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.

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.

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.

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

A lanthipeptide library used to identify a protein-protein interaction inhibitor.

PubMed

Yang, Xiao; Lennard, Katherine R; He, Chang; Walker, Mark C; Ball, Andrew T; Doigneaux, Cyrielle; Tavassoli, Ali; van der Donk, Wilfred A

2018-04-01

In this article we describe the production and screening of a genetically encoded library of 10 6 lanthipeptides in Escherichia coli using the substrate-tolerant lanthipeptide synthetase ProcM. This plasmid-encoded library was combined with a bacterial reverse two-hybrid system for the interaction of the HIV p6 protein with the UEV domain of the human TSG101 protein, which is a critical protein-protein interaction for HIV budding from infected cells. Using this approach, we identified an inhibitor of this interaction from the lanthipeptide library, whose activity was verified in vitro and in cell-based virus-like particle-budding assays. Given the variety of lanthipeptide backbone scaffolds that may be produced with ProcM, this method may be used for the generation of genetically encoded libraries of natural product-like lanthipeptides containing substantial structural diversity. Such libraries may be combined with any cell-based assay to identify lanthipeptides with new biological activities.

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

Enhanced gene disruption by programmable nucleases delivered by a minicircle vector.

PubMed

Dad, A-B K; Ramakrishna, S; Song, M; Kim, H

2014-11-01

Targeted genetic modification using programmable nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) is of great value in biomedical research, medicine and biotechnology. Minicircle vectors, which lack extraneous bacterial sequences, have several advantages over conventional plasmids for transgene delivery. Here, for the first time, we delivered programmable nucleases into human cells using transient transfection of a minicircle vector and compared the results with those obtained using a conventional plasmid. Surrogate reporter assays and T7 endonuclease analyses revealed that cells in the minicircle vector group displayed significantly higher mutation frequencies at the target sites than those in the conventional plasmid group. Quantitative PCR and reverse transcription-PCR showed higher vector copy number and programmable nuclease transcript levels, respectively, in 293T cells after minicircle versus conventional plasmid vector transfection. In addition, tryphan blue staining and flow cytometry after annexin V and propidium iodide staining showed that cell viability was also significantly higher in the minicircle group than in the conventional plasmid group. Taken together, our results show that gene disruption using minicircle vector-mediated delivery of ZFNs and TALENs is a more efficient, safer and less toxic method than using a conventional plasmid, and indicate that the minicircle vector could serve as an advanced delivery method for programmable nucleases.

Current strategies for mobilome research.

PubMed

Jørgensen, Tue S; Kiil, Anne S; Hansen, Martin A; Sørensen, Søren J; Hansen, Lars H

2014-01-01

Mobile genetic elements (MGEs) are pivotal for bacterial evolution and adaptation, allowing shuffling of genes even between distantly related bacterial species. The study of these elements is biologically interesting as the mode of genetic propagation is kaleidoscopic and important, as MGEs are the main vehicles of the increasing bacterial antibiotic resistance that causes thousands of human deaths each year. The study of MGEs has previously focused on plasmids from individual isolates, but the revolution in sequencing technology has allowed the study of mobile genomic elements of entire communities using metagenomic approaches. The problem in using metagenomic sequencing for the study of MGEs is that plasmids and other mobile elements only comprise a small fraction of the total genetic content that are difficult to separate from chromosomal DNA based on sequence alone. The distinction between plasmid and chromosome is important as the mobility and regulation of genes largely depend on their genetic context. Several different approaches have been proposed that specifically enrich plasmid DNA from community samples. Here, we review recent approaches used to study entire plasmid pools from complex environments, and point out possible future developments for and pitfalls of these approaches. Further, we discuss the use of the PacBio long-read sequencing technology for MGE discovery.

The mouse Pol I terminator is more efficient than the hepatitis delta virus ribozyme in generating influenza-virus-like RNAs with precise 3' ends in a plasmid-only-based virus rescue system.

PubMed

Feng, Liqiang; Li, Feng; Zheng, Xuehua; Pan, Weiqi; Zhou, Kai; Liu, Yichu; He, Hongxuan; Chen, Ling

2009-01-01

Reverse genetics systems for generating recombinant influenza viruses are based on two different mechanisms for obtaining the 3' end of the viral RNA: one uses the self-cleaving hepatitis delta virus ribozyme (HDVR), and the other uses the murine RNA polymerase I (Pol I) terminator. In this study, we employed EGFP and Renilla luciferase reporter constructs to compare the efficiency of both methods. Our results indicate that the murine Pol I terminator was more efficient than the HDVR, which will be helpful in choosing an influenza virus rescue system, as well as in establishing other RNA virus rescue systems.

Development of a Reverse Genetic System for Infectious Salmon Anemia Virus: Rescue of Recombinant Fluorescent Virus by Using Salmon Internal Transcribed Spacer Region 1 as a Novel Promoter

PubMed Central

Toro-Ascuy, Daniela; Tambley, Carolina; Beltran, Carolina; Mascayano, Carolina; Sandoval, Nicolas; Olivares, Eduardo; Medina, Rafael A.; Spencer, Eugenio

2014-01-01

Infectious salmon anemia (ISA) is a serious disease of marine-farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV), belonging to the genus Isavirus, family Orthomyxoviridae. There is an urgent need to understand the virulence factors and pathogenic mechanisms of ISAV and to develop new vaccine approaches. Using a recombinant molecular biology approach, we report the development of a plasmid-based reverse genetic system for ISAV, which includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). Salmon cells cotransfected with pSS-URG-based vectors expressing the eight viral RNA segments and four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex allowed the generation of infectious recombinant ISAV (rISAV). We generated three recombinant viruses, wild-type rISAV901_09 and rISAVrS6-NotI-HPR containing a NotI restriction site and rISAVS6/EGFP-HPR harboring the open reading frame of enhanced green fluorescent protein (EGFP), both within the highly polymorphic region (HPR) of segment 6. All rescued viruses showed replication activity and cytopathic effect in Atlantic salmon kidney-infected cells. The fluorescent recombinant viruses also showed a characteristic cytopathic effect in salmon cells, and the viruses replicated to a titer of 6.5 × 105 PFU/ml, similar to that of the wild-type virus. This novel reverse genetics system offers a powerful tool to study the molecular biology of ISAV and to develop a new generation of ISAV vaccines to prevent and mitigate ISAV infection, which has had a profound effect on the salmon industry. PMID:25480750

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.

A Modified Shuttle Plasmid Facilitates Expression of a Flavin Mononucleotide-Based Fluorescent Protein in Treponema denticola ATCC 35405

PubMed Central

Godovikova, Valentina; Goetting-Minesky, M. Paula; Shin, Jae M.; Kapila, Yvonne L.; Rickard, Alexander H.

2015-01-01

Oral pathogens, including Treponema denticola, initiate the dysregulation of tissue homeostasis that characterizes periodontitis. However, progress of research on the roles of T. denticola in microbe-host interactions and signaling, microbial communities, microbial physiology, and molecular evolution has been hampered by limitations in genetic methodologies. This is typified by an extremely low transformation efficiency and inability to transform the most widely studied T. denticola strain with shuttle plasmids. Previous studies have suggested that robust restriction-modification (R-M) systems in T. denticola contributed to these problems. To facilitate further molecular genetic analysis of T. denticola behavior, we optimized existing protocols such that shuttle plasmid transformation efficiency was increased by >100-fold over prior reports. Here, we report routine transformation of T. denticola ATCC 35405 with shuttle plasmids, independently of both plasmid methylation status and activity of the type II restriction endonuclease encoded by TDE0911. To validate the utility of this methodological advance, we demonstrated expression and activity in T. denticola of a flavin mononucleotide-based fluorescent protein (FbFP) that is active under anoxic conditions. Addition of routine plasmid-based fluorescence labeling to the Treponema toolset will enable more-rigorous and -detailed studies of the behavior of this organism. PMID:26162875

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.

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.

Development of Genetically Stable Escherichia coli Strains for Poly(3-Hydroxypropionate) Production

PubMed Central

Gao, Yongqiang; Liu, Changshui; Ding, Yamei; Sun, Chao; Zhang, Rubing; Xian, Mo; Zhao, Guang

2014-01-01

Poly(3-hydroxypropionate) (P3HP) is a biodegradable and biocompatible thermoplastic. In our previous study, a pathway for P3HP production was constructed in recombinant Esecherichia coli. Seven exogenous genes in P3HP synthesis pathway were carried by two plasmid vectors. However, the P3HP production was severely suppressed by strain instability due to plasmid loss. In this paper, two strategies, chromosomal gene integration and plasmid addiction system (PAS) based on amino acid anabolism, were applied to construct a genetically stable strain. Finally, a combination of those two methods resulted in the best results. The resultant strain carried a portion of P3HP synthesis genes on chromosome and the others on plasmid, and also brought a tyrosine-auxotrophy based PAS. In aerobic fed-batch fermentation, this strain produced 25.7 g/L P3HP from glycerol, about 2.5-time higher than the previous strain with two plasmids. To the best of our knowledge, this is the highest P3HP production from inexpensive carbon sources. PMID:24837211

Current strategies for mobilome research

PubMed Central

Jørgensen, Tue S.; Kiil, Anne S.; Hansen, Martin A.; Sørensen, Søren J.; Hansen, Lars H.

2015-01-01

Mobile genetic elements (MGEs) are pivotal for bacterial evolution and adaptation, allowing shuffling of genes even between distantly related bacterial species. The study of these elements is biologically interesting as the mode of genetic propagation is kaleidoscopic and important, as MGEs are the main vehicles of the increasing bacterial antibiotic resistance that causes thousands of human deaths each year. The study of MGEs has previously focused on plasmids from individual isolates, but the revolution in sequencing technology has allowed the study of mobile genomic elements of entire communities using metagenomic approaches. The problem in using metagenomic sequencing for the study of MGEs is that plasmids and other mobile elements only comprise a small fraction of the total genetic content that are difficult to separate from chromosomal DNA based on sequence alone. The distinction between plasmid and chromosome is important as the mobility and regulation of genes largely depend on their genetic context. Several different approaches have been proposed that specifically enrich plasmid DNA from community samples. Here, we review recent approaches used to study entire plasmid pools from complex environments, and point out possible future developments for and pitfalls of these approaches. Further, we discuss the use of the PacBio long-read sequencing technology for MGE discovery. PMID:25657641

The reversed terminator of octopine synthase gene on the Agrobacterium Ti plasmid has a weak promoter activity in prokaryotes.

PubMed

Shao, Jun-Li; Long, Yue-Sheng; Chen, Gu; Xie, Jun; Xu, Zeng-Fu

2010-06-01

Agrobacterium tumefaciens transfers DNA from its Ti plasmid to plant host cells. The genes located within the transferred DNA of Ti plasmid including the octopine synthase gene (OCS) are expressed in plant host cells. The 3'-flanking region of OCS gene, known as OCS terminator, is widely used as a transcriptional terminator of the transgenes in plant expression vectors. In this study, we found the reversed OCS terminator (3'-OCS-r) could drive expression of hygromycin phosphotransferase II gene (hpt II) and beta-glucuronidase gene in Escherichia coli, and expression of hpt II in A. tumefaciens. Furthermore, reverse transcription-polymerase chain reaction analysis revealed that an open reading frame (ORF12) that is located downstream to the 3'-OCS-r was transcribed in A. tumefaciens, which overlaps in reverse with the coding region of the OCS gene in octopine Ti plasmid.

DNA-launched live-attenuated vaccines for biodefense applications

PubMed Central

Pushko, Peter; Lukashevich, Igor S.; Weaver, Scott C.; Tretyakova, Irina

2016-01-01

Summary A novel vaccine platform uses DNA immunization to launch live-attenuated virus vaccines in vivo. This technology has been applied for vaccine development against positive-strand RNA viruses with global public health impact including alphaviruses and flaviviruses. The DNA-launched vaccine represents the recombinant plasmid that encodes the full-length genomic RNA of live-attenuated virus downstream from a eukaryotic promoter. When administered in vivo, the genomic RNA of live-attenuated virus is transcribed. The RNA initiates limited replication of a genetically defined, live-attenuated vaccine virus in the tissues of the vaccine recipient, thereby inducing a protective immune response. This platform combines the strengths of reverse genetics, DNA immunization and the advantages of live-attenuated vaccines, resulting in a reduced chance of genetic reversions, increased safety, and improved immunization. With this vaccine technology, the field of DNA vaccines is expanded from those that express subunit antigens to include a novel type of DNA vaccines that launch live-attenuated viruses. PMID:27055100

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.

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

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

Genomic and Biological Characterization of Aggressive and Docile Strains of LCMV Rescued from a Plasmid-Based Reverse Genetics System

PubMed Central

Chen, Minjie; Lan, Shuiyun; Ou, Rong; Price, Graeme E.; Jiang, Hong; de la Torre, Juan Carlos; Moskophidis, Demetrius

2008-01-01

Arenaviruses include several causative agents of hemorrhagic fever disease in humans. In addition, the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a superb model for the study of virus-host interactions, including the basis of viral persistence and associated diseases. The molecular mechanisms concerning the regulation and specific role of viral proteins in modulating arenavirus-host cell interactions associated either with an acute or persistent infection and associated disease remain little understood. Here we report the genomic and biological characterization of LCMV strains Docile (persistent) and Aggressive (not persistent) recovered from cloned cDNA via reverse genetics. Our results confirmed that the cloned viruses accurately recreated the in vivo phenotypes associated with the corresponding natural Docile and Aggressive viral isolates. In addition, we provide evidence that the ability of the Docile strain to persist is determined by the nature of both S and L RNA segments. Thus, our findings provide the foundation for studies aimed at gaining a detailed understanding of viral determinants of LCMV persistence in its natural host that may aid in the development of vaccines to prevent or treat the diseases caused by arenaviruses in humans. PMID:18474558

pTcGW plasmid vectors 1.1 version: a versatile tool for Trypanosoma cruzi gene characterisation

PubMed Central

Kugeratski, Fernanda G; Batista, Michel; Inoue, Alexandre Haruo; Ramos, Bruno Dias; Krieger, Marco Aurelio; Marchini/, Fabricio K

2015-01-01

The functional characterisation of thousands of Trypanosoma cruzi genes remains a challenge. Reverse genetics approaches compatible with high-throughput cloning strategies can provide the tool needed to tackle this challenge. We previously published the pTcGW platform, composed by plasmid vectors carrying different options of N-terminal fusion tags based on Gateway® technology. Here, we present an improved 1.1 version of pTcGW vectors, which is characterised by a fully flexible structure allowing an easy customisation of each element of the vectors in a single cloning step. Additionally, both N and C-terminal fusions are available with new tag options for protein complexes purification. Three of the newly created vectors were successfully used to determine the cellular localisation of four T. cruzi proteins. The 1.1 version of pTcGW platform can be used in a variety of assays, such as protein overexpression, identification of protein-protein interaction and protein localisation. This powerful and versatile tool allows adding valuable functional information to T. cruzi genes and is freely available for scientific community. PMID:26200713

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.

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.

Genetic manipulation of clostridium acetobutylicum for enhanced butanol production

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

Blaschek, H.P.; Holt, S.

Recent developments in the genetic manipulation of the acetone-butanol-ethanol fermentation microorganism, Clostridium acetobutylicum will be discussed. This specifically involves the characterization of an M13-like genetic system for C. acetobutylicum based on the pCAK1 phagemid, as well as the development of a plasmid-based vector based on the indigenous pDM11 plasmid recovered from C. acetobutylicum NCIB 6443. In addition, a macrorestriction map of the C. acetobutylicum ATCC 824 genome was constructed by utilizing two-dimensional transverse alternating field electrophoresis combined with reciprocal enzyme digestions and hybridization with previously cloned genes. We also describe the genetic engineering of a C. acetobutylicum strain with amplifiedmore » encloglucanase activity and to development and characterization of C. acetobutylicum hyper-amylolytic mutants with enhanced potential for commercial processes and evaluate their ability to produce butanol under batch and continuous culture conditions.« less

BioShuttle-mediated Plasmid Transfer

PubMed Central

Braun, Klaus; von Brasch, Leonie; Pipkorn, Ruediger; Ehemann, Volker; Jenne, Juergen; Spring, Herbert; Debus, Juergen; Didinger, Bernd; Rittgen, Werner; Waldeck, Waldemar

2007-01-01

An efficient gene transfer into target tissues and cells is needed for safe and effective treatment of genetic diseases like cancer. In this paper, we describe the development of a transport system and show its ability for transporting plasmids. This non-viral peptide-based BioShuttle-mediated transfer system consists of a nuclear localization address sequence realizing the delivery of the plasmid phNIS-IRES-EGFP coding for two independent reporter genes into nuclei of HeLa cells. The quantification of the transfer efficiency was achieved by measurements of the sodium iodide symporter activity. EGFP gene expression was measured with Confocal Laser Scanning Microscopy and quantified with biostatistical methods by analysis of the frequency of the amplitude distribution in the CLSM images. The results demonstrate that the “BioShuttle”-Technology is an appropriate tool for an effective transfer of genetic material carried by a plasmid. PMID:18026568

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.

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.

Remarkable Diversity of Escherichia coli Carrying mcr-1 from Hospital Sewage with the Identification of Two New mcr-1 Variants.

PubMed

Zhao, Feifei; Feng, Yu; Lü, Xiaoju; McNally, Alan; Zong, Zhiyong

2017-01-01

The plasmid-borne colistin-resistant gene mcr-1 has rapidly become a worldwide public health concern. This study aims to determine the host bacterial strains, plasmids, and genetic contexts of mcr-1 in hospital sewage. A 1-ml hospital sewage sample was cultured. Colistin-resistant bacterial colonies were selected on agar plates and were subjected to whole genome sequencing and subsequent analysis. The transfer of mcr-1 between bacterial strains was tested using conjugation. New variants of mcr-1 were cloned to test the impact of variations on the function of mcr-1 . Plasmids carrying mcr-1 were retrieved from GenBank for comparison based on concatenated backbone genes. In the sewage sample, we observed that mcr-1 was located in various genetic contexts on the chromosome, or plasmids of four different replicon types (IncHI2, IncI2, IncP, and IncX4), in Klebsiella pneumoniae, Kluyvera spp. and seven Escherichia coli strains of six different sequence types (ST10, ST34, ST48, ST1196, ST7086, and ST7087). We also identified two new variants of mcr-1, mcr-1.4 and mcr-1.7 , both of which encode an amino acid variation from mcr-1 . mcr-1 -carrying IncX4 plasmids, which have a global distribution across the Enterobacteriaceae , are the result of global dissemination of a single common plasmid, while IncI2 mcr-1 plasmids appear to acquire mcr-1 in multiple events. In conclusion, the unprecedented remarkable diversity of species, strains, plasmids, and genetic contexts carrying mcr-1 present in a single sewage sample from a single healthcare site highlights the continued evolution and dynamic transmission of mcr-1 in healthcare-associated environments.

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.

Versatile plasmid-based expression systems for Gram-negative bacteria--General essentials exemplified with the bacterium Ralstonia eutropha H16.

PubMed

Gruber, Steffen; Schwab, Helmut; Koefinger, Petra

2015-12-25

The Gram-negative bacterium Escherichia coli is currently the most efficient and widely used prokaryotic host for recombinant protein and metabolite production. However, due to some limitations and to various interesting features of other Gram-negative bacteria efficient vector systems applicable to a broad range are desired. Basic building blocks for plasmid-based vectors include besides the need for a suitable selection marker in the first line a proper replication and maintenance system. In addition to these basic requirements, further elements are needed for Gram-negative bacteria beyond E. coli, such as Pseudomonas pudita, Ralstonia eutropha, Burkholderia glumae or Acinetobacter sp.. Established building blocks have to be adapted and new building blocks providing the desired functions need to be identified and exploited. This minireview addresses so far described and used genetic elements for broad host range replication, efficient plasmid maintenance, and conjugative plasmid transfer as well as expression elements and protein secretion signals. The industrially important bacterium R. eutropha H16 was chosen as a model organism to provide specific data on the effectivity and utility of building blocks based on such genetic elements. Copyright © 2015 Elsevier B.V. All rights reserved.

☆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.

Arenavirus reverse genetics for vaccine development

PubMed Central

Ortiz-Riaño, Emilio; Cheng, Benson Yee Hin; Carlos de la Torre, Juan

2013-01-01

Arenaviruses are important human pathogens with no Food and Drug Administration (FDA)-licensed vaccines available and current antiviral therapy being limited to an off-label use of the nucleoside analogue ribavirin of limited prophylactic efficacy. The development of reverse genetics systems represented a major breakthrough in arenavirus research. However, rescue of recombinant arenaviruses using current reverse genetics systems has been restricted to rodent cells. In this study, we describe the rescue of recombinant arenaviruses from human 293T cells and Vero cells, an FDA-approved line for vaccine development. We also describe the generation of novel vectors that mediate synthesis of both negative-sense genome RNA and positive-sense mRNA species of lymphocytic choriomeningitis virus (LCMV) directed by the human RNA polymerases I and II, respectively, within the same plasmid. This approach reduces by half the number of vectors required for arenavirus rescue, which could facilitate virus rescue in cell lines approved for human vaccine production but that cannot be transfected at high efficiencies. We have shown the feasibility of this approach by rescuing both the Old World prototypic arenavirus LCMV and the live-attenuated vaccine Candid#1 strain of the New World arenavirus Junín. Moreover, we show the feasibility of using these novel strategies for efficient rescue of recombinant tri-segmented both LCMV and Candid#1. PMID:23364194

Reversible entrapment of plasmid deoxyribonucleic acid on different chromatographic supports.

PubMed

Gabor, Boštjan; Černigoj, Urh; Barut, Miloš; Štrancar, Aleš

2013-10-11

HPLC based analytical assay is a powerful technique that can be used to efficiently monitor plasmid DNA (pDNA) purity and quantity throughout the entire purification process. Anion exchange monolithic and non-porous particle based stationary phases were used to study the recovery of the different pDNA isoforms from the analytical column. Three differently sized pDNA molecules of 3.0kbp, 5.2kbp and 14.0kbp were used. Plasmid DNA was injected onto columns under the binding conditions and the separation of the isoforms took place by increasing the ionic strength of the elution buffer. While there was no substantial decrease of the recovered supercoiled and linear isoforms of the pDNA with the increase of the plasmid size and with the increase of the flow rate (recoveries in all cases larger than 75%), a pronounced decrease of the oc isoform recovery was observed. The entrapment of the oc pDNA isoform occurred under non-binding conditions as well. The partial oc isoform elution from the column could be achieved by decreasing the flow rate of the elution mobile phase. The results suggested a reversible entrapment of the oc isoform in the restrictions within the pores of the monolithic material as well as within the intra-particle space of the non-porous particles. This phenomenon was observed on both types of the stationary phase morphologies and could only be connected to the size of a void space through which the pDNA needs to migrate. A prediction of reversible pDNA entrapment was successfully estimated with the calculation of Peclet numbers, Pe, which defines the ratio between a convective and diffusive mass transport. Copyright © 2013 Elsevier B.V. All rights reserved.

A Rapid and Improved Method to Generate Recombinant Dengue Virus Vaccine Candidates

PubMed Central

Govindarajan, Dhanasekaran; Guan, Liming; Meschino, Steven; Fridman, Arthur; Bagchi, Ansu; Pak, Irene; ter Meulen, Jan; Casimiro, Danilo R.; Bett, Andrew J.

2016-01-01

Dengue is one of the most important mosquito-borne infections accounting for severe morbidity and mortality worldwide. Recently, the tetravalent chimeric live attenuated Dengue vaccine Dengvaxia® was approved for use in several dengue endemic countries. In general, live attenuated vaccines (LAV) are very efficacious and offer long-lasting immunity against virus-induced disease. Rationally designed LAVs can be generated through reverse genetics technology, a method of generating infectious recombinant viruses from full length cDNA contained in bacterial plasmids. In vitro transcribed (IVT) viral RNA from these infectious clones is transfected into susceptible cells to generate recombinant virus. However, the generation of full-length dengue virus cDNA clones can be difficult due to the genetic instability of viral sequences in bacterial plasmids. To circumvent the need for a single plasmid containing a full length cDNA, in vitro ligation of two or three cDNA fragments contained in separate plasmids can be used to generate a full-length dengue viral cDNA template. However, in vitro ligation of multiple fragments often yields low quality template for IVT reactions, resulting in inconsistent low yield RNA. These technical difficulties make recombinant virus recovery less efficient. In this study, we describe a simple, rapid and efficient method of using LONG-PCR to recover recombinant chimeric Yellow fever dengue (CYD) viruses as potential dengue vaccine candidates. Using this method, we were able to efficiently generate several viable recombinant viruses without introducing any artificial mutations into the viral genomes. We believe that the techniques reported here will enable rapid and efficient recovery of recombinant flaviviruses for evaluation as vaccine candidates and, be applicable to the recovery of other RNA viruses. PMID:27008550

A Rapid and Improved Method to Generate Recombinant Dengue Virus Vaccine Candidates.

PubMed

Govindarajan, Dhanasekaran; Guan, Liming; Meschino, Steven; Fridman, Arthur; Bagchi, Ansu; Pak, Irene; ter Meulen, Jan; Casimiro, Danilo R; Bett, Andrew J

2016-01-01

Dengue is one of the most important mosquito-borne infections accounting for severe morbidity and mortality worldwide. Recently, the tetravalent chimeric live attenuated Dengue vaccine Dengvaxia® was approved for use in several dengue endemic countries. In general, live attenuated vaccines (LAV) are very efficacious and offer long-lasting immunity against virus-induced disease. Rationally designed LAVs can be generated through reverse genetics technology, a method of generating infectious recombinant viruses from full length cDNA contained in bacterial plasmids. In vitro transcribed (IVT) viral RNA from these infectious clones is transfected into susceptible cells to generate recombinant virus. However, the generation of full-length dengue virus cDNA clones can be difficult due to the genetic instability of viral sequences in bacterial plasmids. To circumvent the need for a single plasmid containing a full length cDNA, in vitro ligation of two or three cDNA fragments contained in separate plasmids can be used to generate a full-length dengue viral cDNA template. However, in vitro ligation of multiple fragments often yields low quality template for IVT reactions, resulting in inconsistent low yield RNA. These technical difficulties make recombinant virus recovery less efficient. In this study, we describe a simple, rapid and efficient method of using LONG-PCR to recover recombinant chimeric Yellow fever dengue (CYD) viruses as potential dengue vaccine candidates. Using this method, we were able to efficiently generate several viable recombinant viruses without introducing any artificial mutations into the viral genomes. We believe that the techniques reported here will enable rapid and efficient recovery of recombinant flaviviruses for evaluation as vaccine candidates and, be applicable to the recovery of other RNA viruses.

Adsorption behavior of plasmid DNA onto perfusion chromatographic matrix.

PubMed

Limonta, Miladys; Zumalacárregui, Lourdes; Soler, Dayana

2012-05-01

Anion exchange chromatography is the most popular chromatographic method for plasmid separation. POROS RI 50 is a perfusion chromatographic support which is a reversed phase matrix and is an alternative to conventional ones due to its mass transfer properties. The adsorption and elution of the pIDKE2 plasmid onto reversed phase POROS R1 50 was studied. Langmuir isotherm model was adjusted in order to get the maximum adsorption capacity and the dissociation constant for POROS R1 50-plasmid DNA (pDNA) system. Breakthrough curves were obtained for volumetric flows between 0.69-3.33 mL/min, given dynamic capacity up to 2.3 times higher than those reported for ionic exchange matrix used during the purification process of plasmids with similar size to that of pIDKE2. The efficiency was less than 45% for the flow conditions and initial concentration studied, which means that the support will not be operated under saturation circumstances.

Persistence of Animal and Human Glycopeptide-Resistant Enterococci on Two Norwegian Poultry Farms Formerly Exposed to Avoparcin Is Associated with a Widespread Plasmid-Mediated vanA Element within a Polyclonal Enterococcus faecium Population

PubMed Central

Johnsen, P. J.; Østerhus, J. I.; Sletvold, H.; Sørum, M.; Kruse, H.; Nielsen, K.; Simonsen, G. S.; Sundsfjord, A.

2005-01-01

The evolutionary processes responsible for the long-term persistence of glycopeptide-resistant Enterococcus faecium (GREF) in nonselective environments were addressed by genetic analyses of E. faecium populations in animals and humans on two Norwegian poultry farms that were previously exposed to avoparcin. A total of 222 fecal GREF (n = 136) and glycopeptide-susceptible (n = 86) E. faecium (GSEF) isolates were obtained from farmers and poultry on three separate occasions in 1998 and 1999. Pulsed-field gel electrophoresis (PFGE) and plasmid DNA analyses discerned 22 GREF and 32 GSEF PFGE types within shifting polyclonal animal and human E. faecium populations and indicated the presence of transferable plasmid-mediated vanA resistance, respectively. Examples of dominant, persistent GREF PFGE types supported the notion that environmentally well-adapted GREF types may counteract the reversal of resistance. PFGE analyses, sequencing of the purK housekeeping gene, and partial typing of vanA-containing Tn1546 suggested a common animal and human reservoir of glycopeptide resistance. Inverse PCR amplification and sequence analyses targeting the right end of the Tn1546-plasmid junction fragment strongly indicated the presence of a common single Tn1546-plasmid-mediated element in 20 of 22 GREF PFGE types. This observation was further strengthened by vanY-vanZ hybridization analyses of plasmid DNAs as well as the finding of a physical linkage between Tn1546 and a putative postsegregation killing system for seven GREF PFGE types. In conclusion, our observations suggest that the molecular unit of persistence of glycopeptide resistance is a common mobile plasmid-mediated vanA-containing element within a polyclonal GREF population that changes over time. In addition, we propose that “plasmid addiction systems” may contribute to the persistence of GREF in nonselective environments. PMID:15640183

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.

A Java-based tool for the design of classification microarrays.

PubMed

Meng, Da; Broschat, Shira L; Call, Douglas R

2008-08-04

Classification microarrays are used for purposes such as identifying strains of bacteria and determining genetic relationships to understand the epidemiology of an infectious disease. For these cases, mixed microarrays, which are composed of DNA from more than one organism, are more effective than conventional microarrays composed of DNA from a single organism. Selection of probes is a key factor in designing successful mixed microarrays because redundant sequences are inefficient and limited representation of diversity can restrict application of the microarray. We have developed a Java-based software tool, called PLASMID, for use in selecting the minimum set of probe sequences needed to classify different groups of plasmids or bacteria. The software program was successfully applied to several different sets of data. The utility of PLASMID was illustrated using existing mixed-plasmid microarray data as well as data from a virtual mixed-genome microarray constructed from different strains of Streptococcus. Moreover, use of data from expression microarray experiments demonstrated the generality of PLASMID. In this paper we describe a new software tool for selecting a set of probes for a classification microarray. While the tool was developed for the design of mixed microarrays-and mixed-plasmid microarrays in particular-it can also be used to design expression arrays. The user can choose from several clustering methods (including hierarchical, non-hierarchical, and a model-based genetic algorithm), several probe ranking methods, and several different display methods. A novel approach is used for probe redundancy reduction, and probe selection is accomplished via stepwise discriminant analysis. Data can be entered in different formats (including Excel and comma-delimited text), and dendrogram, heat map, and scatter plot images can be saved in several different formats (including jpeg and tiff). Weights generated using stepwise discriminant analysis can be stored for analysis of subsequent experimental data. Additionally, PLASMID can be used to construct virtual microarrays with genomes from public databases, which can then be used to identify an optimal set of probes.

Preclinical development of BCG.HIVA2auxo.int, harboring an integrative expression vector, for a HIV-TB Pediatric vaccine. Enhancement of stability and specific HIV-1 T-cell immunity.

PubMed

Mahant, Aakash; Saubi, Narcís; Eto, Yoshiki; Guitart, Núria; Gatell, Josep Ma; Hanke, Tomáš; Joseph, Joan

2017-08-03

One of the critical issues that should be addressed in the development of a BCG-based HIV vaccine is genetic plasmid stability. Therefore, to address this issue we have considered using integrative vectors and the auxotrophic mutant of BCG complemented with a plasmid carrying a wild-type complementing gene. In this study, we have constructed an integrative E. coli-mycobacterial shuttle plasmid, p2auxo.HIVA int , expressing the HIV-1 clade A immunogen HIVA. This shuttle vector uses an antibiotic resistance-free mechanism for plasmid selection and maintenance. It was first transformed into a glycine auxotrophic E. coli strain and subsequently transformed into a lysine auxotrophic Mycobacterium bovis BCG strain to generate the vaccine BCG.HIVA 2auxo.int . Presence of the HIVA gene sequence and protein expression was confirmed. We demonstrated that the in vitro stability of the integrative plasmid p2auxo.HIVA int was increased 4-fold, as compared with the BCG strain harboring the episomal plasmid, and was genetically and phenotypically characterized. The BCG.HIVA 2auxo.int vaccine in combination with modified vaccinia virus Ankara (MVA).HIVA was found to be safe and induced HIV-1 and Mycobacterium tuberculosis-specific interferon-γ-producing T-cell responses in adult BALB/c mice. We have engineered a more stable and immunogenic BCG-vectored vaccine using the prototype immunogen HIVA. Thus, the use of integrative expression vectors and the antibiotic-free plasmid selection system based on "double" auxotrophic complementation are likely to improve the mycobacterial vaccine stability in vivo and immunogenicity to develop not only recombinant BCG-based vaccines expressing second generation of HIV-1 immunogens but also other major pediatric pathogens to prime protective responses shortly following birth.

Construction of small plasmid vectors for use in genetic improvement of the extremely acidophilic Acidithiobacillus caldus.

PubMed

Meng, Jianzhou; Wang, Huiyan; Liu, Xiangmei; Lin, Jianqun; Pang, Xin; Lin, Jianqiang

2013-10-01

The genetic improvement of biomining bacteria including Acidithiobacillus caldus could facilitate the bioleaching process of sulfur-containing minerals. However, the available vectors for use in A. caldus are very scanty and limited to relatively large broad-host-range IncQ plasmids. In this study, a set of small, mobilizable plasmid vectors (pBBR1MCS-6, pMSD1 and pMSD2) were constructed based on plasmid pBBR1MCS-2, which does not belong to the IncQ, IncW, or IncP groups. The function of the tac promoter on 5.8-kb pMSD2 was determined by inserting a kanamycin-resistant reporter gene. The resulting recombinant pMSD2-Km was successfully transferred by conjugation into A. caldus MTH-04 with transfer frequency of 1.38±0.64×10(-5). The stability and plasmid copy number of pMSD2-Km in A. caldus MTH-04 were 75±2.7% and 5-6 copies per cell, respectively. By inserting an arsABC operon into pMSD2, an arsenic-resistant recombinant pMSD2-As was constructed and transferred into A. caldus MTH-04 by conjugation. The arsenic tolerance of A. caldus MTH-04 containing pMSD2-As was obviously increased up to 45mM of NaAsO2. These vectors could be applied in genetic improvement of A. caldus as well as other bioleaching bacteria. Copyright © 2013 Elsevier GmbH. All rights reserved.

Transcriptional analysis of the Escherichia coli ColV-Ia plasmid pS88 during growth in human serum and urine.

PubMed

Lemaître, Chloé; Bidet, Philippe; Bingen, Edouard; Bonacorsi, Stéphane

2012-06-21

The sequenced O45:K1:H7 Escherichia coli meningitis strain S88 harbors a large virulence plasmid. To identify possible genetic determinants of pS88 virulence, we examined the transcriptomes of 88 plasmidic ORFs corresponding to known and putative virulence genes, and 35 ORFs of unknown function. Quantification of plasmidic transcripts was obtained by quantitative real-time reverse transcription of extracted RNA, normalized on three housekeeping genes. The transcriptome of E. coli strain S88 grown in human serum and urine ex vivo were compared to that obtained during growth in Luria Bertani broth, with and without iron depletion. We also analyzed the transcriptome of a pS88-like plasmid recovered from a neonate with urinary tract infection. The transcriptome obtained after ex vivo growth in serum and urine was very similar to those obtained in iron-depleted LB broth. Genes encoding iron acquisition systems were strongly upregulated. ShiF and ORF 123, two ORFs encoding protein with hypothetical function and physically linked to aerobactin and salmochelin loci, respectively, were also highly expressed in iron-depleted conditions and may correspond to ancillary iron acquisition genes. Four ORFs were induced ex vivo, independently of the iron concentration. Other putative virulence genes such as iss, etsC, ompTp and hlyF were not upregulated in any of the conditions studied. Transcriptome analysis of the pS88-like plasmid recovered in vivo showed a similar pattern of induction but at much higher levels. We identify new pS88 genes potentially involved in the growth of E. coli meningitis strain S88 in human serum and urine.

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

Universal Influenza B Virus Genomic Amplification Facilitates Sequencing, Diagnostics, and Reverse Genetics

PubMed Central

Zhou, Bin; Lin, Xudong; Wang, Wei; Halpin, Rebecca A.; Bera, Jayati; Stockwell, Timothy B.; Barr, Ian G.

2014-01-01

Although human influenza B virus (IBV) is a significant human pathogen, its great genetic diversity has limited our ability to universally amplify the entire genome for subsequent sequencing or vaccine production. The generation of sequence data via next-generation approaches and the rapid cloning of viral genes are critical for basic research, diagnostics, antiviral drugs, and vaccines to combat IBV. To overcome the difficulty of amplifying the diverse and ever-changing IBV genome, we developed and optimized techniques that amplify the complete segmented negative-sense RNA genome from any IBV strain in a single tube/well (IBV genomic amplification [IBV-GA]). Amplicons for >1,000 diverse IBV genomes from different sample types (e.g., clinical specimens) were generated and sequenced using this robust technology. These approaches are sensitive, robust, and sequence independent (i.e., universally amplify past, present, and future IBVs), which facilitates next-generation sequencing and advanced genomic diagnostics. Importantly, special terminal sequences engineered into the optimized IBV-GA2 products also enable ligation-free cloning to rapidly generate reverse-genetics plasmids, which can be used for the rescue of recombinant viruses and/or the creation of vaccine seed stock. PMID:24501036

Mechanisms of Evolution in High-Consequence Drug Resistance Plasmids.

PubMed

He, Susu; Chandler, Michael; Varani, Alessandro M; Hickman, Alison B; Dekker, John P; Dyda, Fred

2016-12-06

The dissemination of resistance among bacteria has been facilitated by the fact that resistance genes are usually located on a diverse and evolving set of transmissible plasmids. However, the mechanisms generating diversity and enabling adaptation within highly successful resistance plasmids have remained obscure, despite their profound clinical significance. To understand these mechanisms, we have performed a detailed analysis of the mobilome (the entire mobile genetic element content) of a set of previously sequenced carbapenemase-producing Enterobacteriaceae (CPE) from the National Institutes of Health Clinical Center. This analysis revealed that plasmid reorganizations occurring in the natural context of colonization of human hosts were overwhelmingly driven by genetic rearrangements carried out by replicative transposons working in concert with the process of homologous recombination. A more complete understanding of the molecular mechanisms and evolutionary forces driving rearrangements in resistance plasmids may lead to fundamentally new strategies to address the problem of antibiotic resistance. The spread of antibiotic resistance among Gram-negative bacteria is a serious public health threat, as it can critically limit the types of drugs that can be used to treat infected patients. In particular, carbapenem-resistant members of the Enterobacteriaceae family are responsible for a significant and growing burden of morbidity and mortality. Here, we report on the mechanisms underlying the evolution of several plasmids carried by previously sequenced clinical Enterobacteriaceae isolates from the National Institutes of Health Clinical Center (NIH CC). Our ability to track genetic rearrangements that occurred within resistance plasmids was dependent on accurate annotation of the mobile genetic elements within the plasmids, which was greatly aided by access to long-read DNA sequencing data and knowledge of their mechanisms. Mobile genetic elements such as transposons and integrons have been strongly associated with the rapid spread of genes responsible for antibiotic resistance. Understanding the consequences of their actions allowed us to establish unambiguous evolutionary relationships between plasmids in the analysis set. Copyright © 2016 He et al.

Nucleotide sequences, genetic organization, and distribution of pEU30 and pEL60 from Erwinia amylovora.

PubMed

Foster, Gayle C; McGhee, Gayle C; Jones, Alan L; Sundin, George W

2004-12-01

The nucleotide sequences, genetic organization, and distribution of plasmids pEU30 (30,314 bp) and pEL60 (60,145 bp) from the plant pathogen Erwinia amylovora are described. The newly characterized pEU30 and pEL60 plasmids inhabited strains isolated in the western United States and Lebanon, respectively. The gene content of pEU30 resembled plasmids found in plant-associated bacteria, while that of pEL60 was most similar to IncL/M plasmids inhabiting enteric bacteria.

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

Genetic tools for the investigation of Roseobacter clade bacteria

PubMed Central

2009-01-01

Background The Roseobacter clade represents one of the most abundant, metabolically versatile and ecologically important bacterial groups found in marine habitats. A detailed molecular investigation of the regulatory and metabolic networks of these organisms is currently limited for many strains by missing suitable genetic tools. Results Conjugation and electroporation methods for the efficient and stable genetic transformation of selected Roseobacter clade bacteria including Dinoroseobacter shibae, Oceanibulbus indolifex, Phaeobacter gallaeciensis, Phaeobacter inhibens, Roseobacter denitrificans and Roseobacter litoralis were tested. For this purpose an antibiotic resistance screening was performed and suitable genetic markers were selected. Based on these transformation protocols stably maintained plasmids were identified. A plasmid encoded oxygen-independent fluorescent system was established using the flavin mononucleotide-based fluorescent protein FbFP. Finally, a chromosomal gene knockout strategy was successfully employed for the inactivation of the anaerobic metabolism regulatory gene dnr from D. shibae DFL12T. Conclusion A genetic toolbox for members of the Roseobacter clade was established. This provides a solid methodical basis for the detailed elucidation of gene regulatory and metabolic networks underlying the ecological success of this group of marine bacteria. PMID:20021642

Genetic engineering of Lactobacillus diolivorans.

PubMed

Pflügl, Stefan; Marx, Hans; Mattanovich, Diethard; Sauer, Michael

2013-07-01

In this study, we developed a toolbox for genetic manipulation of Lactobacillus diolivorans, a promising production organism for 1,3-propanediol from glycerol. Two major findings play a key role for successful transformation of this organism: (1) the absence of a native plasmid, because a native plasmid is a major obstacle for transformation of L. diolivorans, and (2) the absence of DNA methylation. A suitable expression plasmid, pSHM, for homologous and heterologous protein expression in L. diolivorans was constructed. This plasmid is based on the replication origin repA of L. diolivorans. The native glyceraldehyde-3-phosphate dehydrogenase promoter is used for constitutive expression of the genes of interest. Functional expression of genes in L. diolivorans was shown with two examples: production of green fluorescent protein resulted in a 40- to 60-fold higher fluorescence of the obtained clones compared with the wild-type strain. Finally, the homologous overexpression of a putatively NADPH-dependent 1,3-propanediol oxidoreductase improved 1,3-propanediol production by 20% in batch cultures. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Engineering Foot-and-Mouth Disease Viruses with Improved Growth Properties for Vaccine Development

PubMed Central

Zheng, Haixue; Guo, Jianhong; Jin, Ye; Yang, Fan; He, Jijun; Lv, Lv; Zhang, Kesan; Wu, Qiong; Liu, Xiangtao; Cai, Xuepeng

2013-01-01

Background No licensed vaccine is currently available against serotype A foot-and-mouth disease (FMD) in China, despite the isolation of A/WH/CHA/09 in 2009, partly because this strain does not replicate well in baby hamster kidney (BHK) cells. Methodology/Principal Findings A novel plasmid-based reverse genetics system was used to construct a chimeric strain by replacing the P1 gene in the vaccine strain O/CHA/99 with that from the epidemic stain A/WH/CHA/09. The chimeric virus displayed growth kinetics similar to those of O/CHA/99 and was selected for use as a candidate vaccine strain after 12 passages in BHK cells. Cattle were vaccinated with the inactivated vaccine and humoral immune responses were induced in most of the animals on day 7. A challenge infection with A/WH/CHA/09 on day 28 indicated that the group given a 4-µg dose was fully protected and neither developed viremia nor seroconverted to a 3ABC antigen. Conclusions/Significance Our data demonstrate that the chimeric virus not only propagates well in BHK cells and has excellent antigenic matching against serotype A FMD, but is also a potential marker vaccine to distinguish infection from vaccination. These results suggest that reverse genetics technology is a useful tool for engineering vaccines for the prevention and control of FMD. PMID:23372840

Crosstalk between vertical and horizontal gene transfer: plasmid replication control by a conjugative relaxase

PubMed Central

Lorenzo-Díaz, Fabián; Fernández-López, Cris; Lurz, Rudi

2017-01-01

Abstract Horizontal gene transfer is a key process in the evolution of bacteria and also represents a source of genetic variation in eukaryotes. Among elements participating in gene transfer, thousands of small (<10 kb) mobile bacterial plasmids that replicate by the rolling circle mechanism represent a driving force in the spread of antibiotic resistances. In general, these plasmids are built as genetic modules that encode a replicase, an antibiotic-resistance determinant, and a relaxase that participates in their conjugative mobilization. Further, they control their relatively high copy number (∼30 copies per genome equivalent) by antisense RNAs alone or combined with a repressor protein. We report here that the MobM conjugative relaxase encoded by the promiscuous plasmid pMV158 participates in regulation of the plasmid copy number by transcriptional repression of the antisense RNA, thus increasing the number of plasmid molecules ready to be horizontally transferred (mobilization) and/or vertically inherited (replication). This type of crosstalk between genetic modules involved in vertical and horizontal gene flow has not been reported before. PMID:28525572

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.

Hybridizing bacteria, crossing methods, cross-checking arguments: the transition from episomes to plasmids (1961-1969).

PubMed

Grote, Mathias

2008-01-01

Plasmids are non-chromosomal hereditary determinants, mostly found in prokaryotes. Whereas Joshua Lederberg coined the term "plasmid" as early as 1952, today's concept was not established until the early 1970s. In this eclipse period, the plasmid's place was taken by the episome, following the 1958 publication of Elie Wollman and François Jacob. This paper analyzes the transition from the episome to a renewed plasmid concept both on the experimental and the conceptual level. It will become clear that intergeneric transfer experiments were central to this development. These studies rely on conjugational transfer of extrachromosomal hereditary determinants between different bacterial genera. First, experimental systems employing intergeneric transfer shaped the new plasmid by enabling its representation as a species of circular DNA. Moreover, they had a destabilizing effect on the episome, leading to a crisis in the concepts of microbial genetics towards the end of the 1960s. The new plasmid then became one of the cornerstones of recombinant DNA technologies. In an historic perspective, intergeneric transfer experiments indicate a gradual transition of molecular biology from its early "analytic" to the "synthetic" phase of genetic engineering. Hence, the construction of genetic hybrids in vivo as epitomized in the studies shown here marks an intermediate state that one could designate as "recombinant DNA avant la lettre".

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.

Detection and validation of a small broad-host-range plasmid pBBR1MCS-2 for use in genetic manipulation of the extremely acidophilic Acidithiobacillus sp.

PubMed

Hao, Likai; Liu, Xiangmei; Wang, Huiyan; Lin, Jianqun; Pang, Xin; Lin, Jianqiang

2012-09-01

An efficient genetic system for introducing genes into biomining microorganisms is essential not only to experimentally determine the functions of genes predicted based on bioinformatic analysis, but also for their genetic breeding. In this study, a small broad-host-range vector named pBBR1MCS-2, which does not belong to the IncQ, IncW, or IncP groups, was studied for the feasibility of its use in conjugative gene transfer into extremely acidophilic strains of Acidithiobacillus. To do this, a recombinant plasmid pBBR-tac-Sm, a derivative of pBBR1MCS-2, was constructed and the streptomycin resistant gene (Sm(r)) was used as the reporter gene. Using conjugation, pBBR-tac-Sm was successfully transferred into three tested strains of Acidithiobacillus. Then we measured its transfer frequency, its stability in Acidithiobacillus cells, and the level of resistance to streptomycin of the transconjugants and compared this with the IncQ plasmid pJRD215 control. Our results indicate that pBBR1MCS-2 provides a new and useful tool in the genetic manipulation of Acidithiobacillus strains. Copyright © 2012 Elsevier B.V. All rights reserved.

The Acquisition of Indigenous Plasmids by a Genetically Marked Pseudomonad Population Colonizing the Sugar Beet Phytosphere Is Related to Local Environmental Conditions

PubMed Central

Lilley, A. K.; Bailey, M. J.

1997-01-01

The transfer of naturally occurring conjugative plasmids from the indigenous microflora to a genetically modified population of bacteria colonizing the phytospheres of plants has been observed. The marked strain (Pseudomonas fluorescens SBW25EeZY6KX) was introduced as a seed dressing to sugar beets (Beta vulgaris var. Amethyst) as part of a field experiment to assess the ecology and genetic stability of deliberately released bacterial inocula. The sustained populations of the introduced strain, which colonized the phytosphere, were assessed throughout the growing season for the acquisition of plasmids conferring mercury resistance (Hg(supr)). Transconjugants were isolated only from root and leaf samples collected within a narrow temporal window coincident with the midseason maturation of the crop. Conjugal-transfer events were recorded during this defined period in two separate field release experiments conducted over consecutive years. On one occasion seven of nine individual plants sampled supported transconjugant P. fluorescens SBW25EeZY6KX, demonstrating that conjugative gene transfer between bacterial populations in the phytosphere may be a common event under specific environmental conditions. The plasmids acquired in situ by the colonizing inocula were identified as natural variants of restriction digest pattern group I, III, or IV plasmids from five genetically distinct groups of large, conjugative mercury resistance plasmids known to persist in the phytospheres of sugar beets at the field site. These data demonstrate not only that gene transfer may be a common event but also that the genetic and phenotypic stability of inocula released into the natural environment cannot be predicted. PMID:16535580

Evaluation of plasmid and genomic DNA calibrants used for the quantification of genetically modified organisms.

PubMed

Caprioara-Buda, M; Meyer, W; Jeynov, B; Corbisier, P; Trapmann, S; Emons, H

2012-07-01

The reliable quantification of genetically modified organisms (GMOs) by real-time PCR requires, besides thoroughly validated quantitative detection methods, sustainable calibration systems. The latter establishes the anchor points for the measured value and the measurement unit, respectively. In this paper, the suitability of two types of DNA calibrants, i.e. plasmid DNA and genomic DNA extracted from plant leaves, for the certification of the GMO content in reference materials as copy number ratio between two targeted DNA sequences was investigated. The PCR efficiencies and coefficients of determination of the calibration curves as well as the measured copy number ratios for three powder certified reference materials (CRMs), namely ERM-BF415e (NK603 maize), ERM-BF425c (356043 soya), and ERM-BF427c (98140 maize), originally certified for their mass fraction of GMO, were compared for both types of calibrants. In all three systems investigated, the PCR efficiencies of plasmid DNA were slightly closer to the PCR efficiencies observed for the genomic DNA extracted from seed powders rather than those of the genomic DNA extracted from leaves. Although the mean DNA copy number ratios for each CRM overlapped within their uncertainties, the DNA copy number ratios were significantly different using the two types of calibrants. Based on these observations, both plasmid and leaf genomic DNA calibrants would be technically suitable as anchor points for the calibration of the real-time PCR methods applied in this study. However, the most suitable approach to establish a sustainable traceability chain is to fix a reference system based on plasmid DNA.

“Direct cloning in Lactobacillus plantarum: Electroporation with non-methylated plasmid DNA enhances transformation efficiency and makes shuttle vectors obsolete”

PubMed Central

2012-01-01

Background Lactic acid bacteria (LAB) play an important role in agricultural as well as industrial biotechnology. Development of improved LAB strains using e.g. library approaches is often limited by low transformation efficiencies wherefore one reason could be differences in the DNA methylation patterns between the Escherichia coli intermediate host for plasmid amplification and the final LAB host. In the present study, we examined the influence of DNA methylation on transformation efficiency in LAB and developed a direct cloning approach for Lactobacillus plantarum CD033. Therefore, we propagated plasmid pCD256 in E. coli strains with different dam/dcm-methylation properties. The obtained plasmid DNA was purified and transformed into three different L. plantarum strains and a selection of other LAB species. Results Best transformation efficiencies were obtained using the strain L. plantarum CD033 and non-methylated plasmid DNA. Thereby we achieved transformation efficiencies of ~ 109 colony forming units/μg DNA in L. plantarum CD033 which is in the range of transformation efficiencies reached with E. coli. Based on these results, we directly transformed recombinant expression vectors received from PCR/ligation reactions into L. plantarum CD033, omitting plasmid amplification in E. coli. Also this approach was successful and yielded a sufficient number of recombinant clones. Conclusions Transformation efficiency of L. plantarum CD033 was drastically increased when non-methylated plasmid DNA was used, providing the possibility to generate expression libraries in this organism. A direct cloning approach, whereby ligated PCR-products where successfully transformed directly into L. plantarum CD033, obviates the construction of shuttle vectors containing E. coli-specific sequences, as e.g. a ColEI origin of replication, and makes amplification of these vectors in E. coli obsolete. Thus, plasmid constructs become much smaller and occasional structural instability or mutagenesis during E. coli propagation is excluded. The results of our study provide new genetic tools for L. plantarum which will allow fast, forward and systems based genetic engineering of this species. PMID:23098256

Agrobacterium uses a unique ligand-binding mode for trapping opines and acquiring a competitive advantage in the niche construction on plant host.

PubMed

Lang, Julien; Vigouroux, Armelle; Planamente, Sara; El Sahili, Abbas; Blin, Pauline; Aumont-Nicaise, Magali; Dessaux, Yves; Moréra, Solange; Faure, Denis

2014-10-01

By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour), a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP) NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and α-ketoglurate) and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (K(D) of 0.6 µM) greater than that for nopaline (KD of 3.7 µM). Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline-assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to support the niche construction paradigm in bacterial pathogens.

Agrobacterium Uses a Unique Ligand-Binding Mode for Trapping Opines and Acquiring A Competitive Advantage in the Niche Construction on Plant Host

PubMed Central

Planamente, Sara; El Sahili, Abbas; Blin, Pauline; Aumont-Nicaise, Magali; Dessaux, Yves; Moréra, Solange; Faure, Denis

2014-01-01

By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour), a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP) NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and α-ketoglurate) and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (KD of 0.6 µM) greater than that for nopaline (KD of 3.7 µM). Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline-assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to support the niche construction paradigm in bacterial pathogens. PMID:25299655

Impact of Mutation Type and Amplicon Characteristics on Genetic Diversity Measures Generated Using a High-Resolution Melting Diversity Assay

PubMed Central

Cousins, Matthew M.; Donnell, Deborah; Eshleman, Susan H.

2013-01-01

We adapted high-resolution melting (HRM) technology to measure genetic diversity without sequencing. Diversity is measured as a single numeric HRM score. Herein, we determined the impact of mutation types and amplicon characteristics on HRM diversity scores. Plasmids were generated with single-base changes, insertions, and deletions. Different primer sets were used to vary the position of mutations within amplicons. Plasmids and plasmid mixtures were analyzed to determine the impact of mutation type, position, and concentration on HRM scores. The impact of amplicon length and G/C content on HRM scores was also evaluated. Different mutation types affected HRM scores to varying degrees (1-bp deletion < 1-bp change < 3-bp insertion < 9-bp insertion). The impact of mutations on HRM scores was influenced by amplicon length and the position of the mutation within the amplicon. Mutations were detected at concentrations of 5% to 95%, with the greatest impact at 50%. The G/C content altered melting temperature values of amplicons but had no impact on HRM scores. These data are relevant to the design of assays that measure genetic diversity using HRM technology. PMID:23178437

Plasmid Stability in Dried Cells of the Desert Cyanobacterium Chroococcidiopsis and its Potential for GFP Imaging of Survivors on Earth and in Space

NASA Astrophysics Data System (ADS)

Billi, Daniela

2012-06-01

Two GFP-based plasmids, namely pTTQ18-GFP-pDU1mini and pDUCA7-GFP, of about 7 kbp and 15 kbp respectively, able to replicate in Chroococcidiopsis sp. CCMEE 029 and CCMEE 123, were developed. Both plasmids were maintained in Chroococcidiopsis cells after 18 months of dry storage as demonstrated by colony PCR, plasmid restriction analysis, GFP imaging and colony-forming ability under selection of dried transformants; thus suggesting that strategies employed by this cyanobacterium to stabilize dried chromosomal DNA, must have protected plasmid DNA. The suitability of pDU1mini-plasmid for GFP tagging in Chroococcidiopsis was investigated by using the RecA homolog of Synechocystis sp. PCC 6803. After 2 months of dry storage, the presence of dried cells with a GFP-RecASyn distribution resembling that of hydrated cells, supported its capability of preventing desiccation-induced genome damage, whereas the rewetted cells with filamentous GFP-RecASyn structures revealed sub-lethal DNA damage. The long-term stability of plasmid DNA in dried Chroococcidiopsis has implication for space research, for example when investigating the recovery of dried cells after Martian and space simulations or when developing life support systems based on phototrophs with genetically enhanced stress tolerance and stored in the dry state for prolonged periods.

Plasmid stability in dried cells of the desert cyanobacterium Chroococcidiopsis and its potential for GFP imaging of survivors on Earth and in space.

PubMed

Billi, Daniela

2012-06-01

Two GFP-based plasmids, namely pTTQ18-GFP-pDU1(mini) and pDUCA7-GFP, of about 7 kbp and 15 kbp respectively, able to replicate in Chroococcidiopsis sp. CCMEE 029 and CCMEE 123, were developed. Both plasmids were maintained in Chroococcidiopsis cells after 18 months of dry storage as demonstrated by colony PCR, plasmid restriction analysis, GFP imaging and colony-forming ability under selection of dried transformants; thus suggesting that strategies employed by this cyanobacterium to stabilize dried chromosomal DNA, must have protected plasmid DNA. The suitability of pDU1(mini)-plasmid for GFP tagging in Chroococcidiopsis was investigated by using the RecA homolog of Synechocystis sp. PCC 6803. After 2 months of dry storage, the presence of dried cells with a GFP-RecA(Syn) distribution resembling that of hydrated cells, supported its capability of preventing desiccation-induced genome damage, whereas the rewetted cells with filamentous GFP-RecA(Syn) structures revealed sub-lethal DNA damage. The long-term stability of plasmid DNA in dried Chroococcidiopsis has implication for space research, for example when investigating the recovery of dried cells after Martian and space simulations or when developing life support systems based on phototrophs with genetically enhanced stress tolerance and stored in the dry state for prolonged periods.

One Novel Multiple-Target Plasmid Reference Molecule Targeting Eight Genetically Modified Canola Events for Genetically Modified Canola Detection.

PubMed

Li, Zhuqing; Li, Xiang; Wang, Canhua; Song, Guiwen; Pi, Liqun; Zheng, Lan; Zhang, Dabing; Yang, Litao

2017-09-27

Multiple-target plasmid DNA reference materials have been generated and utilized as good substitutes of matrix-based reference materials in the analysis of genetically modified organisms (GMOs). Herein, we report the construction of one multiple-target plasmid reference molecule, pCAN, which harbors eight GM canola event-specific sequences (RF1, RF2, MS1, MS8, Topas 19/2, Oxy235, RT73, and T45) and a partial sequence of the canola endogenous reference gene PEP. The applicability of this plasmid reference material in qualitative and quantitative PCR assays of the eight GM canola events was evaluated, including the analysis of specificity, limit of detection (LOD), limit of quantification (LOQ), and performance of pCAN in the analysis of various canola samples, etc. The LODs are 15 copies for RF2, MS1, and RT73 assays using pCAN as the calibrator and 10 genome copies for the other events. The LOQ in each event-specific real-time PCR assay is 20 copies. In quantitative real-time PCR analysis, the PCR efficiencies of all event-specific and PEP assays are between 91% and 97%, and the squared regression coefficients (R 2 ) are all higher than 0.99. The quantification bias values varied from 0.47% to 20.68% with relative standard deviation (RSD) from 1.06% to 24.61% in the quantification of simulated samples. Furthermore, 10 practical canola samples sampled from imported shipments in the port of Shanghai, China, were analyzed employing pCAN as the calibrator, and the results were comparable with those assays using commercial certified materials as the calibrator. Concluding from these results, we believe that this newly developed pCAN plasmid is one good candidate for being a plasmid DNA reference material in the detection and quantification of the eight GM canola events in routine analysis.

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

Certification of reference materials for detection of the human prothrombin gene G20210A sequence variant.

PubMed

Gancberg, David; Corbisier, Philippe; Meeus, Nele; Marki-Zay, Janos; Mannhalter, Christine; Schimmel, Heinz

2008-01-01

There is a need for reference materials (RMs) in the field of genetic testing for verification of test results obtained in patients and probands. For the frequent genetic variation G20210A in the prothrombin gene, it has been shown that purified plasmids containing the gene fragment harbouring the mutation constitute good candidate RMs. Plasmid-type RMs were characterised for homogeneity, stability, sequence identity and fitness for purpose. Their certification required the use of different real-time PCR methods for genotyping and quantification of the plasmid copy number. Homogeneity, stability and fitness for the purpose of the plasmids could be demonstrated. The long-term stability (up to 24 months) of the materials was confirmed by highly sensitive and specific quantitative real-time PCR methods. New types of certified RMs (CRMs) for genetic testing of the human prothrombin gene G20210A sequence variant are available. Their fitness for purpose was demonstrated and no evidence was found that they would not work with other methods as long as these are targeting the whole or parts of the prothrombin gene fragment inserted into the plasmids. The described CRMs support the efforts of the international community in development, validation and harmonisation of tests for molecular genetic testing.

Protective efficacy of a high-growth reassortant swine H3N2 inactivated vaccine constructed by reverse genetic manipulation

PubMed Central

Wen, Feng; Ma, Ji-Hong; Yang, Fu-Ru; Huang, Meng; Zhou, Yan-Jun; Li, Ze-Jun

2014-01-01

Novel reassortant H3N2 swine influenza viruses (SwIV) with the matrix gene from the 2009 H1N1 pandemic virus have been isolated in many countries as well as during outbreaks in multiple states in the United States, indicating that H3N2 SwIV might be a potential threat to public health. Since southern China is the world's largest producer of pigs, efficient vaccines should be developed to prevent pigs from acquiring H3N2 subtype SwIV infections, and thus limit the possibility of SwIV infection at agricultural fairs. In this study, a high-growth reassortant virus (GD/PR8) was generated by plasmid-based reverse genetics and tested as a candidate inactivated vaccine. The protective efficacy of this vaccine was evaluated in mice by challenging them with another H3N2 SwIV isolate [A/Swine/Heilongjiang/1/05 (H3N2) (HLJ/05)]. Prime and booster inoculation with GD/PR8 vaccine yielded high-titer serum hemagglutination inhibiting antibodies and IgG antibodies. Complete protection of mice against H3N2 SwIV was observed, with significantly reduced lung lesion and viral loads in vaccine-inoculated mice relative to mock-vaccinated controls. These results suggest that the GD/PR8 vaccine may serve as a promising candidate for rapid intervention of H3N2 SwIV outbreaks in China. PMID:24675833

The Tcp conjugation system of Clostridium perfringens.

PubMed

Wisniewski, Jessica A; Rood, Julian I

2017-05-01

The Gram-positive pathogen Clostridium perfringens possesses a family of large conjugative plasmids that is typified by the tetracycline resistance plasmid pCW3. Since these plasmids may carry antibiotic resistance genes or genes encoding extracellular or sporulation-associated toxins, the conjugative transfer of these plasmids appears to be important for the epidemiology of C. perfringens-mediated diseases. Sequence analysis of members of this plasmid family identified a highly conserved 35kb region that encodes proteins with various functions, including plasmid replication and partitioning. The tcp conjugation locus also was identified in this region, initially based on low-level amino acid sequence identity to conjugation proteins from the integrative conjugative element Tn916. Genetic studies confirmed that the tcp locus is required for conjugative transfer and combined with biochemical and structural analyses have led to the development of a functional model of the Tcp conjugation apparatus. This review summarises our current understanding of the Tcp conjugation system, which is now one of the best-characterized conjugation systems in Gram-positive bacteria. Copyright © 2017 Elsevier Inc. All rights reserved.

Construction of an easy-to-use CRISPR-Cas9 system by patching a newly designed EXIT circuit.

PubMed

Tang, Qiang; Lou, Chunbo; Liu, Shuang-Jiang

2017-01-01

Plasmid-borne genetic editing tools, including the widely used CRISPR-Cas9 system, have greatly facilitated bacterial programming to obtain novel functionalities. However, the lack of effective post-editing plasmid elimination methods impedes follow-up genetic manipulation or application. Conventional strategies including exposure to physical and chemical treatments, or exploiting temperature-sensitive replication origins have several drawbacks (e.g., they are limited for efficiency and are time-consuming). Therefore, the demand is apparent for easy and rapid elimination of the tool plasmids from their bacterial hosts after genetic manipulation. To bridge this gap, we designed a novel EXIT circuit with the homing endonuclease, which can be exploited for rapid and efficient elimination of various plasmids with diverse replication origins. As a proof of concept, we validated the EXIT circuit in Escherichia coli by harnessing homing endonuclease I- Sce I and its cleavage site. When integrated into multiple plasmids with different origins, the EXIT circuit allowed them to be eliminated from the host cells, simultaneously. By combining the widely used plasmid-borne CRISPR-Cas9 system and the EXIT circuit, we constructed an easy-to-use CRISPR-Cas9 system that eliminated the Cas9- and the single-guide RNA (sgRNA)-encoding plasmids in one-step. Within 3 days, we successfully constructed an atrazine-degrading E. coli strain, thus further demonstrating the advantage of this new CRISPR-Cas9 system for bacterial genome editing. Our novel EXIT circuit, which exploits the homing endonuclease I- Sce I, enables plasmid(s) with different replication origins to be eliminated from their host cells rapidly and efficiently. We also developed an easy-to-use CRISPR-Cas9 system with the EXIT circuit, and this new system can be widely applied to bacterial genome editing.

