates away from contaminating RNA and protein: CsCl/ethidium bromide density gradient centrifugation, polyethylene glycol (PEG) precipitation, anion-exchange chromatography, and size-exclusion chromatography.

The selfish yeast plasmid uses the nuclear motor Kip1p but not Cin8p for its localization and equal segregation

PubMed Central

Cui, Hong; Ghosh, Santanu K.

2009-01-01

The 2 micron plasmid of Saccharomyces cerevisiae uses the Kip1 motor, but not the functionally redundant Cin8 motor, for its precise nuclear localization and equal segregation. The timing and lifetime of Kip1p association with the plasmid partitioning locus STB are consistent with Kip1p being an authentic component of the plasmid partitioning complex. Kip1–STB association is not blocked by disassembling the mitotic spindle. Lack of Kip1p disrupts recruitment of the cohesin complex at STB and cohesion of replicated plasmid molecules. Colocalization of a 2 micron reporter plasmid with Kip1p in close proximity to the spindle pole body is reminiscent of that of a CEN reporter plasmid. Absence of Kip1p displaces the plasmid from this nuclear address, where it has the potential to tether to a chromosome or poach chromosome segregation factors. Exploiting Kip1p, which is subsidiary to Cin8p for chromosome segregation, to direct itself to a “partitioning center” represents yet another facet of the benign parasitism of the yeast plasmid. PMID:19364922

Closely related NDM-1-encoding plasmids from Escherichia coli and Klebsiella pneumoniae in Taiwan.

PubMed

Chen, Chao-Ju; Wu, Tsu-Lan; Lu, Po-Liang; Chen, Ying-Tsong; Fung, Chang-Phone; Chuang, Yin-Ching; Lin, Jung-Chung; Siu, L Kristopher

2014-01-01

Two plasmids carrying blaNDM-1 isolated from carbapenem-resistant Klebsiella pneumoniae (CR-KP) and carbapenem-resistant Escherichia coli (CR-EC) were sequenced. CR-KP and CR-EC were isolated from two Taiwanese patients without travel histories. Complete sequencing of the plasmids (pLK75 and pLK78) was conducted using a shotgun approach. Annotation of the contigs was performed using the RAST Server, followed by manual inspection and correction. These similar plasmids were obtained from two patients with overlapping stays at the same hospital. The pLK75 and pLK78 plasmids were 56,489-bp and 56,072-bp in length, respectively. Plasmid annotation revealed a common backbone similar to the IncN plasmid pR46. The regions flanking the blaNDM-1 genes in these plasmids were very similar to plasmid pNDM-HU01 in Japan, which contains a complex class 1 integron located next to an ISCR1 element. The ISCR1 element has been suggested to provide a powerful mechanism for mobilising antibiotic resistance genes. Two indigenous NDM-1-producing Enterobacteriaceae cases were identified for the first time in Taiwan, highlighting the alarming introduction of NDM-1-producing Enterobacteriaceae in this region.

Genomic and functional characterisation of IncX3 plasmids encoding blaSHV-12 in Escherichia coli from human and animal origin.

PubMed

Liakopoulos, Apostolos; van der Goot, Jeanet; Bossers, Alex; Betts, Jonathan; Brouwer, Michael S M; Kant, Arie; Smith, Hilde; Ceccarelli, Daniela; Mevius, Dik

2018-05-16

The bla SHV-12 β-lactamase gene is one of the most prevalent genes conferring resistance to extended-spectrum β-lactams in Enterobacteriaceae disseminating within and between reservoirs, mostly via plasmid-mediated horizontal gene transfer. Yet, studies regarding the biology of plasmids encoding bla SHV-12 are very limited. In this study, we revealed the emergence of IncX3 plasmids alongside IncI1α/γ in bla SHV-12 in animal-related Escherichia coli isolates. Four representative bla SHV-12 -encoding IncX3 plasmids were selected for genome sequencing and further genetic and functional characterization. We report here the first complete sequences of IncX3 plasmids of animal origin and show that IncX3 plasmids exhibit remarkable synteny in their backbone, while the major differences lie in their bla SHV-12 -flanking region. Our findings indicate that plasmids of this subgroup are conjugative and highly stable, while they exert no fitness cost on their bacterial host. These favourable features might have contributed to the emergence of IncX3 amongst SHV-12-producing E. coli in the Netherlands, highlighting the epidemic potential of these plasmids.

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

PubMed Central

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

2011-01-01

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

Evidence that compatibility of closely related replicons in Clostridium perfringens depends on linkage to parMRC-like partitioning systems of different subfamilies.

PubMed

Watts, Thomas D; Johanesen, Priscilla A; Lyras, Dena; Rood, Julian I; Adams, Vicki

2017-05-01

Clostridium perfringens produces an extensive repertoire of toxins and extracellular enzymes, many of which are intimately involved in the progression of disease and are encoded by genes on conjugative plasmids. In addition, many C. perfringens strains can carry up to five of these conjugative toxin or antimicrobial resistance plasmids, each of which has a similar 35kb backbone. This conserved backbone includes the tcp conjugation locus and the central control region (CCR), which encodes genes involved in plasmid regulation, replication and partitioning, including a parMRC partitioning locus. Most conjugative plasmids in C. perfringens have a conserved replication protein, raising questions as to how multiple, closely related plasmids are maintained within a single strain. Bioinformatics analysis has highlighted the presence of at least 10 different parMRC partitioning system families (parMRC A-J ) in these plasmids, with differences in amino acid sequence identity between each ParM family ranging from 15% to 54%. No two plasmids that encode genes belonging to the same partitioning family have been observed in a single strain, suggesting that these families represent the basis for plasmid incompatibility. In an attempt to validate the proposed parMRC incompatibility groups, genetically marked C. perfringens plasmids encoding identical parMRC C or parMRC D homologues or different combinations of parMRC A , parMRC C and parMRC D family homologues were introduced into a single strain via conjugation. The stability of each plasmid was determined using an incompatibility assay in which the plasmid profile of each strain was monitored over the course of two days in the absence of direct selection. The results showed that plasmids with identical parMRC C or parMRC D homologues were incompatible and could not coexist in the absence of external selection. By contrast, plasmids that encoded different parMRC homologues were compatible and could coexist in the same cell in the absence of selection, with the exception of strains housing parMRC C and parMRC D combinations, which showed a minor incompatibility phenotype. In conclusion, we have provided the first direct evidence of plasmid incompatibility in Clostridium spp. and have shown experimentally that the compatibility of conjugative C. perfringens plasmids correlates with the presence of parMRC-like partitioning systems of different phylogenetic subfamilies. Copyright © 2017 Elsevier Inc. All rights reserved.

Prevalence and characterization of plasmids carrying sulfonamide resistance genes among Escherichia coli from pigs, pig carcasses and human.

PubMed

Wu, Shuyu; Dalsgaard, Anders; Hammerum, Anette M; Porsbo, Lone J; Jensen, Lars B

2010-07-30

Sulfonamide resistance is very common in Escherichia coli. The aim of this study was to characterize plasmids carrying sulfonamide resistance genes (sul1, sul2 and sul3) in E. coli isolated from pigs and humans with a specific objective to assess the genetic diversity of plasmids involved in the mobility of sul genes. A total of 501 E. coli isolates from pig feces, pig carcasses and human stools were tested for their susceptibility to selected antimicrobial. Multiplex PCR was conducted to detect the presence of three sul genes among the sulfonamide-resistant E. coli isolates. Fifty-seven sulfonamide-resistant E. coli were selected based on presence of sul resistance genes and subjected to conjugation and/or transformation experiments. S1 nuclease digestion followed by pulsed-field gel electrophoresis was used to visualize and determine the size of plasmids. Plasmids carrying sul genes were characterized by PCR-based replicon typing to allow a comparison of the types of sul genes, the reservoir and plasmid present. A total of 109/501 isolates exhibited sulfonamide resistance. The relative prevalences of sul genes from the three reservoirs (pigs, pig carcasses and humans) were 65%, 45% and 12% for sul2, sul1, and sul3, respectively. Transfer of resistance through conjugation was observed in 42/57 isolates. Resistances to streptomycin, ampicillin and trimethoprim were co-transferred in most strains. Class 1 integrons were present in 80% of sul1-carrying plasmids and 100% of sul3-carrying plasmids, but only in 5% of sul2-carrying plasmids. The sul plasmids ranged from 33 to 160-kb in size and belonged to nine different incompatibility (Inc) groups: FII, FIB, I1, FIA, B/O, FIC, N, HI1 and X1. IncFII was the dominant type in sul2-carrying plasmids (52%), while IncI1 was the most common type in sul1 and sul3-carrying plasmids (33% and 45%, respectively). Multireplicons were found associated with all three sul genes. Sul genes were distributed widely in E. coli isolated from pigs and humans with sul2 being most prevalent. Sul-carrying plasmids belonged to diverse replicon types, but most of detected plasmids were conjugative enabling horizontal transfer. IncFII seems to be the dominant replicon type in sul2-carrying plasmids from all three sources.

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.

Type 3 Fimbriae Encoded on Plasmids Are Expressed from a Unique Promoter without Affecting Host Motility, Facilitating an Exceptional Phenotype That Enhances Conjugal Plasmid Transfer

PubMed Central

Madsen, Jonas Stenløkke; Riber, Leise; Kot, Witold; Basfeld, Alrun; Burmølle, Mette; Hansen, Lars Hestbjerg; Sørensen, Søren Johannes

2016-01-01

Horizontal gene transfer (HGT), the transmission of genetic material to a recipient that is not the progeny of the donor, is fundamental in bacterial evolution. HGT is often mediated by mobile genetic elements such as conjugative plasmids, which may be in conflict with the chromosomal elements of the genome because they are independent replicons that may petition their own evolutionary strategy. Here we study differences between type 3 fimbriae encoded on wild type plasmids and in chromosomes. Using known and newly characterized plasmids we show that the expression of type 3 fimbriae encoded on plasmids is systematically different, as MrkH, a c-di-GMP dependent transcriptional activator is not needed for strong expression of the fimbriae. MrkH is required for expression of type 3 fimbriae of the Klebsiella pneumoniae chromosome, wherefrom the fimbriae operon (mrkABCDF) of plasmids is believed to have originated. We find that mrkABCDFs of plasmids are highly expressed via a unique promoter that differs from the original Klebsiella promoter resulting in fundamental behavioral consequences. Plasmid associated mrkABCDFs did not influence the swimming behavior of the host, that hereby acquired an exceptional phenotype being able to both actively swim (planktonic behavior) and express biofilm associated fimbriae (sessile behavior). We show that this exceptional phenotype enhances the conjugal transfer of the plasmid. PMID:27627107

Microneedle-mediated transcutaneous immunization with plasmid DNA coated on cationic PLGA nanoparticles.

PubMed

Kumar, Amit; Wonganan, Piyanuch; Sandoval, Michael A; Li, Xinran; Zhu, Saijie; Cui, Zhengrong

2012-10-28

Previously, it was shown that microneedle-mediated transcutaneous immunization with plasmid DNA can potentially induce a stronger immune response than intramuscular injection of the same plasmid DNA. In the present study, we showed that the immune responses induced by transcutaneous immunization by applying plasmid DNA onto a skin area pretreated with solid microneedles were significantly enhanced by coating the plasmid DNA on the surface of cationic nanoparticles. In addition, the net surface charge of the DNA-coated nanoparticles significantly affected their in vitro skin permeation and their ability to induce immune responses in vivo. Transcutaneous immunization with plasmid DNA-coated net positively charged nanoparticles elicited a stronger immune response than with plasmid DNA-coated net negatively charged nanoparticles or by intramuscular immunization with plasmid DNA alone. Transcutaneous immunization with plasmid DNA-coated net positively charged nanoparticles induced comparable immune responses as intramuscular injection of them, but transcutaneous immunization was able to induce specific mucosal immunity and a more balanced T helper type 1 and type 2 response. The ability of the net positively charged DNA-coated nanoparticles to induce a strong immune response through microneedle-mediated transcutaneous immunization may be attributed to their ability to increase the expression of the antigen gene encoded by the plasmid and to more effectively stimulate the maturation of antigen-presenting cells. Copyright © 2012 Elsevier B.V. All rights reserved.

Plasmid Vectors for Xylella fastidiosa Utilizing a Toxin-Antitoxin System for Stability in the Absence of Antibiotic Selection.

PubMed

Burbank, Lindsey P; Stenger, Drake C

2016-08-01

The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacterial genetics but there are only a limited number of plasmid vectors available that replicate in X. fastidiosa, and even fewer that are retained without antibiotic selection. Two plasmids are described here that show stable replication in X. fastidiosa and are effective for gene complementation both in vitro and in planta. Plasmid maintenance is facilitated by incorporation of the PemI/PemK plasmid addiction system, consisting of PemK, an endoribonuclease toxin, and its cognate antitoxin, PemI. Vector pXf20pemIK utilizes a native X. fastidiosa replication origin as well as a high-copy-number pUC origin for propagation in Escherichia coli cloning strains. Broad-host-range vector pBBR5pemIK is a medium- to low-copy-number plasmid based on the pBBR1 backbone. Both plasmids are maintained for extended periods of time in the absence of antibiotic selection, as well as up to 14 weeks in grapevine, without affecting bacterial fitness. These plasmids present an alternative to traditional complementation and expression vectors which rely on antibiotic selection for plasmid retention.

Two novel families of plasmids from hyperthermophilic archaea encoding new families of replication proteins

PubMed Central

Soler, Nicolas; Marguet, Evelyne; Cortez, Diego; Desnoues, Nicole; Keller, Jenny; van Tilbeurgh, Herman; Sezonov, Guennadi; Forterre, Patrick

2010-01-01

Thermococcales (phylum Euryarchaeota) are model organisms for physiological and molecular studies of hyperthermophiles. Here we describe three new plasmids from Thermococcales that could provide new tools and model systems for genetic and molecular studies in Archaea. The plasmids pTN2 from Thermococcus nautilus sp. 30-1 and pP12-1 from Pyrococcus sp. 12-1 belong to the same family. They have similar size (∼12 kb) and share six genes, including homologues of genes encoded by the virus PAV1 from Pyrococcus abyssi. The plasmid pT26-2 from Thermococcus sp. 26-2 (21.5 kb), that corresponds to another plasmid family, encodes many proteins having homologues in virus-like elements integrated in several genomes of Thermococcales and Methanococcales. Our analyses confirm that viruses and plasmids are evolutionary related and co-evolve with their hosts. Whereas all plasmids previously isolated from Thermococcales replicate by the rolling circle mechanism, the three plasmids described here probably replicate by the theta mechanism. The plasmids pTN2 and pP12-1 encode a putative helicase of the SFI superfamily and a new family of DNA polymerase, whose activity was demonstrated in vitro, whereas pT26-2 encodes a putative new type of helicase. This strengthens the idea that plasmids and viruses are a reservoir of novel protein families involved in DNA replication. PMID:20403814

Megaplasmids encode differing combinations of lantibiotics in Streptococcus salivarius.

PubMed

Wescombe, Philip A; Burton, Jeremy P; Cadieux, Peter A; Klesse, Nikolai A; Hyink, Otto; Heng, Nicholas C K; Chilcott, Chris N; Reid, Gregor; Tagg, John R

2006-10-01

Streptococcus salivarius strains commonly produce bacteriocins as putative anti-competitor or signalling molecules. Here we report that bacteriocin production by the oral probiotic strain S. salivarius K12 is encoded by a large (ca. 190 kb) plasmid. Oral cavity transmission of the plasmid from strain K12 to a plasmid-negative variant of this bacterium was demonstrated in two subjects. Tests of additional S. salivarius strains showed large (up to ca. 220 kb) plasmids present in bacteriocin-producing isolates. Various combinations (up to 3 per plasmid) of loci encoding the known streptococcal lantibiotics salivaricin A, salivaricin B, streptin and SA-FF22 were localised to these plasmids. Since all bacteriocin-producing strains of S. salivarius tested to date appear to harbour plasmids, it appears that they may function as mobile repositories for bacteriocin loci, especially those of the lantibiotic class.

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

PubMed

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

2015-01-01

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

Occurrence of small Hsd plasmids in Salmonella typhi, Shigella boydii, and Escherichia coli.

PubMed Central

Yoshida, Y; Mise, K

1986-01-01

The natural occurrence of small Hsd (host specificity for DNA) plasmids was demonstrated in restriction endonuclease-producing strains of Salmonella typhi, Shigella boydii, and Escherichia coli. The five Hsd plasmids isolated were between 5.0 and 12.2 kilobases long. The copy number of all the Hsd plasmids was high (more than 10 copies per cell). Introduction of these small plasmids into E. coli strain 0 drastically lowered the efficiency of plating of the lambda.0 phages (the efficiency of plating was less than 5 X 10(-5) PFU-1). High restriction endonuclease activities were detected in the Hsd plasmid-positive strains because of the elevated copy numbers of the hsdR+ gene. The advantages of using E. coli strains containing the small Hsd plasmids for purification of type II restriction endonucleases are discussed. Images PMID:3003023

Complementation of Conjugation Functions of Streptomyces lividans Plasmid pIJ101 by the Related Streptomyces Plasmid pSB24.2

PubMed Central

Pettis, Gregg S.; Prakash, Shubha

1999-01-01

A database search revealed extensive sequence similarity between Streptomyces lividans plasmid pIJ101 and Streptomyces plasmid pSB24.2, which is a deletion derivative of Streptomyces cyanogenus plasmid pSB24.1. The high degree of relatedness between the two plasmids allowed the construction of a genetic map of pSB24.2, consisting of putative transfer and replication loci. Two pSB24.2 loci, namely, the cis-acting locus for transfer (clt) and the transfer-associated korB gene, were shown to be capable of complementing the pIJ101 clt and korB functions, respectively, a result that is consistent with the notion that pIJ101 and the parental plasmid pSB24.1 encode highly similar, if not identical, conjugation systems. PMID:10419972

Selfish restriction modification genes: resistance of a resident R/M plasmid to displacement by an incompatible plasmid mediated by host killing.

PubMed

Naito, Y; Naito, T; Kobayashi, I

1998-01-01

Previous work from this laboratory demonstrated that plasmids carrying a type II restriction-modification gene complex are not easily lost from their bacterial host because plasmid-free segregant cells are killed through chromosome cleavage. Here, we have followed the course of events that takes place when an Escherichia coli rec BC sbcA strain carrying a plasmid coding for the PaeR7I restriction-modification (R/M) gene complex is transformed by a plasmid with an identical origin of replication. The number of transformants that appeared was far fewer than with the restriction-minus (r-) control. Most of the transformants were very small. After prolonged incubation, the number and the size of the colonies increased, but this increase never attained the level of the r- control. Most of the transformed colonies retained the drug-resistance of the resident, r+ m+ plasmid. These results indicate that post-segregational host killing occurs when a plasmid bearing an R/M gene complex is displaced by an incompatible plasmid. Such cell killing eliminates the competitor plasmid along with the host and, thus, would allow persistence of the R/M plasmid in the neighboring, clonal host cells in nature. This phenomenon is reminiscent of mammalian apoptosis and other forms of altruistic cell death strategy against infection. This type of resistance to displacement was also studied in a wild type Escherichia coli strain that was normal for homologous recombination (rec+). A number of differences between the recBC sbcA strain and the rec+ strain were observed and these will be discussed.

Evolution and comparative genomics of pAQU-like conjugative plasmids in Vibrio species.

PubMed

Li, Ruichao; Ye, Lianwei; Wong, Marcus Ho Yin; Zheng, Zhiwei; Chan, Edward Wai Chi; Chen, Sheng

2017-09-01

To investigate a set of MDR conjugative plasmids found in Vibrio species and characterize the underlying evolution process. pAQU-type plasmids from Vibrio species were sequenced using both Illumina and PacBio platforms. Bioinformatics tools were utilized to analyse the typical MDR regions and core genes in the plasmids. The nine pAQU-type plasmids ranged from ∼160 to 206 kb in size and were found to harbour as many as 111 core genes encoding conjugative, replication and maintenance functions. Eight plasmids were found to carry a typical MDR region, which contained various accessory and resistance genes, including ISCR1-blaPER-1-bearing complex class 1 integrons, ISCR2-floR, ISCR2-tet(D)-tetR-ISCR2, qnrVC6, a Tn10-like structure and others associated with mobile elements. Comparison between a plasmid without resistance genes and different MDR plasmids showed that integration of different mobile elements, such as IS26, ISCR1, ISCR2, IS10 and IS6100, into the plasmid backbone was the key mechanism by which foreign resistance genes were acquired during the evolution process. This study identified pAQU-type plasmids as emerging MDR conjugative plasmids among important pathogens from different origins in Asia. These findings suggest that aquatic bacteria constitute a major reservoir of resistance genes, which may be transmissible to other human pathogens during food production and processing. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Development of 3D in vitro platform technology to engineer mesenchymal stem cells.

PubMed

Hosseinkhani, Hossein; Hong, Po-Da; Yu, Dah-Shyong; Chen, Yi-Ru; Ickowicz, Diana; Farber, Ira-Yudovin; Domb, Abraham J

2012-01-01

This study aims to develop a three-dimensional in vitro culture system to genetically engineer mesenchymal stem cells (MSC) to express bone morphogenic protein-2. We employed nanofabrication technologies borrowed from the spinning industry, such as electrospinning, to mass-produce identical building blocks in a variety of shapes and sizes to fabricate electrospun nanofiber sheets comprised of composites of poly (glycolic acid) and collagen. Homogenous nanoparticles of cationic biodegradable natural polymer were formed by simple mixing of an aqueous solution of plasmid DNA encoded bone morphogenic protein-2 with the same volume of cationic polysaccharide, dextran-spermine. Rat bone marrow MSC were cultured on electrospun nanofiber sheets comprised of composites of poly (glycolic acid) and collagen prior to the incorporation of the nanoparticles into the nanofiber sheets. Bone morphogenic protein-2 was significantly detected in MSC cultured on nanofiber sheets incorporated with nanoparticles after 2 days compared with MSC cultured on nanofiber sheets incorporated with naked plasmid DNA. We conclude that the incorporation of nanoparticles into nanofiber sheets is a very promising strategy to genetically engineer MSC and can be used for further applications in regenerative medicine therapy.

Genetic and Physiological Studies of Bacillus anthracis Related to Development of an Improved Vaccine

DTIC Science & Technology

1987-07-01

nontransformable Bacillus species such as B. anthracis. Our results suggest that plasmid pLS20 of Bacillus subtilis ( natto ), which promotes transfer of the...mobilizing pBC16, pLS20 mediates transfer of the B. subtills ( natto ) plasmid pLS19 and the Staphylococcus aureus plasmid pUB110. To facilitate direct...and (v) transformation of B. cereus and B. anthracis with plasmid DNA. The 55-kb plasmid, pLS20, of Bacillus subtilis ( natto ) 3335 promotes tr msfer

Exploring the Role of Ubiquitination in Progesterone Receptor Transcriptional Activation and Turnover in Breast Cancer Cells

DTIC Science & Technology

2006-06-01

factors. T47DY cells were cotransfected with a PR construct, a PRE- luciferase plasmid and a renilla plasmid, for transfection control. The cells...PR-B or S294A PR-B, PRE-luciferase reporter constructs and a Renilla control plasmid. Cells were treated for 24hrs with or without R5020 (10nM...plasmid and a plasmid constitutively expressing renilla luciferase for transfection control. Cell were starved for one day and treated with or without

Properties of new Escherichia coli Hfr strains constructed by integration of pSC101-derived conjugative plasmids.

PubMed Central

François, V; Conter, A; Louarn, J M

1990-01-01

Conjugative temperature-sensitive plasmids were derived from pSC101. These plasmids are useful in genetic analysis for two reasons: (i) they render possible the construction of new Hfr lines by plasmid integration at predetermined chromosomal loci via Tn10 inverse transposition, and (ii) the Hfr characters are transducible via bacteriophage P1. We also showed that replication from pSC101 origin is deleterious for the plasmid-chromosome fusion. PMID:2155201

Efficient production of free fatty acids from ionic liquid-based acid- or enzyme-catalyzed bamboo hydrolysate.

PubMed

Mi, Le; Qin, Dandan; Cheng, Jie; Wang, Dan; Li, Sha; Wei, Xuetuan

2017-03-01

Two engineered Escherichia coli strains, DQ101 (MG1655 fadD - )/pDQTES and DQ101 (MG1655 fadD - )/pDQTESZ were constructed to investigate the free fatty acid production using ionic liquid-based acid- or enzyme-catalyzed bamboo hydrolysate as carbon source in this study. The plasmid, pDQTES, carrying an acyl-ACP thioesterase 'TesA of E. coli in pTrc99A was constructed firstly, and then (3R)-hydroxyacyl-ACP dehydratase was ligated after the TesA to give the plasmid pDQTESZ. These two strains exhibited efficient fatty acid production when glucose was used as the sole carbon source, with a final concentration of 2.45 and 3.32 g/L, respectively. The free fatty acid production of the two strains on xylose is not as efficient as that on glucose, which was 2.32 and 2.96 g/L, respectively. For mixed sugars, DQ101 (MG1655 fadD - )-based strains utilized glucose and pentose sequentially under the carbon catabolite repression (CCR) regulation. The highest total FFAs concentration from the mixed sugar culture reached 2.81 g/L by DQ101 (MG1655 fadD - )/pDQTESZ. Furthermore, when ionic liquid-based enzyme-catalyzed bamboo hydrolysate was used as the carbon source, the strain DQ101 (MG1655 fadD - )/pDQTESZ could produce 1.23 g/L FFAs with a yield of 0.13 g/g, and while it just produced 0.65 g/L free fatty acid with the ionic liquid-based acid-catalyzed bamboo hydrolysate as the feedstock. The results suggested that enzymatic catalyzed bamboo hydrolysate with ionic liquid pretreatment could serve as an efficient feedstock for free fatty acid production.

Towards the construction of high-quality mutagenesis libraries.

PubMed

Li, Heng; Li, Jing; Jin, Ruinan; Chen, Wei; Liang, Chaoning; Wu, Jieyuan; Jin, Jian-Ming; Tang, Shuang-Yan

2018-07-01

To improve the quality of mutagenesis libraries in directed evolution strategy. In the process of library transformation, transformants which have been shown to take up more than one plasmid might constitute more than 20% of the constructed library, thereby extensively impairing the quality of the library. We propose a practical transformation method to prevent the occurrence of multiple-plasmid transformants while maintaining high transformation efficiency. A visual library model containing plasmids expressing different fluorescent proteins was used. Multiple-plasmid transformants can be reduced through optimizing plasmid DNA amount used for transformation based on the positive correlation between the occurrence frequency of multiple-plasmid transformants and the logarithmic ratio of plasmid molecules to competent cells. This method provides a simple solution for a seemingly common but often neglected problem, and should be valuable for improving the quality of mutagenesis libraries to enhance the efficiency of directed evolution strategies.

Stable transformation of a mosquito cell line results in extraordinarily high copy numbers of the plasmid.

PubMed Central

Monroe, T J; Muhlmann-Diaz, M C; Kovach, M J; Carlson, J O; Bedford, J S; Beaty, B J

1992-01-01

Stable incorporation of high copy numbers (greater than 10,000 per cell) of a plasmid vector containing a gene conferring resistance to the antibiotic hygromycin was achieved in a cell line derived from the Aedes albopictus mosquito. Plasmid sequences were readily observed by ethidium bromide staining of cellular DNA after restriction endonuclease digestion and agarose gel electrophoresis. The plasmid was demonstrated by in situ hybridization to be present in large arrays integrated in metaphase chromosomes and in minute and double-minute replicating elements. In one subclone, approximately 60,000 copies of the plasmid were organized in a large array that resembles a chromosome, morphologically and in the segregation of its chromatids during anaphase. The original as well as modified versions of the plasmid were rescued by transformation of Escherichia coli using total cellular DNA. Southern blot analyses of recovered plasmids indicate the presence of mosquito-derived sequences. Images PMID:1631052

Nucleotide sequences of the tet(M) genes from the American and Dutch type tetracycline resistance plasmids of Neisseria gonorrhoeae.

PubMed

Gascoyne-Binzi, D M; Heritage, J; Hawkey, P M

1993-11-01

High-level tetracycline-resistant Neisseria gonorrhoeae (TRNG) has been associated with the presence of a plasmid approximately 25.2 MDa in size which carries a Tet M tetracycline resistance determinant. Two different plasmid types, American and Dutch, have previously been described, based on the restriction endonuclease digestion pattern. In this study, the tet(M) genes from the two plasmid types have been amplified by the polymerase chain reaction (PCR) and then sequenced. The gene sequences from the two plasmids shared 96.8% identity, and showed similarities with different segments of the tet(M) gene sequences from Tn1545, Tn916 and Ureaplasma urealyticum. The data suggest that it is highly likely that the Tet M determinant found in the American type plasmid has a different origin from that present in the Dutch plasmid.

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

USDA-ARS?s Scientific Manuscript database

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

Analysis of Heat-Labile Sites Generated by Reactions of Depleted Uranium and Ascorbate in Plasmid DNA

PubMed Central

Wilson, Janice; Young, Ashley; Civitello, Edgar R.

2013-01-01

The goal of this study was to characterize how depleted uranium (DU) causes DNA damage. Procedures were developed to assess the ability of organic and inorganic DNA adducts to convert to single strand breaks (SSB) in pBR322 plasmid DNA in the presence of heat or piperidine. DNA adducts formed by methyl methanesulfonate (MMS), cis-platin (cis-Pt), and chromic chloride were compared to those formed by reaction of uranyl acetate (UA) and ascorbate (Asc). Uranyl ion in the presence of Asc produced U-DNA adducts that converted to SSB upon heating. Piperidine, which acted on DNA methylated by MMS to convert methyl-DNA adducts to SSB, served in the opposite fashion with U-DNA adducts by decreasing SSB. The observation that piperidine also decreased the gel shift for metal-DNA adducts formed by monofunctional cis-Pt and chromic chloride was interpreted to suggest that piperidine served to remove U-DNA adducts. Radical scavengers did not affect formation of U-induced SSB, suggesting that SSB arose from the presence of U-DNA adducts and not from free radicals. A model is proposed to predict how U-DNA adducts may serve as initial lesions that convert to SSB or AP sites. Results suggest that DU can act as a chemical genotoxin that does not require radiation for its mode of action. Characterizing the DNA lesions formed by DU is necessary to assess the relative importance of different DNA lesions in the formation of DU-induced mutations. Understanding mechanisms of formation of DU-induced mutations may contribute to identification of biomarkers of DU exposures in humans. PMID:24218036

Identification and characterization of plasmids from the western aster yellows mycoplasmalike organism.

PubMed Central

Kuske, C R; Kirkpatrick, B C

1990-01-01

Supercoiled double-stranded DNA molecules (plasmids) were isolated from plants infected with three laboratory strains of western aster yellows mycoplasma-like organism (AY-MLO) by using cesium chloride-ethidium bromide density gradients. Southern blot analysis, using plasmids from the severe strain of AY-MLO (SAY-MLO) as the probe, identified at least four plasmids in celery, aster, and periwinkle plants and in Macrosteles severini leafhopper vectors infected with either the dwarf AY-MLO, Tulelake AY-MLO, or SAY-MLO strain. Plasmids were also detected in two California field isolates of AY-MLO but not in plants infected with the beet leafhopper-transmitted virescence agent, western X, or elm yellows MLOs. SAY-MLO plasmids were 5.2, 4.9, 3.4, and 1.7 kilobase pairs in size. Plasmids isolated from dwarf AY- and Tulelake AY-MLOs were 7.4, 5.1, 3.5, and 1.7 kilobase pairs in size. No evidence was obtained for integration of SAY-MLO plasmids into the MLO chromosome. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 FIG. 5 PMID:2307660

Evidence for autonomous replication and stabilization of recombinant plasmids in the transformants of yeast Hansenula polymorpha.