Promiscuous plasmid replication in thermophiles: Use of a novel hyperthermophilic replicon for genetic manipulation of Clostridium thermocellum at its optimum growth temperature

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

Groom, Joseph; Chung, Daehwan; Olson, Daniel G.

2016-01-29

Clostridium thermocellum is a leading candidate for the consolidated bioprocessing of lignocellulosic biomass for the production of fuels and chemicals. A limitation to the engineering of this strain is the availability of stable replicating plasmid vectors for homologous and heterologous expression of genes that provide improved and/or novel pathways for fuel production. Current vectors relay on replicons from mesophilic bacteria and are not stable at the optimum growth temperature of C. thermocellum. To develop more thermostable genetic tools for C. thermocellum, we constructed vectors based on the hyperthermophilic Caldicellulosiruptor bescii replicon pBAS2. Autonomously replicating shuttle vectors based on pBAS2 reproduciblymore » transformed C. thermocellum at 60 °C and were maintained in multiple copy. Promoters, selectable markers and plasmid replication proteins from C. bescii were functional in C. thermocellum. Phylogenetic analyses of the proteins contained on pBAS2 revealed that the replication initiation protein RepL is unique among thermophiles. Lastly, these results suggest that pBAS2 may be a broadly useful replicon for other thermophilic Firmicutes.« less

Production of Mutated Porcine Embryos Using Zinc Finger Nucleases and a Reporter-based Cell Enrichment System.

PubMed

Koo, Ok Jae; Park, Sol Ji; Lee, Choongil; Kang, Jung Taek; Kim, Sujin; Moon, Joon Ho; Choi, Ji Yei; Kim, Hyojin; Jang, Goo; Kim, Jin-Soo; Kim, Seokjoong; Lee, Byeong-Chun

2014-03-01

To facilitate the construction of genetically-modified pigs, we produced cloned embryos derived from porcine fibroblasts transfected with a pair of engineered zinc finger nuclease (ZFN) plasmids to create targeted mutations and enriched using a reporter plasmid system. The reporter expresses RFP and eGFP simultaneously when ZFN-mediated site-specific mutations occur. Thus, double positive cells (RFP(+)/eGFP(+)) were selected and used for somatic cell nuclear transfer. Two types of reporter based enrichment systems were used in this study; the cloned embryos derived from cells enriched using a magnetic sorting-based system showed better developmental competence than did those derived from cells enriched by flow cytometry. Mutated sequences, such as insertions, deletions, or substitutions, together with the wild-type sequence, were found in the cloned porcine blastocysts. Therefore, genetic mutations can be achieved in cloned porcine embryos reconstructed with ZFN-treated cells that were enriched by a reporter-based system.

Plasmid-based genetic modification of human bone marrow-derived stromal cells: analysis of cell survival and transgene expression after transplantation in rat spinal cord

PubMed Central

Ronsyn, Mark W; Daans, Jasmijn; Spaepen, Gie; Chatterjee, Shyama; Vermeulen, Katrien; D'Haese, Patrick; Van Tendeloo, Viggo FI; Van Marck, Eric; Ysebaert, Dirk; Berneman, Zwi N; Jorens, Philippe G; Ponsaerts, Peter

2007-01-01

Background Bone marrow-derived stromal cells (MSC) are attractive targets for ex vivo cell and gene therapy. In this context, we investigated the feasibility of a plasmid-based strategy for genetic modification of human (h)MSC with enhanced green fluorescent protein (EGFP) and neurotrophin (NT)3. Three genetically modified hMSC lines (EGFP, NT3, NT3-EGFP) were established and used to study cell survival and transgene expression following transplantation in rat spinal cord. Results First, we demonstrate long-term survival of transplanted hMSC-EGFP cells in rat spinal cord under, but not without, appropriate immune suppression. Next, we examined the stability of EGFP or NT3 transgene expression following transplantation of hMSC-EGFP, hMSC-NT3 and hMSC-NT3-EGFP in rat spinal cord. While in vivo EGFP mRNA and protein expression by transplanted hMSC-EGFP cells was readily detectable at different time points post-transplantation, in vivo NT3 mRNA expression by hMSC-NT3 cells and in vivo EGFP protein expression by hMSC-NT3-EGFP cells was, respectively, undetectable or declined rapidly between day 1 and 7 post-transplantation. Further investigation revealed that the observed in vivo decline of EGFP protein expression by hMSC-NT3-EGFP cells: (i) was associated with a decrease in transgenic NT3-EGFP mRNA expression as suggested following laser capture micro-dissection analysis of hMSC-NT3-EGFP cell transplants at day 1 and day 7 post-transplantation, (ii) did not occur when hMSC-NT3-EGFP cells were transplanted subcutaneously, and (iii) was reversed upon re-establishment of hMSC-NT3-EGFP cell cultures at 2 weeks post-transplantation. Finally, because we observed a slowly progressing tumour growth following transplantation of all our hMSC cell transplants, we here demonstrate that omitting immune suppressive therapy is sufficient to prevent further tumour growth and to eradicate malignant xenogeneic cell transplants. Conclusion In this study, we demonstrate that genetically modified hMSC lines can survive in healthy rat spinal cord over at least 3 weeks by using adequate immune suppression and can serve as vehicles for transgene expression. However, before genetically modified hMSC can potentially be used in a clinical setting to treat spinal cord injuries, more research on standardisation of hMSC culture and genetic modification needs to be done in order to prevent tumour formation and transgene silencing in vivo. PMID:18078525

Plasmid-based genetic modification of human bone marrow-derived stromal cells: analysis of cell survival and transgene expression after transplantation in rat spinal cord.

PubMed

Ronsyn, Mark W; Daans, Jasmijn; Spaepen, Gie; Chatterjee, Shyama; Vermeulen, Katrien; D'Haese, Patrick; Van Tendeloo, Viggo Fi; Van Marck, Eric; Ysebaert, Dirk; Berneman, Zwi N; Jorens, Philippe G; Ponsaerts, Peter

2007-12-14

Bone marrow-derived stromal cells (MSC) are attractive targets for ex vivo cell and gene therapy. In this context, we investigated the feasibility of a plasmid-based strategy for genetic modification of human (h)MSC with enhanced green fluorescent protein (EGFP) and neurotrophin (NT)3. Three genetically modified hMSC lines (EGFP, NT3, NT3-EGFP) were established and used to study cell survival and transgene expression following transplantation in rat spinal cord. First, we demonstrate long-term survival of transplanted hMSC-EGFP cells in rat spinal cord under, but not without, appropriate immune suppression. Next, we examined the stability of EGFP or NT3 transgene expression following transplantation of hMSC-EGFP, hMSC-NT3 and hMSC-NT3-EGFP in rat spinal cord. While in vivo EGFP mRNA and protein expression by transplanted hMSC-EGFP cells was readily detectable at different time points post-transplantation, in vivo NT3 mRNA expression by hMSC-NT3 cells and in vivo EGFP protein expression by hMSC-NT3-EGFP cells was, respectively, undetectable or declined rapidly between day 1 and 7 post-transplantation. Further investigation revealed that the observed in vivo decline of EGFP protein expression by hMSC-NT3-EGFP cells: (i) was associated with a decrease in transgenic NT3-EGFP mRNA expression as suggested following laser capture micro-dissection analysis of hMSC-NT3-EGFP cell transplants at day 1 and day 7 post-transplantation, (ii) did not occur when hMSC-NT3-EGFP cells were transplanted subcutaneously, and (iii) was reversed upon re-establishment of hMSC-NT3-EGFP cell cultures at 2 weeks post-transplantation. Finally, because we observed a slowly progressing tumour growth following transplantation of all our hMSC cell transplants, we here demonstrate that omitting immune suppressive therapy is sufficient to prevent further tumour growth and to eradicate malignant xenogeneic cell transplants. In this study, we demonstrate that genetically modified hMSC lines can survive in healthy rat spinal cord over at least 3 weeks by using adequate immune suppression and can serve as vehicles for transgene expression. However, before genetically modified hMSC can potentially be used in a clinical setting to treat spinal cord injuries, more research on standardisation of hMSC culture and genetic modification needs to be done in order to prevent tumour formation and transgene silencing in vivo.

Adaptation of genetically monomorphic bacteria: evolution of copper resistance through multiple horizontal gene transfers of complex and versatile mobile genetic elements.

PubMed

Richard, D; Ravigné, V; Rieux, A; Facon, B; Boyer, C; Boyer, K; Grygiel, P; Javegny, S; Terville, M; Canteros, B I; Robène, I; Vernière, C; Chabirand, A; Pruvost, O; Lefeuvre, P

2017-04-01

Copper-based antimicrobial compounds are widely used to control plant bacterial pathogens. Pathogens have adapted in response to this selective pressure. Xanthomonas citri pv. citri, a major citrus pathogen causing Asiatic citrus canker, was first reported to carry plasmid-encoded copper resistance in Argentina. This phenotype was conferred by the copLAB gene system. The emergence of resistant strains has since been reported in Réunion and Martinique. Using microsatellite-based genotyping and copLAB PCR, we demonstrated that the genetic structure of the copper-resistant strains from these three regions was made up of two distant clusters and varied for the detection of copLAB amplicons. In order to investigate this pattern more closely, we sequenced six copper-resistant X. citri pv. citri strains from Argentina, Martinique and Réunion, together with reference copper-resistant Xanthomonas and Stenotrophomonas strains using long-read sequencing technology. Genes involved in copper resistance were found to be strain dependent with the novel identification in X. citri pv. citri of copABCD and a cus heavy metal efflux resistance-nodulation-division system. The genes providing the adaptive trait were part of a mobile genetic element similar to Tn3-like transposons and included in a conjugative plasmid. This indicates the system's great versatility. The mining of all available bacterial genomes suggested that, within the bacterial community, the spread of copper resistance associated with mobile elements and their plasmid environments was primarily restricted to the Xanthomonadaceae family. © 2017 John Wiley & Sons Ltd.

Development of genetic techniques for the psychrotrophic fish pathogen Flavobacterium psychrophilum.

PubMed

Alvarez, B; Secades, P; McBride, M J; Guijarro, J A

2004-01-01

Flavobacterium psychrophilum, a member of the Cytophaga-Flavobacterium-Bacteroides group, is an important pathogen of salmonid fish. Previous attempts to develop genetic techniques for this fastidious, psychrotrophic bacterium have met with failure. Here we describe the development of techniques for the genetic manipulation of F. psychrophilum and the identification of plasmids, selectable markers, a reporter system, and a transposon that function in several isolates of this fish pathogen. The antibiotic resistance genes ermF, cfxA, and tetQ function in F. psychrophilum. Cloning vectors based on the F. psychrophilum cryptic plasmid pCP1 which carried these selectable markers were introduced by conjugation from E. coli, resulting in antibiotic-resistant colonies of F. psychrophilum. Conjugative transfer of DNA into F. psychrophilum was strain dependent. Efficient transfer was observed for two of the seven strains tested (THC02-90 and THC04-90). E. coli lacZY functioned in F. psychrophilum when expressed from a pCP1 promoter, allowing its development as a reporter for studies of gene expression. Plasmids isolated from F. psychrophilum were efficiently introduced into F. psychrophilum by electroporation, but plasmids isolated from E. coli were not suitable for transfer by this route, suggesting the presence of a restriction barrier. DNA isolated from F. psychrophilum was resistant to digestion by Sau3AI and BamHI, indicating that a Sau3AI-like restriction modification system may constitute part of this barrier. Tn4351 was introduced into F. psychrophilum from E. coli and transposed with apparent randomness, resulting in erythromycin-resistant colonies. The techniques developed in this study allow for genetic manipulation and analysis of this important fish pathogen.

Reverse genetics studies on the filamentous morphology of influenza A virus.

PubMed

Bourmakina, Svetlana V; García-Sastre, Adolfo

2003-03-01

We have investigated the genetic determinants responsible for the filamentous morphology of influenza A viruses, a property characteristic of primary virus isolates. A plasmid-based reverse genetics system was used to transfer the M segment of influenza A/Udorn/72 (H3N2) virus into influenza A/WSN/33 (H1N1) virus. While WSN virions display spherical morphology, recombinant WSN-Mud virus acquired the ability of the parental Udorn strain to form filamentous virus particles. This was determined by immunofluorescence studies in infected MDCK cells and by electron microscopy of purified virus particles. To determine the gene product within the M segment responsible for filamentous virus morphology, we generated four recombinant viruses carrying different sets of M1 and M2 genes from WSN or Udorn strains in a WSN background. These studies revealed that the M1 gene of Udorn, independently of the origin of the M2 gene, conferred filamentous budding properties and filamentous virus morphology to the recombinant viruses. We also constructed two WSN viruses encoding chimeric M1 proteins containing the amino-terminal 1-162 amino acids or the carboxy-terminal 163-252 amino acids of the Udorn M1 protein. Neither of these two viruses acquired filamentous phenotypes, indicating that both amino- and carboxy-terminal domains of the M1 protein contribute to filamentous virus morphology. We next rescued seven mutant WSN-M1ud viruses containing Udorn M1 proteins carrying single amino acid substitutions corresponding to the seven amino acid differences with the M1 protein of WSN virus. Characterization of these recombinant viruses revealed that amino acid residues 95 and 204 are critical in determining filamentous virus particle formation.

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

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.

Mechanisms of Evolution in High-Consequence Drug Resistance Plasmids

PubMed Central

He, Susu; Chandler, Michael; Varani, Alessandro M.; Hickman, Alison B.; Dekker, John P.

2016-01-01

ABSTRACT The dissemination of resistance among bacteria has been facilitated by the fact that resistance genes are usually located on a diverse and evolving set of transmissible plasmids. However, the mechanisms generating diversity and enabling adaptation within highly successful resistance plasmids have remained obscure, despite their profound clinical significance. To understand these mechanisms, we have performed a detailed analysis of the mobilome (the entire mobile genetic element content) of a set of previously sequenced carbapenemase-producing Enterobacteriaceae (CPE) from the National Institutes of Health Clinical Center. This analysis revealed that plasmid reorganizations occurring in the natural context of colonization of human hosts were overwhelmingly driven by genetic rearrangements carried out by replicative transposons working in concert with the process of homologous recombination. A more complete understanding of the molecular mechanisms and evolutionary forces driving rearrangements in resistance plasmids may lead to fundamentally new strategies to address the problem of antibiotic resistance. PMID:27923922

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

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

Genome sequence of Ensifer adhaerens OV14 provides insights into its ability as a novel vector for the genetic transformation of plant genomes.

PubMed

Rudder, Steven; Doohan, Fiona; Creevey, Christopher J; Wendt, Toni; Mullins, Ewen

2014-04-07

Recently it has been shown that Ensifer adhaerens can be used as a plant transformation technology, transferring genes into several plant genomes when equipped with a Ti plasmid. For this study, we have sequenced the genome of Ensifer adhaerens OV14 (OV14) and compared it with those of Agrobacterium tumefaciens C58 (C58) and Sinorhizobium meliloti 1021 (1021); the latter of which has also demonstrated a capacity to genetically transform crop genomes, albeit at significantly reduced frequencies. The 7.7 Mb OV14 genome comprises two chromosomes and two plasmids. All protein coding regions in the OV14 genome were functionally grouped based on an eggNOG database. No genes homologous to the A. tumefaciens Ti plasmid vir genes appeared to be present in the OV14 genome. Unexpectedly, OV14 and 1021 were found to possess homologs to chromosomal based genes cited as essential to A. tumefaciens T-DNA transfer. Of significance, genes that are non-essential but exert a positive influence on virulence and the ability to genetically transform host genomes were identified in OV14 but were absent from the 1021 genome. This study reveals the presence of homologs to chromosomally based Agrobacterium genes that support T-DNA transfer within the genome of OV14 and other alphaproteobacteria. The sequencing and analysis of the OV14 genome increases our understanding of T-DNA transfer by non-Agrobacterium species and creates a platform for the continued improvement of Ensifer-mediated transformation (EMT).

Genome sequence of Ensifer adhaerens OV14 provides insights into its ability as a novel vector for the genetic transformation of plant genomes

PubMed Central

2014-01-01

Background Recently it has been shown that Ensifer adhaerens can be used as a plant transformation technology, transferring genes into several plant genomes when equipped with a Ti plasmid. For this study, we have sequenced the genome of Ensifer adhaerens OV14 (OV14) and compared it with those of Agrobacterium tumefaciens C58 (C58) and Sinorhizobium meliloti 1021 (1021); the latter of which has also demonstrated a capacity to genetically transform crop genomes, albeit at significantly reduced frequencies. Results The 7.7 Mb OV14 genome comprises two chromosomes and two plasmids. All protein coding regions in the OV14 genome were functionally grouped based on an eggNOG database. No genes homologous to the A. tumefaciens Ti plasmid vir genes appeared to be present in the OV14 genome. Unexpectedly, OV14 and 1021 were found to possess homologs to chromosomal based genes cited as essential to A. tumefaciens T-DNA transfer. Of significance, genes that are non-essential but exert a positive influence on virulence and the ability to genetically transform host genomes were identified in OV14 but were absent from the 1021 genome. Conclusions This study reveals the presence of homologs to chromosomally based Agrobacterium genes that support T-DNA transfer within the genome of OV14 and other alphaproteobacteria. The sequencing and analysis of the OV14 genome increases our understanding of T-DNA transfer by non-Agrobacterium species and creates a platform for the continued improvement of Ensifer-mediated transformation (EMT). PMID:24708309

Versatile Gene-Specific Sequence Tags for Arabidopsis Functional Genomics: Transcript Profiling and Reverse Genetics Applications

PubMed Central

Hilson, Pierre; Allemeersch, Joke; Altmann, Thomas; Aubourg, Sébastien; Avon, Alexandra; Beynon, Jim; Bhalerao, Rishikesh P.; Bitton, Frédérique; Caboche, Michel; Cannoot, Bernard; Chardakov, Vasil; Cognet-Holliger, Cécile; Colot, Vincent; Crowe, Mark; Darimont, Caroline; Durinck, Steffen; Eickhoff, Holger; de Longevialle, Andéol Falcon; Farmer, Edward E.; Grant, Murray; Kuiper, Martin T.R.; Lehrach, Hans; Léon, Céline; Leyva, Antonio; Lundeberg, Joakim; Lurin, Claire; Moreau, Yves; Nietfeld, Wilfried; Paz-Ares, Javier; Reymond, Philippe; Rouzé, Pierre; Sandberg, Goran; Segura, Maria Dolores; Serizet, Carine; Tabrett, Alexandra; Taconnat, Ludivine; Thareau, Vincent; Van Hummelen, Paul; Vercruysse, Steven; Vuylsteke, Marnik; Weingartner, Magdalena; Weisbeek, Peter J.; Wirta, Valtteri; Wittink, Floyd R.A.; Zabeau, Marc; Small, Ian

2004-01-01

Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics. PMID:15489341

CRISPR: a Versatile Tool for Both Forward and Reverse Genetics Research

PubMed Central

Gurumurthy, Channabasavaiah B.; Grati, M'hamed; Ohtsuka, Masato; Schilit, Samantha L.P.; Quadros, Rolen M.; Liu, Xue Zhong

2016-01-01

Human genetics research employs the two opposing approaches of forward and reverse genetics. While forward genetics identifies and links a mutation to an observed disease etiology, reverse genetics induces mutations in model organisms to study their role in disease. In most cases, causality for mutations identified by forward genetics is confirmed by reverse genetics through the development of genetically engineered animal models and an assessment of whether the model can recapitulate the disease. While many technological advances have helped improve these approaches, some gaps still remain. CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, which has emerged as a revolutionary genetic engineering tool, holds great promise for closing such gaps. By combining the benefits of forward and reverse genetics, it has dramatically expedited human genetics research. We provide a perspective on the power of CRISPR-based forward and reverse genetics tools in human genetics and discuss its applications using some disease examples. PMID:27384229

Development of oriC-Based Plasmids for Mesoplasma florum.

PubMed

Matteau, Dominick; Pepin, Marie-Eve; Baby, Vincent; Gauthier, Samuel; Arango Giraldo, Mélissa; Knight, Thomas F; Rodrigue, Sébastien

2017-04-01

The near-minimal bacterium Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. However, the lack of genetic engineering tools for this microorganism has limited our capacity to understand its basic biology and modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first generation of artificial plasmids able to replicate in this bacterium. Selected regions of the predicted M. florum chromosomal origin of replication ( oriC ) were used to create different plasmid versions that were tested for their transformation frequency and stability. Using polyethylene glycol-mediated transformation, we observed that plasmids harboring both rpmH-dnaA and dnaA-dnaN intergenic regions, interspaced or not with a copy of the dnaA gene, resulted in a frequency of ∼4.1 × 10 -6 transformants per viable cell and were stably maintained throughout multiple generations. In contrast, plasmids containing only one M. florum oriC intergenic region or the heterologous oriC region of Mycoplasma capricolum , Mycoplasma mycoides , or Spiroplasma citri failed to produce any detectable transformants. We also developed alternative transformation procedures based on electroporation and conjugation from Escherichia coli , reaching frequencies up to 7.87 × 10 -6 and 8.44 × 10 -7 transformants per viable cell, respectively. Finally, we demonstrated the functionality of antibiotic resistance genes active against tetracycline, puromycin, and spectinomycin/streptomycin in M. florum Taken together, these valuable genetic tools will facilitate efforts toward building an M. florum -based near-minimal cellular chassis for synthetic biology. IMPORTANCE Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. M. florum is closely related to the mycoides cluster of mycoplasmas, which has become a model for whole-genome cloning, genome transplantation, and genome minimization. However, M. florum shows higher growth rates than other Mollicutes , has no known pathogenic potential, and possesses a significantly smaller genome that positions this species among some of the simplest free-living organisms. So far, the lack of genetic engineering tools has limited our capacity to understand the basic biology of M. florum in order to modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first artificial plasmids and transformation methods for this bacterium. This represents a strong basis for ongoing genome engineering efforts using this near-minimal microorganism. Copyright © 2017 American Society for Microbiology.

Comparative Genomics of the Listeria monocytogenes ST204 Subgroup

PubMed Central

Fox, Edward M.; Allnutt, Theodore; Bradbury, Mark I.; Fanning, Séamus; Chandry, P. Scott

2016-01-01

The ST204 subgroup of Listeria monocytogenes is among the most frequently isolated in Australia from a range of environmental niches. In this study we provide a comparative genomics analysis of food and food environment isolates from geographically diverse sources. Analysis of the ST204 genomes showed a highly conserved core genome with the majority of variation seen in mobile genetic elements such as plasmids, transposons and phage insertions. Most strains (13/15) harbored plasmids, which although varying in size contained highly conserved sequences. Interestingly 4 isolates contained a conserved plasmid of 91,396 bp. The strains examined were isolated over a period of 12 years and from different geographic locations suggesting plasmids are an important component of the genetic repertoire of this subgroup and may provide a range of stress tolerance mechanisms. In addition to this 4 phage insertion sites and 2 transposons were identified among isolates, including a novel transposon. These genetic elements were highly conserved across isolates that harbored them, and also contained a range of genetic markers linked to stress tolerance and virulence. The maintenance of conserved mobile genetic elements in the ST204 population suggests these elements may contribute to the diverse range of niches colonized by ST204 isolates. Environmental stress selection may contribute to maintaining these genetic features, which in turn may be co-selecting for virulence markers relevant to clinical infection with ST204 isolates. PMID:28066377

Comparative Genomics of the Listeria monocytogenes ST204 Subgroup.

PubMed

Fox, Edward M; Allnutt, Theodore; Bradbury, Mark I; Fanning, Séamus; Chandry, P Scott

2016-01-01

The ST204 subgroup of Listeria monocytogenes is among the most frequently isolated in Australia from a range of environmental niches. In this study we provide a comparative genomics analysis of food and food environment isolates from geographically diverse sources. Analysis of the ST204 genomes showed a highly conserved core genome with the majority of variation seen in mobile genetic elements such as plasmids, transposons and phage insertions. Most strains (13/15) harbored plasmids, which although varying in size contained highly conserved sequences. Interestingly 4 isolates contained a conserved plasmid of 91,396 bp. The strains examined were isolated over a period of 12 years and from different geographic locations suggesting plasmids are an important component of the genetic repertoire of this subgroup and may provide a range of stress tolerance mechanisms. In addition to this 4 phage insertion sites and 2 transposons were identified among isolates, including a novel transposon. These genetic elements were highly conserved across isolates that harbored them, and also contained a range of genetic markers linked to stress tolerance and virulence. The maintenance of conserved mobile genetic elements in the ST204 population suggests these elements may contribute to the diverse range of niches colonized by ST204 isolates. Environmental stress selection may contribute to maintaining these genetic features, which in turn may be co-selecting for virulence markers relevant to clinical infection with ST204 isolates.

The genetic basis of the fitness costs of antimicrobial resistance: a meta-analysis approach.

PubMed

Vogwill, Tom; MacLean, R Craig

2015-03-01

The evolution of antibiotic resistance carries a fitness cost, expressed in terms of reduced competitive ability in the absence of antibiotics. This cost plays a key role in the dynamics of resistance by generating selection against resistance when bacteria encounter an antibiotic-free environment. Previous work has shown that the cost of resistance is highly variable, but the underlying causes remain poorly understood. Here, we use a meta-analysis of the published resistance literature to determine how the genetic basis of resistance influences its cost. We find that on average chromosomal resistance mutations carry a larger cost than acquiring resistance via a plasmid. This may explain why resistance often evolves by plasmid acquisition. Second, we find that the cost of plasmid acquisition increases with the breadth of its resistance range. This suggests a potentially important limit on the evolution of extensive multidrug resistance via plasmids. We also find that epistasis can significantly alter the cost of mutational resistance. Overall, our study shows that the cost of antimicrobial resistance can be partially explained by its genetic basis. It also highlights both the danger associated with plasmidborne resistance and the need to understand why resistance plasmids carry a relatively low cost.

Functions and origin of plasmids in Erwinia species that are pathogenic to or epiphytically associated with pome fruit trees.

PubMed

Llop, Pablo; Barbé, Silvia; López, María M

The genus Erwinia includes plant-associated pathogenic and non-pathogenic species. Among them, all species pathogenic to pome fruit trees ( E. amylovora, E. pyrifoliae, E. piriflorinigrans, Erwinia sp. from Japan) cause similar symptoms, but differ in their degrees of aggressiveness, i.e. in symptoms, host range or both. The presence of plasmids of similar size, in the range of 30 kb, is a common characteristic that they possess. Besides, they share some genetic content with high homology in several genes associated with exopolysaccharide production and hence, with virulence, as well as in some other genes. Knowledge of the content of these plasmids and comparative genetic analyses may provide interesting new clues to understanding the origin and evolution of these pathogens and the level of symptoms they produce. Furthermore, genetic similarities observed among some of the plasmids (and genomes) from the above indicated pathogenic species and E. tasmaniensis or E. billingiae , which are epiphytic on the same hosts, may reveal associations that could expose the mechanisms of origin of pathogens. A summary of the current information on their plasmids and the relationships among them is presented here.

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.

Genetic manipulation of Bacillus methanolicus, a gram-positive, thermotolerant methylotroph.

PubMed Central

Cue, D; Lam, H; Dillingham, R L; Hanson, R S; Flickinger, M C

1997-01-01

We report the fist genetic transformation system, shuttle vectors, and integrative vectors for the thermotolerant, methylotrophic bacterium Bacillus methanolicus. By using a polyethylene glycol-mediated transformation procedure, we have successfully transformed B. methanolicus with both integrative and multicopy plasmids. For plasmids with a single BmeTI recognition site, dam methylation of plasmid DNA (in vivo or in vitro) was found to enhance transformation efficiency from 7- to 11-fold. Two low-copy-number Escherichia coli-B, methanolicus shuttle plasmids, pDQ507 and pDQ508, are described. pDQ508 caries the replication origin cloned from a 17-kb endogenous B. methanolicus plasmid, pBM1. pDQ507 carries a cloned B. methanolicus DNA fragment, pmr-1, possibly of chromosomal origin, that supports maintenance of pDQ507 as a circular, extrachromosomal DNA molecule. Deletion analysis of pDQ507 indicated two regions required for replication, i.e., a 90-bp AT-rich segment containing a 46-bp imperfect, inverted repeat sequence and a second region 65% homologous to the B. subtilis dpp operon. We also evaluated two E. coli-B. subtilis vectors, pEN1 and pHP13, for use as E. coli-B. methanolicus shuttle vectors. The plasmids pHP13, pDQ507, and pDQ508 were segregationally and structurally stable in B. methanolicus for greater than 60 generations of growth under nonselective conditions; pEN1 was segregationally unstable. Single-stranded plasmid DNA was detected in B. methanolicus transformants carrying either pEN1, pHP13, or pDQ508, suggesting that pDQ508, like the B. subtilis plasmids, is replicated by a rolling-circle mechanism. These studies provide the basic tools for the genetic manipulation of B. methanolicus. PMID:9097439

Genetic manipulation of Bacillus methanolicus, a gram-positive, thermotolerant methylotroph.

PubMed

Cue, D; Lam, H; Dillingham, R L; Hanson, R S; Flickinger, M C

1997-04-01

We report the fist genetic transformation system, shuttle vectors, and integrative vectors for the thermotolerant, methylotrophic bacterium Bacillus methanolicus. By using a polyethylene glycol-mediated transformation procedure, we have successfully transformed B. methanolicus with both integrative and multicopy plasmids. For plasmids with a single BmeTI recognition site, dam methylation of plasmid DNA (in vivo or in vitro) was found to enhance transformation efficiency from 7- to 11-fold. Two low-copy-number Escherichia coli-B, methanolicus shuttle plasmids, pDQ507 and pDQ508, are described. pDQ508 caries the replication origin cloned from a 17-kb endogenous B. methanolicus plasmid, pBM1. pDQ507 carries a cloned B. methanolicus DNA fragment, pmr-1, possibly of chromosomal origin, that supports maintenance of pDQ507 as a circular, extrachromosomal DNA molecule. Deletion analysis of pDQ507 indicated two regions required for replication, i.e., a 90-bp AT-rich segment containing a 46-bp imperfect, inverted repeat sequence and a second region 65% homologous to the B. subtilis dpp operon. We also evaluated two E. coli-B. subtilis vectors, pEN1 and pHP13, for use as E. coli-B. methanolicus shuttle vectors. The plasmids pHP13, pDQ507, and pDQ508 were segregationally and structurally stable in B. methanolicus for greater than 60 generations of growth under nonselective conditions; pEN1 was segregationally unstable. Single-stranded plasmid DNA was detected in B. methanolicus transformants carrying either pEN1, pHP13, or pDQ508, suggesting that pDQ508, like the B. subtilis plasmids, is replicated by a rolling-circle mechanism. These studies provide the basic tools for the genetic manipulation of B. methanolicus.

Genetic islands in pome fruit pathogenic and non-pathogenic Erwinia species and related plasmids

PubMed Central

Llop, Pablo

2015-01-01

New pathogenic bacteria belonging to the genus Erwinia associated with pome fruit trees (Erwinia, E. piriflorinigrans, E. uzenensis) have been increasingly described in the last years, and comparative analyses have found that all these species share several genetic characteristics. Studies at different level (whole genome comparison, virulence genes, plasmid content, etc.) show a high intraspecies homogeneity (i.e., among E. amylovora strains) and also abundant similarities appear between the different Erwinia species: presence of plasmids of similar size in the pathogenic species; high similarity in several genes associated with exopolysaccharide production and hence, with virulence, as well as in some other genes, in the chromosomes. Many genetic similarities have been observed also among some of the plasmids (and genomes) from the pathogenic species and E. tasmaniensis or E. billingiae, two epiphytic species on the same hosts. The amount of genetic material shared in this genus varies from individual genes to clusters, genomic islands and genetic material that even may constitute a whole plasmid. Recent research on evolution of erwinias point out the horizontal transfer acquisition of some genomic islands that were subsequently lost in some species and several pathogenic traits that are still present. How this common material has been obtained and is efficiently maintained in different species belonging to the same genus sharing a common ecological niche provides an idea of the origin and evolution of the pathogenic Erwinia and the interaction with non-pathogenic species present in the same niche, and the role of the genes that are conserved in all of them. PMID:26379649

Gene Transfer in Leptolyngbya sp. Strain BL0902, a Cyanobacterium Suitable for Production of Biomass and Bioproducts

PubMed Central

Taton, Arnaud; Lis, Ewa; Adin, Dawn M.; Dong, Guogang; Cookson, Scott; Kay, Steve A.; Golden, Susan S.; Golden, James W.

2012-01-01

Current cyanobacterial model organisms were not selected for their growth traits or potential for the production of renewable biomass, biofuels, or other products. The cyanobacterium strain BL0902 emerged from a search for strains with superior growth traits. Morphology and 16S rRNA sequence placed strain BL0902 in the genus Leptolyngbya. Leptolyngbya sp. strain BL0902 (hereafter Leptolyngbya BL0902) showed robust growth at temperatures from 22°C to 40°C and tolerated up to 0.5 M NaCl, 32 mM urea, high pH, and high solar irradiance. Its growth rate under outdoor conditions rivaled Arthrospira (“pirulina” strains. Leptolyngbya BL0902 accumulated higher lipid content and a higher proportion of monounsaturated fatty acids than Arthrospira strains. In addition to these desirable qualities, Leptolyngbya BL0902 is amenable to genetic engineering that is reliable, efficient, and stable. We demonstrated conjugal transfer from Escherichia coli of a plasmid based on RSF1010 and expression of spectinomycin/streptomycin resistance and yemGFP reporter transgenes. Conjugation efficiency was investigated in biparental and triparental matings with and without a “elper”plasmid that carries DNA methyltransferase genes, and with two different conjugal plasmids. We also showed that Leptolyngbya BL0902 is amenable to transposon mutagenesis with a Tn5 derivative. To facilitate genetic manipulation of Leptolyngbya BL0902, a conjugal plasmid vector was engineered to carry a trc promoter upstream of a Gateway recombination cassette. These growth properties and genetic tools position Leptolyngbya BL0902 as a model cyanobacterial production strain. PMID:22292073

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

Chan, Chi Ho; Levar, Caleb E.; Zacharoff, Lori

Metal reduction by members of the Geobacteraceae is encoded by multiple gene clusters, and the study of extracellular electron transfer often requires biofilm development on surfaces. Genetic tools that utilize polar antibiotic cassette insertions limit mutant construction and complementation. In addition, unstable plasmids create metabolic burdens that slow growth, and the presence of antibiotics such as kanamycin can interfere with the rate and extent of Geobacter biofilm growth. We report here genetic system improvements for the model anaerobic metal-reducing bacterium Geobacter sulfurreducens. A motile strain of G. sulfurreducens was constructed by precise removal of a transposon interrupting the fgrM flagellarmore » regulator gene using SacB/sucrose counterselection, and Fe(III) citrate reduction was eliminated by deletion of the gene encoding the inner membrane cytochrome imcH. We also show that RK2-based plasmids were maintained in G. sulfurreducens for over 15 generations in the absence of antibiotic selection in contrast to unstable pBBR1 plasmids. Therefore, we engineered a series of new RK2 vectors containing native constitutive Geobacter promoters, and modified one of these promoters for VanR-dependent induction by the small aromatic carboxylic acid vanillate. Inducible plasmids fully complemented Δ imcH mutants for Fe(III) reduction, Mn(IV) oxide reduction, and growth on poised electrodes. A real-time, high-throughput Fe(III) citrate reduction assay is described that can screen numerous G. sulfurreducens strain constructs simultaneously and shows the sensitivity of imcH expression by the vanillate system. Lastly, these tools will enable more sophisticated genetic studies in G. sulfurreducens without polar insertion effects or need for multiple antibiotics.« less

Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with Various Plasmids

PubMed Central

Serror, Pascale; Sasaki, Takashi; Ehrlich, S. Dusko; Maguin, Emmanuelle

2002-01-01

We describe, for the first time, a detailed electroporation procedure for Lactobacillus delbrueckii. Three L. delbrueckii strains were successfully transformed. Under optimal conditions, the transformation efficiency was 104 transformants per μg of DNA. Using this procedure, we identified several plasmids able to replicate in L. delbrueckii and integrated an integrative vector based on phage integrative elements into the L. delbrueckii subsp. bulgaricus chromosome. These vectors provide a good basis for developing molecular tools for L. delbrueckii and open the field of genetic studies in L. delbrueckii. PMID:11772607

Genetic transformation of the fungal pathogen responsible for rice blast disease

PubMed Central

Parsons, Kenneth A.; Chumley, Forrest G.; Valent, Barbara

1987-01-01

The analysis of complex genetic determinants that control the ability of a fungus to colonize its host has been impaired by the lack of sophisticated genetic tools for characterizing important pathogens. We have developed a system for the genetic transformation of Magnaporthe grisea, the causal agent of rice blast disease, to overcome this limitation. A M. grisea arginine auxotroph was shown to contain a mutation (arg3-12) that abolishes ornithine carbamoyltransferase activity. M. grisea strains that contain arg3-12 were used as recipients in transformation experiments with plasmid pMA2, which carries the ArgB+ gene from Aspergillus nidulans. Stable prototrophic transformants arose at a frequency of about 35 per microgram of plasmid DNA. Integration of single or multiple plasmid copies occurred at a single site in the genome of each transformant; rearrangements were often created during integration. When M. grisea genomic segments were incorporated into pMA2, the presence of any one of five different M. grisea segments did not greatly affect the efficiency of transformation. Integration via homologous recombination occurred when the donor plasmid was linearized by cleaving at a unique restriction site within the M. grisea segment. Images PMID:16593854

Single-Vector, Single-Injection Recombinant Vesicular Stomatitis Virus Vaccines Against High-Containment Viruses.

PubMed

Whitt, Michael A; Geisbert, Thomas W; Mire, Chad E

2016-01-01

There are many avenues for making an effective vaccine against viruses. Depending on the virus these can include one of the following: inactivation of whole virions; attenuation of viruses; recombinant viral proteins; non-replication-competent virus particles; or surrogate virus vector systems such as vesicular stomatitis virus (VSV). VSV is a prototypic enveloped animal virus that has been used for over four decades to study virus replication, entry, and assembly due to its ability to replicate to high titers in a wide variety of mammalian and insect cells. The use of reverse genetics to recover infectious and single-cycle replicating VSV from plasmid DNA transfected in cell culture began a revolution in the study of recombinant VSV (rVSV). This platform can be manipulated to study the viral genetic sequences and proteins important in the virus life cycle. Additionally, foreign genes can be inserted between naturally occurring or generated start/stop signals and polyadenylation sites within the VSV genome. VSV has a tolerance for foreign gene expression which has led to numerous rVSVs reported in the literature. Of particular interest are the very effective single-dose rVSV vaccine vectors against high-containment viruses such as filoviruses, henipaviruses, and arenaviruses. Herein we describe the methods for selecting foreign antigenic genes, selecting the location within the VSV genome for insertion, generation of rVSV using reverse genetics, and proper vaccine study designs.

3G vector-primer plasmid for constructing full-length-enriched cDNA libraries.

PubMed

Zheng, Dong; Zhou, Yanna; Zhang, Zidong; Li, Zaiyu; Liu, Xuedong

2008-09-01

We designed a 3G vector-primer plasmid for the generation of full-length-enriched complementary DNA (cDNA) libraries. By employing the terminal transferase activity of reverse transcriptase and the modified strand replacement method, this plasmid (assembled with a polydT end and a deoxyguanosine [dG] end) combines priming full-length cDNA strand synthesis and directional cDNA cloning. As a result, the number of steps involved in cDNA library preparation is decreased while simplifying downstream gene manipulation, sequencing, and subcloning. The 3G vector-primer plasmid method yields fully represented plasmid primed libraries that are equivalent to those made by the SMART (switching mechanism at 5' end of RNA transcript) approach.

Identification of three extra-chromosomal replicons in Leptospira pathogenic strain and development of new shuttle vectors.

PubMed

Zhu, Weinan; Wang, Jin; Zhu, Yongzhang; Tang, Biao; Zhang, Yunyi; He, Ping; Zhang, Yan; Liu, Boyu; Guo, Xiaokui; Zhao, Guoping; Qin, Jinhong

2015-02-15

The genome of pathogenic Leptospira interrogans contains two chromosomes. Plasmids and prophages are known to play specific roles in gene transfer in bacteria and can potentially serve as efficient genetic tools in these organisms. Although plasmids and prophage remnants have recently been reported in Leptospira species, their characteristics and potential applications in leptospiral genetic transformation systems have not been fully evaluated. Three extrachromosomal replicons designated lcp1 (65,732 bp), lcp2 (56,757 bp), and lcp3 (54,986 bp) in the L. interrogans serovar Linhai strain 56609 were identified through whole genome sequencing. All three replicons were stable outside of the bacterial chromosomes. Phage particles were observed in the culture supernatant of 56609 after mitomycin C induction, and lcp3, which contained phage-related genes, was considered to be an inducible prophage. L. interrogans-Escherichia coli shuttle vectors, constructed with the predicted replication elements of single rep or rep combined with parAB loci from the three plasmids were shown to successfully transform into both saprophytic and pathogenic Leptospira species, suggesting an essential function for rep genes in supporting auto-replication of the plasmids. Additionally, a wide distribution of homologs of the three rep genes was identified in L. interrogans isolates, and correlation tests showed that the transformability of the shuttle vectors in L. interrogans isolates depended, to certain extent, on genetic compatibility between the rep sequences of both plasmid and host. Three extrachromosomal replicons co-exist in L. interrogans, one of which we consider to be an inducible prophage. The vectors constructed with the rep genes of the three replicons successfully transformed into saprophytic and pathogenic Leptospira species alike, but this was partly dependent on genetic compatibility between the rep sequences of both plasmid and host.

Construction of new cloning, lacZ reporter and scarless-markerless suicide vectors for genetic studies in Aggregatibacter actinomycetemcomitans

PubMed Central

Juárez-Rodríguez, María Dolores; Torres-Escobar, Ascención; Demuth, Donald R.

2013-01-01

To elucidate the putative function of a gene, effective tools are required for genetic characterization that facilitate its inactivation, deletion or modification on the bacterial chromosome. In the present study, the nucleotide sequence of the Escherichia coli/Aggregatibacter actinomycetemcomitans shuttle vector pYGK was determined, allowing us to redesign and construct a new shuttle cloning vector, pJT4, and promoterless lacZ transcriptional/translational fusion plasmids, pJT3 and pJT5. Plasmids pJT4 and pJT5 contain the origin of replication necessary to maintain shuttle vector replication. In addition, a new suicide vector, pJT1, was constructed for the generation of scarless and markerless deletion mutations of genes in the oral pathogen A. actinomycetemcomitans. Plasmid pJT1 is a pUC-based suicide vector that is counter-selectable for sucrose sensitivity. This vector does not leave antibiotic markers or scars on the chromosome after gene deletion and thus provides the option to combine several mutations in the same genetic background. The effectiveness of pJT1 was demonstrated by the construction of A. actinomycetemcomitans isogenic qseB single deletion (ΔqseB) mutant and lsrRK double deletion mutants (ΔlsrRK). These new vectors may offer alternatives for genetic studies in A. actinomycetemcomitans and other members of the HACEK (Haemophilus spp., A. actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) group of Gram-negative bacteria. PMID:23353051

Genome-Based Genetic Tool Development for Bacillus methanolicus: Theta- and Rolling Circle-Replicating Plasmids for Inducible Gene Expression and Application to Methanol-Based Cadaverine Production.

PubMed

Irla, Marta; Heggeset, Tonje M B; Nærdal, Ingemar; Paul, Lidia; Haugen, Tone; Le, Simone B; Brautaset, Trygve; Wendisch, Volker F

2016-01-01

Bacillus methanolicus is a thermophilic methylotroph able to overproduce amino acids from methanol, a substrate not used for human or animal nutrition. Based on our previous RNA-seq analysis a mannitol inducible promoter and a putative mannitol activator gene mtlR were identified. The mannitol inducible promoter was applied for controlled gene expression using fluorescent reporter proteins and a flow cytometry analysis, and improved by changing the -35 promoter region and by co-expression of the mtlR regulator gene. For independent complementary gene expression control, the heterologous xylose-inducible system from B. megaterium was employed and a two-plasmid gene expression system was developed. Four different replicons for expression vectors were compared with respect to their copy number and stability. As an application example, methanol-based production of cadaverine was shown to be improved from 11.3 to 17.5 g/L when a heterologous lysine decarboxylase gene cadA was expressed from a theta-replicating rather than a rolling-circle replicating vector. The current work on inducible promoter systems and compatible theta- or rolling circle-replicating vectors is an important extension of the poorly developed B. methanolicus genetic toolbox, valuable for genetic engineering and further exploration of this bacterium.

Genome-Based Genetic Tool Development for Bacillus methanolicus: Theta- and Rolling Circle-Replicating Plasmids for Inducible Gene Expression and Application to Methanol-Based Cadaverine Production

PubMed Central

Irla, Marta; Heggeset, Tonje M. B.; Nærdal, Ingemar; Paul, Lidia; Haugen, Tone; Le, Simone B.; Brautaset, Trygve; Wendisch, Volker F.

2016-01-01

Bacillus methanolicus is a thermophilic methylotroph able to overproduce amino acids from methanol, a substrate not used for human or animal nutrition. Based on our previous RNA-seq analysis a mannitol inducible promoter and a putative mannitol activator gene mtlR were identified. The mannitol inducible promoter was applied for controlled gene expression using fluorescent reporter proteins and a flow cytometry analysis, and improved by changing the -35 promoter region and by co-expression of the mtlR regulator gene. For independent complementary gene expression control, the heterologous xylose-inducible system from B. megaterium was employed and a two-plasmid gene expression system was developed. Four different replicons for expression vectors were compared with respect to their copy number and stability. As an application example, methanol-based production of cadaverine was shown to be improved from 11.3 to 17.5 g/L when a heterologous lysine decarboxylase gene cadA was expressed from a theta-replicating rather than a rolling-circle replicating vector. The current work on inducible promoter systems and compatible theta- or rolling circle-replicating vectors is an important extension of the poorly developed B. methanolicus genetic toolbox, valuable for genetic engineering and further exploration of this bacterium. PMID:27713731

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

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.

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.

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.

Plasmid vectors for Xylella fastidiosa utilizing a toxin-antitoxin system for plasmid stability in the absence of antibiotic selection

USDA-ARS?s Scientific Manuscript database

The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacte...

Engineering new mycobacterial vaccine design for HIV–TB pediatric vaccine vectored by lysine auxotroph of BCG

PubMed Central

Saubi, Narcís; Gea-Mallorquí, Ester; Ferrer, Pau; Hurtado, Carmen; Sánchez-Úbeda, Sara; Eto, Yoshiki; Gatell, Josep M; Hanke, Tomáš; Joseph, Joan

2014-01-01

In this study, we have engineered a new mycobacterial vaccine design by using an antibiotic-free plasmid selection system. We assembled a novel Escherichia coli (E. coli)–mycobacterial shuttle plasmid p2auxo.HIVA, expressing the HIV-1 clade A immunogen HIVA. This shuttle vector employs an antibiotic resistance-free mechanism for plasmid selection and maintenance based on glycine complementation in E. coli and lysine complementation in mycobacteria. This plasmid was first transformed into glycine auxotroph of E. coli strain and subsequently transformed into lysine auxotroph of Mycobacterium bovis BCG strain to generate vaccine BCG.HIVA2auxo. We demonstrated that the episomal plasmid p2auxo.HIVA was stable in vivo over a 7-week period and genetically and phenotypically characterized the BCG.HIVA2auxo vaccine strain. The BCG.HIVA2auxo vaccine in combination with modified vaccinia virus Ankara (MVA). HIVA was safe and induced HIV-1 and Mycobacterium tuberculosis-specific interferon-γ-producing T-cell responses in adult BALB/c mice. Polyfunctional HIV-1-specific CD8+ T cells, which produce interferon-γ and tumor necrosis factor-α and express the degranulation marker CD107a, were induced. Thus, we engineered a novel, safer, good laboratory practice–compatible BCG-vectored vaccine using prototype immunogen HIVA. This antibiotic-free plasmid selection system based on “double” auxotrophic complementation might be a new mycobacterial vaccine platform to develop not only recombinant BCG-based vaccines expressing second generation of HIV-1 immunogens but also other major pediatric pathogens to prime protective response soon after birth. PMID:26015961

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.

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.

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

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.

CRISPR/Cas9-mediated gene knockout screens and target identification via whole-genome sequencing uncover host genes required for picornavirus infection.

PubMed

Kim, Heon Seok; Lee, Kyungjin; Bae, Sangsu; Park, Jeongbin; Lee, Chong-Kyo; Kim, Meehyein; Kim, Eunji; Kim, Minju; Kim, Seokjoong; Kim, Chonsaeng; Kim, Jin-Soo

2017-06-23

Several groups have used genome-wide libraries of lentiviruses encoding small guide RNAs (sgRNAs) for genetic screens. In most cases, sgRNA expression cassettes are integrated into cells by using lentiviruses, and target genes are statistically estimated by the readout of sgRNA sequences after targeted sequencing. We present a new virus-free method for human gene knockout screens using a genome-wide library of CRISPR/Cas9 sgRNAs based on plasmids and target gene identification via whole-genome sequencing (WGS) confirmation of authentic mutations rather than statistical estimation through targeted amplicon sequencing. We used 30,840 pairs of individually synthesized oligonucleotides to construct the genome-scale sgRNA library, collectively targeting 10,280 human genes ( i.e. three sgRNAs per gene). These plasmid libraries were co-transfected with a Cas9-expression plasmid into human cells, which were then treated with cytotoxic drugs or viruses. Only cells lacking key factors essential for cytotoxic drug metabolism or viral infection were able to survive. Genomic DNA isolated from cells that survived these challenges was subjected to WGS to directly identify CRISPR/Cas9-mediated causal mutations essential for cell survival. With this approach, we were able to identify known and novel genes essential for viral infection in human cells. We propose that genome-wide sgRNA screens based on plasmids coupled with WGS are powerful tools for forward genetics studies and drug target discovery. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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

AFLP fingerprinting: an efficient technique for detecting genetic variation of Xanthomonas axonopodis pv. manihotis.

PubMed

Restrepo, S; Duque, M; Tohme, J; Verdier, V

1999-01-01

Xanthomonas axonopodis pv. manihotis (Xam) is the causative agent of cassava bacterial blight (CBB), a worldwide disease that is particularly destructive in South America and Africa. CBB is controlled essentially through the use of resistant varieties. To develop an appropriate disease management strategy, the genetic diversity of the pathogen's populations must be assessed. Until now, the genetic diversity of Xam was characterized by RFLP analyses using ribotyping, and plasmid and genomic Xam probes. We used AFLP (amplified fragment length polymorphism), a novel PCR-based technique, to characterize the genetic diversity of Colombian Xam isolates. Six Xam strains were tested with 65 AFLP primer combinations to identify the best selective primers. Eight primer combinations were selected according to their reproducibility, number of polymorphic bands and polymorphism detected between Xam strains. Forty-seven Xam strains, originating from different Colombian ecozones, were analysed with the selected combinations. Results obtained with AFLP are consistent with those obtained with RFLP, using plasmid DNA as a probe. Some primer combinations differentiated Xam strains that were not distinguished by RFLP analyses, thus AFLP fingerprinting allowed a better definition of the genetic relationships between Xam strains.

Mobilome and genetic modification of bifidobacteria.

PubMed

Guglielmetti, S; Mayo, B; Álvarez-Martín, P

2013-06-01

Until recently, proper development of molecular studies in Bifidobacterium species has been hampered by growth difficulties, because of their exigent nutritive requirements, oxygen sensitivity and lack of efficient genetic tools. These studies, however, are critical to uncover the cross-talk between bifidobacteria and their hosts' cells and to prove unequivocally the supposed beneficial effects provided through the endogenous bifidobacterial populations or after ingestion as probiotics. The genome sequencing projects of different bifidobacterial strains have provided a wealth of genetic data that will be of much help in deciphering the molecular basis of the physiological properties of bifidobacteria. To this end, the purposeful development of stable cloning and expression vectors based on robust replicons - either from temperate phages or resident plasmids - is still needed. This review addresses the current knowledge on the mobile genetic elements of bifidobacteria (prophages, plasmids and transposons) and summarises the different types of vectors already available, together with the transformation procedures for introducing DNA into the cells. It also covers recent molecular studies performed with such vectors and incipient results on the genetic modification of these organisms, establishing the basis that would allow the use of bifidobacteria for future biotechnological applications.

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

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

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

High Diversity of Extended-Spectrum β-Lactamases in Escherichia coli Isolates from Italian Broiler Flocks ▿

PubMed Central

Bortolaia, Valeria; Guardabassi, Luca; Trevisani, Marcello; Bisgaard, Magne; Venturi, Luciano; Bojesen, Anders Miki

2010-01-01

We characterized 67 Escherichia coli isolates with reduced susceptibility to cefotaxime or ceftiofur obtained from healthy broilers housed in five Italian farms. The blaCTX-M-1, blaCTX-M-32 and blaSHV-12 β-lactamase genes were identified on IncI1, IncN, or IncFIB plasmids. Considerable genetic diversity was detected among the extended-spectrum β-lactamase (ESBL)-producing isolates, and we identified indistinguishable strains in unrelated farms and indistinguishable plasmids in genetically unrelated strains. The detection of highly mobile plasmids suggests a potential animal reservoir for β-lactamase genes. PMID:20100875

Bacterial plasmid transfer under space flight conditions: The Mobilisatsia experience

NASA Astrophysics Data System (ADS)

de Boever, P.; Ilyin, V.; Mahillon, J.; Mergeay, M.

Background Microorganisms are subject to a genetic evolution which may lead to the capacity to colonize new environments and to cause infections Central players in this evolutionary process are mobile genetic elements phages plasmids and transposons The latter help to mobilize and reorganize genes be it within a given genome intragenomic mobility or between bacterial cells intercellular mobility Confined environment and space flight related factors such as microgravity and cosmic radiation may influence the frequency with which mobile genetic elements are exchanged between microorganisms Aim Within the frame of the Mobilisatsia experiment a triparental microbial plasmid transfer was promoted aboard the International Space Station ISS The efficiency of the plasmid exchange process was compared with a synchronously performed ground control experiment An experiment was carried out with well-characterized Gram-negative test strains and one experiment was done with Gram-positive test strains Results The experiment took place during the Soyouz Mission 8 to the ISS from April 19th until April 30th 2004 Liquid cultures of the bacterial strains Cupriavidus metallidurans AE815 final recipient Escherichia coli CM1962 carrying a mobilisable vector with a nickel-resistance marker and E coli CM140 carrying the Broad Host Range plasmid RP4 for the Gram-negative experiment and Bacillus thuringiensis Bti AND931 carrying the conjugative plasmid pXO16 Bti 4Q7 with mobilisable vector pC194 carrying a resistance to chloramphenicol and Bti GBJ002

Dcm methylation is detrimental to plasmid transformation in Clostridium thermocellum

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

Guss, Adam M; Olson, Daniel G.; Caiazza, Nicky

2012-01-01

BACKGROUND: Industrial production of biofuels and other products by cellulolytic microorganisms is of interest but hindered by the nascent state of genetic tools. Although a genetic system for Clostridium thermocellum DSM1313 has recently been developed, available methods achieve relatively low efficiency and similar plasmids can transform C. thermocellum at dramatically different efficiencies. RESULTS: We report an increase in transformation efficiency of C. thermocellum for a variety of plasmids by using DNA that has been methylated by Escherichia coli Dam but not Dcm methylases. When isolated from a dam+ dcm+ E. coli strain, pAMG206 transforms C. thermocellum 100-fold better than themore » similar plasmid pAMG205, which contains an additional Dcm methylation site in the pyrF gene. Upon removal of Dcm methylation, transformation with pAMG206 showed a four- to seven-fold increase in efficiency; however, transformation efficiency of pAMG205 increased 500-fold. Removal of the Dcm methylation site from the pAM205 pyrF gene via silent mutation resulted in increased transformation efficiencies equivalent to that of pAMG206. Upon proper methylation, transformation efficiency of plasmids bearing the pMK3 and pB6A origins of replication increased ca. three orders of magnitude. CONCLUSION: E. coli Dcm methylation decreases transformation efficiency in C. thermocellum DSM1313. The use of properly methylated plasmid DNA should facilitate genetic manipulation of this industrially relevant bacterium.« less

Blue genes: An integrative laboratory to differentiate genetic transformation from gene mutation for underclassmen.

PubMed

Militello, Kevin T; Chang, Ming-Mei; Simon, Robert D; Lazatin, Justine C

2016-01-01

The ability of students to understand the relationship between genotype and phenotype, and the mechanisms by which genotypes and phenotypes can change is essential for students studying genetics. To this end, we have developed a four-week laboratory called Blue Genes, which is designed to help novice students discriminate between two mechanisms by which the genetic material can be altered: genetic transformation and gene mutation. In the first week of the laboratory, students incubate a plasmid DNA with calcium chloride-treated Escherichia coli JM109 cells and observe a phenotype change from ampicillin sensitive to ampicillin resistant and from white color to blue color on plates containing 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) and isopropyl β-D-thiogalactopyranoside (IPTG). Over the course of the next three weeks, students use a battery of approaches including plasmid DNA isolation experiments, restriction maps, and PCR to differentiate between mutation and transformation. The students ultimately come to the conclusion that the changes in phenotypes are due to genetic transformation and not mutation based on the evidence generated over the four-week period. Pre-laboratory tests and post-laboratory tests indicate that this set of exercises is successful in helping students differentiate between transformation and mutation. The laboratory is designed for underclassmen and is a good prerequisite for an apprentice-based research opportunity, although it is not designed as a class based research experience. Potential modifications and future directions of the laboratory based upon student experiences and assessment are presented. © 2015 The International Union of Biochemistry and Molecular Biology.

Diversity of Clostridium perfringens isolates from various sources and prevalence of conjugative plasmids.

PubMed

Park, Miseon; Deck, Joanna; Foley, Steven L; Nayak, Rajesh; Songer, J Glenn; Seibel, Janice R; Khan, Saeed A; Rooney, Alejandro P; Hecht, David W; Rafii, Fatemeh

2016-04-01

Clostridium perfringens is an important pathogen, causing food poisoning and other mild to severe infections in humans and animals. Some strains of C. perfringens contain conjugative plasmids, which may carry antimicrobial resistance and toxin genes. We studied genomic and plasmid diversity of 145 C. perfringens type A strains isolated from soils, foods, chickens, clinical samples, and domestic animals (porcine, bovine and canine), from different geographic areas in the United States between 1994 and 2006, using multiple-locus variable-number tandem repeat analysis (MLVA) and/or pulsed-field gel electrophoresis (PFGE). MLVA detected the genetic diversity in a majority of the isolates. PFGE, using SmaI and KspI, confirmed the MLVA results but also detected differences among the strains that could not be differentiated by MLVA. All of the PFGE profiles of the strains were different, except for a few of the epidemiologically related strains, which were identical. The PFGE profiles of strains isolated from the same domestic animal species were clustered more closely with each other than with other strains. However, a variety of C. perfringens strains with distinct genetic backgrounds were found among the clinical isolates. Variation was also observed in the size and number of plasmids in the strains. Primers for the internal fragment of a conjugative tcpH gene of C. perfringens plasmid pCPF4969 amplified identical size fragments from a majority of strains tested; and this gene hybridized to the various-sized plasmids of these strains. The sequences of the PCR-amplified tcpH genes from 12 strains showed diversity among the tcpH genes. Regardless of the sources of the isolates, the genetic diversity of C. perfringens extended to the plasmids carrying conjugative genes. Published by Elsevier Ltd.

Plasmidome Interchange between Clostridium botulinum, Clostridium novyi and Clostridium haemolyticum Converts Strains of Independent Lineages into Distinctly Different Pathogens

PubMed Central

Skarin, Hanna; Segerman, Bo

2014-01-01

Clostridium botulinum (group III), Clostridium novyi and Clostridium haemolyticum are well-known pathogens causing animal botulism, gas gangrene/black disease, and bacillary hemoglobinuria, respectively. A close genetic relationship exists between the species, which has resulted in the collective term C. novyi sensu lato. The pathogenic traits in these species, e.g., the botulinum neurotoxin and the novyi alpha toxin, are mainly linked to a large plasmidome consisting of plasmids and circular prophages. The plasmidome of C. novyi sensu lato has so far been poorly characterized. In this study we explored the genomic relationship of a wide range of strains of C. novyi sensu lato with a special focus on the dynamics of the plasmidome. Twenty-four genomes were sequenced from strains selected to represent as much as possible the genetic diversity in C. novyi sensu lato. Sixty-one plasmids were identified in these genomes and 28 of them were completed. The genomic comparisons revealed four separate lineages, which did not strictly correlate with the species designations. The plasmids were categorized into 13 different plasmid groups on the basis of their similarity and conservation of plasmid replication or partitioning genes. The plasmid groups, lineages and species were to a large extent entwined because plasmids and toxin genes had moved across the lineage boundaries. This dynamic process appears to be primarily driven by phages. We here present a comprehensive characterization of the complex species group C. novyi sensu lato, explaining the intermixed genetic properties. This study also provides examples how the reorganization of the botulinum toxin and the novyi alpha toxin genes within the plasmidome has affected the pathogenesis of the strains. PMID:25254374

Plasmidome interchange between Clostridium botulinum, Clostridium novyi and Clostridium haemolyticum converts strains of independent lineages into distinctly different pathogens.

PubMed

Skarin, Hanna; Segerman, Bo

2014-01-01

Clostridium botulinum (group III), Clostridium novyi and Clostridium haemolyticum are well-known pathogens causing animal botulism, gas gangrene/black disease, and bacillary hemoglobinuria, respectively. A close genetic relationship exists between the species, which has resulted in the collective term C. novyi sensu lato. The pathogenic traits in these species, e.g., the botulinum neurotoxin and the novyi alpha toxin, are mainly linked to a large plasmidome consisting of plasmids and circular prophages. The plasmidome of C. novyi sensu lato has so far been poorly characterized. In this study we explored the genomic relationship of a wide range of strains of C. novyi sensu lato with a special focus on the dynamics of the plasmidome. Twenty-four genomes were sequenced from strains selected to represent as much as possible the genetic diversity in C. novyi sensu lato. Sixty-one plasmids were identified in these genomes and 28 of them were completed. The genomic comparisons revealed four separate lineages, which did not strictly correlate with the species designations. The plasmids were categorized into 13 different plasmid groups on the basis of their similarity and conservation of plasmid replication or partitioning genes. The plasmid groups, lineages and species were to a large extent entwined because plasmids and toxin genes had moved across the lineage boundaries. This dynamic process appears to be primarily driven by phages. We here present a comprehensive characterization of the complex species group C. novyi sensu lato, explaining the intermixed genetic properties. This study also provides examples how the reorganization of the botulinum toxin and the novyi alpha toxin genes within the plasmidome has affected the pathogenesis of the strains.

Characterization of a collection of plasmid-containing bacteria isolated from an on-farm biopurification system used for pesticide removal.

PubMed

Martini, María Carla; Albicoro, Francisco Javier; Nour, Eman; Schlüter, Andreas; van Elsas, Jan Dirk; Springael, Dirk; Smalla, Kornelia; Pistorio, Mariano; Lagares, Antonio; Del Papa, María Florencia

2015-07-01

Biopurification systems (BPS) are complex soil-related and artificially-generated environments usually designed for the removal of toxic compounds from contaminated wastewaters. The present study has been conducted to isolate and characterize a collection of cultivable plasmid-carrying bacterial isolates recovered from a BPS established for the decontamination of wastewater generated in a farmyard. Out of 1400 isolates, a collection of 75 plasmid-containing bacteria was obtained, of which 35 representative isolates comprising in total at least 50 plasmids were chosen for further characterization. Bacterial hosts were taxonomically assigned by 16S ribosomal RNA gene sequencing and phenotypically characterized according to their ability to grow in presence of different antibiotics and heavy metals. The study demonstrated that a high proportion of the isolates was tolerant to antibiotics and/or heavy metals, highlighting the on-farm BPS enrichment in such genetic traits. Several plasmids conferring such resistances in the bacterial collection were detected to be either mobilizable or selftransmissible. Occurrence of broad host range plasmids of the incompatibility groups IncP, IncQ, IncN and IncW was examined with positive results only for the first group. Presence of the IS1071 insertion sequence, frequently associated with xenobiotics degradation genes, was detected in DNA obtained from 24 of these isolates, strongly suggesting the presence of yet-hidden catabolic activities in the collection of isolates. The results showed a remarkable diversity in the plasmid mobilome of cultivable bacteria in the BPS with the presence of abundant resistance markers of different types, thus providing a suitable environment to investigate the genetic structure of the mobile genetic pool in a model on-farm biofilter for wastewater decontamination in intensive agricultural production. Copyright © 2015 Elsevier Inc. All rights reserved.

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

Chauhan, Archana; Layton, Alice; Williams, Daniel W

Pseudomonas fluorescens strain HK44 (DSM 6700) is a genetically engineered lux-based bioluminescent bioreporter. Here we report the draft genome sequence of strain HK44. Annotation of {approx}6.1 Mb sequence indicates that 30% of the traits are unique and distributed over 5 genomic islands, a prophage and two plasmids.

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.

Multiplexed precision genome editing with trackable genomic barcodes in yeast.

PubMed

Roy, Kevin R; Smith, Justin D; Vonesch, Sibylle C; Lin, Gen; Tu, Chelsea Szu; Lederer, Alex R; Chu, Angela; Suresh, Sundari; Nguyen, Michelle; Horecka, Joe; Tripathi, Ashutosh; Burnett, Wallace T; Morgan, Maddison A; Schulz, Julia; Orsley, Kevin M; Wei, Wu; Aiyar, Raeka S; Davis, Ronald W; Bankaitis, Vytas A; Haber, James E; Salit, Marc L; St Onge, Robert P; Steinmetz, Lars M

2018-07-01

Our understanding of how genotype controls phenotype is limited by the scale at which we can precisely alter the genome and assess the phenotypic consequences of each perturbation. Here we describe a CRISPR-Cas9-based method for multiplexed accurate genome editing with short, trackable, integrated cellular barcodes (MAGESTIC) in Saccharomyces cerevisiae. MAGESTIC uses array-synthesized guide-donor oligos for plasmid-based high-throughput editing and features genomic barcode integration to prevent plasmid barcode loss and to enable robust phenotyping. We demonstrate that editing efficiency can be increased more than fivefold by recruiting donor DNA to the site of breaks using the LexA-Fkh1p fusion protein. We performed saturation editing of the essential gene SEC14 and identified amino acids critical for chemical inhibition of lipid signaling. We also constructed thousands of natural genetic variants, characterized guide mismatch tolerance at the genome scale, and ascertained that cryptic Pol III termination elements substantially reduce guide efficacy. MAGESTIC will be broadly useful to uncover the genetic basis of phenotypes in yeast.