PubMed

Tikhomirova, L P; Ikonomova, R N; Kuznetsova, E N

1986-01-01

For the transformation of the yeast Hansenula polymorpha we have constructed a set of hybrid plasmids carrying the LEU2 gene of Saccharomyces cerevisiae as a selective marker and fragments of mitochondrial DNA of Candida utilis and H. polymorpha or chromosomal DNA fragments of H. polymorpha as replicator sequences. The replication properties of chimeric plasmids in the yeast H. polymorpha were investigated. We showed that for plasmids propagated autonomously in this yeast the plasmid monomers could be detected in the transformants only during the immediate time after the transformation event. Further growth under selective conditions led to the selection of polymeric forms of plasmid DNA as it was clearly shown for transformants carrying cosmid pL2 with mtDNA fragment of C. utilis. Such transformants carrying polymerized plasmids showed a remarkably increased stability of the transformed phenotype. Cosmid pL2 was able to shuttle between Escherichia coli, S. cerevisiae and H. polymorpha, whereas plasmids with DNA fragments from H. polymorpha did not transform S. cerevisiae effectively.

Yeast cohesin complex embraces 2 micron plasmid sisters in a tri-linked catenane complex

PubMed Central

Ghosh, Santanu K.; Huang, Chu-Chun; Hajra, Sujata; Jayaram, Makkuni

2010-01-01

Sister chromatid cohesion, crucial for faithful segregation of replicated chromosomes in eukaryotes, is mediated by the multi-subunit protein complex cohesin. The Saccharomyces cerevisiae plasmid 2 micron circle mimics chromosomes in assembling cohesin at its partitioning locus. The plasmid is a multi-copy selfish DNA element that resides in the nucleus and propagates itself stably, presumably with assistance from cohesin. In metaphase cell lysates, or fractions enriched for their cohesed state by sedimentation, plasmid molecules are trapped topologically by the protein ring formed by cohesin. They can be released from cohesin’s embrace either by linearizing the DNA or by cleaving a cohesin subunit. Assays using two distinctly tagged cohesin molecules argue against the hand-cuff (an associated pair of monomeric cohesin rings) or the bracelet (a dimeric cohesin ring) model as responsible for establishing plasmid cohesion. Our cumulative results most easily fit a model in which a single monomeric cohesin ring, rather than a series of such rings, conjoins a pair of sister plasmids. These features of plasmid cohesion account for its sister-to-sister mode of segregation by cohesin disassembly during anaphase. The mechanistic similarities of cohesion between mini-chromosome sisters and 2 micron plasmid sisters suggest a potential kinship between the plasmid partitioning locus and centromeres. PMID:19920123

Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping

NASA Astrophysics Data System (ADS)

Müller, Vilhelm; Rajer, Fredrika; Frykholm, Karolin; Nyberg, Lena K.; Quaderi, Saair; Fritzsche, Joachim; Kristiansson, Erik; Ambjörnsson, Tobias; Sandegren, Linus; Westerlund, Fredrik

2016-12-01

Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single experiment, we obtain the number of different plasmids in the sample, the size of each plasmid, an optical barcode that can be used to identify and trace the plasmid of interest and information about which plasmid that carries a specific resistance gene. Gene identification is done using CRISPR/Cas9 loaded with a guide-RNA (gRNA) complementary to the gene of interest that linearizes the circular plasmids at a specific location that is identified using the optical DNA maps. We demonstrate the principle on clinically relevant extended spectrum beta-lactamase (ESBL) producing isolates. We discuss how the gRNA sequence can be varied to obtain the desired information. The gRNA can either be very specific to identify a homogeneous group of genes or general to detect several groups of genes at the same time. Finally, we demonstrate an example where we use a combination of two gRNA sequences to identify carbapenemase-encoding genes in two previously not characterized clinical bacterial samples.

Quorum-quenching limits quorum-sensing exploitation by signal-negative invaders

NASA Astrophysics Data System (ADS)

Tannières, Mélanie; Lang, Julien; Barnier, Claudie; Shykoff, Jacqui A.; Faure, Denis

2017-01-01

Some bacteria produce and perceive quorum-sensing (QS) signals that coordinate several behaviours, including the costly processes that are exoenzyme production and plasmid transfer. In the case of plasmid transfer, the emergence of QS signal-altered invaders and their policing are poorly documented. In Agrobacterium tumefaciens, the virulence Ti-plasmid encodes both synthesis and sensing of QS-signals, which promote its transfer from a donor to a recipient cell. Here, we reported that QS-altered A. tumefaciens mutants arose during experimental evolution. All showed improved growth compared to their ancestor. Genome sequencing revealed that, though some had lost the Ti-plasmid, most were defective for QS-signal synthesis and Ti-plasmid conjugation (traR mutations) and one exhibited a QS-signal exploitation behaviour, using signal produced by other cells to enhance its own Ti-plasmid transfer. We explored mechanisms that can limit this QS-hijacking. We showed that the A. tumefaciens capacity to inactivate QS-signals by expressing QS-degrading enzyme could attenuate dissemination of the QS signal-negative Ti-plasmids. This work shows that enzymatic QS-disruption whether encoded by the QS-producing Ti-plasmid itself, by a companion plasmid in the same donor cells, or by one in the recipient cells, in all cases can serve as a mechanism for controlling QS exploitation by QS signal-negative mutants.

Evaluation of the effect of non-B DNA structures on plasmid integrity via accelerated stability studies.

PubMed

Ribeiro, S C; Monteiro, G A; Prazeres, D M F

2009-04-01

Plasmid biopharmaceuticals are a new class of medicines with an enormous potential. Attempts to increase the physical stability of highly purified supercoiled (SC) plasmid DNA in pharmaceutical aqueous solutions have relied on: (i) changing the DNA sequence, (ii) improving manufacturing to reduce deleterious impurities and initial DNA damage, and (iii) controlling the storage medium characteristics. In this work we analyzed the role of secondary structures on the degradation of plasmid molecules. Accelerated stability experiments were performed with SC, open circular (OC) and linear (L) isoforms of three plasmids which differed only in the "single-strandlike" content of their polyadenylation (poly A) signals. We have proved that the presence of more altered or interrupted (non-B) DNA secondary structures did not directly translate into an easier strand scission of the SC isoforms. Rather, those unusual structures imposed a lower degree of SC in the plasmids, leading to an increase in their resistance to thermal degradation. However, this behavior was reversed when the relaxed or L isoforms were tested, in which case the absence of SC rendered the plasmids essentially double-stranded. Overall, this work suggests that plasmid DNA sequence and secondary structures should be taken into account in future investigations of plasmid stability during prolonged storage.

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

PubMed Central

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

2014-01-01

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

Degenerate primer MOB typing of multiresistant clinical isolates of E. coli uncovers new plasmid backbones.

PubMed

Garcillán-Barcia, M Pilar; Ruiz del Castillo, Belén; Alvarado, Andrés; de la Cruz, Fernando; Martínez-Martínez, Luis

2015-01-01

Degenerate Primer MOB Typing is a PCR-based protocol for the classification of γ-proteobacterial transmissible plasmids in five phylogenetic relaxase MOB families. It was applied to a multiresistant E. coli collection, previously characterized by PCR-based replicon-typing, in order to compare both methods. Plasmids from 32 clinical isolates of multiresistant E. coli (19 extended spectrum beta-lactamase producers and 13 non producers) and their transconjugants were analyzed. A total of 95 relaxases were detected, at least one per isolate, underscoring the high potential of these strains for antibiotic-resistance transmission. MOBP12 and MOBF12 plasmids were the most abundant. Most MOB subfamilies detected were present in both subsets of the collection, indicating a shared mobilome among multiresistant E. coli. The plasmid profile obtained by both methods was compared, which provided useful data upon which decisions related to the implementation of detection methods in the clinic could be based. The phylogenetic depth at which replicon and MOB-typing classify plasmids is different. While replicon-typing aims at plasmid replication regions with non-degenerate primers, MOB-typing classifies plasmids into relaxase subfamilies using degenerate primers. As a result, MOB-typing provides a deeper phylogenetic depth than replicon-typing and new plasmid groups are uncovered. Significantly, MOB typing identified 17 plasmids and an integrative and conjugative element, which were not detected by replicon-typing. Four of these backbones were different from previously reported elements. Copyright © 2014 Elsevier Inc. All rights reserved.

Epidemiology of virulence-associated plasmids and outer membrane protein patterns within seven common Salmonella serotypes.

PubMed

Helmuth, R; Stephan, R; Bunge, C; Hoog, B; Steinbeck, A; Bulling, E

1985-04-01

Antibiotic-sensitive Salmonella isolates belonging to seven common serotypes and originating from 29 different countries from all continents were investigated for their plasmid DNA content (337 isolates) and their outer membrane protein profiles (216 isolates). Of the S. typhimurium, S. enteritidis, S. dublin, and S. choleraesuis isolates, 90% or more carried a serotype-specific plasmid. The molecular sizes of the plasmids were 60 megadaltons (Md) for S. typhimurium, 37 Md for S. enteritidis, 56 Md for S. dublin, and 30 Md for S. choleraesuis. The outer membrane protein profiles were homogeneous within each of the seven serotypes, except that a minority of S. enteritidis and S. dublin strains were lacking one major outer membrane protein. Virulence studies were performed with 39 representative strains by measuring the 50% lethal doses (LD50S) after oral infection of mice. The LD50 values obtained for plasmid-positive strains of S. typhimurium, S. enteritidis, and S. dublin were up to 10(6)-fold lower than the values obtained for the plasmid-free strains of the same serotype. Only the plasmid-positive strains could invade the livers of orally infected mice, and only they were resistant to the bactericidal activity of 90% guinea pig serum. Strains of S. infantis were generally plasmid free, whereas S. panama and S. heidelberg isolates carried heterogeneous plasmid populations. The virulence properties of the latter three serotypes could not be correlated with the predominant plasmids found in these strains.

Epidemiology of virulence-associated plasmids and outer membrane protein patterns within seven common Salmonella serotypes.

PubMed Central

Helmuth, R; Stephan, R; Bunge, C; Hoog, B; Steinbeck, A; Bulling, E

1985-01-01

Antibiotic-sensitive Salmonella isolates belonging to seven common serotypes and originating from 29 different countries from all continents were investigated for their plasmid DNA content (337 isolates) and their outer membrane protein profiles (216 isolates). Of the S. typhimurium, S. enteritidis, S. dublin, and S. choleraesuis isolates, 90% or more carried a serotype-specific plasmid. The molecular sizes of the plasmids were 60 megadaltons (Md) for S. typhimurium, 37 Md for S. enteritidis, 56 Md for S. dublin, and 30 Md for S. choleraesuis. The outer membrane protein profiles were homogeneous within each of the seven serotypes, except that a minority of S. enteritidis and S. dublin strains were lacking one major outer membrane protein. Virulence studies were performed with 39 representative strains by measuring the 50% lethal doses (LD50S) after oral infection of mice. The LD50 values obtained for plasmid-positive strains of S. typhimurium, S. enteritidis, and S. dublin were up to 10(6)-fold lower than the values obtained for the plasmid-free strains of the same serotype. Only the plasmid-positive strains could invade the livers of orally infected mice, and only they were resistant to the bactericidal activity of 90% guinea pig serum. Strains of S. infantis were generally plasmid free, whereas S. panama and S. heidelberg isolates carried heterogeneous plasmid populations. The virulence properties of the latter three serotypes could not be correlated with the predominant plasmids found in these strains. Images PMID:3980081

Plasmid-Mediated Bioaugmentation for the Bioremediation of Contaminated Soils

PubMed Central

Garbisu, Carlos; Garaiyurrebaso, Olatz; Epelde, Lur; Grohmann, Elisabeth; Alkorta, Itziar

2017-01-01

Bioaugmentation, or the inoculation of microorganisms (e.g., bacteria harboring the required catabolic genes) into soil to enhance the rate of contaminant degradation, has great potential for the bioremediation of soils contaminated with organic compounds. Regrettably, cell bioaugmentation frequently turns into an unsuccessful initiative, owing to the rapid decrease of bacterial viability and abundance after inoculation, as well as the limited dispersal of the inoculated bacteria in the soil matrix. Genes that encode the degradation of organic compounds are often located on plasmids and, consequently, they can be spread by horizontal gene transfer into well-established, ecologically competitive, indigenous bacterial populations. Plasmid-mediated bioaugmentation aims to stimulate the spread of contaminant degradation genes among indigenous soil bacteria by the introduction of plasmids, located in donor cells, harboring such genes. But the acquisition of plasmids by recipient cells can affect the host’s fitness, a crucial aspect for the success of plasmid-mediated bioaugmentation. Besides, environmental factors (e.g., soil moisture, temperature, organic matter content) can play important roles for the transfer efficiency of catabolic plasmids, the expression of horizontally acquired genes and, finally, the contaminant degradation activity. For plasmid-mediated bioaugmentation to be reproducible, much more research is needed for a better selection of donor bacterial strains and accompanying plasmids, together with an in-depth understanding of indigenous soil bacterial populations and the environmental conditions that affect plasmid acquisition and the expression and functioning of the catabolic genes of interest. PMID:29062312

Gene expression promoted by the SV40 DNA targeting sequence and the hypoxia-responsive element under normoxia and hypoxia.

PubMed

Sacramento, C B; Moraes, J Z; Denapolis, P M A; Han, S W

2010-08-01

The main objective of the present study was to find suitable DNA-targeting sequences (DTS) for the construction of plasmid vectors to be used to treat ischemic diseases. The well-known Simian virus 40 nuclear DTS (SV40-DTS) and hypoxia-responsive element (HRE) sequences were used to construct plasmid vectors to express the human vascular endothelial growth factor gene (hVEGF). The rate of plasmid nuclear transport and consequent gene expression under normoxia (20% O2) and hypoxia (less than 5% O2) were determined. Plasmids containing the SV40-DTS or HRE sequences were constructed and used to transfect the A293T cell line (a human embryonic kidney cell line) in vitro and mouse skeletal muscle cells in vivo. Plasmid transport to the nucleus was monitored by real-time PCR, and the expression level of the hVEGF gene was measured by ELISA. The in vitro nuclear transport efficiency of the SV40-DTS plasmid was about 50% lower under hypoxia, while the HRE plasmid was about 50% higher under hypoxia. Quantitation of reporter gene expression in vitro and in vivo, under hypoxia and normoxia, confirmed that the SV40-DTS plasmid functioned better under normoxia, while the HRE plasmid was superior under hypoxia. These results indicate that the efficiency of gene expression by plasmids containing DNA binding sequences is affected by the concentration of oxygen in the medium.

An Enterobacter plasmid as a new genetic background for the transposon Tn1331

PubMed Central

Alavi, Mohammad R; Antonic, Vlado; Ravizee, Adrien; Weina, Peter J; Izadjoo, Mina; Stojadinovic, Alexander

2011-01-01

Background Genus Enterobacter includes important opportunistic nosocomial pathogens that could infect complex wounds. The presence of antibiotic resistance genes in these microorganisms represents a challenging clinical problem in the treatment of these wounds. In the authors’ screening of antibiotic-resistant bacteria from complex wounds, an Enterobacter species was isolated that harbors antibiotic-resistant plasmids conferring resistance to Escherichia coli. The aim of this study was to identify the resistance genes carried by one of these plasmids. Methods The plasmids from the Enterobacter isolate were propagated in E. coli and one of the plasmids, designated as pR23, was sequenced by the Sanger method using fluorescent dyeterminator chemistry on a genetic analyzer. The assembled sequence was annotated by search of the GenBank database. Results Plasmid pR23 is composed of the transposon Tn1331 and a backbone plasmid that is identical to the plasmid pPIGDM1 from Enterobacter agglomerans. The multidrug-resistance transposon Tn1331, which confers resistance to aminoglycoside and beta lactam antibiotics, has been previously isolated only from Klebsiella. The Enterobacter plasmid pPIGDM1, which carries a ColE1-like origin of replication and has no apparent selective marker, appears to provide a backbone for propagation of Tn1331 in Enterobacter. The recognition sequence of Tn1331 transposase for insertion into pPIGDM1 is the pentanucleotide TATTA, which occurs only once throughout the length of this plasmid. Conclusion Transposition of Tn1331 into the Enterobacter plasmid pPIGDM1 enables this transposon to propagate in this Enterobacter. Since Tn1331 was previously isolated only from Klebsiella, this report suggests horizontal transfer of this transposon between the two bacterial genera. PMID:22259249

Elimination of the cryptic plasmid in Leuconostoc citreum by CRISPR/Cas9 system.

PubMed

Jang, Ye-Ji; Seo, Seung-Oh; Kim, Seul-Ah; Li, Ling; Kim, Tae-Jip; Kim, Sun Chang; Jin, Yong-Su; Han, Nam Soo

2017-06-10

Leuconostoc spp. are important lactic acid bacteria for the fermentation of foods. In particular, L. citreum strains isolated from various foods have been used as host strains for genetic and metabolic engineering studies. In order to develop a food-grade genetic engineering system, L. citreum CB2567 was isolated from Kimchi. However, the isolated bacterium contained a cryptic plasmid which was difficult to eliminate. As the existence of the plasmid might hinder strain engineering, we eliminated the plasmid using an RNA-guided DNA endonuclease CRISPR/Cas9 system. We demonstrated that a plasmid-free L. citreum CB2567 host strain could be efficiently constructed through a two-step procedure: 1) transformation of the "killer" plasmid expressing Cas9 endonuclease and a guide RNA (gRNA) targeting for a specific sequence in the cryptic plasmid, and 2) serial subculture without antibiotics for curing the killer plasmid. When the crude extract of L. citreum expressing Cas9 and the guide RNA was incubated with a PCR fragment containing the specific sequence recognized by the guide RNA, the PCR fragment was cleaved. Also, the cryptic plasmid pCB42 was successfully eliminated from the host strain after transforming the plasmid harboring Cas9 and the guide RNA. The Cas9 and gRNA expression plasmid used in this study can be applied for genome engineering purposes by additionally introducing an editing DNA template to repair the double strand DNA breakage caused by Cas9 in the genome of L. citreum. This study demonstrates the feasibility of developing CRISPR/Cas9-based genetic engineering tools to develop a safe host strain and construct food-grade lactic acid bacteria without residual antibiotic markers. Copyright © 2017 Elsevier B.V. All rights reserved.

The Role of Clonal Interference in the Evolutionary Dynamics of Plasmid-Host Adaptation

PubMed Central

Hughes, Julie M.; Lohman, Brian K.; Deckert, Gail E.; Nichols, Eric P.; Settles, Matt; Abdo, Zaid; Top, Eva M.

2012-01-01

ABSTRACT Promiscuous plasmids replicate in a wide range of bacteria and therefore play a key role in the dissemination of various host-beneficial traits, including antibiotic resistance. Despite the medical relevance, little is known about the evolutionary dynamics through which drug resistance plasmids adapt to new hosts and thereby persist in the absence of antibiotics. We previously showed that the incompatibility group P-1 (IncP-1) minireplicon pMS0506 drastically improved its stability in novel host Shewanella oneidensis MR-1 after 1,000 generations under antibiotic selection for the plasmid. The only mutations found were those affecting the N terminus of the plasmid replication initiation protein TrfA1. Our aim in this study was to gain insight into the dynamics of plasmid evolution. Changes in stability and genotype frequencies of pMS0506 were monitored in evolving populations of MR-1 (pMS0506). Genotypes were determined by sequencing trfA1 amplicons from individual clones and by 454 pyrosequencing of whole plasmids from entire populations. Stability of pMS0506 drastically improved by generation 200. Many evolved plasmid genotypes with point mutations as well as in-frame and frameshift deletions and duplications in trfA1 were observed in all lineages with both sequencing methods. Strikingly, multiple genotypes were simultaneously present at high frequencies (>10%) in each population. Their relative abundances changed over time, but after 1,000 generations only one or two genotypes dominated the populations. This suggests that hosts with different plasmid genotypes were competing with each other, thus affecting the evolutionary trajectory. Plasmids can thus rapidly improve their stability, and clonal interference plays a significant role in plasmid-host adaptation dynamics. PMID:22761390

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Casjens S. R.; Dunn J.; Mongodin, E. F.

2012-03-14

Lyme disease is the most common tick-borne human illness in North America. In order to understand the molecular pathogenesis, natural diversity, population structure and epizootic spread of the North American Lyme agent, Borrelia burgdorferi sensu stricto, a much better understanding of the natural diversity of its genome will be required. Towards this end we present a comparative analysis of the nucleotide sequences of the numerous plasmids of B. burgdorferi isolates B31, N40, JD1 and 297. These strains were chosen because they include the three most commonly studied laboratory strains, and because they represent different major genetic lineages and so aremore » informative regarding the genetic diversity and evolution of this organism. A unique feature of Borrelia genomes is that they carry a large number of linear and circular plasmids, and this work shows that strains N40, JD1, 297 and B31 carry related but non-identical sets of 16, 20, 19 and 21 plasmids, respectively, that comprise 33-40% of their genomes. We deduce that there are at least 28 plasmid compatibility types among the four strains. The B. burgdorferi {approx}900 Kbp linear chromosomes are evolutionarily exceptionally stable, except for a short {le}20 Kbp plasmid-like section at the right end. A few of the plasmids, including the linear lp54 and circular cp26, are also very stable. We show here that the other plasmids, especially the linear ones, are considerably more variable. Nearly all of the linear plasmids have undergone one or more substantial inter-plasmid rearrangements since their last common ancestor. In spite of these rearrangements and differences in plasmid contents, the overall gene complement of the different isolates has remained relatively constant.« less

pA506, a Conjugative Plasmid of the Plant Epiphyte Pseudomonas fluorescens A506

PubMed Central

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.

2013-01-01

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

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

PubMed Central

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

2012-01-01

Lyme disease is the most common tick-borne human illness in North America. In order to understand the molecular pathogenesis, natural diversity, population structure and epizootic spread of the North American Lyme agent, Borrelia burgdorferi sensu stricto, a much better understanding of the natural diversity of its genome will be required. Towards this end we present a comparative analysis of the nucleotide sequences of the numerous plasmids of B. burgdorferi isolates B31, N40, JD1 and 297. These strains were chosen because they include the three most commonly studied laboratory strains, and because they represent different major genetic lineages and so are informative regarding the genetic diversity and evolution of this organism. A unique feature of Borrelia genomes is that they carry a large number of linear and circular plasmids, and this work shows that strains N40, JD1, 297 and B31 carry related but non-identical sets of 16, 20, 19 and 21 plasmids, respectively, that comprise 33–40% of their genomes. We deduce that there are at least 28 plasmid compatibility types among the four strains. The B. burgdorferi ∼900 Kbp linear chromosomes are evolutionarily exceptionally stable, except for a short ≤20 Kbp plasmid-like section at the right end. A few of the plasmids, including the linear lp54 and circular cp26, are also very stable. We show here that the other plasmids, especially the linear ones, are considerably more variable. Nearly all of the linear plasmids have undergone one or more substantial inter-plasmid rearrangements since their last common ancestor. In spite of these rearrangements and differences in plasmid contents, the overall gene complement of the different isolates has remained relatively constant. PMID:22432010

A rapid matrix-assisted laser desorption ionization-time of flight mass spectrometry-based method for single-plasmid tracking in an outbreak of carbapenem-resistant Enterobacteriaceae.

PubMed

Lau, Anna F; Wang, Honghui; Weingarten, Rebecca A; Drake, Steven K; Suffredini, Anthony F; Garfield, Mark K; Chen, Yong; Gucek, Marjan; Youn, Jung-Ho; Stock, Frida; Tso, Hanna; DeLeo, Jim; Cimino, James J; Frank, Karen M; Dekker, John P

2014-08-01

Carbapenem-resistant Enterobacteriaceae (CRE) have spread globally and represent a serious and growing threat to public health. Rapid methods for tracking plasmids carrying carbapenemase genes could greatly benefit infection control efforts. Here, we demonstrate that real-time, direct tracking of a single plasmid in a bacterial strain responsible for an outbreak is possible using a commercial matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system. In this case, we retrospectively tracked the bla(KPC) carbapenemase gene-bearing pKpQIL plasmid responsible for a CRE outbreak that occurred at the NIH Clinical Center in 2011. An ∼ 11,109-Da MS peak corresponding to a gene product of the bla(KPC) pKpQIL plasmid was identified and characterized using a combination of proteomics and molecular techniques. This plasmid peak was present in spectra from retrospectively analyzed K. pneumoniae outbreak isolates, concordant with results from whole-genome sequencing, and absent from a diverse control set of bla(KPC)-negative clinical Enterobacteriaceae isolates. Notably, the gene characterized here is located adjacent to the bla(KPC) Tn4401 transposon on the pKpQIL plasmid. Sequence analysis demonstrates the presence of this gene in other bla(KPC) Tn4401-containing plasmids and suggests that this signature MS peak may be useful in tracking other plasmids conferring carbapenem resistance. Plasmid identification using this MALDI-TOF MS method was accomplished in as little as 10 min from isolated colonies and 30 min from positive (spiked) blood cultures, demonstrating the potential clinical utility for real-time plasmid tracking in an outbreak. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

A Rapid Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry-Based Method for Single-Plasmid Tracking in an Outbreak of Carbapenem-Resistant Enterobacteriaceae

PubMed Central

Lau, Anna F.; Wang, Honghui; Weingarten, Rebecca A.; Drake, Steven K.; Suffredini, Anthony F.; Garfield, Mark K.; Chen, Yong; Gucek, Marjan; Youn, Jung-Ho; Stock, Frida; Tso, Hanna; DeLeo, Jim; Cimino, James J.; Frank, Karen M.

2014-01-01

Carbapenem-resistant Enterobacteriaceae (CRE) have spread globally and represent a serious and growing threat to public health. Rapid methods for tracking plasmids carrying carbapenemase genes could greatly benefit infection control efforts. Here, we demonstrate that real-time, direct tracking of a single plasmid in a bacterial strain responsible for an outbreak is possible using a commercial matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) system. In this case, we retrospectively tracked the blaKPC carbapenemase gene-bearing pKpQIL plasmid responsible for a CRE outbreak that occurred at the NIH Clinical Center in 2011. An ∼11,109-Da MS peak corresponding to a gene product of the blaKPC pKpQIL plasmid was identified and characterized using a combination of proteomics and molecular techniques. This plasmid peak was present in spectra from retrospectively analyzed K. pneumoniae outbreak isolates, concordant with results from whole-genome sequencing, and absent from a diverse control set of blaKPC-negative clinical Enterobacteriaceae isolates. Notably, the gene characterized here is located adjacent to the blaKPC Tn4401 transposon on the pKpQIL plasmid. Sequence analysis demonstrates the presence of this gene in other blaKPC Tn4401-containing plasmids and suggests that this signature MS peak may be useful in tracking other plasmids conferring carbapenem resistance. Plasmid identification using this MALDI-TOF MS method was accomplished in as little as 10 min from isolated colonies and 30 min from positive (spiked) blood cultures, demonstrating the potential clinical utility for real-time plasmid tracking in an outbreak. PMID:24850353

NetF-producing Clostridium perfringens: Clonality and plasmid pathogenicity loci analysis.

PubMed

Mehdizadeh Gohari, Iman; Kropinski, Andrew M; Weese, Scott J; Whitehead, Ashley E; Parreira, Valeria R; Boerlin, Patrick; Prescott, John F

2017-04-01

Clostridium perfringens is an important cause of foal necrotizing enteritis and canine acute hemorrhagic diarrhea. A major virulence determinant of the strains associated with these diseases appears to be a beta-sheet pore-forming toxin, NetF, encoded within a pathogenicity locus (NetF locus) on a large tcp-conjugative plasmid. Strains producing NetF also produce the putative toxin NetE, encoded within the same pathogenicity locus, as well as CPE enterotoxin and CPB2 on a second plasmid, and sometimes the putative toxin NetG within a pathogenicity locus (NetG locus) on another separate large conjugative plasmid. Previous genome sequences of two netF-positive C. perfringens showed that they both shared three similar plasmids, including the NetF/NetE and CPE/CPB2 toxins-encoding plasmids mentioned above and a putative bacteriocin-encoding plasmid. The main purpose of this study was to determine whether all NetF-producing strains share this common plasmid profile and whether their distinct NetF and CPE pathogenicity loci are conserved. To answer this question, 15 equine and 15 canine netF-positive isolates of C. perfringens were sequenced using Illumina Hiseq2000 technology. In addition, the clonal relationships among the NetF-producing strains were evaluated by core genome multilocus sequence typing (cgMLST). The data obtained showed that all NetF-producing strains have a common plasmid profile and that the defined pathogenicity loci on the plasmids are conserved in all these strains. cgMLST analysis showed that the NetF-producing C. perfringens strains belong to two distinct clonal complexes. The pNetG plasmid was absent from isolates of one of the clonal complexes, and there were minor but consistent differences in the NetF/NetE and CPE/CPB2 plasmids between the two clonal complexes. Copyright © 2017 Elsevier B.V. All rights reserved.

Phenotypic and molecular characterization of conjugative antibiotic resistance plasmids isolated from bacterial communities of activated sludge.

PubMed

Dröge, M; Pühler, A; Selbitschka, W

2000-04-01

In order to isolate antibiotic resistance plasmids from bacterial communities found in activated sludge, derivatives of the 3-chlorobenzoate-degrading strain Pseudomonas sp. B13, tagged with the green fluorescent protein as an identification marker, were used as recipients in filter crosses. Transconjugants were selected on agar plates containing 3-chlorobenzoate as the sole carbon source and the antibiotic tetracycline, streptomycin or spectinomycin, and were recovered at frequencies in the range of 10(-5) to 10(-8) per recipient. A total of 12 distinct plasmids, designated pB1-pB12, was identified. Their sizes ranged between 41 to 69 kb and they conferred various patterns of antibiotic resistance on their hosts. Two of the plasmids, pB10 and pB11, also mediated resistance to inorganic mercury. Seven of the 12 plasmids were identified as broad-host-range plasmids, displaying extremely high transfer frequencies in filter crosses, ranging from 10(-1) to 10(-2) per recipient cell. Ten of the 12 plasmids belonged to the IncP incompatibility group, based on replicon typing using IncP group-specific PCR primers. DNA sequencing of PCR amplification products further revealed that eight of the 12 plasmids belonged to the IncPbeta subgroup, whereas two plasmids were identified as IncPalpha plasmids. Analysis of the IncP-specific PCR products revealed considerable differences among the IncPbeta plasmids at the DNA sequence level. In order to characterize the gene "load" of the IncP plasmids, restriction fragments were cloned and their DNA sequences established. A remarkable diversity of putative proteins encoded by these fragments was identified. Besides transposases and proteins involved in antibiotic resistance, two putative DNA invertases belonging to the Din family, a methyltransferase of a type I restriction/modification system, a superoxide dismutase, parts of a putative efflux system belonging to the RND family, and proteins of unknown function were identified.

Complete sequences of IncHI1 plasmids carrying blaCTX-M-1 and qnrS1 in equine Escherichia coli provide new insights into plasmid evolution.

PubMed

Dolejska, Monika; Villa, Laura; Minoia, Marco; Guardabassi, Luca; Carattoli, Alessandra

2014-09-01

To determine the structure of two multidrug-resistant IncHI1 plasmids carrying blaCTX-M-1 in Escherichia coli isolates disseminated in an equine clinic in the Czech Republic. A complete nucleotide sequencing of 239 kb IncHI1 (pEQ1) and 287 kb IncHI1/X1 (pEQ2) plasmids was performed using the 454-Genome Sequencer FLX system. The sequences were compared using bioinformatic tools with other sequenced IncHI1 plasmids. A comparative analysis of pEQ1 and pEQ2 identified high nucleotide identity with the IncHI1 type 2 plasmids. A novel 24 kb module containing an operon involved in short-chain fructooligosaccharide uptake and metabolism was found in the pEQ backbones. The role of the pEQ plasmids in the metabolism of short-chain fructooligosaccharides was demonstrated by studying the growth of E. coli cells in the presence of these sugars. The module containing the blaCTX-M-1 gene was formed by a truncated macrolide resistance cluster and flanked by IS26 as previously observed in IncI1 and IncN plasmids. The IncHI1 plasmid changed size and gained the quinolone resistance gene qnrS1 as a result of IS26-mediated fusion with an IncX1 plasmid. Our data highlight the structure and evolution of IncHI1 from equine E. coli. A plasmid-mediated sugar metabolic element could play a key role in strain fitness, contributing to the successful dissemination and maintenance of these plasmids in the intestinal microflora of horses. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Addition of DNA to CrVI and Cytochrome b5 Containing Proteoliposomes Leads to Generation of DNA Strand Breaks and CrIII Complexes

PubMed Central

Borthiry, Griselda R.; Antholine, William E.; Myers, Judith M.; Myers, Charles R.

2009-01-01

Chromium (Cr) is a cytotoxic metal that can be associated with a variety of types of DNA damage, including Cr-DNA adducts and strand breaks. Prior studies with purified human cytochrome b5 and NADPH :P450 reductase in reconstituted proteoliposomes (PLs) demonstrated rapid reduction of CrVI (hexavalent chromium, as CrO42− ), and the generation of CrV, superoxide (O2·−) , and hydroxyl radical (HO˙). Studies reported here examined the potential for the species produced by this system to interact with DNA. Strand breaks of purified plasmid DNA increased over time aerobically, but were not observed in the absence of O2. CrV is formed under both conditions, so the breaks are not mediated directly by CrV. The aerobic strand breaks were significantly prevented by catalase and EtOH, but not by the metal chelator diethylenetriaminepentaacetic acid (DTPA), suggesting that they are largely due to HO˙ from Cr-mediated redox cycling. EPR was used to assess the formation of Cr-DNA complexes. Following a 10-min incubation of PLs, CrO42− , and plasmid DNA, intense EPR signals at g = 5.7and g = 5.0 were observed. These signals are attributed to specific CrIII complexes with large zero field splitting (ZFS). Without DNA, the signals in the g = 5 region were weak. The large ZFS signals were not seen, when CrIIICl3 was incubated with DNA, suggesting that the CrIII–DNA interactions are different when generated by the PLs. After 24 h, a broad signal at g = 2 is attributed to CrIII complexes with a small ZFS. This g = 2 signal was observed without DNA, but it was different from that seen with plasmid. It is concluded that EPR can detect specific CrIII complexes that depend on the presence of plasmid DNA and the manner in which the CrIII is formed. PMID:18729091

Characterization of extended-spectrum cephalosporin resistant Salmonella enterica serovar Heidelberg isolated from food animals, retail meat, and humans in the United States 2009

PubMed Central

Folster, J. P.; Pecic, G.; Singh, A.; Duval, B.; Rickert, R.; Ayers, S.; Abbott, J.; McGlinchey, B.; Bauer-Turpin, J.; Haro, J.; Hise, K.; Zhao, S.; Fedorka-Cray, P. J.; Whichard, J.; McDermott, P. F.

2015-01-01

Salmonella enterica is one of the most common causes of foodborne illness in the United States. Although salmonellosis is usually self-limiting, severe infections typically require antimicrobial treatment and ceftriaxone, an extended-spectrum cephalosporin, is commonly used in both adults and children. Surveillance conducted by the National Antimicrobial Resistance Monitoring System (NARMS) has shown a recent increase in extended-spectrum cephalosporin (ESC) resistance among Salmonella Heidelberg isolated from food animals at slaughter, retail meat, and humans. ESC resistance among Salmonella in the United States is usually mediated by a plasmid-encoded blaCMY β-lactamase. In 2009, we identified 47 ESC resistant blaCMY-positive Heidelberg isolates from humans (n=18), food animals at slaughter (n=16), and retail meats (n=13) associated with a spike in the prevalence of this serovar. Almost 90% (26/29) of the animal and meat isolates were isolated from chicken carcasses or retail chicken meat. We screened NARMS isolates for the presence of blaCMY, determined whether the gene was plasmid-encoded, examined pulsed-field gel electrophoresis patterns to assess the genetic diversities of the isolates, and categorized the blaCMY plasmids by plasmid incompatibility groups and plasmid multi-locus sequence typing. All 47 blaCMY genes were found to be plasmid encoded. Incompatibility/replicon typing demonstrated that 41 were IncI1 plasmids, 40 of which only conferred blaCMY associated resistance. Six were IncA/C plasmids that carried additional resistance genes. Plasmid multi-locus sequence typing (pMLST) of the IncI1-blaCMY plasmids showed that 27 (65.8%) were sequence type (ST) 12, the most common ST among blaCMY-IncI1 plasmids from Heidelberg isolated from humans. Ten plasmids had a new ST profile, ST66, a type very similar to ST12. This work showed that the 2009 increase in ESC resistance among Salmonella Heidelberg was caused mainly by the dissemination of blaCMY on IncI1 and IncA/C plasmids in a variety of genetic backgrounds, and likely not the result of clonal expansion. PMID:22755514

Tracking inter-institutional spread of NDM and identification of a novel NDM-positive plasmid, pSg1-NDM, using next-generation sequencing approaches.

PubMed

Khong, Wei Xin; Marimuthu, Kalisvar; Teo, Jeanette; Ding, Yichen; Xia, Eryu; Lee, Jia Jun; Ong, Rick Twee-Hee; Venkatachalam, Indumathi; Cherng, Benjamin; Pada, Surinder Kaur; Choong, Weng Lam; Smitasin, Nares; Ooi, Say Tat; Deepak, Rama Narayana; Kurup, Asok; Fong, Raymond; Van La, My; Tan, Thean Yen; Koh, Tse Hsien; Lin, Raymond Tzer Pin; Tan, Eng Lee; Krishnan, Prabha Unny; Singh, Siddharth; Pitout, Johann D; Teo, Yik-Ying; Yang, Liang; Ng, Oon Tek

2016-11-01

Owing to gene transposition and plasmid conjugation, New Delhi metallo-β-lactamase (NDM) is typically identified among varied Enterobacteriaceae species and STs. We used WGS to characterize the chromosomal and plasmid molecular epidemiology of NDM transmission involving four institutions in Singapore. Thirty-three Enterobacteriaceae isolates (collection years 2010-14) were sequenced using short-read sequencing-by-synthesis and analysed. Long-read single molecule, real-time sequencing (SMRTS) was used to characterize genetically a novel plasmid pSg1-NDM carried on Klebsiella pneumoniae ST147. In 20 (61%) isolates, bla NDM was located on the pNDM-ECS01 plasmid in the background of multiple bacterial STs, including eight K. pneumoniae STs and five Escherichia coli STs. In six (18%) isolates, a novel bla NDM -positive plasmid, pSg1-NDM, was found only in K. pneumoniae ST147. The pSg1-NDM-K. pneumoniae ST147 clone (Sg1-NDM) was fully sequenced using SMRTS. pSg1-NDM, a 90 103 bp IncR plasmid, carried genes responsible for resistance to six classes of antimicrobials. A large portion of pSg1-NDM had no significant homology to any known plasmids in GenBank. pSg1-NDM had no conjugative transfer region. Combined chromosomal-plasmid phylogenetic analysis revealed five clusters of clonal bacterial NDM-positive plasmid transmission, of which two were inter-institution clusters. The largest inter-institution cluster involved six K. pneumoniae ST147-pSg1-NDM isolates. Fifteen patients were involved in transmission clusters, of which four had ward contact, six had hospital contact and five had an unknown transmission link. A combined sequencing-by-synthesis and SMRTS approach can determine effectively the transmission clusters of bla NDM and genetically characterize novel plasmids. Plasmid molecular epidemiology is important to understanding NDM spread as bla NDM -positive plasmids can conjugate extensively across species and STs. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

The Completely Sequenced Plasmid pEST4011 Contains a Novel IncP1 Backbone and a Catabolic Transposon Harboring tfd Genes for 2,4-Dichlorophenoxyacetic Acid Degradation

PubMed Central

Vedler, Eve; Vahter, Merle; Heinaru, Ain

2004-01-01

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium Achromobacter xylosoxidans subsp. denitrificans strain EST4002 contains plasmid pEST4011. This plasmid ensures its host a stable 2,4-D+ phenotype. We determined the complete 76,958-bp nucleotide sequence of pEST4011. This plasmid is a deletion and duplication derivative of pD2M4, the 95-kb highly unstable laboratory ancestor of pEST4011, and was self-generated during different laboratory manipulations performed to increase the stability of the 2,4-D+ phenotype of the original strain, strain D2M4(pD2M4). The 47,935-bp catabolic region of pEST4011 forms a transposon-like structure with identical copies of the hybrid insertion element IS1071::IS1471 at the two ends. The catabolic regions of pEST4011 and pJP4, the best-studied 2,4-D-degradative plasmid, both contain homologous, tfd-like genes for complete 2,4-D degradation, but they have little sequence similarity other than that. The backbone genes of pEST4011 are most similar to the corresponding genes of broad-host-range self-transmissible IncP1 plasmids. The backbones of the other three IncP1 catabolic plasmids that have been sequenced (the 2,4-D-degradative plasmid pJP4, the haloacetate-catabolic plasmid pUO1, and the atrazine-catabolic plasmid pADP-1) are nearly identical to the backbone of R751, the archetype plasmid of the IncP1 β subgroup. We show that despite the overall similarity in plasmid organization, the pEST4011 backbone is sufficiently different (51 to 86% amino acid sequence identity between individual backbone genes) from the backbones of members of the three IncP1 subgroups (the α, β, and γ subgroups) that it belongs to a new IncP1subgroup, the δ subgroup. This conclusion was also supported by a phylogenetic analysis of the trfA2, korA, and traG gene products of different IncP1 plasmids. PMID:15489427

Femtosecond laser assisted photo-transfection and differentiation of mouse embryonic stem cells

NASA Astrophysics Data System (ADS)

Thobakgale, Lebogang; Manoto, Sello; Ombinda Lemboumba, Satuurnin; Maaza, Malik; Mthunzi-Kufa, Patience

2018-02-01

In tissue engineering research, stem cells have been used as starting material in the synthesis of mammalian cells for the treatment of various cell based diseases. This is done by manipulating the DNA content of the cells to induce a specific effect such as increased proliferation or developing a new cell type through the process of differentiation. Such controlled gene expression of stem cells is achieved by the method of transfection, where exogenous plasmid deoxyribonucleic acid (pDNA) is inserted into a stem cell using chemical, viral or physical methods. In this research, we used femtosecond (fs) laser pulses from a home-build microscope system to perforate the cellular membrane and allow entry of selected pDNA to alter the behaviour of mouse embryonic stem cells (mESCs). In one set of experiments, we induce fluorescence on mESCs using green fluorescence protein plasmid (pGFP) while in other tests; differentiation of mESCs into endoderm cells is performed using Sox-17 plasmid DNA (pSox-17). Primitive endoderm formation was thereafter confirmed using polymerase chain reactions (PCR) and the Sox-17 primer. Cell viability studies using adenosine triphosphate were also conducted. From the data, it was concluded that the photo-transfection method is biocompatible since it was able to induce fluorescence in mESCs. Secondly, it was confirmed that Sox-17 was photo-transfected successfully using 6 μW laser power, 128 fs pulses and 1kHz pulse repetition rate.

Transient blockade of the inducible costimulator pathway generates long-term tolerance to factor VIII after nonviral gene transfer into hemophilia A mice.

PubMed

Peng, Baowei; Ye, Peiqing; Blazar, Bruce R; Freeman, Gordon J; Rawlings, David J; Ochs, Hans D; Miao, Carol H

2008-09-01

Formation of inhibitory antibodies is a common problem encountered in clinical treatment for hemophilia. Human factor VIII (hFVIII) plasmid gene therapy in hemophilia A mice also leads to strong humoral responses. We demonstrate that short-term therapy with an anti-ICOS monoclonal antibody to transiently block the inducible costimulator/inducible costimulator ligand (ICOS/ICOSL) signaling pathway led to sustained tolerance to hFVIII in hFVIII plasmid-treated hemophilia A mice and allowed persistent, high-level FVIII functional activity (100%-300% of normal). Anti-ICOS treatment resulted in depletion of ICOS(+)CD4(+) T cells and activation of CD25(+)Foxp3(+) Tregs in the peripheral blood, spleen, and lymph nodes. CD4(+) T cells from anti-ICOS-treated mice did not proliferate in response to hFVIII stimulation and produced high levels of regulatory cytokines, including interleukin-10 and transforming growth factor-beta. Moreover, CD4(+)CD25(+) Tregs from tolerized mice adoptively transferred dominant tolerance in syngeneic hFVIII plasmid-treated hemophilia A mice and reduced the production of antibodies against FVIII. Anti-ICOS-treated mice tolerized to hFVIII generated normal primary and secondary antibody responses after immunization with the T-dependent antigen, bacteriophage Phix 174, indicating maintenance of immune competency. Our data indicate that transient anti-ICOS monoclonal antibody treatment represents a novel single-agent immunomodulatory strategy to overcome the immune responses against transgene product after gene therapy.

Processing of Nonconjugative Resistance Plasmids by Conjugation Nicking Enzyme of Staphylococci

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pollet, Rebecca M.; Ingle, James D.; Hymes, Jeff P.

Antimicrobial resistance inStaphylococcus aureuspresents an increasing threat to human health. This resistance is often encoded on mobile plasmids, such as pSK41; however, the mechanism of transfer of these plasmids is not well understood. In this study, we first examine key protein-DNA interactions formed by the relaxase enzyme, NES, which initiates and terminates the transfer of the multidrug resistance plasmid pSK41. Two loops on the NES protein, hairpin loops 1 and 2, form extensive contacts with the DNA hairpin formed at theoriTregion of pSK41, and here we establish that these contacts are essential for proper DNA cleavage and religation by themore » full 665-residue NES proteinin vitro. Second, pSK156 and pCA347 are nonconjugativeStaphylococcus aureusplasmids that contain sequences similar to theoriTregion of pSK41 but differ in the sequence predicted to form a DNA hairpin. We show that pSK41-encoded NES is able to bind, cleave, and religate theoriTsequences of these nonconjugative plasmidsin vitro. Although pSK41 could mobilize a coresident plasmid harboring its cognateoriT, it was unable to mobilize plasmids containing the pSK156 and pCA347 variantoriTmimics, suggesting that an accessory protein like that previously shown to confer specificity in the pWBG749 system may also be involved in transmission of plasmids containing a pSK41-likeoriT. These data indicate that the conjugative relaxase intransmechanism recently described for the pWBG749 family of plasmids also applies to the pSK41 family of plasmids, further heightening the potential significance of this mechanism in the horizontal transfer of staphylococcal plasmids. IMPORTANCEUnderstanding the mechanism of antimicrobial resistance transfer in bacteria such asStaphylococcus aureusis an important step toward potentially slowing the spread of antimicrobial-resistant infections. This work establishes protein-DNA interactions essential for the transfer of theStaphylococcus aureusmultiresistance plasmid pSK41 by its relaxase, NES. This enzyme also processed variantoriT-like sequences found on numerous plasmids previously considered nontransmissible, suggesting that in conjunction with an uncharacterized accessory protein, these plasmids may be transferred horizontally via a relaxase intransmechanism. These findings have important implications for our understanding of staphylococcal resistance plasmid evolution.« less

Prevalence and characterization of plasmids carrying sulfonamide resistance genes among Escherichia coli from pigs, pig carcasses and human

PubMed Central

2010-01-01

Background Sulfonamide resistance is very common in Escherichia coli. The aim of this study was to characterize plasmids carrying sulfonamide resistance genes (sul1, sul2 and sul3) in E. coli isolated from pigs and humans with a specific objective to assess the genetic diversity of plasmids involved in the mobility of sul genes. Methods A total of 501 E. coli isolates from pig feces, pig carcasses and human stools were tested for their susceptibility to selected antimicrobial. Multiplex PCR was conducted to detect the presence of three sul genes among the sulfonamide-resistant E. coli isolates. Fifty-seven sulfonamide-resistant E. coli were selected based on presence of sul resistance genes and subjected to conjugation and/or transformation experiments. S1 nuclease digestion followed by pulsed-field gel electrophoresis was used to visualize and determine the size of plasmids. Plasmids carrying sul genes were characterized by PCR-based replicon typing to allow a comparison of the types of sul genes, the reservoir and plasmid present. Results A total of 109/501 isolates exhibited sulfonamide resistance. The relative prevalences of sul genes from the three reservoirs (pigs, pig carcasses and humans) were 65%, 45% and 12% for sul2, sul1, and sul3, respectively. Transfer of resistance through conjugation was observed in 42/57 isolates. Resistances to streptomycin, ampicillin and trimethoprim were co-transferred in most strains. Class 1 integrons were present in 80% of sul1-carrying plasmids and 100% of sul3-carrying plasmids, but only in 5% of sul2-carrying plasmids. The sul plasmids ranged from 33 to 160-kb in size and belonged to nine different incompatibility (Inc) groups: FII, FIB, I1, FIA, B/O, FIC, N, HI1 and X1. IncFII was the dominant type in sul2-carrying plasmids (52%), while IncI1 was the most common type in sul1 and sul3-carrying plasmids (33% and 45%, respectively). Multireplicons were found associated with all three sul genes. Conclusions Sul genes were distributed widely in E. coli isolated from pigs and humans with sul2 being most prevalent. Sul-carrying plasmids belonged to diverse replicon types, but most of detected plasmids were conjugative enabling horizontal transfer. IncFII seems to be the dominant replicon type in sul2-carrying plasmids from all three sources. PMID:20670455

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

PubMed Central

Kaplan, Ella; Sela, Noa; Doron-Faigenboim, Adi; Navon-Venezia, Shiri; Jurkevitch, Edouard; Cytryn, Eddie

2015-01-01

Municipal wastewater treatment facilities are considered to be “hotspots” for antibiotic resistance, since they conjoin high densities of environmental and fecal bacteria with selective pressure in the form of sub-therapeutic concentrations of antibiotics. Discharged effluents and biosolids from these facilities can disseminate antibiotic resistant genes to terrestrial and aquatic environments, potentially contributing to the increasing global trend in antibiotic resistance. This phenomenon is especially pertinent when resistance genes are associated with mobile genetic elements such as conjugative plasmids, which can be transferred between bacterial phyla. Fluoroquinolones are among the most abundant antibiotic compounds detected in wastewater treatment facilities, especially in biosolids, where due to their hydrophobic properties they accumulate to concentrations that may exceed 40 mg/L. Although fluoroquinolone resistance is traditionally associated with mutations in the gyrA/topoisomerase IV genes, there is increasing evidence of plasmid-mediated quinolone resistance, which is primarily encoded on qnr genes. In this study, we sequenced seven qnr-harboring plasmids from a diverse collection of Klebsiella strains, isolated from dewatered biosolids from a large wastewater treatment facility in Israel. One of the plasmids, termed pKPSH-11XL was a large (185.4 kbp), multi-drug resistance, IncF-type plasmid that harbored qnrB and 10 additional antibiotic resistance genes that conferred resistance to five different antibiotic families. It was highly similar to the pKPN3-like plasmid family that has been detected in multidrug resistant clinical Klebsiella isolates. In contrast, the six additional plasmids were much smaller (7–9 Kbp) and harbored a qnrS -type gene. These plasmids were highly similar to each other and closely resembled pGNB2, a plasmid isolated from a German wastewater treatment facility. Comparative genome analyses of pKPSH-11XL and other pKPN3-like plasmids concomitant to phylogenetic analysis of housekeeping genes from host Klebsiella strains, revealed that these plasmids are limited to a predominantly human-associated sub-clade of Klebsiella, suggesting that their host range is very narrow. Conversely, the pGNB2-like plasmids had a much broader host range and appeared to be associated with Klebsiella residing in natural environments. This study suggests that: (A) qnrB-harboring multidrug-resistant pKPN3-like plasmids can endure the rigorous wastewater treatment process and may therefore be disseminated to downstream environments; and (B) that small qnrS-harboring pGNB2-like plasmids are ubiquitous in wastewater treatment facilities and are most likely environmental in origin. PMID:26696974

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

PubMed

Kaplan, Ella; Sela, Noa; Doron-Faigenboim, Adi; Navon-Venezia, Shiri; Jurkevitch, Edouard; Cytryn, Eddie

2015-01-01

Municipal wastewater treatment facilities are considered to be "hotspots" for antibiotic resistance, since they conjoin high densities of environmental and fecal bacteria with selective pressure in the form of sub-therapeutic concentrations of antibiotics. Discharged effluents and biosolids from these facilities can disseminate antibiotic resistant genes to terrestrial and aquatic environments, potentially contributing to the increasing global trend in antibiotic resistance. This phenomenon is especially pertinent when resistance genes are associated with mobile genetic elements such as conjugative plasmids, which can be transferred between bacterial phyla. Fluoroquinolones are among the most abundant antibiotic compounds detected in wastewater treatment facilities, especially in biosolids, where due to their hydrophobic properties they accumulate to concentrations that may exceed 40 mg/L. Although fluoroquinolone resistance is traditionally associated with mutations in the gyrA/topoisomerase IV genes, there is increasing evidence of plasmid-mediated quinolone resistance, which is primarily encoded on qnr genes. In this study, we sequenced seven qnr-harboring plasmids from a diverse collection of Klebsiella strains, isolated from dewatered biosolids from a large wastewater treatment facility in Israel. One of the plasmids, termed pKPSH-11XL was a large (185.4 kbp), multi-drug resistance, IncF-type plasmid that harbored qnrB and 10 additional antibiotic resistance genes that conferred resistance to five different antibiotic families. It was highly similar to the pKPN3-like plasmid family that has been detected in multidrug resistant clinical Klebsiella isolates. In contrast, the six additional plasmids were much smaller (7-9 Kbp) and harbored a qnrS -type gene. These plasmids were highly similar to each other and closely resembled pGNB2, a plasmid isolated from a German wastewater treatment facility. Comparative genome analyses of pKPSH-11XL and other pKPN3-like plasmids concomitant to phylogenetic analysis of housekeeping genes from host Klebsiella strains, revealed that these plasmids are limited to a predominantly human-associated sub-clade of Klebsiella, suggesting that their host range is very narrow. Conversely, the pGNB2-like plasmids had a much broader host range and appeared to be associated with Klebsiella residing in natural environments. This study suggests that: (A) qnrB-harboring multidrug-resistant pKPN3-like plasmids can endure the rigorous wastewater treatment process and may therefore be disseminated to downstream environments; and (B) that small qnrS-harboring pGNB2-like plasmids are ubiquitous in wastewater treatment facilities and are most likely environmental in origin.

A molecular model for illegitimate recombination in Bacillus subtilis.

PubMed

Temeyer, K B; Hopkins, K M; Chapman, L F

1991-01-01

The recombinant DNA junctions at which pUB110 and Bacillus subtilis chromosomal DNA were joined to form the plasmid pKBT1 were cloned and sequenced. From the sequencing data we conclude that the pUB110 sequence is intact in the pair of cloned pKBT1 fragments and pTL12 sequences are not present. A molecular model for the formation of pKBT1 based on structural motifs characteristic of the joint sites is presented.

Isolation and characterization of two novel groups of Kanamycin-resistance ColE1-like plasmids in Salmonella enterica serotypes from food animals

USDA-ARS?s Scientific Manuscript database

While antimicrobial resistance in Salmonella enterica is largely attributed to large plasmids, small plasmids may also harbor antimicrobial resistance genes. Previously, three major groups of ColE1-like plasmids conferring kanamycin-resistance (KanR) in various S. enterica serotypes from diagnostic...

Proposed model for the high rate of rearrangement and rapid migration observed in some IncA/C plasmid lineages

USDA-ARS?s Scientific Manuscript database

IncA/C plasmids are a class of plasmids from Enterobacteraciae that are relatively large (49 to >180 kbp), are readily transferred by conjugation, and carry multiple antimicrobial resistance genes. Reconstruction of the phylogeny of these plasmids has been difficult because of the high rate of remo...

Effect of Dissemination of 2,4-Dichlorophenoxyacetic Acid (2,4-D) Degradation Plasmids on 2,4-D Degradation and on Bacterial Community Structure in Two Different Soil Horizons

PubMed Central

Dejonghe, Winnie; Goris, Johan; El Fantroussi, Saïd; Höfte, Monica; De Vos, Paul; Verstraete, Willy; Top, Eva M.

2000-01-01

Transfer of the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids pEMT1 and pJP4 from an introduced donor strain, Pseudomonas putida UWC3, to the indigenous bacteria of two different horizons (A horizon, depth of 0 to 30 cm; B horizon, depth of 30 to 60 cm) of a 2,4-D-contaminated soil was investigated as a means of bioaugmentation. When the soil was amended with nutrients, plasmid transfer and enhanced degradation of 2,4-D were observed. These findings were most striking in the B horizon, where the indigenous bacteria were unable to degrade any of the 2,4-D (100 mg/kg of soil) during at least 22 days but where inoculation with either of the two plasmid donors resulted in complete 2,4-D degradation within 14 days. In contrast, in soils not amended with nutrients, inoculation of donors in the A horizon and subsequent formation of transconjugants (105 CFU/g of soil) could not increase the 2,4-D degradation rate compared to that of the noninoculated soil. However, donor inoculation in the nonamended B-horizon soil resulted in complete degradation of 2,4-D within 19 days, while no degradation at all was observed in noninoculated soil during 89 days. With plasmid pEMT1, this enhanced degradation seemed to be due only to transconjugants (105 CFU/g of soil), since the donor was already undetectable when degradation started. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes showed that inoculation of the donors was followed by a shift in the microbial community structure of the nonamended B-horizon soils. The new 16S rRNA gene fragments in the DGGE profile corresponded with the 16S rRNA genes of 2,4-D-degrading transconjugant colonies isolated on agar plates. This result indicates that the observed change in the community was due to proliferation of transconjugants formed in soil. Overall, this work clearly demonstrates that bioaugmentation can constitute an effective strategy for cleanup of soils which are poor in nutrients and microbial activity, such as those of the B horizon. PMID:10919784

Oxidation of Naphthenoaromatic and Methyl-Substituted Aromatic Compounds by Naphthalene 1,2-Dioxygenase

PubMed Central

Selifonov, S. A.; Grifoll, M.; Eaton, R. W.; Chapman, P. J.

1996-01-01

Oxidation of acenaphthene, acenaphthylene, and fluorene was examined with recombinant strain Pseudomonas aeruginosa PAO1(pRE695) expressing naphthalene dioxygenase genes cloned from plasmid NAH7. Acenaphthene underwent monooxygenation to 1-acenaphthenol with subsequent conversion to 1-acenaphthenone and cis- and trans-acenaphthene-1,2-diols, while acenaphthylene was dioxygenated to give cis-acenaphthene-1,2-diol. Nonspecific dehydrogenase activities present in the host strain led to the conversion of both of the acenaphthene-1,2-diols to 1,2-acenaphthoquinone. The latter was oxidized spontaneously to naphthalene-1,8-dicarboxylic acid. No aromatic ring dioxygenation products were detected from acenaphthene and acenaphthylene. Mixed monooxygenase and dioxygenase actions of naphthalene dioxygenase on fluorene yielded products of benzylic 9-monooxygenation, aromatic ring dioxygenation, or both. The action of naphthalene dioxygenase on a variety of methyl-substituted aromatic compounds, including 1,2,4-trimethylbenzene and isomers of dimethylnaphthalene, resulted in the formation of benzylic alcohols, i.e., methyl group monooxygenation products, which were subsequently converted to the corresponding carboxylic acids by dehydrogenase(s) in the host strain. Benzylic monooxygenation of methyl groups was strongly predominant over aromatic ring dioxygenation and essentially nonspecific with respect to the substitution pattern of the aromatic substrates. In addition to monooxygenating benzylic methyl and methylene groups, naphthalene dioxygenase behaved as a sulfoxygenase, catalyzing monooxygenation of the sulfur heteroatom of 3-methylbenzothiophene. PMID:16535238

Toxin Kid uncouples DNA replication and cell division to enforce retention of plasmid R1 in Escherichia coli cells.

PubMed

Pimentel, Belén; Nair, Radhika; Bermejo-Rodríguez, Camino; Preston, Mark A; Agu, Chukwuma A; Wang, Xindan; Bernal, Juan A; Sherratt, David J; de la Cueva-Méndez, Guillermo

2014-02-18

Worldwide dissemination of antibiotic resistance in bacteria is facilitated by plasmids that encode postsegregational killing (PSK) systems. These produce a stable toxin (T) and a labile antitoxin (A) conditioning cell survival to plasmid maintenance, because only this ensures neutralization of toxicity. Shortage of antibiotic alternatives and the link of TA pairs to PSK have stimulated the opinion that premature toxin activation could be used to kill these recalcitrant organisms in the clinic. However, validation of TA pairs as therapeutic targets requires unambiguous understanding of their mode of action, consequences for cell viability, and function in plasmids. Conflicting with widespread notions concerning these issues, we had proposed that the TA pair kis-kid (killing suppressor-killing determinant) might function as a plasmid rescue system and not as a PSK system, but this remained to be validated. Here, we aimed to clarify unsettled mechanistic aspects of Kid activation, and of the effects of this for kis-kid-bearing plasmids and their host cells. We confirm that activation of Kid occurs in cells that are about to lose the toxin-encoding plasmid, and we show that this provokes highly selective restriction of protein outputs that inhibits cell division temporarily, avoiding plasmid loss, and stimulates DNA replication, promoting plasmid rescue. Kis and Kid are conserved in plasmids encoding multiple antibiotic resistance genes, including extended spectrum β-lactamases, for which therapeutic options are scarce, and our findings advise against the activation of this TA pair to fight pathogens carrying these extrachromosomal DNAs.

Toxin Kid uncouples DNA replication and cell division to enforce retention of plasmid R1 in Escherichia coli cells

PubMed Central

Pimentel, Belén; Nair, Radhika; Bermejo-Rodríguez, Camino; Preston, Mark A.; Agu, Chukwuma A.; Wang, Xindan; Bernal, Juan A.; Sherratt, David J.; de la Cueva-Méndez, Guillermo

2014-01-01

Worldwide dissemination of antibiotic resistance in bacteria is facilitated by plasmids that encode postsegregational killing (PSK) systems. These produce a stable toxin (T) and a labile antitoxin (A) conditioning cell survival to plasmid maintenance, because only this ensures neutralization of toxicity. Shortage of antibiotic alternatives and the link of TA pairs to PSK have stimulated the opinion that premature toxin activation could be used to kill these recalcitrant organisms in the clinic. However, validation of TA pairs as therapeutic targets requires unambiguous understanding of their mode of action, consequences for cell viability, and function in plasmids. Conflicting with widespread notions concerning these issues, we had proposed that the TA pair kis-kid (killing suppressor-killing determinant) might function as a plasmid rescue system and not as a PSK system, but this remained to be validated. Here, we aimed to clarify unsettled mechanistic aspects of Kid activation, and of the effects of this for kis-kid–bearing plasmids and their host cells. We confirm that activation of Kid occurs in cells that are about to lose the toxin-encoding plasmid, and we show that this provokes highly selective restriction of protein outputs that inhibits cell division temporarily, avoiding plasmid loss, and stimulates DNA replication, promoting plasmid rescue. Kis and Kid are conserved in plasmids encoding multiple antibiotic resistance genes, including extended spectrum β-lactamases, for which therapeutic options are scarce, and our findings advise against the activation of this TA pair to fight pathogens carrying these extrachromosomal DNAs. PMID:24449860

The PL6-Family Plasmids of Haloquadratum Are Virus-Related.

PubMed

Dyall-Smith, Mike; Pfeiffer, Friedhelm

2018-01-01

Plasmids PL6A and PL6B are both carried by the C23 T strain of the square archaeon Haloquadratum walsbyi , and are closely related (76% nucleotide identity), circular, about 6 kb in size, and display the same gene synteny. They are unrelated to other known plasmids and all of the predicted proteins are cryptic in function. Here we describe two additional PL6-related plasmids, pBAJ9-6 and pLT53-7, each carried by distinct isolates of Haloquadratum walsbyi that were recovered from hypersaline waters in Australia. A third PL6-like plasmid, pLTMV-6, was assembled from metavirome data from Lake Tyrell, a salt-lake in Victoria, Australia. Comparison of all five plasmids revealed a distinct plasmid family with strong conservation of gene content and synteny, an average size of 6.2 kb (range 5.8-7.0 kb) and pairwise similarities between 61-79%. One protein (F3) was closely similar to a protein carried by betapleolipoviruses while another (R6) was similar to a predicted AAA-ATPase of His 1 halovirus (His1V_gp16). Plasmid pLT53-7 carried a gene for a FkbM family methyltransferase that was not present in any of the other plasmids. Comparative analysis of all PL6-like plasmids provided better resolution of conserved sequences and coding regions, confirmed the strong link to haloviruses, and showed that their sequences are highly conserved among examples from Haloquadratum isolates and metagenomic data that collectively cover geographically distant locations, indicating that these genetic elements are widespread.