Plasmid Vectors and Molecular Building Blocks for the Development of Genetic Manipulation Tools for Trypanosoma cruzi

PubMed Central

Bouvier, León A.; Cámara, María de los Milagros; Canepa, Gaspar E.; Miranda, Mariana R.; Pereira, Claudio A.

2013-01-01

The post genomic era revealed the need for developing better performing, easier to use and more sophisticated genetic manipulation tools for the study of Trypanosoma cruzi, the etiological agent of Chagas disease. In this work a series of plasmids that allow genetic manipulation of this protozoan parasite were developed. First of all we focused on useful tools to establish selection strategies for different strains and which can be employed as expression vectors. On the other hand molecular building blocks in the form of diverse selectable markers, modifiable fluorescent protein and epitope-tag coding sequences were produced. Both types of modules were harboured in backbone molecules conceived to offer multiple construction and sub-cloning strategies. These can be used to confer new properties to already available genetic manipulation tools or as starting points for whole novel designs. The performance of each plasmid and building block was determined independently. For illustration purposes, some simple direct practical applications were conducted. PMID:24205392

Scarless genome editing and stable inducible expression vectors for Geobacter sulfurreducens

DOE PAGES

Chan, Chi Ho; Levar, Caleb E.; Zacharoff, Lori; ...

2015-08-07

Metal reduction by members of the Geobacteraceae is encoded by multiple gene clusters, and the study of extracellular electron transfer often requires biofilm development on surfaces. Genetic tools that utilize polar antibiotic cassette insertions limit mutant construction and complementation. In addition, unstable plasmids create metabolic burdens that slow growth, and the presence of antibiotics such as kanamycin can interfere with the rate and extent of Geobacter biofilm growth. We report here genetic system improvements for the model anaerobic metal-reducing bacterium Geobacter sulfurreducens. A motile strain of G. sulfurreducens was constructed by precise removal of a transposon interrupting the fgrM flagellarmore » regulator gene using SacB/sucrose counterselection, and Fe(III) citrate reduction was eliminated by deletion of the gene encoding the inner membrane cytochrome imcH. We also show that RK2-based plasmids were maintained in G. sulfurreducens for over 15 generations in the absence of antibiotic selection in contrast to unstable pBBR1 plasmids. Therefore, we engineered a series of new RK2 vectors containing native constitutive Geobacter promoters, and modified one of these promoters for VanR-dependent induction by the small aromatic carboxylic acid vanillate. Inducible plasmids fully complemented Δ imcH mutants for Fe(III) reduction, Mn(IV) oxide reduction, and growth on poised electrodes. A real-time, high-throughput Fe(III) citrate reduction assay is described that can screen numerous G. sulfurreducens strain constructs simultaneously and shows the sensitivity of imcH expression by the vanillate system. Lastly, these tools will enable more sophisticated genetic studies in G. sulfurreducens without polar insertion effects or need for multiple antibiotics.« less

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

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

Multilevel population genetic analysis of vanA and vanB Enterococcus faecium causing nosocomial outbreaks in 27 countries (1986-2012).

PubMed

Freitas, Ana R; Tedim, Ana P; Francia, Maria V; Jensen, Lars B; Novais, Carla; Peixe, Luísa; Sánchez-Valenzuela, Antonio; Sundsfjord, Arnfinn; Hegstad, Kristin; Werner, Guido; Sadowy, Ewa; Hammerum, Anette M; Garcia-Migura, Lourdes; Willems, Rob J; Baquero, Fernando; Coque, Teresa M

2016-12-01

Vancomycin-resistant Enterococcus faecium (VREfm) have been increasingly reported since the 1980s. Despite the high number of published studies about VRE epidemiology, the dynamics and evolvability of these microorganisms are still not fully understood. A multilevel population genetic analysis of VREfm outbreak strains since 1986, representing the first comprehensive characterization of plasmid content in E. faecium, was performed to provide a detailed view of potential transmissible units. From a comprehensive MeSH search, we identified VREfm strains causing hospital outbreaks (1986-2012). In total, 53 VanA and 18 VanB isolates (27 countries, 5 continents) were analysed and 82 vancomycin-susceptible E. faecium (VSEfm) were included for comparison. Clonal relatedness was established by PFGE and MLST (goeBURST/Bayesian Analysis of Population Structure, BAPS). Characterization of van transposons (PCR mapping, RFLP, sequencing), plasmids (transfer, ClaI-RFLP, PCR typing of relaxases, replication-initiation proteins and toxin-antitoxin systems, hybridization, sequencing), bacteriocins and virulence determinants (PCR, hybridization, sequencing) was performed. VREfm were mainly associated with major human lineages ST17, ST18 and ST78. VREfm and VSEfm harboured plasmids of different families [RCR, small theta plasmids, RepA_N (pRUM/pLG1) and Inc18] able to yield mosaic elements. Tn1546-vanA was mainly located on pRUM/Axe-Txe (USA) and Inc18-pIP186 (Europe) plasmids. The VanB2 type (Tn5382/Tn1549) was predominant among VanB strains (chromosome and plasmids). Both strains and plasmids contributed to the spread and persistence of vancomycin resistance among E. faecium. Horizontal gene transfer events among genetic elements from different clonal lineages (same or different species) result in chimeras with different stability and host range, complicating the surveillance of epidemic plasmids. © 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.

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.

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

Retroviral mutation rates and A-to-G hypermutations during different stages of retroviral replication.

PubMed Central

Kim, T; Mudry, R A; Rexrode, C A; Pathak, V K

1996-01-01

Retroviruses mutate at a high rate in vivo during viral replication. Mutations may occur during proviral transcription by RNA polymerase II, during minus-strand DNA synthesis (RNA template) by viral reverse transcriptase, or during plus-strand DNA synthesis (DNA template) by reverse transcriptase. To determine the contributions of different stages of replication to the retroviral mutation rates, we developed a spleen necrosis virus-based in vivo system to selectively identify mutations occurring during the early stage (RNA transcription plus minus-strand synthesis) and the late stage (plus-strand synthesis plus DNA repair). A lacZalpha reporter gene was inserted into the long terminal repeat (LTR) of a spleen necrosis virus shuttle vector, and proviruses were recovered from infected cells as plasmids containing either one or both LTRs. Plasmids containing both LTRs generated a mutant phenotype only if the lacZalpha genes in both LTRs were mutated, which is most likely to occur during the early stage. Mutant phenotypes were identified from plasmids containing one LTR regardless of the stage at which the mutations occurred. Thus, mutant frequencies obtained after recovery of plasmids containing both LTRs or one LTR provided early-stage and total mutation rates, respectively. Analysis of 56,409 proviruses suggested that the retroviral mutation rates during the early and late stages of replication were equal or within twofold of each other. In addition, two mutants with A-to-G hypermutations were discovered, suggesting a role for mammalian double-stranded RNA adenosine deaminase enzyme in retroviral mutations. These experiments provide a system to selectively identify mutations in the early stage of retroviral replication and to provide upper and lower limits to the in vivo mutation rates during minus-strand and plus-strand synthesis, respectively. PMID:8892879

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

Rescue of a Porcine Anellovirus (Torque Teno Sus Virus 2) from Cloned Genomic DNA in Pigs

PubMed Central

Huang, Yao-Wei; Patterson, Abby R.; Opriessnig, Tanja; Dryman, Barbara A.; Gallei, Andreas; Harrall, Kylie K.; Vaughn, Eric M.; Roof, Michael B.

2012-01-01

Anelloviruses are a group of single-stranded circular DNA viruses infecting humans and other animal species. Animal models combined with reverse genetic systems of anellovirus have not been developed. We report here the construction and initial characterization of full-length DNA clones of a porcine anellovirus, torque teno sus virus 2 (TTSuV2), in vitro and in vivo. We first demonstrated that five cell lines, including PK-15 cells, are free of TTSuV1 or TTSuV2 contamination, as determined by a real-time PCR and an immunofluorescence assay (IFA) using anti-TTSuV antibodies. Recombinant plasmids harboring monomeric or tandem-dimerized genomic DNA of TTSuV2 from the United States and Germany were constructed. Circular TTSuV2 genomic DNA with or without introduced genetic markers and tandem-dimerized TTSuV2 plasmids were transfected into PK-15 cells, respectively. Splicing of viral mRNAs was identified in transfected cells. Expression of TTSuV2-specific open reading frame 1 (ORF1) in cell nuclei, especially in nucleoli, was detected by IFA. However, evidence of productive TTSuV2 infection was not observed in 12 different cell lines transfected with the TTSuV2 DNA clones. Transfection with circular DNA from a TTSuV2 deletion mutant did not produce ORF1 protein, suggesting that the observed ORF1 expression is driven by TTSuV2 DNA replication in cells. Pigs inoculated with either the tandem-dimerized clones or circular genomic DNA of U.S. TTSuV2 developed viremia, and the introduced genetic markers were retained in viral DNA recovered from the sera of infected pigs. The availability of an infectious DNA clone of TTSuV2 will facilitate future study of porcine anellovirus pathogenesis and biology. PMID:22491450

Study of the genetics and regulation of methane oxidation. Progress report, first year, August 1, 1980-July 31, 1981. [Methylobacterium ethanolicum

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

Not Available

The plasmids present in M. ethanolicum have been characterized as to size, relatedness, and curing rate. Auxotrophs have been isolated and are being tested for the ability of several plasmids to promote mobilization of these markers. A cloning vector has been identified which can be used not only to clone the genes of interest but to isolate mutants in these genes and place a selectable marker on each of the plasmids. Isolation of a series of methane and methanol mutants, determination of which of the plasmids carries the mtn gene(s), and identification of methane-specific proteins on two-dimensonal O'Farrell gels shouldmore » all be completed shortly. In addition, the cloning of the methane genes and development of a genetic system should be well underway. A more detailed appraisal of future experiments is presented in the accompanying renewal proposal. (ERB)« less

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

Expression Plasmids for Use in Candida glabrata

PubMed Central

Zordan, Rebecca E.; Ren, Yuxia; Pan, Shih-Jung; Rotondo, Giuseppe; Peñas, Alejandro De Las; Iluore, Joseph; Cormack, Brendan P.

2013-01-01

We describe a series of CEN/ARS episomal plasmids containing different Candida glabrata promoters, allowing for a range of constitutive or regulated expression of proteins in C. glabrata. The set of promoters includes three constitutive promoters (EGD2pr, HHT2pr, PDC1pr), two macrophage/phagocytosis-induced promoters (ACO2pr, LYS21pr), and one nutritionally regulated promoter (MET3pr). Each promoter was cloned into two plasmid backbones that differ in their selectable marker, URA3, or the dominant-selectable NAT1 gene, which confers resistance to the drug nourseothricin. Expression from the 12 resulting plasmids was assessed using GFP as a reporter and flow cytometry or quantitative reverse-transcription polymerase chain reaction to assess expression levels. Together this set of plasmids expands the toolkit of expression vectors available for use with C. glabrata. PMID:23934995

Biochemistry and genetics of autotrophy in Methanococcus. Progress report

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

Whitman, W.B.

In the last two years of this research, the most exciting results have come from the work on the genetics of methanococci. First, the author demonstrated that the cryptic plasmid from Methanococcus maripaludis C5, pURB500, could be transformed into Methanococcus maripaludis JJ. Strain JJ is the type strain of M. maripaludis and has only about 65% DNA:DNA hybridization to strain C5. Because of the low relatedness of these strains, it was not obvious that pURB500 could be transferred between them. This goal was achieved by first transforming strain C5 with a series of suicide plasmids containing the pac cassette, whichmore » possessed the selectable puromycin resistance marker, and different cloned fragments of pURB500. From the puromycin-resistant transformants, a plasmid was isolated that transformed strain JJ. However, when this plasmid was electroporated into E. coli, only rearrangement products were obtained that contained small portions of the original pURB500. These plasmids no longer transformed Methanococcus. While these experiments did not yield a shuttle vector, they demonstrated that pURB500 could replicate in strain JJ.« less

Construction of a self-cloning sake yeast that overexpresses alcohol acetyltransferase gene by a two-step gene replacement protocol.

PubMed

Hirosawa, I; Aritomi, K; Hoshida, H; Kashiwagi, S; Nishizawa, Y; Akada, R

2004-07-01

The commercial application of genetically modified industrial microorganisms has been problematic due to public concerns. We constructed a "self-cloning" sake yeast strain that overexpresses the ATF1 gene encoding alcohol acetyltransferase, to improve the flavor profile of Japanese sake. A constitutive yeast overexpression promoter, TDH3p, derived from the glyceraldehyde-3-phosphate dehydrogenase gene from sake yeast was fused to ATF1; and the 5' upstream non-coding sequence of ATF1 was further fused to TDH3p-ATF1. The fragment was placed on a binary vector, pGG119, containing a drug-resistance marker for transformation and a counter-selection marker for excision of unwanted DNA. The plasmid was integrated into the ATF1 locus of a sake yeast strain. This integration constructed tandem repeats of ATF1 and TDH3p-ATF1 sequences, between which the plasmid was inserted. Loss of the plasmid, which occurs through homologous recombination between either the TDH3p downstream ATF1 repeats or the TDH3p upstream repeat sequences, was selected by growing transformants on counter-selective medium. Recombination between the downstream repeats led to reversion to a wild type strain, but that between the upstream repeats resulted in a strain that possessed TDH3p-ATF1 without the extraneous DNA sequences. The self-cloning TDH3p-ATF1 yeast strain produced a higher amount of isoamyl acetate. This is the first expression-controlled self-cloning industrial yeast.

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

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

Gemini surfactants mediate efficient mitochondrial gene delivery and expression.

PubMed

Cardoso, Ana M; Morais, Catarina M; Cruz, A Rita; Cardoso, Ana L; Silva, Sandra G; do Vale, M Luísa; Marques, Eduardo F; Pedroso de Lima, Maria C; Jurado, Amália S

2015-03-02

Gene delivery targeting mitochondria has the potential to transform the therapeutic landscape of mitochondrial genetic diseases. Taking advantage of the nonuniversal genetic code used by mitochondria, a plasmid DNA construct able to be specifically expressed in these organelles was designed by including a codon, which codes for an amino acid only if read by the mitochondrial ribosomes. In the present work, gemini surfactants were shown to successfully deliver plasmid DNA to mitochondria. Gemini surfactant-based DNA complexes were taken up by cells through a variety of routes, including endocytic pathways, and showed propensity for inducing membrane destabilization under acidic conditions, thus facilitating cytoplasmic release of DNA. Furthermore, the complexes interacted extensively with lipid membrane models mimicking the composition of the mitochondrial membrane, which predicts a favored interaction of the complexes with mitochondria in the intracellular environment. This work unravels new possibilities for gene therapy toward mitochondrial diseases.

Development of transgenic strains for the biological control of the Mexican fruit fly, Anastrepha ludens

USDA-ARS?s Scientific Manuscript database

The Mexican fruit fly, Anastrepha ludens, is a highly significant agricultural pest species that has been genetically transformed with a piggyBac¬-based transposon vector system using independent vector and transposase helper plasmids. Estimated germ-line transformation frequencies were approximate...

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

Phytotracker, an information management system for easy recording and tracking of plants, seeds and plasmids

PubMed Central

2012-01-01

Background A large number of different plant lines are produced and maintained in a typical plant research laboratory, both as seed stocks and in active growth. These collections need careful and consistent management to track and maintain them properly, and this is a particularly pressing issue in laboratories undertaking research involving genetic manipulation due to regulatory requirements. Researchers and PIs need to access these data and collections, and therefore an easy-to-use plant-oriented laboratory information management system that implements, maintains and displays the information in a simple and visual format would be of great help in both the daily work in the lab and in ensuring regulatory compliance. Results Here, we introduce ‘Phytotracker’, a laboratory management system designed specifically to organise and track plasmids, seeds and growing plants that can be used in mixed platform environments. Phytotracker is designed with simplicity of user operation and ease of installation and management as the major factor, whilst providing tracking tools that cover the full range of activities in molecular genetics labs. It utilises the cross-platform Filemaker relational database, which allows it to be run as a stand-alone or as a server-based networked solution available across all workstations in a lab that can be internet accessible if desired. It can also be readily modified or customised further. Phytotracker provides cataloguing and search functions for plasmids, seed batches, seed stocks and plants growing in pots or trays, and allows tracking of each plant from seed sowing, through harvest to the new seed batch and can print appropriate labels at each stage. The system enters seed information as it is transferred from the previous harvest data, and allows both selfing and hybridization (crossing) to be defined and tracked. Transgenic lines can be linked to their plasmid DNA source. This ease of use and flexibility helps users to reduce their time needed to organise their plants, seeds and plasmids and to maintain laboratory continuity involving multiple workers. Conclusion We have developed and used Phytotracker for over five years and have found it has been an intuitive, powerful and flexible research tool in organising our plasmid, seed and plant collections requiring minimal maintenance and training for users. It has been developed in an Arabidopsis molecular genetics environment, but can be readily adapted for almost any plant laboratory research. PMID:23062011

Phytotracker, an information management system for easy recording and tracking of plants, seeds and plasmids.

PubMed

Nieuwland, Jeroen; Sornay, Emily; Marchbank, Angela; de Graaf, Barend Hj; Murray, James Ah

2012-10-13

A large number of different plant lines are produced and maintained in a typical plant research laboratory, both as seed stocks and in active growth. These collections need careful and consistent management to track and maintain them properly, and this is a particularly pressing issue in laboratories undertaking research involving genetic manipulation due to regulatory requirements. Researchers and PIs need to access these data and collections, and therefore an easy-to-use plant-oriented laboratory information management system that implements, maintains and displays the information in a simple and visual format would be of great help in both the daily work in the lab and in ensuring regulatory compliance. Here, we introduce 'Phytotracker', a laboratory management system designed specifically to organise and track plasmids, seeds and growing plants that can be used in mixed platform environments. Phytotracker is designed with simplicity of user operation and ease of installation and management as the major factor, whilst providing tracking tools that cover the full range of activities in molecular genetics labs. It utilises the cross-platform Filemaker relational database, which allows it to be run as a stand-alone or as a server-based networked solution available across all workstations in a lab that can be internet accessible if desired. It can also be readily modified or customised further. Phytotracker provides cataloguing and search functions for plasmids, seed batches, seed stocks and plants growing in pots or trays, and allows tracking of each plant from seed sowing, through harvest to the new seed batch and can print appropriate labels at each stage. The system enters seed information as it is transferred from the previous harvest data, and allows both selfing and hybridization (crossing) to be defined and tracked. Transgenic lines can be linked to their plasmid DNA source. This ease of use and flexibility helps users to reduce their time needed to organise their plants, seeds and plasmids and to maintain laboratory continuity involving multiple workers. We have developed and used Phytotracker for over five years and have found it has been an intuitive, powerful and flexible research tool in organising our plasmid, seed and plant collections requiring minimal maintenance and training for users. It has been developed in an Arabidopsis molecular genetics environment, but can be readily adapted for almost any plant laboratory research.

DNA packaging by the Bacillus subtilis defective bacteriophage PBSX.

PubMed Central

Anderson, L M; Bott, K F

1985-01-01

Defective bacteriophage PBSX, a resident of all Bacillus subtilis 168 chromosomes, packages fragments of DNA from all portions of the host chromosome when induced by mitomycin C. In this study, the physical process for DNA packaging of both chromosomal and plasmid DNAs was examined. Discrete 13-kilobase (kb) lengths of DNA were packaged by wild-type phage, and the process was DNase I resistant and probably occurred by a head-filling mechanism. Genetically engineered isogenic host strains having a chloramphenicol resistance determinant integrated as a genetic flag at two different regions of the chromosome were used to monitor the packaging of specific chromosomal regions. No dramatic selectivity for these regions could be documented. If the wild-type strain 168 contains autonomously replicating plasmids, especially pC194, the mitomycin C induces an increase in size of resident plasmid DNA, which is then packaged as 13-kb pieces into phage heads. In strain RB1144, which lacks substantial portions of the PBSX resident phage region, mitomycin C treatment did not affect the structure of resident plasmids. Induction of PBSX started rolling circle replication on plasmids, which then became packaged as 13-kb fragments. This alteration or cannibalization of plasmid replication resulting from mitomycin C treatment requires for its function some DNA within the prophage deletion of strain RB1144. Images PMID:3923209

NDM-1 encoded by a pNDM-BJ01-like plasmid p3SP-NDM in clinical Enterobacter aerogenes

PubMed Central

Chen, Zhenhong; Li, Hongxia; Feng, Jiao; Li, Yuxue; Chen, Xin; Guo, Xuemin; Chen, Weijun; Wang, Li; Lin, Lei; Yang, Huiying; Yang, Wenhui; Wang, Jie; Zhou, Dongsheng; Liu, Changting; Yin, Zhe

2015-01-01

A carbapenem-nonsusceptible Enterobacter aerogenes strain named 3-SP was isolated from a human case of pneumonia in a Chinese teaching hospital. NDM-1 carbapenemase is produced by a pNDM-BJ01-like conjugative plasmid designated p3SP-NDM to account for carbapenem resistance of 3-SP. p3SP-NDM was fully sequenced and compared with all publically available pNDM-BJ01-like plasmids. The genetic differences between p3SP-NDM and pNDM-BJ01 include only 18 single nucleotide polymorphisms, a 1 bp deletion and a 706 bp deletion. p3SP-NDM and pNDM-BJ01 harbor an identical Tn125 element organized as ISAba125, blaNDM−1, bleMBL, ΔtrpF, dsbC, cutA, ΔgroES, groEL, ISCR27, and ISAba125. The blaNDM−1 surrounding regions in these pNDM-BJ01-like plasmids have a conserved linear organization ISAba14-aphA6-Tn125-unknown IS, with considerable genetic differences identified within or immediately downstream of Tn125. All reported pNDM-BJ01-like plasmids are exclusively found in Acinetobacter, whereas this is the first report of identification of a pNDM-BJ01-like plasmid in Enterobacteriaceae. PMID:25926823

NDM-1 encoded by a pNDM-BJ01-like plasmid p3SP-NDM in clinical Enterobacter aerogenes.

PubMed

Chen, Zhenhong; Li, Hongxia; Feng, Jiao; Li, Yuxue; Chen, Xin; Guo, Xuemin; Chen, Weijun; Wang, Li; Lin, Lei; Yang, Huiying; Yang, Wenhui; Wang, Jie; Zhou, Dongsheng; Liu, Changting; Yin, Zhe

2015-01-01

A carbapenem-nonsusceptible Enterobacter aerogenes strain named 3-SP was isolated from a human case of pneumonia in a Chinese teaching hospital. NDM-1 carbapenemase is produced by a pNDM-BJ01-like conjugative plasmid designated p3SP-NDM to account for carbapenem resistance of 3-SP. p3SP-NDM was fully sequenced and compared with all publically available pNDM-BJ01-like plasmids. The genetic differences between p3SP-NDM and pNDM-BJ01 include only 18 single nucleotide polymorphisms, a 1 bp deletion and a 706 bp deletion. p3SP-NDM and pNDM-BJ01 harbor an identical Tn125 element organized as ISAba125, bla NDM-1, ble MBL, ΔtrpF, dsbC, cutA, ΔgroES, groEL, ISCR27, and ISAba125. The bla NDM-1 surrounding regions in these pNDM-BJ01-like plasmids have a conserved linear organization ISAba14-aphA6-Tn125-unknown IS, with considerable genetic differences identified within or immediately downstream of Tn125. All reported pNDM-BJ01-like plasmids are exclusively found in Acinetobacter, whereas this is the first report of identification of a pNDM-BJ01-like plasmid in Enterobacteriaceae.

Identification of dfrA14 in two distinct plasmids conferring trimethoprim resistance in Actinobacillus pleuropneumoniae.

PubMed

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

2015-08-01

The objective of this study was to determine the distribution and genetic basis of trimethoprim resistance in Actinobacillus pleuropneumoniae isolates from pigs in England. 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. 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. 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. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.

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.

Genetic Characterization and Comparison of Clostridium botulinum Isolates from Botulism Cases in Japan between 2006 and 2011

PubMed Central

Sekizuka, Tsuyoshi; Yamamoto, Akihiko; Iwaki, Masaaki; Komiya, Takako; Hatakeyama, Takashi; Nakajima, Hiroshi; Takahashi, Motohide; Kuroda, Makoto; Shibayama, Keigo

2014-01-01

Genetic characterization was performed for 10 group I Clostridium botulinum strains isolated from botulism cases in Japan between 2006 and 2011. Of these, 1 was type A, 2 were type B, and 7 were type A(B) {carrying a silent bont/B [bont/(B)] gene} serotype strains, based on botulinum neurotoxin (BoNT) production. The type A strain harbored the subtype A1 BoNT gene (bont/A1), which is associated with the ha gene cluster. The type B strains carried bont/B5 or bont/B6 subtype genes. The type A(B) strains carried bont/A1 identical to that of type A(B) strain NCTC2916. However, bont/(B) genes in these strains showed single-nucleotide polymorphisms (SNPs) among strains. SNPs at 2 nucleotide positions of bont/(B) enabled classification of the type A(B) strains into 3 groups. Pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeat analysis (MLVA) also provided consistent separation results. In addition, the type A(B) strains were separated into 2 lineages based on their plasmid profiles. One lineage carried a small plasmid (5.9 kb), and another harbored 21-kb plasmids. To obtain more detailed genetic information about the 10 strains, we sequenced their genomes and compared them with 13 group I C. botulinum genomes in a database using whole-genome SNP analysis. This analysis provided high-resolution strain discrimination and enabled us to generate a refined phylogenetic tree that provides effective traceability of botulism cases, as well as bioterrorism materials. In the phylogenetic tree, the subtype B6 strains, Okayama2011 and Osaka05, were distantly separated from the other strains, indicating genomic divergence of subtype B6 strains among group I strains. PMID:25192986

Ultrasound-Mediated DNA Transformation in Thermophilic Gram-Positive Anaerobes

PubMed Central

Ji, Yuetong; He, Zhili; Pu, Yunting; Zhou, Jizhong; Xu, Jian

2010-01-01

Background Thermophilic, Gram-positive, anaerobic bacteria (TGPAs) are generally recalcitrant to chemical and electrotransformation due to their special cell-wall structure and the low intrinsic permeability of plasma membranes. Methodology/Principal Findings Here we established for any Gram-positive or thermophiles an ultrasound-based sonoporation as a simple, rapid, and minimally invasive method to genetically transform TGPAs. We showed that by applying a 40 kHz ultrasound frequency over a 20-second exposure, Texas red-conjugated dextran was delivered with 27% efficiency into Thermoanaerobacter sp. X514, a TGPA that can utilize both pentose and hexose for ethanol production. Experiments that delivered plasmids showed that host-cell viability and plasmid DNA integrity were not compromised. Via sonoporation, shuttle vectors pHL015 harboring a jellyfish gfp gene and pIKM2 encoding a Clostridium thermocellum β-1,4-glucanase gene were delivered into X514 with an efficiency of 6×102 transformants/µg of methylated DNA. Delivery into X514 cells was confirmed via detecting the kanamycin-resistance gene for pIKM2, while confirmation of pHL015 was detected by visualization of fluorescence signals of secondary host-cells following a plasmid-rescue experiment. Furthermore, the foreign β-1,4-glucanase gene was functionally expressed in X514, converting the host into a prototypic thermophilic consolidated bioprocessing organism that is not only ethanologenic but cellulolytic. Conclusions/Significance In this study, we developed an ultrasound-based sonoporation method in TGPAs. This new DNA-delivery method could significantly improve the throughput in developing genetic systems for TGPAs, many of which are of industrial interest yet remain difficult to manipulate genetically. PMID:20838444

Isocost Lines Describe the Cellular Economy of Genetic Circuits

PubMed Central

Gyorgy, Andras; Jiménez, José I.; Yazbek, John; Huang, Hsin-Ho; Chung, Hattie; Weiss, Ron; Del Vecchio, Domitilla

2015-01-01

Genetic circuits in living cells share transcriptional and translational resources that are available in limited amounts. This leads to unexpected couplings among seemingly unconnected modules, which result in poorly predictable circuit behavior. In this study, we determine these interdependencies between products of different genes by characterizing the economy of how transcriptional and translational resources are allocated to the production of proteins in genetic circuits. We discover that, when expressed from the same plasmid, the combinations of attainable protein concentrations are constrained by a linear relationship, which can be interpreted as an isocost line, a concept used in microeconomics. We created a library of circuits with two reporter genes, one constitutive and the other inducible in the same plasmid, without a regulatory path between them. In agreement with the model predictions, experiments reveal that the isocost line rotates when changing the ribosome binding site strength of the inducible gene and shifts when modifying the plasmid copy number. These results demonstrate that isocost lines can be employed to predict how genetic circuits become coupled when sharing resources and provide design guidelines for minimizing the effects of such couplings. PMID:26244745

Biotechnology by Design: An Introductory Level, Project-Based, Synthetic Biology Laboratory Program for Undergraduate Students.

PubMed

Beach, Dale L; Alvarez, Consuelo J

2015-12-01

Synthetic biology offers an ideal opportunity to promote undergraduate laboratory courses with research-style projects, immersing students in an inquiry-based program that enhances the experience of the scientific process. We designed a semester-long, project-based laboratory curriculum using synthetic biology principles to develop a novel sensory device. Students develop subject matter knowledge of molecular genetics and practical skills relevant to molecular biology, recombinant DNA techniques, and information literacy. During the spring semesters of 2014 and 2015, the Synthetic Biology Laboratory Project was delivered to sophomore genetics courses. Using a cloning strategy based on standardized BioBrick genetic "parts," students construct a "reporter plasmid" expressing a reporter gene (GFP) controlled by a hybrid promoter regulated by the lac-repressor protein (lacI). In combination with a "sensor plasmid," the production of the reporter phenotype is inhibited in the presence of a target environmental agent, arabinose. When arabinose is absent, constitutive GFP expression makes cells glow green. But the presence of arabinose activates a second promoter (pBAD) to produce a lac-repressor protein that will inhibit GFP production. Student learning was assessed relative to five learning objectives, using a student survey administered at the beginning (pre-survey) and end (post-survey) of the course, and an additional 15 open-ended questions from five graded Progress Report assignments collected throughout the course. Students demonstrated significant learning gains (p < 0.05) for all learning outcomes. Ninety percent of students indicated that the Synthetic Biology Laboratory Project enhanced their understanding of molecular genetics. The laboratory project is highly adaptable for both introductory and advanced courses.

Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone.

PubMed

Weger-Lucarelli, James; Duggal, Nisha K; Bullard-Feibelman, Kristen; Veselinovic, Milena; Romo, Hannah; Nguyen, Chilinh; Rückert, Claudia; Brault, Aaron C; Bowen, Richard A; Stenglein, Mark; Geiss, Brian J; Ebel, Gregory D

2017-01-01

Zika virus (ZIKV; family Flaviviridae, genus Flavivirus) is a rapidly expanding global pathogen that has been associated with severe clinical manifestations, including devastating neurological disease in infants. There are currently no molecular clones of a New World ZIKV available that lack significant attenuation, hindering progress toward understanding determinants of transmission and pathogenesis. Here we report the development and characterization of a novel ZIKV reverse genetics system based on a 2015 isolate from Puerto Rico (PRVABC59). We generated a two-plasmid infectious clone system from which infectious virus was rescued that replicates in human and mosquito cells with growth kinetics representative of wild-type ZIKV. Infectious clone-derived virus initiated infection and transmission rates in Aedes aegypti mosquitoes comparable to those of the primary isolate and displayed similar pathogenesis in AG129 mice. This infectious clone system provides a valuable resource to the research community to explore ZIKV molecular biology, vaccine development, antiviral development, diagnostics, vector competence, and disease pathogenesis. ZIKV is a rapidly spreading mosquito-borne pathogen that has been linked to Guillain-Barré syndrome in adults and congenital microcephaly in developing fetuses and infants. ZIKV can also be sexually transmitted. The viral molecular determinants of any of these phenotypes are not well understood. There is no reverse genetics system available for the current epidemic virus that will allow researchers to study ZIKV immunity, develop novel vaccines, or develop antiviral drugs. Here we provide a novel infectious clone system generated from a recent ZIKV isolated from a patient infected in Puerto Rico. This infectious clone produces virus with in vitro and in vivo characteristics similar to those of the primary isolate, providing a critical tool to study ZIKV infection and disease. Copyright © 2016 American Society for Microbiology.

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.

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.

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

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.

Archaeal Extrachromosomal Genetic Elements

PubMed Central

Wang, Haina; Peng, Nan; Shah, Shiraz A.

2015-01-01

SUMMARY Research on archaeal extrachromosomal genetic elements (ECEs) has progressed rapidly in the past decade. To date, over 60 archaeal viruses and 60 plasmids have been isolated. These archaeal viruses exhibit an exceptional diversity in morphology, with a wide array of shapes, such as spindles, rods, filaments, spheres, head-tails, bottles, and droplets, and some of these new viruses have been classified into one order, 10 families, and 16 genera. Investigation of model archaeal viruses has yielded important insights into mechanisms underlining various steps in the viral life cycle, including infection, DNA replication and transcription, and virion egression. Many of these mechanisms are unprecedented for any known bacterial or eukaryal viruses. Studies of plasmids isolated from different archaeal hosts have also revealed a striking diversity in gene content and innovation in replication strategies. Highly divergent replication proteins are identified in both viral and plasmid genomes. Genomic studies of archaeal ECEs have revealed a modular sequence structure in which modules of DNA sequence are exchangeable within, as well as among, plasmid families and probably also between viruses and plasmids. In particular, it has been suggested that ECE-host interactions have shaped the coevolution of ECEs and their archaeal hosts. Furthermore, archaeal hosts have developed defense systems, including the innate restriction-modification (R-M) system and the adaptive CRISPR (clustered regularly interspaced short palindromic repeats) system, to restrict invasive plasmids and viruses. Together, these interactions permit a delicate balance between ECEs and their hosts, which is vitally important for maintaining an innovative gene reservoir carried by ECEs. In conclusion, while research on archaeal ECEs has just started to unravel the molecular biology of these genetic entities and their interactions with archaeal hosts, it is expected to accelerate in the next decade. PMID:25694123

Vertical transmission of highly similar blaCTX-M-1-harboring IncI1 plasmids in Escherichia coli with different MLST types in the poultry production pyramid

PubMed Central

Zurfluh, Katrin; Wang, Juan; Klumpp, Jochen; Nüesch-Inderbinen, Magdalena; Fanning, Séamus; Stephan, Roger

2014-01-01

Objectives: The purpose of this study was to characterize sets of extended-spectrum β-lactamases (ESBL)-producing Enterobacteriaceae collected longitudinally from different flocks of broiler breeders, meconium of 1-day-old broilers from theses breeder flocks, as well as from these broiler flocks before slaughter. Methods: Five sets of ESBL-producing Escherichia coli were studied by multi-locus sequence typing (MLST), phylogenetic grouping, PCR-based replicon typing and resistance profiling. The blaCTX-M-1-harboring plasmids of one set (pHV295.1, pHV114.1, and pHV292.1) were fully sequenced and subjected to comparative analysis. Results: Eleven different MLST sequence types (ST) were identified with ST1056 the predominant one, isolated in all five sets either on the broiler breeder or meconium level. Plasmid sequencing revealed that blaCTX-M-1 was carried by highly similar IncI1/ST3 plasmids that were 105 076 bp, 110 997 bp, and 117 269 bp in size, respectively. Conclusions: The fact that genetically similar IncI1/ST3 plasmids were found in ESBL-producing E. coli of different MLST types isolated at the different levels in the broiler production pyramid provides strong evidence for a vertical transmission of these plasmids from a common source (nucleus poultry flocks). PMID:25324838

Vertical transmission of highly similar bla CTX-M-1-harboring IncI1 plasmids in Escherichia coli with different MLST types in the poultry production pyramid.

PubMed

Zurfluh, Katrin; Wang, Juan; Klumpp, Jochen; Nüesch-Inderbinen, Magdalena; Fanning, Séamus; Stephan, Roger

2014-01-01

The purpose of this study was to characterize sets of extended-spectrum β-lactamases (ESBL)-producing Enterobacteriaceae collected longitudinally from different flocks of broiler breeders, meconium of 1-day-old broilers from theses breeder flocks, as well as from these broiler flocks before slaughter. Five sets of ESBL-producing Escherichia coli were studied by multi-locus sequence typing (MLST), phylogenetic grouping, PCR-based replicon typing and resistance profiling. The bla CTX-M-1-harboring plasmids of one set (pHV295.1, pHV114.1, and pHV292.1) were fully sequenced and subjected to comparative analysis. Eleven different MLST sequence types (ST) were identified with ST1056 the predominant one, isolated in all five sets either on the broiler breeder or meconium level. Plasmid sequencing revealed that bla CTX-M-1 was carried by highly similar IncI1/ST3 plasmids that were 105 076 bp, 110 997 bp, and 117 269 bp in size, respectively. The fact that genetically similar IncI1/ST3 plasmids were found in ESBL-producing E. coli of different MLST types isolated at the different levels in the broiler production pyramid provides strong evidence for a vertical transmission of these plasmids from a common source (nucleus poultry flocks).

Error baseline rates of five sample preparation methods used to characterize RNA virus populations.

PubMed

Kugelman, Jeffrey R; Wiley, Michael R; Nagle, Elyse R; Reyes, Daniel; Pfeffer, Brad P; Kuhn, Jens H; Sanchez-Lockhart, Mariano; Palacios, Gustavo F

2017-01-01

Individual RNA viruses typically occur as populations of genomes that differ slightly from each other due to mutations introduced by the error-prone viral polymerase. Understanding the variability of RNA virus genome populations is critical for understanding virus evolution because individual mutant genomes may gain evolutionary selective advantages and give rise to dominant subpopulations, possibly even leading to the emergence of viruses resistant to medical countermeasures. Reverse transcription of virus genome populations followed by next-generation sequencing is the only available method to characterize variation for RNA viruses. However, both steps may lead to the introduction of artificial mutations, thereby skewing the data. To better understand how such errors are introduced during sample preparation, we determined and compared error baseline rates of five different sample preparation methods by analyzing in vitro transcribed Ebola virus RNA from an artificial plasmid-based system. These methods included: shotgun sequencing from plasmid DNA or in vitro transcribed RNA as a basic "no amplification" method, amplicon sequencing from the plasmid DNA or in vitro transcribed RNA as a "targeted" amplification method, sequence-independent single-primer amplification (SISPA) as a "random" amplification method, rolling circle reverse transcription sequencing (CirSeq) as an advanced "no amplification" method, and Illumina TruSeq RNA Access as a "targeted" enrichment method. The measured error frequencies indicate that RNA Access offers the best tradeoff between sensitivity and sample preparation error (1.4-5) of all compared methods.

Error baseline rates of five sample preparation methods used to characterize RNA virus populations

PubMed Central

Kugelman, Jeffrey R.; Wiley, Michael R.; Nagle, Elyse R.; Reyes, Daniel; Pfeffer, Brad P.; Kuhn, Jens H.; Sanchez-Lockhart, Mariano; Palacios, Gustavo F.

2017-01-01

Individual RNA viruses typically occur as populations of genomes that differ slightly from each other due to mutations introduced by the error-prone viral polymerase. Understanding the variability of RNA virus genome populations is critical for understanding virus evolution because individual mutant genomes may gain evolutionary selective advantages and give rise to dominant subpopulations, possibly even leading to the emergence of viruses resistant to medical countermeasures. Reverse transcription of virus genome populations followed by next-generation sequencing is the only available method to characterize variation for RNA viruses. However, both steps may lead to the introduction of artificial mutations, thereby skewing the data. To better understand how such errors are introduced during sample preparation, we determined and compared error baseline rates of five different sample preparation methods by analyzing in vitro transcribed Ebola virus RNA from an artificial plasmid-based system. These methods included: shotgun sequencing from plasmid DNA or in vitro transcribed RNA as a basic “no amplification” method, amplicon sequencing from the plasmid DNA or in vitro transcribed RNA as a “targeted” amplification method, sequence-independent single-primer amplification (SISPA) as a “random” amplification method, rolling circle reverse transcription sequencing (CirSeq) as an advanced “no amplification” method, and Illumina TruSeq RNA Access as a “targeted” enrichment method. The measured error frequencies indicate that RNA Access offers the best tradeoff between sensitivity and sample preparation error (1.4−5) of all compared methods. PMID:28182717

Enhanced production of recombinant proteins with Corynebacterium glutamicum by deletion of insertion sequences (IS elements).

PubMed

Choi, Jae Woong; Yim, Sung Sun; Kim, Min Jeong; Jeong, Ki Jun

2015-12-29

In most bacteria, various jumping genetic elements including insertion sequences elements (IS elements) cause a variety of genetic rearrangements resulting in harmful effects such as genome and recombinant plasmid instability. The genetic stability of a plasmid in a host is critical for high-level production of recombinant proteins, and in this regard, the development of an IS element-free strain could be a useful strategy for the enhanced production of recombinant proteins. Corynebacterium glutamicum, which is a workhorse in the industrial-scale production of various biomolecules including recombinant proteins, also has several IS elements, and it is necessary to identify the critical IS elements and to develop IS element deleted strain. From the cultivation of C. glutamicum harboring a plasmid for green fluorescent protein (GFP) gene expression, non-fluorescent clones were isolated by FACS (fluorescent activated cell sorting). All the isolated clones had insertions of IS elements in the GFP coding region, and two major IS elements (ISCg1 and ISCg2 families) were identified. By co-cultivating cells harboring either the isolated IS element-inserted plasmid or intact plasmid, it was clearly confirmed that cells harboring the IS element-inserted plasmids became dominant during the cultivation due to their growth advantage over cells containing intact plasmids, which can cause a significant reduction in recombinant protein production during cultivation. To minimize the harmful effects of IS elements on the expression of heterologous genes in C. glutamicum, two IS element free C. glutamicum strains were developed in which each major IS element was deleted, and enhanced productivity in the engineered C. glutamicum strain was successfully demonstrated with three models: GFP, poly(3-hydroxybutyrate) [P(3HB)] and γ-aminobutyrate (GABA). Our findings clearly indicate that the hopping of IS elements could be detrimental to the production of recombinant proteins in C. glutamicum, emphasizing the importance of developing IS element free host strains.

Genetic and Physiological Studies of Bacillus Anthracis Related to Development of An Improved Vaccine

DTIC Science & Technology

1988-07-01

to replicate at all in B. anthracis. Such mutants al currently being tested as vehicles for transposon mutagenesis in B. anthracis. B. subtilis ( natto ...subtilis ( natto ) 3335 harbors a plasmid, pLS20, which encodes functions responsible for conjugal transfer of plasmids among genetically re- lated and...Cultures were grown for 16 hours in BHI broth supplemented with 0.1% glycerol, o- in LG broth for B. subtilis natto . After cell pellets were suspended in

Optimization of supercoiled HPV-16 E6/E7 plasmid DNA purification with arginine monolith using design of experiments.

PubMed

Almeida, A M; Queiroz, J A; Sousa, F; Sousa, A

2015-01-26

The progress of DNA vaccines is dependent on the development of suitable chromatographic procedures to successfully purify genetic vectors, such as plasmid DNA. Human Papillomavirus is associated with the development of tumours due to the oncogenic power of E6 and E7 proteins, produced by this virus. The supercoiled HPV-16 E6/E7 plasmid-based vaccine was recently purified with the arginine monolith, with 100% of purity, but only 39% of recovery was achieved. Therefore, the present study describes the application of experimental design tools, a newly explored methodology in preparative chromatography, in order to improve the supercoiled plasmid DNA recovery with the arginine monolith, maintaining the high purity degree. In addition, the importance and influence of pH in the pDNA retention to the arginine ligand was also demonstrated. The Composite Central Face design was validated and the recovery of the target molecule was successfully improved from 39% to 83.5%, with an outstanding increase of more than double, while maintaining 100% of purity. Copyright © 2014 Elsevier B.V. All rights reserved.

The cost of copy number in a selfish genetic element: the 2-μm plasmid of Saccharomyces cerevisiae.

PubMed

Harrison, Ellie; Koufopanou, V; Burt, A; MacLean, R C

2012-11-01

Many autonomously replicating genetic elements exist as multiple copies within the cell. The copy number of these elements is often assumed to have important fitness consequences for both element and host, yet the forces shaping its evolution are not well understood. The 2 μm is a multicopy plasmid of Saccharomyces yeasts, encoding just four genes that are solely involved in plasmid replication. One simple model for the fitness relationship between yeasts and 2 μm is that plasmid copy number evolves as a trade-off between selection for increased vertical transmission, favouring high copy number, and selection for decreased virulence, favouring low copy number. To test this model, we experimentally manipulated the copy number of the plasmid and directly measured the fitness cost, in terms of growth rate reduction, associated with high plasmid copy number. We find that the fitness burden imposed by the 2 μm increases with plasmid copy number, such that each copy imposes a fitness burden of 0.17% (± 0.008%), greatly exceeding the cost expected for it to be stably maintained in yeast populations. Our results demonstrate the crucial importance of copy number in the evolution of yeast per 2 μm associations and pave the way for future studies examining how selection can shape the cost of multicopy elements. © 2012 The Authors. Journal of Evolutionary Biology © 2012 European Society For Evolutionary Biology.

Insights into the bovine rumen plasmidome

PubMed Central

Kav, Aya Brown; Sasson, Goor; Jami, Elie; Doron-Faigenboim, Adi; Benhar, Itai; Mizrahi, Itzhak

2012-01-01

Plasmids are self-replicating genetic elements capable of mobilization between different hosts. Plasmids often serve as mediators of lateral gene transfer, a process considered to be a strong and sculpting evolutionary force in microbial environments. Our aim was to characterize the overall plasmid population in the environment of the bovine rumen, which houses a complex and dense microbiota that holds enormous significance for humans. We developed a procedure for the isolation of total rumen plasmid DNA, termed rumen plasmidome, and subjected it to deep sequencing using the Illumina paired-end protocol and analysis using public and custom-made bioinformatics tools. A large number of plasmidome contigs aligned with plasmids of rumen bacteria isolated from different locations and at various time points, suggesting that not only the bacterial taxa, but also their plasmids, are defined by the ecological niche. The bacterial phylum distribution of the plasmidome was different from that of the rumen bacterial taxa. Nevertheless, both shared a dominance of the phyla Firmicutes, Bacteroidetes, and Proteobacteria. Evidently, the rumen plasmidome is of a highly mosaic nature that can cross phyla. Interestingly, when we compared the functional profile of the rumen plasmidome to two plasmid databases and two recently published rumen metagenomes, it became apparent that the rumen plasmidome codes for functions, which are enriched in the rumen ecological niche and could confer advantages to their hosts, suggesting that the functional profiles of mobile genetic elements are associated with their environment, as has been previously implied for viruses. PMID:22431592

Strains and stressors: an analysis of touchscreen learning in genetically diverse mouse strains.

PubMed

Graybeal, Carolyn; Bachu, Munisa; Mozhui, Khyobeni; Saksida, Lisa M; Bussey, Timothy J; Sagalyn, Erica; Williams, Robert W; Holmes, Andrew

2014-01-01

Touchscreen-based systems are growing in popularity as a tractable, translational approach for studying learning and cognition in rodents. However, while mouse strains are well known to differ in learning across various settings, performance variation between strains in touchscreen learning has not been well described. The selection of appropriate genetic strains and backgrounds is critical to the design of touchscreen-based studies and provides a basis for elucidating genetic factors moderating behavior. Here we provide a quantitative foundation for visual discrimination and reversal learning using touchscreen assays across a total of 35 genotypes. We found significant differences in operant performance and learning, including faster reversal learning in DBA/2J compared to C57BL/6J mice. We then assessed DBA/2J and C57BL/6J for differential sensitivity to an environmental insult by testing for alterations in reversal learning following exposure to repeated swim stress. Stress facilitated reversal learning (selectively during the late stage of reversal) in C57BL/6J, but did not affect learning in DBA/2J. To dissect genetic factors underlying these differences, we phenotyped a family of 27 BXD strains generated by crossing C57BL/6J and DBA/2J. There was marked variation in discrimination, reversal and extinction learning across the BXD strains, suggesting this task may be useful for identifying underlying genetic differences. Moreover, different measures of touchscreen learning were only modestly correlated in the BXD strains, indicating that these processes are comparatively independent at both genetic and phenotypic levels. Finally, we examined the behavioral structure of learning via principal component analysis of the current data, plus an archival dataset, totaling 765 mice. This revealed 5 independent factors suggestive of "reversal learning," "motivation-related late reversal learning," "discrimination learning," "speed to respond," and "motivation during discrimination." Together, these findings provide a valuable reference to inform the choice of strains and genetic backgrounds in future studies using touchscreen-based tasks.

Highly Effective Polyphosphate Synthesis, Phosphate Removal, and Concentration Using Engineered Environmental Bacteria Based on a Simple Solo Medium-Copy Plasmid Strategy.

PubMed

Wang, Xin; Wang, Xiaomeng; Hui, Kaimin; Wei, Wei; Zhang, Wen; Miao, Aijun; Xiao, Lin; Yang, Liuyan

2018-01-02

Microbial polyphosphate (polyP) production is vital to the removal of phosphate from wastewater. However, to date, engineered polyP synthesis using genetically accessible environmental bacteria remains a challenge. This study develops a simple solo medium-copy plasmid-based polyphosphate kinase (PPK1) overexpression strategy for achieving maximum intracellular polyphosphate accumulation by environmental bacteria. The polyP content of the subsequently engineered Citrobacter freundii (CPP) could reach as high as 12.7% of its dry weight. The biomass yield of CPP was also guaranteed because of negligible metabolic burden effects resulting from the medium plasmid copy number. Consequently, substantial removal of phosphate (P i ) from the ambient environment was achieved simultaneously. Because of the need for exogenous P i for in vivo ATP regeneration, CPP could thoroughly remove P i from synthetic municipal wastewater when it was applied for the "one-step" removal of P i with a bench-scale sequence batch membrane reactor. Almost all the phosphorus except for that assimilated by CPP for cellular growth could be recovered in the form of more concentrated P i . Overall, engineering environmental bacteria to overexpress PPK1 via a solo medium-copy plasmid strategy may represent a valuable general option for not only biotechnological research based on sufficient intracellular polyP production but also removal of P i from wastewater and P i enrichment.

Influence of Space Flight Factors on the Genetic Properties of Streptomyces Lividans 66 (PIJ702)

NASA Technical Reports Server (NTRS)

Tabakov, V. Yu.; Voeikova, T. A.; Tairbekov, M. G.; Goins, T. L.; Martinson, V. G.; Pyle, B. H.

2006-01-01

Gram-positive Streptomyces bacteria display genetic instability in response to external factors. Strain S. lividans 66 harbors the multicopy plasmid pIJ702 with selective and differential marker genes for antibiotic thiostrepton resistance and melanin production. Culture plates of modified ISP agar medium with and without thiostrepton were flown on Foton-M2. Suboptimal flight temperatures, which were simulated for asynchronous ground controls, resulted in slow growth and failure to differentiate and sporulate. Flight samples and asynchronous controls showed a high frequency of failing to express plasmid markers compared to laboratory controls. This was associated with loss of plasmid DNA and likely resulted from suboptimal temperatures for flight cultures and controls. Neither restriction fragment length polymorphism, nor polymerase chain reaction amplification coupled with denaturing gradient gel electrophoresis, revealed differences between pIJ702 DNA from flight vs. control clones. Mutations of the plasmid marker genes resulting from specific spaceflight factors, e.g., microgravity and radiation, were not detected.

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.

Development of an expression plasmid and its use in genetic manipulation of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (higher Basidiomycetes).

PubMed

Yu, Xuya; Ji, Sen-Lin; He, Yi-Long; Ren, Meng-Fei; Xu, Jun-Wei

2014-01-01

We report the construction of a plasmid, pJW-EXP, designed for the expression of homologous and heterologous genes in Ganoderma lucidum. pJW-EXP was generated from the plasmid pMD19-T by inserting the G. lucidum glyceraldehyde-3-phosphate dehydrogenase gene promoter, the G. lucidum iron-sulfur protein subunit of succinate dehydrogenase gene terminator and the homologous carboxin-resistance gene as selection marker. This expression plasmid can be efficiently transformed into Ganoderma through polyethylene glycol-mediated protoplast transformation. Southern blot analysis showed that most of the integrated DNA appeared as multiple copies in the genome. The applicability of the constructed plasmid was tested by expression of the truncated G. lucidum 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene that encodes the catalytic domain of HMGR. Overexpression of the truncated HMGR gene, which is a key gene in the biosynthetic pathway of the antitumor compounds, ganoderic acids, increased the transcription of the HMGR gene and enhanced ganoderic acid accumulation. pJW-EXP can serve as a useful tool in the genetic improvement and metabolic engineering of Ganoderma.

High-level generation of polyclonal antibodies by genetic immunization.

PubMed

Chambers, Ross S; Johnston, Stephen Albert

2003-09-01

Antibodies are important tools for investigating the proteome, but current methods for producing them have become a rate-limiting step. A primary obstacle in most methods for generating antibodies or antibody-like molecules is the requirement for at least microgram quantities of purified protein. We have developed a technology for producing antibodies using genetic immunization. Genetic immunization-based antibody production offers several advantages, including high throughput and high specificity. Moreover, antibodies produced from genetically immunized animals are more likely to recognize the native protein. Here we show that a genetic immunization-based system can be used to efficiently raise useful antibodies to a wide range of antigens. We accomplished this by linking the antigen gene to various elements that enhance antigenicity and by codelivering plasmids encoding genetic adjuvants. Our system, which was tested by immunizing mice with >130 antigens, has shown a final success rate of 84%.

A plasmid containing the human metallothionein II gene can function as an antibody-assisted electrophoretic biosensor for heavy metals.

PubMed

Wooten, Dennis C; Starr, Clarise R; Lyon, Wanda J

2016-01-01

Different forms of heavy metals affect biochemical systems in characteristic ways that cannot be detected with typical metal analysis methods like atomic absorption spectrometry. Further, using living systems to analyze interaction of heavy metals with biochemical systems can be laborious and unreliable. To generate a reliable easy-to-use biologically-based biosensor system, the entire human metallothionein-II (MT-II) gene was incorporated into a plasmid (pUC57-MT) easily replicated in Escherichia coli. In this system, a commercial polyclonal antibody raised against human metal-responsive transcription factor-1 protein (MTF-1 protein) could modify the electrophoretic migration patterns (i.e. cause specific decreases in agarose gel electrophoretic mobility) of the plasmid in the presence or absence of heavy metals other than zinc (Zn). In the study here, heavy metals, MTF-1 protein, and polyclonal anti-MTF-1 antibody were used to assess pUC57-MT plasmid antibody-assisted electrophoretic mobility. Anti-MTF-1 antibody bound both MTF-1 protein and pUC57-MT plasmid in a non-competitive fashion such that it could be used to differentiate specific heavy metal binding. The results showed that antibody-inhibited plasmid migration was heavy metal level-dependent. Zinc caused a unique mobility shift pattern opposite to that of other metals tested, i.e. Zn blocked the antibody ability to inhibit plasmid migration, despite a greatly increased affinity for DNA by the antibody when Zn was present. The Zn effect was reversed/modified by adding MTF-1 protein. Additionally, antibody inhibition of plasmid mobility was resistant to heat pre-treatment and trypsinization, indicating absence of residual DNA extraction-resistant bacterial DNA binding proteins. DNA binding by anti-DNA antibodies may be commonly enhanced by xenobiotic heavy metals and elevated levels of Zn, thus making them potentially effective tools for assessment of heavy metal bioavailability in aqueous solutions and fluid obtained from metal implant sites.

Description of two new plasmids isolated from Francisella philomiragia strains and construction of shuttle vectors for the study of Francisella tularensis.

PubMed

Le Pihive, E; Blaha, D; Chenavas, S; Thibault, F; Vidal, D; Valade, E

2009-11-01

Francisella tularensis is the causative agent of tularemia, a zoonotic disease often transmitted to humans by infected animals. The lack of useful specific genetic tools has long hampered the study of F. tularensis subspecies. We identified and characterized two new plasmids, pF242 and pF243, isolated from Francisella philomiragia strains ATCC 25016 and ATCC 25017, respectively. Sequence analysis revealed that pF242 and pF243 are closely related to pC194 and pFNL10 plasmids, respectively. Two generations of pF242- and pF243-based shuttle vectors, harboring several antibiotic resistance markers, were developed. We used the first generation to compare transformation efficiencies in two virulent F. tularensis subspecies. We found that electroporation was more efficient than cryotransformation: almost all vectors tested were successfully introduced by electroporation into Francisella strains with a high level of efficiency. The second generation of shuttle vectors, containing a multiple cloning site and/or gfp gene downstream of Francisella groES promotor, was used for GFP production in F. tularensis. The development of new shuttle vectors offers new perspectives in the genetic manipulation of F. tularensis, helping to elucidate the mechanisms underlying its virulence.

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

Quantification of genetically modified soybeans using a combination of a capillary-type real-time PCR system and a plasmid reference standard.

PubMed

Toyota, Akie; Akiyama, Hiroshi; Sugimura, Mitsunori; Watanabe, Takahiro; Kikuchi, Hiroyuki; Kanamori, Hisayuki; Hino, Akihiro; Esaka, Muneharu; Maitani, Tamio

2006-04-01

Because the labeling of grains and feed- and foodstuffs is mandatory if the genetically modified organism (GMO) content exceeds a certain level of approved genetically modified varieties in many countries, there is a need for a rapid and useful method of GMO quantification in food samples. In this study, a rapid detection system was developed for Roundup Ready Soybean (RRS) quantification using a combination of a capillary-type real-time PCR system, a LightCycler real-time PCR system, and plasmid DNA as the reference standard. In addition, we showed for the first time that the plasmid and genomic DNA should be similar in the established detection system because the PCR efficiencies of using plasmid DNA and using genomic DNA were not significantly different. The conversion factor (Cf) to calculate RRS content (%) was further determined from the average value analyzed in three laboratories. The accuracy and reproducibility of this system for RRS quantification at a level of 5.0% were within a range from 4.46 to 5.07% for RRS content and within a range from 2.0% to 7.0% for the relative standard deviation (RSD) value, respectively. This system rapidly monitored the labeling system and had allowable levels of accuracy and precision.

Improvement of a yeast self-excising integrative vector by prevention of expression leakage of the intronated Cre recombinase gene during plasmid maintenance in Escherichia coli.

PubMed

Agaphonov, Michael O

2017-12-01

The use of plasmids possessing a regulatable gene coding for a site-specific recombinase together with its recognition sequences significantly facilitates genome manipulations since it allows self-excision of the portion of the genetic construct integrated into the host genome. Stable maintenance of such plasmids in Escherichia coli, which is used for plasmid preparation, requires prevention of recombinase synthesis in this host, which can be achieved by interrupting the recombinase gene with an intron. Based on this approach, Saccharomyces cerevisiae and Hansenula polymorpha self-excising vectors possessing intronated gene for Cre recombinase and its recognition sites (LoxP) were previously constructed. However, this work shows instability of the H. polymorpha vectors during plasmid maintenance in E. coli cells. This could be due to recombination between the loxP sites caused by residual expression of the cre gene. Prevention of translation reinitiation on an internal methionine codon completely solved this problem. A similar modification was made in a self-excising vector designed for S. cerevisiae. Apart from substantial improvement of yeast self-excising vectors, the obtained results also narrow down the essential part of Cre sequence. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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.

An efficient and rapid influenza gene cloning strategy for reverse genetics system.

PubMed

Shao, Hongxia; Fan, Zhonglei; Wan, Zhimin; Tian, Xiaoyan; Chen, Hongjun; Perez, Daniel R; Qin, Aijian; Ye, Jianqiang

2015-09-15

Influenza reverse genetics plays vital roles in understanding influenza molecular characteristics and vaccine development. However, current influenza reverse genetics heavily depends on restriction enzyme and ligation for gene cloning. The traditional cloning process of influenza eight fragments for virus rescuing generally requires considerable work. To simplify and increase the pace of gene cloning for influenza reverse genetics system, we developed a rapid restriction enzyme-free ExnaseTM II-based in vitro recombination approach for influenza gene cloning. We used this strategy rapidly and successfully to clone influenza eight genes both from viruses PR8 and H9N2 for virus rescuing. Our data demonstrate that the strategy developed here can accelerate the process of influenza gene cloning into reverse genetics system, and shows high potential for applications in both influenza basic and applied research. Copyright © 2015 Elsevier B.V. All rights reserved.

Sequence-based analysis of pQBR103; a representative of a unique, transfer-proficient mega plasmid resident in the microbial community of sugar beet

PubMed Central

Tett, Adrian; Spiers, Andrew J; Crossman, Lisa C; Ager, Duane; Ciric, Lena; Dow, J Maxwell; Fry, John C; Harris, David; Lilley, Andrew; Oliver, Anna; Parkhill, Julian; Quail, Michael A; Rainey, Paul B; Saunders, Nigel J; Seeger, Kathy; Snyder, Lori AS; Squares, Rob; Thomas, Christopher M; Turner, Sarah L; Zhang, Xue-Xian; Field, Dawn; Bailey, Mark J

2009-01-01

The plasmid pQBR103 was found within Pseudomonas populations colonizing the leaf and root surfaces of sugar beet plants growing at Wytham, Oxfordshire, UK. At 425 kb it is the largest self-transmissible plasmid yet sequenced from the phytosphere. It is known to enhance the competitive fitness of its host, and parts of the plasmid are known to be actively transcribed in the plant environment. Analysis of the complete sequence of this plasmid predicts a coding sequence (CDS)-rich genome containing 478 CDSs and an exceptional degree of genetic novelty; 80% of predicted coding sequences cannot be ascribed a function and 60% are orphans. Of those to which function could be assigned, 40% bore greatest similarity to sequences from Pseudomonas spp, and the majority of the remainder showed similarity to other c-proteobacterial genera and plasmids. pQBR103 has identifiable regions presumed responsible for replication and partitioning, but despite being tra+ lacks the full complement of any previously described conjugal transfer functions. The DNA sequence provided few insights into the functional significance of plant-induced transcriptional regions, but suggests that 14% of CDSs may be expressed (11 CDSs with functional annotation and 54 without), further highlighting the ecological importance of these novel CDSs. Comparative analysis indicates that pQBR103 shares significant regions of sequence with other plasmids isolated from sugar beet plants grown at the same geographic location. These plasmid sequences indicate there is more novelty in the mobile DNA pool accessible to phytosphere pseudomonas than is currently appreciated or understood. PMID:18043644

Genomic Epidemiology of NDM-1-Encoding Plasmids in Latin American Clinical Isolates Reveals Insights into the Evolution of Multidrug Resistance

PubMed Central

Marquez-Ortiz, Ricaurte Alejandro; Haggerty, Leanne; Olarte, Narda; Duarte, Carolina; Garza-Ramos, Ulises; Silva-Sanchez, Jesus; Castro, Betsy E.; Sim, Eby M.; Beltran, Mauricio; Moncada, María V.; Valderrama, Alberto; Castellanos, Jaime E.; Charles, Ian G.; Vanegas, Natasha

2017-01-01

Abstract Bacteria that produce the broad-spectrum Carbapenem antibiotic New Delhi Metallo-β-lactamase (NDM) place a burden on health care systems worldwide, due to the limited treatment options for infections caused by them and the rapid global spread of this antibiotic resistance mechanism. Although it is believed that the associated resistance gene blaNDM-1 originated in Acinetobacter spp., the role of Enterobacteriaceae in its dissemination remains unclear. In this study, we used whole genome sequencing to investigate the dissemination dynamics of blaNDM-1-positive plasmids in a set of 21 clinical NDM-1-positive isolates from Colombia and Mexico (Providencia rettgeri, Klebsiella pneumoniae, and Acinetobacter baumannii) as well as six representative NDM-1-positive Escherichia coli transconjugants. Additionally, the plasmids from three representative P. rettgeri isolates were sequenced by PacBio sequencing and finished. Our results demonstrate the presence of previously reported plasmids from K. pneumoniae and A. baumannii in different genetic backgrounds and geographically distant locations in Colombia. Three new previously unclassified plasmids were also identified in P. rettgeri from Colombia and Mexico, plus an interesting genetic link between NDM-1-positive P. rettgeri from distant geographic locations (Canada, Mexico, Colombia, and Israel) without any reported epidemiological links was discovered. Finally, we detected a relationship between plasmids present in P. rettgeri and plasmids from A. baumannii and K. pneumoniae. Overall, our findings suggest a Russian doll model for the dissemination of blaNDM-1 in Latin America, with P. rettgeri playing a central role in this process, and reveal new insights into the evolution and dissemination of plasmids carrying such antibiotic resistance genes. PMID:28854628

Development of plasmid vector and electroporation condition for gene transfer in sporogenic lactic acid bacterium, Bacillus coagulans.