Sequencing and diversity analyses reveal extensive similarities between some epsilon-toxin-encoding plasmids and the pCPF5603 Clostridium perfringens enterotoxin plasmid.

PubMed

Miyamoto, Kazuaki; Li, Jihong; Sayeed, Sameera; Akimoto, Shigeru; McClane, Bruce A

2008-11-01

Clostridium perfringens type B and D isolates produce epsilon-toxin, the third most potent clostridial toxin. The epsilon-toxin gene (etx) is plasmid borne in type D isolates, but etx genetics have been poorly studied in type B isolates. This study reports the first sequencing of any etx plasmid, i.e., pCP8533etx, from type B strain NCTC8533. This etx plasmid is 64.7 kb, carries tcp conjugative transfer genes, and encodes additional potential virulence factors including beta2-toxin, sortase, and collagen adhesin but not beta-toxin. Interestingly, nearly 80% of pCP8533etx open reading frames (ORFs) are also present on pCPF5603, an enterotoxin-encoding plasmid from type A isolate F5603. Pulsed-field gel electrophoresis and overlapping PCR indicated that a pCP8533etx-like etx plasmid is also present in most, if not all, other type B isolates and some beta2-toxin-positive, cpe-negative type D isolates, while other type D isolates carry different etx plasmids. Sequences upstream of the etx gene vary between type B isolates and some type D isolates that do not carry a pCP8533etx-like etx plasmid. However, nearly all type B and D isolates have an etx locus with an upstream IS1151, and those etx loci typically reside near a dcm ORF. These results suggest that pCPF5603 and pCP8533etx evolved from insertion of mobile genetic elements carrying enterotoxin or etx genes, respectively, onto a common progenitor plasmid.

Population structure of plasmid-containing strains of Streptococcus mutans, a member of the human indigenous biota.

PubMed

Caufield, Page W; Saxena, Deepak; Fitch, David; Li, Yihong

2007-02-01

There are suggestions that the phylogeny of Streptococcus mutans, a member of the human indigenous biota that is transmitted mostly mother to child, might parallel the evolutionary history of its human host. The relatedness and phylogeny of plasmid-containing strains of S. mutans were examined based on chromosomal DNA fingerprints (CDF), a hypervariable region (HVR) of a 5.6-kb plasmid, the rRNA gene intergenic spacer region (IGSR), serotypes, and the genotypes of mutacin I and II. Plasmid-containing strains were studied because their genetic diversity was twice as great as that of plasmid-free strains. The CDF of S. mutans from unrelated human hosts were unique, except those from Caucasians, which were essentially identical. The evolutionary history of the IGSR, with or without the serotype and mutacin characters, clearly delineated an Asian clade. Also, a continuous association with mutacin II could be reconstructed through an evolutionary lineage with the IGSR, but not for serotype e. DNA sequences from the HVR of the plasmid produced a well-resolved phylogeny that differed from the chromosomal phylogeny, indicating that the horizontal transfer of the plasmid may have occurred multiple times. The plasmid phylogeny was more congruent with serotype e than with mutacin II evolution, suggesting a possible functional correlation. Thus, the history of this three-tiered relationship between human, bacterium, and plasmid supported both coevolution and independent evolution.

Application of methylation in improving plasmid transformation into Helicobacter pylori.

PubMed

Zhao, Huilin; Xu, Linlin; Rong, Qianyu; Xu, Zheng; Ding, Yunfei; Zhang, Ying; Wu, Yulong; Li, Boqing; Ji, Xiaofei

2018-05-23

Helicobacter pylori is an important gastrointestinal pathogen. Its strains possess different levels of powerful restriction modification systems, which are significant barriers to genetic tools used for studying the role of functional genes in its pathogenesis. Methylating vectors in vitro was reported as an alternative to overcome this barrier in several bacteria. In this study we used two H. pylori-E. coli shuttle plasmids and several single/double-crossover homologous recombination gene-targeting plasmids, to test the role of methylation in H. pylori transformation. According to our results, transformants could be obtained only after shuttle plasmids were methylated before transformation. It is helpful in gene complementation and over-expression although at a low frequency. The frequency of gene-targeting transformation was also increased after methylation, especially for the single-crossover recombination plasmids, the transformants of which could only be obtained after methylation. For the double-crossover recombination targeting plasmids, the initial yield of transformants was 0.3-0.8 × 10 2 CFUs per microgram plasmid DNA. With the help of methylation, the yield was increased to 0.4-1.3 × 10 2 CFUs per microgram plasmid DNA. These results suggest that in vitro methylation can improve H. pylori transformation by different plasmids, which will benefit the pathogenic mechanism research. Copyright © 2018. Published by Elsevier B.V.

Reporter gene expression in dendritic cells after gene gun administration of plasmid DNA.

PubMed

Watkins, Craig; Hopkins, John; Harkiss, Gordon

2005-07-21

Dendritic cells (DC) play an integral role in plasmid DNA vaccination. However, the interaction between plasmid DNA and DC in vivo is incompletely understood. In this report, we utilise the sheep pseudoafferent cannulation model to examine the interaction between plasmid DNA encoding enhanced green fluorescent protein (pEGFP) and afferent lymph DC (ALDC) following gene gun administration. The results show that peaks of fluorescent ALDC tended to appear around days 1-4 and 9-13, then erratically thereafter for up to 2 months. Phenotypic analysis showed that EGFP+ ALDC expressed MHC class II, WC6, CD1b, and SIRPalpha markers. Plasmid, detected by PCR, was found in lymph cells and cell-free plasma on a daily basis, and was present variably for up to 2 months. Plasmid was also detected in purified CD1b+ ALDC, but the presence of plasmid did not correlate with EGFP expression by ALDC. Free EGFP in afferent lymph plasma was detectable by luminometry only after three administrations of the plasmid. The results show that gene gun administered pEGFP persisted for extended periods after a single administration, leeching out of skin on a daily basis. The plasmid was associated with both the cellular and fluid components of afferent lymph. EGFP protein appeared in afferent lymph in a pulsatile manner, but associated only with ALDC.

Complete sequence of Enterococcus faecium pVEF3 and the detection of an omega-epsilon-zeta toxin-antitoxin module and an ABC transporter.

PubMed

Sletvold, H; Johnsen, P J; Hamre, I; Simonsen, G S; Sundsfjord, A; Nielsen, K M

2008-07-01

Glycopeptide resistant Enterococcus faecium (GREF) persists on Norwegian poultry farms despite the ban on the growth promoter avoparcin. The biological basis for long-term persistence of avoparcin resistance is not fully understood. This study presents the complete DNA sequence of the E. faecium R-plasmid pVEF3 and functional studies of some plasmid-encoded traits (a toxin-antitoxin (TA) system and an ABC transporter) that may be of importance for plasmid persistence. The pVEF3 (63.1 kbp), isolated from an E. faecium strain of poultry origin sampled in Norway in 1999, has 71 coding sequences including the vanA avoparcin/vancomycin resistance encoding gene cluster. pVEF3 encodes the TA system omega-epsilon-zeta, and plasmid stability tests and transcription analysis show that omega-epsilon-zeta is functional in Enterococcus faecalis OGIX, although with decreasing effect over time. The predicted ABC transporter was not found to confer reduced susceptibility to any of the 28 substances tested. The TA system identified in the pVEF-type plasmids may contribute to vanA plasmid persistence on Norwegian poultry farms. However, size and compositional heterogeneity among E. faecium vanA plasmids suggest that additional plasmid maintenance systems in combination with host specific factors and frequent horizontal gene transfer and rearrangement causes the observed plasmid composition and distribution patterns.

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

USDA-ARS?s Scientific Manuscript database

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

Structural and physiological studies of the Escherichia coli histidine operon inserted into plasmid vectors.

PubMed Central

Bruni, C B; Musti, A M; Frunzio, R; Blasi, F

1980-01-01

A fragment of deoxyribonucleic acid 5,300 base paris long and containing the promoter-proximal portion of the histidine operon of Escherichia coli K-12, has been cloned in plasmid pBR313 (plasmids pCB2 and pCB3). Restriction mapping, partial nucleotide sequencing, and studies on functional expression in vivo and on protein synthesis in minicells have shown that the fragment contains the regulatory region of the operon, the hisG, hisD genes, and part of the hisC gene. Another plasmid (pCB5) contained the hisG gene and part of the hisD gene. Expression of the hisG gene in the latter plasmid was under control of the tetracycline promoter of the pBR313 plasmid. The in vivo expression of the two groups of plasmids described above, as well as their effect on the expression of the histidine genes not carried by the plasmids but present on the host chromosome, has been studied. The presence of multiple copies of pCB2 or pCB3, but not of pCB5, prevented derepression of the chromosomal histidine operon. Possible interpretations of this phenomenon are discussed. Images PMID:6246067

In vivo induction of interferon gamma expression in grey horses with metastatic melanoma resulting from direct injection of plasmid DNA coding for equine interleukin 12.

PubMed

Müller, J-M V; Wissemann, J; Meli, M L; Dasen, G; Lutz, H; Heinzerling, L; Feige, K

2011-11-01

Whole blood pharmacokinetics of intratumourally injected naked plasmid DNA coding for equine Interleukin 12 (IL-12) was assessed as a means of in vivo gene transfer in the treatment of melanoma in grey horses. The expression of induced interferon gamma (IFN-g) was evaluated in order to determine the pharmacodynamic properties of in vivo gene transduction. Seven grey horses bearing melanoma were injected intratumourally with 250 µg naked plasmid DNA coding for IL-12. Peripheral blood and biopsies from the injection site were taken at 13 time points until day 14 post injection (p.i.). Samples were analysed using quantitative real-time PCR. Plasmid DNA was quantified in blood samples and mRNA expression for IFN-g in tissue samples. Plasmid DNA showed fast elimination kinetics with more than 99 % of the plasmid disappearing within 36 hours. IFN-g expression increased quickly after IL-12 plasmid injection, but varied between individual horses. Intratumoural injection of plasmid DNA is a feasible method for inducing transgene expression in vivo. Biological activity of the transgene IL-12 was confirmed by measuring expression of IFN-g.

Sequence determination and analysis of three plasmids of Pseudomonas sp. GLE121, a psychrophile isolated from surface ice of Ecology Glacier (Antarctica).

PubMed

Dziewit, Lukasz; Grzesiak, Jakub; Ciok, Anna; Nieckarz, Marta; Zdanowski, Marek K; Bartosik, Dariusz

2013-09-01

Pseudomonas sp. GLE121 (a psychrophilic Antarctic strain) carries three plasmids: pGLE121P1 (6899 bp), pGLE121P2 (8330 bp) and pGLE121P3 (39,583 bp). Plasmids pGLE121P1 and pGLE121P2 show significant sequence similarity to members of the IncP-9 and IncP-7 incompatibility groups, respectively, while the largest replicon, pGLE121P3, is highly related to plasmid pNCPPB880-40 of Pseudomonas syringae pathovar tomato NCPPB880. All three plasmids have a narrow host range, limited to members of the genus Pseudomonas. Plasmid pGLE121P3 encodes a conjugal transfer system, while pGLE121P1 carries only a putative MOB module, conserved in many mobilizable plasmids. Plasmid pGLE121P3 contains an additional load of genetic information, including a pair of genes with homology to the rulAB operon, responsible for ultraviolet radiation (UVR) tolerance. Given the increasing UV exposure in Antarctic regions, the expression of these genes is likely to be an important adaptive response. Copyright © 2013 Elsevier Inc. All rights reserved.

Mobilization of the relaxable Staphylococcus aureus plasmid pC221 by the conjugative plasmid pGO1 involves three pC221 loci.

PubMed Central

Projan, S J; Archer, G L

1989-01-01

The Staphylococcus aureus plasmid pC221, a 4.6-kilobase multicopy chloramphenicol resistance plasmid that forms plasmid-protein relaxation complexes, was mobilized for transfer by the conjugative plasmid pGO1. Two open reading frames on the pC221 genome, now designated mobA and mobB, as well as a cis-acting locus, the putative oriT, were shown to be in involved in pC221 mobilization. The mobA (but not mobB) and oriT loci were required for pC221 relaxation, and relaxation was necessary but not sufficient for pC221 mobilization by pGO1. oriT was cloned onto a pE194 derivative and complemented in trans for both relaxation and mobilization. Mobilization of relaxable plasmids in S. aureus appears to be analogous to mobilization by donation observed in gram-negative bacteria. Images PMID:2703461

Plasmid-mediated resistance to protein biosynthesis inhibitors in staphylococci.

PubMed

Schwarz, Stefan; Fessler, Andrea T; Hauschild, Tomasz; Kehrenberg, Corinna; Kadlec, Kristina

2011-12-01

Protein biosynthesis inhibitors (PBIs) represent powerful antimicrobial agents for the control of bacterial infections. In staphylococci, numerous resistance genes are known to be involved in resistance to PBIs, most of which mediate resistance to a specific class/subclass of PBIs, though a few genes do confer a multidrug resistance phenotype-up to five classes/subclasses of PBIs. Plasmids play a key role in the dissemination of PBI resistance among staphylococci, as they primarily carry plasmid-borne PBI resistance genes; however, plasmids also can be vectors for transposon-borne PBI resistance genes. Small plasmids that carry single PBI resistance genes are widespread among staphylococci of human and animal origin. Various mechanisms exist by which they can recombine, form cointegrates, or integrate into chromosomal DNA or larger plasmids. We provide an overview of the current knowledge of plasmid-mediated PBI resistance in staphylococci, with particular reference to the currently known PBI resistance genes, their association with mobile genetic elements, and the recombination/integration processes that control their mobility. © 2011 New York Academy of Sciences.

Plasmid fermentation process for DNA immunization applications.

PubMed

Carnes, Aaron E; Williams, James A

2014-01-01

Plasmid DNA for immunization applications must be of the highest purity and quality. The ability of downstream purification to efficiently produce a pure final product is directly influenced by the performance of the upstream fermentation process. While several clinical manufacturing facilities already have validated fermentation processes in place to manufacture plasmid DNA for use in humans, a simple and inexpensive laboratory-scale fermentation process can be valuable for in-house production of plasmid DNA for use in animal efficacy studies. This chapter describes a simple fed-batch fermentation process for producing bacterial cell paste enriched with high-quality plasmid DNA. A constant feeding strategy results in a medium cell density culture with continuously increasing plasmid amplification towards the end of the process. Cell banking and seed culture preparation protocols, which can dramatically influence final product yield and quality, are also described. These protocols are suitable for production of research-grade plasmid DNA at the 100 mg-to-1.5 g scale from a typical 10 L laboratory benchtop fermentor.

Plasmid ColE1 as a Molecular Vehicle for Cloning and Amplification of DNA

PubMed Central

Hershfield, Vickers; Boyer, Herbert W.; Yanofsky, Charles; Lovett, Michael A.; Helinski, Donald R.

1974-01-01

DNA fragments obtained from EcoRI endonuclease digestion of bacteriophage ϕ80pt190 (trp+) and the plasmid ColE1 were covalently joined with polynucleotide ligase. Transformation of Escherichia coli trp- strains to tryptophan independence with the recombined DNA selected for reconstituted ColE1 plasmids containing the tryptophan operon and the ϕ80 immunity region. Similarly, an EcoRI endonuclease generated fragment of plasmid pSC105 DNA containing the genetic determinant of kanamycin resistance was inserted into the ColE1 plasmid and recovered in E. coli. The plasmids containing the trp operon (ColE1-trp) and the kanamycin resistance gene were maintained under logarithmic growth conditions at a level of 25-30 copies per cell and accumulate to the extent of several hundred copies per cell in the presence of chloramphenicol. Cells carrying the ColE1-trp plasmid determined the production of highly elevated levels of trp operon-specific mRNA and tryptophan biosynthetic enzymes. Images PMID:4610576

Characterization of a cryptic plasmid from an alpha-proteobacterial endosymbiont of Amoeba proteus.

PubMed

Park, Miey; Kim, Min-Soo; Lee, Kyung-Min; Hwang, Sue-Yun; Ahn, Tae In

2009-01-01

A new cryptic plasmid pAP3.9 was discovered in symbiotic alpha-proteobacteria present in the cytoplasm of Amoeba proteus. The plasmid is 3869bp with a GC content of 34.66% and contains replication origins for both double-strand (dso) and single-strand (sso). It has three putative ORFs encoding Mob, Rep and phosphoglycolate phosphatase (PGPase). The pAP3.9 plasmid appears to propagate by the conjugative rolling-circle replication (RCR), since it contains all required factors such as Rep, sso and dso. Mob and Rep showed highest similarities to those of the cryptic plasmid pBMYdx in Bacillus mycoides. The PGPase was homologous to that of Bacillus cereus and formed a clade with those of Bacillus sp. in molecular phylogeny. These results imply that the pAP3.9 plasmid evolved by the passage through Bacillus species. We hypothesize that the plasmid-encoded PGPase may have contributed to the establishment of bacterial symbiosis within the hostile environment of amoeba cytoplasm.

Induction of micronuclei by HTLV-I Tax: a cellular assay for function.

PubMed

Majone, F; Semmes, O J; Jeang, K T

1993-03-01

Cellular chromosomal damage is ubiquitously seen in HTLV-I-transformed lymphocytes. It is also characteristic of cells that have been exposed to mutagens. A sensitive measurement for mutagen-induced DNA damage is the formation of micronuclei in treated cells. Because current evidence suggests that HTLV-I Tax is etiologically linked to transformation, we tested for its activity in inducing micronuclei. We show here that transfection into cells of a Tax-producing plasmid rapidly induced the formation of micronuclei. This effect cooperated with that of a mutagen (mitomycin C) and was correlated with the inherent trans-activation capacity of Tax. These findings suggest that a commonly used mutagen assay could be a quick biological test for putatively oncogenic proteins.

Experimental study on effects of geologic heterogeneity in enhancing dissolution trapping of supercritical CO2

NASA Astrophysics Data System (ADS)

Agartan, Elif; Trevisan, Luca; Cihan, Abdullah; Birkholzer, Jens; Zhou, Quanlin; Illangasekare, Tissa H.

2015-03-01

Dissolution trapping is one of the primary mechanisms that enhance the storage security of supercritical carbon dioxide (scCO2) in saline geologic formations. When scCO2 dissolves in formation brine produces an aqueous solution that is denser than formation brine, which leads to convective mixing driven by gravitational instabilities. Convective mixing can enhance the dissolution of CO2 and thus it can contribute to stable trapping of dissolved CO2. However, in the presence of geologic heterogeneities, diffusive mixing may also contribute to dissolution trapping. The effects of heterogeneity on mixing and its contribution to stable trapping are not well understood. The goal of this experimental study is to investigate the effects of geologic heterogeneity on mixing and stable trapping of dissolved CO2. Homogeneous and heterogeneous media experiments were conducted in a two-dimensional test tank with various packing configurations using surrogates for scCO2 (water) and brine (propylene glycol) under ambient pressure and temperature conditions. The results show that the density-driven flow in heterogeneous formations may not always cause significant convective mixing especially in layered systems containing low-permeability zones. In homogeneous formations, density-driven fingering enhances both storage in the deeper parts of the formation and contact between the host rock and dissolved CO2 for the potential mineralization. On the other hand, for layered systems, dissolved CO2 becomes immobilized in low-permeability zones with low-diffusion rates, which reduces the risk of leakage through any fault or fracture. Both cases contribute to the permanence of the dissolved plume in the formation.

Plasmid-borne Tn5 insertion mutation resulting in accumulation of gentisate from salicylate.

PubMed Central

Monticello, D J; Bakker, D; Schell, M; Finnerty, W R

1985-01-01

Plasmid-borne Tn5 insertion mutants of a Pseudomonas species which accumulated 2,5-dihydroxybenzoate (gentisate) following growth on 2-hydroxybenzoate (salicylate) were obtained from a pool of mutants that were unable to grow on naphthalene. One such mutant was characterized further. The ability of this mutant to oxidize gentisate was 100-fold less than the ability of a Nah+ Sal+ strain harboring the unmutagenized plasmid, although both strains oxidized and grew on salicylate. These bacteria were presumably able to metabolize salicylate via catechol, since they possessed an inducible, plasmid-encoded catechol 2,3-dioxygenase. Our results suggest that there is an alternate, plasmid-encoded route of salicylate degradation via gentisate and that some plasmid-associated relationship between this pathway and naphthalene oxidation exists. PMID:2988437

The Impact of Test Dimensionality, Common-Item Set Format, and Scale Linking Methods on Mixed-Format Test Equating

ERIC Educational Resources Information Center

Öztürk-Gübes, Nese; Kelecioglu, Hülya

2016-01-01

The purpose of this study was to examine the impact of dimensionality, common-item set format, and different scale linking methods on preserving equity property with mixed-format test equating. Item response theory (IRT) true-score equating (TSE) and IRT observed-score equating (OSE) methods were used under common-item nonequivalent groups design.…

Transfected Cell Microarrays for the Expression of Membrane-Displayed Single-Chain Antibodies

DTIC Science & Technology

2011-01-01

v) yeast extract, 0.005% (w/v) NaCl, and 50 μg/ml kanamycin. The broth was stored at 4◦C for up to 3 months. 4. QIAGEN Plasmid Midi kit (Qiagen) or...ampi- cillin. The broth was stored at 4◦C for up to 3 months. 16. QIAprep spin miniprep kit and QIAGEN Plasmid Midi kit (Qiagen) or PureYield Plasmid...was stored at 4◦C for up to 3 months. 8. QIAGEN Plasmid Midi kit (Qiagen) or PureYield Plasmid Midiprep System (Promega Corp.) was stored at room tem

Successful Establishment of Plasmids R1 and pMV158 in a New Host Requires the Relief of the Transcriptional Repression of Their Essential rep Genes

PubMed Central

Ruiz-Masó, José Á.; Luengo, Luis M.; Moreno-Córdoba, Inmaculada; Díaz-Orejas, Ramón; del Solar, Gloria

2017-01-01

Although differing in size, encoded traits, host range, and replication mechanism, both narrow-host-range theta-type conjugative enterobacterial plasmid R1 and promiscuous rolling-circle-type mobilizable streptococcal plasmid pMV158 encode a transcriptional repressor protein, namely CopB in R1 and CopG in pMV158, involved in replication control. The gene encoding CopB or CopG is cotranscribed with a downstream gene that encodes the replication initiator Rep protein of the corresponding plasmid. However, whereas CopG is an auto-repressor that inhibits transcription of the entire copG-repB operon, CopB is expressed constitutively and represses a second, downstream promoter that directs transcription of repA. As a consequence of the distinct regulatory pathways implied by CopB and CopG, these repressor proteins play a different role in control of plasmid replication during the steady state: while CopB has an auxiliary role by keeping repressed the regulated promoter whenever the plasmid copy number is above a low threshold, CopG plays a primary role by acting coordinately with RNAII. Here, we have studied the role of the regulatory circuit mediated by these transcriptional repressors during the establishment of these two plasmids in a new host cell, and found that excess Cop repressor molecules in the recipient cell result in a severe decrease in the frequency and/or the velocity of appearance of transformant colonies for the cognate plasmid but not for unrelated plasmids. Using the pMV158 replicon as a model system, together with highly sensitive real-time qPCR and inverse PCR methods, we have also analyzed the effect of CopG on the kinetics of repopulation of the plasmid in Streptococcus pneumoniae. We show that, whereas in the absence of CopG pMV158 repopulation occurs mainly during the first 45 min following plasmid transfer, the presence of the transcriptional repressor in the recipient cell severely impairs the replicon repopulation and makes the plasmid replicate at approximately the same rate as the chromosome at any time after transformation, which results in maximal plasmid loss rate in the absence of selection. Overall, these findings indicate that unrepressed activity of the Cop-regulated promoter is crucial for the successful colonization of the recipient bacterial cells by the plasmid. PMID:29250051

F14:A-:B- and IncX4 Inc group cfr-positive plasmids circulating in Escherichia coli of animal origin in Northeast China.

PubMed

Wang, Xiumei; Zhu, Yao; Hua, Xin; Chen, Fuguang; Wang, Changzhen; Zhang, Yanhe; Liu, Siguo; Zhang, Wanjiang

2018-04-01

The objective of this study was to investigate the prevalence of the cfr gene in Escherichia coli isolates from domestic animals in Northeast China and to characterize the cfr-containing plasmids. Between June 2015 and April 2016, 370 E. coli isolates were collected from pigs, chickens, and dairy cows in Northeast China. Among these, 111 were florfenicol resistant, including 109 isolates carrying the floR gene and 6 positives for cfr. The prevalence of cfr in E. coli isolates from the four northeast provinces in China was 1.6% (6/370), which was higher than that previously reported (0.08% and 0.5%). All six cfr-containing E. coli isolates were highly resistant to florfenicol (100%), cefotaxime (100%), and fosfomycin (100%). Complete sequence analysis of two cfr-carrying plasmids revealed high homology of the IncX4-type pEC14cfr plasmid with two other cfr-harboring plasmids, pSD11 and pGXEC6, found in swine E. coli isolates from southern China. pEC14cfr-like plasmids have been isolated in five provinces in southern and northern China. The isolation sites were up to 2700 kilometers apart, implying that pEC14cfr-like plasmids are likely to be national epidemic cfr-carrying plasmids that mediate the dissemination of cfr in China. Moreover, the genetic structure (IS26-IS26-cfr-rec-pre/mob-ramA-IS26) of the second cfr-carrying plasmid, IncF14:A-:B- pEC295cfr, represents a novel genetic environment for cfr identified for the first time in the present study. Sequence homology analysis indicated that the cfr-carrying element was most likely introduced into a cfr-negative pEC12 plasmid backbone, which evolved into the cfr-carrying vector, pEC295cfr. Moreover, isolation of the IncF14:A-:B- pEC295cfr plasmid harboring cfr suggests that IncFII plasmids maybe have become additional effective vehicles for cfr dissemination. These results highlight the importance of surveying the prevalence of IncX4 and IncFII plasmids in gram-negative bacteria, especially in swine E. coli isolates. Copyright © 2018 Elsevier B.V. All rights reserved.

Characterization of blaCMY plasmids and their possible role in source attribution of Salmonella enterica serotype Typhimurium infections.

PubMed

Folster, Jason P; Tolar, Beth; Pecic, Gary; Sheehan, Deborah; Rickert, Regan; Hise, Kelley; Zhao, Shaohua; Fedorka-Cray, Paula J; McDermott, Patrick; Whichard, Jean M

2014-04-01

Salmonella is an important cause of foodborne illness; however, identifying the source of these infections can be difficult. This is especially true for Salmonella serotype Typhimurium, which is found in diverse agricultural niches. Extended-spectrum cephalosporins (ESC) are one of the primary treatment choices for complicated Salmonella infections. In Salmonella, ESC resistance in the United States is mainly mediated by blaCMY genes carried on various plasmids. In this study, we examined whether the characterization of blaCMY plasmids, along with additional information, can help us identify potential sources of infection by Salmonella, and used serotype Typhimurium as a model. In the United States, monitoring of retail meat, food animals, and ill persons for antimicrobial-resistant Salmonella is conducted by the National Antimicrobial Resistance Monitoring System. In 2008, 70 isolates (70/581; 12.0%) (34 isolates from retail meat, 23 food animal, and 13 human) were resistant to ceftriaxone and amoxicillin/clavulanic acid. All were polymerase chain reaction (PCR)-positive for blaCMY and 59/70 (84.3%) of these genes were plasmid encoded. PCR-based replicon typing identified 42/59 (71.2%) IncI1-blaCMY plasmids and 17/59 (28.8%) IncA/C-blaCMY plasmids. Isolates from chickens or chicken products with blaCMY plasmids primarily had IncI1-blaCMY plasmids (37/40; 92.5%), while all isolates from cattle had IncA/C-blaCMY plasmids. Isolates from humans had either IncA/C- blaCMY (n=8/12; [66.7%]) or IncI1- blaCMY (n=4/12 [33.3%]) plasmids. All of the IncI1-blaCMY plasmids were ST12 or were closely related to ST12. Antimicrobial susceptibility patterns (AST) and pulsed-field gel electrophoresis (PFGE) patterns of the isolates were also compared and differences were identified between isolate sources. When the source of a Typhimurium outbreak or sporadic illness is unknown, characterizing the outbreak isolate's blaCMY plasmids, AST, and PFGE patterns may help identify it.

Characterization of blaCMY Plasmids and Their Possible Role in Source Attribution of Salmonella enterica Serotype Typhimurium Infections

PubMed Central

Folster, J.P.; Tolar, B.; Pecic, G.; Sheehan, D.; Rickert, R.; Hise, K.; Zhao, S.; Fedorka-Cray, P. J.; McDermott, P.; Whichard, J.M.

2015-01-01

Salmonella is an important cause of foodborne illness; however, identifying the source of these infections can be difficult. This is especially true for Salmonella serotype Typhimurium which is found in diverse agricultural niches. Extended spectrum cephalosporins (ESC) are one of the primary treatment choices for complicated Salmonella infections. In Salmonella, ESC resistance in the U.S. is mainly mediated by blaCMY genes carried on various plasmids. In this study, we examined whether the characterization of blaCMY plasmids, along with additional information, can help us identify potential sources of infection by Salmonella, and use serotype Typhimurium as a model. In the U.S., monitoring of retail meat, food animals, and ill persons for antimicrobial resistant Salmonella is conducted by the National Antimicrobial Resistance Monitoring System (NARMS). In 2008, 70 isolates (70/581;12.0 %) (34 isolates from retail meat, 23 food animal, and 13 human) were resistant to ceftriaxone and amoxicillin/clavulanic acid. All were PCR-positive for blaCMY and 59/70 (84.3%) of these genes were plasmid-encoded. PCR-based replicon typing (PBRT) identified 42/59 (71.2%) IncI1-blaCMY plasmids and 17/59 (28.8%) IncA/C-blaCMY plasmids. Isolates from chickens or chicken products with blaCMY plasmids primarily had IncI1-blaCMY plasmids (37/40; 92.5%), while all isolates from cattle had IncA/C-blaCMY plasmids. Isolates from humans had either IncA/C- blaCMY (n = 8/12; [66.7%]) or IncI1- blaCMY (n = 4/12 [33.3%]) plasmids. All of the IncI1-blaCMY plasmids were ST12 or were closely related to ST12. Antimicrobial susceptibility patterns (AST) and pulsed-field gel electrophoresis (PFGE) patterns of the isolates were also compared and differences were identified between isolate sources. When the source of a Typhimurium outbreak or sporadic illness is unknown, characterizing outbreak isolate’s blaCMY plasmids, AST, and PFGE patterns may help identify it. PMID:24484290

Characterization of extended-spectrum cephalosporin-resistant Salmonella enterica serovar Heidelberg isolated from food animals, retail meat, and humans in the United States 2009.

PubMed

Folster, J P; Pecic, G; Singh, A; Duval, B; Rickert, R; Ayers, S; Abbott, J; McGlinchey, B; Bauer-Turpin, J; Haro, J; Hise, K; Zhao, S; Fedorka-Cray, P J; Whichard, J; McDermott, P F

2012-07-01

Salmonella enterica is one of the most common causes of foodborne illness in the United States. Although salmonellosis is usually self-limiting, severe infections typically require antimicrobial treatment, and ceftriaxone, an extended-spectrum cephalosporin (ESC), is commonly used in both adults and children. Surveillance conducted by the National Antimicrobial Resistance Monitoring System (NARMS) has shown a recent increase in ESC resistance among Salmonella Heidelberg isolated from food animals at slaughter, retail meat, and humans. ESC resistance among Salmonella in the United States is usually mediated by a plasmid-encoded bla(CMY) β-lactamase. In 2009, we identified 47 ESC-resistant bla(CMY)-positive Heidelberg isolates from humans (n=18), food animals at slaughter (n=16), and retail meats (n=13) associated with a spike in the prevalence of this serovar. Almost 90% (26/29) of the animal and meat isolates were isolated from chicken carcasses or retail chicken meat. We screened NARMS isolates for the presence of bla(CMY), determined whether the gene was plasmid-encoded, examined pulsed-field gel electrophoresis patterns to assess the genetic diversities of the isolates, and categorized the bla(CMY) plasmids by plasmid incompatibility groups and plasmid multi-locus sequence typing (pMLST). All 47 bla(CMY) genes were found to be plasmid encoded. Incompatibility/replicon typing demonstrated that 41 were IncI1 plasmids, 40 of which only conferred bla(CMY)-associated resistance. Six were IncA/C plasmids that carried additional resistance genes. pMLST of the IncI1-bla(CMY) plasmids showed that 27 (65.8%) were sequence type (ST) 12, the most common ST among bla(CMY)-IncI1 plasmids from Heidelberg isolated from humans. Ten plasmids had a new ST profile, ST66, a type very similar to ST12. This work showed that the 2009 increase in ESC resistance among Salmonella Heidelberg was caused mainly by the dissemination of bla(CMY) on IncI1 and IncA/C plasmids in a variety of genetic backgrounds, and is likely not the result of clonal expansion.

Characterization of Four Multidrug Resistance Plasmids Captured from the Sediments of an Urban Coastal Wetland

PubMed Central

Botts, Ryan T.; Apffel, Brooke A.; Walters, C. J.; Davidson, Kelly E.; Echols, Ryan S.; Geiger, Michael R.; Guzman, Victoria L.; Haase, Victoria S.; Montana, Michal A.; La Chat, Chip A.; Mielke, Jenna A.; Mullen, Kelly L.; Virtue, Cierra C.; Brown, Celeste J.; Top, Eva M.; Cummings, David E.

2017-01-01

Self-transmissible and mobilizable plasmids contribute to the emergence and spread of multidrug-resistant bacteria by enabling the horizontal transfer of acquired antibiotic resistance. The objective of this study was to capture and characterize self-transmissible and mobilizable resistance plasmids from a coastal wetland impacted by urban stormwater runoff and human wastewater during the rainy season. Four plasmids were captured, two self-transmissible and two mobilizable, using both mating and enrichment approaches. Plasmid genomes, sequenced with either Illumina or PacBio platforms, revealed representatives of incompatibility groups IncP-6, IncR, IncN3, and IncF. The plasmids ranged in size from 36 to 144 kb and encoded known resistance genes for most of the major classes of antibiotics used to treat Gram-negative infections (tetracyclines, sulfonamides, β-lactams, fluoroquinolones, aminoglycosides, and amphenicols). The mobilizable IncP-6 plasmid pLNU-11 was discovered in a strain of Citrobacter freundii enriched from the wetland sediments with tetracycline and nalidixic acid, and encodes a novel AmpC-like β-lactamase (blaWDC-1), which shares less than 62% amino acid sequence identity with the PDC class of β-lactamases found in Pseudomonas aeruginosa. Although the IncR plasmid pTRE-1611 was captured by mating wetland bacteria with P. putida KT2440 as recipient, it was found to be mobilizable rather than self-transmissible. Two self-transmissible multidrug-resistance plasmids were also captured: the small (48 kb) IncN3 plasmid pTRE-131 was captured by mating wetland bacteria with Escherichia coli HY842 where it is seemed to be maintained at nearly 240 copies per cell, while the large (144 kb) IncF plasmid pTRE-2011, which was isolated from a cefotaxime-resistant environmental strain of E. coli ST744, exists at just a single copy per cell. Furthermore, pTRE-2011 bears the globally epidemic blaCTX-M-55 extended-spectrum β-lactamase downstream of ISEcp1. Our results indicate that urban coastal wetlands are reservoirs of diverse self-transmissible and mobilizable plasmids of relevance to human health. PMID:29067005

[Isolation and characterization of petroleum catabolic broad-host-range plasmids from Shen-Fu wastewater irrigation zone].

PubMed

Wang, Ya-Fei; Wang, Ya-Fei; Li, Hui; Li, Xiao-Bin

2013-11-01

Based on triparental mating, we isolated a total of eight broad host range (BHR) petroleum hydrocarbon catabolic plasmids from the soils, sediments, and wastewater samples in the Shen-Fu irrigation zone. The antibiotic resistance of the plasmids was tested, and then, the plasmids were transferred to Escherichia coli EC100. The plasmids carrying no antibiotic resistance were tagged by miniTn5 transposon consisting of antibiotic resistant genes. The PCR-based incompatibility test revealed that the pS3-2C and pS4-6G belonged to Inc P group, the pS3-2G, pW22-3G, and pA15-7G belonged to Inc N group, the pS7-2G was identified as Inc W plasmid, and the pA23-1G and pA10-1C were placed into Inc Q group. By adopting the reported PCR amplification methods of petroleum hydrocarbon-degrading catabolic genes, the petroleum-degrading capability of these BHR plasmids were preliminarily analyzed. The plasmids pS3-2G, pS7-2G, pA23-1G, pW22-3G, and pA10-1C carried aromatic ring- hydroxylating dioxygenase gene phdA and toluene monooxygenase gene touA; the plasmid pA15-7G carried touA and toluene dioxygenase gene tod; the plasmid pS3-2C carried ben, phdA, and tod; whereas the pS4-6G only carried ben. The host range test showed that all the isolated plasmids except pS3-2C could be transferred and maintained stably in the representative strains Agrobacterium tumefaciens C58, Cupriavidus necator JMP228, and E. coli EC100 of the alpha-, beta-, and gamma-Proteobacteria, respectively.

Restriction Fragment Length Polymorphisms of Virulence Plasmids in Rhodococcus equi

PubMed Central

Takai, Shinji; Shoda, Masato; Sasaki, Yukako; Tsubaki, Shiro; Fortier, Guillaume; Pronost, Stephane; Rahal, Karim; Becu, Teotimo; Begg, Angela; Browning, Glenn; Nicholson, Vivian M.; Prescott, John F.

1999-01-01

Virulent Rhodococcus equi, which is a well-known cause of pyogranulomatous pneumonia in foals, possesses a large plasmid encoding virulence-associated 15- to 17-kDa antigens. Foal and soil isolates from five countries—Argentina, Australia, Canada, France, and Japan—were investigated for the presence of 15- to 17-kDa antigens by colony blotting, using the monoclonal antibody 10G5, and the gene coding for 15- to 17-kDa antigens by PCR. Plasmid DNAs extracted from positive isolates were digested with restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII, and the digestion patterns that resulted divided the plasmids of virulent isolates into five closely related types. Three of the five types had already been reported in Canadian and Japanese isolates, and the two new types had been found in French and Japanese isolates. Therefore, we tentatively designated these five types 85-kb type I (pREAT701), 85-kb type II (a new type), 87-kb type I (EcoRI and BamHI type 2 [V. M. Nicholson and J. F. Prescott, J. Clin. Microbiol. 35:738–740, 1997]), 87-kb type II (a new type), and 90-kb (pREL1) plasmids. The 85-kb type I plasmid was found in isolates from Argentina, Australia, Canada, and France. Plasmid 87-kb type I was isolated in specimens from Argentina, Canada, and France. The 85-kb type II plasmid appeared in isolates from France. On the other hand, plasmids 87-kb type II and 90-kb were found only in isolates from Japan. These results revealed geographic differences in the distribution of the virulence plasmids found in the five countries and suggested that the restriction fragment length polymorphism of virulence plasmids might be useful to elucidate the molecular epidemiology of virulent R. equi in the world. PMID:10488224

Plasmid Characterization and Chromosome Analysis of Two netF+ Clostridium perfringens Isolates Associated with Foal and Canine Necrotizing Enteritis.

PubMed

Mehdizadeh Gohari, Iman; Kropinski, Andrew M; Weese, Scott J; Parreira, Valeria R; Whitehead, Ashley E; Boerlin, Patrick; Prescott, John F

2016-01-01

The recent discovery of a novel beta-pore-forming toxin, NetF, which is strongly associated with canine and foal necrotizing enteritis should improve our understanding of the role of type A Clostridium perfringens associated disease in these animals. The current study presents the complete genome sequence of two netF-positive strains, JFP55 and JFP838, which were recovered from cases of foal necrotizing enteritis and canine hemorrhagic gastroenteritis, respectively. Genome sequencing was done using Single Molecule, Real-Time (SMRT) technology-PacBio and Illumina Hiseq2000. The JFP55 and JFP838 genomes include a single 3.34 Mb and 3.53 Mb chromosome, respectively, and both genomes include five circular plasmids. Plasmid annotation revealed that three plasmids were shared by the two newly sequenced genomes, including a NetF/NetE toxins-encoding tcp-conjugative plasmid, a CPE/CPB2 toxins-encoding tcp-conjugative plasmid and a putative bacteriocin-encoding plasmid. The putative beta-pore-forming toxin genes, netF, netE and netG, were located in unique pathogenicity loci on tcp-conjugative plasmids. The C. perfringens JFP55 chromosome carries 2,825 protein-coding genes whereas the chromosome of JFP838 contains 3,014 protein-encoding genes. Comparison of these two chromosomes with three available reference C. perfringens chromosome sequences identified 48 (~247 kb) and 81 (~430 kb) regions unique to JFP55 and JFP838, respectively. Some of these divergent genomic regions in both chromosomes are phage- and plasmid-related segments. Sixteen of these unique chromosomal regions (~69 kb) were shared between the two isolates. Five of these shared regions formed a mosaic of plasmid-integrated segments, suggesting that these elements were acquired early in a clonal lineage of netF-positive C. perfringens strains. These results provide significant insight into the basis of canine and foal necrotizing enteritis and are the first to demonstrate that netF resides on a large and unique plasmid-encoded locus.

Novel conjugative plasmids from the natural isolate Lactococcus lactis subspecies cremoris DPC3758: a repository of genes for the potential improvement of dairy starters.

PubMed

Fallico, V; Ross, R P; Fitzgerald, G F; McAuliffe, O

2012-07-01

A collection of 17 natural lactococcal isolates from raw milk cheeses were studied in terms of their plasmid distribution, content, and diversity. All strains in the collection harbored an abundance of plasmids, including Lactococcus lactis ssp. cremoris DPC3758, whose 8-plasmid complement was selected for sequencing. The complete sequences of pAF22 (22,388 kb), pAF14 (14,419 kb), pAF12 (12,067 kb), pAF07 (7,435 kb), and pAF04 (3,801 kb) were obtained, whereas gene functions of technological interest were mapped to pAF65 (65 kb) and pAF45 (45 kb) by PCR. The plasmids of L. lactis DPC3758 were found to encode many genes with the potential to improve the technological properties of dairy starters. These included 3 anti-phage restriction/modification (R/M) systems (1 of type I and 2 of type II) and genes for immunity/resistance to nisin, lacticin 481, cadmium, and copper. Regions encoding conjugative/mobilization functions were present in 6 of the 8 plasmids, including those containing the R/M systems, thus enabling the food-grade transfer of these mechanisms to industrial strains. Using cadmium selection, the sequential stacking of the R/M plasmids into a plasmid-free host provided the recipient with increased protection against 936- and c2-type phages. The association of food-grade selectable markers and mobilization functions on L. lactis DPC3758 plasmids will facilitate their exploitation to obtain industrial strains with enhanced phage protection and robustness. These natural plasmids also provide another example of the major role of plasmids in contributing to host fitness and preservation within its ecological niche. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Identification of two new genes, mukE and mukF, involved in chromosome partitioning in Escherichia coli.

PubMed

Yamanaka, K; Ogura, T; Niki, H; Hiraga, S

1996-02-25

We have previously reported that the MukB protein is essential for chromosome partitioning in Escherichia coli and that mukB mutants produce anucleate cells and are temperature-sensitive for colony formation. The mukB gene maps at 21 min on the E. coli chromosome and smtA-mukF-mukE-mukB genes might comprise an operon, which is transcribed in a clockwise direction. Here, we report that mukF and mukE null mutants are both temperature-sensitive for colony formation and produce anucleate cells even at the permissive temperature. These phenotypes are the same as those observed in the mukB null mutant. The primary sequence of MukF includes a leucine zipper structure and an acidic domain. Mutational analysis revealed that both are required for MukF function. When the MukF protein was overproduced in the wild-type strain, anucleate cells were produced. In contrast, overproduction of either MukE or MukB did not cause the defect. In null mutants for the mukF, mukE, and mukB genes, the synchronous initiation of chromosome replication was not affected. The mini-F plasmid was as stably maintained in these mutants as in the wild-type strain. These results indicate that the MukF, MukE, and MukB proteins are involved in the chromosome partitioning steps, but are not required for mini-F plasmid partitioning.

Characterization of blaCTX-M IncFII plasmids and clones of Escherichia coli from pets in France.

PubMed

Dahmen, Safia; Haenni, Marisa; Châtre, Pierre; Madec, Jean-Yves

2013-12-01

To characterize bla(CTX-M) IncFII plasmids and clones of Escherichia coli from cats and dogs and to compare them with bla(CTX-M) IncFII plasmids reported in humans. From December 2006 to April 2010, 518 E. coli isolates from clinical infections in cats and dogs were screened for extended-spectrum β-lactamase (ESBL) production. Antimicrobial susceptibility was performed by disc diffusion and resistance genes were identified by PCR and sequencing. Plasmids were characterized using PCR-based replicon typing and sub-typing schemes, restriction fragment length polymorphism analysis, S1-PFGE and Southern hybridization. Isolates were characterized by PFGE, phylogenetic grouping, O25b typing and multilocus sequence typing. Nineteen E. coli isolates (3.7%) produced ESBLs, of which 14 (74%) carried bla(CTX-M) IncFII plasmids. The bla(CTX-M) gene was predominant and located on F31:A4:B1, F36:A4:B1 or F36:A1:B20 plasmids, abundantly reported in humans. The bla(CTX-M) F22:A1:B20 or F2:A2:B20 plasmids were also found. Different sequence types (STs) were identified, such as ST10, ST410, ST359, ST617 and ST224. Only one E. coli isolate belonged to the ST131 E. coli clone and carried a bla(CTX-M) F2:A2:B20 plasmid. This is the first known extensive study on ESBL-producing E. coli isolates from pets in France. The ST131 clone was rare. However, the predominance of human-like bla(CTX-M) IncFII plasmids suggests exchanges of these plasmids with the human reservoir.

Deletion of the Clostridium thermocellum recA gene reveals that it is required for thermophilic plasmid replication but not plasmid integration at homologous DNA sequences.

PubMed

Groom, Joseph; Chung, Daehwan; Kim, Sun-Ki; Guss, Adam; Westpheling, Janet

2018-05-28

A limitation to the engineering of cellulolytic thermophiles is the availability of functional, thermostable (≥ 60 °C) replicating plasmid vectors for rapid expression and testing of genes that provide improved or novel fuel molecule production pathways. A series of plasmid vectors for genetic manipulation of the cellulolytic thermophile Caldicellulosiruptor bescii has recently been extended to Clostridium thermocellum, another cellulolytic thermophile that very efficiently solubilizes plant biomass and produces ethanol. While the C. bescii pBAS2 replicon on these plasmids is thermostable, the use of homologous promoters, signal sequences and genes led to undesired integration into the bacterial chromosome, a result also observed with less thermostable replicating vectors. In an attempt to overcome undesired plasmid integration in C. thermocellum, a deletion of recA was constructed. As expected, C. thermocellum ∆recA showed impaired growth in chemically defined medium and an increased susceptibility to UV damage. Interestingly, we also found that recA is required for replication of the C. bescii thermophilic plasmid pBAS2 in C. thermocellum, but it is not required for replication of plasmid pNW33N. In addition, the C. thermocellum recA mutant retained the ability to integrate homologous DNA into the C. thermocellum chromosome. These data indicate that recA can be required for replication of certain plasmids, and that a recA-independent mechanism exists for the integration of homologous DNA into the C. thermocellum chromosome. Understanding thermophilic plasmid replication is not only important for engineering of these cellulolytic thermophiles, but also for developing genetic systems in similar new potentially useful non-model organisms.

Adsorption of bacterial plasmids in pure mineral mixtures

NASA Astrophysics Data System (ADS)

Zhang, L.; Cochran, J. P.; Seaman, J. C.; Parrott, B.

2017-12-01

Microorganisms play an important role in controlling the fate and transport of subsurface contaminants through the direct degradation of organic contaminants to the control of chemical redox conditions that impact the speciation and partitioning of inorganic contaminants. Genes that control these processes, including the relative tolerance associated with direct exposure to toxic contaminants, are found within the bacteria's chromosomal DNA and also within distinct, circular DNA elements called plasmids. Plasmids are mobile genetic elements that can be exchanged with other bacterial species through horizontal gene transfer (HGT). The frequency of HGT in soil is influenced by several factors, with the physicochemical characteristics of soil possibly being a primary factor. Thus, the objective for our research was to determine the movement and persistence of bacterial plasmids within soil. Our current study focuses on batch sorption experiments designed to evaluate the partitioning of bacterial plasmids in idealized mineral mixtures that represent the clay mineralogy of highly weathered soils of the Southeastern US. Specifically, we compared plasmid adsorption among pure goethite, kaolinite, and a mixture of goethite and kaolinite. We also determined the adsorption of plasmids on the above minerals over increasing pH (3 to 10). Our results show that adsorption decreased in the following order: goethite > kaolinite > mixture of goethite and kaolinite. We also found that plasmids adsorption was higher at lower pH levels, with pH 3 having the adsorption maximum. However, at pH 3, DNA denaturing may have occurred, leading to aggregation or precipitation of plasmids on the mineral surfaces. Our study was the first steps in determining the influence of soil properties on plasmid adsorption. Our future goals are to determine the adsorption in other pure minerals and in natural soils.

Deletion of the Clostridium thermocellum recA Gene Reveals that it is Required for Thermophilic Plasmid Replication but not Plasmid Integration at Homologous DNA Sequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chung, Daehwan; Groom, Joseph; Kim, Sun-Ki

A limitation to the engineering of cellulolytic thermophiles is the availability of functional, thermostable (>/= 60 degrees C) replicating plasmid vectors for rapid expression and testing of genes that provide improved or novel fuel molecule production pathways. A series of plasmid vectors for genetic manipulation of the cellulolytic thermophile Caldicellulosiruptor bescii has recently been extended to Clostridium thermocellum, another cellulolytic thermophile that very efficiently solubilizes plant biomass and produces ethanol. While the C. bescii pBAS2 replicon on these plasmids is thermostable, the use of homologous promoters, signal sequences and genes led to undesired integration into the bacterial chromosome, a resultmore » also observed with less thermostable replicating vectors. In an attempt to overcome undesired plasmid integration in C. thermocellum, a deletion of recA was constructed. As expected, C. thermocellum ..delta..recA showed impaired growth in chemically defined medium and an increased susceptibility to UV damage. Interestingly, we also found that recA is required for replication of the C. bescii thermophilic plasmid pBAS2 in C. thermocellum, but it is not required for replication of plasmid pNW33N. In addition, the C. thermocellum recA mutant retained the ability to integrate homologous DNA into the C. thermocellum chromosome. These data indicate that recA can be required for replication of certain plasmids, and that a recA-independent mechanism exists for the integration of homologous DNA into the C. thermocellum chromosome. Understanding thermophilic plasmid replication is not only important for engineering of these cellulolytic thermophiles, but also for developing genetic systems in similar new potentially useful non-model organisms.« less

Evaluation of droplet digital PCR for characterizing plasmid reference material used for quantifying ammonia oxidizers and denitrifiers.

PubMed

Dong, Lianhua; Meng, Ying; Wang, Jing; Liu, Yingying

2014-02-01

DNA reference materials of certified value have a critical function in many analytical processes of DNA measurement. Quantification of amoA genes in ammonia oxidizing bacteria (AOB) and archaea (AOA), and of nirS and nosZ genes in the denitrifiers is very important for determining their distribution and abundance in the natural environment. A plasmid reference material containing nirS, nosZ, amoA-AOB, and amoA-AOA is developed to provide a DNA standard with copy number concentration for ensuring comparability and reliability of quantification of these genes. Droplet digital PCR (ddPCR) was evaluated for characterization of the plasmid reference material. The result revealed that restriction endonuclease digestion of plasmids can improve amplification efficiency and minimize the measurement bias of ddPCR. Compared with the conformation of the plasmid, the size of the DNA fragment containing the target sequence and the location of the restriction site relative to the target sequence are not significant factors affecting plasmid quantification by ddPCR. Liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) was used to provide independent data for quantifying the plasmid reference material. The copy number concentration of the digested plasmid determined by ddPCR agreed well with that determined by LC-IDMS, improving both the accuracy and reliability of the plasmid reference material. The reference value, with its expanded uncertainty (k = 2), of the plasmid reference material was determined to be (5.19 ± 0.41) × 10(9) copies μL(-1) by averaging the results of two independent measurements. Consideration of the factors revealed in this study can improve the reliability and accuracy of ddPCR; thus, this method has the potential to accurately quantify DNA reference materials.

The Composite 259-kb Plasmid of Martelella mediterranea DSM 17316T–A Natural Replicon with Functional RepABC Modules from Rhodobacteraceae and Rhizobiaceae

PubMed Central

Bartling, Pascal; Brinkmann, Henner; Bunk, Boyke; Overmann, Jörg; Göker, Markus; Petersen, Jörn

2017-01-01

A multipartite genome organization with a chromosome and many extrachromosomal replicons (ECRs) is characteristic for Alphaproteobacteria. The best investigated ECRs of terrestrial rhizobia are the symbiotic plasmids for legume root nodulation and the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens. RepABC plasmids represent the most abundant alphaproteobacterial replicon type. The currently known homologous replication modules of rhizobia and Rhodobacteraceae are phylogenetically distinct. In this study, we surveyed type-strain genomes from the One Thousand Microbial Genomes (KMG-I) project and identified a roseobacter-specific RepABC-type operon in the draft genome of the marine rhizobium Martelella mediterranea DSM 17316T. PacBio genome sequencing demonstrated the presence of three circular ECRs with sizes of 593, 259, and 170-kb. The rhodobacteral RepABC module is located together with a rhizobial equivalent on the intermediate sized plasmid pMM259, which likely originated in the fusion of a pre-existing rhizobial ECR with a conjugated roseobacter plasmid. Further evidence for horizontal gene transfer (HGT) is given by the presence of a roseobacter-specific type IV secretion system on the 259-kb plasmid and the rhodobacteracean origin of 62% of the genes on this plasmid. Functionality tests documented that the genuine rhizobial RepABC module from the Martelella 259-kb plasmid is only maintained in A. tumefaciens C58 (Rhizobiaceae) but not in Phaeobacter inhibens DSM 17395 (Rhodobacteraceae). Unexpectedly, the roseobacter-like replication system is functional and stably maintained in both host strains, thus providing evidence for a broader host range than previously proposed. In conclusion, pMM259 is the first example of a natural plasmid that likely mediates genetic exchange between roseobacters and rhizobia. PMID:28983283

Evidence for Transfer of CMY-2 AmpC β-Lactamase Plasmids between Escherichia coli and Salmonella Isolates from Food Animals and Humans

PubMed Central

Winokur, P. L.; Vonstein, D. L.; Hoffman, L. J.; Uhlenhopp, E. K.; Doern, G. V.

2001-01-01

Escherichia coli is an important pathogen that shows increasing antimicrobial resistance in isolates from both animals and humans. Our laboratory recently described Salmonella isolates from food animals and humans that expressed an identical plasmid-mediated, AmpC-like β-lactamase, CMY-2. In the present study, 59 of 377 E. coli isolates from cattle and swine (15.6%) and 6 of 1,017 (0.6%) isolates of human E. coli from the same geographic region were resistant to both cephamycins and extended-spectrum cephalosporins. An ampC gene could be amplified with CMY-2 primers in 94.8% of animal and 33% of human isolates. Molecular epidemiological studies of chromosomal DNA revealed little clonal relatedness among the animal and human E. coli isolates harboring the CMY-2 gene. The ampC genes from 10 animal and human E. coli isolates were sequenced, and all carried an identical CMY-2 gene. Additionally, all were able to transfer a plasmid containing the CMY-2 gene to a laboratory strain of E. coli. CMY-2 plasmids demonstrated two different plasmid patterns that each showed strong similarities to previously described Salmonella CMY-2 plasmids. Additionally, Southern blot analyses using a CMY-2 probe demonstrated conserved fragments among many of the CMY-2 plasmids identified in Salmonella and E. coli isolates from food animals and humans. These data demonstrate that common plasmids have been transferred between animal-associated Salmonella and E. coli, and identical CMY-2 genes carried by similar plasmids have been identified in humans, suggesting that the CMY-2 plasmid has undergone transfer between different bacterial species and may have been transmitted between food animals and humans. PMID:11557460

Dissemination of plasmid-encoded AmpC β-lactamases in antimicrobial resistant Salmonella serotypes originating from humans, pigs and the swine environment.

PubMed

Keelara, Shivaramu; Thakur, Siddhartha

2014-09-17

The aim of this study was to characterize and determine the inter-serovar exchange of AmpC β-lactamase conferring plasmids isolated from humans, pigs and the swine environment. Plasmids isolated from a total of 21 antimicrobial resistant (AMR) Salmonella isolates representing human clinical cases (n=6), pigs (n=6) and the swine farm environment (n=9) were characterized by replicon typing and restriction digestion, inter-serovar transferability by conjugation, and presence of AmpC β-lactamase enzyme encoding gene blaCMY-2 by southern hybridization. Based on replicon typing, the majority (17/21, 81%) of the plasmids belonged to the I1-Iγ Inc group and were between 70 and 103kb. The potential for inter-serovar plasmid transfer was further confirmed by the PCR detection of AMR genes on the plasmids isolated from trans-conjugants. Plasmids from Salmonella serovars Anatum, Ouakam, Johannesburg and Typhimurium isolated from the same cohort of pigs and their environment and S. Heidelberg from a single human clinical isolate had identical plasmids based on digestion with multiple restriction enzymes (EcoRI, HindIII and PstI) and southern blotting. We demonstrated likely horizontal inter-serovar exchange of plasmid-encoding AmpC β-lactamases resistance among MDR Salmonella serotypes isolated from pigs, swine farm environment and clinical human cases. This study provides valuable information on the role of the swine farm environment and by extension other livestock farm environments, as a potential reservoir of resistant bacterial strains that potentially transmit resistance determinants to livestock, in this case, swine, humans and possibly other hosts by horizontal exchange of plasmids. Copyright © 2014 Elsevier B.V. All rights reserved.

Erwinia amylovora Novel Plasmid pEI70: Complete Sequence, Biogeography, and Role in Aggressiveness in the Fire Blight Phytopathogen

PubMed Central

Llop, Pablo; Cabrefiga, Jordi; Smits, Theo H. M.; Dreo, Tanja; Barbé, Silvia; Pulawska, Joanna; Bultreys, Alain; Blom, Jochen; Duffy, Brion; Montesinos, Emilio; López, María M.

2011-01-01

Comparative genomics of several strains of Erwinia amylovora, a plant pathogenic bacterium causal agent of fire blight disease, revealed that its diversity is primarily attributable to the flexible genome comprised of plasmids. We recently identified and sequenced in full a novel 65.8 kb plasmid, called pEI70. Annotation revealed a lack of known virulence-related genes, but found evidence for a unique integrative conjugative element related to that of other plant and human pathogens. Comparative analyses using BLASTN showed that pEI70 is almost entirely included in plasmid pEB102 from E. billingiae, an epiphytic Erwinia of pome fruits, with sequence identities superior to 98%. A duplex PCR assay was developed to survey the prevalence of plasmid pEI70 and also that of pEA29, which had previously been described in several E. amylovora strains. Plasmid pEI70 was found widely dispersed across Europe with frequencies of 5–92%, but it was absent in E. amylovora analyzed populations from outside of Europe. Restriction analysis and hybridization demonstrated that this plasmid was identical in at least 13 strains. Curing E. amylovora strains of pEI70 reduced their aggressiveness on pear, and introducing pEI70 into low-aggressiveness strains lacking this plasmid increased symptoms development in this host. Discovery of this novel plasmid offers new insights into the biogeography, evolution and virulence determinants in E. amylovora. PMID:22174857

Erwinia amylovora novel plasmid pEI70: complete sequence, biogeography, and role in aggressiveness in the fire blight phytopathogen.

PubMed

Llop, Pablo; Cabrefiga, Jordi; Smits, Theo H M; Dreo, Tanja; Barbé, Silvia; Pulawska, Joanna; Bultreys, Alain; Blom, Jochen; Duffy, Brion; Montesinos, Emilio; López, María M

2011-01-01

Comparative genomics of several strains of Erwinia amylovora, a plant pathogenic bacterium causal agent of fire blight disease, revealed that its diversity is primarily attributable to the flexible genome comprised of plasmids. We recently identified and sequenced in full a novel 65.8 kb plasmid, called pEI70. Annotation revealed a lack of known virulence-related genes, but found evidence for a unique integrative conjugative element related to that of other plant and human pathogens. Comparative analyses using BLASTN showed that pEI70 is almost entirely included in plasmid pEB102 from E. billingiae, an epiphytic Erwinia of pome fruits, with sequence identities superior to 98%. A duplex PCR assay was developed to survey the prevalence of plasmid pEI70 and also that of pEA29, which had previously been described in several E. amylovora strains. Plasmid pEI70 was found widely dispersed across Europe with frequencies of 5-92%, but it was absent in E. amylovora analyzed populations from outside of Europe. Restriction analysis and hybridization demonstrated that this plasmid was identical in at least 13 strains. Curing E. amylovora strains of pEI70 reduced their aggressiveness on pear, and introducing pEI70 into low-aggressiveness strains lacking this plasmid increased symptoms development in this host. Discovery of this novel plasmid offers new insights into the biogeography, evolution and virulence determinants in E. amylovora.

Characterization of Resistance Genes and Plasmids from Outbreaks and Illness Clusters Caused by Salmonella Resistant to Ceftriaxone in the United States, 2011–2012

PubMed Central

Folster, Jason P.; Grass, Julian E.; Bicknese, Amelia; Taylor, Julia; Friedman, Cindy R.; Whichard, Jean M.

2017-01-01

Salmonella is an important cause of foodborne illness; however, quickly identifying the source of these infections can be difficult, and source identification is a crucial step in preventing additional illnesses. Although most infections are self-limited, invasive salmonellosis may require antimicrobial treatment. Ceftriaxone, an extended-spectrum cephalosporin, is commonly used for treatment of salmonellosis. Previous studies have identified a correlation between the food animal/retail meat source of ceftriaxone-resistant Salmonella and the type of resistance gene and plasmid it carries. In this study, we examined seven outbreaks of ceftriaxone-resistant Salmonella infections, caused by serotypes Typhimurium, Newport, Heidelberg, and Infantis. All isolates were positive for a plasmid-encoded blaCMY gene. Plasmid incompatibility typing identified five IncI1 and two IncA/C plasmids. Both outbreaks containing blaCMY-IncA/C plasmids were linked to consumption of cattle products. Three of five outbreaks with blaCMY-IncI1 (ST12) plasmids were linked to a poultry source. The remaining IncI1 outbreaks were associated with ground beef (ST20) and tomatoes (ST12). Additionally, we examined isolates from five unsolved clusters of ceftriaxone-resistant Salmonella infections and used our plasmid encoded gene findings to predict the source. Overall, we identified a likely association between the source of ceftriaxone-resistant Salmonella outbreaks and the type of resistance gene/plasmid it carries. PMID:27828730

[Construction, identification and expression of three kinds of shuttle plasmids of adenovirus expression vector of hepatitis C virus structure gene].