PubMed

Rhee, Mun Su; Kim, Jin-Woo; Qian, Yilei; Ingram, L O; Shanmugam, K T

2007-07-01

Bacillus coagulans is a sporogenic lactic acid bacterium that ferments glucose and xylose, major components of plant biomass, a potential feedstock for cellulosic ethanol. The temperature and pH for optimum rate of growth of B. coagulans (50 to 55 degrees C, pH 5.0) are very similar to that of commercially developed fungal cellulases (50 degrees C; pH 4.8). Due to this match, simultaneous saccharification and fermentation (SSF) of cellulose to products by B. coagulans is expected to require less cellulase than needed if the SSF is conducted at a sub-optimal temperature, such as 30 degrees C, the optimum for yeast, the main biocatalyst used by the ethanol industry. To fully exploit B. coagulans as a platform organism, we have developed an electroporation method to transfer plasmid DNA into this genetically recalcitrant bacterium. We also constructed a B. coagulans/E. coli shuttle vector, plasmid pMSR10 that contains the rep region from a native plasmid (pMSR0) present in B. coagulans strain P4-102B. The native plasmid, pMSR0 (6823bp), has 9 ORFs, and replicates by rolling-circle mode of replication. Plasmid pNW33N, developed for Geobacillus stearothermophilus, was also transformed into this host and stably maintained while several other Bacillus/Escherichia coli shuttle vector plasmids were not transformed into B. coagulans. The transformation efficiency of B. coagulans strain P4-102B using the plasmids pNW33N or pMSR10 was about 1.5x10(16) per mole of DNA. The availability of shuttle vectors and an electroporation method is expected to aid in genetic and metabolic engineering of B. coagulans.

Induction of multiple pleiotropic drug resistance genes in yeast engineered to produce an increased level of anti-malarial drug precursor, artemisinic acid.

PubMed

Ro, Dae-Kyun; Ouellet, Mario; Paradise, Eric M; Burd, Helcio; Eng, Diana; Paddon, Chris J; Newman, Jack D; Keasling, Jay D

2008-11-04

Due to the global occurrence of multi-drug-resistant malarial parasites (Plasmodium falciparum), the anti-malarial drug most effective against malaria is artemisinin, a natural product (sesquiterpene lactone endoperoxide) extracted from sweet wormwood (Artemisia annua). However, artemisinin is in short supply and unaffordable to most malaria patients. Artemisinin can be semi-synthesized from its precursor artemisinic acid, which can be synthesized from simple sugars using microorganisms genetically engineered with genes from A. annua. In order to develop an industrially competent yeast strain, detailed analyses of microbial physiology and development of gene expression strategies are required. Three plant genes coding for amorphadiene synthase, amorphadiene oxidase (AMO or CYP71AV1), and cytochrome P450 reductase, which in concert divert carbon flux from farnesyl diphosphate to artemisinic acid, were expressed from a single plasmid. The artemisinic acid production in the engineered yeast reached 250 microg mL(-1) in shake-flask cultures and 1 g L(-1) in bio-reactors with the use of Leu2d selection marker and appropriate medium formulation. When plasmid stability was measured, the yeast strain synthesizing amorphadiene alone maintained the plasmid in 84% of the cells, whereas the yeast strain synthesizing artemisinic acid showed poor plasmid stability. Inactivation of AMO by a point-mutation restored the high plasmid stability, indicating that the low plasmid stability is not caused by production of the AMO protein but by artemisinic acid synthesis or accumulation. Semi-quantitative reverse-transcriptase (RT)-PCR and quantitative real time-PCR consistently showed that pleiotropic drug resistance (PDR) genes, belonging to the family of ATP-Binding Cassette (ABC) transporter, were massively induced in the yeast strain producing artemisinic acid, relative to the yeast strain producing the hydrocarbon amorphadiene alone. Global transcriptional analysis by yeast microarray further demonstrated that the induction of drug-resistant genes such as ABC transporters and major facilitator superfamily (MSF) genes is the primary cellular stress-response; in addition, oxidative and osmotic stress responses were observed in the engineered yeast. The data presented here suggest that the engineered yeast producing artemisinic acid suffers oxidative and drug-associated stresses. The use of plant-derived transporters and optimizing AMO activity may improve the yield of artemisinic acid production in the engineered yeast.

Genetic plasticity of the Shigella virulence plasmid is mediated by intra- and inter-molecular events between insertion sequences.

PubMed

Pilla, Giulia; McVicker, Gareth; Tang, Christoph M

2017-09-01

Acquisition of a single copy, large virulence plasmid, pINV, led to the emergence of Shigella spp. from Escherichia coli. The plasmid encodes a Type III secretion system (T3SS) on a 30 kb pathogenicity island (PAI), and is maintained in a bacterial population through a series of toxin:antitoxin (TA) systems which mediate post-segregational killing (PSK). The T3SS imposes a significant cost on the bacterium, and strains which have lost the plasmid and/or genes encoding the T3SS grow faster than wild-type strains in the laboratory, and fail to bind the indicator dye Congo Red (CR). Our aim was to define the molecular events in Shigella flexneri that cause loss of Type III secretion (T3S), and to examine whether TA systems exert positional effects on pINV. During growth at 37°C, we found that deletions of regions of the plasmid including the PAI lead to the emergence of CR-negative colonies; deletions occur through intra-molecular recombination events between insertion sequences (ISs) flanking the PAI. Furthermore, by repositioning MvpAT (which belongs to the VapBC family of TA systems) near the PAI, we demonstrate that the location of this TA system alters the rearrangements that lead to loss of T3S, indicating that MvpAT acts both globally (by reducing loss of pINV through PSK) as well as locally (by preventing loss of adjacent sequences). During growth at environmental temperatures, we show for the first time that pINV spontaneously integrates into different sites in the chromosome, and this is mediated by inter-molecular events involving IS1294. Integration leads to reduced PAI gene expression and impaired secretion through the T3SS, while excision of pINV from the chromosome restores T3SS function. Therefore, pINV integration provides a reversible mechanism for Shigella to circumvent the metabolic burden imposed by pINV. Intra- and inter-molecular events between ISs, which are abundant in Shigella spp., mediate plasticity of S. flexneri pINV.

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.

Intricate interactions between the bloom-forming cyanobacterium Microcystis aeruginosa and foreign genetic elements, revealed by diversified clustered regularly interspaced short palindromic repeat (CRISPR) signatures.

PubMed

Kuno, Sotaro; Yoshida, Takashi; Kaneko, Takakazu; Sako, Yoshihiko

2012-08-01

Clustered regularly interspaced short palindromic repeats (CRISPR) confer sequence-dependent, adaptive resistance in prokaryotes against viruses and plasmids via incorporation of short sequences, called spacers, derived from foreign genetic elements. CRISPR loci are thus considered to provide records of past infections. To describe the host-parasite (i.e., cyanophages and plasmids) interactions involving the bloom-forming freshwater cyanobacterium Microcystis aeruginosa, we investigated CRISPR in four M. aeruginosa strains and in two previously sequenced genomes. The number of spacers in each locus was larger than the average among prokaryotes. All spacers were strain specific, except for a string of 11 spacers shared in two closely related strains, suggesting diversification of the loci. Using CRISPR repeat-based PCR, 24 CRISPR genotypes were identified in a natural cyanobacterial community. Among 995 unique spacers obtained, only 10 sequences showed similarity to M. aeruginosa phage Ma-LMM01. Of these, six spacers showed only silent or conservative nucleotide mutations compared to Ma-LMM01 sequences, suggesting a strategy by the cyanophage to avert CRISPR immunity dependent on nucleotide identity. These results imply that host-phage interactions can be divided into M. aeruginosa-cyanophage combinations rather than pandemics of population-wide infectious cyanophages. Spacer similarity also showed frequent exposure of M. aeruginosa to small cryptic plasmids that were observed only in a few strains. Thus, the diversification of CRISPR implies that M. aeruginosa has been challenged by diverse communities (almost entirely uncharacterized) of cyanophages and plasmids.

Intricate Interactions between the Bloom-Forming Cyanobacterium Microcystis aeruginosa and Foreign Genetic Elements, Revealed by Diversified Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Signatures

PubMed Central

Kuno, Sotaro; Kaneko, Takakazu; Sako, Yoshihiko

2012-01-01

Clustered regularly interspaced short palindromic repeats (CRISPR) confer sequence-dependent, adaptive resistance in prokaryotes against viruses and plasmids via incorporation of short sequences, called spacers, derived from foreign genetic elements. CRISPR loci are thus considered to provide records of past infections. To describe the host-parasite (i.e., cyanophages and plasmids) interactions involving the bloom-forming freshwater cyanobacterium Microcystis aeruginosa, we investigated CRISPR in four M. aeruginosa strains and in two previously sequenced genomes. The number of spacers in each locus was larger than the average among prokaryotes. All spacers were strain specific, except for a string of 11 spacers shared in two closely related strains, suggesting diversification of the loci. Using CRISPR repeat-based PCR, 24 CRISPR genotypes were identified in a natural cyanobacterial community. Among 995 unique spacers obtained, only 10 sequences showed similarity to M. aeruginosa phage Ma-LMM01. Of these, six spacers showed only silent or conservative nucleotide mutations compared to Ma-LMM01 sequences, suggesting a strategy by the cyanophage to avert CRISPR immunity dependent on nucleotide identity. These results imply that host-phage interactions can be divided into M. aeruginosa-cyanophage combinations rather than pandemics of population-wide infectious cyanophages. Spacer similarity also showed frequent exposure of M. aeruginosa to small cryptic plasmids that were observed only in a few strains. Thus, the diversification of CRISPR implies that M. aeruginosa has been challenged by diverse communities (almost entirely uncharacterized) of cyanophages and plasmids. PMID:22636003

Generation of recombinant European bat lyssavirus type 1 and inter-genotypic compatibility of lyssavirus genotype 1 and 5 antigenome promoters.

PubMed

Orbanz, Jeannette; Finke, Stefan

2010-10-01

Bat lyssaviruses (Fam. Rhabdoviridae) represent a source for the infection of terrestial mammals and the development of rabies disease. Molecular differences in the replication of bat and non-bat lyssaviruses and their contribution to pathogenicity, however, are unknown. One reason for this is the lack of reverse genetics systems for bat-restricted lyssaviruses. To investigate bat lyssavirus replication and host adaptation, we developed a reverse genetics system for European bat lyssavirus type 1 (EBLV-1; genotype 5). This was achieved by co-transfection of HEK-293T cells with a full-length EBLV-1 genome cDNA and expression plasmids for EBLV-1 proteins, resulting in recombinant EBLV-1 (rEBLV-1). Replication of rEBLV-1 was comparable to that of parental virus, showing that rEBLV-1 is a valid tool to investigate EBLV-1 replication functions. In a first approach, we tested whether the terminal promoter sequences of EBLV-1 are genotype-specific. Although genotype 1 (rabies virus) minigenomes were successfully amplified by EBLV-1 helper virus, in the context of the complete virus, only the antigenome promoter (AGP) sequence of EBLV-1 was replaceable, as indicated by comparable replication of rEBLV-1 and the chimeric virus. These analyses demonstrate that the terminal AGPs of genotype 1 and genotype 5 lyssaviruses are compatible with those of the heterologous genotype.

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.

Isocost Lines Describe the Cellular Economy of Genetic Circuits.

PubMed

Gyorgy, Andras; Jiménez, José I; Yazbek, John; Huang, Hsin-Ho; Chung, Hattie; Weiss, Ron; Del Vecchio, Domitilla

2015-08-04

Genetic circuits in living cells share transcriptional and translational resources that are available in limited amounts. This leads to unexpected couplings among seemingly unconnected modules, which result in poorly predictable circuit behavior. In this study, we determine these interdependencies between products of different genes by characterizing the economy of how transcriptional and translational resources are allocated to the production of proteins in genetic circuits. We discover that, when expressed from the same plasmid, the combinations of attainable protein concentrations are constrained by a linear relationship, which can be interpreted as an isocost line, a concept used in microeconomics. We created a library of circuits with two reporter genes, one constitutive and the other inducible in the same plasmid, without a regulatory path between them. In agreement with the model predictions, experiments reveal that the isocost line rotates when changing the ribosome binding site strength of the inducible gene and shifts when modifying the plasmid copy number. These results demonstrate that isocost lines can be employed to predict how genetic circuits become coupled when sharing resources and provide design guidelines for minimizing the effects of such couplings. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Marburg Virus Reverse Genetics Systems

PubMed Central

Schmidt, Kristina Maria; Mühlberger, Elke

2016-01-01

The highly pathogenic Marburg virus (MARV) is a member of the Filoviridae family and belongs to the group of nonsegmented negative-strand RNA viruses. Reverse genetics systems established for MARV have been used to study various aspects of the viral replication cycle, analyze host responses, image viral infection, and screen for antivirals. This article provides an overview of the currently established MARV reverse genetic systems based on minigenomes, infectious virus-like particles and full-length clones, and the research that has been conducted using these systems. PMID:27338448

Marburg Virus Reverse Genetics Systems.

PubMed

Schmidt, Kristina Maria; Mühlberger, Elke

2016-06-22

The highly pathogenic Marburg virus (MARV) is a member of the Filoviridae family and belongs to the group of nonsegmented negative-strand RNA viruses. Reverse genetics systems established for MARV have been used to study various aspects of the viral replication cycle, analyze host responses, image viral infection, and screen for antivirals. This article provides an overview of the currently established MARV reverse genetic systems based on minigenomes, infectious virus-like particles and full-length clones, and the research that has been conducted using these systems.

Direct Cloning of Yeast Genes from an Ordered Set of Lambda Clones in Saccharomyces Cerevisiae by Recombination in Vivo

PubMed Central

Erickson, J. R.; Johnston, M.

1993-01-01

We describe a technique that facilitates the isolation of yeast genes that are difficult to clone. This technique utilizes a plasmid vector that rescues lambda clones as yeast centromere plasmids. The source of these lambda clones is a set of clones whose location in the yeast genome has been determined by L. Riles et al. in 1993. The Esherichia coli-yeast shuttle plasmid carries URA3, ARS4 and CEN6, and contains DNA fragments from the lambda vector that flank the cloned yeast insert. When yeast is cotransformed with linearized plasmid and lambda clone DNA, Ura(+) transformants are obtained by a recombination event between the lambda clone and the plasmid vector that generates an autonomously replicating plasmid containing the cloned yeast DNA sequences. Genes whose genetic map positions are known can easily be identified and recovered in this plasmid by testing only those lambda clones that map to the relevant region of the yeast genome for their ability to complement the mutant phenotype. This technique facilitates the isolation of yeast genes that resist cloning either because (1) they are underrepresented in yeast genomic libraries amplified in E. coli, (2) they provide phenotypes that are too marginal to allow selection of the gene by genetic complementation or (3) they provide phenotypes that are laborious to score. We demonstrate the utility of this technique by isolating three genes, GAL83, SSN2 and MAK7, each of which presents one of these problems for cloning. PMID:8514124

Functional characterization of Pol III U6 promoters for gene knockdown and knockout in Plutella xylostella.

PubMed

Huang, Yuping; Wang, Yajun; Zeng, Baosheng; Liu, Zhaoxia; Xu, Xuejiao; Meng, Qian; Huang, Yongping; Yang, Guang; Vasseur, Liette; Gurr, Geoff M; You, Minsheng

2017-10-01

RNA polymerase type III (Pol-III) promoters such as U6 are commonly used to express small RNAs, including short hairpin RNAs (shRNAs) and single guide RNAs (sgRNAs). Functional U6 promoters are widely used in CRISPR systems, and their characterization can facilitate genome editing of non-model organisms. In the present study, six U6 small nuclear RNA (snRNA) promoters containing two conserved elements of a proximal sequence element (PSEA) and a TATA box, were identified and characterized in the diamondback moth (Plutella xylostella) genome. Relative efficiency of the U6 promoters to express shRNA induced EGFP knockdown was tested in a P. xylostella cell line, revealing that the PxU6:3 promoter had the strongest expression effect. Further work with the PxU6:3 promoter showed its efficacy in EGFP knockout using CRISPR/Cas9 system in the cells. The expression plasmids with versatile Pxabd-A gene specific sgRNA driven by the PxU6:3 promoter, combined with Cas9 mRNA, could induce mutagenesis at specific genomic loci in vivo. The phenotypes induced by sgRNA expression plasmids were similar to those done in vitro transcription sgRNAs. A plasmid with two tandem arranged PxU6:3:sgRNA expression cassettes targeting Pxabd-A loci was generated, which caused a 28,856 bp fragment deletion, suggesting that the multi-sgRNA expression plasmid can be used for multi-targeting. Our work indicates that U6 snRNA promoters can be used for functional studies of genes with the approach of reverse genetics in P. xylostella. These essential promoters also provide valuable potential for CRISPR-derived gene drive as a tactic for population control in this globally significant pest. Copyright © 2017 Elsevier Ltd. All rights reserved.

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 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

Development and evaluation of event-specific quantitative PCR method for genetically modified soybean A2704-12.

PubMed

Takabatake, Reona; Akiyama, Hiroshi; Sakata, Kozue; Onishi, Mari; Koiwa, Tomohiro; Futo, Satoshi; Minegishi, Yasutaka; Teshima, Reiko; Mano, Junichi; Furui, Satoshi; Kitta, Kazumi

2011-01-01

A novel real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) soybean event; A2704-12. During the plant transformation, DNA fragments derived from pUC19 plasmid were integrated in A2704-12, and the region was found to be A2704-12 specific. The pUC19-derived DNA sequences were used as primers for the specific detection of A2704-12. We first tried to construct a standard plasmid for A2704-12 quantification using pUC19. However, non-specific signals appeared with both qualitative and quantitative PCR analyses using the specific primers with pUC19 as a template, and we then constructed a plasmid using pBR322. The conversion factor (C(f)), which is required to calculate the amount of the genetically modified organism (GMO), was experimentally determined with two real-time PCR instruments, the Applied Biosystems 7900HT and the Applied Biosystems 7500. The determined C(f) values were both 0.98. The quantitative method was evaluated by means of blind tests in multi-laboratory trials using the two real-time PCR instruments. The limit of quantitation for the method was estimated to be 0.1%. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSD(R)), and the determined bias and RSD(R) values for the method were each less than 20%. These results suggest that the developed method would be suitable for practical analyses for the detection and quantification of A2704-12.

Development of Nested PCR-Based Specific Markers for Detection of Peach Rosette Mosaic Virus in Plant Quarantine.

PubMed

Lee, S; Kim, C S; Shin, Y G; Kim, J H; Kim, Y S; Jheong, W H

2016-03-01

The Peach rosette mosaic virus (PRMV) is a plant pathogen of the genus Nepovirus, and has been designated as a controlled quarantine virus in Korea. In this study, a specific reverse transcription (RT)-PCR marker set, nested PCR marker set, and modified-plasmid positive control were developed to promptly and accurately diagnose PRMV at plant-quarantine sites. The final selected PRMV-specific RT-PCR marker was PRMV-N10/C70 (967 bp), and the nested PCR product of 419 bp was finally amplified. The modified-plasmid positive control, in which the SalI restriction-enzyme region (GTCGAC) was inserted, verified PRMV contamination in a comparison with the control, enabling a more accurate diagnosis. It is expected that the developed method will continuously contribute to the plant-quarantine process in Korea.

Comparative genomic and plasmid analysis of beer-spoiling and non-beer-spoiling Lactobacillus brevis isolates.

PubMed

Bergsveinson, Jordyn; Ziola, Barry

2017-12-01

Beer-spoilage-related lactic acid bacteria (BSR LAB) belong to multiple genera and species; however, beer-spoilage capacity is isolate-specific and partially acquired via horizontal gene transfer within the brewing environment. Thus, the extent to which genus-, species-, or environment- (i.e., brewery-) level genetic variability influences beer-spoilage phenotype is unknown. Publicly available Lactobacillus brevis genomes were analyzed via BlAst Diagnostic Gene findEr (BADGE) for BSR genes and assessed for pangenomic relationships. Also analyzed were functional coding capacities of plasmids of LAB inhabiting extreme niche environments. Considerable genetic variation was observed in L. brevis isolated from clinical samples, whereas 16 candidate genes distinguish BSR and non-BSR L. brevis genomes. These genes are related to nutrient scavenging of gluconate or pentoses, mannose, and metabolism of pectin. BSR L. brevis isolates also have higher average nucleotide identity and stronger pangenome association with one another, though isolation source (i.e., specific brewery) also appears to influence the plasmid coding capacity of BSR LAB. Finally, it is shown that niche-specific adaptation and phenotype are plasmid-encoded for both BSR and non-BSR LAB. The ultimate combination of plasmid-encoded genes dictates the ability of L. brevis to survive in the most extreme beer environment, namely, gassed (i.e., pressurized) beer.

Applicability of plasmid calibrant pTC1507 in quantification of TC1507 maize: an interlaboratory study.

PubMed

Meng, Yanan; Liu, Xin; Wang, Shu; Zhang, Dabing; Yang, Litao

2012-01-11

To enforce the labeling regulations of genetically modified organisms (GMOs), the application of DNA plasmids as calibrants is becoming essential for the practical quantification of GMOs. This study reports the construction of plasmid pTC1507 for a quantification assay of genetically modified (GM) maize TC1507 and the collaborative ring trial in international validation of its applicability as a plasmid calibrant. pTC1507 includes one event-specific sequence of TC1507 maize and one unique sequence of maize endogenous gene zSSIIb. A total of eight GMO detection laboratories worldwide were invited to join the validation process, and test results were returned from all eight participants. Statistical analysis of the returned results showed that real-time PCR assays using pTC1507 as calibrant in both GM event-specific and endogenous gene quantifications had high PCR efficiency (ranging from 0.80 to 1.15) and good linearity (ranging from 0.9921 to 0.9998). In a quantification assay of five blind samples, the bias between the test values and true values ranged from 2.6 to 24.9%. All results indicated that the developed pTC1507 plasmid is applicable for the quantitative analysis of TC1507 maize and can be used as a suitable substitute for dried powder certified reference materials (CRMs).

Purification and characterization of enterocin FH 99 produced by a faecal isolate Enterococcus faecium FH 99.

PubMed

Gupta, H; Malik, R K; Bhardwaj, A; Kaur, G; De, S; Kaushik, J K

2010-06-01

Enterococcus faecium FH 99 was isolated from human faeces and selected because of its broad spectrum of inhibitory activity against several Gram-positive foodborne spoilage and pathogenic bacteria. Ent. faecium FH 99 accumulates enterocin in large number in early stationary phase of the growth. The enterocin FH 99 was stable over a wide pH range (2-10) and recovered activity even after treatment at high temperatures (10 min at 100°C). The enterocin was subjected to different purification techniques viz., gel filteration, cation exchange chromatography and reverse-phase high-performance liquid chromatography. The activity was eluted as one individual active fraction. SDSPAGE revealed a molecular weight of less than 6.5 kDa. Studies carried out to identify the genetic determinants for bacteriocin production showed that this trait may be plasmid encoded as loss in both of the plasmids (size>chromosomal DNA) led to loss in bacteriocin production by Ent. faecium FH 99. Ent. faecium strain FH 99 is a newly discovered high bacteriocin producer with Activity Units 1.8 × 10(5) AU ml(-1) and its characteristics indicate that it may have strong potential for application as a protective agent against pathogens and spoilage bacteria in foods.

Strains and Stressors: An Analysis of Touchscreen Learning in Genetically Diverse Mouse Strains

PubMed Central

Graybeal, Carolyn; Bachu, Munisa; Mozhui, Khyobeni; Saksida, Lisa M.; Bussey, Timothy J.; Sagalyn, Erica; Williams, Robert W.; Holmes, Andrew

2014-01-01

Touchscreen-based systems are growing in popularity as a tractable, translational approach for studying learning and cognition in rodents. However, while mouse strains are well known to differ in learning across various settings, performance variation between strains in touchscreen learning has not been well described. The selection of appropriate genetic strains and backgrounds is critical to the design of touchscreen-based studies and provides a basis for elucidating genetic factors moderating behavior. Here we provide a quantitative foundation for visual discrimination and reversal learning using touchscreen assays across a total of 35 genotypes. We found significant differences in operant performance and learning, including faster reversal learning in DBA/2J compared to C57BL/6J mice. We then assessed DBA/2J and C57BL/6J for differential sensitivity to an environmental insult by testing for alterations in reversal learning following exposure to repeated swim stress. Stress facilitated reversal learning (selectively during the late stage of reversal) in C57BL/6J, but did not affect learning in DBA/2J. To dissect genetic factors underlying these differences, we phenotyped a family of 27 BXD strains generated by crossing C57BL/6J and DBA/2J. There was marked variation in discrimination, reversal and extinction learning across the BXD strains, suggesting this task may be useful for identifying underlying genetic differences. Moreover, different measures of touchscreen learning were only modestly correlated in the BXD strains, indicating that these processes are comparatively independent at both genetic and phenotypic levels. Finally, we examined the behavioral structure of learning via principal component analysis of the current data, plus an archival dataset, totaling 765 mice. This revealed 5 independent factors suggestive of “reversal learning,” “motivation-related late reversal learning,” “discrimination learning,” “speed to respond,” and “motivation during discrimination.” Together, these findings provide a valuable reference to inform the choice of strains and genetic backgrounds in future studies using touchscreen-based tasks. PMID:24586288

Detection and characterization of pXFSL21, a novel single-copy plasmid from Xylella fastidiosa Strain Stag’s Leap

USDA-ARS?s Scientific Manuscript database

Xylella fastidiosa Strain Stag’s Leap, originally isolated from Napa Valley, California, is highly virulent in causing Pierce’s Disease (PD) of grapevine. Plasmids are extrachromosomal genetic elements associated with bacterial environmental adaptation such as virulence development. In this study, t...

A simplified and accurate detection of the genetically modified wheat MON71800 with one calibrator plasmid.

PubMed

Kim, Jae-Hwan; Park, Saet-Byul; Roh, Hyo-Jeong; Park, Sunghoon; Shin, Min-Ki; Moon, Gui Im; Hong, Jin-Hwan; Kim, Hae-Yeong

2015-06-01

With the increasing number of genetically modified (GM) events, unauthorized GMO releases into the food market have increased dramatically, and many countries have developed detection tools for them. This study described the qualitative and quantitative detection methods of unauthorized the GM wheat MON71800 with a reference plasmid (pGEM-M71800). The wheat acetyl-CoA carboxylase (acc) gene was used as the endogenous gene. The plasmid pGEM-M71800, which contains both the acc gene and the event-specific target MON71800, was constructed as a positive control for the qualitative and quantitative analyses. The limit of detection in the qualitative PCR assay was approximately 10 copies. In the quantitative PCR assay, the standard deviation and relative standard deviation repeatability values ranged from 0.06 to 0.25 and from 0.23% to 1.12%, respectively. This study supplies a powerful and very simple but accurate detection strategy for unauthorized GM wheat MON71800 that utilizes a single calibrator plasmid. Copyright © 2014 Elsevier Ltd. All rights reserved.

A prototype stable RNA identification cassette for monitoring plasmids of genetically engineered microorganisms

NASA Technical Reports Server (NTRS)

Hedenstierna, K. O.; Lee, Y. H.; Yang, Y.; Fox, G. E.

1993-01-01

A prototype stable RNA identification cassette for monitoring genetically engineered plasmids carried by strains of Escherichia coli has been developed. The cassette consists of a Vibrio proteolyticus 5S ribosomal RNA (rRNA) gene surrounded by promoters and terminators from the rrnB operon of Escherischia coli. The identifier RNA is expressed and successfully processed so that approximately 30% of the 5S rRNA isolated from either whole cells or 70S ribosomes is of the V. proteolyticus type. Cells carrying the identifier are readily detectable by hybridization. Accurate measurements show that the identification cassette has little effect on fitness compared to a strain containing an analogous plasmid carrying wild type E. coli 5S rRNA, and the V. proteolyticus 5S rRNA gene is not inactivated after prolonged growth. These results demonstrate the feasibility of developing small standardized identification cassettes that can utilize already existing highly sensitive rRNA detection methods. Cassettes of this type could in principle be incorporated into either the engineered regions of recombinant plasmids or their hosts.

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.

Networking in microbes: conjugative elements and plasmids in the genus Alteromonas.

PubMed

López-Pérez, Mario; Ramon-Marco, Nieves; Rodriguez-Valera, Francisco

2017-01-05

To develop evolutionary models for the free living bacterium Alteromonas the genome sequences of isolates of the genus have been extensively analyzed. However, the main genetic exchange drivers in these microbes, conjugative elements (CEs), have not been considered in detail thus far. In this work, CEs have been searched in several complete Alteromonas genomes and their sequence studied to understand their role in the evolution of this genus. Six genomes are reported here for the first time. We have found nine different plasmids of sizes ranging from 85 to 600 Kb, most of them were found in a single strain. Networks of gene similarity could be established among six of the plasmids that were also connected with another cluster of plasmids found in Shewanella strains. The cargo genes found in these plasmids included cassettes found before in chromosome flexible genomic islands of Alteromonas strains. We describe also the plasmids pAMCP48-600 and pAMCP49-600, the largest found in Alteromonas thus far (ca. 600 Kb) and containing all the hallmarks to be classified as chromids. We found in them some housekeeping genes and a cluster that code for an exocellular polysaccharide. They could represent the transport vectors for the previously described replacement flexible genomic islands. Integrative and conjugative elements (ICEs) were more common than plasmids and showed similar patterns of variation with cargo genes coding for components of additive flexible genomic islands. A nearly identical ICE was found in A. mediterranea MED64 and Vibrio cholera AHV1003 isolated from a human pathogen, indicating the potential exchange of these genes across phylogenetic distances exceeding the family threshold. We have seen evidence of how CEs can be vectors to transfer gene cassettes acquired in the chromosomal flexible genomic islands, both of the additive and replacement kind. These CEs showed evidence of how genetic material is exchanged among members of the same species but also (albeit less frequently) across genus and family barriers. These gradients of exchange frequency are probably one of the main drivers of species origin and maintenance in prokaryotes and also provide these taxa with large genetic diversity.

Occurrence and genetic characteristics of third-generation cephalosporin-resistant Escherichia coli in Swiss retail meat.

PubMed

Vogt, Debora; Overesch, Gudrun; Endimiani, Andrea; Collaud, Alexandra; Thomann, Andreas; Perreten, Vincent

2014-10-01

Prevalence and genetic relatedness were determined for third-generation cephalosporin-resistant Escherichia coli (3GC-R-Ec) detected in Swiss beef, veal, pork, and poultry retail meat. Samples from meat-packing plants (MPPs) processing 70% of the slaughtered animals in Switzerland were purchased at different intervals between April and June 2013 and analyzed. Sixty-nine 3GC-R-Ec isolates were obtained and characterized by microarray, PCR/DNA sequencing, Multi Locus Sequence Typing (MLST), and plasmid replicon typing. Plasmids of selected strains were transformed by electroporation into E. coli TOP10 cells and analyzed by plasmid MLST. The prevalence of 3GC-R-Ec was 73.3% in chicken and 2% in beef meat. No 3GC-R-Ec were found in pork and veal. Overall, the bla(CTX-M-1) (79.4%), bla(CMY-2) (17.6%), bla(CMY-4) (1.5%), and bla(SHV-12) (1.5%) β-lactamase genes were detected, as well as other genes conferring resistance to chloramphenicol (cmlA1-like), sulfonamides (sul), tetracycline (tet), and trimethoprim (dfrA). The 3GC-R-Ec from chicken meat often harbored virulence genes associated with avian pathogens. Plasmid incompatibility (Inc) groups IncI1, IncFIB, IncFII, and IncB/O were the most frequent. A high rate of clonality (e.g., ST1304, ST38, and ST93) among isolates from the same MPPs suggests that strains persist at the plant and spread to meat at the carcass-processing stage. Additionally, the presence of the blaCTX-M-1 gene on an IncI1 plasmid sequence type 3 (IncI1/pST3) in genetically diverse strains indicates interstrain spread of an epidemic plasmid. The bla(CMY-2) and bla(CMY-4) genes were located on IncB/O plasmids. This study represents the first comprehensive assessment of 3GC-R-Ec in meat in Switzerland. It demonstrates the need for monitoring contaminants and for the adaptation of the Hazard Analysis and Critical Control Point concept to avoid the spread of multidrug-resistant bacteria through the food chain.

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.

A stable shuttle vector system for efficient genetic complementation of Helicobacter pylori strains by transformation and conjugation.

PubMed

Heuermann, D; Haas, R

1998-03-01

A versatile plasmid shuttle vector system was constructed, which is useful for genetic complementation of Helicobacter pylori strains or mutants with cloned genes of homologous or heterologous origin. The individual plasmid vectors consist of the minimal essential genetic elements, including an origin of replication for Escherichia coli, a H. pylori-specific replicon originally identified on a small cryptic H. pylori plasmid, an oriT sequence and a multiple cloning site. Shuttle plasmid pHel2 carries a chloramphenicol resistance cassette (catGC) and pHel3 contains a kanamycin resistance gene (aphA-3) as the selectable marker; both are functional in E. coli and H. pylori. The shuttle plasmids were introduced into the H. pylori strain P1 by natural transformation. A efficiency of 7.0 x 10(-7) and 4.7 x 10(-7) transformants per viable recipient was achieved with pHel2 and pHel3, respectively, and both vectors showed stable, autonomous replication in H. pylori. An approximately 100-fold higher H. pylori transformation rate was obtained when the shuttle vectors for transformation were isolated from the homologous H. pylori strain, rather than E. coli, indicating that DNA restriction and modification mechanisms play a crucial role in plasmid transformation. Interestingly, both shuttle vectors could also be mobilized efficiently from E. coli into different H. pylori recipients, with pHel2 showing an efficiency of 2.0 x 10(-5) transconjugants per viable H. pylori P1 recipient. Thus, DNA restriction seems to be strongly reduced or absent during conjugal transfer. The functional complementation of a recA-deficient H. pylori mutant by the cloned H. pylori recA+ gene, and the expression of the heterologous green fluorescent protein (GFP) in H. pylori demonstrate the general usefulness of this system, which will significantly facilitate the molecular analysis of H. pylori virulence factors in the future.

Genetic linkage between resistance to quaternary ammonium compounds and beta-lactam antibiotics in food-related Staphylococcus spp.

PubMed

Sidhu, M S; Heir, E; Sørum, H; Holck, A

2001-01-01

Little is known about the occurrence of antimicrobial resistance determinants in staphylococci isolated from food and food processing industries. Quaternary ammonium compound (QAC)-resistant coagulase-negative staphylococci (CNS) isolated from food and food-processing industries were investigated for the presence of genetic determinants (qacA/B and qacC/smr) encoding resistance to the QAC benzalkonium chloride (BC), several antibiotic resistance genes, and staphylococcal insertion sequences IS257 and IS256. Six qacA/B-harboring strains were resistant to penicillin and hybridized to a blaZ probe. The qacA/B and blaZ probes hybridized to plasmids of similar size in three isolates. Molecular and genetic characterization of the 23-kb plasmid (pST6) of Staphylococcus epidermidis St.6 revealed the presence of qacB adjacent to an incomplete beta-lactamase transposon Tn552 encoding the gene cluster blaZ, blaR, and blaI. Sequence analysis of flanking regions and the intergenic region between blaZ and qacB revealed the presence of IS257 downstream of blaZ as well as sin and binR between blaZ and qacB. In the three other BC and penicillin-resistant strains, the qacA/B and blaZ genes were located on separate plasmids. A qacC harboring S. epidermidis strain (St.17) also hybridized to tetK (tetracycline resistance) and ermB (erythromycin resistance) genes. The individual genes were located on separate plasmids, suggesting no linkage between QAC and antibiotic resistance determinants. Plasmid-free Staphylococcus aureus RN4220 allowed uptake of the pST6 plasmid DNA, indicating that the resistance genes could potentially be transferred to pathogens under selective stress. In conclusion, presence of both resistance determinants could lead to co-selection during antimicrobial therapy or disinfection in hospitals or in food industries.

H-NS-like nucleoid-associated proteins, mobile genetic elements and horizontal gene transfer in bacteria.

PubMed

Dorman, Charles J

2014-09-01

Horizontal gene transfer plays an important role in the evolution of bacterial species, conferring new genetic traits on the recipient bacterium that extend its range of phenotypes and plasmids make important contributions to this process. However, the inappropriate expression of newly acquired genes may lead to a loss of competitive fitness, resulting in the elimination of the new gene-bacterium combination. It is thought that transcriptional silencing of horizontally acquired genes offers a route out of this dilemma and that nucleoid-associated proteins, especially those related to the H-NS protein, play a particularly important role in the silencing process. The discovery that many plasmids express orthologues of nucleoid-associated proteins adds an interesting dimension to current models of regulatory integration following lateral transfer of DNA. Other horizontally acquired genetic elements, such as genomic islands, also express nucleoid-associated proteins of their own. Here the interactions of H-NS-like nucleoid-associated proteins encoded by the core genome, genomic islands and plasmids are described. Copyright © 2014 Elsevier Inc. All rights reserved.

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

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.

Multiple Antibiotic Resistance Plasmids Allow Scalable,
PCR-Mediated DNA Manipulation and Near-Zero Background Cloning

PubMed Central

Arnak, Remigiusz; Altun, Burcin; Tosato, Valentina

2016-01-01

Summary We have constructed two plasmids that can be used for cloning as templates for PCR- -based gene disruption, mutagenesis and the construction of DNA chromosome translocation cassettes. To our knowledge, these plasmids are the first vectors that confer resistance to ampicillin, kanamycin and hygromycin B in bacteria, and to geneticin (G418) and hygromycin B in Saccharomyces cerevisiae simultaneously. The option of simultaneously using up to three resistance markers provides a highly stringent control of recombinant selection and the almost complete elimination of background resistance, while unique restriction sites allow easy cloning of chosen genetic material. Moreover, we successfully used these new vectors as PCR templates for the induction of chromosome translocation in budding yeast by the bridge-induced translocation system. Cells in which translocation was induced carried chromosomal rearrangements as expected and exhibited resistance to both, G418 and hygromycin B. These features make our constructs very handy tools for many molecular biology applications. PMID:27956856

Shifts in Abundance and Diversity of Mobile Genetic Elements after the Introduction of Diverse Pesticides into an On-Farm Biopurification System over the Course of a Year

PubMed Central

Dealtry, Simone; Holmsgaard, Peter N.; Dunon, Vincent; Jechalke, Sven; Ding, Guo-Chun; Krögerrecklenfort, Ellen; Heuer, Holger; Hansen, Lars H.; Springael, Dirk; Zühlke, Sebastian; Sørensen, Søren J.

2014-01-01

Biopurification systems (BPS) are used on farms to control pollution by treating pesticide-contaminated water. It is assumed that mobile genetic elements (MGEs) carrying genes coding for enzymes involved in degradation might contribute to the degradation of pesticides. Therefore, the composition and shifts of MGEs, in particular, of IncP-1 plasmids carried by BPS bacterial communities exposed to various pesticides, were monitored over the course of an agricultural season. PCR amplification of total community DNA using primers targeting genes specific to different plasmid groups combined with Southern blot hybridization indicated a high abundance of plasmids belonging to IncP-1, IncP-7, IncP-9, IncQ, and IncW, while IncU and IncN plasmids were less abundant or not detected. Furthermore, the integrase genes of class 1 and 2 integrons (intI1, intI2) and genes encoding resistance to sulfonamides (sul1, sul2) and streptomycin (aadA) were detected and seasonality was revealed. Amplicon pyrosequencing of the IncP-1 trfA gene coding for the replication initiation protein revealed high IncP-1 plasmid diversity and an increase in the abundance of IncP-1β and a decrease in the abundance of IncP-1ε over time. The data of the chemical analysis showed increasing concentrations of various pesticides over the course of the agricultural season. As an increase in the relative abundances of bacteria carrying IncP-1β plasmids also occurred, this might point to a role of these plasmids in the degradation of many different pesticides. PMID:24771027

[Comparative study of aromatic ring meta-cleavage enzymes in Pseudomonas strains with plasmid and chromosomal genetic control of the catabolism of biphenyl and m-toluate].

PubMed

Selifonov, S A; Starozoĭtov, I I

1990-12-01

It was shown that two different enzymes of aromatic ring oxidative meta-cleavage (2,3-dihydroxybiphenyl-1,2-dioxygenase), DBO and catechol-2,3-dioxygenase, C230) function in Pseudomonas strains with a plasmid and chromosomal genetic control of biphenyl and toluate catabolism. A comparative analysis of DBO's and C230's expressed by the pBS241 biphenyl degradative plasmid in P. putida BS893, pBS311 in P. putida U83, chromosomal genes in P. putida BF and C230 from P. putida PaW160 (pWWO) was carried out. It was found that the DBO's of all strains under study are highly specialized enzymes in respect of 2,3-dihydroxybiphenyl cleavage and are also able to cleave 3-methyl-catechol and catechol (but not 4-methylcatechol) at low rates. In contrast with DBO's, in Pseudomonas strains the substrate specificities of all C230's are variable. The C230's expressed by the D-plasmids pBS241 and pBC311 have a moderate affinity for catechol, 3-methyl- and 4-methylcatechol, but are unable to cleave 2,3-dihydroxybiphenyl. The C230 which is encoded by the chromosomal structure gene from P. putida BF is very similar to C230 which codes for the TOL-plasmid pWWO. These plasmid differ from C230's expressed by biphenyl D-plasmids due to their capability to cleave 2,3-dihydroxybiphenyl in addition to catechol cleavage. All DBO's and C230's under study possess a number of properties that are typical for the enzymes having an oxidative meta-cleaving effect. The different roles of these enzymes in biphenyl and toluate catabolism in Pseudomonas strains are discussed.

Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

PubMed Central

2011-01-01

Background The genome of a number of species of malaria parasites (Plasmodium spp.) has been sequenced in the hope of identifying new drug and vaccine targets. However, almost one-half of predicted Plasmodium genes are annotated as hypothetical and are difficult to analyse in bulk due to the inefficiency of current reverse genetic methodologies for Plasmodium. Recently, it has been shown that the transposase piggyBac integrates at random into the genome of the human malaria parasite P. falciparum offering the possibility to develop forward genetic screens to analyse Plasmodium gene function. This study reports the development and application of the piggyBac transposition system for the rodent malaria parasite P. berghei and the evaluation of its potential as a tool in forward genetic studies. P. berghei is the most frequently used malaria parasite model in gene function analysis since phenotype screens throughout the complete Plasmodium life cycle are possible both in vitro and in vivo. Results We demonstrate that piggyBac based gene inactivation and promoter-trapping is both easier and more efficient in P. berghei than in the human malaria parasite, P. falciparum. Random piggyBac-mediated insertion into genes was achieved after parasites were transfected with the piggyBac donor plasmid either when transposase was expressed either from a helper plasmid or a stably integrated gene in the genome. Characterization of more than 120 insertion sites demonstrated that more than 70 most likely affect gene expression classifying their protein products as non-essential for asexual blood stage development. The non-essential nature of two of these genes was confirmed by targeted gene deletion one of which encodes P41, an ortholog of a human malaria vaccine candidate. Importantly for future development of whole genome phenotypic screens the remobilization of the piggyBac element in parasites that stably express transposase was demonstrated. Conclusion These data demonstrate that piggyBac behaved as an efficient and random transposon in P. berghei. Remobilization of piggyBac element shows that with further development the piggyBac system can be an effective tool to generate random genome-wide mutation parasite libraries, for use in large-scale phenotype screens in vitro and in vivo. PMID:21418605

Rhizobium etli and Rhizobium gallicum Nodulate Common Bean (Phaseolus vulgaris) in a Traditionally Managed Milpa Plot in Mexico: Population Genetics and Biogeographic Implications

PubMed Central

Silva, Claudia; Vinuesa, Pablo; Eguiarte, Luis E.; Martínez-Romero, Esperanza; Souza, Valeria

2003-01-01

The stability of the genetic structure of rhizobial populations nodulating Phaseolus vulgaris cultivated in a traditionally managed milpa plot in Mexico was studied over three consecutive years. The set of molecular markers analyzed (including partial rrs, glnII, nifH, and nodB sequences), along with host range experiments, placed the isolates examined in Rhizobium etli bv. phaseoli and Rhizobium gallicum bv. gallicum. Cluster analysis of multilocus enzyme electrophoresis and plasmid profile data separated the two species and identified numerically dominant clones within each of them. Population genetic analyses showed that there was high genetic differentiation between the two species and that there was low intrapopulation differentiation of the species over the 3 years. The results of linkage disequilibrium analyses are consistent with an epidemic genetic structure for both species, with frequent genetic exchange taking place within conspecific populations but not between the R. etli and R. gallicum populations. A subsample of isolates was selected and used for 16S ribosomal DNA PCR-restriction fragment length polymorphism analysis, nifH copy number determination, and host range experiments. Plasmid profiles and nifH hybridization patterns also revealed the occurrence of lateral plasmid transfer among distinct multilocus genotypes within species but not between species. Both species were recovered from nodules of the same plants, indicating that mechanisms other than host, spatial, or temporal isolation may account for the genetic barrier between the species. The biogeographic implications of finding an R. gallicum bv. gallicum population nodulating common bean in America are discussed. PMID:12571008

First report on rapid screening of nanomaterial-based antimicrobial agents against β-lactamase resistance using pGLO plasmid transformed Escherichia coli HB 101 K-12

NASA Astrophysics Data System (ADS)

Raj, M. Alpha; Muralidhar, Y.; Sravanthi, M.; Prasad, T. N. V. K. V.; Nissipriya, M.; Reddy, P. Sirisha; Neelima, T. Shoba; Reddy, G. Dilip; Adilaxmamma, K.; Kumar, P. Anand; Krishna, T. Giridhara

2016-08-01

Combating antibiotic resistance requires discovery of novel antimicrobials effective against resistant bacteria. Herein, we present for the first time, pGLO plasmid transformed Escherichia coli HB 101 K 12 as novel model for screening of nanomaterial-based antimicrobial agents against β-lactamase resistance. E. coli HB 101 was transformed by pGLO plasmid in the presence of calcium chloride (50 mM; pH 6.1) aided by heat shock (0-42-0 °C). The transformed bacteria were grown on Luria-Bertani agar containing ampicillin (amp) and arabinose (ara). The transformed culture was able to grow in the presence of ampicillin and also exhibited fluorescence under UV light. Both untransformed and transformed bacteria were used for screening citrate-mediated nanosilver (CNS), aloin-mediated nanosilver (ANS), 11-α-keto-boswellic acid (AKBA)-mediated nanosilver (BNS); nanozinc oxide, nanomanganese oxide (NMO) and phytochemicals such as aloin and AKBA. Minimum inhibitory concentrations (MIC) were obtained by microplate method using ρ-iodo nitro tetrazolium indicator. All the compounds were effective against transformed bacteria except NMO and AKBA. Transformed bacteria exhibited reverse cross resistance against aloin. ANS showed the highest antibacterial activity with a MIC of 0.32 ppm followed by BNS (10.32 ppm), CNS (20.64 ppm) and NZO (34.83 ppm). Thus, pGLO plasmid can be used to induce resistance against β-lactam antibiotics and the model can be used for rapid screening of new antibacterial agents effective against resistant bacteria.

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.

Insights into heliobacterial photosynthesis and physiology from the genome of Heliobacterium modesticaldum.

PubMed

Sattley, W Matthew; Blankenship, Robert E

2010-06-01

The complete annotated genome sequence of Heliobacterium modesticaldum strain Ice1 provides our first glimpse into the genetic potential of the Heliobacteriaceae, a unique family of anoxygenic phototrophic bacteria. H. modesticaldum str. Ice1 is the first completely sequenced phototrophic representative of the Firmicutes, and heliobacteria are the only phototrophic members of this large bacterial phylum. The H. modesticaldum genome consists of a single 3.1-Mb circular chromosome with no plasmids. Of special interest are genomic features that lend insight to the physiology and ecology of heliobacteria, including the genetic inventory of the photosynthesis gene cluster. Genes involved in transport, photosynthesis, and central intermediary metabolism are described and catalogued. The obligately heterotrophic metabolism of heliobacteria is a key feature of the physiology and evolution of these phototrophs. The conspicuous absence of recognizable genes encoding the enzyme ATP-citrate lyase prevents autotrophic growth via the reverse citric acid cycle in heliobacteria, thus being a distinguishing differential characteristic between heliobacteria and green sulfur bacteria. The identities of electron carriers that enable energy conservation by cyclic light-driven electron transfer remain in question.

Stable transfection of babesia bigemina and babesia bovis using a single plasmid containing homologous and heterologous insertion and gene regulatory sequences

USDA-ARS?s Scientific Manuscript database

Bovine babesiosis caused by Babesia bovis and B. bigemina is a severe, often fatal, tick-borne disease of cattle for which improved methods for control are urgently needed. One of our long term goals is to develop effective vaccines based on genetically modfied B. bigemina and B. bovis parasites. Al...

Counterselection method based on conditional silencing of antitoxin genes in Escherichia coli.

PubMed

Tsukuda, Miyuki; Nakashima, Nobutaka; Miyazaki, Kentaro

2015-11-01

Counterselection is a genetic engineering technique to eliminate specific genetic fragments containing selectable marker genes. Although the technique is widely used in bacterial genome engineering and plasmid curing experiments, the repertoire of the markers usable in Escherichia coli is limited. Here we developed a novel counterselection method in E. coli based on antisense RNA (asRNA) technology directed against toxin-antitoxin (TA) modules. Under normal conditions, excess antitoxin neutralizes its cognate toxin and thus the module is stably maintained in the genome. We hypothesised that repression of an antitoxin gene would perturb cell growth due to the toxin being released. We designed asRNAs corresponding to all 19 type II antitoxins encoded in the E. coli genome. asRNAs were then conditionally expressed; repression of MqsA in the MqsR/MqsA module had the greatest inhibitory effect, followed by RnlB in the RnlA/RnlB module. The utility of asRNA(MqsA) as a counterselection marker was demonstrated by efficient plasmid curing and strain improvement experiments. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Advanced Design of Dumbbell-shaped Genetic Minimal Vectors Improves Non-coding and Coding RNA Expression.

PubMed

Jiang, Xiaoou; Yu, Han; Teo, Cui Rong; Tan, Genim Siu Xian; Goh, Sok Chin; Patel, Parasvi; Chua, Yiqiang Kevin; Hameed, Nasirah Banu Sahul; Bertoletti, Antonio; Patzel, Volker

2016-09-01

Dumbbell-shaped DNA minimal vectors lacking nontherapeutic genes and bacterial sequences are considered a stable, safe alternative to viral, nonviral, and naked plasmid-based gene-transfer systems. We investigated novel molecular features of dumbbell vectors aiming to reduce vector size and to improve the expression of noncoding or coding RNA. We minimized small hairpin RNA (shRNA) or microRNA (miRNA) expressing dumbbell vectors in size down to 130 bp generating the smallest genetic expression vectors reported. This was achieved by using a minimal H1 promoter with integrated transcriptional terminator transcribing the RNA hairpin structure around the dumbbell loop. Such vectors were generated with high conversion yields using a novel protocol. Minimized shRNA-expressing dumbbells showed accelerated kinetics of delivery and transcription leading to enhanced gene silencing in human tissue culture cells. In primary human T cells, minimized miRNA-expressing dumbbells revealed higher stability and triggered stronger target gene suppression as compared with plasmids and miRNA mimics. Dumbbell-driven gene expression was enhanced up to 56- or 160-fold by implementation of an intron and the SV40 enhancer compared with control dumbbells or plasmids. Advanced dumbbell vectors may represent one option to close the gap between durable expression that is achievable with integrating viral vectors and short-term effects triggered by naked RNA.

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.

Genetic and Physiological Studies of Bacillus anthracis Related to Development of an Improved Vaccine

DTIC Science & Technology

1986-07-01

demonstrated that plasmid pLS20 of B. subtilis ( natto ) is capa le of promoting the transfer of pBC16 from B. subtilis to a variety of Bacillus s ecies...anthracis. Hofetver, results of recent experiments demonstrate that pL320, a 34-megadalton plasmid of B. subtilis ( natto ), is capable of promoting the...plasmid in Bacillus subtilis ( natto ).20 IV. Determination of the size of pX02 by restriction analysis-....... 24 V. Transfer of pXO1 by the B

Plasmids from Food Lactic Acid Bacteria: Diversity, Similarity, and New Developments

PubMed Central

Cui, Yanhua; Hu, Tong; Qu, Xiaojun; Zhang, Lanwei; Ding, Zhongqing; Dong, Aijun

2015-01-01

Plasmids are widely distributed in different sources of lactic acid bacteria (LAB) as self-replicating extrachromosomal genetic materials, and have received considerable attention due to their close relationship with many important functions as well as some industrially relevant characteristics of the LAB species. They are interesting with regard to the development of food-grade cloning vectors. This review summarizes new developments in the area of lactic acid bacteria plasmids and aims to provide up to date information that can be used in related future research. PMID:26068451

Use of AFLP, plasmid typing and phenotyping in a comparative study to assess genetic diversity of Shigella flexneri strains.

PubMed Central

Herrera, S.; Cabrera, R.; Ramirez, M. M.; Usera, M. A.; Echeita, M. A.

2002-01-01

Shigella flexneri infections are one of the main causes of acute diarrhoea in Cuba. Twenty strains isolated from sporadic cases in nine different Cuban provinces were characterized. Serotyping, antibiotic-resistance typing, plasmid-typing and AFLP-typing were used to determine their suitability for use in epidemiological studies of S. flexneri. The predominant serotypes were serotype 6 (35%) and serotype 2 (35%). Eleven different plasmid profiles were detected (Diversity Index = 0.92). AFLP-typing discriminated 12 different patterns (DI = 0.95), these patterns were not coincident with plasmid-typing patterns. Both techniques combined distinguished 14 patterns among the 20 studied strains (DI = 0.99). There was no consistent relationship between plasmid-typing and AFLP-typing patterns or antibiotic-resistance typing patterns. Ninety-five percent of S. flexneri strains were multiresistant. PMID:12558326

Development of a Gene Cloning System in Methanogens.

DTIC Science & Technology

1987-03-27

Genetic transfer via DNA-dependent natural transformation was achieved for two markers, 5-fluorouracil-resistance, and 6- mercaptopurine resistance...resistance genes, and genes coding for enzymes that produce colored products will be tested as markers for plasmid transformation. A functional plasmid...clones, which include resistances to mercaptopurine , azahypoxanthine, diazauracil, kanamycin, mitomycin C, and fluorouracil- mercaptopurine and

[Preventive and therapeutic effect of genetic vaccine based on recombinant alpha virus against mouse mastocytoma P815].

PubMed

Ni, Bing; Yang, Ri-gao; Li, Yan-qiu; Wu, Yu-zhang

2004-01-01

To explore the immunological effect of genetic vaccine based on alpha-virus and to seek out better forms of gene vaccines. Expression plasmid P1A/pSMART2a and packaging plasmid helper were cotransfected into mammalian 293 cells by calcium phosphate precipitation method and high level of recombinant alpha-virus P1A/SFV was prepared. Following identification of rSFV and its expression, BALB/c mice were inoculated with rSFV, and the production of antigen-specific antibody and the cytotoxic effect of CTLs were determined. In the preventive and therapeutic experiments, the percents of tumor-free and of survival mice immunized with rSFV were observed. The recombinant SFV could express correctly in cultured cells. After being inoculated into the mice, rSFV could prime stronger CTL response than that in control mice. When the ratio of E/T cells was 100:1, the (51)Cr release rate reached 75%. No antibody could be detected in mice from all groups. The immunological effect of P1A/SFV among all groups was the best in both preventive and therapeutic experiment within experimental deadline. On 60th day in preventive experiment, the percent of tumor-free animal in P1A/SFV group reached 60%, whereas that was only 20% in P1A/pCI-neogroup. On 60th day in therapeutic experiment, survival rate of mice in P1A/SFV group reached 50%, but only 10% mice could survive in all control groups. Compared with common gene vaccines, the genetic vaccine based on recombinant SFV has the best immunological effect, which provides some new strategies for clinical genetic therapy of tumors.

Coexistence of blaOXA-48 and Truncated blaNDM-1 on Different Plasmids in a Klebsiella pneumoniae Isolate in China.

PubMed

Xie, Lianyan; Dou, Yi; Zhou, Kaixin; Chen, Yue; Han, Lizhong; Guo, Xiaokui; Sun, Jingyong

2017-01-01

Objectives: To describe the genetic environment, transferability, and antibiotic susceptibility of one clinical Klebsiella pneumoniae isolate harboring both bla OXA-48 and bla NDM-1 on different plasmids from a Chinese hospital. Methods: The isolate was subjected to antimicrobial susceptibility testing and multilocus sequence typing using Etest and PCR. The plasmids harboring bla OXA-48 and bla NDM-1 were analyzed through conjugation experiments, S1-nuclease pulsed-field gel electrophoresis, and hybridization with specific probes. Plasmid DNA was sequenced using Pacbio RS II and annotated using RAST. Results: K. pneumoniae RJ119, carrying both bla OXA-48 and bla NDM-1 , was resistant to almost all carbapenems, cephalosporins, fluoroquinolone, and aminoglycosides and belonged to ST307. bla OXA-48 was located on a 61,748-bp IncL/M conjugative plasmid, which displayed overall nucleotide identity (99%) to pKPN-E1-Nr.7. bla NDM-1 was located on a 335,317-bp conjugative plasmid, which was a fusion of a bla NDM-1 -harboring InA/C plasmid pNDM-US (140,825 bp, 99% identity) and an IncFIB plasmid pKPN-c22 (178,563 bp, 99% identity). The transconjugant RJ119-1 harboring bla NDM-1 was susceptible to carbapenem, and there was an insertion of IS 10 into the bla NDM-1 gene. Conclusion: This is the first report of the coexistence of bla OXA-48 and bla NDM-1 in one K. pneumoniae clinical isolate in China. OXA-48 in RJ119 contributed to the majority to its high resistance to carbapenems, whereas NDM-1 remained unexpressed, most likely due to the insertion of IS 10 . Our results provide new insight for the relationship between genetic diagnosis and clinical treatment. They also indicate that increased surveillance of bla OXA-48 is urgently needed in China.

Antimicrobial drug resistance and genetic properties of Salmonella enterica serotype Enteritidis circulating in chicken farms in Tunisia.

PubMed

Ben Salem, Rakia; Abbassi, Mohamed S; García, Vanesa; García-Fierro, Raquel; Fernández, Javier; Kilani, Hajer; Jaouani, Imen; Khayeche, Monia; Messadi, Lilia; Rodicio, María R

This study focused on 77 isolates of Salmonella enterica serotype Enteritidis collected during 2009 to 2013 from healthy and sick chickens and environmental farm samples in Tunisia. Resistance to 14 antimicrobials and the encoding genes were analyzed. 66, 26, 6.5, 3.9 and 1.3% were pan-susceptible or showed resistance to nalidixic acid (Asp87 to Tyr and Asp87 to Asn substitutions in GyrA), ampicillin (bla TEM-1-like and bla SHV ), sulfonamides (sul1and sul3) and streptomycin (strB), respectively. A single isolate with intermediate susceptibility to ciprofloxacin was positive for qnrB, whereas qnrA, qnrS or aac(6')-Ib-cr were not detected. The virulotype of the isolates was established by testing ten virulence genes. The orgA, ssaQ, mgtC, siiD, sopB genes, located on Salmonella pathogenicity islands, and spvC of the serotype-specific virulence plasmid, were common to all isolates. In contrast, the prophage-associated sopE-1, sodC1 and gipA genes and the fimbrial bcfC gene were variably represented. All isolates except one contained the virulence plasmid, which appeared either alone or together with one or more additional plasmids. One isolate carried a single plasmid of ca. 90Kb which may be derived from the virulence plasmid (60Kb). Overall, seven resistotypes, six virulotypes and six plasmid profiles were identified. XbaI-PFGE revealed four related pulsotypes (X1-X4), with 80% of the isolates sharing the X1 pattern. The latter isolates exhibited different resistance, virulence and plasmid profiles, suggesting that mobile genetic elements, particularly prophages and plasmids, are of central importance for the evolution and adaptation of S. Enteritidis circulating in chicken farms in Tunisia. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

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.

Virulence Phenotypes of Legionella pneumophila Associated with Noncoding RNA lpr0035

PubMed Central

Jayakumar, Deepak; Early, Julie V.

2012-01-01

The Philadelphia-1 strain of Legionella pneumophila, the causative organism of Legionnaires' disease, contains a recently discovered noncoding RNA, lpr0035. lpr0035 straddles the 5′ chromosomal junction of a 45-kbp mobile genetic element, pLP45, which can exist as an episome or integrated in the bacterial chromosome. A 121-bp deletion was introduced in strain JR32, a Philadelphia-1 derivative. The deletion inactivated lpr0035, removed the 49-bp direct repeat at the 5′ junction of pLP45, and locked pLP45 in the chromosome. Intracellular multiplication of the deletion mutant was decreased by nearly 3 orders of magnitude in Acanthamoeba castellanii amoebae and nearly 2 orders of magnitude in J774 mouse macrophages. Entry of the deletion mutant into amoebae and macrophages was decreased by >70%. The level of entry in both hosts was restored to that in strain JR32 by plasmid copies of two open reading frames immediately downstream of the 5′ junction and plasmid lpr0035 driven by its endogenous promoter. When induced from a tac promoter, plasmid lpr0035 completely reversed the intracellular multiplication defect in macrophages but was without effect in amoebae. These data are the first evidence of a role for noncoding RNA lpr0035, which has homologs in six other Legionella genomes, in entry of L. pneumophila into amoebae and macrophages and in host-specific intracellular multiplication. The data also demonstrate that deletion of a direct-repeat sequence restricts the mobility of pLP45 and is a means of studying the role of pLP45 mobility in Legionella virulence phenotypes. PMID:22966048

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.

International Spread and Persistence of TEM-24 Is Caused by the Confluence of Highly Penetrating Enterobacteriaceae Clones and an IncA/C2 Plasmid Containing Tn1696::Tn1 and IS5075-Tn21▿

PubMed Central

Novais, Ângela; Baquero, Fernando; Machado, Elisabete; Cantón, Rafael; Peixe, Luísa; Coque, Teresa M.

2010-01-01

TEM-24 remains one of the most widespread TEM-type extended-spectrum β-lactamases (ESBLs) among Enterobacteriaceae. To analyze the reasons influencing its spread and persistence, a multilevel population genetics study was carried out on 28 representative TEM-24 producers from Belgium, France, Portugal, and Spain (13 Enterobacter aerogenes isolates, 6 Escherichia coli isolates, 6 Klebsiella pneumoniae isolates, 2 Proteus mirabilis isolates, and 1 Klebsiella oxytoca isolate, from 1998 to 2004). Clonal relatedness (XbaI pulsed-field gel electrophoresis [PFGE] and E. coli phylogroups) and antibiotic susceptibility were determined by standard procedures. Plasmid analysis included determination of the incompatibility group (by PCR, hybridization, and/or sequencing) and comparison of restriction fragment length polymorphism (RFLP) patterns. Characterization of genetic elements conferring antibiotic resistance included integrons (classes 1, 2, and 3) and transposons (Tn3, Tn21, and Tn402). Similar PFGE patterns were identified among E. aerogenes, K. pneumoniae, and P. mirabilis isolates, while E. coli strains were diverse (phylogenetic groups A, B2, and D). Highly related 180-kb IncA/C2 plasmids conferring resistance to kanamycin, tobramycin, chloramphenicol, trimethoprim, and sulfonamides were identified. Each plasmid contained defective In0-Tn402 (dfrA1-aadA1, aacA4, or aacA4-aacC1-orfE-aadA2-cmlA1) and In4-Tn402 (aacA4 or dfrA1-aadA1) variants. These integrons were located within Tn21, Tn1696, or hybrids of these transposons, with IS5075 interrupting their IRtnp and IRmer. In all cases, blaTEM-24 was part of an IS5075-ΔTn1 transposon within tnp1696, mimicking other genetic elements containing blaTEM-2 and blaTEM-3 variants. The international dissemination of TEM-24 is fuelled by an IncA/C2 plasmid acquired by different enterobacterial clones which seem to evolve by gaining diverse genetic elements. This work highlights the risks of a confluence between highly penetrating clones and highly promiscuous plasmids in the spread of antibiotic resistance, and it contributes to the elucidation of the origin and evolution of TEM-2 ESBL derivatives. PMID:19995930

Metabolic transcription analysis of engineered Escherichia coli strains that overproduce L-phenylalanine

PubMed Central

Báez-Viveros, José Luis; Flores, Noemí; Juárez, Katy; Castillo-España, Patricia; Bolivar, Francisco; Gosset, Guillermo

2007-01-01

Background The rational design of L-phenylalanine (L-Phe) overproducing microorganisms has been successfully achieved by combining different genetic strategies such as inactivation of the phosphoenolpyruvate: phosphotransferase transport system (PTS) and overexpression of key genes (DAHP synthase, transketolase and chorismate mutase-prephenate dehydratase), reaching yields of 0.33 (g-Phe/g-Glc), which correspond to 60% of theoretical maximum. Although genetic modifications introduced into the cell for the generation of overproducing organisms are specifically targeted to a particular pathway, these can trigger unexpected transcriptional responses of several genes. In the current work, metabolic transcription analysis (MTA) of both L-Phe overproducing and non-engineered strains using Real-Time PCR was performed, allowing the detection of transcriptional responses to PTS deletion and plasmid presence of genes related to central carbon metabolism. This MTA included 86 genes encoding enzymes of glycolysis, gluconeogenesis, pentoses phosphate, tricarboxylic acid cycle, fermentative and aromatic amino acid pathways. In addition, 30 genes encoding regulatory proteins and transporters for aromatic compounds and carbohydrates were also analyzed. Results MTA revealed that a set of genes encoding carbohydrate transporters (galP, mglB), gluconeogenic (ppsA, pckA) and fermentative enzymes (ldhA) were significantly induced, while some others were down-regulated such as ppc, pflB, pta and ackA, as a consequence of PTS inactivation. One of the most relevant findings was the coordinated up-regulation of several genes that are exclusively gluconeogenic (fbp, ppsA, pckA, maeB, sfcA, and glyoxylate shunt) in the best PTS- L-Phe overproducing strain (PB12-ev2). Furthermore, it was noticeable that most of the TCA genes showed a strong up-regulation in the presence of multicopy plasmids by an unknown mechanism. A group of genes exhibited transcriptional responses to both PTS inactivation and the presence of plasmids. For instance, acs-ackA, sucABCD, and sdhABCD operons were up-regulated in PB12 (PTS mutant that carries an arcB- mutation). The induction of these operons was further increased by the presence of plasmids in PB12-ev2. Some genes involved in the shikimate and specific aromatic amino acid pathways showed down-regulation in the L-Phe overproducing strains, might cause possible metabolic limitations in the shikimate pathway. Conclusion The identification of potential rate-limiting steps and the detection of transcriptional responses in overproducing microorganisms may suggest "reverse engineering" strategies for the further improvement of L-Phe production strains. PMID:17880710

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

Molecular Characterization of OXA-198 Carbapenemase-Producing Pseudomonas aeruginosa Clinical Isolates.