PubMed

Cao, Yi-zhan; Hao, Chun-qiu; Feng, Zhi-hua; Zhou, Yong-xing; Li, Jin-ge; Jia, Zhan-sheng; Wang, Ping-zhong

2003-02-01

To construct three recombinant shuttle plasmids of adenovirus expression vector which can express hepatitis C virus(HCV) different structure genes(C, C+E1, C+E1+E2) in order to pack adenovirus expression vectors which can express HCV different structure gene effectively. The different HCV structure genes derived from the plasmid pBRTM/HCV1-3011 by using polymerase chain reaction (PCR) were inserted into the backward position of cytomegalovirus(CMV) immediate early promotor element of shuttle plasmid(pAd.CMV-Link.1) of adenovirus expression vector respectively, then the three recombinant plasmids (pAd.HCV-C, pAd.HCV-CE1, pAd.HCV-S) were obtained. The recombinant plasmids were identified by endonuclease, PCR and sequencing. HCV structure genes were expressed transiently with Lipofectamine 2000 coated in HepG2 cells which were confirmed by immunofluorescence and Western-Blot. Insert DNAs of the three recombinant plasmids' were confirmed to be HCV different structure genes by endonuclease, PCR and sequencing. The three recombinant plasmids can express HCV structure gene (C, C+E1, C+E1+E2) transiently in HepG2 cells which were confirmed by immunofluorescence and Western-Blot. The three recombinant shuttle plasmids of adenovirus expression vector can express HCV structure gene(C, C+E1, C+E1+E2) transiently. This should be useful to pack adenovirus expression vector which can express HCV structure genes.

Eco-Evolutionary Dynamics of Episomes among Ecologically Cohesive Bacterial Populations

DOE PAGES

Xue, Hong; Cordero, Otto X.; Camas, Francisco M.; ...

2015-05-05

Although plasmids and other episomes are recognized as key players in horizontal gene transfer among microbes, their diversity and dynamics among ecologically structured host populations in the wild remain poorly understood. Here, we show that natural populations of marine Vibrionaceae bacteria host large numbers of families of episomes, consisting of plasmids and a surprisingly high fraction of plasmid-like temperate phages. Episomes are unevenly distributed among host populations, and contrary to the notion that high-density communities in biofilms act as hot spots of gene transfer, we identified a strong bias for episomes to occur in free-living as opposed to particle-attached cells.more » Mapping of episomal families onto host phylogeny shows that, with the exception of all phage and a few plasmid families, most are of recent evolutionary origin and appear to have spread rapidly by horizontal transfer. Such high eco-evolutionary turnover is particularly surprising for plasmids that are, based on previously suggested categorization, putatively nontransmissible, indicating that this type of plasmid is indeed frequently transferred by currently unknown mechanisms. Finally, analysis of recent gene transfer among plasmids reveals a network of extensive exchange connecting nearly all episomes. Genes functioning in plasmid transfer and maintenance are frequently exchanged, suggesting that plasmids can be rapidly transformed from one category to another. The broad distribution of episomes among distantly related hosts and the observed promiscuous recombination patterns show how episomes can offer their hosts rapid assembly and dissemination of novel functions.« less

Characterization of Resistance Genes and Plasmids from Outbreaks and Illness Clusters Caused by Salmonella Resistant to Ceftriaxone in the United States, 2011-2012.

PubMed

Folster, Jason P; Grass, Julian E; Bicknese, Amelia; Taylor, Julia; Friedman, Cindy R; Whichard, Jean M

2017-03-01

Salmonella is an important cause of foodborne illness; however, quickly identifying the source of these infections can be difficult, and source identification is a crucial step in preventing additional illnesses. Although most infections are self-limited, invasive salmonellosis may require antimicrobial treatment. Ceftriaxone, an extended-spectrum cephalosporin, is commonly used for treatment of salmonellosis. Previous studies have identified a correlation between the food animal/retail meat source of ceftriaxone-resistant Salmonella and the type of resistance gene and plasmid it carries. In this study, we examined seven outbreaks of ceftriaxone-resistant Salmonella infections, caused by serotypes Typhimurium, Newport, Heidelberg, and Infantis. All isolates were positive for a plasmid-encoded bla CMY gene. Plasmid incompatibility typing identified five IncI1 and two IncA/C plasmids. Both outbreaks containing bla CMY -IncA/C plasmids were linked to consumption of cattle products. Three of five outbreaks with bla CMY -IncI1 (ST12) plasmids were linked to a poultry source. The remaining IncI1 outbreaks were associated with ground beef (ST20) and tomatoes (ST12). In addition, we examined isolates from five unsolved clusters of ceftriaxone-resistant Salmonella infections and used our plasmid-encoded gene findings to predict the source. Overall, we identified a likely association between the source of ceftriaxone-resistant Salmonella outbreaks and the type of resistance gene/plasmid it carries.

Precise determination, cross-recognition, and functional analysis of the double-strand origins of the rolling-circle replication plasmids in haloarchaea.

PubMed

Zhou, Ligang; Zhou, Meixian; Sun, Chaomin; Han, Jing; Lu, Qiuhe; Zhou, Jian; Xiang, Hua

2008-08-01

The precise nick site in the double-strand origin (DSO) of pZMX201, a 1,668-bp rolling-circle replication (RCR) plasmid from the haloarchaeon Natrinema sp. CX2021, was determined by electron microscopy and DSO mapping. In this plasmid, DSO nicking occurred between residues C404 and G405 within a heptanucleotide sequence (TCTC/GGC) located in the stem region of an imperfect hairpin structure. This nick site sequence was conserved among the haloarchaeal RCR plasmids, including pNB101, suggesting that the DSO nick site might be the same for all members of this plasmid family. Interestingly, the DSOs of pZMX201 and pNB101 were found to be cross-recognized in RCR initiation and termination in a hybrid plasmid system. Mutation analysis of the DSO from pZMX201 (DSO(Z)) in this hybrid plasmid system revealed that: (i) the nucleotides in the middle of the conserved TCTCGGC sequence play more-important roles in the initiation and termination process; (ii) the left half of the hairpin structure is required for initiation but not for termination; and (iii) a 36-bp sequence containing TCTCGGC and the downstream sequence is essential and sufficient for termination. In conclusion, these haloarchaeal plasmids, with novel features that are different from the characteristics of both single-stranded DNA phages and bacterial RCR plasmids, might serve as a good model for studying the evolution of RCR replicons.

A novel type 1/2 hybrid IncC plasmid carrying fifteen antimicrobial resistance genes recovered from Proteus mirabilis in China.

PubMed

Lei, Chang-Wei; Kong, Ling-Han; Ma, Su-Zhen; Liu, Bi-Hui; Chen, Yan-Peng; Zhang, An-Yun; Wang, Hong-Ning

2017-09-01

IncC plasmids are of great concern as vehicles of broad-spectrum cephalosporins and carbapenems resistance genes bla CMY and bla NDM . The aim of this study was to sequence and characterize a multidrug resistance (MDR) IncC plasmid (pPm14C18) recovered from Proteus mirabilis. pPm14C18 was identified in a CMY-2-producing P. mirabilis isolate from chicken in China in 2014, and could be transferred to Escherichia coli conferring an MDR phenotype. Whole genome sequencing confirmed pPm14C18 was a novel type 1/2 hybrid IncC plasmid 165,992bp in size, containing fifteen antimicrobial resistance genes. It harboured a novel MDR mosaic region comprised of a hybrid Tn21 tnp -pDU mer , in which bla CTX-M-65 , dfrA32 and ereA were firstly reported in IncC plasmid. Phylogenetic relationship reconstruction based on the nucleotide sequences of the 52 IncC backbones showed all type 1 IncC plasmids were clustered into one clade, and then merged with pPm14C18 and finally with the type 2 IncC plasmids and another type 1/2 hybrid IncC plasmid pYR1. The MDR IncC plasmids in P. mirabilis of animal origin might threaten public health, which should be drawn more attention. Copyright © 2017 Elsevier Inc. All rights reserved.

P62 plasmid can alleviate diet-induced obesity and metabolic dysfunctions.

PubMed

Halenova, Tatiana; Savchuk, Oleksii; Ostapchenko, Ludmila; Chursov, Andrey; Fridlyand, Nathan; Komissarov, Andrey B; Venanzi, Franco; Kolesnikov, Sergey I; Sufianov, Albert A; Sherman, Michael Y; Gabai, Vladimir L; Shneider, Alexander M

2017-08-22

A high-calorie diet (HCD) induces two mutually exacerbating effects contributing to diet-induced obesity (DIO): impaired glucose metabolism and increased food consumption. A link between the metabolic and behavioral manifestations is not well understood yet. We hypothesized that chronic inflammation induced by HCD plays a key role in linking together the two components of diet-induced pathology. Based on this hypothesis, we tested if a plasmid (DNA vaccine) encoding p62 (SQSTM1) would alleviate DIO including its metabolic and/or food consumption abnormalities. Previously we reported that injections of the p62 plasmid reduce chronic inflammation during ovariectomy-induced osteoporosis. Here we found that the p62 plasmid reduced levels of pro-inflammatory cytokines IL-1β, IL-12, and INFγ and increased levels of anti-inflammatory cytokines IL-4, IL-10 and TGFβ in HCD-fed animals. Due to this anti-inflammatory response, we further tested whether the plasmid can alleviate HCD-induced obesity and associated metabolic and feeding impairments. Indeed, p62 plasmid significantly reversed effects of HCD on the body mass index (BMI), levels of glucose, insulin and glycosylated hemoglobin (HbA1c). Furthermore, p62 plasmid partially restored levels of the satiety hormone, serotonin, and tryptophan, simultaneously reducing activity of monoamine oxidase (MAO) in the brain affected by the HCD. Finally, the plasmid partially reversed increased food consumption caused by HCD. Therefore, the administering of p62 plasmid alleviates both metabolic and behavioral components of HCD-induced obesity.

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.

The mechanism and control of DNA transfer by the conjugative relaxase of resistance plasmid pCU1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nash, Rebekah Potts; Habibi, Sohrab; Cheng, Yuan

2010-11-15

Bacteria expand their genetic diversity, spread antibiotic resistance genes, and obtain virulence factors through the highly coordinated process of conjugative plasmid transfer (CPT). A plasmid-encoded relaxase enzyme initiates and terminates CPT by nicking and religating the transferred plasmid in a sequence-specific manner. We solved the 2.3 {angstrom} crystal structure of the relaxase responsible for the spread of the resistance plasmid pCU1 and determined its DNA binding and nicking capabilities. The overall fold of the pCU1 relaxase is similar to that of the F plasmid and plasmid R388 relaxases. However, in the pCU1 structure, the conserved tyrosine residues (Y18,19,26,27) that aremore » required for DNA nicking and religation were displaced up to 14 {angstrom} out of the relaxase active site, revealing a high degree of mobility in this region of the enzyme. In spite of this flexibility, the tyrosines still cleaved the nic site of the plasmid's origin of transfer, and did so in a sequence-specific, metal-dependent manner. Unexpectedly, the pCU1 relaxase lacked the sequence-specific DNA binding previously reported for the homologous F and R388 relaxase enzymes, despite its high sequence and structural similarity with both proteins. In summary, our work outlines novel structural and functional aspects of the relaxase-mediated conjugative transfer of plasmid pCU1.« less

Integration of narrow-host-range vectors from Escherichia coli into the genomes of amino acid-producing corynebacteria after intergeneric conjugation.

PubMed

Mateos, L M; Schäfer, A; Kalinowski, J; Martin, J F; Pühler, A

1996-10-01

Conjugative transfer of mobilizable derivatives of the Escherichia coli narrow-host-range plasmids pBR322, pBR325, pACYC177, and pACYC184 from E. coli to species of the gram-positive genera Corynebacterium and Brevibacterium resulted in the integration of the plasmids into the genomes of the recipient bacteria. Transconjugants appeared at low frequencies and reproducibly with a delay of 2 to 3 days compared with matings with replicative vectors. Southern analysis of corynebacterial transconjugants and nucleotide sequences from insertion sites revealed that integration occurs at different locations and that different parts of the vector are involved in the process. Integration is not dependent on indigenous insertion sequence elements but results from recombination between very short homologous DNA segments (8 to 12 bp) present in the vector and in the host DNA. In the majority of the cases (90%), integration led to cointegrate formation, and in some cases, deletions or rearrangements occurred during the recombination event. Insertions were found to be quite stable even in the absence of selective pressure.

Effects of TGF-β1 and VEGF-A transgenes on the osteogenic potential of bone marrow stromal cells in vitro and in vivo

PubMed Central

Sumner, Dale R; Virdi, Amarjit S

2012-01-01

An exogenous supply of growth factors and bioreplaceable scaffolds may help bone regeneration. The aim of this study was to examine the effects of TGF-β1 and VEGF-A transgenes on the osteogenic potential of bone marrow stromal cells. Rat bone marrow stromal cells were transfected with plasmids encoding mouse TGF-β1 and/or VEGF-A complementary DNAs and cultured for up to 28 days. Furthermore, collagen scaffolds carrying combinations of the plasmids-transfected cells were implanted subcutaneously in rats. The transgenes increased alkaline phosphatase activity, enhanced mineralized nodule formation, and elevated osteogenic gene expressions in vitro. In vivo, messenger RNA expression of osteogenic genes such as BMPs and Runx2 elevated higher by the transgenes. The data indicate that exogenous TGF-β1 and VEGF-A acted synergistically and could induce osteoblastic differentiation of bone marrow stromal cells in both cell culture and an animal model. The results may provide valuable information to optimize protocols for transgene-and-cell-based tissue engineering. PMID:22962632

Structural basis of a histidine-DNA nicking/joining mechanism for gene transfer and promiscuous spread of antibiotic resistance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pluta, Radoslaw; Boer, D. Roeland; Lorenzo-Diaz, Fabian

Relaxases are metal-dependent nucleases that break and join DNA for the initiation and completion of conjugative bacterial gene transfer. Conjugation is the main process through which antibiotic resistance spreads among bacteria, with multidrug-resistant staphylococci and streptococci infections posing major threats to human health. The MOB V family of relaxases accounts for approximately 85% of all relaxases found in Staphylococcus aureus isolates. Here, we present six structures of the MOB V relaxase MobM from the promiscuous plasmid pMV158 in complex with several origin of transfer DNA fragments. A combined structural, biochemical, and computational approach reveals that MobM follows a previously uncharacterizedmore » histidine/metal-dependent DNA processing mechanism, which involves the formation of a covalent phosphoramidate histidine-DNA adduct for cell-to-cell transfer. In conclusion, we discuss how the chemical features of the high-energy phosphorus-nitrogen bond shape the dominant position of MOB V histidine relaxases among small promiscuous plasmids and their preference toward Gram-positive bacteria.« less

Structural basis of a histidine-DNA nicking/joining mechanism for gene transfer and promiscuous spread of antibiotic resistance

DOE PAGES

Pluta, Radoslaw; Boer, D. Roeland; Lorenzo-Diaz, Fabian; ...

2017-07-24

Relaxases are metal-dependent nucleases that break and join DNA for the initiation and completion of conjugative bacterial gene transfer. Conjugation is the main process through which antibiotic resistance spreads among bacteria, with multidrug-resistant staphylococci and streptococci infections posing major threats to human health. The MOB V family of relaxases accounts for approximately 85% of all relaxases found in Staphylococcus aureus isolates. Here, we present six structures of the MOB V relaxase MobM from the promiscuous plasmid pMV158 in complex with several origin of transfer DNA fragments. A combined structural, biochemical, and computational approach reveals that MobM follows a previously uncharacterizedmore » histidine/metal-dependent DNA processing mechanism, which involves the formation of a covalent phosphoramidate histidine-DNA adduct for cell-to-cell transfer. In conclusion, we discuss how the chemical features of the high-energy phosphorus-nitrogen bond shape the dominant position of MOB V histidine relaxases among small promiscuous plasmids and their preference toward Gram-positive bacteria.« less

Sequestration of cAMP response element-binding proteins by transcription factor decoys causes collateral elaboration of regenerating Aplysia motor neuron axons.

PubMed

Dash, P K; Tian, L M; Moore, A N

1998-07-07

Axonal injury increases intracellular Ca2+ and cAMP and has been shown to induce gene expression, which is thought to be a key event for regeneration. Increases in intracellular Ca2+ and/or cAMP can alter gene expression via activation of a family of transcription factors that bind to and modulate the expression of CRE (Ca2+/cAMP response element) sequence-containing genes. We have used Aplysia motor neurons to examine the role of CRE-binding proteins in axonal regeneration after injury. We report that axonal injury increases the binding of proteins to a CRE sequence-containing probe. In addition, Western blot analysis revealed that the level of ApCREB2, a CRE sequence-binding repressor, was enhanced as a result of axonal injury. The sequestration of CRE-binding proteins by microinjection of CRE sequence-containing plasmids enhanced axon collateral formation (both number and length) as compared with control plasmid injections. These findings show that Ca2+/cAMP-mediated gene expression via CRE-binding transcription factors participates in the regeneration of motor neuron axons.

Integration of narrow-host-range vectors from Escherichia coli into the genomes of amino acid-producing corynebacteria after intergeneric conjugation.

PubMed Central

Mateos, L M; Schäfer, A; Kalinowski, J; Martin, J F; Pühler, A

1996-01-01

Conjugative transfer of mobilizable derivatives of the Escherichia coli narrow-host-range plasmids pBR322, pBR325, pACYC177, and pACYC184 from E. coli to species of the gram-positive genera Corynebacterium and Brevibacterium resulted in the integration of the plasmids into the genomes of the recipient bacteria. Transconjugants appeared at low frequencies and reproducibly with a delay of 2 to 3 days compared with matings with replicative vectors. Southern analysis of corynebacterial transconjugants and nucleotide sequences from insertion sites revealed that integration occurs at different locations and that different parts of the vector are involved in the process. Integration is not dependent on indigenous insertion sequence elements but results from recombination between very short homologous DNA segments (8 to 12 bp) present in the vector and in the host DNA. In the majority of the cases (90%), integration led to cointegrate formation, and in some cases, deletions or rearrangements occurred during the recombination event. Insertions were found to be quite stable even in the absence of selective pressure. PMID:8824624

Cold atmospheric pressure plasma jets: Interaction with plasmid DNA and tailored electron heating using dual-frequency excitation

NASA Astrophysics Data System (ADS)

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

2012-05-01

Recent progress in plasma science and technology has enabled the development of a new generation of stable cold non-equilibrium plasmas operating at ambient atmospheric pressure. This opens horizons for new plasma technologies, in particular in the emerging field of plasma medicine. These non-equilibrium plasmas are very efficient sources for energy transport through reactive neutral particles (radicals and metastables), charged particles (ions and electrons), UV radiation, and electro-magnetic fields. The effect of a cold radio frequency-driven atmospheric pressure plasma jet on plasmid DNA has been investigated. The formation of double strand breaks correlates well with the atomic oxygen density. Taken with other measurements, this indicates that neutral components in the jet are effective in inducing double strand breaks. Plasma manipulation techniques for controlled energy delivery are highly desirable. Numerical simulations are employed for detailed investigations of the electron dynamics, which determines the generation of reactive species. New concepts based on nonlinear power dissipation promise superior strategies to control energy transport for tailored technological exploitations.

Enhancing Malaria Vaccine Development by the Naval Medical Research Center

DTIC Science & Technology

2003-03-01

optimized in Milestone 1 of this Phase II project. Reduction in particle size of the biopolymeric carrier was sufficient for intramuscular administration of...glycolide) (PLGA) with incorporated DNA plasmid were developed for systemic administration of DNA plasmids for use as a malaria vaccine. Objectives in...with incorporated DNA plasmid were developed for systemic administration of DNA plasmids for use as a malaria vaccine. Objectives in Milestone 1

Development of Novel Peptide Inhibitors of the Estrogen Receptor

DTIC Science & Technology

1997-10-01

plasmids used for the transfection experiments described below included pERE-TK- CAT , an estrogen responsive chloramphenicol acetylase reporter plasmid...The inhibitory potential of expressed fragments of ER were assessed by measuring the activity of chloramphenicol acetyltransferase ( CAT ) enzyme...with an ER expression plasmid (pCMV-ER) and an estrogen-responsive reporter plasmid (pERE-TK- CAT ) in order to look for inhibition of an ER mediated

DNA sequence analysis of the composite plasmid pTC conferring virulence and antimicrobial resistance for porcine enterotoxigenic Escherichia coli.

PubMed

Fekete, Péter Z; Brzuszkiewicz, Elzbieta; Blum-Oehler, Gabriele; Olasz, Ferenc; Szabó, Mónika; Gottschalk, Gerhard; Hacker, Jörg; Nagy, Béla

2012-01-01

In this study the plasmid pTC, a 90 kb self-conjugative virulence plasmid of the porcine enterotoxigenic Escherichia coli (ETEC) strain EC2173 encoding the STa and STb heat-stable enterotoxins and tetracycline resistance, has been sequenced in two steps. As a result we identified five main distinct regions of pTC: (i) the maintenance region responsible for the extreme stability of the plasmid, (ii) the TSL (toxin-specific locus comprising the estA and estB genes) which is unique and characteristic for pTC, (iii) a Tn10 transposon, encoding tetracycline resistance, (iv) the tra (plasmid transfer) region, and (v) the colE1-like origin of replication. It is concluded that pTC is a self-transmissible composite plasmid harbouring antibiotic resistance and virulence genes. pTC belongs to a group of large conjugative E. coli plasmids represented by NR1 with a widespread tra backbone which might have evolved from a common ancestor. This is the first report of a completely sequenced animal ETEC virulence plasmid containing an antimicrobial resistance locus, thereby representing a selection advantage for spread of pathogenicity in the presence of antimicrobials leading to increased disease potential. Copyright © 2011. Published by Elsevier GmbH.

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

Tailor-made fibroblast-specific and antibiotic-free interleukin 12 plasmid for gene electrotransfer-mediated cancer immunotherapy.

PubMed

Kamensek, Urska; Tesic, Natasa; Sersa, Gregor; Kos, Spela; Cemazar, Maja

2017-01-01

Electrotransfer mediated delivery of interleukin-12 (IL-12) gene, encoded on a plasmid vector, has already been demonstrated to have a potent antitumor efficacy and great potential for clinical application. In the present study, our aim was to construct an optimized IL-12-encoding plasmid that is safe from the regulatory point of view. In light of previous studies demonstrating that IL-12 should be released in a tumor localized manner for optimal efficacy, the strong ubiquitous promoter was replaced with a weak endogenous promoter of the collagen 2 gene, which is specific for fibroblasts. Next, to comply with increasing regulatory demands for clinically used plasmids, the expression cassette was cloned in a plasmid lacking the antibiotic resistance gene. The constructed fibroblast-specific and antibiotic-free IL-12 plasmid was demonstrated to support low IL-12 expression after gene electrotransfer in selected cell lines. Furthermore, the removal of antibiotic resistance did not affect the plasmid expression profile and lowered its cytotoxicity. With optimal IL-12 expression and minimal transgene non-specific effects, i.e., low cytotoxicity, the constructed plasmid could be especially valuable for different modern immunological approaches to achieve localized boosting of the host's immune system. Copyright © 2016 Elsevier Inc. All rights reserved.

Construction of conjugative gene transfer system between E. coli and moderately thermophilic, extremely acidophilic Acidithiobacillus caldus MTH-04.

PubMed

Liu, Xiangmei; Lin, Jianqun; Zhang, Zheng; Bian, Jiang; Zhao, Qing; Liu, Ying; Lin, Jianqiang; Yan, Wangming

2007-01-01

A genetic transfer system for introducing foreign genes to biomining microorganisms is urgently needed. Thus, a conjugative gene transfer system was investigated for a moderately thermophilic, extremely acidophilic biomining bacterium, Acidithiobacillus caldus MTH-04. The broad-host-range IncP plasmids RP4 and R68.45 were transferred directly into A. caldus MTH-04 from Escherichia coli by conjugation at relatively high frequencies. Additionally the broad-host-range IncQ plasmids pJRD215, pVLT33, and pVLT35 were also transferred into A. caldus MTH-04 with the help of plasmid RP4 or strains with plasmid RP4 integrated into their chromosome, such as E. coli SM10. The Km(r) and Sm(r) selectable markers from these plasmids were successfully expressed in A. caldus MTH-04. Futhermore, the IncP and IncQ plasmids were transferred back into E. coli cells from A. caldus MTH-04, thereby confirming the initial transfer of these plasmids from E. coli to A. caldus MTH-04. All the IncP and IncQ plasmids studied were stable in A. caldus MTH-04. Consequently, this development of a conjugational system for A. caldus MTH-04 will greatly facilitate its genetic study.

An 'instant gene bank' method for gene cloning by mutant complementation.

PubMed

Gems, D; Aleksenko, A; Belenky, L; Robertson, S; Ramsden, M; Vinetski, Y; Clutterbuck, A J

1994-02-01

We describe a new method of gene cloning by complementation of mutant alleles which obviates the need for construction of a gene library in a plasmid vector in vitro and its amplification in Escherichia coli. The method involves simultaneous transformation of mutant strains of the fungus Aspergillus nidulans with (i) fragmented chromosomal DNA from a donor species and (ii) DNA of a plasmid without a selectable marker gene, but with a fungal origin of DNA replication ('helper plasmid'). Transformant colonies appear as the result of the joining of chromosomal DNA fragments carrying the wild-type copies of the mutant allele with the helper plasmid. Joining may occur either by ligation (if the helper plasmid is in linear form) or recombination (if it is cccDNA). This event occurs with high efficiency in vivo, and generates an autonomously replicating plasmid cointegrate. Transformants containing Penicillium chrysogenum genomic DNA complementing A. nidulans niaD, nirA and argB mutations have been obtained. While some of these cointegrates were evidently rearranged or consisted only of unaltered replicating plasmid, in other cases plasmids could be recovered into E. coli and were subsequently shown to contain the selected gene. The utility of this "instant gene bank" technique is demonstrated here by the molecular cloning of the P. canescens trpC gene.

Efficient generation of complete sequences of MDR-encoding plasmids by rapid assembly of MinION barcoding sequencing data.

PubMed

Li, Ruichao; Xie, Miaomiao; Dong, Ning; Lin, Dachuan; Yang, Xuemei; Wong, Marcus Ho Yin; Chan, Edward Wai-Chi; Chen, Sheng

2018-03-01

Multidrug resistance (MDR)-encoding plasmids are considered major molecular vehicles responsible for transmission of antibiotic resistance genes among bacteria of the same or different species. Delineating the complete sequences of such plasmids could provide valuable insight into the evolution and transmission mechanisms underlying bacterial antibiotic resistance development. However, due to the presence of multiple repeats of mobile elements, complete sequencing of MDR plasmids remains technically complicated, expensive, and time-consuming. Here, we demonstrate a rapid and efficient approach to obtaining multiple MDR plasmid sequences through the use of the MinION nanopore sequencing platform, which is incorporated in a portable device. By assembling the long sequencing reads generated by a single MinION run according to a rapid barcoding sequencing protocol, we obtained the complete sequences of 20 plasmids harbored by multiple bacterial strains. Importantly, single long reads covering a plasmid end-to-end were recorded, indicating that de novo assembly may be unnecessary if the single reads exhibit high accuracy. This workflow represents a convenient and cost-effective approach for systematic assessment of MDR plasmids responsible for treatment failure of bacterial infections, offering the opportunity to perform detailed molecular epidemiological studies to probe the evolutionary and transmission mechanisms of MDR-encoding elements.

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

PubMed Central

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

2014-01-01

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

Small Universal Bacteria and Plasmid Computing Systems.

PubMed

Wang, Xun; Zheng, Pan; Ma, Tongmao; Song, Tao

2018-05-29

Bacterial computing is a known candidate in natural computing, the aim being to construct "bacterial computers" for solving complex problems. In this paper, a new kind of bacterial computing system, named the bacteria and plasmid computing system (BP system), is proposed. We investigate the computational power of BP systems with finite numbers of bacteria and plasmids. Specifically, it is obtained in a constructive way that a BP system with 2 bacteria and 34 plasmids is Turing universal. The results provide a theoretical cornerstone to construct powerful bacterial computers and demonstrate a concept of paradigms using a "reasonable" number of bacteria and plasmids for such devices.

Construction of Stable Fluorescent Reporter Plasmids for Use in Staphylococcus aureus

PubMed Central

Rodriguez, Michelle D.; Paul, Zubin; Wood, Charles E.; Rice, Kelly C.; Triplett, Eric W.

2017-01-01

Here, the genes encoding three different fluorescent proteins were cloned into the stably maintained Staphylococcus aureus shuttle vector pKK30. The resulting plasmids were transformed into two S. aureus strains; SH1000 and RN4220. Stability assays illustrated that the three recombinant plasmids retained near 100% maintenance in vitro for 160 generations. S. aureus strain SH1000 expressing green fluorescent protein was then inoculated in an ovine model and in vivo stability for 6 days was demonstrated. In essence, these reporter plasmids represent a useful set of tools for dynamic imaging studies in S. aureus. These three reporter plasmids are available through BEI Resources. PMID:29312199

Construction of Stable Fluorescent Reporter Plasmids for Use in Staphylococcus aureus.

PubMed

Rodriguez, Michelle D; Paul, Zubin; Wood, Charles E; Rice, Kelly C; Triplett, Eric W

2017-01-01

Here, the genes encoding three different fluorescent proteins were cloned into the stably maintained Staphylococcus aureus shuttle vector pKK30. The resulting plasmids were transformed into two S. aureus strains; SH1000 and RN4220. Stability assays illustrated that the three recombinant plasmids retained near 100% maintenance in vitro for 160 generations. S. aureus strain SH1000 expressing green fluorescent protein was then inoculated in an ovine model and in vivo stability for 6 days was demonstrated. In essence, these reporter plasmids represent a useful set of tools for dynamic imaging studies in S. aureus . These three reporter plasmids are available through BEI Resources.

Supercoiled plasmid DNA purification by integrating membrane technology with a monolithic chromatography.

PubMed

Nunes, Catherine; Sousa, Angela; Nunes, José C; Morão, António M; Sousa, Fani; Queiroz, João A

2014-06-01

The present study describes the integration of membrane technology with monolithic chromatography to obtain plasmid DNA with high quality. Isolation and clarification of plasmid DNA lysate were first conducted by a microfiltration step, by using a hydrophilic nylon microfiltration membrane, avoiding the need of centrifugation. For the total elimination of the remaining impurities, a suitable purification step is required. Monolithic stationary phases have been successfully applied as an alternative to conventional supports. Thus, the sample recovered from the membrane process was applied into a nongrafted CarbonylDiImidazole disk. Throughout the global procedure, a reduced level of impurities such as proteins and RNA was obtained, and no genomic DNA was detectable in the plasmid DNA sample. The chromatographic process demonstrated an efficient performance on supercoiled plasmid DNA purity and recovery (100 and 84.44%, respectively). Thereby, combining the membrane technology to eliminate some impurities from lysate sample with an efficient chromatographic strategy to purify the supercoiled plasmid DNA arises as a powerful approach for industrial-scale systems aiming at plasmid DNA purification. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bacteriological water quality in school's drinking fountains and detection antibiotic resistance genes.

PubMed

Gomes Freitas, Denize; Silva, Rassan Dyego Romão; Bataus, Luis Artur Mendes; Barbosa, Mônica Santiago; da Silva Bitencourt Braga, Carla Afonso; Carneiro, Lilian Carla

2017-02-08

The fecal coliform can contaminate water of human consumption causing problems to public health. Many of these microorganisms may contain plasmid and transfer them to other bacteria. This genetic material may confer selective advantages, among them resistance to antibiotics. The objectives of this study were to analyze the presence of fecal coliforms in water and at drinker surface, to identify the existence of plasmid, conducting studies of resistance to antibiotics, plasmid stability and capacity of bacterial conjugation. Were collected microorganisms in water of drinker surface and were used specific culture media and biochemical tests for identification of organisms, tests were performed by checking the resistance to antibiotics (ampicillin 10 μg, tetracycline 30 μg, and ciprofloxacin 5 μg), was performed extraction of plasmid DNA, plasmid stability and bacterial conjugation. Was obtained results of 31% of Salmonella spp. and 51% for other coliforms. Among the samples positive for coliforms, 27 had plasmid stable and with the ability to perform conjugation. The plasmids had similar forms, suggesting that the resistance in some bacteria may be linked to those genes extra chromosomal.

Molecular Characterization of Plasmid-Mediated Oxytetracycline Resistance in Aeromonas salmonicida

PubMed Central

Adams, C. A.; Austin, B.; Meaden, P. G.; McIntosh, D.