PubMed

Bonnin, Rémy A; Bogaerts, Pierre; Girlich, Delphine; Huang, Te-Din; Dortet, Laurent; Glupczynski, Youri; Naas, Thierry

2018-06-01

Carbapenemase-producing Pseudomonadaceae have increasingly been reported worldwide, with an ever-increasing heterogeneity of carbapenem resistance mechanisms, depending on the bacterial species and the geographical location. OXA-198 is a plasmid-encoded class D β-lactamase involved in carbapenem resistance in one Pseudomonas aeruginosa isolate from Belgium. In the setting of a multicenter survey of carbapenem resistance in P. aeruginosa strains in Belgian hospitals in 2013, three additional OXA-198-producing P. aeruginosa isolates originating from patients hospitalized in one hospital were detected. To reveal the molecular mechanism underlying the reduced susceptibility to carbapenems, MIC determinations, whole-genome sequencing, and PCR analyses to confirm the genetic organization were performed. The plasmid harboring the bla OXA-198 gene was characterized, along with the genetic relatedness of the four P. aeruginosa isolates. The bla OXA-198 gene was harbored on a class 1 integron carried by an ∼49-kb IncP-type plasmid proposed as IncP-11. The same plasmid was present in all four P. aeruginosa isolates. Multilocus sequence typing revealed that the isolates all belonged to sequence type 446, and single-nucleotide polymorphism analysis revealed only a few differences between the isolates. This report describes the structure of a 49-kb plasmid harboring the bla OXA-198 gene and presents the first description of OXA-198-producing P. aeruginosa isolates associated with a hospital-associated cluster episode. Copyright © 2018 American Society for Microbiology.

Massive increase, spread, and exchange of extended spectrum β-lactamase-encoding genes among intestinal Enterobacteriaceae in hospitalized children with severe acute malnutrition in Niger.

PubMed

Woerther, Paul-Louis; Angebault, Cécile; Jacquier, Hervé; Hugede, Henri-Charles; Janssens, Ann-Carole; Sayadi, Sani; El Mniai, Assiya; Armand-Lefèvre, Laurence; Ruppé, Etienne; Barbier, François; Raskine, Laurent; Page, Anne-Laure; de Rekeneire, Nathalie; Andremont, Antoine

2011-10-01

From the time of CTX-M emergence, extended-spectrum β-lactamase-producing enterobacteria (ESBL-E) have spread worldwide in community settings as well as in hospitals, particularly in developing countries. Although their dissemination appears linked to Escherichia coli intestinal carriage, precise paths of this dynamic are largely unknown. Children from a pediatric renutrition center were prospectively enrolled in a fecal carriage study. Antibiotic exposure was recorded. ESBL-E strains were isolated using selective media from fecal samples obtained at admission and, when negative, also at discharge. ESBL-encoding genes were identified, their environments and plasmids were characterized, and clonality was assessed with polymerase chain reaction-based methods and pulsed-field gel electrophoresis for E. coli and Klebsiella pneumoniae. E. coli strains were subjected to multilocus sequence typing. The ESBL-E carriage rate was 31% at admission in the 55 children enrolled. All children enrolled received antibiotics during hospitalization. Among the ESBL-E-negative children, 16 were resampled at discharge, and the acquisition rate was 94%. The bla(CTX-M-15) gene was found in >90% of the carriers. Genetic environments and plasmid characterization evidenced the roles of a worldwide, previously described, multidrug-resistant region and of IncF plasmids in CTX-M-15 E. coli dissemination. Diversity of CTX-M-15-carrying genetic structures and clonality of acquired ESBL E. coli suggested horizontal genetic transfer and underlined the potential of some ST types for nosocomial cross-transmission. Cross-transmission and high selective pressure lead to very high acquisition of ESBL-E carriage, contributing to dissemination in the community. Strict hygiene measures as well as careful balancing of benefit-risk ratio of current antibiotic policies need to be reevaluated.

Elimination of endogenous aberrant kappa chain transcripts from sp2/0-derived hybridoma cells by specific ribozyme cleavage: utility in genetic therapy of HIV-1 infections.

PubMed Central

Duan, L; Pomerantz, R J

1994-01-01

The pooled degenerate-primer polymerase chain reaction (PCR) technology is now widely used in the amplification and cloning of murine hybridoma-specific immunoglobulin gene cDNAs. The design of primers is mainly based on the highly conserved 5' terminus of immunoglobulin gene variable regions and the constant region in the 3' terminus. Of note, most murine hybridoma cell lines are derived from the Sp2/0 cell line, which is demonstrated to express endogenous aberrant kappa chains (abV kappa). This high-level endogenous abV kappa mixes with specific kappa chains in the hybridomas and interferes with the efficiency of the reverse transcriptase (RT)-PCR cloning strategy. In this report, during the cloning of murine anti-human immunodeficiency virus type I (HIV-1) hybridoma immunoglobulin cDNAs, a specific primer-PCR screening system was developed, based on the abV kappa complementarity-defining region (CDR), to eliminate abV kappa-carrying plasmids. Furthermore, an abV kappa sequence-specific derived ribozyme was developed and packaged in a retroviral expression vector system. This abV kappa ribozyme can be transduced into different murine hybridomas, and expressed intracellularly to potently eliminate endogenous abV kappa RNA. Images PMID:7816635

RNAi-mediated resistance to whitefly (Bemisia tabaci) in genetically engineered lettuce (Lactuca sativa).

PubMed

Ibrahim, Abdulrazak B; Monteiro, Tatiane R; Cabral, Glaucia B; Aragão, Francisco J L

2017-10-01

RNA interference (RNAi)-based transgenic technologies have evolved as potent biochemical tools for silencing specific genes of plant pathogens and pests. The approach has been demonstrated to be useful in silencing genes in insect species. Here, we report on the successful construction of RNAi-based plasmid containing an interfering cassette designed to generate dsRNAs that target a novel v-ATPase transcript in whitefly (Bemisia tabaci), an important agricultural pest in tropical and sub-tropical regions. The presence of the transgene was confirmed in T 0 and T 1 generations of transgenic lettuce lines, segregating in a Mendelian fashion. Seven lines were infested with whiteflies and monitored over a period of 32 days. Analysis of mortality showed that within five days of feeding, insects on transgenic plants showed a mortality rate of 83.8-98.1%. In addition, a reduced number of eggs (95 fold less) was observed in flies feeding on transgenic lettuce plants than insects on control lines. Quantitative reverse transcription PCR showed decreased expression level of endogenous v-ATPase gene in whiteflies feeding on transgenic plants. This technology is a foundation for the production of whitefly-resistant commercial crops, improving agricultural sustainability and food security, reducing the use of more environmentally aggressive methods of pest control.

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

Sequence analysis of the lactococcal plasmid pNP40: a mobile replicon for coping with environmental hazards.

PubMed

O'Driscoll, Jonathan; Glynn, Frances; Fitzgerald, Gerald F; van Sinderen, Douwe

2006-09-01

The conjugative lactococcal plasmid pNP40, identified in Lactococcus lactis subsp. diacetylactis DRC3, possesses a potent complement of bacteriophage resistance systems, which has stimulated its application as a fitness-improving, food-grade genetic element for industrial starter cultures. The complete sequence of this plasmid allowed the mapping of previously known functions including replication, conjugation, bacteriocin resistance, heavy metal tolerance, and bacteriophage resistance. In addition, functions for cold shock adaptation and DNA damage repair were identified, further confirming pNP40's contribution to environmental stress protection. A plasmid cointegration event appears to have been part of the evolution of pNP40, resulting in a "stockpiling" of bacteriophage resistance systems.

Construction of upp deletion mutant strains of Lactobacillus casei and Lactococcus lactis based on counterselective system using temperature-sensitive plasmid.

PubMed

Song, Li; Cui, Hongyu; Tang, Lijie; Qiao, Xinyuan; Liu, Min; Jiang, Yanping; Cui, Wen; Li, Yijing

2014-07-01

Integration plasmids are often used in constructing chromosomal mutations, as it enables the alternation of genes at any location by integration or replacement. Food-grade integration vectors can integrate into the host genome without introducing any selectable markers or residual bases, and the recombination often happens in non-coding region. In this study we used the temperature-sensitive pWV01 replicon to construct 2 chloramphenicol-resistant integration plasmids (pGBHC32-upp) containing the uracil phosphoribosyl transferase (upp) gene as a counterselective marker for Lactobacillus casei (L. casei) ATCC393 and Lactococcus lactis (L. lactis) MG1363. We then ligated the designed homologous arms to the pGBHC32-upp plasmids to allow their integration to the bacterial chromosome, and selected upp deletion mutants of L. casei ATCC393 and L. lactis MG1363 in the presence of 5-fluorouracil (5-FU). Analysis of genetic stability, growth curve, carbon utilization and scanning electronic microscopy showed that, except for 5-FU resistance, there were no significant differences between the wild type and mutant lactic acid bacteria. The integration system and the upp deletion strains could be used in the insertion or deletion of genes at any location of the chromosome of both L. casei ATCC 393 and L. lactis MG1363, and the homologous recombination would not introduce any selectable markers or residual bases. These mutant strains can be further investigated for heterologous protein expression and construction of a live mucosal vaccine carrier. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification of facultatively heterotrophic, N/sub 2/-fixing cyanobacteria able to receive plasmid vectors from Escherichia coli by conjugation. [Anabaena spp; Nostoc

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

Flores, E.; Wolk, C.P.

1985-06-01

Plasmid vectors transferable by conjugation from Escherichia coli to obligately photoautotrophic strains of Anabaena spp. are also transferred to and maintained in heterotrophic, filamentous cyanobacteria of the genus Nostoc. These organisms can be used for the genetic analysis of oxygenic photosynthesis, chromatic adaptation, nitrogen fixation, and heterocyst development.

Analysis of protein function in clinical C. albicans isolates

PubMed Central

Gerami-Nejad, Maryam; Forche, Anja; McClellan, Mark; Berman, Judith

2012-01-01

Clinical isolates are prototrophic and hence are not amenable to genetic manipulation using nutritional markers. Here we describe a new set of plasmids carrying the NAT1 (nourseothricin) drug resistance marker (Shen et al., 2005) that can be used both in clinical isolates and in laboratory strains. We constructed novel plasmids containing HA-NAT1 or MYC-NAT1 cassettes to facilitate PCR-mediated construction of strains with C-terminal epitope-tagged proteins and a NAT1-pMet3-GFP plasmid to enable conditional expression of proteins with or without the green fluorescent protein fused at the N-terminus. Furthermore, for proteins that require both the endogenous N- and C-termini for function, we have constructed a GF-NAT1-FP cassette carrying truncated alleles that facilitate insertion of an intact, single copy of GFP internal to the coding sequence. In addition, GFP-NAT1, RFP-NAT1, and M-Cherry-NAT1 plasmids were constructed expressing two differently labeled gene products for the study of protein co-expression and co-localization in vivo. Together, these vectors provide a useful set of genetic tools for studying diverse aspects of gene function in C. albicans clinical as well as laboratory strains. PMID:22777821

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.

Cloning-independent plasmid construction for genetic studies in streptococci

PubMed Central

Xie, Zhoujie; Qi, Fengxia; Merritt, Justin

2013-01-01

Shuttle plasmids are among the few routinely utilized tools in the Streptococcus mutans genetic system that still require the use of classical cloning methodologies and intermediate hosts for genetic manipulation. Accordingly, it typically requires considerably less time and effort to introduce mutations onto the S. mutans chromosome than it does to construct shuttle vectors for expressing genes in trans. Occasionally, shuttle vector constructs also exhibit toxicity in E. coli, which prevents their proper assembly. To circumvent these limitations, we modified a prolonged overlap extension PCR (POE-PCR) protocol to facilitate direct plasmid assembly in S. mutans. Using solely PCR, we created the reporter vector pZX7, which contains a single minimal streptococcal replication origin and harbors a spectinomycin resistance cassette and the gusA gene encoding β-glucuronidase. We compared the efficiency of pZX7 assembly using multiple strains of S. mutans and were able to obtain from 5×103 – 2×105 CFU/μg PCR product. Likewise, we used pZX7 to further demonstrate that Streptococcus sanguinis and Streptococcus gordonii are also excellent hosts for cloning-independent plasmid assembly, which suggests that this system is likely to function in numerous other streptococci. Consequently, it should be possible to completely forgo the use of E. coli – Streptococcus shuttle vectors in many streptococcal species, thereby decreasing the time and effort required to assemble constructs and eliminating any toxicity issues associated with intermediate hosts. PMID:23673081

Cloning-independent plasmid construction for genetic studies in streptococci.

PubMed

Xie, Zhoujie; Qi, Fengxia; Merritt, Justin

2013-08-01

Shuttle plasmids are among the few routinely utilized tools in the Streptococcus mutans genetic system that still require the use of classical cloning methodologies and intermediate hosts for genetic manipulation. Accordingly, it typically requires considerably less time and effort to introduce mutations onto the S. mutans chromosome than it does to construct shuttle vectors for expressing genes in trans. Occasionally, shuttle vector constructs also exhibit toxicity in Escherichia coli, which prevents their proper assembly. To circumvent these limitations, we modified a prolonged overlap extension PCR (POE-PCR) protocol to facilitate direct plasmid assembly in S. mutans. Using solely PCR, we created the reporter vector pZX7, which contains a single minimal streptococcal replication origin and harbors a spectinomycin resistance cassette and the gusA gene encoding β-glucuronidase. We compared the efficiency of pZX7 assembly using multiple strains of S. mutans and were able to obtain from 5 × 10³ to 2 × 10⁵ CFU/μg PCR product. Likewise, we used pZX7 to further demonstrate that Streptococcus sanguinis and Streptococcus gordonii are also excellent hosts for cloning-independent plasmid assembly, which suggests that this system is likely to function in numerous other streptococci. Consequently, it should be possible to completely forgo the use of E. coli-Streptococcus shuttle vectors in many streptococcal species, thereby decreasing the time and effort required to assemble constructs and eliminating any toxicity issues associated with intermediate hosts. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

Construction and sequencing of an infectious clone of the goose embryo-adapted Muscovy duck parvovirus vaccine strain FZ91-30.

PubMed

Wang, Jianye; Huang, Yu; Zhou, Mingxu; Hardwidge, Philip R; Zhu, Guoqiang

2016-06-21

Muscovy duck parvovirus (MDPV) is the etiological agent of Muscovy duckling parvoviral disease, which is characterized by diarrhea, locomotive dysfunction, stunting, and death in young ducklings, and causes substantial economic losses in the Muscovy duck industry worldwide. FZ91-30 is an attenuated vaccine strain that is safe and immunogenic to ducklings, but the genomic information and molecular mechanism underlining the attenuation are not understood. The FZ91-30 strain was propagated in 11-day-old embryonated goose eggs, and viral particles were purified from the pooled allantoic fluid by differential centrifugation and ultracentrifugation. Single-stranded genomic DNA was extracted and annealed to form double-stranded DNA. The dsDNA digested with NcoI resulted two sub-genomic fragments, which were then cloned into the modified plasmid pBluescript II SK, respectively, generating plasmid pBSKNL and pBSKNR. The sub-genomic plasmid clones were sequenced and further combined to construct the plasmid pFZ that contained the entire genome of strain FZ91-30. The complete genome sequences of strain FM and YY and partial genome sequences of other strains were retrieved from GenBank for sequence comparison. The plasmid pFZ containing the entire genome of FZ91-30 was transfected in 11-day-old embryonated goose eggs via the chorioallantoic membranes route to rescue infectious virus. A genetic marker was introduced into the rescued virus to discriminate from its parental virus. The genome of FZ91-30 consists of 5,131 nucleotides and has 98.9 % similarity to the FM strain. The inverted terminal repeats (ITR) are 456 nucleotides in length, 14 nucleotides longer than that of Goose parvovirus (GPV). The exterior 415 nucleotides of the ITR form a hairpin structure, and the interior 41 nucleotides constitute the D sequence, a reverse complement of the D' sequence at the 3' ITR. Amino acid sequence alignment of the VP1 proteins between FZ91-30 and five pathogenic MDPV strains revealed that FZ91-30 had five mutations; two in the unique region of the VP1 protein (VP1u) and three in VP3. Sequence alignment of the Rep1 proteins revealed two amino acid alterations for FZ91-30, both of which were conserved for two pathogenic strains YY and P. Transfection of the plasmid pFZ in 11-day-old embryonated goose eggs resulted in generation of infectious virus with similar biological properties as compared with the parental strain. The amino acid mutations identified in the VP1 and Rep1 protein may contribute to the attenuation of FZ91-30 in Muscovy ducklings. Plasmid transfection in embryonated goose eggs was suitable for rescue of infectious MDPV.

Use of a novel virus inactivation method for a multicenter avian influenza real-time reverse transcriptase-polymerase chain reaction proficiency study.

PubMed

Spackman, Erica; Suarez, David L

2005-01-01

Proficiency assessments are important elements in quality control for diagnostic laboratories. Traditionally, proficiency testing for polymerase chain reaction (PCR)-based assays has involved the use of clinical samples, samples "spiked" with live agents or DNA plasmids. Because of government regulations and biosecurity concerns, distribution of live high-consequence pathogens of livestock and poultry, such as avian influenza, is not possible, and DNA plasmids are not technically suitable for evaluating RNA virus detection. Therefore, a proficiency testing panel using whole avian influenza in a diluent containing a phenolic disinfectant that inactivates the virus while preserving the RNA for at least 8 weeks at -70 C was developed and used in a multicenter proficiency assessment for a type A influenza real-time reverse transcriptase (RT)-PCR test. The test, which was highly standardized, except for variation in the real-time RT-PCR equipment used, was shown to be highly reproducible by proficiency testing in 12 laboratories in the United States, Canada, and Hong Kong. Variation in cycle threshold values among 35 data sets and 490 samples was minimal (CV = 5.19%), and sample identifications were highly accurate (96.7% correct identifications) regardless of real-time PCR instrumentation.

Isolation and molecular characterization of Salmonella enterica serovar Enteritidis from poultry house and clinical samples during 2010.

PubMed

Mezal, Ezat H; Sabol, Ashley; Khan, Mariam A; Ali, Nawab; Stefanova, Rossina; Khan, Ashraf A

2014-04-01

A total of 60 Salmonella enterica serovar (ser.) Enteritidis isolates, 28 from poultry houses and 32 from clinical samples, were isolated during 2010. These isolates were subjected to testing and analyzed for antibiotic resistance, virulence genes, plasmids and plasmid replicon types. To assess genetic diversity, pulsed-field gel electrophoresis (PFGE) fingerprinting, using the XbaI restriction enzyme, Multiple-Locus Variable-Number Tandem Repeat Analysis (MLVA) and plasmid profiles were performed. All isolates from poultry, and 10 out of 32 clinical isolates were sensitive to ampicillin, chloramphenicol, gentamicin, kanamycin, nalidixic acid, sulfisoxazole, streptomycin, and tetracycline. Twenty-one of thirty-two clinical isolates were resistant to ampicillin and tetracycline, and one isolate was resistant to nalidixic acid. PFGE typing of sixty ser. Enteritidis isolates by XbaI resulted in 10-12 bands and grouped into six clusters each with similarity from 95% to 81%. The MLVA analysis of sixty isolates gave 18 allele profiles with the majority of isolates displayed in three groups, and two clinical isolates found to be new in the PulseNet national MLVA database. All isolates were positive for 12 or more of the 17 virulence genes mostly found in S. enterica (spvB, spiA, pagC, msgA, invA, sipB, prgH, spaN, orgA, tolC, iroN, sitC, IpfC, sifA, sopB, and pefA) and negative for one gene (cdtB). All isolates carried a typical 58 kb plasmid, type Inc/FIIA. Three poultry isolates and one clinical isolate carried small plasmids with 3.8, 6, 7.6 and 11.5 kb. Ten of the clinical isolates carried plasmids, with sizes 36 and 38 kb, types IncL/M and IncN, and one isolate carried an 81 kb plasmid, type IncI. Southern hybridization of a plasmid with an Inc/FIIA gene probe hybridized one large 58 kb plasmid in all isolates. Several large and small plasmids from poultry isolates were not typed by our PCR-based method. These results confirmed that PFGE fingerprinting has limited discriminatory power for ser. Enteritidis in both poultry and clinical sources. However, the plasmid and MLVA allele profiles were a useful and important epidemiology tool to discriminate outbreak strains of ser. Enteritidis from poultry and clinical samples. Published by Elsevier Ltd.

The behavioural consequences of sex reversal in dragons

PubMed Central

Li, Hong; Holleley, Clare E.; Elphick, Melanie; Georges, Arthur

2016-01-01

Sex differences in morphology, physiology, and behaviour are caused by sex-linked genes, as well as by circulating sex-steroid levels. Thus, a shift from genotypic to environmental sex determination may create an organism that exhibits a mixture of male-like and female-like traits. We studied a lizard species (Central Bearded Dragon, Pogona vitticeps), in which the high-temperature incubation of eggs transforms genetically male individuals into functional females. Although they are reproductively female, sex-reversed dragons (individuals with ZZ genotype reversed to female phenotype) resemble genetic males rather than females in morphology (relative tail length), general behaviour (boldness and activity level), and thermoregulatory tactics. Indeed, sex-reversed ‘females’ are more male-like in some behavioural traits than are genetic males. This novel phenotype may impose strong selection on the frequency of sex reversal within natural populations, facilitating rapid shifts in sex-determining systems. A single period of high incubation temperatures (generating thermally induced sex reversal) can produce functionally female individuals with male-like (or novel) traits that enhance individual fitness, allowing the new temperature-dependent sex-determining system to rapidly replace the previous genetically based one.

pSTM6-275, a Conjugative IncHI2 Plasmid of Salmonella enterica That Confers Antibiotic and Heavy-Metal Resistance under Changing Physiological Conditions.

PubMed

Billman-Jacobe, Helen; Liu, Yuhong; Haites, Ruth; Weaver, Tom; Robinson, Lily; Marenda, Marc; Dyall-Smith, Mike

2018-05-01

Detailed annotation of an IncHI2 plasmid, pSTM6-275, from Salmonella enterica serotype 1,4,5,12:i:- strain TW-Stm6 revealed a composite structure, including antimicrobial resistance genes on mobile genetic elements. The plasmid was thermosensitive for transfer to Escherichia coli and conferred reduced susceptibility to antibiotics, copper sulfate, and silver nitrate. Metal ion susceptibility was dependent on physiological conditions, giving an insight into the environments where this trait might confer a fitness advantage. Copyright © 2018 Billman-Jacobe et al.

Gene editing in clinical isolates of Candida parapsilosis using CRISPR/Cas9.

PubMed

Lombardi, Lisa; Turner, Siobhán A; Zhao, Fang; Butler, Geraldine

2017-08-14

Candida parapsilosis is one of the most common causes of candidiasis, particularly in the very young and the very old. Studies of gene function are limited by the lack of a sexual cycle, the diploid genome, and a paucity of molecular tools. We describe here the development of a plasmid-based CRISPR-Cas9 system for gene editing in C. parapsilosis. A major advantage of the system is that it can be used in any genetic background, which we showed by editing genes in 20 different isolates. Gene editing is carried out in a single transformation step. The CAS9 gene is expressed only when the plasmid is present, and it can be removed easily from transformed strains. There is theoretically no limit to the number of genes that can be edited in any strain. Gene editing is increased by homology-directed repair in the presence of a repair template. Editing by non-homologous end joining (NHEJ) also occurs in some genetic backgrounds. Finally, we used the system to introduce unique tags at edited sites.

Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria

PubMed Central

Taton, Arnaud; Unglaub, Federico; Wright, Nicole E.; Zeng, Wei Yue; Paz-Yepes, Javier; Brahamsha, Bianca; Palenik, Brian; Peterson, Todd C.; Haerizadeh, Farzad; Golden, Susan S.; Golden, James W.

2014-01-01

Inspired by the developments of synthetic biology and the need for improved genetic tools to exploit cyanobacteria for the production of renewable bioproducts, we developed a versatile platform for the construction of broad-host-range vector systems. This platform includes the following features: (i) an efficient assembly strategy in which modules released from 3 to 4 donor plasmids or produced by polymerase chain reaction are assembled by isothermal assembly guided by short GC-rich overlap sequences. (ii) A growing library of molecular devices categorized in three major groups: (a) replication and chromosomal integration; (b) antibiotic resistance; (c) functional modules. These modules can be assembled in different combinations to construct a variety of autonomously replicating plasmids and suicide plasmids for gene knockout and knockin. (iii) A web service, the CYANO-VECTOR assembly portal, which was built to organize the various modules, facilitate the in silico construction of plasmids, and encourage the use of this system. This work also resulted in the construction of an improved broad-host-range replicon derived from RSF1010, which replicates in several phylogenetically distinct strains including a new experimental model strain Synechocystis sp. WHSyn, and the characterization of nine antibiotic cassettes, four reporter genes, four promoters, and a ribozyme-based insulator in several diverse cyanobacterial strains. PMID:25074377

A Search for a General Phenomenon of Adaptive Mutability

PubMed Central

Galitski, T.; Roth, J. R.

1996-01-01

The most prominent systems for the study of adaptive mutability depend on the specialized activities of genetic elements like bacteriophage Mu and the F plasmid. Searching for general adaptive mutability, we have investigated the behavior of Salmonella typhimurium strains with chromosomal lacZ mutations. We have studied 30 revertible nonsense, missense, frameshift, and insertion alleles. One-third of the mutants produced >=10 late revertant colonies (appearing three to seven days after plating on selective medium). For the prolific mutants, the number of late revertants showed rank correlation with the residual β-galactosidase activity; for the same mutants, revertant number showed no correlation with the nonselective reversion rate (from fluctuation tests). Leaky mutants, which grew slowly on selective medium, produced late revertants whereas tight nongrowing mutants generally did not produce late revertants. However, the number of late revertants was not proportional to residual growth. Using total residual growth and the nonselective reversion rate, the expected number of late revertants was calculated. For several leaky mutants, the observed revertant number exceeded the expected number. We suggest that excess late revertants from these mutants arise from general adaptive mutability available to any chromosomal gene. PMID:8725216

Use of Staby® technology for development and production of DNA vaccines free of antibiotic resistance gene

PubMed Central

Reschner, Anca; Scohy, Sophie; Vandermeulen, Gaëlle; Daukandt, Marc; Jacques, Céline; Michel, Benjamin; Nauwynck, Hans; Xhonneux, Florence; Préat, Véronique; Vanderplasschen, Alain; Szpirer, Cédric

2013-01-01

The appearance of new viruses and the cost of developing certain vaccines require that new vaccination strategies now have to be developed. DNA vaccination seems to be a particularly promising method. For this application, plasmid DNA is injected into the subject (man or animal). This plasmid DNA encodes an antigen that will be expressed by the cells of the subject. In addition to the antigen, the plasmid also encodes a resistance to an antibiotic, which is used during the construction and production steps of the plasmid. However, regulatory agencies (FDA, USDA and EMA) recommend to avoid the use of antibiotics resistance genes. Delphi Genetics developed the Staby® technology to replace the antibiotic-resistance gene by a selection system that relies on two bacterial genes. These genes are small in size (approximately 200 to 300 bases each) and consequently encode two small proteins. They are naturally present in the genomes of bacteria and on plasmids. The technology is already used successfully for production of recombinant proteins to achieve higher yields and without the need of antibiotics. In the field of DNA vaccines, we have now the first data validating the innocuousness of this Staby® technology for eukaryotic cells and the feasibility of an industrial production of an antibiotic-free DNA vaccine. Moreover, as a proof of concept, mice have been successfully vaccinated with our antibiotic-free DNA vaccine against a deadly disease, pseudorabies (induced by Suid herpesvirus-1). PMID:24051431

Use of Staby(®) technology for development and production of DNA vaccines free of antibiotic resistance gene.

PubMed

Reschner, Anca; Scohy, Sophie; Vandermeulen, Gaëlle; Daukandt, Marc; Jacques, Céline; Michel, Benjamin; Nauwynck, Hans; Xhonneux, Florence; Préat, Véronique; Vanderplasschen, Alain; Szpirer, Cédric

2013-10-01

The appearance of new viruses and the cost of developing certain vaccines require that new vaccination strategies now have to be developed. DNA vaccination seems to be a particularly promising method. For this application, plasmid DNA is injected into the subject (man or animal). This plasmid DNA encodes an antigen that will be expressed by the cells of the subject. In addition to the antigen, the plasmid also encodes a resistance to an antibiotic, which is used during the construction and production steps of the plasmid. However, regulatory agencies (FDA, USDA and EMA) recommend to avoid the use of antibiotics resistance genes. Delphi Genetics developed the Staby(®) technology to replace the antibiotic-resistance gene by a selection system that relies on two bacterial genes. These genes are small in size (approximately 200 to 300 bases each) and consequently encode two small proteins. They are naturally present in the genomes of bacteria and on plasmids. The technology is already used successfully for production of recombinant proteins to achieve higher yields and without the need of antibiotics. In the field of DNA vaccines, we have now the first data validating the innocuousness of this Staby(®) technology for eukaryotic cells and the feasibility of an industrial production of an antibiotic-free DNA vaccine. Moreover, as a proof of concept, mice have been successfully vaccinated with our antibiotic-free DNA vaccine against a deadly disease, pseudorabies (induced by Suid herpesvirus-1).

CRISPR-based screening of genomic island excision events in bacteria.

PubMed

Selle, Kurt; Klaenhammer, Todd R; Barrangou, Rodolphe

2015-06-30

Genomic analysis of Streptococcus thermophilus revealed that mobile genetic elements (MGEs) likely contributed to gene acquisition and loss during evolutionary adaptation to milk. Clustered regularly interspaced short palindromic repeats-CRISPR-associated genes (CRISPR-Cas), the adaptive immune system in bacteria, limits genetic diversity by targeting MGEs including bacteriophages, transposons, and plasmids. CRISPR-Cas systems are widespread in streptococci, suggesting that the interplay between CRISPR-Cas systems and MGEs is one of the driving forces governing genome homeostasis in this genus. To investigate the genetic outcomes resulting from CRISPR-Cas targeting of integrated MGEs, in silico prediction revealed four genomic islands without essential genes in lengths from 8 to 102 kbp, totaling 7% of the genome. In this study, the endogenous CRISPR3 type II system was programmed to target the four islands independently through plasmid-based expression of engineered CRISPR arrays. Targeting lacZ within the largest 102-kbp genomic island was lethal to wild-type cells and resulted in a reduction of up to 2.5-log in the surviving population. Genotyping of Lac(-) survivors revealed variable deletion events between the flanking insertion-sequence elements, all resulting in elimination of the Lac-encoding island. Chimeric insertion sequence footprints were observed at the deletion junctions after targeting all of the four genomic islands, suggesting a common mechanism of deletion via recombination between flanking insertion sequences. These results established that self-targeting CRISPR-Cas systems may direct significant evolution of bacterial genomes on a population level, influencing genome homeostasis and remodeling.

Recovery of Recombinant Crimean Congo Hemorrhagic Fever Virus Reveals a Function for Non-structural Glycoproteins Cleavage by Furin.

PubMed

Bergeron, Éric; Zivcec, Marko; Chakrabarti, Ayan K; Nichol, Stuart T; Albariño, César G; Spiropoulou, Christina F

2015-05-01

Crimean Congo hemorrhagic fever virus (CCHFV) is a negative-strand RNA virus of the family Bunyaviridae (genus: Nairovirus). In humans, CCHFV causes fever, hemorrhage, severe thrombocytopenia, and high fatality. A major impediment in precisely determining the basis of CCHFV's high pathogenicity has been the lack of methodology to produce recombinant CCHFV. We developed a reverse genetics system based on transfecting plasmids into BSR-T7/5 and Huh7 cells. In our system, bacteriophage T7 RNA polymerase produced complementary RNA copies of the viral S, M, and L segments that were encapsidated with the support, in trans, of CCHFV nucleoprotein and L polymerase. The system was optimized to systematically recover high yields of infectious CCHFV. Additionally, we tested the ability of the system to produce specifically designed CCHFV mutants. The M segment encodes a polyprotein that is processed by host proprotein convertases (PCs), including the site-1 protease (S1P) and furin-like PCs. S1P and furin cleavages are necessary for producing the non-structural glycoprotein GP38, while S1P cleavage yields structural Gn. We studied the role of furin cleavage by rescuing a recombinant CCHFV encoding a virus glycoprotein precursor lacking a functional furin cleavage motif (RSKR mutated to ASKA). The ASKA mutation blocked glycoprotein precursor's maturation to GP38, and Gn precursor's maturation to Gn was slightly diminished. Furin cleavage was not essential for replication, as blocking furin cleavage resulted only in transient reduction of CCHFV titers, suggesting that either GP38 and/or decreased Gn maturation accounted for the reduced virion production. Our data demonstrate that nairoviruses can be produced by reverse genetics, and the utility of our system uncovered a function for furin cleavage. This viral rescue system could be further used to study the CCHFV replication cycle and facilitate the development of efficacious vaccines to counter this biological and public health threat.

Recovery of Recombinant Crimean Congo Hemorrhagic Fever Virus Reveals a Function for Non-structural Glycoproteins Cleavage by Furin

PubMed Central

Bergeron, Éric; Zivcec, Marko; Chakrabarti, Ayan K.; Nichol, Stuart T.; Albariño, César G.; Spiropoulou, Christina F.

2015-01-01

Crimean Congo hemorrhagic fever virus (CCHFV) is a negative-strand RNA virus of the family Bunyaviridae (genus: Nairovirus). In humans, CCHFV causes fever, hemorrhage, severe thrombocytopenia, and high fatality. A major impediment in precisely determining the basis of CCHFV’s high pathogenicity has been the lack of methodology to produce recombinant CCHFV. We developed a reverse genetics system based on transfecting plasmids into BSR-T7/5 and Huh7 cells. In our system, bacteriophage T7 RNA polymerase produced complementary RNA copies of the viral S, M, and L segments that were encapsidated with the support, in trans, of CCHFV nucleoprotein and L polymerase. The system was optimized to systematically recover high yields of infectious CCHFV. Additionally, we tested the ability of the system to produce specifically designed CCHFV mutants. The M segment encodes a polyprotein that is processed by host proprotein convertases (PCs), including the site-1 protease (S1P) and furin-like PCs. S1P and furin cleavages are necessary for producing the non-structural glycoprotein GP38, while S1P cleavage yields structural Gn. We studied the role of furin cleavage by rescuing a recombinant CCHFV encoding a virus glycoprotein precursor lacking a functional furin cleavage motif (RSKR mutated to ASKA). The ASKA mutation blocked glycoprotein precursor’s maturation to GP38, and Gn precursor’s maturation to Gn was slightly diminished. Furin cleavage was not essential for replication, as blocking furin cleavage resulted only in transient reduction of CCHFV titers, suggesting that either GP38 and/or decreased Gn maturation accounted for the reduced virion production. Our data demonstrate that nairoviruses can be produced by reverse genetics, and the utility of our system uncovered a function for furin cleavage. This viral rescue system could be further used to study the CCHFV replication cycle and facilitate the development of efficacious vaccines to counter this biological and public health threat. PMID:25933376

A Novel Multiplex PCR Discriminates Bacillus anthracis and Its Genetically Related Strains from Other Bacillus cereus Group Species

PubMed Central

Ogawa, Hirohito; Fujikura, Daisuke; Ohnuma, Miyuki; Ohnishi, Naomi; Hang'ombe, Bernard M.; Mimuro, Hitomi; Ezaki, Takayuki; Mweene, Aaron S.; Higashi, Hideaki

2015-01-01

Anthrax is an important zoonotic disease worldwide that is caused by Bacillus anthracis, a spore-forming pathogenic bacterium. A rapid and sensitive method to detect B. anthracis is important for anthrax risk management and control in animal cases to address public health issues. However, it has recently become difficult to identify B. anthracis by using previously reported molecular-based methods because of the emergence of B. cereus, which causes severe extra-intestinal infection, as well as the human pathogenic B. thuringiensis, both of which are genetically related to B. anthracis. The close genetic relation of chromosomal backgrounds has led to complexity of molecular-based diagnosis. In this study, we established a B. anthracis multiplex PCR that can screen for the presence of B. anthracis virulent plasmids and differentiate B. anthracis and its genetically related strains from other B. cereus group species. Six sets of primers targeting a chromosome of B. anthracis and B. anthracis-like strains, two virulent plasmids, pXO1 and pXO2, a bacterial gene, 16S rRNA gene, and a mammalian gene, actin-beta gene, were designed. The multiplex PCR detected approximately 3.0 CFU of B. anthracis DNA per PCR reaction and was sensitive to B. anthracis. The internal control primers also detected all bacterial and mammalian DNAs examined, indicating the practical applicability of this assay as it enables monitoring of appropriate amplification. The assay was also applied for detection of clinical strains genetically related to B. anthracis, which were B. cereus strains isolated from outbreaks of hospital infections in Japan, and field strains isolated in Zambia, and the assay differentiated B. anthracis and its genetically related strains from other B. cereus group strains. Taken together, the results indicate that the newly developed multiplex PCR is a sensitive and practical method for detecting B. anthracis. PMID:25774512

Heterologous Expression of Gene of Interest Using the Marine Protozoan Perkinsus marinus

NASA Astrophysics Data System (ADS)

Cold, E. R.

2016-02-01

Perkinsus marinus is a marine protozoan parasite that causes "Dermo" disease in eastern oysters (Crassostrea virginica). P. marinus is closely related to Plasmodium falciparum which causes malaria. A recent study has showed that P. marinus causes no pathology damage but an immune response in humanized mouse, providing the bases for a genetically modified P. marinus expressing Plasmodium genes to be used as a vaccination delivery system for malaria and other pathogenic diseases. A modified plasmid vector (pMOE-GFP) based on highly expressed gene tagged with green fluorescence protein and targeted to P. marinus cell wall was used to clone MSP8 and HAP2. MSP8 encodes for merozoite surface in P. falciparum and HAP2 is essential for fusion of male and female gametes; genetic disruption of the HAP2 locus revealed that parasite fertilization is prevented. Using electroporation, MSP8 and HAP2 plasmid were introduced into the P. marinus trophozoites. As controls pMOE-GFP was transfected into P. mediterraneus, P. atlanticus and P. chesapeaki. Transfection conditions included 5x107 Perkinsus trophozoites and 10 µg of plasmid using Nucleofector® technology (D-023 program). The cells were recovered in 3 mL of Perkinsus culture media and transfected trophozoites were examined for green fluorescence. To facilitate subcloning of cells expressing GFP, we optimized a DME: HAM's F12 -5% FBS -containing agar solid medium for plating Perkinsus. Examination of all transfected cells indicates expression of both MSP8 and HAP2. This is the first time that genes of a protozoan parasite have been expressed in a marine protozoan. It was also concluded that P. mediterraneus, P. atlanticus and P. chesapeaki were stable mutation and can be isolated for further research.

GENETIC ENGINEERING OF ENHANCED MICROBIAL NITRIFICATION

EPA Science Inventory

Experiments were conducted to introduce genetic information in the form of antibiotic or mercuric ion resistance genes into Nitrobacter hamburgensis strain X14. The resistance genes were either stable components of broad host range plasmids or transposable genes on methods for p...

Fluorescent dye technique as an alternative to gfp-labeled plasmid for visualization of Escherichia coli O157:H7 cells on romaine lettuce leaves following sanitizer treatment

USDA-ARS?s Scientific Manuscript database

The task of imaging Escherichia coli O157:H7 cells on artificially inoculated produce often requires genetic modification of the cells through the introduction of gfp-labeled plasmid. However, these modified cells do not behave as the parent cells and the auto fluorescence of lettuce leaves interfe...

Plasmids of Legionella Species.

DTIC Science & Technology

1982-06-18

LEGIONELLA SPECIES *PERRY MIKESELL, CPT GREGORY B. KNUDSON, PhD U.S. ARMY MEDICAL RESEARCH INSTITUTE OF INFECTIOUS DISEASES FORT DETRICK, FREDERICK, MARYLAND...sponsible for metabolism, resistan to metals and fertility factors , plasmids also encode for druKg_-sistance factors . These latter genetic elements are an...3). The etiological agent was identified as a fastidious, aerobic, gram-nega- tive bacterium and given the name Legionella pneumophila. The number of

Mobility and generation of mosaic non-autonomous transposons by Tn3-derived inverted-repeat miniature elements (TIMEs).

PubMed

Szuplewska, Magdalena; Ludwiczak, Marta; Lyzwa, Katarzyna; Czarnecki, Jakub; Bartosik, Dariusz

2014-01-01

Functional transposable elements (TEs) of several Pseudomonas spp. strains isolated from black shale ore of Lubin mine and from post-flotation tailings of Zelazny Most in Poland, were identified using a positive selection trap plasmid strategy. This approach led to the capture and characterization of (i) 13 insertion sequences from 5 IS families (IS3, IS5, ISL3, IS30 and IS1380), (ii) isoforms of two Tn3-family transposons--Tn5563a and Tn4662a (the latter contains a toxin-antitoxin system), as well as (iii) non-autonomous TEs of diverse structure, ranging in size from 262 to 3892 bp. The non-autonomous elements transposed into AT-rich DNA regions and generated 5- or 6-bp sequence duplications at the target site of transposition. Although these TEs lack a transposase gene, they contain homologous 38-bp-long terminal inverted repeat sequences (IRs), highly conserved in Tn5563a and many other Tn3-family transposons. The simplest elements of this type, designated TIMEs (Tn3 family-derived Inverted-repeat Miniature Elements) (262 bp), were identified within two natural plasmids (pZM1P1 and pLM8P2) of Pseudomonas spp. It was demonstrated that TIMEs are able to mobilize segments of plasmid DNA for transposition, which results in the generation of more complex non-autonomous elements, resembling IS-driven composite transposons in structure. Such transposon-like elements may contain different functional genetic modules in their core regions, including plasmid replication systems. Another non-autonomous element "captured" with a trap plasmid was a TIME derivative containing a predicted resolvase gene and a res site typical for many Tn3-family transposons. The identification of a portable site-specific recombination system is another intriguing example confirming the important role of non-autonomous TEs of the TIME family in shuffling genetic information in bacterial genomes. Transposition of such mosaic elements may have a significant impact on diversity and evolution, not only of transposons and plasmids, but also of other types of mobile genetic elements.

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

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.

Random mutagenesis by error-prone pol plasmid replication in Escherichia coli.

PubMed

Alexander, David L; Lilly, Joshua; Hernandez, Jaime; Romsdahl, Jillian; Troll, Christopher J; Camps, Manel

2014-01-01

Directed evolution is an approach that mimics natural evolution in the laboratory with the goal of modifying existing enzymatic activities or of generating new ones. The identification of mutants with desired properties involves the generation of genetic diversity coupled with a functional selection or screen. Genetic diversity can be generated using PCR or using in vivo methods such as chemical mutagenesis or error-prone replication of the desired sequence in a mutator strain. In vivo mutagenesis methods facilitate iterative selection because they do not require cloning, but generally produce a low mutation density with mutations not restricted to specific genes or areas within a gene. For this reason, this approach is typically used to generate new biochemical properties when large numbers of mutants can be screened or selected. Here we describe protocols for an advanced in vivo mutagenesis method that is based on error-prone replication of a ColE1 plasmid bearing the gene of interest. Compared to other in vivo mutagenesis methods, this plasmid-targeted approach allows increased mutation loads and facilitates iterative selection approaches. We also describe the mutation spectrum for this mutagenesis methodology in detail, and, using cycle 3 GFP as a target for mutagenesis, we illustrate the phenotypic diversity that can be generated using our method. In sum, error-prone Pol I replication is a mutagenesis method that is ideally suited for the evolution of new biochemical activities when a functional selection is available.

Genomics of high molecular weight plasmids isolated from an on-farm biopurification system.

PubMed

Martini, María C; Wibberg, Daniel; Lozano, Mauricio; Torres Tejerizo, Gonzalo; Albicoro, Francisco J; Jaenicke, Sebastian; van Elsas, Jan Dirk; Petroni, Alejandro; Garcillán-Barcia, M Pilar; de la Cruz, Fernando; Schlüter, Andreas; Pühler, Alfred; Pistorio, Mariano; Lagares, Antonio; Del Papa, María F

2016-06-20

The use of biopurification systems (BPS) constitutes an efficient strategy to eliminate pesticides from polluted wastewaters from farm activities. BPS environments contain a high microbial density and diversity facilitating the exchange of information among bacteria, mediated by mobile genetic elements (MGEs), which play a key role in bacterial adaptation and evolution in such environments. Here we sequenced and characterized high-molecular-weight plasmids from a bacterial collection of an on-farm BPS. The high-throughput-sequencing of the plasmid pool yielded a total of several Mb sequence information. Assembly of the sequence data resulted in six complete replicons. Using in silico analyses we identified plasmid replication genes whose encoding proteins represent 13 different Pfam families, as well as proteins involved in plasmid conjugation, indicating a large diversity of plasmid replicons and suggesting the occurrence of horizontal gene transfer (HGT) events within the habitat analyzed. In addition, genes conferring resistance to 10 classes of antimicrobial compounds and those encoding enzymes potentially involved in pesticide and aromatic hydrocarbon degradation were found. Global analysis of the plasmid pool suggest that the analyzed BPS represents a key environment for further studies addressing the dissemination of MGEs carrying catabolic genes and pathway assembly regarding degradation capabilities.

Deep sequencing revealed genome-wide single-nucleotide polymorphism and plasmid content of Erwinia amylovora strains isolated in Middle Atlas, Morocco.

PubMed

Hannou, Najat; Mondy, Samuel; Planamente, Sara; Moumni, Mohieddine; Llop, Pablo; López, María; Manceau, Charles; Barny, Marie-Anne; Faure, Denis

2013-10-01

Erwinia amylovora causes economic losses that affect pear and apple production in Morocco. Here, we report comparative genomics of four Moroccan E. amylovora strains with the European strain CFBP1430 and North-American strain ATCC49946. Analysis of single nucleotide polymorphisms (SNPs) revealed genetic homogeneity of Moroccan's strains and their proximity to the European strain CFBP1430. Moreover, the collected sequences allowed the assembly of a 65 kpb plasmid, which is highly similar to the plasmid pEI70 harbored by several European E. amylovora isolates. This plasmid was found in 33% of the 40 E. amylovora strains collected from several host plants in 2009 and 2010 in Morocco. Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Plasmid-Mediated Antibiotic Resistance and Virulence in Gram-Negatives: the Klebsiella pneumoniae Paradigm.

PubMed

Ramirez, Maria S; Traglia, German M; Lin, David L; Tran, Tung; Tolmasky, Marcelo E

2014-10-01

Plasmids harbor genes coding for specific functions including virulence factors and antibiotic resistance that permit bacteria to survive the hostile environment found in the host and resist treatment. Together with other genetic elements such as integrons and transposons, and using a variety of mechanisms, plasmids participate in the dissemination of these traits, resulting in the virtual elimination of barriers among different kinds of bacteria. In this article we review the current information about the physiology of plasmids and their role in virulence and antibiotic resistance from the Gram-negative opportunistic pathogen Klebsiella pneumoniae. This bacterium has acquired multidrug resistance and is the causative agent of serious community- and hospital-acquired infections. It is also included in the recently defined ESKAPE group of bacteria that cause most U.S. hospital infections.

Plasmid-Mediated Antibiotic Resistance and Virulence in Gram-negatives: the Klebsiella pneumoniae Paradigm.

PubMed

Ramirez, Maria S; Traglia, German M; Lin, David L; Tran, Tung; Tolmasky, Marcelo E

Plasmids harbor genes coding for specific functions including virulence factors and antibiotic resistance that permit bacteria to survive the hostile environment found in the host and resist treatment. Together with other genetic elements such as integrons and transposons, and using a variety of mechanisms, plasmids participate in the dissemination of these traits resulting in the virtual elimination of barriers among different kinds of bacteria. In this article we review the current information about physiology and role in virulence and antibiotic resistance of plasmids from the gram-negative opportunistic pathogen Klebsiella pneumoniae . This bacterium has acquired multidrug resistance and is the causative agent of serious communityand hospital-acquired infections. It is also included in the recently defined ESKAPE group of bacteria that cause most of US hospital infections.

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.

SapTrap, a Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans.

PubMed

Schwartz, Matthew L; Jorgensen, Erik M

2016-04-01

In principle, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 allows genetic tags to be inserted at any locus. However, throughput is limited by the laborious construction of repair templates and guide RNA constructs and by the identification of modified strains. We have developed a reagent toolkit and plasmid assembly pipeline, called "SapTrap," that streamlines the production of targeting vectors for tag insertion, as well as the selection of modified Caenorhabditis elegans strains. SapTrap is a high-efficiency modular plasmid assembly pipeline that produces single plasmid targeting vectors, each of which encodes both a guide RNA transcript and a repair template for a particular tagging event. The plasmid is generated in a single tube by cutting modular components with the restriction enzyme SapI, which are then "trapped" in a fixed order by ligation to generate the targeting vector. A library of donor plasmids supplies a variety of protein tags, a selectable marker, and regulatory sequences that allow cell-specific tagging at either the N or the C termini. All site-specific sequences, such as guide RNA targeting sequences and homology arms, are supplied as annealed synthetic oligonucleotides, eliminating the need for PCR or molecular cloning during plasmid assembly. Each tag includes an embedded Cbr-unc-119 selectable marker that is positioned to allow concurrent expression of both the tag and the marker. We demonstrate that SapTrap targeting vectors direct insertion of 3- to 4-kb tags at six different loci in 10-37% of injected animals. Thus SapTrap vectors introduce the possibility for high-throughput generation of CRISPR/Cas9 genome modifications. Copyright © 2016 by the Genetics Society of America.

Metal chelate affinity precipitation of RNA and purification of plasmid DNA

NASA Technical Reports Server (NTRS)

Balan, Sindhu; Murphy, Jason; Galaev, Igor; Kumar, Ashok; Fox, George E.; Mattiasson, Bo; Willson, Richard C.

2003-01-01

The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine 'tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.

Construction of a novel porcine circovirus type 2 infectious clone as a basis for the development of a PCV2 iDNA vaccine.

PubMed

Wang, Wei-Cheng; Zeng, Zhi-Yong; Tang, De-Yuan; Liang, Hai-Ying; Liu, Zhao; Dai, Zhen-Jiang

2015-08-01

Porcine circovirus-associated disease is a highly contagious disease that has significant economic consequences. The disease is prevalent in many countries and regions. To generate a genetic marker strain of PCV2, a Sal I restriction enzyme site was inserted into the PCV2 clone as a genetic marker by applying iDNA infectious clone technology. The iDNA represents plasmids that encode the full-length DNA genome of PCV2 assembled in a pcDNA3.1-based vectors. The mutant PCV2 was rescued by transfecting an infectious clone into PK-15 cells and was characterised by an immunoperoxidase monolayer assay (IPMA). The viral genome could be differentiated from the wild-type parent by PCR and restriction fragment length polymorphism (PCR-RFLP). Kunming mice were inoculated with the PCV2 infectious clone or rescued virus via intranasal and intraperitoneal routes. Seroconversion to PCV2-specific antibody appeared in the majority of mice from the two inoculated groups at 7 days postinoculation (DPI), and the specific antibody level was steady for at least 42 days. Viraemia, beginning at 7 DPI and lasting 4 weeks, was detected in the majority of the pigs from the two inoculated groups. The animal experiments revealed that the PCV2 infectious clone and rescued virus both could replicate in mice and induce mice to generate anti-PCV2 antibodies. The infectious clones of PCV2 will be useful for further research investigating a potential tractable iDNA vaccine by reverse genetics technology for attenuated virulance. Copyright © 2015 Elsevier B.V. All rights reserved.

First report of plasmid-mediated quinolone resistance qnrA1 gene in Klebsiella pneumoniae isolate of animal origin.

PubMed

Yue, Lei; Chen, Xueying; Li, Shujuan; Liao, Xiaoping; Zhuang, Na; Zhang, Yue; Liu, Ya-Hong

2011-04-01

One QnrA1-producing Klebsiella pneumoniae isolate GDKA1 from chicken was detected. The qnrA1 gene on plasmid pGDKA1 was located in a genetic environment similar to that in In36 on plasmid pHSH1 and could be cotransferred to Escherichia coli J53 Az(R) with other resistances by a conjugation experiment. Upstream of the qnrA1 gene, there was a class I integron with the dfrA27 and aadA2 cassettes. Similar genetic environments of qnrA1 in Enterobacteriaceae isolates from both human and animal origin might, to some extent, demonstrate similar mechanisms of qnrA distribution. The presence of qnrA1 in health animal commensal bacteria should be worthy of note. This is the first report of qnrA1 in K. pneumoniae and dfrA27 in an Enterobacteriaceae isolate of animal origin. © Mary Ann Liebert, Inc.

Characterization of Tn3000, a Transposon Responsible for blaNDM-1 Dissemination among Enterobacteriaceae in Brazil, Nepal, Morocco, and India.

PubMed

Campos, Juliana Coutinho; da Silva, Maria José Félix; dos Santos, Paulo Roberto Nascimento; Barros, Elaine Menezes; Pereira, Mayne de Oliveira; Seco, Bruna Mara Silva; Magagnin, Cibele Massotti; Leiroz, Leonardo Kalab; de Oliveira, Théo Gremen Mimary; de Faria-Júnior, Célio; Cerdeira, Louise Teixeira; Barth, Afonso Luís; Sampaio, Suely Carlos Ferreira; Zavascki, Alexandre Prehn; Poirel, Laurent; Sampaio, Jorge Luiz Mello

2015-12-01

In Enterobacteriaceae, the blaNDM genes have been found in many different genetic contexts, and a wide diversity of plasmid scaffolds bearing those genes has been found. In August 2013, we identified NDM-1-producing Escherichia coli and Enterobacter hormaechei strains from a single rectal swab sample from a patient hospitalized in Rio de Janeiro, Brazil, who had no history of travel abroad. Complete DNA sequencing using the Illumina platform and annotation of the two plasmids harboring the blaNDM-1 gene, one from each strain, showed that they belonged to incompatibility groups IncFIIK and IncX3 and harbored a novel transposon named Tn3000. Similar genetic structures have been identified among other isolates in Brazil but also on plasmids from other continents. Our findings suggest that the blaNDM-1 gene may be transmitted by Tn3000 in different parts of the world. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Interplay of a non-conjugative integrative element and a conjugative plasmid in the spread of antibiotic resistance via suicidal plasmid transfer from an aquaculture Vibrio isolate.

PubMed

Nonaka, Lisa; Yamamoto, Tatsuya; Maruyama, Fumito; Hirose, Yuu; Onishi, Yuki; Kobayashi, Takeshi; Suzuki, Satoru; Nomura, Nobuhiko; Masuda, Michiaki; Yano, Hirokazu

2018-01-01

The capture of antimicrobial resistance genes (ARGs) by mobile genetic elements (MGEs) plays a critical role in resistance acquisition for human-associated bacteria. Although aquaculture environments are recognized as important reservoirs of ARGs, intra- and intercellular mobility of MGEs discovered in marine organisms is poorly characterized. Here, we show a new pattern of interspecies ARGs transfer involving a 'non-conjugative' integrative element. To identify active MGEs in a Vibrio ponticus isolate, we conducted whole-genome sequencing of a transconjugant obtained by mating between Escherichia coli and Vibrio ponticus. This revealed integration of a plasmid (designated pSEA1) into the chromosome, consisting of a self-transmissible plasmid backbone of the MOBH group, ARGs, and a 13.8-kb integrative element Tn6283. Molecular genetics analysis suggested a two-step gene transfer model. First, Tn6283 integrates into the recipient chromosome during suicidal plasmid transfer, followed by homologous recombination between the Tn6283 copy in the chromosome and that in the newly transferred pSEA1. Tn6283 is unusual among integrative elements in that it apparently does not encode transfer function and its excision barely generates unoccupied donor sites. Thus, its movement is analogous to the transposition of insertion sequences rather than to that of canonical integrative and conjugative elements. Overall, this study reveals the presence of a previously unrecognized type of MGE in a marine organism, highlighting diversity in the mode of interspecies gene transfer.

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

Dissemination of imipenem-resistant Acinetobacter baumannii with new plasmid-borne bla(OXA-72) in Taiwan.

PubMed

Kuo, Shu-Chen; Yang, Su-Pen; Lee, Yi-Tzu; Chuang, Han-Chuan; Chen, Chien-Pei; Chang, Chi-Ling; Chen, Te-Li; Lu, Po-Liang; Hsueh, Po-Ren; Fung, Chang-Phone

2013-07-13

The systemic surveillance of imipenem-resistant Acinetobacter baumannii (IRAB) from multicenters in Taiwan revealed the emergence of isolates with bla(OXA-72). This study described their genetic makeup, mechanism of spread, and contribution to carbapenem resistance. Two hundred and ninety-one non-repetitive isolates of A. baumannii were collected from 10 teaching hospitals from different geographical regions in Taiwan from June 2007 to September 2007. Minimal inhibitory concentrations (MICs) were determined by agar dilution. Clonality was determined by pulsed-field gel electrophoresis. Plasmid was extracted and digested by restriction enzymes, and subsequently analyzed by electrophoresis and Southern blot for bla(OXA-72). The flanking regions of bla(OXA-72) were determined by inverse PCR. The contribution of bla(OXA-72) to imipenem MIC was determined by transforming plasmids carrying bla(OXA-72) into imipenem-susceptible A. baumannii. Among 142 IRAB in Taiwan, 27 harbored bla(OXA-72); 22 originated from Southern Taiwan, 5 from Central Taiwan, and none from Northern Taiwan. There were two major clones. The bla(OXA-72) was identified in the plasmids of all isolates. Two genetic structures flanking plasmid-borne bla(OXA-72) were identified and shared identical sequences in certain regions; the one described in previous literature was present in only one isolate, and the new one was present in the remaining isolates. Introduction of bla(OXA-72) resulted in an increase of imipenem MIC in the transformants. The overexpression of bla(OXA-72) mRNA in response to imipenem further supported the contribution of bla(OXA-72). In conclusion, isolates with new plasmid-borne blaOXA-72 were found to be disseminated successfully in Southern Taiwan. The spread of the resistance gene depended on clonal spread and dissemination of a new plasmid. Bla(OXA-72) in these isolates directly led to their imipenem-resistance.

Engineering Complex Microbial Phenotypes with Continuous Genetic Integration and Plasmid Based Multi-gene Library

DTIC Science & Technology

2013-10-09

have desirable traits. We aim to enlarge the E. coli genome using Lactobacillusplantarum genes to build cells tolerant to EtOH and BT. L. plantarum is...chemicals III. Approach Objective 1 & la: Integrated heterologous (L. plantarum ) DNA into the E. coli chromosome and selected for insertions that...developed in combination with genes identified from screening L. plantarum libraries. Additionally, we have screened heterologous libraries for

Bioluminescence-based system for rapid detection of natural transformation.

PubMed

Santala, Ville; Karp, Matti; Santala, Suvi

2016-07-01

Horizontal gene transfer plays a significant role in bacterial evolution and has major clinical importance. Thus, it is vital to understand the mechanisms and kinetics of genetic transformations. Natural transformation is the driving mechanism for horizontal gene transfer in diverse genera of bacteria. Our study introduces a simple and rapid method for the investigation of natural transformation. This highly sensitive system allows the detection of a transformation event directly from a bacterial population without any separation step or selection of cells. The system is based on the bacterial luciferase operon from Photorhabdus luminescens The studied molecular tools consist of the functional modules luxCDE and luxAB, which involve a replicative plasmid and an integrative gene cassette. A well-established host for bacterial genetic investigations, Acinetobacter baylyi ADP1, is used as the model bacterium. We show that natural transformation followed by homologous recombination or plasmid recircularization can be readily detected in both actively growing and static biofilm-like cultures, including very rare transformation events. The system allows the detection of natural transformation within 1 h of introducing sample DNA into the culture. The introduced method provides a convenient means to study the kinetics of natural transformation under variable conditions and perturbations. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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

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

Characterization of the genetic elements required for site-specific integration of plasmid pSE211 in Saccharopolyspora erythraea.

PubMed Central

Brown, D P; Idler, K B; Katz, L

1990-01-01

The 18.1-kilobase plasmid pSE211 integrates into the chromosome of Saccharopolyspora erythraea at a specific attB site. Restriction analysis of the integrated plasmid, pSE211int, and adjacent chromosomal sequences allowed identification of attP, the plasmid attachment site. Nucleotide sequencing of attP, attB, attL, and attR revealed a 57-base-pair sequence common to all sites with no duplications of adjacent plasmid or chromosomal sequences in the integrated state, indicating that integration takes place through conservative, reciprocal strand exchange. An analysis of the sequences indicated the presence of a putative gene for Phe-tRNA at attB which is preserved at attL after integration has occurred. A comparison of the attB site for a number of actinomycete plasmids is presented. Integration at attB was also observed when a 2.4-kilobase segment of pSE211 containing attP and the adjacent plasmid sequence was used to transform a pSE211- host. Nucleotide sequencing of this segment revealed the presence of two complete open reading frames (ORFs) and a segment of a third ORF. The ORF adjacent to attP encodes a putative polypeptide 437 amino acids in length that shows similarity, at its C-terminal domain, to sequences of site-specific recombinases of the integrase family. The adjacent ORF encodes a putative 98-amino-acid basic polypeptide that contains a helix-turn-helix motif at its N terminus which corresponds to domains in the Xis proteins of a number of bacteriophages. A proposal for the function of this polypeptide is presented. The deduced amino acid sequence of the third ORF did not reveal similarities to polypeptide sequences in the current data banks. Images FIG. 2 FIG. 3 PMID:2180909

Dissemination and Persistence of blaCTX-M-9 Are Linked to Class 1 Integrons Containing CR1 Associated with Defective Transposon Derivatives from Tn402 Located in Early Antibiotic Resistance Plasmids of IncHI2, IncP1-α, and IncFI Groups

PubMed Central

Novais, Ângela; Cantón, Rafael; Valverde, Aránzazu; Machado, Elisabete; Galán, Juan-Carlos; Peixe, Luísa; Carattoli, Alessandra; Baquero, Fernando; Coque, Teresa M.

2006-01-01

This study analyzes the diversity of In60, a class 1 integron bearing CR1 and containing blaCTX-M-9, and its association with Tn402, Tn21, and classical conjugative plasmids among 45 CTX-M-9-producing clinical strains (41 Escherichia coli strains, 2 Klebsiella pneumoniae strains, 1 Salmonella enterica strain, and 1 Enterobacter cloacae strain). Forty-five patients in a Spanish tertiary care hospital were studied (1996 to 2003). The diversity of In60 and association of In60 with Tn402 or mercury resistance transposons were investigated by overlapping PCR assays and/or hybridization. Plasmid characterization included comparison of restriction fragment length polymorphism patterns and determination of incompatibility group by PCR-based replicon typing, sequencing, and hybridization. CTX-M-9 plasmids belonged to IncHI2 (n = 26), IncP-1α (n = 10), IncFI (n = 4), and IncI (n = 1) groups. Genetic platforms containing blaCTX-M-9 were classified in six types in relation to the In60 backbone and in eight subtypes in relation to Tn402 derivatives. They were associated with Tn21 sequences when located in IncP-1α or IncHI2 plasmids. Our study identified blaCTX-M-9 in a high diversity of CR1-bearing class 1 integrons linked to different Tn402 derivatives, often to Tn21, highlighting the role of recombination events in the evolution of antibiotic resistance plasmids. The presence of blaCTX-M-9 on broad-host-range IncP-1α plasmids might contribute to its dissemination to hosts that were not members of the family Enterobacteriaceae. PMID:16870767

Virulence plasmid (pYV)-associated expression of phenotypic virulent determinants in pathogenic Yersinia species: a convenient method for monitoring the presence of pYV under culture conditions and its application for...food

USDA-ARS?s Scientific Manuscript database

In Yersinia pestis, Y. pseudotuberculosis, and Y, enterocolitica, phenotypic expression of virulence plasmid (pYV: 70-kb)-associated genetic determinants may include low calcium response (Lcr, pin point colony, size = 0.36 mm), colony morphology (size = 1.13 mm), crystal violet (CV) binding (dark-v...

Virulence plasmid (pYV)-associated expression of phenotypic virulent determinants in pathogenic Yersinia species: a convenient method for monitoring the presence of pYV under culture conditions and its application for....food

USDA-ARS?s Scientific Manuscript database

In Yersinia pestis, Y. pseudotuberculosis, and Y, enterocolitica, phenotypic expression of several virulence plasmid (pYV: 70-kb)-associated genetic determinants may include low calcium response (Lcr, pin point colony, size = 0.36 mm), colony morphology (size = 1.13 mm), crystal violet (CV) binding...

Identification of Essential Genetic Baculoviral Elements for Recombinant Protein Expression by Transactivation in Sf21 Insect Cells.

PubMed

Bleckmann, Maren; Schürig, Margitta; Chen, Fang-Fang; Yen, Zen-Zen; Lindemann, Nils; Meyer, Steffen; Spehr, Johannes; van den Heuvel, Joop

2016-01-01

The Baculovirus Expression Vector System (BEVS) is widely used to produce high amounts of recombinant proteins. Nevertheless, generating recombinant baculovirus in high quality is rather time-consuming and labor-intensive. Alternatively, virus-free expression in insect cells did not achieve similar expression levels for most proteins so far. The transactivation method is a promising approach for protein expression in Sf21 cells. It combines advantages of BEVS and plasmid-based expression by activating strong virus-dependent promoters on a transfected plasmid by baculoviral coinfection. Here, we identified expression elements required for transactivation. Therefore, we designed several vectors comprising different viral promoters or promoter combinations and tested them for eGFP expression using the automated BioLector microcultivation system. Remarkably, only the combination of the very late promoter p10 together with the homologous region 5 (hr5) could boost expression during transactivation. Other elements, like p10 alone or the late viral promoter polH, did not respond to transactivation. A new combination of hr5 and p10 with the strongest immediate early OpMNPV viral promoter OpIE2 improved the yield of eGFP by ~25% in comparison to the previous applied hr5-IE1-p10 expression cassette. Furthermore, we observed a strong influence of the transcription termination sequence and vector backbone on the level of expression. Finally, the expression levels for transactivation, BEVS and solely plasmid-based expression were compared for the marker protein eGFP, underlining the potential of transactivation for fast recombinant protein expression in Sf21 cells. In conclusion, essential elements for transactivation could be identified. The optimal elements were applied to generate an improved vector applicable in virus-free plasmid-based expression, transactivation and BEVS.

Coexistence of Two blaNDM-5 Genes on an IncF Plasmid as Revealed by Nanopore Sequencing.

PubMed

Feng, Yu; Liu, Lu; McNally, Alan; Zong, Zhiyong

2018-05-01

In a carbapenem-resistant Escherichia coli clinical isolate of sequence type 167, two copies of bla NDM-5 were found on a 144,225-bp IncF self-transmissible plasmid of the F36:A4:B - type. Both bla NDM-5 genes were located in 11,065-bp regions flanked by two copies of IS 26 The two regions were identical in sequence but were present at different locations on the plasmid, suggesting a duplication of the same region. This study highlights the complex genetic contexts of bla NDM-5 . Copyright © 2018 American Society for Microbiology.

Sequencing cDNAs: An Introduction to DNA Sequence Analysis in the Undergraduate Molecular Genetics Course.

ERIC Educational Resources Information Center

Galewsky, Samuel

2000-01-01

Introduces a series of molecular genetics laboratories where students pick a single colony from a Drosophila melanogester embryo cDNA library and purify the plasmid, then analyze the insert through restriction digests and gel electrophoresis. (Author/YDS)

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

Genetic Stability of Streptomyces Lividans pIJ702 in Response to Spaceflight

NASA Astrophysics Data System (ADS)

Lim, K. S.; Goins, T. L.; Voeikova, T. A.; Pyle, B. H.

2008-06-01

Streptomyces lividans carrying plasmid pIJ702 encoding genes for thiostrepton resistance (tsr-) and melanin production (mel+) was plated on agar and flown on the Russian satellite Foton-M3 for 16 days. The percentage loss of plasmid expression in flight samples was lower than that in ground samples when both samples were grown in enriched (ISP) media. Differences in media content also affect plasmid expression rate; ISP media have a higher loss of plasmid expression than samples in minimum media when both were grown on ground conditions. Results suggest that stress resulted in the increased expression of plasmid pIJ702 by S. lividans. Screening of thiostrepton resistant white (tsr+ mel-) mutants showed similar proportions of variants in ground samples and flight samples. To determine if there are mutations in the mel gene, DNA extracted from flight and control white mutants was amplified and gel electrophoresis of amplified products show no major mutation in the products. Sequencing of amplified products is required to identify mutations resulting in loss of pigmentation.

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

Genetic Tools for the Industrially Promising Methanotroph Methylomicrobium buryatense

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

Puri, AW; Owen, S; Chu, F

2015-02-10

Aerobic methanotrophs oxidize methane at ambient temperatures and pressures and are therefore attractive systems for methane-based bioconversions. In this work, we developed and validated genetic tools for Methylomicrobium buryatense, a haloalkaliphilic gammaproteobacterial (type I) methanotroph. M. buryatense was isolated directly on natural gas and grows robustly in pure culture with a 3-h doubling time, enabling rapid genetic manipulation compared to many other methanotrophic species. As a proof of concept, we used a sucrose counterselection system to eliminate glycogen production in M. buryatense by constructing unmarked deletions in two redundant glycogen synthase genes. We also selected for a more genetically tractablemore » variant strain that can be conjugated with small incompatibility group P (IncP)-based broad-host-range vectors and determined that this capability is due to loss of the native plasmid. These tools make M. buryatense a promising model system for studying aerobic methanotroph physiology and enable metabolic engineering in this bacterium for industrial biocatalysis of methane.« less

Extrachromosomal genetic elements in Micrococcus.

PubMed

Dib, Julián Rafael; Liebl, Wolfgang; Wagenknecht, Martin; Farías, María Eugenia; Meinhardt, Friedhelm

2013-01-01

Micrococci are Gram-positive G + C-rich, nonmotile, nonspore-forming actinomycetous bacteria. Micrococcus comprises ten members, with Micrococcus luteus being the type species. Representatives of the genus play important roles in the biodegradation of xenobiotics, bioremediation processes, production of biotechnologically important enzymes or bioactive compounds, as test strains in biological assays for lysozyme and antibiotics, and as infective agents in immunocompromised humans. The first description of plasmids dates back approximately 28 years, when several extrachromosomal elements ranging in size from 1.5 to 30.2 kb were found in Micrococcus luteus. Up to the present, a number of circular plasmids conferring antibiotic resistance, the ability to degrade aromatic compounds, and osmotolerance are known, as well as cryptic elements with unidentified functions. Here, we review the Micrococcus extrachromosomal traits reported thus far including phages and the only quite recently described large linear extrachromosomal genetic elements, termed linear plasmids, which range in size from 75 kb (pJD12) to 110 kb (pLMA1) and which confer putative advantageous capabilities, such as antibiotic or heavy metal resistances (inferred from sequence analyses and curing experiments). The role of the extrachromosomal elements for the frequently proven ecological and biotechnological versatility of the genus will be addressed as well as their potential for the development and use as genetic tools.

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.

Heat Resistance Mediated by pLM58 Plasmid-Borne ClpL in Listeria monocytogenes

PubMed Central

Aalto-Araneda, Mariella; Lindström, Miia; Korkeala, Hannu

2017-01-01

ABSTRACT Listeria monocytogenes is one of the most heat-resistant non-spore-forming food-borne pathogens and poses a notable risk to food safety, particularly when mild heat treatments are used in food processing and preparation. While general heat stress properties and response mechanisms of L. monocytogenes have been described, accessory mechanisms providing particular L. monocytogenes strains with the advantage of enhanced heat resistance are unknown. Here, we report plasmid-mediated heat resistance of L. monocytogenes for the first time. This resistance is mediated by the ATP-dependent protease ClpL. We tested the survival of two wild-type L. monocytogenes strains—both of serotype 1/2c, sequence type ST9, and high sequence identity—at high temperatures and compared their genome composition in order to identify genetic mechanisms involved in their heat survival phenotype. L. monocytogenes AT3E was more heat resistant (0.0 CFU/ml log10 reduction) than strain AL4E (1.4 CFU/ml log10 reduction) after heating at 55°C for 40 min. A prominent difference in the genome compositions of the two strains was a 58-kb plasmid (pLM58) harbored by the heat-resistant AT3E strain, suggesting plasmid-mediated heat resistance. Indeed, plasmid curing resulted in significantly decreased heat resistance (1.1 CFU/ml log10 reduction) at 55°C. pLM58 harbored a 2,115-bp open reading frame annotated as an ATP-dependent protease (ClpL)-encoding clpL gene. Introducing the clpL gene into a natively heat-sensitive L. monocytogenes strain (1.2 CFU/ml log10 reduction) significantly increased the heat resistance of the recipient strain (0.4 CFU/ml log10 reduction) at 55°C. Plasmid-borne ClpL is thus a potential predictor of elevated heat resistance in L. monocytogenes. IMPORTANCE Listeria monocytogenes is a dangerous food pathogen causing the severe illness listeriosis that has a high mortality rate in immunocompromised individuals. Although destroyed by pasteurization, L. monocytogenes is among the most heat-resistant non-spore-forming bacteria. This poses a risk to food safety, as listeriosis is commonly associated with ready-to-eat foods that are consumed without thorough heating. However, L. monocytogenes strains differ in their ability to survive high temperatures, and comprehensive understanding of the genetic mechanisms underlying these differences is still limited. Whole-genome-sequence analysis and phenotypic characterization allowed us to identify a novel plasmid, designated pLM58, and a plasmid-borne ATP-dependent protease (ClpL), which mediated heat resistance in L. monocytogenes. As the first report on plasmid-mediated heat resistance in L. monocytogenes, our study sheds light on the accessory genetic mechanisms rendering certain L. monocytogenes strains particularly capable of surviving high temperatures—with plasmid-borne ClpL being a potential predictor of elevated heat resistance. PMID:29104933

A New Suite of Plasmid Vectors for Fluorescence-Based Imaging of Root Colonizing Pseudomonads

DOE PAGES

Wilton, Rosemarie; Ahrendt, Angela J.; Shinde, Shalaka; ...

2018-02-01

In the terrestrial ecosystem, plant-microbe symbiotic associations are ecologically and economically important processes. To better understand these associations at structural and functional levels, different molecular and biochemical tools are applied. In this study, we have constructed a suite of vectors that incorporates several new elements into the rhizosphere stable, broad-host vector pME6031. The new vectors are useful for studies requiring multi-color tagging and visualization of plant-associated, Gram negative bacterial strains such as Pseudomonas plant growth promotion and biocontrol strains. A number of genetic elements, including constitutive promoters and signal peptides that target secretion to the periplasm, have been evaluated. Severalmore » next generation fluorescent proteins, namely mTurquoise2, mNeonGreen, mRuby2, DsRed-Express2 and E2-Crimson have been incorporated into the vectors for whole cell labeling or protein tagging. Secretion of mTurquoise2 and mNeonGreen into the periplasm of Pseudomonas fluorescens SBW25 has also been demonstrated, providing a vehicle for tagging proteins in the periplasmic compartment. A higher copy number version of select plasmids has been produced by introduction of a previously described repA mutation, affording an increase in protein expression levels. The utility of these plasmids for fluorescence-based imaging is demonstrated by root colonization of Solanum lycopersicum seedlings by P. fluorescens SBW25 in a hydroponic growth system. As a result, the plasmids are stably maintained during root colonization in the absence of selective pressure for more than two weeks.« less

A New Suite of Plasmid Vectors for Fluorescence-Based Imaging of Root Colonizing Pseudomonads

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

Wilton, Rosemarie; Ahrendt, Angela J.; Shinde, Shalaka

In the terrestrial ecosystem, plant-microbe symbiotic associations are ecologically and economically important processes. To better understand these associations at structural and functional levels, different molecular and biochemical tools are applied. In this study, we have constructed a suite of vectors that incorporates several new elements into the rhizosphere stable, broad-host vector pME6031. The new vectors are useful for studies requiring multi-color tagging and visualization of plant-associated, Gram negative bacterial strains such as Pseudomonas plant growth promotion and biocontrol strains. A number of genetic elements, including constitutive promoters and signal peptides that target secretion to the periplasm, have been evaluated. Severalmore » next generation fluorescent proteins, namely mTurquoise2, mNeonGreen, mRuby2, DsRed-Express2 and E2-Crimson have been incorporated into the vectors for whole cell labeling or protein tagging. Secretion of mTurquoise2 and mNeonGreen into the periplasm of Pseudomonas fluorescens SBW25 has also been demonstrated, providing a vehicle for tagging proteins in the periplasmic compartment. A higher copy number version of select plasmids has been produced by introduction of a previously described repA mutation, affording an increase in protein expression levels. The utility of these plasmids for fluorescence-based imaging is demonstrated by root colonization of Solanum lycopersicum seedlings by P. fluorescens SBW25 in a hydroponic growth system. As a result, the plasmids are stably maintained during root colonization in the absence of selective pressure for more than two weeks.« 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

Identification and characterization of plasmid-mediated quinolone resistance determinants in Enterobacteriaceae isolated from healthy poultry in Brazil.

PubMed

Ferreira, Joseane Cristina; Penha Filho, Rafael Antonio Casarin; Kuaye, Ana Paula Yorika; Andrade, Leonardo Neves; Berchieri Junior, Angelo; Darini, Ana Lúcia da Costa

2018-06-01

The expression of plasmid-mediated quinolone resistance (PMQR) genes confers low-level quinolone and fluoroquinolones resistance alone. However, the association to chromosomal resistance mechanisms determines an expressively higher resistance in Enterobacteriaceae. These mechanisms are horizontally disseminated within plasmids and have contributed to the emergence of bacteria with reduced susceptibility or resistant to therapies worldwide. The epidemiological characterization of PMQR dissemination is highly relevant in the scientific and medical context, to investigate the dissemination within enterobacteria, from different populations, including humans and food-producing animals. In the present study, 200 Enterobacteriaceae isolates were harvested from poultry with cloacal swabs and identified as Escherichia coli (90.5%), Escherichia fergusonii (5.5%), Klebsiella oxytoca (2.5%) and Klebsiella pneumoniae (1.5%). Among isolates evaluated, 46 (23%) harboured PMQR genes including qnrB (43/200), qnrS (2/200) and aac(6')-Ib-cr (1/200). All isolates carrying PMQR genes showed multidrug-resistance phenotype. The 36 E. coli isolates showed 18 different PFGE types. All E. fergusonii isolates showed the same PFGE type. The two Klebsiella oxytoca belonged to two different PFGE types. The phylogenetic groups A, B1, and D were found among the E. coli harboring PMQR genes. Based on the phylogenetic analysis and PFGE, the population structure of E. coli isolates was diverse, even within the same farm. All isolates carrying qnrB and qnrS genes also harboured ColE-like plasmids. The Southern blot hybridization using the S1-PFGE revealed that the qnrB genes were located on low molecular weight plasmids, smaller than 10Kb. Resistance plasmids were sequenced and showed 100% identity with plasmid pPAB19-3. The association of PMQR genes with mobile genetic elements, such as transferable plasmids, favours the selection and dissemination of (fluoro) quinolones resistant bacteria among food-producing animals, and may play an important role in the current increased prevalence of resistant bacteria in different environments reported worldwide. Copyright © 2018. Published by Elsevier B.V.

Detection and molecular characterization of Escherichia coli CTX-M-15 and Klebsiella pneumoniae SHV-12 β-lactamases from bovine mastitis isolates in the United Kingdom.

PubMed

Timofte, Dorina; Maciuca, Iuliana E; Evans, Nicholas J; Williams, Helen; Wattret, Andrew; Fick, Jenny C; Williams, Nicola J

2014-01-01

Recent reports raised concerns about the role that farm stock may play in the dissemination of extended-spectrum β-lactamase (ESBL)-producing bacteria. This study characterized the ESBLs in two Escherichia coli and three Klebsiella pneumoniae subsp. pneumoniae isolates from cases of clinical bovine mastitis in the United Kingdom. Bacterial culture and sensitivity testing of bovine mastitic milk samples identified Gram-negative cefpodoxime-resistant isolates, which were assessed for their ESBL phenotypes. Conjugation experiments and PCR-based replicon typing (PBRT) were used for characterization of transferable plasmids. E. coli isolates belonged to sequence type 88 (ST88; determined by multilocus sequence typing) and carried blaCTX-M-15 and blaTEM-1, while K. pneumoniae subsp. pneumoniae isolates carried blaSHV-12 and blaTEM-1. Conjugation experiments demonstrated that blaCTX-M-15 and blaTEM-1 were carried on a conjugative plasmid in E. coli, and PBRT identified this to be an IncI1 plasmid. The resistance genes were nontransferable in K. pneumoniae subsp. pneumoniae isolates. Moreover, in the E. coli isolates, an association of ISEcp1 and IS26 with blaCTX-M-15 was found where the IS26 element was inserted upstream of both ISEcp1 and the blaCTX-M promoter, a genetic arrangement highly similar to that described in some United Kingdom human isolates. We report the first cases in Europe of bovine mastitis due to E. coli CTX-M-15 and also of bovine mastitis due to K. pneumoniae subsp. pneumoniae SHV-12 β-lactamases in the United Kingdom. We also describe the genetic environment of blaCTX-M-15 and highlight the role that IncI1 plasmids may play in the spread and dissemination of ESBL genes, which have been described in both human and cattle isolates.

Removal of N-6-methyladenine by the nucleotide excision repair pathway triggers the repair of mismatches in yeast gap-repair intermediates.

PubMed

Guo, Xiaoge; Jinks-Robertson, Sue

2013-12-01

Gap-repair assays have been an important tool for studying the genetic control of homologous recombination in yeast. Sequence analysis of recombination products derived when a gapped plasmid is diverged relative to the chromosomal repair template additionally has been used to infer structures of strand-exchange intermediates. In the absence of the canonical mismatch repair pathway, mismatches present in these intermediates are expected to persist and segregate at the next round of DNA replication. In a mismatch repair defective (mlh1Δ) background, however, we have observed that recombination-generated mismatches are often corrected to generate gene conversion or restoration events. In the analyses reported here, the source of the aberrant mismatch removal during gap repair was examined. We find that most mismatch removal is linked to the methylation status of the plasmid used in the gap-repair assay. Whereas more than half of Dam-methylated plasmids had patches of gene conversion and/or restoration interspersed with unrepaired mismatches, mismatch removal was observed in less than 10% of products obtained when un-methylated plasmids were used in transformation experiments. The methylation-linked removal of mismatches in recombination intermediates was due specifically to the nucleotide excision repair pathway, with such mismatch removal being partially counteracted by glycosylases of the base excision repair pathway. These data demonstrate that nucleotide excision repair activity is not limited to bulky, helix-distorting DNA lesions, but also targets removal of very modest perturbations in DNA structure. In addition to its effects on mismatch removal, methylation reduced the overall gap-repair efficiency, but this reduction was not affected by the status of excision repair pathways. Finally, gel purification of DNA prior to transformation reduced gap-repair efficiency four-fold in a nucleotide excision repair-defective background, indicating that the collateral introduction of UV damage can potentially compromise genetic interpretations. Copyright © 2013 Elsevier B.V. All rights reserved.

Detection and Molecular Characterization of Escherichia coli CTX-M-15 and Klebsiella pneumoniae SHV-12 β-Lactamases from Bovine Mastitis Isolates in the United Kingdom

PubMed Central

Maciuca, Iuliana E.; Evans, Nicholas J.; Williams, Helen; Wattret, Andrew; Fick, Jenny C.; Williams, Nicola J.

2014-01-01

Recent reports raised concerns about the role that farm stock may play in the dissemination of extended-spectrum β-lactamase (ESBL)-producing bacteria. This study characterized the ESBLs in two Escherichia coli and three Klebsiella pneumoniae subsp. pneumoniae isolates from cases of clinical bovine mastitis in the United Kingdom. Bacterial culture and sensitivity testing of bovine mastitic milk samples identified Gram-negative cefpodoxime-resistant isolates, which were assessed for their ESBL phenotypes. Conjugation experiments and PCR-based replicon typing (PBRT) were used for characterization of transferable plasmids. E. coli isolates belonged to sequence type 88 (ST88; determined by multilocus sequence typing) and carried blaCTX-M-15 and blaTEM-1, while K. pneumoniae subsp. pneumoniae isolates carried blaSHV-12 and blaTEM-1. Conjugation experiments demonstrated that blaCTX-M-15 and blaTEM-1 were carried on a conjugative plasmid in E. coli, and PBRT identified this to be an IncI1 plasmid. The resistance genes were nontransferable in K. pneumoniae subsp. pneumoniae isolates. Moreover, in the E. coli isolates, an association of ISEcp1 and IS26 with blaCTX-M-15 was found where the IS26 element was inserted upstream of both ISEcp1 and the blaCTX-M promoter, a genetic arrangement highly similar to that described in some United Kingdom human isolates. We report the first cases in Europe of bovine mastitis due to E. coli CTX-M-15 and also of bovine mastitis due to K. pneumoniae subsp. pneumoniae SHV-12 β-lactamases in the United Kingdom. We also describe the genetic environment of blaCTX-M-15 and highlight the role that IncI1 plasmids may play in the spread and dissemination of ESBL genes, which have been described in both human and cattle isolates. PMID:24247146

Removal of N-6-methyladenine by the nucleotide excision repair pathway triggers the repair of mismatches in yeast gap-repair intermediates

PubMed Central

Guo, Xiaoge; Jinks-Robertson, Sue

2013-01-01

Gap-repair assays have been an important tool for studying the genetic control of homologous recombination in yeast. Sequence analysis of recombination products derived when a gapped plasmid is diverged relative to the chromosomal repair template additionally has been used to infer structures of strand-exchange intermediates. In the absence of the canonical mismatch repair pathway, mismatches present in these intermediates are expected to persist and segregate at the next round of DNA replication. In a mismatch repair defective (mlh1Δ) background, however, we have observed that recombination-generated mismatches are often corrected to generate gene conversion or restoration events. In the analyses reported here, the source of the aberrant mismatch removal during gap repair was examined. We find that most mismatch removal is linked to the methylation status of the plasmid used in the gap-repair assay. Whereas more than half of Dam-methylated plasmids had patches of gene conversion and/or restoration interspersed with unrepaired mismatches, mismatch removal was observed in less than 10% of products obtained when un-methylated plasmids were used in transformation experiments. The methylation-linked removal of mismatches in recombination intermediates was due specifically to the nucleotide excision repair pathway, with such mismatch removal being partially counteracted by glycosylases of the base excision repair pathway. These data demonstrate that nucleotide excision repair activity is not limited to bulky, helix-distorting DNA lesions, but also targets removal of very modest perturbations in DNA structure. In addition to its effects on mismatch removal, methylation reduced the overall gap-repair efficiency, but this reduction was not affected by the status of excision repair pathways. Finally, gel purification of DNA prior to transformation reduced gap-repair efficiency four-fold in a nucleotide excision repair-defective background, indicating that the cillateral introduction of UV damage can potentially compromise genetic interpretations. PMID:24120148

Establishment of a Nipah virus rescue system.

PubMed

Yoneda, Misako; Guillaume, Vanessa; Ikeda, Fusako; Sakuma, Yuki; Sato, Hiroki; Wild, T Fabian; Kai, Chieko

2006-10-31

Nipah virus (NiV), a paramyxovirus, was first discovered in Malaysia in 1998 in an outbreak of infection in pigs and humans and incurred a high fatality rate in humans. Fruit bats, living in vast areas extending from India to the western Pacific, were identified as the natural reservoir of the virus. However, the mechanisms that resulted in severe pathogenicity in humans (up to 70% mortality) and that enabled crossing the species barrier were not known. In this study, we established a system that enabled the rescue of replicating NiVs from a cloned DNA by cotransfection of a constructed full-length cDNA clone and supporting plasmids coding virus nucleoprotein, phosphoprotein, and polymerase with the infection of the recombinant vaccinia virus, MVAGKT7, expressing T7 RNA polymerase. The rescued NiV (rNiV), by using the newly developed reverse genetics system, showed properties in vitro that were similar to the parent virus and retained the severe pathogenicity in a previously established animal model by experimental infection. A recombinant NiV was also developed, expressing enhanced green fluorescent protein (rNiV-EGFP). Using the virus, permissibility of NiV was compared with the presence of a known cellular receptor, ephrin B2, in a number of cell lines of different origins. Interestingly, two cell lines expressing ephrin B2 were not susceptible for rNiV-EGFP, indicating that additional factors are clearly required for full NiV replication. The reverse genetics for NiV will provide a powerful tool for the analysis of the molecular mechanisms of pathogenicity and cross-species infection.

Cloning of a promoter-like soybean DNA sequence responding to IAA induction in Escherichia coli K12.

PubMed

Kline, E L; Chiang, S J; Lattora, D; Chaung, W

1992-02-01

We have constructed a soybean genomic DNA library in Escherichia coli K12 strain KC13 using plasmid pPV33, which consists of a promoter-less tetracycline resistance (Tcr) gene. A recombinant clone, KC13(pAU-SB1)+, was obtained by selecting for resistance to tetracycline in the presence of indole-3-acetic acid (IAA). Restriction enzyme cleavage and Southern hybridization analysis revealed that the pAU-SB1 plasmid has a 250 bp soybean DNA insert fused with the Tcr gene. In the presence of a selected group of auxins, induction of the Tcr phenotype and mRNA synthesis of the Tcr gene are observed only in KC13(pAU-SB1)+ cultures. On the other hand, induction of the Tcr phenotype and mRNA synthesis of the Tcr gene are absent in cells harboring the cloning vector pPV33 or a recombinant plasmid containing the 250 bp insert in the reverse orientation, pAU-SB1ro. This demonstrated a need for the insertion of the 250 bp soybean DNA and the specificity of its orientation in response to IAA induction. The start point of mRNA transcription in response to IAA, IBA, IPA, 2,4,5-T, and a-NAP is at base pair -96 or -95 upstream of the translational start site of the Tcr gene and base pair -98 with 2,4-D.

Candicidin Biosynthesis Gene Cluster Is Widely Distributed among Streptomyces spp. Isolated from the Sediments and the Neuston Layer of the Trondheim Fjord, Norway▿ †

PubMed Central

Jørgensen, Hanne; Fjærvik, Espen; Hakvåg, Sigrid; Bruheim, Per; Bredholt, Harald; Klinkenberg, Geir; Ellingsen, Trond E.; Zotchev, Sergey B.

2009-01-01

A large number of Streptomyces bacteria with antifungal activity isolated from samples collected in the Trondheim fjord (Norway) were found to produce polyene compounds. Investigation of polyene-containing extracts revealed that most of the isolates produced the same compound, which had an atomic mass and UV spectrum corresponding to those of candicidin D. The morphological diversity of these isolates prompted us to speculate about the involvement of a mobile genetic element in dissemination of the candicidin biosynthesis gene cluster (can). Eight candicidin-producing isolates were analyzed by performing a 16S rRNA gene-based taxonomic analysis, pulsed-field gel electrophoresis, PCR, and Southern blot hybridization with can-specific probes. These analyses revealed that most of the isolates were related, although they were morphologically diverse, and that all of them contained can genes. The majority of the isolates studied contained large plasmids, and two can-specific probes hybridized to a 250-kb plasmid in one isolate. Incubation of the latter isolate at a high temperature resulted in loss of the can genes and candicidin production, while mating of the “cured” strain with a plasmid-containing donor restored candicidin production. The latter result suggested that the 250-kb plasmid contains the complete can gene cluster and could be responsible for conjugative transfer of this cluster to other streptomycetes. PMID:19286787

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

Fitness costs of various mobile genetic elements in Enterococcus faecium and Enterococcus faecalis

PubMed Central

Starikova, Irina; Al-Haroni, Mohammed; Werner, Guido; Roberts, Adam P.; Sørum, Vidar; Nielsen, Kaare M.; Johnsen, Pål J.

2013-01-01

Objectives To determine the fitness effects of various mobile genetic elements (MGEs) in Enterococcus faecium and Enterococcus faecalis when newly acquired. We also tested the hypothesis that the biological cost of vancomycin resistance plasmids could be mitigated during continuous growth in the laboratory. Methods Different MGEs, including two conjugative transposons (CTns) of the Tn916 family (18 and 33 kb), a pathogenicity island (PAI) of 200 kb and vancomycin-resistance (vanA) plasmids (80–200 kb) of various origins and classes, were transferred into common ancestral E. faecium and E. faecalis strains by conjugation assays and experimentally evolved (vanA plasmids only). Transconjugants were characterized by PFGE, S1 nuclease assays and Southern blotting hybridization analyses. Single specific primer PCR was performed to determine the target sites for the insertion of the CTns. The fitness costs of various MGEs in E. faecium and E. faecalis were estimated in head-to-head competition experiments, and evolved populations were generated in serial transfer assays. Results The biological cost of a newly acquired PAI and two CTns were both host- and insertion-locus-dependent. Newly acquired vanA plasmids may severely reduce host fitness (25%–27%), but these costs were rapidly mitigated after only 400 generations of continuous growth in the absence of antibiotic selection. Conclusions Newly acquired MGEs may impose an immediate biological cost in E. faecium. However, as demonstrated for vanA plasmids, the initial costs of MGE carriage may be mitigated during growth and beneficial plasmid–host association can rapidly emerge. PMID:23833178

Genome Dynamics of Escherichia coli during Antibiotic Treatment: Transfer, Loss, and Persistence of Genetic Elements In situ of the Infant Gut.

PubMed

Porse, Andreas; Gumpert, Heidi; Kubicek-Sutherland, Jessica Z; Karami, Nahid; Adlerberth, Ingegerd; Wold, Agnes E; Andersson, Dan I; Sommer, Morten O A

2017-01-01

Elucidating the adaptive strategies and plasticity of bacterial genomes in situ is crucial for understanding the epidemiology and evolution of pathogens threatening human health. While much is known about the evolution of Escherichia coli in controlled laboratory environments, less effort has been made to elucidate the genome dynamics of E. coli in its native settings. Here, we follow the genome dynamics of co-existing E. coli lineages in situ of the infant gut during the first year of life. One E. coli lineage causes a urinary tract infection (UTI) and experiences several alterations of its genomic content during subsequent antibiotic treatment. Interestingly, all isolates of this uropathogenic E. coli strain carried a highly stable plasmid implicated in virulence of diverse pathogenic strains from all over the world. While virulence elements are certainly beneficial during infection scenarios, their role in gut colonization and pathogen persistence is poorly understood. We performed in vivo competitive fitness experiments to assess the role of this highly disseminated virulence plasmid in gut colonization, but found no evidence for a direct benefit of plasmid carriage. Through plasmid stability assays, we demonstrate that this plasmid is maintained in a parasitic manner, by strong first-line inheritance mechanisms, acting on the single-cell level, rather than providing a direct survival advantage in the gut. Investigating the ecology of endemic accessory genetic elements, in their pathogenic hosts and native environment, is of vital importance if we want to understand the evolution and persistence of highly virulent and drug resistant bacterial isolates.

Fluorescence-Based Reporters for Detection of Mutagenesis in E. coli

PubMed Central

Standley, Melissa; Allen, Jennifer; Cervantes, Layla; Lilly, Joshua; Camps, Manel

2017-01-01

Mutagenesis in model organisms following exposure to chemicals is used as an indicator of genotoxicity. Mutagenesis assays are also used to study mechanisms of DNA homeostasis. The present article focuses on detection of mutagenesis in prokaryotes, which boils down to two approaches: reporter inactivation (forward mutation assay) and reversion of an inactivating mutation (reversion mutation assay). Both methods are labor-intensive, involving visual screening, quantification of colonies on solid media, or determining a Poisson distribution in liquid culture. Here we present two reversion reporters for in vivo mutagenesis that produce a quantitative output, and thus have the potential to greatly reduce the amount of test chemical and labor involved in these assays. This output is obtained by coupling a TEM β lactamase-based reversion assay with GFP fluorescence, either by placing the two genes on the same plasmid or by fusing them translationally and interrupting the N-terminus of the ORF with a stop codon. We also describe a reporter aimed at facilitating the monitoring of continuous mutagenesis in mutator strains. This reporter couples two reversion markers, allowing the temporal separation of mutation events in time, thus providing information about the dynamics of mutagenesis in mutator strains. Here, we describe these reporter systems, provide protocols for use, and demonstrate their key functional features using error-prone Pol I mutagenesis as a source of mutations. PMID:28645368

Fluorescence-Based Reporters for Detection of Mutagenesis in E. coli.

PubMed

Standley, Melissa; Allen, Jennifer; Cervantes, Layla; Lilly, Joshua; Camps, Manel

2017-01-01

Mutagenesis in model organisms following exposure to chemicals is used as an indicator of genotoxicity. Mutagenesis assays are also used to study mechanisms of DNA homeostasis. This chapter focuses on detection of mutagenesis in prokaryotes, which boils down to two approaches: reporter inactivation (forward mutation assay) and reversion of an inactivating mutation (reversion mutation assay). Both methods are labor intensive, involving visual screening, quantification of colonies on solid media, or determining a Poisson distribution in liquid culture. Here, we present two reversion reporters for in vivo mutagenesis that produce a quantitative output, and thus have the potential to greatly reduce the amount of test chemical and labor involved in these assays. This output is obtained by coupling a TEM β lactamase-based reversion assay with GFP fluorescence, either by placing the two genes on the same plasmid or by fusing them translationally and interrupting the N-terminus of the chimeric ORF with a stop codon. We also describe a reporter aimed at facilitating the monitoring of continuous mutagenesis in mutator strains. This reporter couples two reversion markers, allowing the temporal separation of mutation events in time, thus providing information about the dynamics of mutagenesis in mutator strains. Here, we describe these reporter systems, provide protocols for use, and demonstrate their key functional features using error-prone Pol I mutagenesis as a source of mutations. © 2017 Elsevier Inc. All rights reserved.

Common genetic variants of the human UMOD gene are functional on transcription and predict plasma uric acid in two distinct populations

PubMed Central

Han, Jia; Liu, Ying; Rao, Fangwen; Nievergelt, Caroline M.; O’Connor, Daniel T.; Wang, Xingyu; Liu, Lisheng; Bu, Dingfang; Liang, Yu; Wang, Fang; Zhang, Luxia; Zhang, Hong; Chen, Yuqing; Wang, Haiyan

2013-01-01

Uromodulin (UMOD) genetic variants cause familial juvenile hyperuricemic nephropathy, characterized by hyperuricemia, decreased renal excretion of UMOD and uric acid; such findings suggest a role for UMOD in the regulation of plasma uric acid. We screened common variants across the UMOD locus in two populations, one from a community-based Chinese population, the other from California twins and siblings. Transcriptional activity of promoter variants was estimated in luciferase reporter plasmids transfected into HEK293 cells and mlMCD3 cells. By variance components in twin pairs, uric acid concentration and excretion were heritable traits. In the primary population from Beijing, we identified that carriers of haplotype GCC displayed higher plasma uric acid, and 3 UMOD promoter variants associated with plasma uric acid. UMOD promoter variants displayed reciprocal effects on urine uric acid excretion and plasma uric acid concentration, suggesting a primary effect on renal tubular handling of urate. These UMOD genetic marker-on-trait associations for uric acid were replicated in an independent American population sample. Site-directed mutagenesis at trait-associated UMOD promoter variants altered promoter activity in transfected luciferase reporter plasmids. These results suggest that UMOD promoter variants seem to initiate a cascade of transcriptional and biochemical changes influencing UMOD secretion, eventuating in elevation of plasma uric acid. PMID:23344472

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

NASA Astrophysics Data System (ADS)

Alizadeh, Rahman; Afzal, Mohd; Arjmand, Farukh

2014-10-01

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

Materials and methods for efficient succinate and malate production

DOEpatents

Jantama, Kaemwich; Haupt, Mark John; Zhang, Xueli; Moore, Jonathan C; Shanmugam, Keelnatham T; Ingram, Lonnie O'Neal

2014-04-08

Genetically engineered microorganisms have been constructed to produce succinate and malate in mineral salt media in pH-controlled batch fermentations without the addition of plasmids or foreign genes. The subject invention also provides methods of producing succinate and malate comprising the culture of genetically modified microorganisms.

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

Mitochondrial genome rearrangements in glomus species triggered by homologous recombination between distinct mtDNA haplotypes.

PubMed

Beaudet, Denis; Terrat, Yves; Halary, Sébastien; de la Providencia, Ivan Enrique; Hijri, Mohamed

2013-01-01

Comparative mitochondrial genomics of arbuscular mycorrhizal fungi (AMF) provide new avenues to overcome long-lasting obstacles that have hampered studies aimed at understanding the community structure, diversity, and evolution of these multinucleated and genetically polymorphic organisms.AMF mitochondrial (mt) genomes are homogeneous within isolates, and their intergenic regions harbor numerous mobile elements that have rapidly diverged, including homing endonuclease genes, small inverted repeats, and plasmid-related DNA polymerase genes (dpo), making them suitable targets for the development of reliable strain-specific markers. However, these elements may also lead to genome rearrangements through homologous recombination, although this has never previously been reported in this group of obligate symbiotic fungi. To investigate whether such rearrangements are present and caused by mobile elements in AMF, the mitochondrial genomes from two Glomeraceae members (i.e., Glomus cerebriforme and Glomus sp.) with substantial mtDNA synteny divergence,were sequenced and compared with available glomeromycotan mitochondrial genomes. We used an extensive nucleotide/protein similarity network-based approach to investigated podiversity in AMF as well as in other organisms for which sequences are publicly available. We provide strong evidence of dpo-induced inter-haplotype recombination, leading to a reshuffled mitochondrial genome in Glomus sp. These findings raise questions as to whether AMF single spore cultivations artificially underestimate mtDNA genetic diversity.We assessed potential dpo dispersal mechanisms in AMF and inferred a robust phylogenetic relationship with plant mitochondrial plasmids. Along with other indirect evidence, our analyses indicate that members of the Glomeromycota phylum are potential donors of mitochondrial plasmids to plants.

Mitochondrial Genome Rearrangements in Glomus Species Triggered by Homologous Recombination between Distinct mtDNA Haplotypes

PubMed Central

Beaudet, Denis; Terrat, Yves; Halary, Sébastien; de la Providencia, Ivan Enrique; Hijri, Mohamed

2013-01-01

Comparative mitochondrial genomics of arbuscular mycorrhizal fungi (AMF) provide new avenues to overcome long-lasting obstacles that have hampered studies aimed at understanding the community structure, diversity, and evolution of these multinucleated and genetically polymorphic organisms. AMF mitochondrial (mt) genomes are homogeneous within isolates, and their intergenic regions harbor numerous mobile elements that have rapidly diverged, including homing endonuclease genes, small inverted repeats, and plasmid-related DNA polymerase genes (dpo), making them suitable targets for the development of reliable strain-specific markers. However, these elements may also lead to genome rearrangements through homologous recombination, although this has never previously been reported in this group of obligate symbiotic fungi. To investigate whether such rearrangements are present and caused by mobile elements in AMF, the mitochondrial genomes from two Glomeraceae members (i.e., Glomus cerebriforme and Glomus sp.) with substantial mtDNA synteny divergence, were sequenced and compared with available glomeromycotan mitochondrial genomes. We used an extensive nucleotide/protein similarity network-based approach to investigate dpo diversity in AMF as well as in other organisms for which sequences are publicly available. We provide strong evidence of dpo-induced inter-haplotype recombination, leading to a reshuffled mitochondrial genome in Glomus sp. These findings raise questions as to whether AMF single spore cultivations artificially underestimate mtDNA genetic diversity. We assessed potential dpo dispersal mechanisms in AMF and inferred a robust phylogenetic relationship with plant mitochondrial plasmids. Along with other indirect evidence, our analyses indicate that members of the Glomeromycota phylum are potential donors of mitochondrial plasmids to plants. PMID:23925788

CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli

PubMed Central

Díez-Villaseñor, César; Guzmán, Noemí M.; Almendros, Cristóbal; García-Martínez, Jesús; Mojica, Francisco J.M.

2013-01-01

Prokaryotes immunize themselves against transmissible genetic elements by the integration (acquisition) in clustered regularly interspaced short palindromic repeats (CRISPR) loci of spacers homologous to invader nucleic acids, defined as protospacers. Following acquisition, mono-spacer CRISPR RNAs (termed crRNAs) guide CRISPR-associated (Cas) proteins to degrade (interference) protospacers flanked by an adjacent motif in extrachomosomal DNA. During acquisition, selection of spacer-precursors adjoining the protospacer motif and proper orientation of the integrated fragment with respect to the leader (sequence leading transcription of the flanking CRISPR array) grant efficient interference by at least some CRISPR-Cas systems. This adaptive stage of the CRISPR action is poorly characterized, mainly due to the lack of appropriate genetic strategies to address its study and, at least in Escherichia coli, the need of Cas overproduction for insertion detection. In this work, we describe the development and application in Escherichia coli strains of an interference-independent assay based on engineered selectable CRISPR-spacer integration reporter plasmids. By using this tool without the constraint of interference or cas overexpression, we confirmed fundamental aspects of this process such as the critical requirement of Cas1 and Cas2 and the identity of the CTT protospacer motif for the E. coli K12 system. In addition, we defined the CWT motif for a non-K12 CRISPR-Cas variant, and obtained data supporting the implication of the leader in spacer orientation, the preferred acquisition from plasmids harboring cas genes and the occurrence of a sequential cleavage at the insertion site by a ruler mechanism. PMID:23445770

Genomic diversity of necrotic enteritis-associated strains of Clostridium perfringens: a review.

PubMed

Lacey, Jake A; Johanesen, Priscilla A; Lyras, Dena; Moore, Robert J

2016-06-01

The investigation of genomic variation between Clostridium perfringens isolates from poultry has been an important tool to enhance our understanding of the genetic basis of strain pathogenicity and the epidemiology of virulent and avirulent strains within the context of necrotic enteritis (NE). The earliest studies used whole genome profiling techniques such as pulsed-field gel electrophoresis to differentiate isolates and determine their relative levels of relatedness. DNA sequencing has been used to investigate genetic variation in (a) individual genes, such as those encoding the alpha and NetB toxins; (b) panels of housekeeping genes for multi-locus sequence typing and (c) most recently whole genome sequencing to build a more complete picture of genomic differences between isolates. Conclusions drawn from these studies include: differential carriage of large conjugative plasmids accounts for a large proportion of inter-strain differences; plasmid-encoded genes are more highly conserved than chromosomal genes, perhaps indicating a relatively recent origin for the plasmids; isolates from NE-affected birds fall into three distinct sequence-based clades while non-pathogenic isolates from healthy birds tend to be more genomically diverse. Overall, the NE causing strains are closely related to C. perfringens isolates from other birds and other diseases whereas the non-pathogenic poultry strains are generally more remotely related to either the pathogenic strains or the strains from other birds. Genomic analysis has indicated that genes in addition to netB are associated with NE pathogenic isolates. Collectively, this work has resulted in a deeper understanding of the pathogenesis of this important poultry disease.

CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli.

PubMed

Díez-Villaseñor, César; Guzmán, Noemí M; Almendros, Cristóbal; García-Martínez, Jesús; Mojica, Francisco J M

2013-05-01

Prokaryotes immunize themselves against transmissible genetic elements by the integration (acquisition) in clustered regularly interspaced short palindromic repeats (CRISPR) loci of spacers homologous to invader nucleic acids, defined as protospacers. Following acquisition, mono-spacer CRISPR RNAs (termed crRNAs) guide CRISPR-associated (Cas) proteins to degrade (interference) protospacers flanked by an adjacent motif in extrachomosomal DNA. During acquisition, selection of spacer-precursors adjoining the protospacer motif and proper orientation of the integrated fragment with respect to the leader (sequence leading transcription of the flanking CRISPR array) grant efficient interference by at least some CRISPR-Cas systems. This adaptive stage of the CRISPR action is poorly characterized, mainly due to the lack of appropriate genetic strategies to address its study and, at least in Escherichia coli, the need of Cas overproduction for insertion detection. In this work, we describe the development and application in Escherichia coli strains of an interference-independent assay based on engineered selectable CRISPR-spacer integration reporter plasmids. By using this tool without the constraint of interference or cas overexpression, we confirmed fundamental aspects of this process such as the critical requirement of Cas1 and Cas2 and the identity of the CTT protospacer motif for the E. coli K12 system. In addition, we defined the CWT motif for a non-K12 CRISPR-Cas variant, and obtained data supporting the implication of the leader in spacer orientation, the preferred acquisition from plasmids harboring cas genes and the occurrence of a sequential cleavage at the insertion site by a ruler mechanism.

GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules

PubMed Central

Sarrion-Perdigones, Alejandro; Falconi, Erica Elvira; Zandalinas, Sara I.; Juárez, Paloma; Fernández-del-Carmen, Asun; Granell, Antonio; Orzaez, Diego

2011-01-01

Synthetic Biology requires efficient and versatile DNA assembly systems to facilitate the building of new genetic modules/pathways from basic DNA parts in a standardized way. Here we present GoldenBraid (GB), a standardized assembly system based on type IIS restriction enzymes that allows the indefinite growth of reusable gene modules made of standardized DNA pieces. The GB system consists of a set of four destination plasmids (pDGBs) designed to incorporate multipartite assemblies made of standard DNA parts and to combine them binarily to build increasingly complex multigene constructs. The relative position of type IIS restriction sites inside pDGB vectors introduces a double loop (“braid”) topology in the cloning strategy that allows the indefinite growth of composite parts through the succession of iterative assembling steps, while the overall simplicity of the system is maintained. We propose the use of GoldenBraid as an assembly standard for Plant Synthetic Biology. For this purpose we have GB-adapted a set of binary plasmids for A. tumefaciens-mediated plant transformation. Fast GB-engineering of several multigene T-DNAs, including two alternative modules made of five reusable devices each, and comprising a total of 19 basic parts are also described. PMID:21750718

GoldenBraid: an iterative cloning system for standardized assembly of reusable genetic modules.

PubMed

Sarrion-Perdigones, Alejandro; Falconi, Erica Elvira; Zandalinas, Sara I; Juárez, Paloma; Fernández-del-Carmen, Asun; Granell, Antonio; Orzaez, Diego

2011-01-01

Synthetic Biology requires efficient and versatile DNA assembly systems to facilitate the building of new genetic modules/pathways from basic DNA parts in a standardized way. Here we present GoldenBraid (GB), a standardized assembly system based on type IIS restriction enzymes that allows the indefinite growth of reusable gene modules made of standardized DNA pieces. The GB system consists of a set of four destination plasmids (pDGBs) designed to incorporate multipartite assemblies made of standard DNA parts and to combine them binarily to build increasingly complex multigene constructs. The relative position of type IIS restriction sites inside pDGB vectors introduces a double loop ("braid") topology in the cloning strategy that allows the indefinite growth of composite parts through the succession of iterative assembling steps, while the overall simplicity of the system is maintained. We propose the use of GoldenBraid as an assembly standard for Plant Synthetic Biology. For this purpose we have GB-adapted a set of binary plasmids for A. tumefaciens-mediated plant transformation. Fast GB-engineering of several multigene T-DNAs, including two alternative modules made of five reusable devices each, and comprising a total of 19 basic parts are also described.

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.

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

A replicative plasmid vector allows efficient complementation of pathogenic Leptospira strains.

PubMed

Pappas, Christopher J; Benaroudj, Nadia; Picardeau, Mathieu

2015-05-01

Leptospirosis, an emerging zoonotic disease, remains poorly understood because of a lack of genetic manipulation tools available for pathogenic leptospires. Current genetic manipulation techniques include insertion of DNA by random transposon mutagenesis and homologous recombination via suicide vectors. This study describes the construction of a shuttle vector, pMaORI, that replicates within saprophytic, intermediate, and pathogenic leptospires. The shuttle vector was constructed by the insertion of a 2.9-kb DNA segment including the parA, parB, and rep genes into pMAT, a plasmid that cannot replicate in Leptospira spp. and contains a backbone consisting of an aadA cassette, ori R6K, and oriT RK2/RP4. The inserted DNA segment was isolated from a 52-kb region within Leptospira mayottensis strain 200901116 that is not found in the closely related strain L. mayottensis 200901122. Because of the size of this region and the presence of bacteriophage-like proteins, it is possible that this region is a result of a phage-related genomic island. The stability of the pMaORI plasmid within pathogenic strains was tested by passaging cultures 10 times without selection and confirming the presence of pMaORI. Concordantly, we report the use of trans complementation in the pathogen Leptospira interrogans. Transformation of a pMaORI vector carrying a functional copy of the perR gene in a null mutant background restores the expression of PerR and susceptibility to hydrogen peroxide comparable to that of wild-type cells. In conclusion, we demonstrate the replication of a stable plasmid vector in a large panel of Leptospira strains, including pathogens. The shuttle vector described will expand our ability to perform genetic manipulation of Leptospira spp. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Characterization of Isolates of Salmonella enterica Serovar Stanley, a Serovar Endemic to Asia and Associated with Travel

PubMed Central

Le Hello, Simon; Bortolaia, Valeria; Pulsrikarn, Chaiwat; Nielsen, Eva Møller; Pornruangmong, Srirat; Chaichana, Phattharaporn; Svendsen, Christina Aaby; Weill, François-Xavier; Aarestrup, Frank M.

2012-01-01

Salmonella enterica serovar Stanley (S. Stanley) is a common serovar in Southeast Asia and was the second most common serovar implicated in human salmonellosis in Thailand in the years 2002 to 2007. In contrast, this serovar is relatively uncommon in Europe. The objective of this study was to characterize a collection of S. Stanley strains isolated from Thai (n = 62), Danish (n = 39), and French (n = 24) patients to gain a broader understanding of the genetic diversity, population dynamics, and susceptibility to antimicrobials. All isolates were characterized by pulsed-field gel electrophoresis and antimicrobial susceptibility testing. The molecular mechanisms of resistance to extended-spectrum cephalosporins and plasmid-mediated resistance to quinolones were characterized by PCR and sequencing. Plasmid profiling, replicon typing, and microarray analysis were used to characterize the genetic mechanisms of antimicrobial resistance in 10 extended-spectrum cephalosporinase-producing isolates. Considerable genetic diversity was observed among the isolates characterized with 91 unique XbaI pulsed-field gel electrophoresis (PFGE) patterns, including 17 distinct clusters consisting of two to seven indistinguishable isolates. We found some of the S. Stanley isolates isolated from patients in Europe were acquired during travel to Southeast Asia, including Thailand. The presence of multiple plasmid lineages carrying the extended-spectrum cephalosporinase-encoding blaCMY-2 gene in S. Stanley isolates from the central part of Thailand was confirmed. Our results emphasize that Thai authorities, as well as authorities in other countries lacking prudent use of antimicrobials, should improve the ongoing efforts to regulate antimicrobial use in agriculture and in clinical settings to limit the spread of multidrug-resistant Salmonella isolates and plasmids among humans and pigs in Thailand and abroad. PMID:22205822

Characterization of isolates of Salmonella enterica serovar Stanley, a serovar endemic to Asia and associated with travel.

PubMed

Hendriksen, Rene S; Le Hello, Simon; Bortolaia, Valeria; Pulsrikarn, Chaiwat; Nielsen, Eva Møller; Pornruangmong, Srirat; Chaichana, Phattharaporn; Svendsen, Christina Aaby; Weill, François-Xavier; Aarestrup, Frank M

2012-03-01

Salmonella enterica serovar Stanley (S. Stanley) is a common serovar in Southeast Asia and was the second most common serovar implicated in human salmonellosis in Thailand in the years 2002 to 2007. In contrast, this serovar is relatively uncommon in Europe. The objective of this study was to characterize a collection of S. Stanley strains isolated from Thai (n = 62), Danish (n = 39), and French (n = 24) patients to gain a broader understanding of the genetic diversity, population dynamics, and susceptibility to antimicrobials. All isolates were characterized by pulsed-field gel electrophoresis and antimicrobial susceptibility testing. The molecular mechanisms of resistance to extended-spectrum cephalosporins and plasmid-mediated resistance to quinolones were characterized by PCR and sequencing. Plasmid profiling, replicon typing, and microarray analysis were used to characterize the genetic mechanisms of antimicrobial resistance in 10 extended-spectrum cephalosporinase-producing isolates. Considerable genetic diversity was observed among the isolates characterized with 91 unique XbaI pulsed-field gel electrophoresis (PFGE) patterns, including 17 distinct clusters consisting of two to seven indistinguishable isolates. We found some of the S. Stanley isolates isolated from patients in Europe were acquired during travel to Southeast Asia, including Thailand. The presence of multiple plasmid lineages carrying the extended-spectrum cephalosporinase-encoding bla(CMY-2) gene in S. Stanley isolates from the central part of Thailand was confirmed. Our results emphasize that Thai authorities, as well as authorities in other countries lacking prudent use of antimicrobials, should improve the ongoing efforts to regulate antimicrobial use in agriculture and in clinical settings to limit the spread of multidrug-resistant Salmonella isolates and plasmids among humans and pigs in Thailand and abroad.

A Simple And Rapid Minicircle DNA Vector Manufacturing System

PubMed Central

Kay, Mark A; He, Cheng-Yi; Chen, Zhi-Ying

2010-01-01

Minicircle DNA vectors consisting of a circular expression cassette devoid of the bacterial plasmid DNA backbone provides several advantages including sustained transgene expression in quiescent cells/tissues. Their use has been limited by labor-intensive production. We report on a strategy for making multiple genetic modifications in E.coli to construct a producer strain that stably expresses a set of inducible minicircle-assembly enzymes, the øC31-integrase and I-SceI homing-endonuclease. This bacterial strain is capable of producing highly purified minicircle yields in the same time frame as routine plasmid DNA. It is now feasible for minicircle DNA vectors to replace routine plasmids in mammalian transgene expression studies. PMID:21102455

Genetic and Biochemical characterization of OXA-519, a novel OXA-48-like β-lactamase.

PubMed

Dabos, Laura; Bogaerts, Pierre; Bonnin, Remy A; Zavala, Agustin; Sacré, Pierre; Iorga, Bogdan I; Huang, Daniel T; Glupczynski, Youri; Naas, Thierry

2018-06-04

A multidrug-resistant K. pneumoniae 1210 isolate with reduced carbapenem susceptibility revealed the presence of a novel plasmid-encoded bla OXA-48-like gene, named bla OXA-519. The 60.7-kb plasmid (pOXA-519) was similar to the IncL-OXA-48 prototypical plasmid except for a ca. 2-kb deletion due to an IS 1R insertion. OXA-519 differed from OXA-48 by a Val120Leu substitution, which resulted in an overall reduced ß-lactam-hydrolysis profile, except for ertapenem and meropenem that was increased. Thus, detection of OXA-519-producers using biochemical tests monitoring imipenem-hydrolysis will be difficult. Copyright © 2018 American Society for Microbiology.

An abyssal mobilome: viruses, plasmids and vesicles from deep-sea hydrothermal vents.

PubMed

Lossouarn, Julien; Dupont, Samuel; Gorlas, Aurore; Mercier, Coraline; Bienvenu, Nadege; Marguet, Evelyne; Forterre, Patrick; Geslin, Claire

2015-12-01

Mobile genetic elements (MGEs) such as viruses, plasmids, vesicles, gene transfer agents (GTAs), transposons and transpovirions, which collectively represent the mobilome, interact with cellular organisms from all three domains of life, including those thriving in the most extreme environments. While efforts have been made to better understand deep-sea vent microbial ecology, our knowledge of the mobilome associated with prokaryotes inhabiting deep-sea hydrothermal vents remains limited. Here we focus on the abyssal mobilome by reviewing accumulating data on viruses, plasmids and vesicles associated with thermophilic and hyperthermophilic Bacteria and Archaea present in deep-sea hydrothermal vents. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Norwegian patients and retail chicken meat share cephalosporin-resistant Escherichia coli and IncK/blaCMY-2 resistance plasmids.

PubMed

Berg, E S; Wester, A L; Ahrenfeldt, J; Mo, S S; Slettemeås, J S; Steinbakk, M; Samuelsen, Ø; Grude, N; Simonsen, G S; Løhr, I H; Jørgensen, S B; Tofteland, S; Lund, O; Dahle, U R; Sunde, M

2017-06-01

In 2012 and 2014 the Norwegian monitoring programme for antimicrobial resistance in the veterinary and food production sectors (NORM-VET) showed that 124 of a total of 406 samples (31%) of Norwegian retail chicken meat were contaminated with extended-spectrum cephalosporin-resistant Escherichia coli. The aim of this study was to compare selected cephalosporin-resistant E. coli from humans and poultry to determine their genetic relatedness based on whole genome sequencing (WGS). Escherichia coli representing three prevalent cephalosporin-resistant multi-locus sequence types (STs) isolated from poultry (n=17) were selected from the NORM-VET strain collections. All strains carried an IncK plasmid with a bla CMY-2 gene. Clinical E. coli isolates (n=284) with AmpC-mediated resistance were collected at Norwegian microbiology laboratories from 2010 to 2014. PCR screening showed that 29 of the clinical isolates harboured both IncK and bla CMY-2 . All IncK/bla CMY-2 -positive isolates were analysed with WGS-based bioinformatics tools. Analysis of single nucleotide polymorphisms (SNP) in 2.5 Mbp of shared genome sequences showed close relationship, with fewer than 15 SNP differences between five clinical isolates from urinary tract infections (UTIs) and the ST38 isolates from poultry. Furthermore, all of the 29 clinical isolates harboured IncK/bla CMY-2 plasmid variants highly similar to the IncK/bla CMY-2 plasmid present in the poultry isolates. Our results provide support for the hypothesis that clonal transfer of cephalosporin-resistant E. coli from chicken meat to humans may occur, and may cause difficult-to-treat infections. Furthermore, these E. coli can be a source of AmpC-resistance plasmids for opportunistic pathogens in the human microbiota. Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

[Characteristics of natural strains of naphthalene-utilizing bacteria of the genus Pseudomonas].

PubMed

Levchuk, A A; Vasilenko, S L; Bulyga, I M; Titok, M A; Thomas, K M

2005-01-01

Sixty-three strains of bacteria capable of utilizing naphthalene as the sole source of carbon and energy were isolated from 137 samples of soil taken in different sites in Belarus. All isolated bacteria contained extrachromosomal genetic elements of 45 to 150 kb in length. It was found that bacteria of 31 strains contained the IncP-9 incompatibility group plasmids, bacteria of one strain carried a plasmid containing replicons IncP-9 and IncP-7, and bacteria of 31 strains contained unidentified plasmids. Primary identification showed that the hosts of plasmids of naphthalene biodegradation are fluorescent bacteria of the genus Pseudomonas (P. putida and P. aeruginosa; a total of 47 strains) and unidentified nonfluorescent microorganisms (a total of 16 strains). In addition to the ability to utilize naphthalene, some strains exhibited the ability to stimulate the growth and development of the root system of Secale cereale.

Genetic transformation of the plant pathogens Phytophthora capsici and Phytophthora parasitica.

PubMed Central

Bailey, A M; Mena, G L; Herrera-Estrella, L

1991-01-01

Phytophthora capsici and P.parasitica were transformed to hygromycin B resistance using plasmids pCM54 and pHL1, which contain the bacterial hygromycin B phosphotransferase gene (hph) fused to promoter elements of the Ustilago maydis heat shock hsp70 gene. Enzymes Driselase and Novozyme 234 were used to generate protoplasts which were then transformed following exposure to plasmid DNA and polyethylene glycol 6000. Transformation frequencies of over 500 transformants per micrograms of DNA per 1 x 10(6) protoplasts were obtained. Plasmid pCM54 appears to be transmitted in Phytophthora spp. as an extra-chromosomal element through replication, as shown by Southern blot hybridization and by the loss of plasmid methylation. In addition, transformed strains retained their capacity of infecting Serrano pepper seedlings and Mc. Intosh apple fruits, the host plants for P.capsici and P.parasitica, respectively. Images PMID:1651483

Reverse-engineering the genetic circuitry of a cancer cell with predicted intervention in chronic lymphocytic leukemia.

PubMed

Vallat, Laurent; Kemper, Corey A; Jung, Nicolas; Maumy-Bertrand, Myriam; Bertrand, Frédéric; Meyer, Nicolas; Pocheville, Arnaud; Fisher, John W; Gribben, John G; Bahram, Seiamak

2013-01-08

Cellular behavior is sustained by genetic programs that are progressively disrupted in pathological conditions--notably, cancer. High-throughput gene expression profiling has been used to infer statistical models describing these cellular programs, and development is now needed to guide orientated modulation of these systems. Here we develop a regression-based model to reverse-engineer a temporal genetic program, based on relevant patterns of gene expression after cell stimulation. This method integrates the temporal dimension of biological rewiring of genetic programs and enables the prediction of the effect of targeted gene disruption at the system level. We tested the performance accuracy of this model on synthetic data before reverse-engineering the response of primary cancer cells to a proliferative (protumorigenic) stimulation in a multistate leukemia biological model (i.e., chronic lymphocytic leukemia). To validate the ability of our method to predict the effects of gene modulation on the global program, we performed an intervention experiment on a targeted gene. Comparison of the predicted and observed gene expression changes demonstrates the possibility of predicting the effects of a perturbation in a gene regulatory network, a first step toward an orientated intervention in a cancer cell genetic program.

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.

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

Development of a PCR-Based Reverse Genetics System for an Attenuated Duck Tembusu Virus Strain

PubMed Central

Wu, Xiaogang; Shi, Ying; Yan, Dawei; Li, Xuesong; Yan, Pixi; Gao, Xuyuan; Zhang, Yuee; Yu, Lei; Ren, Chaochao; Li, Guoxin; Yan, Liping; Teng, Qiaoyang; Li, Zejun

2016-01-01

The infectious disease caused by the duck Tembusu virus (DTMUV) has resulted in massive economic losses to the Chinese duck industry in China since 2010. Research on the molecular basis of DTMUV pathogenicity has been hampered by the lack of a reliable reverse genetics system for this virus. Here we developed a PCR-based reverse genetics system with high fidelity for the attenuated DTMUV strain FX2010-180P. The rescued virus was characterized by using both indirect immunofluorescence assays (IFA) and whole genome sequencing. The rescued virus (rFX2010-180P) grew to similar titers as compared with the wild-type virus in DF-1 cells, and had similar replication and immunogenicity properties in ducks. To determine whether exogenous proteins could be expressed from DTMUV, both an internal ribosomal entry site (IRES) and the enhanced green fluorescent protein (eGFP) gene were introduced between the NS5 gene and the 3' non-coding sequence of FX2010-180P. A recombinant DTMUV expressing eGFP was rescued, but eGFP expression was unstable after 4 passages in DF-1 cells due to a deletion of 1,294 nucleotides. The establishment of a reliable reverse genetics system for FX2010-180P provides a foundation for future studies of DTMUV. PMID:27248497

Development of a PCR-Based Reverse Genetics System for an Attenuated Duck Tembusu Virus Strain.

PubMed

Wu, Xiaogang; Shi, Ying; Yan, Dawei; Li, Xuesong; Yan, Pixi; Gao, Xuyuan; Zhang, Yuee; Yu, Lei; Ren, Chaochao; Li, Guoxin; Yan, Liping; Teng, Qiaoyang; Li, Zejun

2016-01-01

The infectious disease caused by the duck Tembusu virus (DTMUV) has resulted in massive economic losses to the Chinese duck industry in China since 2010. Research on the molecular basis of DTMUV pathogenicity has been hampered by the lack of a reliable reverse genetics system for this virus. Here we developed a PCR-based reverse genetics system with high fidelity for the attenuated DTMUV strain FX2010-180P. The rescued virus was characterized by using both indirect immunofluorescence assays (IFA) and whole genome sequencing. The rescued virus (rFX2010-180P) grew to similar titers as compared with the wild-type virus in DF-1 cells, and had similar replication and immunogenicity properties in ducks. To determine whether exogenous proteins could be expressed from DTMUV, both an internal ribosomal entry site (IRES) and the enhanced green fluorescent protein (eGFP) gene were introduced between the NS5 gene and the 3' non-coding sequence of FX2010-180P. A recombinant DTMUV expressing eGFP was rescued, but eGFP expression was unstable after 4 passages in DF-1 cells due to a deletion of 1,294 nucleotides. The establishment of a reliable reverse genetics system for FX2010-180P provides a foundation for future studies of DTMUV.

Draft Genome Sequences of Three European Laboratory Derivatives from Enterohemorrhagic Escherichia coli O157:H7 Strain EDL933, Including Two Plasmids

PubMed Central

Fellner, Lea; Huptas, Christopher; Simon, Svenja; Mühlig, Anna; Neuhaus, Klaus

2016-01-01

Escherichia coli O157:H7 EDL933, isolated in 1982 in the United States, was the first enterohemorrhagic E. coli (EHEC) strain sequenced. Unfortunately, European labs can no longer receive the original strain. We checked three European EDL933 derivatives and found major genetic deviations (deletions, inversions) in two strains. All EDL933 strains contain the cryptic EHEC-plasmid, not reported before. PMID:27056239

An Enterobacter Plasmid as a New Genetic Background for the Transposon Tn1331

DTIC Science & Technology

2011-11-25

determined to be 99% similar to E. cloacae by both 16S rDNA and Phoenix analysis and was designated Enterobacter sp W001. Enterobacter sp W001 was...adolescents. JAMA. 2002;287(23):3096–3102. 9. Foster TJ. Plasmid- determined resistance to antimicrobial drugs and toxic metal ions in bacteria. Microbiol...mediated type II dihydrofolate reductase gene among trimethoprim -resistant urinary pathogens in Greek hospitals. J Antimicrob Chemother. 1992;29

Nucleotide excision repair pathway assessment in DNA exposed to low-intensity red and infrared lasers.

PubMed

Fonseca, A S; Campos, V M A; Magalhães, L A G; Paoli, F

2015-10-01

Low-intensity lasers are used for prevention and management of oral mucositis induced by anticancer therapy, but the effectiveness of treatment depends on the genetic characteristics of affected cells. This study evaluated the survival and induction of filamentation of Escherichia coli cells deficient in the nucleotide excision repair pathway, and the action of T4endonuclease V on plasmid DNA exposed to low-intensity red and near-infrared laser light. Cultures of wild-type (strain AB1157) E. coli and strain AB1886 (deficient in uvrA protein) were exposed to red (660 nm) and infrared (808 nm) lasers at various fluences, powers and emission modes to study bacterial survival and filamentation. Also, plasmid DNA was exposed to laser light to study DNA lesions produced in vitro by T4endonuclease V. Low-intensity lasers:i) had no effect on survival of wild-type E. coli but decreased the survival of uvrA protein-deficient cells,ii) induced bacterial filamentation, iii) did not alter the electrophoretic profile of plasmids in agarose gels, andiv) did not alter the electrophoretic profile of plasmids incubated with T4 endonuclease V. These results increase our understanding of the effects of laser light on cells with various genetic characteristics, such as xeroderma pigmentosum cells deficient in nucleotide excision pathway activity in patients with mucositis treated by low-intensity lasers.

Nucleotide excision repair pathway assessment in DNA exposed to low-intensity red and infrared lasers

PubMed Central

Fonseca, A.S.; Campos, V.M.A.; Magalhães, L.A.G.; Paoli, F.