1998-01-01

Using broth conjugation, we found that 19 of 29 (66%) oxytetracycline (OT)-resistant isolates of Aeromonas salmonicida transferred the OT resistance phenotype to Escherichia coli. The OT resistance phenotype was encoded by high-molecular-weight R-plasmids that were capable of transferring OT resistance to both environmental and clinical isolates of Aeromonas spp. The molecular basis for antibiotic resistance in OT-resistant isolates of A. salmonicida was determined. The OT resistance determinant from one plasmid (pASOT) of A. salmonicida was cloned and used in Southern blotting and hybridization experiments as a probe. The determinant was identified on a 5.4-kb EcoRI fragment on R-plasmids from the 19 OT-resistant isolates of A. salmonicida. Hybridization with plasmids encoding the five classes (classes A to E) of OT resistance determinants demonstrated that the OT resistance plasmids of the 19 A. salmonicida isolates carried the class A resistance determinant. Analysis of data generated from restriction enzyme digests showed that the OT resistance plasmids were not identical; three profiles were characterized, two of which showed a high degree of homology. PMID:9797265

Recombination between bacteriophage lambda and plasmid pBR322 in Escherichia coli.

PubMed Central

Pogue-Geile, K L; Dassarma, S; King, S R; Jaskunas, S R

1980-01-01

Recombinant lambda phages were isolated that resulted from recombination between the lambda genome and plasmid pBR322 in Escherichia coli, even though these deoxyribonucleic acids (DNAs) did not share extensive regions of homology. The characterization of these recombinant DNAs by heteroduplex analysis and restriction endonucleases is described. All but one of the recombinants appeared to have resulted from reciprocal recombination between a site on lambda DNA and a site on the plasmid. In general, there were two classes of recombinants. One class appeared to have resulted from recombination at the phage attachment site that probably resulted from lambda integration into secondary attachment sites on the plasmid. Seven different secondary attachment sites on pBR322 were found. The other class resulted from plasmid integration at other sites that were widely scattered on the lambda genome. For this second class of recombinants, more than one site on the plasmid could recombine with lambda DNA. Thus, the recombination did not appear to be site specific with respect to lambda or the plasmid. Possible mechanisms for generating these recombinants are discussed. Images PMID:6247334

Molecular and phenotypic characterization of penicillinase-producing Neisseria gonorrhoeae from Canadian sources.

PubMed Central

Dillon, J R; Duck, P; Thomas, D Y

1981-01-01

The incidence of penicillinase-producing Neisseria gonorrhoeae (PPNG) infections has increased in Canada during the past 2 years. Most of these cases were imported from abroad. The PPNG strains from these cases were characterized with respect to susceptibility to 11 antibiotics, auxotype, and plasmid content. Rosaramicin and cefuroxime proved to be the most potent of the antibiotics tested. The molecular characterization of the isolates indicated that all carried a 2.6-megadalton cryptic plasmid. Most of the PPNG isolates (87%) harbored a 4.5-megadalton penicillinase-producing plasmid, whereas only 13% harbored the 3.2-megadalton penicillinase-producing plasmid. In those cases where contact tracing was possible, the correlation linking strains of Far Eastern etiology with carriage of the 4.5-megadalton plasmid was upheld. The penicillinase-producing strains were typed auxanographically in either the proline-requiring (57%) or prototrophic groups (42%). Substrate hydrolysis profiles and analytical isoelectric focusing of crude beta-lactamase extracts of several isolates has reconfirmed that these strains elaborate a type TEM-1 enzyme. Several of the penicillinase-producing plasmids were also examined for plasmid stability. PMID:6791587

Toward Contactless Biology: Acoustophoretic DNA Transfection

NASA Astrophysics Data System (ADS)

Vasileiou, Thomas; Foresti, Daniele; Bayram, Adem; Poulikakos, Dimos; Ferrari, Aldo

2016-02-01

Acoustophoresis revolutionized the field of container-less manipulation of liquids and solids by enabling mixing procedures which avoid contamination and loss of reagents due to the contact with the support. While its applications to chemistry and engineering are straightforward, additional developments are needed to obtain reliable biological protocols in a contactless environment. Here, we provide a first, fundamental step towards biological reactions in air by demonstrating the acoustophoretic DNA transfection of mammalian cells. We developed an original acoustophoretic design capable of levitating, moving and mixing biological suspensions of living mammalians cells and of DNA plasmids. The precise and sequential delivery of the mixed solutions into tissue culture plates is actuated by a novel mechanism based on the controlled actuation of the acoustophoretic force. The viability of the contactless procedure is tested using a cellular model sensitive to small perturbation of neuronal differentiation pathways. Additionally, the efficiency of the transfection procedure is compared to standard, container-based methods for both single and double DNA transfection and for different cell types including adherent growing HeLa cancer cells, and low adhesion neuron-like PC12 cells. In all, this work provides a proof of principle which paves the way to the development of high-throughput acoustophoretic biological reactors.

Toward Contactless Biology: Acoustophoretic DNA Transfection.

PubMed

Vasileiou, Thomas; Foresti, Daniele; Bayram, Adem; Poulikakos, Dimos; Ferrari, Aldo

2016-02-01

Acoustophoresis revolutionized the field of container-less manipulation of liquids and solids by enabling mixing procedures which avoid contamination and loss of reagents due to the contact with the support. While its applications to chemistry and engineering are straightforward, additional developments are needed to obtain reliable biological protocols in a contactless environment. Here, we provide a first, fundamental step towards biological reactions in air by demonstrating the acoustophoretic DNA transfection of mammalian cells. We developed an original acoustophoretic design capable of levitating, moving and mixing biological suspensions of living mammalians cells and of DNA plasmids. The precise and sequential delivery of the mixed solutions into tissue culture plates is actuated by a novel mechanism based on the controlled actuation of the acoustophoretic force. The viability of the contactless procedure is tested using a cellular model sensitive to small perturbation of neuronal differentiation pathways. Additionally, the efficiency of the transfection procedure is compared to standard, container-based methods for both single and double DNA transfection and for different cell types including adherent growing HeLa cancer cells, and low adhesion neuron-like PC12 cells. In all, this work provides a proof of principle which paves the way to the development of high-throughput acoustophoretic biological reactors.

Toward Contactless Biology: Acoustophoretic DNA Transfection

PubMed Central

Vasileiou, Thomas; Foresti, Daniele; Bayram, Adem; Poulikakos, Dimos; Ferrari, Aldo

2016-01-01

Acoustophoresis revolutionized the field of container-less manipulation of liquids and solids by enabling mixing procedures which avoid contamination and loss of reagents due to the contact with the support. While its applications to chemistry and engineering are straightforward, additional developments are needed to obtain reliable biological protocols in a contactless environment. Here, we provide a first, fundamental step towards biological reactions in air by demonstrating the acoustophoretic DNA transfection of mammalian cells. We developed an original acoustophoretic design capable of levitating, moving and mixing biological suspensions of living mammalians cells and of DNA plasmids. The precise and sequential delivery of the mixed solutions into tissue culture plates is actuated by a novel mechanism based on the controlled actuation of the acoustophoretic force. The viability of the contactless procedure is tested using a cellular model sensitive to small perturbation of neuronal differentiation pathways. Additionally, the efficiency of the transfection procedure is compared to standard, container-based methods for both single and double DNA transfection and for different cell types including adherent growing HeLa cancer cells, and low adhesion neuron-like PC12 cells. In all, this work provides a proof of principle which paves the way to the development of high-throughput acoustophoretic biological reactors. PMID:26828312

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

PubMed

Olukoya, D K; Asielue, J O; Olasupo, N A; Ikea, J K

1995-06-01

In an investigation into the problems of infections due to Staphylococcus aureus in Nigeria, 100 strains were isolated from various hospitals in Lagos. The strains were screened for the presence of plasmids and for susceptibility to antimicrobial agents. Plasmids were extracted by modification of the method of Takahashi and Nagono[1]. The plasmids were diverse in nature. The strains were found to be highly resistant to commonly prescribed antibiotics.

Plasmids of corynebacteria.

PubMed

Deb, J K; Nath, N

1999-06-01

Corynebacteria are pleomorphic, asporogenous, Gram-positive bacteria. Included in this group are nonpathogenic soil corynebacteria, which are widely used for the industrial production of amino acids and detergents, and in biotransformation of steroids. Other members of this group are plant and animal pathogens. This review summarizes the current information available about the plasmids of corynebacteria. The emphasis is mainly on the small plasmids, which have been used for construction of vectors for expression of genes in these bacteria. Moreover, considerable information is now available on their nucleotide sequence, gene organization and modes of replication, which would make it possible to further manipulate these plasmids. Other plasmid properties, such as incompatibility and host range, are also discussed. Finally, use of these plasmids as cloning vectors for the expression of heterologous proteins using corynebacteria as hosts is also summarized to highlight the potential of these bacteria as hosts for recombinant DNA.

Plasmid and surface antigen markers of endemic and epidemic Legionella pneumophila strains.

PubMed Central

Brown, A; Vickers, R M; Elder, E M; Lema, M; Garrity, G M

1982-01-01

Environmental and clinical isolates of Legionella pneumophila obtained from the Pittsburgh Veterans Administration Medical Center were studied for the presence of plasmids and for unique surface antigens. The majority of environmental isolates contained a single 80-megadalton plasmid. After an epidemic of nosocomial Legionnaires disease subsided in the Spring of 1981, plasmid-bearing environmental isolates persisted in the environment. Whereas L. pneumophila could not be reisolated from most sites with plasmidless isolates. During this epidemic the attack rate was highest on wards with plasmidless isolates. All clinical isolates were plasmidless. Strains were serotyped by the indirect immunofluorescence method with serum from a single immunized rat which was used both without absorption and after absorption with various plasmid-bearing and plasmidless isolates. These studies suggested that a plasmid-associated surface antigen was present and that the most common plasmidless environmental serotype was similar to the epidemic clinical serotype. Images PMID:7119096

Live, attenuated Salmonella typhimurium vectoring Campylobacter antigens.

PubMed

Sizemore, Donata R; Warner, Beth; Lawrence, Julie; Jones, Amy; Killeen, Kevin P

2006-05-01

We describe the evaluation of three live, attenuated deltaphoP/Q Salmonella enteric serovar Typhimurium strains expressing PEB1 minus its signal sequence (PEB1-ss) from three different plasmids: a pBR-based asd plasmid, an arabinose-based runaway plasmid, which each expressed PEB1-ss in the bacterial cytosol, and a PEB1::HlyA fusion plasmid that directs secretion of PEB1-ss into the extracellular milieu. Serum IgG responses specific for PEB1-ss were induced by pBR-derived and runaway plasmids, with 100 and 90% seroconversion, respectively, at a 1:500 dilution of anti-sera as measured by Western blot analysis, while the PEB1-ss::HlyA fusion plasmid induced serum IgG in only 20% of the mice. Although significant levels of anti-PEB serum IgG were induced, no protection against oral Campylobacter jejuni challenge was observed.

Imipenem-resistance in Serratia marcescens is mediated by plasmid expression of KPC-2.

PubMed

Su, W-Q; Zhu, Y-Q; Deng, N-M; Li, L

2017-04-01

Imipenem is a broad-spectrum carbapenem antibiotic with applications against severe bacterial infections. Here, we describe the identification of imipenem-resistant Serratia marcescens in our hospital and the role of plasmid-mediated KPC-2 expression in imipenem resistance. We used the modified Hodge test to detect carbapenemase produced in imipenem-resistant strains. His resistance can be transferred to E. coli in co-culture tests, which implicates the plasmid in imipenem resistance. PCR amplification from the plasmid identified two products consistent with KPC-2 of 583 and 1050 bp that were also present in E. coli after co-culture. The restriction pattern for both plasmids was identical, supporting the transfer from the S. marcescens isolate to E. coli. Finally, gene sequencing confirmed KPC-2 in the plasmid. Due to the presence of KPC-2 in the imipenem-resistant S. marcescens, we propose that KPC-2 mediates antibiotic resistance in the S. marcescens isolate.

Fire-scar formation in Jeffrey pine - mixed conifer forests in the Sierra San Pedro Martir, Mexico

Treesearch

Scott L. Stephens; Danny L. Fry; Brandon M. Collins; Carl N. Skinner; Ernesto Franco-Vizcaino; Travis J. Freed

2010-01-01

Little is known about the probability of fire-scar formation. In this study, we examined all mixed conifer trees for fire-scar formation in a 16 ha watershed that burned as part of a 2003 wildfire in Sierra San Pedro Ma´rtir National Park (SSPM), Mexico. In addition, we examine the probability of fire-scar formation in relation to the previous fire interval in forests...

Genetic Dissection of Tropodithietic Acid Biosynthesis by Marine Roseobacters▿ ‡

PubMed Central

Geng, Haifeng; Bruhn, Jesper Bartholin; Nielsen, Kristian F.; Gram, Lone; Belas, Robert

2008-01-01

The symbiotic association between the roseobacter Silicibacter sp. strain TM1040 and the dinoflagellate Pfiesteria piscicida involves bacterial chemotaxis to dinoflagellate-produced dimethylsulfoniopropionate (DMSP), DMSP demethylation, and ultimately a biofilm on the surface of the host. Biofilm formation is coincident with the production of an antibiotic and a yellow-brown pigment. In this report, we demonstrate that the antibiotic is a sulfur-containing compound, tropodithietic acid (TDA). Using random transposon insertion mutagenesis, 12 genes were identified as critical for TDA biosynthesis by the bacteria, and mutation in any one of these results in a loss of antibiotic activity (Tda−) and pigment production. Unexpectedly, six of the genes, referred to as tdaA-F, could not be found on the annotated TM1040 genome and were instead located on a previously unidentified plasmid (ca. 130 kb; pSTM3) that exhibited a low frequency of spontaneous loss. Homologs of tdaA and tdaB from Silicibacter sp. strain TM1040 were identified by mutagenesis in another TDA-producing roseobacter, Phaeobacter sp. strain 27-4, which also possesses two large plasmids (ca. 60 and ca. 70 kb, respectively), and tda genes were found by DNA-DNA hybridization in 88% of a diverse collection of nine roseobacters with known antibiotic activity. These data suggest that roseobacters may use a common pathway for TDA biosynthesis that involves plasmid-encoded proteins. Using metagenomic library databases and a bioinformatics approach, differences in the biogeographical distribution between the critical TDA synthesis genes were observed. The implications of these results to roseobacter survival and the interaction between TM1040 and its dinoflagellate host are discussed. PMID:18192410

Addition of six-His-tagged peptide to the C terminus of adeno-associated virus VP3 does not affect viral tropism or production.

PubMed

Zhang, Huang-Ge; Xie, Jinfu; Dmitriev, Igor; Kashentseva, Elena; Curiel, David T; Hsu, Hui-Chen; Mountz, John D

2002-12-01

Production of large quantities of recombinant adeno-associated virus (AAV) is difficult and not cost-effective. To overcome this problem, we have explored the feasibility of creating a recombinant AAV encoding a 6xHis tag on the VP3 capsid protein. We generated a plasmid vector containing a six-His (6xHis)-tagged AAV VP3. A second plasmid vector was generated that contained the full-length AAV capsid capable of producing VP1 and VP2, but not VP3 due to a mutation at position 2809 that encodes the start codon for VP3. These plasmids, necessary for production of AAV, were transfected into 293 cells to generate a 6xHis-tagged VP3mutant recombinant AAV. The 6xHis-tagged VP3 did not affect the formation of AAV virus, and the physical properties of the 6xHis-modified AAV were equivalent to those of wild-type particles. The 6xHis-tagged AAV did not affect the production titer of recombinant AAV and could be used to purify the recombinant AAV using an Ni-nitrilotriacetic acid column. Addition of the 6xHis tag did not alter the viral tropism compared to wild-type AAV. These observations demonstrate the feasibility of producing high-titer AAV containing a 6xHis-tagged AAV VP3 capsid protein and to utilize the 6xHis-tagged VP3 capsid to achieve high-affinity purification of this recombinant AAV.

Hydrodynamics based transfection in normal and fibrotic rats

PubMed Central

Yeikilis, Rita; Gal, Shunit; Kopeiko, Natalia; Paizi, Melia; Pines, Mark; Braet, Filip; Spira, Gadi

2006-01-01

AIM: Hydrodynamics based transfection (HBT), the injection of a large volume of naked plasmid DNA in a short time is a relatively simple, efficient and safe method for in vivo transfection of liver cells. Though used for quite some time, the mechanism of gene transfection has not yet been elucidated. METHODS: A luciferase encoding plasmid was injected using the hydrodynamics based procedure into normal and thioacetamide-induced fibrotic Sprague Dawley rats. Scanning and transmission electron microscopy images were taken. The consequence of a dual injection of Ringer solution and luciferase pDNA was followed. Halofuginone, an anti collagen type I inhibitor was used to reduce ECM load in fibrotic rats prior to the hydrodynamic injection. RESULTS: Large endothelial gaps formed as soon as 10’ following hydrodynamic injection; these gradually returned to normal 10 d post injection. Hydrodynamic administration of Ringer 10 or 30 m prior to moderate injection of plasmid did not result in efficient transfection suggesting that endothelial gaps by themselves are not sufficient for gene expression. Gene transfection following hydrodynamic injection in thioacetamide induced fibrotic rats was diminished coinciding with the level of fibrosis. Halofuginone, a specific collagen typeIinhibitor, alleviated this effect. CONCLUSION: The hydrodynamic pressure formed following HBT results in the formation of large endothelial gaps. These gaps, though important in the transfer of DNA molecules from the blood to the space of Disse are not enough to provide the appropriate conditions for hepatocyte transfection. Hydrodynamics based injection is applicable in fibrotic rats provided that ECM load is reduced. PMID:17036386

Codon-optimized filovirus DNA vaccines delivered by intramuscular electroporation protect cynomolgus macaques from lethal Ebola and Marburg virus challenges.

PubMed

Grant-Klein, Rebecca J; Altamura, Louis A; Badger, Catherine V; Bounds, Callie E; Van Deusen, Nicole M; Kwilas, Steven A; Vu, Hong A; Warfield, Kelly L; Hooper, Jay W; Hannaman, Drew; Dupuy, Lesley C; Schmaljohn, Connie S

2015-01-01

Cynomolgus macaques were vaccinated by intramuscular electroporation with DNA plasmids expressing codon-optimized glycoprotein (GP) genes of Ebola virus (EBOV) or Marburg virus (MARV) or a combination of codon-optimized GP DNA vaccines for EBOV, MARV, Sudan virus and Ravn virus. When measured by ELISA, the individual vaccines elicited slightly higher IgG responses to EBOV or MARV than did the combination vaccines. No significant differences in immune responses of macaques given the individual or combination vaccines were measured by pseudovirion neutralization or IFN-γ ELISpot assays. Both the MARV and mixed vaccines were able to protect macaques from lethal MARV challenge (5/6 vs. 6/6). In contrast, a greater proportion of macaques vaccinated with the EBOV vaccine survived lethal EBOV challenge in comparison to those that received the mixed vaccine (5/6 vs. 1/6). EBOV challenge survivors had significantly higher pre-challenge neutralizing antibody titers than those that succumbed.

Plasmid DNA-encapsulating liposomes: effect of a spacer between the cationic head group and hydrophobic moieties of the lipids on gene expression efficiency.

PubMed

Obata, Yosuke; Saito, Shunsuke; Takeda, Naoya; Takeoka, Shinji

2009-05-01

We have synthesized a series of cationic amino acid-based lipids having a spacer between the cationic head group and hydrophobic moieties and examined the influence of the spacer on a liposome gene delivery system. As a comparable spacer, a hydrophobic spacer with a hydrocarbon chain composed of 0, 3, 5, 7, or 11 carbons, and a hydrophilic spacer with an oxyethylene chain (10 carbon and 3 oxygen molecules) were investigated. Plasmid DNA (pDNA)-encapsulating liposomes were prepared by mixing an ethanol solution of the lipids with an aqueous solution of pDNA. The zeta potentials and cellular uptake efficiency of the cationic liposomes containing each synthetic lipid were almost equivalent. However, the cationic lipids with the hydrophobic spacer were subject to fuse with biomembrane-mimicking liposomes. 1,5-Dihexadecyl-N-lysyl-N-heptyl-l-glutamate, having a seven carbon atom spacer, exhibited the highest fusogenic potential among the synthetic lipids. Increased fusion potential correlated with enhanced gene expression efficiency. By contrast, an oxyethylene chain spacer showed low gene expression efficiency. We conclude that a hydrophobic spacer between the cationic head group and hydrophobic moieties is a key component for improving pDNA delivery.

Aspects of nonviral gene therapy: correlation of molecular parameters with lipoplex structure and transfection efficacy in pyridinium-based cationic lipids.

PubMed

Parvizi, Paria; Jubeli, Emile; Raju, Liji; Khalique, Nada Abdul; Almeer, Ahmed; Allam, Hebatalla; Manaa, Maryem Al; Larsen, Helge; Nicholson, David; Pungente, Michael D; Fyles, Thomas M

2014-01-30

This study seeks correlations between the molecular structures of cationic and neutral lipids, the lipid phase behavior of the mixed-lipid lipoplexes they form with plasmid DNA, and the transfection efficacy of the lipoplexes. Synthetic cationic pyridinium lipids were co-formulated (1:1) with the cationic lipid 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), and these lipids were co-formulated (3:2) with the neutral lipids 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or cholesterol. All lipoplex formulations exhibited plasmid DNA binding and a level of protection from DNase I degradation. Composition-dependent transfection (beta-galactosidase and GFP) and cytotoxicity was observed in Chinese hamster ovarian-K1 cells. The most active formulations containing the pyridinium lipids were less cytotoxic but of comparable activity to a Lipofectamine 2000™ control. Molecular structure parameters and partition coefficients were calculated for all lipids using fragment additive methods. The derived shape parameter values correctly correlated with observed hexagonal lipid phase behavior of lipoplexes as derived from small-angle X-ray scattering experiments. A transfection index applicable to hexagonal phase lipoplexes derived from calculated parameters of the lipid mixture (partition coefficient, shape parameter, lipoplex packing) produced a direct correlation with transfection efficiency. Copyright © 2013 Elsevier B.V. All rights reserved.

Isolation and characterization of novel mutations in the pSC101 origin that increase copy number

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thompson, Mitchell G.; Sedaghatian, Nima; Barajas, Jesus F.

pSC101 is a narrow host range, low-copy plasmid commonly used for genetically manipulating Escherichia coli. As a byproduct of a genetic screen for a more sensitive lactam biosensor, we identified multiple novel mutations that increase the copy number of plasmids with the pSC101 origin. All mutations identified in this study occurred on plasmids which also contained at least one mutation localized to the RepA protein encoded within the origin. Homology modelling predicts that many of these mutations occur within the dimerization interface of RepA. Mutant RepA resulted in plasmid copy numbers between ~31 and ~113 copies/cell, relative to ~5 copies/cellmore » in wild-type pSC101 plasmids. Combining the mutations that were predicted to disrupt multiple contacts on the dimerization interface resulted in copy numbers of ~500 copies/cell, while also attenuating growth in host strains. Fluorescent protein production expressed from an arabinose-inducible promoter on mutant origin derived plasmids did correlate with copy number. Plasmids harboring RepA with one of two mutations, E83K and N99D, resulted in fluorescent protein production similar to that from p15a- (~20 copies/cell) and ColE1- (~31 copies/cell) based plasmids, respectively. The mutant copy number variants retained compatibility with p15a, pBBR, and ColE1 origins of replication. Thus, these pSC101 variants may be useful in future metabolic engineering efforts that require medium or high-copy vectors compatible with p15a- and ColE1-based plasmids.« less

A cell engineering strategy to enhance supercoiled plasmid DNA production for gene therapy.

PubMed

Hassan, Sally; Keshavarz-Moore, Eli; Ward, John

2016-09-01

With the recent revival of the promise of plasmid DNA vectors in gene therapy, a novel synthetic biology approach was used to enhance the quantity, (yield), and quality of the plasmid DNA. Quality was measured by percentage supercoiling and supercoiling density, as well as improving segregational stability in fermentation. We examined the hypothesis that adding a Strong Gyrase binding Site (SGS) would increase DNA gyrase-mediated plasmid supercoiling. SGS from three different replicons, (the Mu bacteriophage and two plasmids, pSC101 and pBR322) were inserted into the plasmid, pUC57. Different sizes of these variants were transformed into E. coli DH5α, and their supercoiling properties and segregational stability measured. A 36% increase in supercoiling density was found in pUC57-SGS, but only when SGS was derived from the Mu phage and was the larger sized version of this fragment. These results were also confirmed at fermentation scale. Total percentage supercoiled monomer was maintained to 85-90%. A twofold increase in plasmid yield was also observed for pUC57-SGS in comparison to pUC57. pUC57-SGS displayed greater segregational stability than pUC57-cer and pUC57, demonstrating a further potential advantage of the SGS site. These findings should augment the potential of plasmid DNA vectors in plasmid DNA manufacture. Biotechnol. Bioeng. 2016;113: 2064-2071. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Isolation and characterization of novel mutations in the pSC101 origin that increase copy number

DOE PAGES

Thompson, Mitchell G.; Sedaghatian, Nima; Barajas, Jesus F.; ...

2018-01-25

pSC101 is a narrow host range, low-copy plasmid commonly used for genetically manipulating Escherichia coli. As a byproduct of a genetic screen for a more sensitive lactam biosensor, we identified multiple novel mutations that increase the copy number of plasmids with the pSC101 origin. All mutations identified in this study occurred on plasmids which also contained at least one mutation localized to the RepA protein encoded within the origin. Homology modelling predicts that many of these mutations occur within the dimerization interface of RepA. Mutant RepA resulted in plasmid copy numbers between ~31 and ~113 copies/cell, relative to ~5 copies/cellmore » in wild-type pSC101 plasmids. Combining the mutations that were predicted to disrupt multiple contacts on the dimerization interface resulted in copy numbers of ~500 copies/cell, while also attenuating growth in host strains. Fluorescent protein production expressed from an arabinose-inducible promoter on mutant origin derived plasmids did correlate with copy number. Plasmids harboring RepA with one of two mutations, E83K and N99D, resulted in fluorescent protein production similar to that from p15a- (~20 copies/cell) and ColE1- (~31 copies/cell) based plasmids, respectively. The mutant copy number variants retained compatibility with p15a, pBBR, and ColE1 origins of replication. Thus, these pSC101 variants may be useful in future metabolic engineering efforts that require medium or high-copy vectors compatible with p15a- and ColE1-based plasmids.« less

Development and Host Compatibility of Plasmids for Two Important Ruminant Pathogens, Mycoplasma bovis and Mycoplasma agalactiae

PubMed Central

Sharma, Shukriti; Citti, Chistine; Sagné, Eveline; Marenda, Marc S.

2015-01-01

Mycoplasma bovis is a cause of pneumonia, mastitis, arthritis and otitis media in cattle throughout the world. However, despite its clinical significance, there is a paucity of tools to genetically manipulate it, impeding our capacity to further explore the molecular basis of its virulence. To address this limitation, we developed a series of homologous and heterologous replicable plasmids from M. bovis and M. agalactiae. The shortest replicable oriC plasmid based on the region downstream of dnaA in M. bovis was 247 bp and contained two DnaA boxes, while oriC plasmids based on the region downstream of dnaA in M. agalactiae strains 5632 and PG2 were 219 bp and 217 bp in length, respectively, and contained only a single DnaA box. The efficiency of transformation in M. bovis and M. agalactiae was inversely correlated with the size of the oriC region in the construct, and, in general, homologous oriC plasmids had a higher transformation efficiency than heterologous oriC plasmids. The larger pWholeoriC45 and pMM21-7 plasmids integrated into the genomic oriC region of M. bovis, while the smaller oriC plasmids remained extrachromosomal for up to 20 serial passages in selective media. Although specific gene disruptions were not be achieved in M. bovis in this study, the oriC plasmids developed here could still be useful as tools in complementation studies and for expression of exogenous genes in both M. bovis and M. agalactiae. PMID:25746296

Identification of dfrA14 in two distinct plasmids conferring trimethoprim resistance in Actinobacillus pleuropneumoniae

PubMed Central

Bossé, Janine T.; Li, Yanwen; Walker, Stephanie; Atherton, Tom; Fernandez Crespo, Roberto; Williamson, Susanna M.; Rogers, Jon; Chaudhuri, Roy R.; Weinert, Lucy A.; Oshota, Olusegun; Holden, Matt T. G.; Maskell, Duncan J.; Tucker, Alexander W.; Wren, Brendan W.; Rycroft, Andrew N.; Langford, Paul R.

2015-01-01

Objectives The objective of this study was to determine the distribution and genetic basis of trimethoprim resistance in Actinobacillus pleuropneumoniae isolates from pigs in England. Methods Clinical isolates collected between 1998 and 2011 were tested for resistance to trimethoprim and sulphonamide. The genetic basis of trimethoprim resistance was determined by shotgun WGS analysis and the subsequent isolation and sequencing of plasmids. Results A total of 16 (out of 106) A. pleuropneumoniae isolates were resistant to both trimethoprim (MIC >32 mg/L) and sulfisoxazole (MIC ≥256 mg/L), and a further 32 were resistant only to sulfisoxazole (MIC ≥256 mg/L). Genome sequence data for the trimethoprim-resistant isolates revealed the presence of the dfrA14 dihydrofolate reductase gene. The distribution of plasmid sequences in multiple contigs suggested the presence of two distinct dfrA14-containing plasmids in different isolates, which was confirmed by plasmid isolation and sequencing. Both plasmids encoded mobilization genes, the sulphonamide resistance gene sul2, as well as dfrA14 inserted into strA, a streptomycin-resistance-associated gene, although the gene order differed between the two plasmids. One of the plasmids further encoded the strB streptomycin-resistance-associated gene. Conclusions This is the first description of mobilizable plasmids conferring trimethoprim resistance in A. pleuropneumoniae and, to our knowledge, the first report of dfrA14 in any member of the Pasteurellaceae. The identification of dfrA14 conferring trimethoprim resistance in A. pleuropneumoniae isolates will facilitate PCR screens for resistance to this important antimicrobial. PMID:25957382

Small, Enigmatic Plasmids of the Nosocomial Pathogen, Acinetobacter baumannii: Good, Bad, Who Knows?

PubMed Central

Lean, Soo Sum; Yeo, Chew Chieng

2017-01-01

Acinetobacter baumannii is a Gram-negative nosocomial pathogen that has become a serious healthcare concern within a span of two decades due to its ability to rapidly acquire resistance to all classes of antimicrobial compounds. One of the key features of the A. baumannii genome is an open pan genome with a plethora of plasmids, transposons, integrons, and genomic islands, all of which play important roles in the evolution and success of this clinical pathogen, particularly in the acquisition of multidrug resistance determinants. An interesting genetic feature seen in majority of A. baumannii genomes analyzed is the presence of small plasmids that usually ranged from 2 to 10 kb in size, some of which harbor antibiotic resistance genes and homologs of plasmid mobilization genes. These plasmids are often overlooked when compared to their larger, conjugative counterparts that harbor multiple antibiotic resistance genes and transposable elements. In this mini-review, we will examine our current knowledge of these small A. baumannii plasmids and look into their genetic diversity and phylogenetic relationships. Some of these plasmids, such as the Rep-3 superfamily group and the pRAY-type, which has no recognizable replicase genes, are quite widespread among diverse A. baumannii clinical isolates worldwide, hinting at their usefulness to the lifestyle of this pathogen. Other small plasmids especially those from the Rep-1 superfamily are truly enigmatic, encoding only hypothetical proteins of unknown function, leading to the question of whether these small plasmids are “good” or “bad” to their host A. baumannii. PMID:28861061

Distribution of Oxytetracycline Resistance Plasmids between Aeromonads in Hospital and Aquaculture Environments: Implication of Tn1721 in Dissemination of the Tetracycline Resistance Determinant Tet A

PubMed Central

Rhodes, Glenn; Huys, Geert; Swings, Jean; Mcgann, Patrick; Hiney, Maura; Smith, Peter; Pickup, Roger W.