2015-01-01

Low-intensity lasers are used for prevention and management of oral mucositis induced by anticancer therapy, but the effectiveness of treatment depends on the genetic characteristics of affected cells. This study evaluated the survival and induction of filamentation of Escherichia coli cells deficient in the nucleotide excision repair pathway, and the action of T4endonuclease V on plasmid DNA exposed to low-intensity red and near-infrared laser light. Cultures of wild-type (strain AB1157) E. coli and strain AB1886 (deficient in uvrA protein) were exposed to red (660 nm) and infrared (808 nm) lasers at various fluences, powers and emission modes to study bacterial survival and filamentation. Also, plasmid DNA was exposed to laser light to study DNA lesions produced in vitro by T4endonuclease V. Low-intensity lasers:i) had no effect on survival of wild-type E. coli but decreased the survival of uvrA protein-deficient cells,ii) induced bacterial filamentation, iii) did not alter the electrophoretic profile of plasmids in agarose gels, andiv) did not alter the electrophoretic profile of plasmids incubated with T4 endonuclease V. These results increase our understanding of the effects of laser light on cells with various genetic characteristics, such as xeroderma pigmentosum cells deficient in nucleotide excision pathway activity in patients with mucositis treated by low-intensity lasers. PMID:26445337

Genetic diversity and antimicrobial resistance of Flavobacterium psychrophilum isolated from cultured rainbow trout, Onchorynchus mykiss (Walbaum), in Spain.

PubMed

Del Cerro, A; Márquez, I; Prieto, J M

2010-04-01

Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease (CWD) and rainbow trout fry syndrome (RTFS) in salmonids. These diseases are a major problem in the aquaculture industry in Spain, and a better understanding of the epidemiology of F. psychrophilum isolates is necessary to improve management strategies. In this study, to investigate genetic variability of this bacterium, pulsed-field gel electrophoresis after DNA digestion with endonuclease StuI, plasmid profiling analysis and antimicrobial susceptibility testing were undertaken with 25 isolates of F. psychrophilum from Spain. These isolates were classified into 17 patterns by PFGE analysis, which were grouped into four clusters and seven independent branches. Twenty isolates (80%) possessed plasmids of 3.5 kb (n = 13) or 5.5 kb (n = 7). No plasmids were associated with antibiotic resistance to oxytetracycline (OTC) or florfenicol (FLO). Twenty isolates (80%) had minimum inhibitory concentrations (MICs) to OTC of between 2.4 and 9.7 microg mL(-1), and all isolates were susceptible to FLO. A relationship between the origin of the isolates and PFGE genotypes was found. Plasmid profile typing correlated with PFGE profile typing, whereas no correlation was found between antimicrobial susceptibility testing and PFGE profiles. These results suggest that the population of F. psychrophilum with pathogenic potential in northern Spain is quite heterogeneous.

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

Genomic epidemiology of global Klebsiella pneumoniae carbapenemase (KPC)-producing Escherichia coli.

PubMed

Stoesser, N; Sheppard, A E; Peirano, G; Anson, L W; Pankhurst, L; Sebra, R; Phan, H T T; Kasarskis, A; Mathers, A J; Peto, T E A; Bradford, P; Motyl, M R; Walker, A S; Crook, D W; Pitout, J D

2017-07-19

The dissemination of carbapenem resistance in Escherichia coli has major implications for the management of common infections. bla KPC , encoding a transmissible carbapenemase (KPC), has historically largely been associated with Klebsiella pneumoniae, a predominant plasmid (pKpQIL), and a specific transposable element (Tn4401, ~10 kb). Here we characterize the genetic features of bla KPC emergence in global E. coli, 2008-2013, using both long- and short-read whole-genome sequencing. Amongst 43/45 successfully sequenced bla KPC -E. coli strains, we identified substantial strain diversity (n = 21 sequence types, 18% of annotated genes in the core genome); substantial plasmid diversity (≥9 replicon types); and substantial bla KPC -associated, mobile genetic element (MGE) diversity (50% not within complete Tn4401 elements). We also found evidence of inter-species, regional and international plasmid spread. In several cases bla KPC was found on high copy number, small Col-like plasmids, previously associated with horizontal transmission of resistance genes in the absence of antimicrobial selection pressures. E. coli is a common human pathogen, but also a commensal in multiple environmental and animal reservoirs, and easily transmissible. The association of bla KPC with a range of MGEs previously linked to the successful spread of widely endemic resistance mechanisms (e.g. bla TEM , bla CTX-M ) suggests that it may become similarly prevalent.

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.

Engineering Complex Microbial Phenotypes with Continuous Genetic Integration and Plasmid Based Multi-Gene Library

DTIC Science & Technology

2010-01-01

genes from strains that have desirable traits. Here, we aim to enlarge the E. coli genome using Lactobacillus plantarum genes to build cells tolerant to...EtOH and BT. L. plantarum is an organism with established high tolerance to alcohols and solvents more broadly. Objective 2: Build a stress...heterologous (here: L. plantarum ; abbreviated as L. pl) DNA into the E. coli chromosome while selecting for insertions that enhance ethanol tolerance (which

Horizontal gene transfer of a ColV plasmid has resulted in a dominant avian clonal type of Salmonella enterica serovar Kentucky.

PubMed

Johnson, Timothy J; Thorsness, Jessica L; Anderson, Cole P; Lynne, Aaron M; Foley, Steven L; Han, Jing; Fricke, W Florian; McDermott, Patrick F; White, David G; Khatri, Mahesh; Stell, Adam L; Flores, Cristian; Singer, Randall S

2010-12-22

Salmonella enterica continues to be a significant cause of foodborne gastrointestinal illness in humans. A wide variety of Salmonella serovars have been isolated from production birds and from retail poultry meat. Recently, though, S. enterica subsp. enterica serovar Kentucky has emerged as one of the prominent Salmonella serovars isolated from broiler chickens. Recent work suggests that its emergence apparently coincides with its acquisition of a ColV virulence plasmid. In the present study, we examined 902 Salmonella isolates belonging to 59 different serovars for the presence of this plasmid. Of the serovars examined, the ColV plasmid was found only among isolates belonging to the serovars Kentucky (72.9%), Typhimurium (15.0%) and Heidelberg (1.7%). We demonstrated that a single PFGE clonal type of S. Kentucky harbors this plasmid, and acquisition of this plasmid by S. Kentucky significantly increased its ability to colonize the chicken cecum and cause extraintestinal disease. Comparison of the completed sequences of three ColV plasmids from S. Kentucky isolated from different geographical locales, timepoints and sources revealed a nearly identical genetic structure with few single nucleotide changes or insertions/deletions. Overall, it appears that the ColV plasmid was recently acquired by a single clonal type S. Kentucky and confers to its host enhanced colonization and fitness capabilities. Thus, the potential for horizontal gene transfer of virulence and fitness factors to Salmonella from other enteric bacteria exists in poultry, representing a potential human health hazard.

Plasmid-cured Chlamydia caviae activates TLR2-dependent signaling and retains virulence in the guinea pig model of genital tract infection.

PubMed

Frazer, Lauren C; Darville, Toni; Chandra-Kuntal, Kumar; Andrews, Charles W; Zurenski, Matthew; Mintus, Margaret; AbdelRahman, Yasser M; Belland, Robert J; Ingalls, Robin R; O'Connell, Catherine M

2012-01-01

Loss of the conserved "cryptic" plasmid from C. trachomatis and C. muridarum is pleiotropic, resulting in reduced innate inflammatory activation via TLR2, glycogen accumulation and infectivity. The more genetically distant C. caviae GPIC is a natural pathogen of guinea pigs and induces upper genital tract pathology when inoculated intravaginally, modeling human disease. To examine the contribution of pCpGP1 to C. caviae pathogenesis, a cured derivative of GPIC, strain CC13, was derived and evaluated in vitro and in vivo. Transcriptional profiling of CC13 revealed only partial conservation of previously identified plasmid-responsive chromosomal loci (PRCL) in C. caviae. However, 2-deoxyglucose (2DG) treatment of GPIC and CC13 resulted in reduced transcription of all identified PRCL, including glgA, indicating the presence of a plasmid-independent glucose response in this species. In contrast to plasmid-cured C. muridarum and C. trachomatis, plasmid-cured C. caviae strain CC13 signaled via TLR2 in vitro and elicited cytokine production in vivo similar to wild-type C. caviae. Furthermore, inflammatory pathology induced by infection of guinea pigs with CC13 was similar to that induced by GPIC, although we observed more rapid resolution of CC13 infection in estrogen-treated guinea pigs. These data indicate that either the plasmid is not involved in expression or regulation of virulence in C. caviae or that redundant effectors prevent these phenotypic changes from being observed in C. caviae plasmid-cured strains.

Plasmid mediated antimicrobial resistance in Ontario isolates of Actinobacillus (Haemophilus) pleuropneumoniae.

PubMed Central

Gilbride, K A; Rosendal, S; Brunton, J L

1989-01-01

The genetic basis of antimicrobial resistance in Ontario isolates of Actinobacillus (Haemophilus) pleuropneumoniae was studied. Two Ontario isolates of A. pleuropneumoniae were found to be resistant to sulfonamides (Su), streptomycin (Sm) and ampicillin (Amp). Resistance to Su and Sm was specified by a 2.3 megadalton (Mdal) plasmid which appeared to be identical to pVM104, which has been described in isolates of A. pleuropneumoniae from South Dakota. Southern hybridization showed that the 2.3 Mdal Su Sm plasmid was highly related to those Hinc II fragments of RSF1010 known to carry the Su Sm genes, but was unrelated to the remainder of this Salmonella resistance plasmid. Resistance to Su and Amp was specified by a 3.5 Mdal plasmid and appeared identical to pVM105 previously reported. The beta-lactamase enzyme had an isoelectric point of approximately 9.0. Southern hybridization showed no relationship to the TEM beta-lactamase. A third isolate of A. pleuropneumoniae was found to be resistant to chloramphenicol (Cm), Su and Sm by virtue of a 3.0 Mdal plasmid which specified a chloramphenicol acetyl transferase. We conclude that resistance to Su, Sm, Amp and Cm is mediated by small plasmids in A. pleuropneumoniae. Although the Su and Sm resistance determinants are highly related to those found in Enterobacteriaceae, the plasmids themselves and the beta-lactamase determinant are different. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:2914226

Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice.

PubMed

Nakagawa, Yoshiko; Sakuma, Tetsushi; Nishimichi, Norihisa; Yokosaki, Yasuyuki; Yanaka, Noriyuki; Takeo, Toru; Nakagata, Naomi; Yamamoto, Takashi

2016-08-15

Current advances in producing genetically modified mice using genome-editing technologies have indicated the need for improvement of limiting factors including zygote collection for microinjection and their cryopreservation. Recently, we developed a novel superovulation technique using inhibin antiserum and equine chorionic gonadotropin to promote follicle growth. This method enabled the increased production of fertilized oocytes via in vitro fertilization compared with the conventional superovulation method. Here, we verify that the ultra-superovulation technique can be used for the efficient generation of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated knockout mice by microinjection of plasmid vector or ribonucleoprotein into zygotes. We also investigated whether single-amino-acid-substituted mice and conditional knockout mice could be generated. Founder mice bearing base substitutions were generated more efficiently by co-microinjection of Cas9 protein, a guide RNA and single-stranded oligodeoxynucleotide (ssODN) than by plasmid microinjection with ssODN. The conditional allele was successfully introduced by the one-step insertion of an ssODN designed to carry an exon flanked by two loxP sequences and homology arms using a double-cut CRISPR-Cas9 strategy. Our study presents a useful method for the CRISPR-Cas9-based generation of genetically modified mice from the viewpoints of animal welfare and work efficiency. © 2016. Published by The Company of Biologists Ltd.

tCRISPRi: tunable and reversible, one-step control of gene expression

NASA Astrophysics Data System (ADS)

Li, Xin-Tian; Jun, Yonggun; Erickstad, Michael J.; Brown, Steven D.; Parks, Adam; Court, Donald L.; Jun, Suckjoon

2016-12-01

The ability to control the level of gene expression is a major quest in biology. A widely used approach employs deletion of a nonessential gene of interest (knockout), or multi-step recombineering to move a gene of interest under a repressible promoter (knockdown). However, these genetic methods are laborious, and limited for quantitative study. Here, we report a tunable CRISPR-cas system, “tCRISPRi”, for precise and continuous titration of gene expression by more than 30-fold. Our tCRISPRi system employs various previous advancements into a single strain: (1) We constructed a new strain containing a tunable arabinose operon promoter PBAD to quantitatively control the expression of CRISPR-(d)Cas protein over two orders of magnitude in a plasmid-free system. (2) tCRISPRi is reversible, and gene expression is repressed under knockdown conditions. (3) tCRISPRi shows significantly less than 10% leaky expression. (4) Most important from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step of oligo recombineering. Our results show that tCRISPRi, in combination with recombineering, provides a simple and easy-to-implement tool for gene expression control, and is ideally suited for construction of both individual strains and high-throughput tunable knockdown libraries.

An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells.

PubMed

Guo, Jianying; Ma, Dacheng; Huang, Rujin; Ming, Jia; Ye, Min; Kee, Kehkooi; Xie, Zhen; Na, Jie

2017-05-01

Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naïve pluripotent gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass (ICM) of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.

Highly Efficient Electroporation-mediated Transformation into Edible Mushroom Flammulina velutipes

PubMed Central

Kim, Jong Kun; Park, Young Jin; Kong, Won Sik

2010-01-01

In this study, we developed an efficient electroporation-mediated transformation system featuring Flammulina velutipes. The flammutoxin (ftx) gene of F. velutipes was isolated by reverse transcription-PCR. pFTXHg plasmid was constructed using the partial ftx gene (410 bp) along with the hygromycin B phosphotransferase gene (hygB) downstream of the glyceraldehydes-3-phosphate dehydrogenase (gpd) promoter. The plasmid was transformed into protoplasts of monokaryotic strain 4019-20 of F. velutipes by electroporation. High transformation efficiency was obtained with an electric-pulse of 1.25 kV/cm by using 177 transformants/µg of DNA in 1 × 107 protoplasts. PCR and Southern blot hybridization indicated that a single copy of the plasmid DNA was inserted at different locations in the F. velutipes genome by non-homologous recombination. Therefore, this transformation system could be used as a useful tool for gene function analysis of F. velutipes. PMID:23956676

Highly Efficient Electroporation-mediated Transformation into Edible Mushroom Flammulina velutipes.

PubMed

Kim, Jong Kun; Park, Young Jin; Kong, Won Sik; Kang, Hee Wan

2010-12-01

In this study, we developed an efficient electroporation-mediated transformation system featuring Flammulina velutipes. The flammutoxin (ftx) gene of F. velutipes was isolated by reverse transcription-PCR. pFTXHg plasmid was constructed using the partial ftx gene (410 bp) along with the hygromycin B phosphotransferase gene (hygB) downstream of the glyceraldehydes-3-phosphate dehydrogenase (gpd) promoter. The plasmid was transformed into protoplasts of monokaryotic strain 4019-20 of F. velutipes by electroporation. High transformation efficiency was obtained with an electric-pulse of 1.25 kV/cm by using 177 transformants/µg of DNA in 1 × 10(7) protoplasts. PCR and Southern blot hybridization indicated that a single copy of the plasmid DNA was inserted at different locations in the F. velutipes genome by non-homologous recombination. Therefore, this transformation system could be used as a useful tool for gene function analysis of F. velutipes.

Engineering bacteria to solve the Burnt Pancake Problem

PubMed Central

Haynes, Karmella A; Broderick, Marian L; Brown, Adam D; Butner, Trevor L; Dickson, James O; Harden, W Lance; Heard, Lane H; Jessen, Eric L; Malloy, Kelly J; Ogden, Brad J; Rosemond, Sabriya; Simpson, Samantha; Zwack, Erin; Campbell, A Malcolm; Eckdahl, Todd T; Heyer, Laurie J; Poet, Jeffrey L

2008-01-01

Background We investigated the possibility of executing DNA-based computation in living cells by engineering Escherichia coli to address a classic mathematical puzzle called the Burnt Pancake Problem (BPP). The BPP is solved by sorting a stack of distinct objects (pancakes) into proper order and orientation using the minimum number of manipulations. Each manipulation reverses the order and orientation of one or more adjacent objects in the stack. We have designed a system that uses site-specific DNA recombination to mediate inversions of genetic elements that represent pancakes within plasmid DNA. Results Inversions (or "flips") of the DNA fragment pancakes are driven by the Salmonella typhimurium Hin/hix DNA recombinase system that we reconstituted as a collection of modular genetic elements for use in E. coli. Our system sorts DNA segments by inversions to produce different permutations of a promoter and a tetracycline resistance coding region; E. coli cells become antibiotic resistant when the segments are properly sorted. Hin recombinase can mediate all possible inversion operations on adjacent flippable DNA fragments. Mathematical modeling predicts that the system reaches equilibrium after very few flips, where equal numbers of permutations are randomly sorted and unsorted. Semiquantitative PCR analysis of in vivo flipping suggests that inversion products accumulate on a time scale of hours or days rather than minutes. Conclusion The Hin/hix system is a proof-of-concept demonstration of in vivo computation with the potential to be scaled up to accommodate larger and more challenging problems. Hin/hix may provide a flexible new tool for manipulating transgenic DNA in vivo. PMID:18492232

A reverse engineering algorithm for neural networks, applied to the subthalamopallidal network of basal ganglia.

PubMed

Floares, Alexandru George

2008-01-01

Modeling neural networks with ordinary differential equations systems is a sensible approach, but also very difficult. This paper describes a new algorithm based on linear genetic programming which can be used to reverse engineer neural networks. The RODES algorithm automatically discovers the structure of the network, including neural connections, their signs and strengths, estimates its parameters, and can even be used to identify the biophysical mechanisms involved. The algorithm is tested on simulated time series data, generated using a realistic model of the subthalamopallidal network of basal ganglia. The resulting ODE system is highly accurate, and results are obtained in a matter of minutes. This is because the problem of reverse engineering a system of coupled differential equations is reduced to one of reverse engineering individual algebraic equations. The algorithm allows the incorporation of common domain knowledge to restrict the solution space. To our knowledge, this is the first time a realistic reverse engineering algorithm based on linear genetic programming has been applied to neural networks.

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

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.

Complete Sequencing of pNDM-HK Encoding NDM-1 Carbapenemase from a Multidrug-Resistant Escherichia coli Strain Isolated in Hong Kong

PubMed Central

Ho, Pak Leung; Lo, Wai U.; Yeung, Man Kiu; Lin, Chi Ho; Chow, Kin Hung; Ang, Irene; Tong, Amy Hin Yan; Bao, Jessie Yun-Juan; Lok, Si; Lo, Janice Yee Chi

2011-01-01

Background The emergence of plasmid-mediated carbapenemases, such as NDM-1 in Enterobacteriaceae is a major public health issue. Since they mediate resistance to virtually all β-lactam antibiotics and there is often co-resistance to other antibiotic classes, the therapeutic options for infections caused by these organisms are very limited. Methodology We characterized the first NDM-1 producing E. coli isolate recovered in Hong Kong. The plasmid encoding the metallo-β-lactamase gene was sequenced. Principal Findings The plasmid, pNDM-HK readily transferred to E. coli J53 at high frequencies. It belongs to the broad host range IncL/M incompatibility group and is 88803 bp in size. Sequence alignment showed that pNDM-HK has a 55 kb backbone which shared 97% homology with pEL60 originating from the plant pathogen, Erwina amylovora in Lebanon and a 28.9 kb variable region. The plasmid backbone includes the mucAB genes mediating ultraviolet light resistance. The 28.9 kb region has a composite transposon-like structure which includes intact or truncated genes associated with resistance to β-lactams (bla TEM-1, bla NDM-1, Δbla DHA-1), aminoglycosides (aacC2, armA), sulphonamides (sul1) and macrolides (mel, mph2). It also harbors the following mobile elements: IS26, ISCR1, tnpU, tnpAcp2, tnpD, ΔtnpATn1 and insL. Certain blocks within the 28.9 kb variable region had homology with the corresponding sequences in the widely disseminated plasmids, pCTX-M3, pMUR050 and pKP048 originating from bacteria in Poland in 1996, in Spain in 2002 and in China in 2006, respectively. Significance The genetic support of NDM-1 gene suggests that it has evolved through complex pathways. The association with broad host range plasmid and multiple mobile genetic elements explain its observed horizontal mobility in multiple bacterial taxa. PMID:21445317

Immune Efficacy of a Genetically Engineered Vaccine against Lymphocystis Disease Virus: Analysis of Different Immunization Strategies

PubMed Central

Zheng, Fengrong; Sun, Xiuqin; Wu, Xing'an; Liu, Hongzhan; Li, Jiye; Wu, Suqi; Zhang, Jinxing

2011-01-01

Here, we report the construction of a vaccine against lymphocystis disease virus (LCDV) using nucleic acid vaccination technology. A fragment of the major capsid protein encoding gene from an LCDV isolated from China (LCDV-cn) was cloned into an eukaryotic expression vector pEGFP-N2, yielding a recombinant plasmid pEGFP-N2-LCDV-cn0.6 kb. This plasmid was immediately expressed after liposomal transfer into the Japanese flounder embryo cell line. The recombinant plasmid was inoculated into Japanese flounder via two routes (intramuscular injection and hypodermic injection) at three doses (0.1, 5, and 15 μg), and then T-lymphopoiesis in different tissues and antibodies raised against LCDV were evaluated. The results indicated that this recombinant plasmid induced unique humoral or cell-mediated immune responses depending on the inoculation route and conferred immune protection. Furthermore, the humoral immune responses and protective effects were significantly increased at higher vaccine doses via the two injection routes. Plasmid pEGFP-N2-LCDV0.6 kb is therefore a promising vaccine candidate against LCDV in Japanese flounder. PMID:21789044

EcoliWiki: a wiki-based community resource for Escherichia coli

PubMed Central

McIntosh, Brenley K.; Renfro, Daniel P.; Knapp, Gwendowlyn S.; Lairikyengbam, Chanchala R.; Liles, Nathan M.; Niu, Lili; Supak, Amanda M.; Venkatraman, Anand; Zweifel, Adrienne E.; Siegele, Deborah A.; Hu, James C.

2012-01-01

EcoliWiki is the community annotation component of the PortEco (http://porteco.org; formerly EcoliHub) project, an online data resource that integrates information on laboratory strains of Escherichia coli, its phages, plasmids and mobile genetic elements. As one of the early adopters of the wiki approach to model organism databases, EcoliWiki was designed to not only facilitate community-driven sharing of biological knowledge about E. coli as a model organism, but also to be interoperable with other data resources. EcoliWiki content currently covers genes from five laboratory E. coli strains, 21 bacteriophage genomes, F plasmid and eight transposons. EcoliWiki integrates the Mediawiki wiki platform with other open-source software tools and in-house software development to extend how wikis can be used for model organism databases. EcoliWiki can be accessed online at http://ecoliwiki.net. PMID:22064863

Plasmid profiling of bacterial isolates from confined environments

NASA Astrophysics Data System (ADS)

van Houdt, Rob; Provoost, Ann; Coninx, Ilse; Leys, Natalie; Mergeay, Max

Plasmid profiling of bacterial isolates from confined environments R. Van Houdt, I. Coninx, A. Provoost, N. Leys, and M. Mergeay Expertise group for Molecular and Cellular Biology, Institute for Environment, Health and Safety, Belgian Nuclear Research Centre (SCK•CEN), Boeretang 200, B-2400 Mol, Belgium. Human exploration of extreme and isolated hostile environments such as space requires special confined small volume habitats to protect and house the crew. However, human confinement in such small volume habitats has restrictions on waste disposal and personal hygiene and inevitably generates a particular community of microorganisms within the habitat. These microorganisms are mainly originating from the crew (skin, mucous membranes, upper respiratory tract, mouth, and gastrointestinal tract) but also include the residing environmental microorganisms. Earth-based confined habitats such as the Antarctic Research Station Concordia are used as test beds for long-duration spaceflights to study the physiologic and psychological adaptation to isolated environments. The dynamics of the environmental microbial population in such a test bed could render additional insights in assessing the potential health risks in long-duration space missions. Not only total bacterial contamination levels are important, but it is essential to identify also the predominant microbial taxa and their mobile genetic elements (MGE). These MGEs could be exchanged between bacteria by horizontal gene transfer and may alter the pathogenic potential since they often carry antibiotic resistance or more in general adaptation-enhancing traits. In this study several bacterial strains isolated in the Concordia research station were examined for their plasmid content. An optimized protocol for extraction of large plasmids showed the present of at least one plasmid in 50% of the strains. For all strains the minimal inhibitory concentration of a range of antibiotics was determined indicating resistance to different classes of antibiotics including aminoglycosides, penicillins, macrolides and chloramphenicol. Whether these antibiotic resistance determinants are plasmid-bound and whether these traits can be transferred to other bacteria is under investigation.

Occurrence and molecular characteristics of ESBL/AmpC-producing Escherichia coli in faecal samples from horses in an equine clinic.

PubMed

Apostolakos, Ilias; Franz, Eelco; van Hoek, Angela H A M; Florijn, Alice; Veenman, Christiaan; Sloet-van Oldruitenborgh-Oosterbaan, Marianne M; Dierikx, Cindy; van Duijkeren, Engeline

2017-07-01

To investigate the occurrence and characteristics of ESBL/AmpC-producing Escherichia coli in faecal samples from horses at one equine clinic in the Netherlands. A total of 91 horses, including residents and patients, were sampled. ESBL/AmpC-producing E. coli were identified by a combination disc diffusion test. Phylogenetic groups and MLST were determined. ESBL/AmpC genes were analysed using PCR and sequencing. Plasmids were characterized by transformation and PCR-based replicon typing. Subtyping of plasmids was done by plasmid MLST. At least one E. coli isolate with a confirmed ESBL/AmpC gene was found in samples from 76 horses (84%). Although phylogenetic group B1 E. coli bla CTX-M-1 predominated, a diverse E. coli population was found, indicating that clonal nosocomial spread was not the only reason for the high occurrence found. MLST analysis revealed the presence of 47 E. coli STs, organized in four clusters of genetically related strains. ST10, ST641, ST1079 and ST1250 were most commonly found. With regard to the genes, bla CTX-M-1 was most prevalent ( n  =   91), followed by bla CTX-M-2 ( n  =   26). The most frequently found plasmid type was IncHI1, but plasmids belonging to the IncF, IncI1 and IncN groups were also identified. A high occurrence of ESBL-producing E. coli in faecal samples was found among horses in an equine clinic and the variety of STs, ESBL genes and plasmid types suggests nosocomial transmission. ESBL E. coli can cause difficult-to-treat infections in horses and prudent use of antimicrobials is warranted. A further assessment of the risks of transmission to persons in close contact with horses, such as caretakers or veterinarians, is crucial. © 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.

Analysis of antimicrobial resistance genes detected in multidrug-resistant Salmonella enterica serovar Typhimurium isolated from food animals.

PubMed

Glenn, LaShanda M; Lindsey, Rebecca L; Frank, Joseph F; Meinersmann, Richard J; Englen, Mark D; Fedorka-Cray, Paula J; Frye, Jonathan G

2011-09-01

Multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium is the most prevalent penta-resistant serovar isolated from animals by the U.S. National Antimicrobial Resistance Monitoring System. Penta-resistant isolates are often resistant to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline. To investigate MDR in Salmonella Typhimurium (including variant 5-), one isolate each from cattle, poultry, and swine with at least the ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline phenotype were selected for each year from 1997 to 2007 (n = 33) for microarray analysis of antimicrobial resistance, incompatibility IncA/C, and HI1 plasmid genes. Cluster analysis based on these data separated 31 of the isolates into two groups A and B (15 and 16 isolates, respectively). Isolates in group A were phage type DT104 or U302 and were mostly swine isolates (7/15). Genes detected included intI1, bla(PSE-1), floR, aadA, sulI, tet(G), and tetR, which are often found in Salmonella Genomic Island I. Isolates in group B had numerous IncA/C plasmid genes detected and were mostly cattle isolates (9/16). Genes detected included bla(CMY-2), floR, aac(3), aadA, aphA1, strA, strB, sulI, sulII, dfrA, dhf, tet(A)(B)(C)(D), and tetR, which are often found on MDR-AmpC IncA/C plasmids. The IncA/C replicon was also detected in all group B isolates. The two remaining isolates did not cluster with any others and both had many HI1 plasmid genes detected. Linkage disequilibrium analysis detected significant associations between plasmid replicon type, phage type, and animal source. These data suggest that MDR in Salmonella Typhimurium is associated with DT104/Salmonella Genomic Island I or IncA/C MDR-AmpC encoding plasmids and these genetic elements have persisted throughout the study period.

Map-based cloning of a gene controlling Omega-3 fatty acid desaturation in Arabidopsis

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

Arondel, V.; Lemieux, B.; Hwang, I.

1992-11-20

A gene from the flowering plant Arabidopsis thaliana that encodes an omega-3 desaturase was cloned on the basis of the genetic map position of a mutation affecting membrane and storage lipid fatty acid composition. Yeast artificial chromosomes covering the genetic locus were identified and used to probe a seed complementary DNA library. A complementary DNA clone for the desaturase was identified and introduced into roots of both wild-type and mutant plants by Ti plasmid-mediated transformation. Transgenic tissues of both mutant and wild-type plants had significantly increased amounts of the fatty acid produced by this desaturase. 24 refs., 2 figs., 1more » tabs.« less

Correction of the lack of commutability between plasmid DNA and genomic DNA for quantification of genetically modified organisms using pBSTopas as a model.

PubMed

Zhang, Li; Wu, Yuhua; Wu, Gang; Cao, Yinglong; Lu, Changming

2014-10-01

Plasmid calibrators are increasingly applied for polymerase chain reaction (PCR) analysis of genetically modified organisms (GMOs). To evaluate the commutability between plasmid DNA (pDNA) and genomic DNA (gDNA) as calibrators, a plasmid molecule, pBSTopas, was constructed, harboring a Topas 19/2 event-specific sequence and a partial sequence of the rapeseed reference gene CruA. Assays of the pDNA showed similar limits of detection (five copies for Topas 19/2 and CruA) and quantification (40 copies for Topas 19/2 and 20 for CruA) as those for the gDNA. Comparisons of plasmid and genomic standard curves indicated that the slopes, intercepts, and PCR efficiency for pBSTopas were significantly different from CRM Topas 19/2 gDNA for quantitative analysis of GMOs. Three correction methods were used to calibrate the quantitative analysis of control samples using pDNA as calibrators: model a, or coefficient value a (Cva); model b, or coefficient value b (Cvb); and the novel model c or coefficient formula (Cf). Cva and Cvb gave similar estimated values for the control samples, and the quantitative bias of the low concentration sample exceeded the acceptable range within ±25% in two of the four repeats. Using Cfs to normalize the Ct values of test samples, the estimated values were very close to the reference values (bias -13.27 to 13.05%). In the validation of control samples, model c was more appropriate than Cva or Cvb. The application of Cf allowed pBSTopas to substitute for Topas 19/2 gDNA as a calibrator to accurately quantify the GMO.

Draft Genome Sequences of Three European Laboratory Derivatives from Enterohemorrhagic Escherichia coli O157:H7 Strain EDL933, Including Two Plasmids.

PubMed

Fellner, Lea; Huptas, Christopher; Simon, Svenja; Mühlig, Anna; Scherer, Siegfried; Neuhaus, Klaus

2016-04-07

Escherichia coliO157:H7 EDL933, isolated in 1982 in the United States, was the first enterohemorrhagicE. coli(EHEC) strain sequenced. Unfortunately, European labs can no longer receive the original strain. We checked three European EDL933 derivatives and found major genetic deviations (deletions, inversions) in two strains. All EDL933 strains contain the cryptic EHEC-plasmid, not reported before. Copyright © 2016 Fellner et al.

Generation of Footprint-Free Induced Pluripotent Stem Cells from Human Fibroblasts Using Episomal Plasmid Vectors.

PubMed

Ovchinnikov, Dmitry A; Sun, Jane; Wolvetang, Ernst J

2015-01-01

Human induced pluripotent stem cells (hiPSCs) have provided novel insights into the etiology of disease and are set to transform regenerative medicine and drug screening over the next decade. The generation of human iPSCs free of a genetic footprint of the reprogramming process is crucial for the realization of these potential uses. Here we describe in detail the generation of human iPSC from control and disease-carrying individuals' fibroblasts using episomal plasmids.

The bacterial segrosome: a dynamic nucleoprotein machine for DNA trafficking and segregation.

PubMed

Hayes, Finbarr; Barillà, Daniela

2006-02-01

The genomes of unicellular and multicellular organisms must be partitioned equitably in coordination with cytokinesis to ensure faithful transmission of duplicated genetic material to daughter cells. Bacteria use sophisticated molecular mechanisms to guarantee accurate segregation of both plasmids and chromosomes at cell division. Plasmid segregation is most commonly mediated by a Walker-type ATPase and one of many DNA-binding proteins that assemble on a cis-acting centromere to form a nucleoprotein complex (the segrosome) that mediates intracellular plasmid transport. Bacterial chromosome segregation involves a multipartite strategy in which several discrete protein complexes potentially participate. Shedding light on the basis of genome segregation in bacteria could indicate new strategies aimed at combating pathogenic and antibiotic-resistant bacteria.

DIY series of genetic cassettes useful in construction of versatile vectors specific for Alphaproteobacteria.

PubMed

Dziewit, Lukasz; Adamczuk, Marcin; Szuplewska, Magdalena; Bartosik, Dariusz

2011-08-01

We have developed a DIY (Do It Yourself) series of genetic cassettes, which facilitate construction of novel versatile vectors for Alphaproteobacteria. All the cassettes are based on defined genetic modules derived from three natural plasmids of Paracoccus aminophilus JCM 7686. We have constructed over 50 DIY cassettes, which differ in structure and specific features. All of them are functional in eight strains representing three orders of Alphaproteobacteria: Rhodobacterales, Rhizobiales and Caulobacterales. Besides various replication and stabilization systems, many of the cassettes also contain selective markers appropriate for Alphaproteobacteria (40 cassettes) and genetic modules responsible for mobilization for conjugal transfer (24 cassettes). All the DIY cassettes are bordered by different types of polylinkers, which facilitate vector construction. Using these DIY cassettes, we have created a set of compatible Escherichia coli-Alphaproteobacteria mobilizable shuttle vectors (high or low copy number in E. coli), which will greatly assist the genetic manipulation of Alphaproteobacteria. Copyright © 2011 Elsevier B.V. All rights reserved.

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

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.

Directed and persistent movement arises from mechanochemistry of the ParA/ParB system.

PubMed

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

2015-12-22

The segregation of DNA before cell division is essential for faithful genetic inheritance. In many bacteria, segregation of low-copy number plasmids involves an active partition system composed of a nonspecific DNA-binding ATPase, ParA, and its stimulator protein ParB. The ParA/ParB system drives directed and persistent movement of DNA cargo both in vivo and in vitro. Filament-based models akin to actin/microtubule-driven motility were proposed for plasmid segregation mediated by ParA. Recent experiments challenge this view and suggest that ParA/ParB system motility is driven by a diffusion ratchet mechanism in which ParB-coated plasmid both creates and follows a ParA gradient on the nucleoid surface. However, the detailed mechanism of ParA/ParB-mediated directed and persistent movement remains unknown. Here, we develop a theoretical model describing ParA/ParB-mediated motility. We show that the ParA/ParB system can work as a Brownian ratchet, which effectively couples the ATPase-dependent cycling of ParA-nucleoid affinity to the motion of the ParB-bound cargo. Paradoxically, this resulting processive motion relies on quenching diffusive plasmid motion through a large number of transient ParA/ParB-mediated tethers to the nucleoid surface. Our work thus sheds light on an emergent phenomenon in which nonmotor proteins work collectively via mechanochemical coupling to propel cargos-an ingenious solution shaped by evolution to cope with the lack of processive motor proteins in bacteria.

The effects of a low-intensity red laser on bacterial growth, filamentation and plasmid DNA

NASA Astrophysics Data System (ADS)

Roos, C.; Santos, J. N.; Guimarães, O. R.; Geller, M.; Paoli, F.; Fonseca, A. S.

2013-07-01

Exposure of nonphotosynthesizing microorganisms to light could increase cell division in cultures, a phenomenon denominated as biostimulation. However, data concerning the importance of the genetic characteristics of cells on this effect are as yet scarce. The aim of this work was to evaluate the effects of a low-intensity red laser on the growth, filamentation and plasmids in Escherichia coli cells proficient and deficient in DNA repair. E. coli cultures were exposed to a laser (658 nm, 10 mW, 1 and 8 J cm-2) to study bacterial growth and filamentation. Also, bacterial cultures hosting pBSK plasmids were exposed to the laser to study DNA topological forms from the electrophoretic profile in agarose gels. Data indicate the low-intensity red laser: (i) had no effect on the growth of E. coli wild type and exonuclease III deficient cells; (ii) induced bacterial filamentation, (iii) led to no alteration in the electrophoretic profile of plasmids from exonuclease III deficient cells, but plasmids from wild type cells were altered. A low-intensity red laser at the low fluences used in phototherapy has no effect on growth, but induces filamentation and alters the topological forms of plasmid DNA in E. coli cultures depending on the DNA repair mechanisms.

Development and application of a general plasmid reference material for GMO screening.

PubMed

Wu, Yuhua; Li, Jun; Wang, Yulei; Li, Xiaofei; Li, Yunjing; Zhu, Li; Li, Jun; Wu, Gang

The use of analytical controls is essential when performing GMO detection through screening tests. Additionally, the presence of taxon-specific sequences is analyzed mostly for quality control during GMO detection. In this study, 11 commonly used genetic elements involving three promoters (P-35S, P-FMV35S and P-NOS), four marker genes (Bar, NPTII, HPT and Pmi), and four terminators (T-NOS, T-35S, T-g7 and T-e9), together with the reference gene fragments from six major crops of maize, soybean, rapeseed, rice, cotton and wheat, were co-integrated into the same single plasmid to construct a general reference plasmid pBI121-Screening. The suitability test of pBI121-Screening plasmid as reference material indicated that the non-target sequence on the pBI121-Screening plasmid did not affect the PCR amplification efficiencies of screening methods and taxon-specific methods. The sensitivity of screening and taxon-specific assays ranged from 5 to 10 copies of pBI121-Screening plasmid, meeting the sensitivity requirement of GMO detection. The construction of pBI121-Screening solves the lack of a general positive control for screening tests, thereby reducing the workload and cost of preparing a plurality of the positive control. Copyright © 2016 Elsevier B.V. All rights reserved.

Global phylogenetic analysis of Escherichia coli and plasmids carrying the mcr-1 gene indicates bacterial diversity but plasmid restriction.

PubMed

Matamoros, Sébastien; van Hattem, Jarne M; Arcilla, Maris S; Willemse, Niels; Melles, Damian C; Penders, John; Vinh, Trung Nguyen; Thi Hoa, Ngo; de Jong, Menno D; Schultsz, Constance

2017-11-10

To understand the dynamics behind the worldwide spread of the mcr-1 gene, we determined the population structure of Escherichia coli and of mobile genetic elements (MGEs) carrying the mcr-1 gene. After a systematic review of the literature we included 65 E. coli whole genome sequences (WGS), adding 6 recently sequenced travel related isolates, and 312 MLST profiles. We included 219 MGEs described in 7 Enterobacteriaceae species isolated from human, animal and environmental samples. Despite a high overall diversity, 2 lineages were observed in the E. coli population that may function as reservoirs of the mcr-1 gene, the largest of which was linked to ST10, a sequence type known for its ubiquity in human faecal samples and in food samples. No genotypic clustering by geographical origin or isolation source was observed. Amongst a total of 13 plasmid incompatibility types, the IncI2, IncX4 and IncHI2 plasmids accounted for more than 90% of MGEs carrying the mcr-1 gene. We observed significant geographical clustering with regional spread of IncHI2 plasmids in Europe and IncI2 in Asia. These findings point towards promiscuous spread of the mcr-1 gene by efficient horizontal gene transfer dominated by a limited number of plasmid incompatibility types.

Molecular Genetics of Mycobacteriophages

PubMed Central

HATFULL, GRAHAM F.

2014-01-01

Mycobacteriophages have provided numerous essential tools for mycobacterial genetics, including delivery systems for transposons, reporter genes, and allelic exchange substrates, and components for plasmid vectors and mutagenesis. Their genetically diverse genomes also reveal insights into the broader nature of the phage population and the evolutionary mechanisms that give rise to it. The substantial advances in our understanding of the biology of mycobacteriophages including a large collection of completely sequenced genomes indicates a rich potential for further contributions in tuberculosis genetics and beyond. PMID:25328854

Antibiotic resistance due to an unusual ColE1-type replicon plasmid in Aeromonas salmonicida.

PubMed

Vincent, Antony T; Emond-Rheault, Jean-Guillaume; Barbeau, Xavier; Attéré, Sabrina A; Frenette, Michel; Lagüe, Patrick; Charette, Steve J

2016-06-01

Aeromonas salmonicida subsp. salmonicida is a fish pathogen known to have a rich plasmidome. In the present study, we discovered an isolate of this bacterium bearing an additional unidentified small plasmid. After having sequenced the DNA of that isolate by next-generation sequencing, it appeared that the new small plasmid is a ColE1-type replicon plasmid, named here pAsa7. This plasmid bears a functional chloramphenicol-acetyltransferase-encoding gene (cat-pAsa7) previously unknown in A. salmonicida and responsible for resistance to chloramphenicol. A comparison of pAsa7 with pAsa2, the only known ColE1-type replicon plasmid usually found in A. salmonicida subsp. salmonicida, revealed that even if both plasmids share a high structural similarity, it is still unclear if pAsa7 is a derivative of pAsa2 since they showed several mutations at the nucleotide level. Transcriptomic analysis revealed that the cat-pAsa4 gene, another chloramphenicol-acetyltransferase-encoding gene, found on the large plasmid pAsa4, was significantly more transcribed than cat-pAsa7. This was correlated with a higher chloramphenicol resistance for isolates bearing pAsa4 compared with the one having pAsa7. Finally, a phylogenetic analysis showed that both CAT-pAsa4 and CAT-pAsa7 proteins were in different clusters. The clustering was supported by the identity of residues involved in the catalytic site. In addition, to give a better understanding of the large drug-resistance panel of A. salmonicida, this study reinforces the hypothesis that A. salmonicida subsp. salmonicida is a considerable reservoir for mobile genetic elements such as plasmids.

The Plasmid Mobilome of the Model Plant-Symbiont Sinorhizobium meliloti: Coming up with New Questions and Answers.

PubMed

Lagares, Antonio; Sanjuán, Juan; Pistorio, Mariano

2014-10-01

Rhizobia are Gram-negative Alpha- and Betaproteobacteria living in the underground which have the ability to associate with legumes for the establishment of nitrogen-fixing symbioses. Sinorhizobium meliloti in particular-the symbiont of Medicago, Melilotus, and Trigonella spp.-has for the past decades served as a model organism for investigating, at the molecular level, the biology, biochemistry, and genetics of a free-living and symbiotic soil bacterium of agricultural relevance. To date, the genomes of seven different S. meliloti strains have been fully sequenced and annotated, and several other draft genomic sequences are also available. The vast amount of plasmid DNA that S. meliloti frequently bears (up to 45% of its total genome), the conjugative ability of some of those plasmids, and the extent of the plasmid diversity has provided researchers with an extraordinary system to investigate functional and structural plasmid molecular biology within the evolutionary context surrounding a plant-associated model bacterium. Current evidence indicates that the plasmid mobilome in S. meliloti is composed of replicons varying greatly in size and having diverse conjugative systems and properties along with different evolutionary stabilities and biological roles. While plasmids carrying symbiotic functions (pSyms) are known to have high structural stability (approaching that of chromosomes), the remaining plasmid mobilome (referred to as the non-pSym, functionally cryptic, or accessory compartment) has been shown to possess remarkable diversity and to be highly active in conjugation. In light of the modern genomic and current biochemical data on the plasmids of S. meliloti, the current article revises their main structural components, their transfer and regulatory mechanisms, and their potential as vehicles in shaping the evolution of the rhizobial genome.

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.

Characterization of clinically isolated thymidine-dependent small-colony variants of Escherichia coli producing extended-spectrum β-lactamase.

PubMed

Negishi, Tatsuya; Matsumoto, Takehisa; Horiuchi, Kazuki; Kasuga, Eriko; Natori, Tatsuya; Matsuoka, Mina; Ogiwara, Naoko; Sugano, Mitsutoshi; Uehara, Takeshi; Nagano, Noriyuki; Honda, Takayuki

2018-01-01

Thymidine-dependent small-colony variants (TD-SCVs) are difficult to detect or test for antimicrobial susceptibility. We investigated the characteristics of clonal TD-SCVs of Escherichia coli, both with and without blaCTX-M-3, isolated from a patient. Mutation in the thyA gene was analysed by sequencing, and morphological abnormalities in the colonies and cells of the isolates were examined. Additionally, conjugational transfer experiments were performed to prove the horizontal transferability of plasmids harbouring resistance genes. The TD-SCVs contained a single nucleotide substitution in the thyA gene, c.62G>A, corresponding to p.Arg21His. Morphologically, their colonies were more translucent and flattened than those of the wild-type strain. In addition, cells of the TD-SCVs were swollen and elongated, sometimes with abnormal and incomplete divisions; a large amount of cell debris was also observed. Changing c.62G>A back to the wild-type sequence reversed these abnormalities. Conjugational transfer experiments showed that the TD-SCV of E. coli with blaCTX-M-3 failed to transfer blaCTX-M-3 to E. coli CSH2. However, the TD-SCV of E. coli without blaCTX-M-3 experimentally received the plasmid encoding blaSHV-18 from Klebsiella pneumoniae ATCC 700603 and transferred it to E. coli CSH2. Mutation in the thyA gene causes morphological abnormalities in the colonies and cells of E. coli, as well as inducing thymidine auxotrophy. In addition, TD-SCVs horizontally transmit plasmids encoding resistance genes. It is important to detect TD-SCVs based on their characteristics because they serve as reservoirs of transferable antibiotic resistance plasmids.

Persistence of transferable extended-spectrum-β-lactamase resistance in the absence of antibiotic pressure.

PubMed

Cottell, Jennifer L; Webber, Mark A; Piddock, Laura J V

2012-09-01

The treatment of infections caused by antibiotic-resistant bacteria is one of the great challenges faced by clinicians in the 21st century. Antibiotic resistance genes are often transferred between bacteria by mobile genetic vectors called plasmids. It is commonly believed that removal of antibiotic pressure will reduce the numbers of antibiotic-resistant bacteria due to the perception that carriage of resistance imposes a fitness cost on the bacterium. This study investigated the ability of the plasmid pCT, a globally distributed plasmid that carries an extended-spectrum-β-lactamase (ESBL) resistance gene (bla(CTX-M-14)), to persist and disseminate in the absence of antibiotic pressure. We investigated key attributes in plasmid success, including conjugation frequencies, bacterial-host growth rates, ability to cause infection, and impact on the fitness of host strains. We also determined the contribution of the bla(CTX-M-14) gene itself to the biology of the plasmid and host bacterium. Carriage of pCT was found to impose no detectable fitness cost on various bacterial hosts. An absence of antibiotic pressure and inactivation of the antibiotic resistance gene also had no effect on plasmid persistence, conjugation frequency, or bacterial-host biology. In conclusion, plasmids such as pCT have evolved to impose little impact on host strains. Therefore, the persistence of antibiotic resistance genes and their vectors is to be expected in the absence of antibiotic selective pressure regardless of antibiotic stewardship. Other means to reduce plasmid stability are needed to prevent the persistence of these vectors and the antibiotic resistance genes they carry.

Horizontal Gene Transfer of a ColV Plasmid Has Resulted in a Dominant Avian Clonal Type of Salmonella enterica Serovar Kentucky

PubMed Central

Johnson, Timothy J.; Thorsness, Jessica L.; Anderson, Cole P.; Lynne, Aaron M.; Foley, Steven L.; Han, Jing; Fricke, W. Florian; McDermott, Patrick F.; White, David G.; Khatri, Mahesh; Stell, Adam L.; Flores, Cristian; Singer, Randall S.

2010-01-01

Salmonella enterica continues to be a significant cause of foodborne gastrointestinal illness in humans. A wide variety of Salmonella serovars have been isolated from production birds and from retail poultry meat. Recently, though, S. enterica subsp. enterica serovar Kentucky has emerged as one of the prominent Salmonella serovars isolated from broiler chickens. Recent work suggests that its emergence apparently coincides with its acquisition of a ColV virulence plasmid. In the present study, we examined 902 Salmonella isolates belonging to 59 different serovars for the presence of this plasmid. Of the serovars examined, the ColV plasmid was found only among isolates belonging to the serovars Kentucky (72.9%), Typhimurium (15.0%) and Heidelberg (1.7%). We demonstrated that a single PFGE clonal type of S. Kentucky harbors this plasmid, and acquisition of this plasmid by S. Kentucky significantly increased its ability to colonize the chicken cecum and cause extraintestinal disease. Comparison of the completed sequences of three ColV plasmids from S. Kentucky isolated from different geographical locales, timepoints and sources revealed a nearly identical genetic structure with few single nucleotide changes or insertions/deletions. Overall, it appears that the ColV plasmid was recently acquired by a single clonal type S. Kentucky and confers to its host enhanced colonization and fitness capabilities. Thus, the potential for horizontal gene transfer of virulence and fitness factors to Salmonella from other enteric bacteria exists in poultry, representing a potential human health hazard. PMID:21203520

Comparable dynamics of linuron catabolic genes and IncP-1 plasmids in biopurification systems (BPSs) as a response to linuron spiking.

PubMed

Nour, Eman H; Elsayed, Tarek R; Springael, Dirk; Smalla, Kornelia

2017-06-01

On-farm biopurification systems (BPSs) represent an efficient technology for treating pesticide-contaminated wastewater. Biodegradation by genetically adapted bacteria has been suggested to perform a major contribution to the removal of pesticides in BPSs. Recently, several studies pointed to the role of IncP-1 plasmids in the degradation of pesticides in BPSs but this was never linked with catabolic markers. Therefore, a microcosm experiment was conducted in order to examine whether changes in mobile genetic element (MGE) abundances in response to the application of phenylurea herbicide linuron are linked with changes in catabolic genes. Denaturing gradient gel electrophoresis (DGGE) fingerprints of 16S ribosomal RNA gene fragments amplified from total community (TC)-DNA suggested significant shifts in the bacterial community composition. PCR-Southern blot-based detection of genes involved in linuron hydrolysis (libA and hylA) or degradation of its metabolite 3,4-dichloroaniline (dcaQ I , dcaQ II , and ccdC) in TC-DNA showed that the abundance of the hylA gene was increased faster and stronger in response to linuron application than that of the libA gene, and that the dcaQ II gene was more abundant than the isofunctional gene dcaQ I 20 and 60 days after linuron addition. Furthermore, a significant increase in the relative abundance of the IncP-1-specific korB gene in response to linuron was recorded. Our data suggest that different bacterial populations bearing isofunctional genes coding for enzymes degrading linuron seemed to be enriched in BPSs in response to linuron and that IncP-1 plasmids might be involved in their dissemination.

Next generation sequencing of DNA-launched Chikungunya vaccine virus

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

Hidajat, Rachmat; Nickols, Brian; Forrester, Naomi

Chikungunya virus (CHIKV) represents a pandemic threat with no approved vaccine available. Recently, we described a novel vaccination strategy based on iDNA® infectious clone designed to launch a live-attenuated CHIKV vaccine from plasmid DNA in vitro or in vivo. As a proof of concept, we prepared iDNA plasmid pCHIKV-7 encoding the full-length cDNA of the 181/25 vaccine. The DNA-launched CHIKV-7 virus was prepared and compared to the 181/25 virus. Illumina HiSeq2000 sequencing revealed that with the exception of the 3′ untranslated region, CHIKV-7 viral RNA consistently showed a lower frequency of single-nucleotide polymorphisms than the 181/25 RNA including at themore » E2-12 and E2-82 residues previously identified as attenuating mutations. In the CHIKV-7, frequencies of reversions at E2-12 and E2-82 were 0.064% and 0.086%, while in the 181/25, frequencies were 0.179% and 0.133%, respectively. We conclude that the DNA-launched virus has a reduced probability of reversion mutations, thereby enhancing vaccine safety. - Highlights: • Chikungunya virus (CHIKV) is an emerging pandemic threat. • In vivo DNA-launched attenuated CHIKV is a novel vaccine technology. • DNA-launched virus was sequenced using HiSeq2000 and compared to the 181/25 virus. • DNA-launched virus has lower frequency of SNPs at E2-12 and E2-82 attenuation loci.« less

Rescue of infectious rift valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene.

PubMed

Ikegami, Tetsuro; Won, Sungyong; Peters, C J; Makino, Shinji

2006-03-01

Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) has a tripartite negative-strand genome, causes a mosquito-borne disease that is endemic in sub-Saharan African countries and that also causes large epidemics among humans and livestock. Furthermore, it is a bioterrorist threat and poses a risk for introduction to other areas. In spite of its danger, neither veterinary nor human vaccines are available. We established a T7 RNA polymerase-driven reverse genetics system to rescue infectious clones of RVFV MP-12 strain entirely from cDNA, the first for any phlebovirus. Expression of viral structural proteins from the protein expression plasmids was not required for virus rescue, whereas NSs protein expression abolished virus rescue. Mutants of MP-12 partially or completely lacking the NSs open reading frame were viable. These NSs deletion mutants replicated efficiently in Vero and 293 cells, but not in MRC-5 cells. In the latter cell line, accumulation of beta interferon mRNA occurred after infection by these NSs deletion mutants, but not after infection by MP-12. The NSs deletion mutants formed larger plaques than MP-12 did in Vero E6 cells and failed to shut off host protein synthesis in Vero cells. An MP-12 mutant carrying a luciferase gene in place of the NSs gene replicated as efficiently as MP-12 did, produced enzymatically active luciferase during replication, and stably retained the luciferase gene after 10 virus passages, representing the first demonstration of foreign gene expression in any bunyavirus. This reverse genetics system can be used to study the molecular virology of RVFV, assess current vaccine candidates, produce new vaccines, and incorporate marker genes into animal vaccines.

An Implementation-Focused Bio/Algorithmic Workflow for Synthetic Biology.

PubMed

Goñi-Moreno, Angel; Carcajona, Marta; Kim, Juhyun; Martínez-García, Esteban; Amos, Martyn; de Lorenzo, Víctor

2016-10-21

As synthetic biology moves away from trial and error and embraces more formal processes, workflows have emerged that cover the roadmap from conceptualization of a genetic device to its construction and measurement. This latter aspect (i.e., characterization and measurement of synthetic genetic constructs) has received relatively little attention to date, but it is crucial for their outcome. An end-to-end use case for engineering a simple synthetic device is presented, which is supported by information standards and computational methods and focuses on such characterization/measurement. This workflow captures the main stages of genetic device design and description and offers standardized tools for both population-based measurement and single-cell analysis. To this end, three separate aspects are addressed. First, the specific vector features are discussed. Although device/circuit design has been successfully automated, important structural information is usually overlooked, as in the case of plasmid vectors. The use of the Standard European Vector Architecture (SEVA) is advocated for selecting the optimal carrier of a design and its thorough description in order to unequivocally correlate digital definitions and molecular devices. A digital version of this plasmid format was developed with the Synthetic Biology Open Language (SBOL) along with a software tool that allows users to embed genetic parts in vector cargoes. This enables annotation of a mathematical model of the device's kinetic reactions formatted with the Systems Biology Markup Language (SBML). From that point onward, the experimental results and their in silico counterparts proceed alongside, with constant feedback to preserve consistency between them. A second aspect involves a framework for the calibration of fluorescence-based measurements. One of the most challenging endeavors in standardization, metrology, is tackled by reinterpreting the experimental output in light of simulation results, allowing us to turn arbitrary fluorescence units into relative measurements. Finally, integration of single-cell methods into a framework for multicellular simulation and measurement is addressed, allowing standardized inspection of the interplay between the carrier chassis and the culture conditions.

Combined chemical and physical transformation method with RbCl and sepiolite for the transformation of various bacterial species.

PubMed

Ren, Jun; Lee, Haram; Yoo, Seung Min; Yu, Myeong-Sang; Park, Hansoo; Na, Dokyun

2017-04-01

DNA transformation that delivers plasmid DNAs into bacterial cells is fundamental in genetic manipulation to engineer and study bacteria. Developed transformation methods to date are optimized to specific bacterial species for high efficiency. Thus, there is always a demand for simple and species-independent transformation methods. We herein describe the development of a chemico-physical transformation method that combines a rubidium chloride (RbCl)-based chemical method and sepiolite-based physical method, and report its use for the simple and efficient delivery of DNA into various bacterial species. Using this method, the best transformation efficiency for Escherichia coli DH5α was 4.3×10 6 CFU/μg of pUC19 plasmid, which is higher than or comparable to the reported transformation efficiencies to date. This method also allowed the introduction of plasmid DNAs into Bacillus subtilis (5.7×10 3 CFU/μg of pSEVA3b67Rb), Bacillus megaterium (2.5×10 3 CFU/μg of pSPAsp-hp), Lactococcus lactis subsp. lactis (1.0×10 2 CFU/μg of pTRKH3-ermGFP), and Lactococcus lactis subsp. cremoris (2.2×10 2 CFU/μg of pMSP3535VA). Remarkably, even when the conventional chemical and physical methods failed to generate transformed cells in Bacillus sp. and Enterococcus faecalis, E. malodoratus and E. mundtii, our combined method showed a significant transformation efficiency (2.4×10 4 , 4.5×10 2 , 2×10 1 , and 0.5×10 1 CFU/μg of plasmid DNA). Based on our results, we anticipate that our simple and efficient transformation method should prove usefulness for introducing DNA into various bacterial species without complicated optimization of parameters affecting DNA entry into the cell. Copyright © 2017. Published by Elsevier B.V.

Development and application of a multi-targeting reference plasmid as calibrator for analysis of five genetically modified soybean events.

PubMed

Pi, Liqun; Li, Xiang; Cao, Yiwei; Wang, Canhua; Pan, Liangwen; Yang, Litao

2015-04-01

Reference materials are important in accurate analysis of genetically modified organism (GMO) contents in food/feeds, and development of novel reference plasmid is a new trend in the research of GMO reference materials. Herein, we constructed a novel multi-targeting plasmid, pSOY, which contained seven event-specific sequences of five GM soybeans (MON89788-5', A2704-12-3', A5547-127-3', DP356043-5', DP305423-3', A2704-12-5', and A5547-127-5') and sequence of soybean endogenous reference gene Lectin. We evaluated the specificity, limit of detection and quantification, and applicability of pSOY in both qualitative and quantitative PCR analyses. The limit of detection (LOD) was as low as 20 copies in qualitative PCR, and the limit of quantification (LOQ) in quantitative PCR was 10 copies. In quantitative real-time PCR analysis, the PCR efficiencies of all event-specific and Lectin assays were higher than 90%, and the squared regression coefficients (R(2)) were more than 0.999. The quantification bias varied from 0.21% to 19.29%, and the relative standard deviations were from 1.08% to 9.84% in simulated samples analysis. All the results demonstrated that the developed multi-targeting plasmid, pSOY, was a credible substitute of matrix reference materials, and could be used as a reliable reference calibrator in the identification and quantification of multiple GM soybean events.

Biochemical and Genetic Characterization of Coagulin, a New Antilisterial Bacteriocin in the Pediocin Family of Bacteriocins, Produced by Bacillus coagulans I4

PubMed Central

Le Marrec, Claire; Hyronimus, Bertrand; Bressollier, Philippe; Verneuil, Bernard; Urdaci, Maria C.

2000-01-01

A plasmid-linked antimicrobial peptide, named coagulin, produced by Bacillus coagulans I4 has recently been reported (B. Hyronimus, C. Le Marrec and M. C. Urdaci, J. Appl. Microbiol. 85:42–50, 1998). In the present study, the complete, unambiguous primary amino acid sequence of the peptide was obtained by a combination of both N-terminal sequencing of purified peptide and the complete sequence deduced from the structural gene harbored by plasmid I4. Data revealed that this peptide of 44 residues has an amino acid sequence similar to that described for pediocins AcH and PA-1, produced by different Pediococcus acidilactici strains and 100% identical. Coagulin and pediocin differed only by a single amino acid at their C terminus. Analysis of the genetic determinants revealed the presence, on the pI4 DNA, of the entire 3.5-kb operon of four genes described for pediocin AcH and PA-1 production. No extended homology was observed between pSMB74 from P. acidilactici and pI4 when analyzing the regions upstream and downstream of the operon. An oppositely oriented gene immediately dowstream of the bacteriocin operon specifies a 474-amino-acid protein which shows homology to Mob-Pre (plasmid recombination enzyme) proteins encoded by several small plasmids extracted from gram-positive bacteria. This is the first report of a pediocin-like peptide appearing naturally in a non-lactic acid bacterium genus. PMID:11097892

Real-Time PCR-Based Quantitation Method for the Genetically Modified Soybean Line GTS 40-3-2.

PubMed

Kitta, Kazumi; Takabatake, Reona; Mano, Junichi

2016-01-01

This chapter describes a real-time PCR-based method for quantitation of the relative amount of genetically modified (GM) soybean line GTS 40-3-2 [Roundup Ready(®) soybean (RRS)] contained in a batch. The method targets a taxon-specific soybean gene (lectin gene, Le1) and the specific DNA construct junction region between the Petunia hybrida chloroplast transit peptide sequence and the Agrobacterium 5-enolpyruvylshikimate-3-phosphate synthase gene (epsps) sequence present in GTS 40-3-2. The method employs plasmid pMulSL2 as a reference material in order to quantify the relative amount of GTS 40-3-2 in soybean samples using a conversion factor (Cf) equal to the ratio of the RRS-specific DNA to the taxon-specific DNA in representative genuine GTS 40-3-2 seeds.

Study of the genetics and regulation of methane oxidation. Progress report, second year and a half, August 1, 1981-January 31, 1983

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

Not Available

The purpose is to develop mutagenesis, gene transfer and cloning systems in methanotrophic bacteria, and use these techniques to study the methane oxidation genes. Although we have been successful in the first part of these objectives, the study of methane oxidation genes has proven difficult. Problems arose due to the discovery that the culture, Methylobacterium ethanolicum, is in reality a stable coculture between two methylotrophs. These partners are Methylocystis POC, an obligate methanotroph and Xanthobacter H4.14, and autotrophic methanolutilizer. The Methylocystis strain contains the three plasmids we had observed previously in methane-grown cultures, while the Xanthobacter strain contains no detectiblemore » plasmids. Therefore, our original approach to studying the methane oxidation genes, that of isolating plasmid mutants, is no longer valid. However, our discovery of the nature of this culture has led to some interesting results which show promise in elucidating the genetic structure of the methane oxidation genes in obligate methanotrophs. In addition, we have been successful in developing mutagenesis, gene transfer and cloning systems that are applicable to a wide variety of methanotrophs.« less

Intracellular integration of synthetic nanostructures with viable cells for controlled biochemical manipulation

NASA Astrophysics Data System (ADS)

McKnight, Timothy E.; Melechko, Anatoli V.; Griffin, Guy D.; Guillorn, Michael A.; Merkulov, Vladimir I.; Serna, Francisco; Hensley, Dale K.; Doktycz, Mitchel J.; Lowndes, Douglas H.; Simpson, Michael L.

2003-05-01

We demonstrate the integration of vertically aligned carbon nanofibre (VACNF) elements with the intracellular domains of viable cells for controlled biochemical manipulation. Deterministically synthesized VACNFs were modified with either adsorbed or covalently-linked plasmid DNA and were subsequently inserted into cells. Post insertion viability of the cells was demonstrated by continued proliferation of the interfaced cells and long-term (> 22 day) expression of the introduced plasmid. Adsorbed plasmids were typically desorbed in the intracellular domain and segregated to progeny cells. Covalently bound plasmids remained tethered to nanofibres and were expressed in interfaced cells but were not partitioned into progeny, and gene expression ceased when the nanofibre was no longer retained. This provides a method for achieving a genetic modification that is non-inheritable and whose extent in time can be directly and precisely controlled. These results demonstrate the potential of VACNF arrays as an intracellular interface for monitoring and controlling subcellular and molecular phenomena within viable cells for applications including biosensors, in vivo diagnostics, and in vivo logic devices.

Correlation between Detection of a Plasmid and High-Level Virulence of Vibrio nigripulchritudo, a Pathogen of the Shrimp Litopenaeus stylirostris▿

PubMed Central

Reynaud, Yann; Saulnier, Denis; Mazel, Didier; Goarant, Cyrille; Le Roux, Frédérique

2008-01-01

Vibrio nigripulchritudo, the etiological agent of Litopenaeus stylirostris summer syndrome, is responsible for mass mortalities of shrimp in New Caledonia. Epidemiological studies led to the suggestion that this disease is caused by an emergent group of pathogenic strains. Genomic subtractive hybridization was carried out between two isolates exhibiting low and high virulence. Our subtraction library was constituted of 521 specific fragments; 55 of these were detected in all virulent isolates from our collection (n = 32), and 13 were detected only in the isolates demonstrating the highest pathogenicity (n = 19), suggesting that they could be used as genetic markers for high virulence capacity. Interestingly, 10 of these markers are carried by a replicon of 11.2 kbp that contains sequences highly similar to those of a plasmid detected in Vibrio shilonii, a coral pathogen. The detection of this plasmid was correlated with the highest pathogenicity status of the isolates from our collection. The origin and consequence of this plasmid acquisition are discussed. PMID:18359828

A novel reference plasmid for the qualitative detection of genetically modified rice in food and feed.