2000-01-01

Oxytetracycline-resistant (OTr) mesophilic aeromonads were recovered from untreated hospital effluent (72 isolates) and from fish farm hatchery tanks (91 isolates) at sites within the English Lake District, Cumbria, England. The transfer of OTr plasmids from these isolates was investigated. Using Escherichia coli J53-1 as a recipient, 11 isolates from the hospital site and 6 isolates from the fish farm site transferred OTr plasmids (designated pFBAOT1 to 17). Original isolates were identified using fatty acid methyl ester and fluorescent amplified fragment length polymorphism comparisons as either Aeromonas hydrophila HG3 (eight isolates), A. veronii b.v. sobria HG8 (six isolates), and A. caviae HGB5 (one isolate). One isolate remained unidentified, and one could not be assigned a taxonomic designation beyond the genus level. Plasmids pFBAOT1 to -17 were screened for the presence of the tetracycline resistance determinants Tet A to E and Tet G. Only determinant Tet A (10 plasmids) was detected in these plasmids, with 7 tet gene determinants remaining unclassified. In all cases, Tet A was located on a 5.5-kb EcoRI restriction fragment. Hybridization with inc-rep probes N, P, Q, W, and U showed pFBAOT3, -4, -5, -6, -7, -9, and -11, from the hospital environment, to be IncU plasmids. Further, restriction fragment length polymorphism (RFLP) analyses and DNA probing demonstrated that pFBAOT plasmids were closely related to IncU OTr plasmids pASOT, pASOT2, pASOT3, pRAS1 (originally isolated from A. salmonicida strains from fish farms in Scotland and Norway, respectively), and pIE420 (isolated from a German hospital E. coli strain). In addition, DNA analyses demonstrated that plasmids pRAS1 and pIE420 had identical RFLP profiles and that all fragments hybridized to each other. The presence of tetracycline resistance transposon Tn1721 in its entirety or in a truncated form in these plasmids was demonstrated. These results provided direct evidence that related tetracycline resistance-encoding plasmids have disseminated between different Aeromonas species and E. coli and between the human and aquaculture environments in distinct geographical locations. Collectively, these findings provide evidence to support the hypothesis that the aquaculture and human compartments of the environment behave as a single interactive compartment. PMID:10966404

Plasmid-encoded amikacin resistance in multiresistant strains of Klebsiella pneumoniae isolated from neonates with meningitis.

PubMed Central

Woloj, M; Tolmasky, M E; Roberts, M C; Crosa, J H

1986-01-01

Two multiresistant Klebsiella pneumoniae strains isolated from cerebrospinal fluid of human neonates were analyzed for their plasmid content. Two of the plasmids harbored by these strains, pJHCMW1 (11 kilobase pairs) and pJHCMW4 (75 kilobase pairs), carried genetic determinants for amikacin resistance. These plasmids also encoded resistance to kanamycin, tobramycin, and ampicillin which could be transferred to Escherichia coli by conjugation. Extracts from transconjugant derivatives carrying pJHCMW4 produced an acetyltransferase activity that acetylated all three aminoglycosides. Transconjugant derivatives carrying pJHCMW1 encoded both acetylating and phosphorylating activities. Southern blot hybridization analysis indicated considerable DNA homology between these two plasmids. Images PMID:3521478

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

PubMed Central

Taylor, D E; Summers, A O

1979-01-01

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

Detection of plasmids and class 1 integrons in Salmonella enterica serovar agona isolated from NARMS slaughter samples collected in the years 1997-2003.

PubMed

Douris, Aphrodite; Fedorka-Cray, Paula J; Jackson, Charlene R

2007-01-01

A total of 60 Salmonella enterica serovar Agona isolates (25 pan-susceptible isolates and 35 isolates resistant to five or more antimicrobials) submitted to the National Antimicrobial Resistance Monitoring System-Enteric Bacteria (NARMS) from 1997 through 2003 were examined for plasmids and class 1 integrons. Samples originated from cattle, turkey, chicken, and swine presented at federally inspected slaughter and processing plants. Large plasmids (33-291 kb) were present in 83% of the isolates resistant to five or more antimicrobials; however, 16% of the pan-susceptible isolates also had large plasmids. The presence of large plasmids did not correspond to the isolate source or the year the isolate was recovered but did appear to correspond to XbaI pulsed-field gel electrophoresis (PFGE) patterns. Two sizes of large plasmids appeared most often: 145.4 kb and 97 kb. Class 1 integrons were not detected on plasmids but were detected on the chromosome of 8% (2/25) of the pan-susceptible isolates and 49% (17/35) of the isolates with multiple drug resistance. Expression of multiple drug resistance among S. Agona isolates occurred regardless of the presence of class 1 integrons, suggesting that plasmids play an equally important role in the development of resistant S. Agona. More research is needed to understand better the mechanisms by which S. Agona acquires, harbors, and transfers resistance determinants.

Shared Features of Cryptic Plasmids from Environmental and Pathogenic Francisella Species

DOE Office of Scientific and Technical Information (OSTI.GOV)

Challacombe, Jean Faust; Pillai, Segaran; Kuske, Cheryl R.

The Francisella genus includes several recognized species, additional potential species, and other representatives that inhabit a range of incredibly diverse ecological niches, but are not closely related to the named species. Francisella species have been obtained from a wide variety of clinical and environmental sources; documented species include highly virulent human and animal pathogens, fish pathogens, opportunistic human pathogens, tick endosymbionts, and free-living isolates inhabiting brackish water. While more than 120 Francisella genomes have been sequenced to date, only a few contain plasmids, and most of these appear to be cryptic, with unknown benefit to the host cell. We havemore » identified several putative cryptic plasmids in the sequenced genomes of three Francisella novicida and F. novicida-like strains (TX07-6608, AZ06-7470, DPG_3A-IS) and two new Francisella species (F. frigiditurris CA97-1460 and F. opportunistica MA06-7296). These plasmids were compared to each other and to previously identified plasmids from other Francisella species. Some of the plasmids encoded functions potentially involved in replication, conjugal transfer and partitioning, environmental survival (transcriptional regulation, signaling, metabolism), and hypothetical proteins with no assignable functions. In conclusion, genomic and phylogenetic comparisons of these new plasmids to the other known Francisella plasmids revealed some similarities that add to our understanding of the evolutionary relationships among the diverse Francisella species.« less

Epsilon-toxin plasmids of Clostridium perfringens type D are conjugative.

PubMed

Hughes, Meredith L; Poon, Rachael; Adams, Vicki; Sayeed, Sameera; Saputo, Juliann; Uzal, Francisco A; McClane, Bruce A; Rood, Julian I

2007-11-01

Isolates of Clostridium perfringens type D produce the potent epsilon-toxin (a CDC/U.S. Department of Agriculture overlap class B select agent) and are responsible for several economically significant enterotoxemias of domestic livestock. It is well established that the epsilon-toxin structural gene, etx, occurs on large plasmids. We show here that at least two of these plasmids are conjugative. The etx gene on these plasmids was insertionally inactivated using a chloramphenicol resistance cassette to phenotypically tag the plasmid. High-frequency conjugative transfer of the tagged plasmids into the C. perfringens type A strain JIR325 was demonstrated, and the resultant transconjugants were shown to act as donors in subsequent mating experiments. We also demonstrated the transfer of "unmarked" native epsilon-toxin plasmids into strain JIR325 by exploiting the high transfer frequency. The transconjugants isolated in these experiments expressed functional epsilon-toxin since their supernatants had cytopathic effects on MDCK cells and were toxic in mice. Using the widely accepted multiplex PCR approach for toxin genotyping, these type A-derived transconjugants were genotypically type D. These findings have significant implications for the C. perfringens typing system since it is based on the toxin profile of each strain. Our study demonstrated the fluid nature of the toxinotypes and their dependence upon the presence or absence of toxin plasmids, some of which have for the first time been shown to be conjugative.

Shared Features of Cryptic Plasmids from Environmental and Pathogenic Francisella Species

DOE PAGES

Challacombe, Jean Faust; Pillai, Segaran; Kuske, Cheryl R.

2017-08-24

The Francisella genus includes several recognized species, additional potential species, and other representatives that inhabit a range of incredibly diverse ecological niches, but are not closely related to the named species. Francisella species have been obtained from a wide variety of clinical and environmental sources; documented species include highly virulent human and animal pathogens, fish pathogens, opportunistic human pathogens, tick endosymbionts, and free-living isolates inhabiting brackish water. While more than 120 Francisella genomes have been sequenced to date, only a few contain plasmids, and most of these appear to be cryptic, with unknown benefit to the host cell. We havemore » identified several putative cryptic plasmids in the sequenced genomes of three Francisella novicida and F. novicida-like strains (TX07-6608, AZ06-7470, DPG_3A-IS) and two new Francisella species (F. frigiditurris CA97-1460 and F. opportunistica MA06-7296). These plasmids were compared to each other and to previously identified plasmids from other Francisella species. Some of the plasmids encoded functions potentially involved in replication, conjugal transfer and partitioning, environmental survival (transcriptional regulation, signaling, metabolism), and hypothetical proteins with no assignable functions. In conclusion, genomic and phylogenetic comparisons of these new plasmids to the other known Francisella plasmids revealed some similarities that add to our understanding of the evolutionary relationships among the diverse Francisella species.« less

Isolation and characterization of two cryptic plasmids in the ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11.

PubMed

Yamagata, A; Kato, J; Hirota, R; Kuroda, A; Ikeda, T; Takiguchi, N; Ohtake, H

1999-06-01

Two plasmids were discovered in the ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11, which was isolated from activated sludge. The plasmids, designated pAYS and pAYL, were relatively small, being approximately 1.9 kb long. They were cryptic plasmids, having no detectable plasmid-linked antibiotic resistance or heavy metal resistance markers. The complete nucleotide sequences of pAYS and pAYL were determined, and their physical maps were constructed. There existed two major open reading frames, ORF1 in pAYS and ORF2 in pAYL, each of which was more than 500 bp long. The predicted product of ORF2 was 28% identical to part of the replication protein of a Bacillus plasmid, pBAA1. However, no significant similarity to any known protein sequences was detected with the predicted product of ORF1. pAYS and pAYL had a highly homologous region, designated HHR, of 262 bp. The overall identity was 98% between the two nucleotide sequences. Interestingly, HHR-homologous sequences were also detected in the genomes of ENI-11 and the plasmidless strain Nitrosomonas europaea IFO14298. Deletion analysis of pAYS and pAYL indicated that HHR, together with either ORF1 or ORF2, was essential for plasmid maintenance in ENI-11. To our knowledge, pAYS and pAYL are the first plasmids found in the ammonia-oxidizing autotrophic bacteria.

pTC Plasmids from Sulfolobus Species in the Geothermal Area of Tengchong, China: Genomic Conservation and Naturally-Occurring Variations as a Result of Transposition by Mobile Genetic Elements

PubMed Central

Xiang, Xiaoyu; Huang, Xiaoxing; Wang, Haina; Huang, Li

2015-01-01

Plasmids occur frequently in Archaea. A novel plasmid (denoted pTC1) containing typical conjugation functions has been isolated from Sulfolobus tengchongensis RT8-4, a strain obtained from a hot spring in Tengchong, China, and characterized. The plasmid is a circular double-stranded DNA molecule of 20,417 bp. Among a total of 26 predicted pTC1 ORFs, 23 have homologues in other known Sulfolobus conjugative plasmids (CPs). pTC1 resembles other Sulfolobus CPs in genome architecture, and is most highly conserved in the genomic region encoding conjugation functions. However, attempts to demonstrate experimentally the capacity of the plasmid for conjugational transfer were unsuccessful. A survey revealed that pTC1 and its closely related plasmid variants were widespread in the geothermal area of Tengchong. Variations of the plasmids at the target sites for transposition by an insertion sequence (IS) and a miniature inverted-repeat transposable element (MITE) were readily detected. The IS was efficiently inserted into the pTC1 genome, and the inserted sequence was inactivated and degraded more frequently in an imprecise manner than in a precise manner. These results suggest that the host organism has evolved a strategy to maintain a balance between the insertion and elimination of mobile genetic elements to permit genomic plasticity while inhibiting their fast spreading. PMID:25686154

Plasmid incidence in bacteria from deep subsurface sediments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fredrickson, J.K.; Hicks, R.J.; Li, S.W.

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 themore » 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.« less

Resistance of Permafrost and Modern Acinetobacter lwoffii Strains to Heavy Metals and Arsenic Revealed by Genome Analysis.

PubMed

Mindlin, Sofia; Petrenko, Anatolii; Kurakov, Anton; Beletsky, Alexey; Mardanov, Andrey; Petrova, Mayya

2016-01-01

We performed whole-genome sequencing of five permafrost strains of Acinetobacter lwoffii (frozen for 15-3000 thousand years) and analyzed their resistance genes found in plasmids and chromosomes. Four strains contained multiple plasmids (8-12), which varied significantly in size (from 4,135 to 287,630 bp) and genetic structure; the fifth strain contained only two plasmids. All large plasmids and some medium-size and small plasmids contained genes encoding resistance to various heavy metals, including mercury, cobalt, zinc, cadmium, copper, chromium, and arsenic compounds. Most resistance genes found in the ancient strains of A . lwoffii had their closely related counterparts in modern clinical A . lwoffii strains that were also located on plasmids. The vast majority of the chromosomal resistance determinants did not possess complete sets of the resistance genes or contained truncated genes. Comparative analysis of various A . lwoffii and of A . baumannii strains discovered a number of differences between them: (i) chromosome sizes in A . baumannii exceeded those in A . lwoffii by about 20%; (ii) on the contrary, the number of plasmids in A . lwoffii and their total size were much higher than those in A . baumannii ; (iii) heavy metal resistance genes in the environmental A . lwoffii strains surpassed those in A . baumannii strains in the number and diversity and were predominantly located on plasmids. Possible reasons for these differences are discussed.

Resistance of Permafrost and Modern Acinetobacter lwoffii Strains to Heavy Metals and Arsenic Revealed by Genome Analysis

PubMed Central

Kurakov, Anton; Beletsky, Alexey; Mardanov, Andrey

2016-01-01

We performed whole-genome sequencing of five permafrost strains of Acinetobacter lwoffii (frozen for 15–3000 thousand years) and analyzed their resistance genes found in plasmids and chromosomes. Four strains contained multiple plasmids (8–12), which varied significantly in size (from 4,135 to 287,630 bp) and genetic structure; the fifth strain contained only two plasmids. All large plasmids and some medium-size and small plasmids contained genes encoding resistance to various heavy metals, including mercury, cobalt, zinc, cadmium, copper, chromium, and arsenic compounds. Most resistance genes found in the ancient strains of A. lwoffii had their closely related counterparts in modern clinical A. lwoffii strains that were also located on plasmids. The vast majority of the chromosomal resistance determinants did not possess complete sets of the resistance genes or contained truncated genes. Comparative analysis of various A. lwoffii and of A. baumannii strains discovered a number of differences between them: (i) chromosome sizes in A. baumannii exceeded those in A. lwoffii by about 20%; (ii) on the contrary, the number of plasmids in A. lwoffii and their total size were much higher than those in A. baumannii; (iii) heavy metal resistance genes in the environmental A. lwoffii strains surpassed those in A. baumannii strains in the number and diversity and were predominantly located on plasmids. Possible reasons for these differences are discussed. PMID:27795957

pTC Plasmids from Sulfolobus Species in the Geothermal Area of Tengchong, China: Genomic Conservation and Naturally-Occurring Variations as a Result of Transposition by Mobile Genetic Elements.

PubMed

Xiang, Xiaoyu; Huang, Xiaoxing; Wang, Haina; Huang, Li

2015-02-12

Plasmids occur frequently in Archaea. A novel plasmid (denoted pTC1) containing typical conjugation functions has been isolated from Sulfolobus tengchongensis RT8-4, a strain obtained from a hot spring in Tengchong, China, and characterized. The plasmid is a circular double-stranded DNA molecule of 20,417 bp. Among a total of 26 predicted pTC1 ORFs, 23 have homologues in other known Sulfolobus conjugative plasmids (CPs). pTC1 resembles other Sulfolobus CPs in genome architecture, and is most highly conserved in the genomic region encoding conjugation functions. However, attempts to demonstrate experimentally the capacity of the plasmid for conjugational transfer were unsuccessful. A survey revealed that pTC1 and its closely related plasmid variants were widespread in the geothermal area of Tengchong. Variations of the plasmids at the target sites for transposition by an insertion sequence (IS) and a miniature inverted-repeat transposable element (MITE) were readily detected. The IS was efficiently inserted into the pTC1 genome, and the inserted sequence was inactivated and degraded more frequently in an imprecise manner than in a precise manner. These results suggest that the host organism has evolved a strategy to maintain a balance between the insertion and elimination of mobile genetic elements to permit genomic plasticity while inhibiting their fast spreading.

76 FR 65544 - Standard Format and Content of License Applications for Mixed Oxide Fuel Fabrication Facilities

Federal Register 2010, 2011, 2012, 2013, 2014

2011-10-21

...The U.S. Nuclear Regulatory Commission (NRC or Commission) is issuing a revision to regulatory guide (RG) 3.39, ``Standard Format and Content of License Applications for Mixed Oxide Fuel Fabrication Facilities.'' This guide endorses the standard format and content for license applications and integrated safety analysis (ISA) summaries described in the current version of NUREG-1718, ``Standard Review Plan for the Review of an Application for a Mixed Oxide (MOX) Fuel Fabrication Facility,'' as a method that the NRC staff finds acceptable for meeting the regulatory requirements of Title 10 of the Code of Federal Regulations (10 CFR) part 70, ``Domestic Licensing of Special Nuclear Material'' for mixed oxide fuel fabrication facilities.

Plasmid Characterization and Chromosome Analysis of Two netF+ Clostridium perfringens Isolates Associated with Foal and Canine Necrotizing Enteritis

PubMed Central

Mehdizadeh Gohari, Iman; Kropinski, Andrew M.; Weese, Scott J.; Parreira, Valeria R.; Whitehead, Ashley E.; Boerlin, Patrick; Prescott, John F.

2016-01-01

The recent discovery of a novel beta-pore-forming toxin, NetF, which is strongly associated with canine and foal necrotizing enteritis should improve our understanding of the role of type A Clostridium perfringens associated disease in these animals. The current study presents the complete genome sequence of two netF-positive strains, JFP55 and JFP838, which were recovered from cases of foal necrotizing enteritis and canine hemorrhagic gastroenteritis, respectively. Genome sequencing was done using Single Molecule, Real-Time (SMRT) technology-PacBio and Illumina Hiseq2000. The JFP55 and JFP838 genomes include a single 3.34 Mb and 3.53 Mb chromosome, respectively, and both genomes include five circular plasmids. Plasmid annotation revealed that three plasmids were shared by the two newly sequenced genomes, including a NetF/NetE toxins-encoding tcp-conjugative plasmid, a CPE/CPB2 toxins-encoding tcp-conjugative plasmid and a putative bacteriocin-encoding plasmid. The putative beta-pore-forming toxin genes, netF, netE and netG, were located in unique pathogenicity loci on tcp-conjugative plasmids. The C. perfringens JFP55 chromosome carries 2,825 protein-coding genes whereas the chromosome of JFP838 contains 3,014 protein-encoding genes. Comparison of these two chromosomes with three available reference C. perfringens chromosome sequences identified 48 (~247 kb) and 81 (~430 kb) regions unique to JFP55 and JFP838, respectively. Some of these divergent genomic regions in both chromosomes are phage- and plasmid-related segments. Sixteen of these unique chromosomal regions (~69 kb) were shared between the two isolates. Five of these shared regions formed a mosaic of plasmid-integrated segments, suggesting that these elements were acquired early in a clonal lineage of netF-positive C. perfringens strains. These results provide significant insight into the basis of canine and foal necrotizing enteritis and are the first to demonstrate that netF resides on a large and unique plasmid-encoded locus. PMID:26859667

Increased Abundance of IncP-1β Plasmids and Mercury Resistance Genes in Mercury-Polluted River Sediments: First Discovery of IncP-1β Plasmids with a Complex mer Transposon as the Sole Accessory Element▿

PubMed Central

Smalla, Kornelia; Haines, Anthony S.; Jones, Karen; Krögerrecklenfort, Ellen; Heuer, Holger; Schloter, Michael; Thomas, Christopher M.

2006-01-01

Although it is generally assumed that mobile genetic elements facilitate the adaptation of microbial communities to environmental stresses, environmental data supporting this assumption are rare. In this study, river sediment samples taken from two mercury-polluted (A and B) and two nonpolluted or less-polluted (C and D) areas of the river Nura (Kazakhstan) were analyzed by PCR for the presence and abundance of mercury resistance genes and of broad-host-range plasmids. PCR-based detection revealed that mercury pollution corresponded to an increased abundance of mercury resistance genes and of IncP-1β replicon-specific sequences detected in total community DNA. The isolation of IncP-1β plasmids from contaminated sediments was attempted in order to determine whether they carry mercury resistance genes and thus contribute to an adaptation of bacterial populations to Hg pollution. We failed to detect IncP-1β plasmids in the genomic DNA of the cultured Hg-resistant bacterial isolates. However, without selection for mercury resistance, three different IncP-1β plasmids (pTP6, pTP7, and pTP8) were captured directly from contaminated sediment slurry in Cupriavidus necator JMP228 based on their ability to mobilize the IncQ plasmid pIE723. These plasmids hybridized with the merRTΔP probe and conferred Hg resistance to their host. A broad host range and high stability under conditions of nonselective growth were observed for pTP6 and pTP7. The full sequence of plasmid pTP6 was determined and revealed a backbone almost identical to that of the IncP-1β plasmids R751 and pB8. However, this is the first example of an IncP-1β plasmid which had acquired only a mercury resistance transposon but no antibiotic resistance or biodegradation genes. This transposon carries a rather complex set of mer genes and is inserted between Tra1 and Tra2. PMID:16980416

Multiple Pathways of Plasmid DNA Transfer in Helicobacter pylori

PubMed Central

Rohrer, Stefanie; Holsten, Lea; Weiss, Evelyn; Benghezal, Mohammed; Fischer, Wolfgang; Haas, Rainer

2012-01-01

Many Helicobacter pylori (Hp) strains carry cryptic plasmids of different size and gene content, the function of which is not well understood. A subgroup of these plasmids (e.g. pHel4, pHel12), contain a mobilisation region, but no cognate type IV secretion system (T4SS) for conjugative transfer. Instead, certain H. pylori strains (e.g. strain P12 carrying plasmid pHel12) can harbour up to four T4SSs in their genome (cag-T4SS, comB, tfs3, tfs4). Here, we show that such indigenous plasmids can be efficiently transferred between H. pylori strains, even in the presence of extracellular DNaseI eliminating natural transformation. Knockout of a plasmid-encoded mobA relaxase gene significantly reduced plasmid DNA transfer in the presence of DNaseI, suggesting a DNA conjugation or mobilisation process. To identify the T4SS involved in this conjugative DNA transfer, each individual T4SS was consecutively deleted from the bacterial chromosome. Using a marker-free counterselectable gene deletion procedure (rpsL counterselection method), a P12 mutant strain was finally obtained with no single T4SS (P12ΔT4SS). Mating experiments using these mutants identified the comB T4SS in the recipient strain as the major mediator of plasmid DNA transfer between H. pylori strains, both in a DNaseI-sensitive (natural transformation) as well as a DNaseI-resistant manner (conjugative transfer). However, transfer of a pHel12::cat plasmid from a P12ΔT4SS donor strain into a P12ΔT4SS recipient strain provided evidence for the existence of a third, T4SS-independent mechanism of DNA transfer. This novel type of plasmid DNA transfer, designated as alternate DNaseI-Resistant (ADR) mechanism, is observed at a rather low frequency under in vitro conditions. Taken together, our study describes for the first time the existence of three distinct pathways of plasmid DNA transfer between H. pylori underscoring the importance of horizontal gene transfer for this species. PMID:23029142

Multiple pathways of plasmid DNA transfer in Helicobacter pylori.

PubMed

Rohrer, Stefanie; Holsten, Lea; Weiss, Evelyn; Benghezal, Mohammed; Fischer, Wolfgang; Haas, Rainer

2012-01-01

Many Helicobacter pylori (Hp) strains carry cryptic plasmids of different size and gene content, the function of which is not well understood. A subgroup of these plasmids (e.g. pHel4, pHel12), contain a mobilisation region, but no cognate type IV secretion system (T4SS) for conjugative transfer. Instead, certain H. pylori strains (e.g. strain P12 carrying plasmid pHel12) can harbour up to four T4SSs in their genome (cag-T4SS, comB, tfs3, tfs4). Here, we show that such indigenous plasmids can be efficiently transferred between H. pylori strains, even in the presence of extracellular DNaseI eliminating natural transformation. Knockout of a plasmid-encoded mobA relaxase gene significantly reduced plasmid DNA transfer in the presence of DNaseI, suggesting a DNA conjugation or mobilisation process. To identify the T4SS involved in this conjugative DNA transfer, each individual T4SS was consecutively deleted from the bacterial chromosome. Using a marker-free counterselectable gene deletion procedure (rpsL counterselection method), a P12 mutant strain was finally obtained with no single T4SS (P12ΔT4SS). Mating experiments using these mutants identified the comB T4SS in the recipient strain as the major mediator of plasmid DNA transfer between H. pylori strains, both in a DNaseI-sensitive (natural transformation) as well as a DNaseI-resistant manner (conjugative transfer). However, transfer of a pHel12::cat plasmid from a P12ΔT4SS donor strain into a P12ΔT4SS recipient strain provided evidence for the existence of a third, T4SS-independent mechanism of DNA transfer. This novel type of plasmid DNA transfer, designated as alternate DNaseI-Resistant (ADR) mechanism, is observed at a rather low frequency under in vitro conditions. Taken together, our study describes for the first time the existence of three distinct pathways of plasmid DNA transfer between H. pylori underscoring the importance of horizontal gene transfer for this species.

The plasmid of Escherichia coli strain S88 (O45:K1:H7) that causes neonatal meningitis is closely related to avian pathogenic E. coli plasmids and is associated with high-level bacteremia in a neonatal rat meningitis model.

PubMed

Peigne, Chantal; Bidet, Philippe; Mahjoub-Messai, Farah; Plainvert, Céline; Barbe, Valérie; Médigue, Claudine; Frapy, Eric; Nassif, Xavier; Denamur, Erick; Bingen, Edouard; Bonacorsi, Stéphane

2009-06-01

A new Escherichia coli virulent clonal group, O45:K1, belonging to the highly virulent subgroup B2(1) was recently identified in France, where it accounts for one-third of E. coli neonatal meningitis cases. Here we describe the sequence, epidemiology and function of the large plasmid harbored by strain S88, which is representative of the O45:K1 clonal group. Plasmid pS88 is 133,853 bp long and contains 144 protein-coding genes. It harbors three different iron uptake systems (aerobactin, salmochelin, and the sitABCD genes) and other putative virulence genes (iss, etsABC, ompT(P), and hlyF). The pS88 sequence is composed of several gene blocks homologous to avian pathogenic E. coli plasmids pAPEC-O2-ColV and pAPEC-O1-ColBM. PCR amplification of 11 open reading frames scattered throughout the plasmid was used to investigate the distribution of pS88 and showed that a pS88-like plasmid is present in other meningitis clonal groups such as O18:K1, O1:K1, and O83:K1. A pS88-like plasmid was also found in avian pathogenic strains and human urosepsis strains belonging to subgroup B2(1). A variant of S88 cured of its plasmid displayed a marked loss of virulence relative to the wild-type strain in a neonatal rat model, with bacteremia more than 2 log CFU/ml lower. The salmochelin siderophore, a known meningovirulence factor, could not alone explain the plasmid's contribution to virulence, as a salmochelin mutant displayed only a minor fall in bacteremia (0.9 log CFU/ml). Thus, pS88 is a major virulence determinant related to avian pathogenic plasmids that has spread not only through meningitis clonal groups but also human urosepsis and avian pathogenic strains.

Bacterial mutation affecting plasmid maintenance in Pseudomonas aeruginosa.

PubMed Central

Chang, B J; Holloway, B W

1977-01-01

A bacterial mutation, risA, in Pseudomonas aeruginosa caused growth inhibition at 43 degrees C of risA strains containing P2 plasmids. Incubation at 43 degrees C resulted in selection for clones that had lost P2 plasmids. PMID:122513

The Ferredoxin-Like Proteins HydN and YsaA Enhance Redox Dye-Linked Activity of the Formate Dehydrogenase H Component of the Formate Hydrogenlyase Complex.

PubMed

Pinske, Constanze

2018-01-01

Formate dehydrogenase H (FDH-H) and [NiFe]-hydrogenase 3 (Hyd-3) form the catalytic components of the hydrogen-producing formate hydrogenlyase (FHL) complex, which disproportionates formate to H 2 and CO 2 during mixed acid fermentation in enterobacteria. FHL comprises minimally seven proteins and little is understood about how this complex is assembled. Early studies identified a ferredoxin-like protein, HydN, as being involved in FDH-H assembly into the FHL complex. In order to understand how FDH-H and its small subunit HycB, which is also a ferredoxin-like protein, attach to the FHL complex, the possible roles of HydN and its paralogue, YsaA, in FHL complex stability and assembly were investigated. Deletion of the hycB gene reduced redox dye-mediated FDH-H activity to approximately 10%, abolished FHL-dependent H 2 -production, and reduced Hyd-3 activity. These data are consistent with HycB being an essential electron transfer component of the FHL complex. The FDH-H activity of the hydN and the ysaA deletion strains was reduced to 59 and 57% of the parental, while the double deletion reduced activity of FDH-H to 28% and the triple deletion with hycB to 1%. Remarkably, and in contrast to the hycB deletion, the absence of HydN and YsaA was without significant effect on FHL-dependent H 2 -production or total Hyd-3 activity; FDH-H protein levels were also unaltered. This is the first description of a phenotype for the E. coli ysaA deletion strain and identifies it as a novel factor required for optimal redox dye-linked FDH-H activity. A ysaA deletion strain could be complemented for FDH-H activity by hydN and ysaA , but the hydN deletion strain could not be complemented. Introduction of these plasmids did not affect H 2 production. Bacterial two-hybrid interactions showed that YsaA, HydN, and HycB interact with each other and with the FDH-H protein. Further novel anaerobic cross-interactions of 10 ferredoxin-like proteins in E. coli were also discovered and described. Together, these data indicate that FDH-H activity measured with the redox dye benzyl viologen is the sum of the FDH-H protein interacting with three independent small subunits and suggest that FDH-H can associate with different redox-protein complexes in the anaerobic cell to supply electrons from formate oxidation.

Characterization of pLAC1, a cryptic plasmid isolated from Lactobacillus acidipiscis and comparative analysis with its related plasmids.

PubMed

Asteri, Ioanna-Areti; Papadimitriou, Konstantinos; Boutou, Effrossyni; Anastasiou, Rania; Pot, Bruno; Vorgias, Constantinos E; Tsakalidou, Effie

2010-07-15

The pLAC1 plasmid of Lactobacillus acidipiscis ACA-DC 1533, a strain isolated from traditional Kopanisti cheese, was characterised. Nucleotide sequence analysis revealed a circular molecule of 3478bp with a G+C content of 37.2%. Ab initio annotation indicated four putative open reading frames (orfs). orf1 and orf4 were found to encode a replication initiation protein (Rep) and a mobilization protein (Mob), respectively. The deduced products of orf2 and orf3 revealed no significant homology to other known proteins. However, in silico examination of the plasmid sequence supported the existence of a novel operon that includes rep, orf2 and orf3 in pLAC1 and that this operon is highly conserved also in plasmids pLB925A02, pSMA23, pLC88 and pC7. RT-PCR experiments allowed us to verify that these three genes are co-transcribed as a single polycistronic mRNA species. Furthermore, phylogenetic analysis of pLAC1 Rep and Mob proteins demonstrated that they may have derived from different plasmid origins, suggesting that pLAC1 is a product of a modular evolution process. Comparative analysis of full length nucleotide sequences of pLAC1 and related Lactobacillus plasmids showed that pLAC1 shares a very similar replication backbone with pLB925A02, pSMA23 and pLC88. In contrast, mob of pLAC1 was almost identical with the respective gene of plasmids pLAB1000, pLB4 and pPB1. These findings lead to the conclusion that pLAC1 acquired mob probably via an ancestral recombination event. Our overall work highlights the importance of characterizing plasmids deriving from non-starter 'wild' isolates in order to better appreciate plasmid divergence and evolution of lactic acid bacteria. 2010 Elsevier B.V. All rights reserved.