PubMed

Li, Liang; Dong, Mei; An, Na; Liang, Lixia; Wan, Yusong; Jin, Wujun

2015-01-01

Rice is one of the most important food crops in the world. Genetically modified (GM) technology has been used in rice to confer herbicide tolerance and pathogen or insect resistance. China invests heavily in research on GM rice. By the end of 2014, at least 250 transgenic rice lines had been developed in China. To monitor the presence of GM rice in food and feed, we collected information on foreign elements from 250 transgenic rice lines and found 5 elements, including the Agrobacterium tumefaciens nopaline synthase terminator (T-NOS), the cauliflower mosaic virus 35S promoter (CaMV35S), the ubiquitin gene (Ubi), the bar gene, and the hygromycin phosphotransferase gene (Hpt), that are commonly present in GM rice. Therefore, we constructed a novel plasmid (pBJGMM001) that contains fragments of these elements and two endogenous reference genes (the sucrose phosphate synthase gene, SPS, and the phosphoenolpyruvate carboxylase gene, PEPC). pBJGMM001 can serve as a standard for detecting 96% of GM rice lines in China. The primers, amplicons, reaction mixture, and PCR program were developed based on Chinese National Standards. The protocol was validated and determined to be suitable for practical use in monitoring and identifying GM rice.

EMMA: An Extensible Mammalian Modular Assembly Toolkit for the Rapid Design and Production of Diverse Expression Vectors.

PubMed

Martella, Andrea; Matjusaitis, Mantas; Auxillos, Jamie; Pollard, Steven M; Cai, Yizhi

2017-07-21

Mammalian plasmid expression vectors are critical reagents underpinning many facets of research across biology, biomedical research, and the biotechnology industry. Traditional cloning methods often require laborious manual design and assembly of plasmids using tailored sequential cloning steps. This process can be protracted, complicated, expensive, and error-prone. New tools and strategies that facilitate the efficient design and production of bespoke vectors would help relieve a current bottleneck for researchers. To address this, we have developed an extensible mammalian modular assembly kit (EMMA). This enables rapid and efficient modular assembly of mammalian expression vectors in a one-tube, one-step golden-gate cloning reaction, using a standardized library of compatible genetic parts. The high modularity, flexibility, and extensibility of EMMA provide a simple method for the production of functionally diverse mammalian expression vectors. We demonstrate the value of this toolkit by constructing and validating a range of representative vectors, such as transient and stable expression vectors (transposon based vectors), targeting vectors, inducible systems, polycistronic expression cassettes, fusion proteins, and fluorescent reporters. The method also supports simple assembly combinatorial libraries and hierarchical assembly for production of larger multigenetic cargos. In summary, EMMA is compatible with automated production, and novel genetic parts can be easily incorporated, providing new opportunities for mammalian synthetic biology.

Genomic Analysis of Phylotype I Strain EP1 Reveals Substantial Divergence from Other Strains in the Ralstonia solanacearum Species Complex

PubMed Central

Li, Peng; Wang, Dechen; Yan, Jinli; Zhou, Jianuan; Deng, Yinyue; Jiang, Zide; Cao, Bihao; He, Zifu; Zhang, Lianhui

2016-01-01

Ralstonia solanacearum species complex is a devastating group of phytopathogens with an unusually wide host range and broad geographical distribution. R. solanacearum isolates may differ considerably in various properties including host range and pathogenicity, but the underlying genetic bases remain vague. Here, we conducted the genome sequencing of strain EP1 isolated from Guangdong Province of China, which belongs to phylotype I and is highly virulent to a range of solanaceous crops. Its complete genome contains a 3.95-Mb chromosome and a 2.05-Mb mega-plasmid, which is considerably bigger than reported genomes of other R. solanacearum strains. Both the chromosome and the mega-plasmid have essential house-keeping genes and many virulence genes. Comparative analysis of strain EP1 with other 3 phylotype I and 3 phylotype II, III, IV strains unveiled substantial genome rearrangements, insertions and deletions. Genome sequences are relatively conserved among the 4 phylotype I strains, but more divergent among strains of different phylotypes. Moreover, the strains exhibited considerable variations in their key virulence genes, including those encoding secretion systems and type III effectors. Our results provide valuable information for further elucidation of the genetic basis of diversified virulences and host range of R. solanacearum species. PMID:27833603

A Novel Reference Plasmid for the Qualitative Detection of Genetically Modified Rice in Food and Feed

PubMed Central

Dong, Mei; An, Na; Liang, Lixia; Wan, Yusong

2015-01-01

Rice is one of the most important food crops in the world. Genetically modified (GM) technology has been used in rice to confer herbicide tolerance and pathogen or insect resistance. China invests heavily in research on GM rice. By the end of 2014, at least 250 transgenic rice lines had been developed in China. To monitor the presence of GM rice in food and feed, we collected information on foreign elements from 250 transgenic rice lines and found 5 elements, including the Agrobacterium tumefaciens nopaline synthase terminator (T-NOS), the cauliflower mosaic virus 35S promoter (CaMV35S), the ubiquitin gene (Ubi), the bar gene, and the hygromycin phosphotransferase gene (Hpt), that are commonly present in GM rice. Therefore, we constructed a novel plasmid (pBJGMM001) that contains fragments of these elements and two endogenous reference genes (the sucrose phosphate synthase gene, SPS, and the phosphoenolpyruvate carboxylase gene, PEPC). pBJGMM001 can serve as a standard for detecting 96% of GM rice lines in China. The primers, amplicons, reaction mixture, and PCR program were developed based on Chinese National Standards. The protocol was validated and determined to be suitable for practical use in monitoring and identifying GM rice. PMID:26495318

Association of virulence plasmid and antibiotic resistance determinants with chromosomal multilocus genotypes in Mexican Salmonella enterica serovar Typhimurium strains

PubMed Central

2009-01-01

Background Bacterial genomes are mosaic structures composed of genes present in every strain of the same species (core genome), and genes present in some but not all strains of a species (accessory genome). The aim of this study was to compare the genetic diversity of core and accessory genes of a Salmonella enterica subspecies enterica serovar Typhimurium (Typhimurium) population isolated from food-animal and human sources in four regions of Mexico. Multilocus sequence typing (MLST) and macrorestriction fingerprints by pulsed-field gel electrophoresis (PFGE) were used to address the core genetic variation, and genes involved in pathogenesis and antibiotic resistance were selected to evaluate the accessory genome. Results We found a low genetic diversity for both housekeeping and accessory genes. Sequence type 19 (ST19) was supported as the founder genotype of STs 213, 302 and 429. We found a temporal pattern in which the derived ST213 is replacing the founder ST19 in the four geographic regions analyzed and a geographic trend in the number of resistance determinants. The distribution of the accessory genes was not random among chromosomal genotypes. We detected strong associations among the different accessory genes and the multilocus chromosomal genotypes (STs). First, the Salmonella virulence plasmid (pSTV) was found mostly in ST19 isolates. Second, the plasmid-borne betalactamase cmy-2 was found only in ST213 isolates. Third, the most abundant integron, IP-1 (dfrA12, orfF and aadA2), was found only in ST213 isolates. Fourth, the Salmonella genomic island (SGI1) was found mainly in a subgroup of ST19 isolates carrying pSTV. The mapping of accessory genes and multilocus genotypes on the dendrogram derived from macrorestiction fingerprints allowed the establishment of genetic subgroups within the population. Conclusion Despite the low levels of genetic diversity of core and accessory genes, the non-random distribution of the accessory genes across chromosomal backgrounds allowed us to discover genetic subgroups within the population. This study provides information about the importance of the accessory genome in generating genetic variability within a bacterial population. PMID:19573249

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

PubMed

Alizadeh, Rahman; Afzal, Mohd; Arjmand, Farukh

2014-10-15

Bicyclic heterocyclic compounds viz. benzothiazoles are key components of deoxyribonucleic acid (DNA) molecules and participate directly in the encoding of genetic information. Benzothiazoles, therefore, represent a potent and selective class of antitumor compounds. The design and synthesis of chiral antitumor chemotherapeutic agents of Cu(II) and Zn(II), L- and -D benzothiazole Schiff base-valine complexes 1a &b and 2a &b, respectively were carried out and thoroughly characterized by spectroscopic and analytical techniques. Interaction of 1a and b and 2a and b with CT DNA by employing UV-vis, florescence, circular dichroic methods and cleavage studies of 1a with pBR322 plasmid, molecular docking were done in order to demonstrate their enantiomeric disposition toward the molecular drug target DNA. Interestingly, these studies unambiguously demonstrated the greater potency of L-enantiomer in comparison to D-enantiomer. Copyright © 2014 Elsevier B.V. All rights reserved.

Biological safety concepts of genetically modified live bacterial vaccines.

PubMed

Frey, Joachim

2007-07-26

Live vaccines possess the advantage of having access to induce cell-mediated and antibody-mediated immunity; thus in certain cases they are able to prevent infection, and not only disease. Furthermore, live vaccines, particularly bacterial live vaccines, are relatively cheap to produce and easy to apply. Hence they are suitable to immunize large communities or herds. The induction of both cell-mediated immunity as well as antibody-mediated immunity, which is particularly beneficial in inducing mucosal immune responses, is obtained by the vaccine-strain's ability to colonize and multiply in the host without causing disease. For this reason, live vaccines require attenuation of virulence of the bacterium to which immunity must be induced. Traditionally attenuation was achieved simply by multiple passages of the microorganism on growth medium, in animals, eggs or cell cultures or by chemical or physical mutagenesis, which resulted in random mutations that lead to attenuation. In contrast, novel molecular methods enable the development of genetically modified organisms (GMOs) targeted to specific genes that are particularly suited to induce attenuation or to reduce undesirable effects in the tissue in which the vaccine strains can multiply and survive. Since live vaccine strains (attenuated by natural selection or genetic engineering) are potentially released into the environment by the vaccinees, safety issues concerning the medical as well as environmental aspects must be considered. These involve (i) changes in cell, tissue and host tropism, (ii) virulence of the carrier through the incorporation of foreign genes, (iii) reversion to virulence by acquisition of complementation genes, (iv) exchange of genetic information with other vaccine or wild-type strains of the carrier organism and (v) spread of undesired genes such as antibiotic resistance genes. Before live vaccines are applied, the safety issues must be thoroughly evaluated case-by-case. Safety assessment includes knowledge of the precise function and genetic location of the genes to be mutated, their genetic stability, potential reversion mechanisms, possible recombination events with dormant genes, gene transfer to other organisms as well as gene acquisition from other organisms by phage transduction, transposition or plasmid transfer and cis- or trans-complementation. For this, GMOs that are constructed with modern techniques of genetic engineering display a significant advantage over random mutagenesis derived live organisms. The selection of suitable GMO candidate strains can be made under in vitro conditions using basic knowledge on molecular mechanisms of pathogenicity of the corresponding bacterial species rather than by in vivo testing of large numbers of random mutants. This leads to a more targeted safety testing on volunteers and to a reduction in the use of animal experimentation.

Exploration for the Salinity Tolerance-Related Genes from Xero-Halophyte Atriplex canescens Exploiting Yeast Functional Screening System

PubMed Central

Li, Jingtao; Sun, Xinhua; Liu, Yanzhi; Wang, Xueliang; Zhang, Hao; Pan, Hongyu

2017-01-01

Plant productivity is limited by salinity stress, both in natural and agricultural systems. Identification of salt stress-related genes from halophyte can provide insights into mechanisms of salt stress tolerance in plants. Atriplex canescens is a xero-halophyte that exhibits optimum growth in the presence of 400 mM NaCl. A cDNA library derived from highly salt-treated A. canescens plants was constructed based on a yeast expression system. A total of 53 transgenic yeast clones expressing enhanced salt tolerance were selected from 105 transformants. Their plasmids were sequenced and the gene characteristics were annotated using a BLASTX search. Retransformation of yeast cells with the selected plasmids conferred salt tolerance to the resulting transformants. The expression patterns of 28 of these stress-related genes were further investigated in A. canescens leaves by quantitative reverse transcription-PCR. In this study, we provided a rapid and robust assay system for large-scale screening of genes for varied abiotic stress tolerance with high efficiency in A. canescens. PMID:29149055

Genetic manipulation of Treponema denticola.

PubMed

Kuramitsu, Howard K; Chi, Bo; Ikegami, Akihiko

2005-07-01

The oral anaerobic spirochete, Treponema denticola, has been implicated in the etiology of human periodontal diseases; however, the molecular basis for the virulence of these organisms is still unclear. Potential pathogenic factors expressed by T. denticola have recently begun to be identified through the development of gene transfer approaches in this organism following electroporetic transformation. Several antibiotic resistance markers have been developed for use in the construction of monospecific mutants in these organisms. In addition, these antibiotic resistance cassettes have been more recently utilized to construct shuttle plasmids for complementation analysis of the mutants. These plasmids were also used to express heterologous spirochete genes in T. denticola. The transformation of other spirochetes such as T. phagedenis with these plasmids further suggests that it should be possible to develop similar gene transfer systems in other cultivable treponemes.

Detection of different β-lactamases encoding genes, including blaNDM, and plasmid-mediated quinolone resistance genes in different water sources from Brazil.

PubMed

Sanchez, Danilo Garcia; de Melo, Fernanda Maciel; Savazzi, Eduardo Angelino; Stehling, Eliana Guedes

2018-06-16

Bacterial resistance occurs by spontaneous mutations or horizontal gene transfer mediated by mobile genetic elements, which represents a great concern. Resistance to β-lactam antibiotics is mainly due to the production of β-lactamases, and an important mechanism of fluoroquinolone resistance is the acquisition plasmid determinants. The aim of this study was to verify the presence of β-lactamase-encoding genes and plasmid-mediated quinolone resistance genes in different water samples obtained from São Paulo state, Brazil. A high level of these resistance genes was detected, being the bla SHV , bla GES , and qnr the most prevalent. Besides that, the bla NDM gene, which codify an important and hazardous metallo-β-lactamase, was detected.

Genetic markers for detection of Escherichia coli K-12 harboring ampicillin-resistance plasmid from an industrial wastewater treatment effluent pond.

PubMed

Simões, G A R; Xavier, M A S; Oliveira, D A; Menezes, E V; Magalhães, S S G; Gandra, J A C D; Xavier, A R E O

2016-06-17

Biotechnology industries that use recombinant DNA technology are potential sources for release of genetically modified organisms to the environment. Antibiotic-resistance marker genes are commonly used for recombinant bacteria selection. One example is the marker gene coding for β-lactamase (bla) in plasmids found in Escherichia coli K-12. The aim of this study was to provide an approach to develop a molecular method for genetic marker detection in E. coli K-12 harboring bla genes from an industrial wastewater treatment effluent pond (IWTEP). For the detection of bla and Achromobacter lyticus protease I (api) genes in samples from IWTEP, we employed multiplex polymerase chain reaction (PCR) using E. coli K-12 genetic marker detection primers, previously described in the literature, and primers designed in our laboratory. The microbiological screening method resulted in 22 bacterial colony-forming units isolated from three different IWTEP harvesting points. The multiplex PCR amplicons showed that five isolates were positive for the bla gene marker and negative for the E. coli K-12 and api genes. The 16S rRNA regions of positive microorganisms carrying the bla gene were genotyped by the MicroSeq®500 system. The bacteria found were Escherichia spp (3/5), Chromobacterium spp (1/5), and Aeromonas spp (1/5). None of the 22 isolated microorganisms presented the molecular pattern of E. coli K-12 harboring the bla gene. The presence of microorganisms positive for the bla gene and negative for E. coli K-12 harboring bla genes at IWTEP suggests that the ampicillin resistance found in the isolated bacteria could be from microorganisms other than the E. coli K-12 strain harboring plasmid.

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.

Development of a novel genetically modified bioluminescent-bacteria-based assay for detection of fluoroquinolones in animal-derived foods.

PubMed

Cheng, Guyue; Dong, Xiaobing; Wang, Yulian; Peng, Dapeng; Wang, Xu; Hao, Haihong; Xie, Shuyu; Qu, Wei; Liu, Zhenli; Yuan, Zonghui

2014-12-01

Fluoroquinolones (FQNs) are broad-spectrum antibacterial agents widely used in animal husbandry and aquaculture. The residues and antimicrobial resistance of such antibiotics are a major public health concern. To realize multianalyte detection of FQN residues, a genetically modified bacterium, Escherichia coli pK12 harboring plasmid pRecAlux3, was constructed in this study to develop a bioluminescent-bacteria-based assay for the detection of FQNs in animal-derived foods. This assay was based on the principle of induction of an SOS response by FQNs via inducing the recA-promoter-fused luciferase reporter gene existing on the plasmid pRecAlux3. E. coli pK12 was able to recognize 11 FQNs: difloxacin, enrofloxacin, ciprofloxacin, sarafloxacin, norfloxacin, danofloxacin, ofloxacin, pefloxacin, lomefloxacin, marbofloxacin, and orbifloxacin. This method could be applied to 11 edible tissues, including milk, fish muscle, and the muscles, livers, and kidneys of cattle, chickens, and pigs, with a very simple and rapid sample extraction procedure using only phosphate-buffered saline. The limits of detection of the FQNs were between 12.5 and 100 μg kg(-1), all of which were lower than the maximum residue limits. Most of the recoveries of the FQNs were in the range from 60 to 120 %, and the interassay coefficients of variation were less than 30 %. This method, confirmed by high-performance liquid chromatography, is reliable and can be used as both a screening test and a semiquantitative assay, when the identity of a single type of FQN is known.

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.

Mobile genetic elements of Pseudomonas aeruginosa isolates from hydrotherapy facility and respiratory infections.

PubMed

Pereira, S G; Cardoso, O

2014-03-01

The content of mobile genetic elements in Pseudomonas aeruginosa isolates of a pristine natural mineral water system associated with healthcare was compared with clinical isolates from respiratory infections. One isolate, from the therapy pool circuit, presented a class 1 integron, with 100% similarity to a class 1 integron contained in plasmid p4800 of the Klebsiella pneumoniae Kp4800 strain, which is the first time it has been reported in P. aeruginosa. Class 1 integrons were found in 25.6% of the clinical isolates. PAGI1 orf3 was more prevalent in environmental isolates, while PAGI2 c105 and PAGI3 sg100 were more prevalent in clinical isolates. Plasmids were not observed in either population. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

CRISPR/Cas9-loxP-Mediated Gene Editing as a Novel Site-Specific Genetic Manipulation Tool.

PubMed

Yang, Fayu; Liu, Changbao; Chen, Ding; Tu, Mengjun; Xie, Haihua; Sun, Huihui; Ge, Xianglian; Tang, Lianchao; Li, Jin; Zheng, Jiayong; Song, Zongming; Qu, Jia; Gu, Feng

2017-06-16

Cre-loxP, as one of the site-specific genetic manipulation tools, offers a method to study the spatial and temporal regulation of gene expression/inactivation in order to decipher gene function. CRISPR/Cas9-mediated targeted genome engineering technologies are sparking a new revolution in biological research. Whether the traditional site-specific genetic manipulation tool and CRISPR/Cas9 could be combined to create a novel genetic tool for highly specific gene editing is not clear. Here, we successfully generated a CRISPR/Cas9-loxP system to perform gene editing in human cells, providing the proof of principle that these two technologies can be used together for the first time. We also showed that distinct non-homologous end-joining (NHEJ) patterns from CRISPR/Cas9-mediated gene editing of the targeting sequence locates at the level of plasmids (episomal) and chromosomes. Specially, the CRISPR/Cas9-mediated NHEJ pattern in the nuclear genome favors deletions (64%-68% at the human AAVS1 locus versus 4%-28% plasmid DNA). CRISPR/Cas9-loxP, a novel site-specific genetic manipulation tool, offers a platform for the dissection of gene function and molecular insights into DNA-repair pathways. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Multiplex engineering of industrial yeast genomes using CRISPRm.

PubMed

Ryan, Owen W; Cate, Jamie H D

2014-01-01

Global demand has driven the use of industrial strains of the yeast Saccharomyces cerevisiae for large-scale production of biofuels and renewable chemicals. However, the genetic basis of desired domestication traits is poorly understood because robust genetic tools do not exist for industrial hosts. We present an efficient, marker-free, high-throughput, and multiplexed genome editing platform for industrial strains of S. cerevisiae that uses plasmid-based expression of the CRISPR/Cas9 endonuclease and multiple ribozyme-protected single guide RNAs. With this multiplex CRISPR (CRISPRm) system, it is possible to integrate DNA libraries into the chromosome for evolution experiments, and to engineer multiple loci simultaneously. The CRISPRm tools should therefore find use in many higher-order synthetic biology applications to accelerate improvements in industrial microorganisms.

Genes involved in transitory recombination between phage M13 and plasmid pHV33.

PubMed Central

Dagert, M; Ehrlich, S D

1984-01-01

Plasmid pHV33 and phage M13 combine in Escherichia coli cells to form a chimera, which decombines to regenerate two parental genomes. Combination can occur via two genetic pathways, one defined by the recBC genes, the other by recA, recF and possibly recL genes. Decombination can also occur via two pathways, one defined again by the recBC genes, the other by a gene not identified, but active only in the absence of the recL gene product. PMID:6323172

Dissemination of blaOXA-58 in Proteus mirabilis isolates from Germany.

PubMed

Lange, Felix; Pfennigwerth, Niels; Gerigk, Sonja; Gohlke, Frank; Oberdorfer, Klaus; Purr, Ingvill; Wohanka, Nikolaus; Roggenkamp, Andreas; Gatermann, Sören G; Kaase, Martin

2017-05-01

Characterization of Proteus mirabilis isolates harbouring bla OXA-58 with emphasis on the genetic environment of this resistance determinant. Strains of P. mirabilis ( n  =   37) isolated from different patients were tested for the presence of bla OXA-58 . The genetic context of bla OXA-58 was determined by WGS of two strains and Sanger sequencing. Clonality of the strains was assessed by PFGE. Susceptibility testing was performed by microdilution according to EUCAST. Four strains isolated in different geographical regions of Germany were positive for bla OXA-58 , and WGS showed that this resistance gene was harboured on a plasmid. Sanger sequencing confirmed the presence of two nearly identical plasmids, 6219 and 6208 bp in size, in all four strains. Upstream of bla OXA-58 an IS Aba 3-like transposase gene was located. The P. mirabilis strains were not clonally related according to PFGE. MICs of meropenem for three of the strains were only just above the EUCAST breakpoint and the Carba NP test was positive for only two of the strains. To our knowledge, this is the first description of bla OXA-58 in the species P. mirabilis . The resistance gene is harboured by almost identical plasmids in strains not clonally related and from different geographical regions. Apart from an IS Aba 3-like transposase gene upstream of bla OXA-58 the genetic context is different from bla OXA-58 harboured on plasmids in the genus Acinetobacter . With MICs of meropenem well below the EUCAST breakpoint or only just above it and equivocal or false negative results from the Carba NP test, bla OXA-58 can be easily overlooked in P. mirabilis . © 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.

Optimization of a one-step heat-inducible in vivo mini DNA vector production system.

PubMed

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

2014-01-01

While safer than their viral counterparts, conventional circular covalently closed (CCC) plasmid DNA vectors offer a limited safety profile. They often result in the transfer of unwanted prokaryotic sequences, antibiotic resistance genes, and bacterial origins of replication that may lead to unwanted immunostimulatory responses. Furthermore, such vectors may impart the potential for chromosomal integration, thus potentiating oncogenesis. Linear covalently closed (LCC), bacterial sequence free DNA vectors have shown promising clinical improvements in vitro and in vivo. However, the generation of such minivectors has been limited by in vitro enzymatic reactions hindering their downstream application in clinical trials. We previously characterized an in vivo temperature-inducible expression system, governed by the phage λ pL promoter and regulated by the thermolabile λ CI[Ts]857 repressor to produce recombinant protelomerase enzymes in E. coli. In this expression system, induction of recombinant protelomerase was achieved by increasing culture temperature above the 37°C threshold temperature. Overexpression of protelomerase led to enzymatic reactions, acting on genetically engineered multi-target sites called "Super Sequences" that serve to convert conventional CCC plasmid DNA into LCC DNA minivectors. Temperature up-shift, however, can result in intracellular stress responses and may alter plasmid replication rates; both of which may be detrimental to LCC minivector production. We sought to optimize our one-step in vivo DNA minivector production system under various induction schedules in combination with genetic modifications influencing plasmid replication, processing rates, and cellular heat stress responses. We assessed different culture growth techniques, growth media compositions, heat induction scheduling and temperature, induction duration, post-induction temperature, and E. coli genetic background to improve the productivity and scalability of our system, achieving an overall LCC DNA minivector production efficiency of ∼ 90%.We optimized a robust technology conferring rapid, scalable, one-step in vivo production of LCC DNA minivectors with potential application to gene transfer-mediated therapeutics.

Genetic background of novel sequence types of CTX-M-8- and CTX-M-15-producing Escherichia coli and Klebsiella pneumoniae from public wastewater treatment plants in São Paulo, Brazil.

PubMed

Dropa, Milena; Lincopan, Nilton; Balsalobre, Livia C; Oliveira, Danielle E; Moura, Rodrigo A; Fernandes, Miriam Rodriguez; da Silva, Quézia Moura; Matté, Glavur R; Sato, Maria I Z; Matté, Maria H

2016-03-01

The release of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae to the environment is a public health issue worldwide. The aim of this study was to investigate the genetic background of genes encoding ESBLs in wastewater treatment plants (WWTPs) in São Paulo, southeastern Brazil. In 2009, during a local surveillance study, seven ESBL-producing Enterobacteriaceae strains were recovered from five WWTPs and screened for ESBL genes and mobile genetic elements. Multilocus sequence typing (MLST) was carried out, and wild plasmids were transformed into electrocompetent Escherichia coli. S1-PFGE technique was used to verify the presence of high molecular weight plasmids in wild-type strains and in bla ESBL-containing E. coli transformants. Strains harbored bla CTX-M-8, bla CTX-M-15, and/or bla SHV-28. Sequencing results showed that bla CTX-M-8 and bla CTX-M-15 genes were associated with IS26. MLST revealed new sequence types for E. coli (ST4401, ST4402, ST4403, and ST4445) and Klebsiella pneumoniae (ST1574), except for one K. pneumoniae from ST307 and Enterobacter cloacae from ST131. PCR and S1-PFGE results showed CTX-M-producing E. coli transformants carried heavy plasmids sizing 48.5-209 kb, which belonged to IncI1, IncF, and IncM1 incompatibility groups. This is the first report of CTX-M-8 and SHV-28 enzymes in environmental samples, and the present results demonstrate the plasmid-mediated spread of CTX-M-encoding genes through five WWTPs in São Paulo, Brazil, suggesting WWTPs are hotspots for the transfer of ESBL genes and confirming the urgent need to improve the management of sewage in order to minimize the dissemination of resistance genes to the environment.

Detection of qnrVC6, within a new genetic context, in a NDM-1-producing Citrobacter freundii clinical isolate from Uruguay.

PubMed

Bado, Inés; Ezdra, Romina Papa; Cordeiro, Nicolás; Outeda, Matilde; Caiata, Leticia; García-Fulgueiras, Virginia; Seija, Verónica; Vignoli, Rafael

2018-03-08

The objective of the present study was to characterise the mechanisms underlying quinolone and oxyimino-cephalosporin resistance in a Citrobacter freundii clinical isolate obtained from the ICU in Uruguay's University Hospital. Citrobacter freundii strain CF638 was isolated from a urine culture. Identification and susceptibility testing were performed using the VITEK ® 2 system, and MIC determination and disc diffusion assay, respectively. Resistance genes and mobile genetic elements were identified, by PCR and sequencing. Plasmid transfer was assessed by conjugation; plasmid, size was estimated by treatment with S1 and PFGE. Plasmid incompatibility, group and toxin-antitoxin systems were sought by PCR. Strain CF638 showed a multi-drug resistant profile, including, resistance to carbapenemes and quinolones. Transconjugant TcCF638, harbouring a ∼200 kb IncA/C plasmid, also showed resistance to all β-lactams, (except for aztreonam), and diminished susceptibility to ciprofloxacin. PCR, assays were positive for bla NDM-1 and qnrVC in CF638 and TcCF638. Two different class 1 integrons were detected, In127 and In907. In127, featured the genetic array: aadA2-ltr2. Conversely, complex In907 featured, two variable regions (VR); VR-1 consisted of aadB-bla OXA-10 -aadA1cc, whereas, VR-2, featured a gene qnrVC6 108 bp downstream from the ISCR1 and 45 bp, upstream from the qacEΔ1. Expression of qnrVC6 would be on account of a, putative promoter region, detected using the Neural Network Promoter, Prediction program. To the best of our knowledge this constitutes the first report of a qnrVC gene within a complex class 1 integron, and the first report as well of the occurrence of such gene in an NDM-1-producing enterobacterial clinical isolate. Copyright © 2018. Published by Elsevier Ltd.

Optimization of a One-Step Heat-Inducible In Vivo Mini DNA Vector Production System

PubMed Central

Wettig, Shawn; Slavcev, Roderick A.

2014-01-01

While safer than their viral counterparts, conventional circular covalently closed (CCC) plasmid DNA vectors offer a limited safety profile. They often result in the transfer of unwanted prokaryotic sequences, antibiotic resistance genes, and bacterial origins of replication that may lead to unwanted immunostimulatory responses. Furthermore, such vectors may impart the potential for chromosomal integration, thus potentiating oncogenesis. Linear covalently closed (LCC), bacterial sequence free DNA vectors have shown promising clinical improvements in vitro and in vivo. However, the generation of such minivectors has been limited by in vitro enzymatic reactions hindering their downstream application in clinical trials. We previously characterized an in vivo temperature-inducible expression system, governed by the phage λ pL promoter and regulated by the thermolabile λ CI[Ts]857 repressor to produce recombinant protelomerase enzymes in E. coli. In this expression system, induction of recombinant protelomerase was achieved by increasing culture temperature above the 37°C threshold temperature. Overexpression of protelomerase led to enzymatic reactions, acting on genetically engineered multi-target sites called “Super Sequences” that serve to convert conventional CCC plasmid DNA into LCC DNA minivectors. Temperature up-shift, however, can result in intracellular stress responses and may alter plasmid replication rates; both of which may be detrimental to LCC minivector production. We sought to optimize our one-step in vivo DNA minivector production system under various induction schedules in combination with genetic modifications influencing plasmid replication, processing rates, and cellular heat stress responses. We assessed different culture growth techniques, growth media compositions, heat induction scheduling and temperature, induction duration, post-induction temperature, and E. coli genetic background to improve the productivity and scalability of our system, achieving an overall LCC DNA minivector production efficiency of ∼90%.We optimized a robust technology conferring rapid, scalable, one-step in vivo production of LCC DNA minivectors with potential application to gene transfer-mediated therapeutics. PMID:24586704

Genetic evolution and clinical impact in extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae.

PubMed

Chong, Yong; Ito, Yoshikiyo; Kamimura, Tomohiko

2011-10-01

The emergence of extended-spectrum β-lactamase (ESBL)-producing bacteria, particularly Escherichia coli and Klebsiella pneumoniae, is now a critical concern for the development of therapies against bacterial infection. ESBLs consist of three major genetic groups: TEM, SHV, and CTX-M types. Nosocomial infections due to TEM and SHV-producing K. pneumoniae strains were frequently documented until the late 1990s. The number of reports on community-acquired infections caused by CTX-M-producing E. coli strains have dramatically increased over the last decade; however, K. pneumoniae strains, of either the TEM or SHV types, are persistent and important ESBL producers. The spread of ESBL genes is associated with various mobile genetic elements, such as transposons, insertion sequences, and integrons. The rapid dissemination of ESBL genes of the CTX-M type may be related to highly complicated genetic structures. These structures harboring ESBL genes and mobile elements are found in a variety of plasmids, which often carry many other antibiotic resistance genes. Multidrug-resistant CTX-M-15-producing E. coli strains disseminate worldwide. Efficient mobile elements and plasmids may have accelerated the genetic diversity and the rapid spread of ESBL genes, and their genetic evolution has caused an emerging threat to the bacteria for which few effective drugs have been identified. Copyright © 2011 Elsevier B.V. All rights reserved.

Non-GMO genetically edited crop plants.

PubMed

Kanchiswamy, Chidananda Nagamangala; Malnoy, Mickael; Velasco, Riccardo; Kim, Jin-Soo; Viola, Roberto

2015-09-01

Direct delivery of purified Cas9 protein with guide RNA into plant cells, as opposed to plasmid-mediated delivery, displays high efficiency and reduced off-target effects. Following regeneration from edited cells, the ensuing plant is also likely to bypass genetically modified organism (GMO) legislation as the genome editing complex is degraded in the recipient cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

An Archaeal Immune System Can Detect Multiple Protospacer Adjacent Motifs (PAMs) to Target Invader DNA*

PubMed Central

Fischer, Susan; Maier, Lisa-Katharina; Stoll, Britta; Brendel, Jutta; Fischer, Eike; Pfeiffer, Friedhelm; Dyall-Smith, Mike; Marchfelder, Anita

2012-01-01

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system provides adaptive and heritable immunity against foreign genetic elements in most archaea and many bacteria. Although this system is widespread and diverse with many subtypes, only a few species have been investigated to elucidate the precise mechanisms for the defense of viruses or plasmids. Approximately 90% of all sequenced archaea encode CRISPR/Cas systems, but their molecular details have so far only been examined in three archaeal species: Sulfolobus solfataricus, Sulfolobus islandicus, and Pyrococcus furiosus. Here, we analyzed the CRISPR/Cas system of Haloferax volcanii using a plasmid-based invader assay. Haloferax encodes a type I-B CRISPR/Cas system with eight Cas proteins and three CRISPR loci for which the identity of protospacer adjacent motifs (PAMs) was unknown until now. We identified six different PAM sequences that are required upstream of the protospacer to permit target DNA recognition. This is only the second archaeon for which PAM sequences have been determined, and the first CRISPR group with such a high number of PAM sequences. Cells could survive the plasmid challenge if their CRISPR/Cas system was altered or defective, e.g. by deletion of the cas gene cassette. Experimental PAM data were supplemented with bioinformatics data on Haloferax and Haloquadratum. PMID:22767603

Building of Reusable Reverse Logistics Model and its Optimization Considering the Decision of Backorder or Next Arrival of Goods

NASA Astrophysics Data System (ADS)

Lee, Jeong-Eun; Gen, Mitsuo; Rhee, Kyong-Gu; Lee, Hee-Hyol

This paper deals with the building of the reusable reverse logistics model considering the decision of the backorder or the next arrival of goods. The optimization method to minimize the transportation cost and to minimize the volume of the backorder or the next arrival of goods occurred by the Just in Time delivery of the final delivery stage between the manufacturer and the processing center is proposed. Through the optimization algorithms using the priority-based genetic algorithm and the hybrid genetic algorithm, the sub-optimal delivery routes are determined. Based on the case study of a distilling and sale company in Busan in Korea, the new model of the reusable reverse logistics of empty bottles is built and the effectiveness of the proposed method is verified.

Identification, Characterization, and Application of the Replicon Region of the Halophilic Temperate Sphaerolipovirus SNJ1.

PubMed

Wang, Yuchen; Sima, Linshan; Lv, Jie; Huang, Suiyuan; Liu, Ying; Wang, Jiao; Krupovic, Mart; Chen, Xiangdong

2016-07-15

The temperate haloarchaeal virus SNJ1 displays lytic and lysogenic life cycles. During the lysogenic cycle, the virus resides in its host, Natrinema sp. strain J7-1, in the form of an extrachromosomal circular plasmid, pHH205. In this study, a 3.9-kb region containing seven predicted genes organized in two operons was identified as the minimal replicon of SNJ1. Only RepA, encoded by open reading frame 11-12 (ORF11-12), was found to be essential for replication, and its expression increased during the lytic cycle. Sequence analysis suggested that RepA is a distant homolog of HUH endonucleases, a superfamily that includes rolling-circle replication initiation proteins from various viruses and plasmids. In addition to RepA, two genetic elements located within both termini of the 3.9-kb replicon were also required for SNJ1 replication. SNJ1 genome and SNJ1 replicon-based shuttle vectors were present at 1 to 3 copies per chromosome. However, the deletion of ORF4 significantly increased the SNJ1 copy number, suggesting that the product of ORF4 is a negative regulator of SNJ1 abundance. Shuttle vectors based on the SNJ1 replicon were constructed and validated for stable expression of heterologous proteins, both in J7 derivatives and in Natrinema pallidum JCM 8980(T), suggesting their broad applicability as genetic tools for Natrinema species. Archaeal viruses exhibit striking morphological diversity and unique gene content. In this study, the minimal replicon of the temperate haloarchaeal virus SNJ1 was identified. A number of ORFs and genetic elements controlling virus genome replication, maintenance, and copy number were characterized. In addition, based on the replicon, a novel expression shuttle vector has been constructed and validated for protein expression and purification in Natrinema sp. CJ7 and Natrinema pallidum JCM 8980(T) This study not only provided mechanistic and functional insights into SNJ1 replication but also led to the development of useful genetic tools to investigate SNJ1 and other viruses infecting Natrinema species as well as their hosts. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Dextransucrase Expression Is Concomitant with that of Replication and Maintenance Functions of the pMN1 Plasmid in Lactobacillus sakei MN1

PubMed Central

Nácher-Vázquez, Montserrat; Ruiz-Masó, José A.; Mohedano, María L.; del Solar, Gloria; Aznar, Rosa; López, Paloma

2017-01-01

The exopolysaccharide synthesized by Lactobacillus sakei MN1 is a dextran with antiviral and immunomodulatory properties of potential utility in aquaculture. In this work we have investigated the genetic basis of dextran production by this bacterium. Southern blot hybridization experiments demonstrated the plasmidic location of the dsrLS gene, which encodes the dextransucrase involved in dextran synthesis. DNA sequencing of the 11,126 kbp plasmid (pMN1) revealed that it belongs to a family which replicates by the theta mechanism, whose prototype is pUCL287. The plasmid comprises the origin of replication, repA, repB, and dsrLS genes, as well as seven open reading frames of uncharacterized function. Lb. sakei MN1 produces dextran when sucrose, but not glucose, is present in the growth medium. Therefore, plasmid copy number and stability, as well as dsrLS expression, were investigated in cultures grown in the presence of either sucrose or glucose. The results revealed that pMN1 is a stable low-copy-number plasmid in both conditions. Gene expression studies showed that dsrLS is constitutively expressed, irrespective of the carbon source present in the medium. Moreover, dsrLS is expressed from a monocistronic transcript as well as from a polycistronic repA-repB-orf1-dsrLS mRNA. To our knowledge, this is the first report of a plasmid-borne dextransucrase-encoding gene, as well as the first time that co-transcription of genes involved in plasmid maintenance and replication with a gene encoding an enzyme has been established. PMID:29209293

PiggyBac transposon-mediated gene delivery efficiently generates stable transfectants derived from cultured primary human deciduous tooth dental pulp cells (HDDPCs) and HDDPC-derived iPS cells.

PubMed

Inada, Emi; Saitoh, Issei; Watanabe, Satoshi; Aoki, Reiji; Miura, Hiromi; Ohtsuka, Masato; Murakami, Tomoya; Sawami, Tadashi; Yamasaki, Youichi; Sato, Masahiro

2015-09-14

The ability of human deciduous tooth dental pulp cells (HDDPCs) to differentiate into odontoblasts that generate mineralized tissue holds immense potential for therapeutic use in the field of tooth regenerative medicine. Realization of this potential depends on efficient and optimized protocols for the genetic manipulation of HDDPCs. In this study, we demonstrate the use of a PiggyBac (PB)-based gene transfer system as a method for introducing nonviral transposon DNA into HDDPCs and HDDPC-derived inducible pluripotent stem cells. The transfection efficiency of the PB-based system was significantly greater than previously reported for electroporation-based transfection of plasmid DNA. Using the neomycin resistance gene as a selection marker, HDDPCs were stably transfected at a rate nearly 40-fold higher than that achieved using conventional methods. Using this system, it was also possible to introduce two constructs simultaneously into a single cell. The resulting stable transfectants, expressing tdTomato and enhanced green fluorescent protein, exhibited both red and green fluorescence. The established cell line did not lose the acquired phenotype over three months of culture. Based on our results, we concluded that PB is superior to currently available methods for introducing plasmid DNA into HDDPCs. There may be significant challenges in the direct clinical application of this method for human dental tissue engineering due to safety risks and ethical concerns. However, the high level of transfection achieved with PB may have significant advantages in basic scientific research for dental tissue engineering applications, such as functional studies of genes and proteins. Furthermore, it is a useful tool for the isolation of genetically engineered HDDPC-derived stem cells for studies in tooth regenerative medicine.

CRISPR-Cas systems target a diverse collection of invasive mobile genetic elements in human microbiomes

PubMed Central

2013-01-01

Background Bacteria and archaea develop immunity against invading genomes by incorporating pieces of the invaders' sequences, called spacers, into a clustered regularly interspaced short palindromic repeats (CRISPR) locus between repeats, forming arrays of repeat-spacer units. When spacers are expressed, they direct CRISPR-associated (Cas) proteins to silence complementary invading DNA. In order to characterize the invaders of human microbiomes, we use spacers from CRISPR arrays that we had previously assembled from shotgun metagenomic datasets, and identify contigs that contain these spacers' targets. Results We discover 95,000 contigs that are putative invasive mobile genetic elements, some targeted by hundreds of CRISPR spacers. We find that oral sites in healthy human populations have a much greater variety of mobile genetic elements than stool samples. Mobile genetic elements carry genes encoding diverse functions: only 7% of the mobile genetic elements are similar to known phages or plasmids, although a much greater proportion contain phage- or plasmid-related genes. A small number of contigs share similarity with known integrative and conjugative elements, providing the first examples of CRISPR defenses against this class of element. We provide detailed analyses of a few large mobile genetic elements of various types, and a relative abundance analysis of mobile genetic elements and putative hosts, exploring the dynamic activities of mobile genetic elements in human microbiomes. A joint analysis of mobile genetic elements and CRISPRs shows that protospacer-adjacent motifs drive their interaction network; however, some CRISPR-Cas systems target mobile genetic elements lacking motifs. Conclusions We identify a large collection of invasive mobile genetic elements in human microbiomes, an important resource for further study of the interaction between the CRISPR-Cas immune system and invaders. PMID:23628424

Purification and Genetic Characterization of Enterocin I from Enterococcus faecium 6T1a, a Novel Antilisterial Plasmid-Encoded Bacteriocin Which Does Not Belong to the Pediocin Family of Bacteriocins

PubMed Central

Floriano, Belén; Ruiz-Barba, José L.; Jiménez-Díaz, Rufino

1998-01-01

Enterocin I (ENTI) is a novel bacteriocin produced by Enterococcus faecium 6T1a, a strain originally isolated from a Spanish-style green olive fermentation. The bacteriocin is active against many olive spoilage and food-borne gram-positive pathogenic bacteria, including clostridia, propionibacteria, and Listeria monocytogenes. ENTI was purified to homogeneity by ammonium sulfate precipitation, binding to an SP-Sepharose fast-flow column, and phenyl-Sepharose CL-4B and C2/C18 reverse-phase chromatography. The purification procedure resulted in a final yield of 954% and a 170,000-fold increase in specific activity. The primary structure of ENTI was determined by amino acid and nucleotide sequencing. ENTI consists of 44 amino acids and does not show significant sequence similarity with any other previously described bacteriocin. Sequencing of the entI structural gene, which is located on the 23-kb plasmid pEF1 of E. faecium 6T1a, revealed the absence of a leader peptide at the N-terminal region of the gene product. A second open reading frame, ORF2, located downstream of entI, encodes a putative protein that is 72.7% identical to ENTI. entI and ORF2 appear to be cotranscribed, yielding an mRNA of ca. 0.35 kb. A gene encoding immunity to ENTI was not identified. However, curing experiments demonstrated that both enterocin production and immunity are conferred by pEF1. PMID:9835578

A Genetic System for the Thermophilic Acetogenic Bacterium Thermoanaerobacter kivui.

PubMed

Basen, Mirko; Geiger, Irina; Henke, Laura; Müller, Volker

2018-02-01

Thermoanaerobacter kivui is one of the very few thermophilic acetogenic microorganisms. It grows optimally at 66°C on sugars but also lithotrophically with H 2 + CO 2 or with CO, producing acetate as the major product. While a genome-derived model of acetogenesis has been developed, only a few physiological or biochemical experiments regarding the function of important enzymes in carbon and energy metabolism have been carried out. To address this issue, we developed a method for targeted markerless gene deletions and for integration of genes into the genome of T. kivui The strain naturally took up plasmid DNA in the exponential growth phase, with a transformation frequency of up to 3.9 × 10 -6 A nonreplicating plasmid and selection with 5-fluoroorotate was used to delete the gene encoding the orotate phosphoribosyltransferase ( pyrE ), resulting in a Δ pyrE uracil-auxotrophic strain, TKV002. Reintroduction of pyrE on a plasmid or insertion of pyrE into different loci within the genome restored growth without uracil. We subsequently studied fructose metabolism in T. kivui The gene fruK (TKV_c23150) encoding 1-phosphofructosekinase (1-PFK) was deleted, using pyrE as a selective marker via two single homologous recombination events. The resulting Δ fruK strain, TKV003, did not grow on fructose; however, growth on glucose (or on mannose) was unaffected. The combination of pyrE as a selective marker and the natural competence of the strain for DNA uptake will be the basis for future studies on CO 2 reduction and energy conservation and their regulation in this thermophilic acetogenic bacterium. IMPORTANCE Acetogenic bacteria are currently the focus of research toward biotechnological applications due to their potential for de novo synthesis of carbon compounds such as acetate, butyrate, or ethanol from H 2 + CO 2 or from synthesis gas. Based on available genome sequences and on biochemical experiments, acetogens differ in their energy metabolism. Thus, there is an urgent need to understand the carbon and electron flows through the Wood-Ljungdahl pathway and their links to energy conservation, which requires genetic manipulations such as deletion or overexpression of genes encoding putative key enzymes. Unfortunately, genetic systems have been reported for only a few acetogenic bacteria. Here, we demonstrate proof of concept for the genetic modification of the thermophilic acetogenic species Thermoanaerobacter kivui The genetic system will be used to study genes involved in biosynthesis and energy metabolism, and may further be applied to metabolically engineer T. kivui to produce fuels and chemicals. Copyright © 2018 American Society for Microbiology.

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.

Development and Evaluation of Event-Specific Quantitative PCR Method for Genetically Modified Soybean MON87701.

PubMed

Tsukahara, Keita; Takabatake, Reona; Masubuchi, Tomoko; Futo, Satoshi; Minegishi, Yasutaka; Noguchi, Akio; Kondo, Kazunari; Nishimaki-Mogami, Tomoko; Kurashima, Takeyo; Mano, Junichi; Kitta, Kazumi

2016-01-01

A real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) soybean event, MON87701. First, a standard plasmid for MON87701 quantification was constructed. The conversion factor (C f ) required to calculate the amount of genetically modified organism (GMO) was experimentally determined for a real-time PCR instrument. The determined C f for the real-time PCR instrument was 1.24. For the evaluation of the developed method, a blind test was carried out in an inter-laboratory trial. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSDr), respectively. The determined biases and the RSDr values were less than 30 and 13%, respectively, at all evaluated concentrations. The limit of quantitation of the method was 0.5%, and the developed method would thus be applicable for practical analyses for the detection and quantification of MON87701.

Exploiting Multisite Gateway and pENFRUIT plasmid collection for fruit genetic engineering.

PubMed

Estornell, Leandro H; Granell, Antonio; Orzaez, Diego

2012-01-01

MultiSite Gateway cloning techniques based on homologous recombination facilitate the combinatorial assembly of basic genetic pieces (i.e., promoters, CDS, and terminators) into gene expression or gene silencing cassettes. pENFRUIT is a collection of MultiSite Triple Gateway Entry vectors dedicated to genetic engineering in fruits. It comprises a number of fruit-operating promoters as well as C-terminal tags adapted to the Gateway standard. In this way, flanking regulatory/labeling sequences can be easily Gateway-assembled with a given gene of interest for its ectopic expression or silencing in fruits. The resulting gene constructs can be analyzed in stable transgenic plants or in transient expression assays, the latter allowing fast testing of the increasing number of combinations arising from MultiSite methodology. A detailed description of the use of MultiSite cloning methodology for the assembly of pENFRUIT elements is presented.

Genetic discovery in Xylella fastidiosa through sequence analysis of selected randomly amplified polymorphic DNAs.

PubMed

Chen, Jianchi; Civerolo, Edwin L; Jarret, Robert L; Van Sluys, Marie-Anne; de Oliveira, Mariana C

2005-02-01

Xylella fastidiosa causes many important plant diseases including Pierce's disease (PD) in grape and almond leaf scorch disease (ALSD). DNA-based methodologies, such as randomly amplified polymorphic DNA (RAPD) analysis, have been playing key roles in genetic information collection of the bacterium. This study further analyzed the nucleotide sequences of selected RAPDs from X. fastidiosa strains in conjunction with the available genome sequence databases and unveiled several previously unknown novel genetic traits. These include a sequence highly similar to those in the phage family of Podoviridae. Genome comparisons among X. fastidiosa strains suggested that the "phage" is currently active. Two other RAPDs were also related to horizontal gene transfer: one was part of a broadly distributed cryptic plasmid and the other was associated with conjugal transfer. One RAPD inferred a genomic rearrangement event among X. fastidiosa PD strains and another identified a single nucleotide polymorphism of evolutionary value.

Induction of a robust immune response against avian influenza virus following transdermal inoculation with H5-DNA vaccine formulated in modified dendrimer-based delivery system in mouse model.

PubMed

Bahadoran, Azadeh; Ebrahimi, Mehdi; Yeap, Swee Keong; Safi, Nikoo; Moeini, Hassan; Hair-Bejo, Mohd; Hussein, Mohd Zobir; Omar, Abdul Rahman

2017-01-01

This study was aimed to evaluate the immunogenicity of recombinant plasmid deoxyribonucleic acid (DNA), pBud-H5-green fluorescent protein (GFP)-interferon-regulatory factor (IRF)3 following delivery using polyamidoamine (PAMAM) dendrimer and transactivator of transcription (TAT)-conjugated PAMAM dendrimer as well as the effect of IRF3 as the genetic adjuvant. BALB/c mice were vaccinated transdermally with pBud-H5-GFP, PAMAM/pBud-H5-GFP, TAT-PAMAM/pBud-H5-GFP, and TAT-PAMAM/pBud-H5-GFP-IRF3. The expression analysis of H5 gene from the blood by using quantitative real-time reverse transcriptase polymerase chain reaction confirmed the ability of PAMAM dendrimer as a carrier for gene delivery, as well as the ability of TAT peptide to enhance the delivery efficiency of PAMAM dendrimer. Mice immunized with modified PAMAM by TAT peptide showed higher hemagglutination inhibition titer, and larger CD3 + /CD4 + T cells and CD3 + /CD8 + T cells population, as well as the production of cytokines, namely, interferon (IFN)-γ, interleukin (IL)-2, IL-15, IL-12, IL-6, and tumor necrosis factor-α compared with those immunized with native PAMAM. These results suggest that the function of TAT peptide as a cell-penetrating peptide is able to enhance the gene delivery, which results in rapid distribution of H5 in the tissues of the immunized mice. Furthermore, pBud-H5-GFP co-expressing IRF3 as a genetic adjuvant demonstrated the highest hemagglutination inhibition titer besides larger CD3 + /CD4 + and CD3 + /CD8 + T cells population, and strong Th1-like cytokine responses among all the systems tested. In conclusion, TAT-PAMAM dendrimer-based delivery system with IRF3 as a genetic adjuvant is an attractive transdermal DNA vaccine delivery system utilized to evaluate the efficacy of the developed DNA vaccine in inducing protection during challenge with virulent H5N1 virus.

Genetic modification of bone-marrow mesenchymal stem cells and hematopoietic cells with human coagulation factor IX-expressing plasmids.

PubMed

Sam, Mohammad Reza; Azadbakhsh, Azadeh Sadat; Farokhi, Farrah; Rezazadeh, Kobra; Sam, Sohrab; Zomorodipour, Alireza; Haddad-Mashadrizeh, Aliakbar; Delirezh, Nowruz; Mokarizadeh, Aram

2016-05-01

Ex-vivo gene therapy of hemophilias requires suitable bioreactors for secretion of hFIX into the circulation and stem cells hold great potentials in this regard. Viral vectors are widely manipulated and used to transfer hFIX gene into stem cells. However, little attention has been paid to the manipulation of hFIX transgene itself. Concurrently, the efficacy of such a therapeutic approach depends on determination of which vectors give maximal transgene expression. With this in mind, TF-1 (primary hematopoietic lineage) and rat-bone marrow mesenchymal stem cells (BMSCs) were transfected with five hFIX-expressing plasmids containing different combinations of two human β-globin (hBG) introns inside the hFIX-cDNA and Kozak element and hFIX expression was evaluated by different methods. In BMSCs and TF-1 cells, the highest hFIX level was obtained from the intron-less and hBG intron-I,II containing plasmids respectively. The highest hFIX activity was obtained from the cells that carrying the hBG intron-I,II containing plasmids. BMSCs were able to produce higher hFIX by 1.4 to 4.7-fold increase with activity by 2.4 to 4.4-fold increase compared to TF-1 cells transfected with the same constructs. BMSCs and TF-1 cells could be effectively bioengineered without the use of viral vectors and hFIX minigene containing hBG introns could represent a particular interest in stem cell-based gene therapy of hemophilias. Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Directed and persistent movement arises from mechanochemistry of the ParA/ParB system

PubMed Central

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

2015-01-01

The segregation of DNA before cell division is essential for faithful genetic inheritance. In many bacteria, segregation of low-copy number plasmids involves an active partition system composed of a nonspecific DNA-binding ATPase, ParA, and its stimulator protein ParB. The ParA/ParB system drives directed and persistent movement of DNA cargo both in vivo and in vitro. Filament-based models akin to actin/microtubule-driven motility were proposed for plasmid segregation mediated by ParA. Recent experiments challenge this view and suggest that ParA/ParB system motility is driven by a diffusion ratchet mechanism in which ParB-coated plasmid both creates and follows a ParA gradient on the nucleoid surface. However, the detailed mechanism of ParA/ParB-mediated directed and persistent movement remains unknown. Here, we develop a theoretical model describing ParA/ParB-mediated motility. We show that the ParA/ParB system can work as a Brownian ratchet, which effectively couples the ATPase-dependent cycling of ParA–nucleoid affinity to the motion of the ParB-bound cargo. Paradoxically, this resulting processive motion relies on quenching diffusive plasmid motion through a large number of transient ParA/ParB-mediated tethers to the nucleoid surface. Our work thus sheds light on an emergent phenomenon in which nonmotor proteins work collectively via mechanochemical coupling to propel cargos—an ingenious solution shaped by evolution to cope with the lack of processive motor proteins in bacteria. PMID:26647183

Genomic Analysis of Hospital Plumbing Reveals Diverse Reservoir of Bacterial Plasmids Conferring Carbapenem Resistance

PubMed Central

2018-01-01

ABSTRACT The hospital environment is a potential reservoir of bacteria with plasmids conferring carbapenem resistance. Our Hospital Epidemiology Service routinely performs extensive sampling of high-touch surfaces, sinks, and other locations in the hospital. Over a 2-year period, additional sampling was conducted at a broader range of locations, including housekeeping closets, wastewater from hospital internal pipes, and external manholes. We compared these data with previously collected information from 5 years of patient clinical and surveillance isolates. Whole-genome sequencing and analysis of 108 isolates provided comprehensive characterization of blaKPC/blaNDM-positive isolates, enabling an in-depth genetic comparison. Strikingly, despite a very low prevalence of patient infections with blaKPC-positive organisms, all samples from the intensive care unit pipe wastewater and external manholes contained carbapenemase-producing organisms (CPOs), suggesting a vast, resilient reservoir. We observed a diverse set of species and plasmids, and we noted species and susceptibility profile differences between environmental and patient populations of CPOs. However, there were plasmid backbones common to both populations, highlighting a potential environmental reservoir of mobile elements that may contribute to the spread of resistance genes. Clear associations between patient and environmental isolates were uncommon based on sequence analysis and epidemiology, suggesting reasonable infection control compliance at our institution. Nonetheless, a probable nosocomial transmission of Leclercia sp. from the housekeeping environment to a patient was detected by this extensive surveillance. These data and analyses further our understanding of CPOs in the hospital environment and are broadly relevant to the design of infection control strategies in many infrastructure settings. PMID:29437920

Detection of New Delhi Metallo-β-Lactamase Variants NDM-4, NDM-5, and NDM-7 in Enterobacter aerogenes Isolated from a Neonatal Intensive Care Unit of a North India Hospital: A First Report.

PubMed

Ahmad, Nayeem; Ali, Syed Manazir; Khan, Asad U

2018-03-01

A total 402 Enterobacteriaceae isolates were recovered from blood and rectal swabs of 1,000 infants admitted to the Neonatal Intensive Care Unit (NICU) of the Jawaharlal Medical College and Hospital Aligarh, India. Carbapenamase producers were determined by Carba NP phenotype biochemical assay. Out of 402 isolates, it was the first time three of the isolates were identified as Enterobacter aerogenes carrying bla NDM-4, bla NDM-5, and bla NDM-7 genes. These genes were identified by polymerase chain reaction (PCR) and sequence analysis. The isolates were further characterized to know the plasmid type and genetic environment features, including integron and IS elements. All the three E. aerogenes isolates (AK-93, AK-95, and AK-96) were resistant to all β-lactams, including carbapenems. The β-lactamase genes bla OXA-1 , bla OXA-9, bla SHV-1 , and bla VIM-2 were also found to be coassociated with bla NDM-4 in AK-93, bla OXA-1 , bla OXA-9, and bla CMY-149 were found to be coexisted with bla NDM-5 in AK-95, and bla OXA-1; bla OXA-9, and bla CMY-145 were also found to be coassociated with bla NDM-7 in AK-96, identified by PCR analysis. Plasmid-based replicon typing revealed plasmids of different incompatibility in E. aerogenes in each of the isolates, AK-93 AK-95, and AK-96, respectively. ERIC-PCR was performed for the analysis of genetic relatedness of the strains. We found bla NDM-4 , bla NDM-5, and bla NDM-7 producing three E. aerogenes strains, which were not clonally related. Genetic environment analysis revealed the presence of bleomycin resistance gene (ble MBL ) to downstream of bla NDM and complete ISAba125 sequence were found upstream of bla NDM in all the three variants of these isolates. This is the first time we have identified bla NDM-4 , bla NDM-5, and bla NDM-7 in E. aerogenes species, isolated from the NICU of a tertiary care hospital in India.

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.

Genetic diversity of human isolates of Salmonella enterica serovar Enteritidis in Malaysia.

PubMed

Bakeri, S A; Yasin, R M; Koh, Y T; Puthucheary, S D; Thong, K L

2003-01-01

The study was undertaken to determine clonal relationship and genetic diversity of the human strains of Salmonella enterica serovar Enteritidis isolated from 1995 to 2002 from different parts of Malaysia. Antimicrobial susceptibility test, plasmid profiling and pulsed-field gel electrophoresis were applied to analyse 65 human isolates of S. Enteritidis obtained over an eight year period from different parts of Malaysia. Four nonhuman isolates were included for comparison. A total of 14 distinct XbaI-pulsed-field profiles (PFPs) were observed, although a single PFP X1 was predominant and this particular clone was found to be endemic in Malaysia. The incidence of drug resistant S. Enteritidis remained relatively low with only 37% of the strains analysed being resistant to one or more antimicrobial agents. All except one resistant strain carried at least one plasmid ranging in size from 3.7 to 62 MDa giving nine plasmid profiles. The three isolates from raw milk and one from well-water had similar PFPs to that of the human isolates. Salmonella Enteritidis strains were more diverse than was previously thought. Fourteen subtypes were noted although one predominant clone persisted in Malaysia. The combination of pulsed-field gel electrophoresis, plasmid profiling and antibiograms provided additional discrimination to the highly clonal strains of S. Enteritidis. This is the first report to assess the genotypes of the predominant clinical S. Enteritidis in different parts of the country. As S. Enteritidis is highly endemic in Malaysia, the data generated would be useful for tracing the source during outbreaks of gastroenteritis in the study area.

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.

A circular genetic map of Erwinia carotovora subsp. atroseptica 3-2

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

Nikolaichik, E.A.; Pesnyakevich, A.G.

1995-08-01

A circular genetic map of Erwinia carotovora subsp. atroseptica 3-2 was constructed on the basis of the R471a plasmid and Tn5 and Tn9 using Hfr-like donors. Forty-six genes, including phytopathogenicity genes, were located on the basis of interrupted mating experiment results and analysis of coinheritance of markers on a map of 183 min in length. The similarity and differences of chromosomal genetic maps of Erwinia genus bacteria are discussed. 23 refs., 2 figs., 4 tabs.

Thermophilic Gram-Positive Biocatalysts for Biomass Conversion to Ethanol

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

Shanmugam, K.T.; Ingram, L.O.; Maupin-Furlow, J.A.

2003-12-01

Production of energy from renewable sources is receiving increased attention due to the finite nature of fossil fuels and the environmental impact associated with the continued large scale use of fossil energy sources. Biomass, a CO2-neutral abundant resource, is an attractive alternate source of energy. Biomass-derived sugars, such as glucose, xylose, and other minor sugars, can be readily fermented to fuel ethanol and commodity chemicals. Extracellular cellulases produced by fungi are commercially developed for depolymerization of cellulose in biomass to glucose for fermentation by appropriate biocatalysts in a simultaneous saccharification and fermentation (SSF) process. Due to the differences in themore » optimum conditions for the activity of the fungal cellulases and the growth and fermentation characteristics of the current industrial biocatalysts, SSF of cellulose is envisioned at conditions that are not optimal for the fungal cellulase activity leading to higher than required cost of cellulase in SSF. We have isolated bacterial biocatalysts whose growth and fermentation requirements match the optimum conditions for commercial fungal cellulase activity (pH 5.0 and 50 deg. C). These isolates fermented both glucose and xylose, major components of cellulose and hemicellulose, respectively, to L(+)-lactic acid. Xylose was metabolized through the pentose-phosphate pathway by these organisms as evidenced by the fermentation profile and analysis of the fermentation products of 13C1-xylose by NMR. As expected for the metabolism of xylose by the pentose-phosphate pathway, 13C-lactate accounted for more than 90% of the total 13C-labeled products. All three strains fermented crystalline cellulose to lactic acid with the addition of fungal cellulase (Spezyme CE) (SSF) at an optimum of about 10 FPU/g cellulose. These isolates also fermented cellulose and sugar cane bagasse hemicellulose acid hydrolysate simultaneously. Based on fatty acid profile and 16S rRNA sequence, these isolates cluster with Bacillus coagulans although B. coagulans type strain, ATCC 7050, failed to utilize xylose as a carbon source. For successful production of ethanol from pyruvate, both pyruvate decarboxylase (PDC) and alcohol dehydrogenase (AHD) need to be produced at optimal levels in these biocatalysts. A plasmid containing the S. ventriculi pdc gene and the adh gene from geobacillus stearothermophilus was constructed using plasmid pWH1520 that was successfully used for expression of pdc in B. megaterium. The resulting portable ethanol (PET) plasmid, pJAM423, was transformed into B. megaterium. After xylose induction, a significant fraction of cell cytoplasm was composed of the S. ventriculi PDC and G. stearothermophilus ADH proteins. In preliminary experiments, the amount of ethanol produced by b. megaterium with plasmid pJAM423 was about twice (20 mM) of the bacterium without the plasmid. These results show that the PET operon is functional in B. megaterium but high level ethanol production needs further genetic and metabolic engineering. A genetic transfer system for the second generation biocatalysts needs to be developed for transferring the plasmid pJAM423 and its derivatives for engineering these organisms for ethanol production from biomass derived sugars and cellulose to ethanol. One of the new biocatalysts, strain P4-102B was found to be transformable with plasmids and the method for introducing plasmid pJAM423 into this strain and expression of the encoded DNA is being optimized. These new second generation biocatalysts have the potential to reduce the cost of SSF by minimizing the amount of fungal cellulases, a significant cost component in the use of biomass as a renewable resource for production of fuels and chemicals.« less

Biodegradable polymers as non-viral carriers for plasmid DNA delivery.

PubMed

Luten, Jordy; van Nostrum, Cornelus F; De Smedt, Stefaan C; Hennink, Wim E

2008-03-03

Gene therapy holds a great promise for the treatment of acquired and inherited diseases with a genetic origin that are currently incurable. Non-viral gene delivery systems are gaining recognition as an alternative to viral gene vectors for their potential in avoiding immunogenicity and toxicity problems inherently associated with the use of viral systems. Many cationic polymers have been studied both in vitro and in vivo for gene delivery purposes. However, in recent years there has been a focus on biodegradable carrier systems. The potential advantage of biodegradable carriers as compared to their non-degradable counterparts is their reduced toxicity and the avoidance of accumulation of the polymer in the cells after repeated administration. Also, the degradation of the polymer can be used as a tool to release the plasmid DNA into the cytosol. In this article the recent results obtained with two classes of degradable gene delivery systems, namely those based on water-soluble cationic polymers and on micro- and nanoparticles will be summarized and discussed.

Genetic redundancy and persistence of plasmid-mediated trimethoprim/sulfamethoxazole resistant effluent and stream water Escherichia coli.

PubMed

Suhartono, Suhartono; Savin, Mary; Gbur, Edward E

2016-10-15

Antibiotic resistant bacteria may persist in effluent receiving surface water in the presence of low (sub-inhibitory) antibiotic concentrations if the bacteria possess multiple genes encoding resistance to the same antibiotic. This redundancy of antibiotic resistance genes may occur in plasmids harboring conjugation and mobilization (mob) and integrase (intI) genes. Plasmids extracted from 76 sulfamethoxazole-trimethoprim resistant Escherichia coli originally isolated from effluent and an effluent-receiving stream were used as DNA template to identify sulfamethoxazole (sul) and trimethoprim (dfr) resistances genes plus detect the presence of intI and mob genes using PCR. Sulfamethoxazole and trimethoprim resistance was plasmid-mediated with three sul (sul1, sul2 and sul3 genes) and four dfr genes (dfrA12, dfrA8, dfrA17, and dfrA1 gene) the most prevalently detected. Approximately half of the plasmids carried class 1 and/or 2 integron and, although unrelated, half were also transmissible. Sampling site in relationship to effluent input significantly affected the number of intI and mob but not the number of sul and dfr genes. In the presence of low (sub-inhibitory) sulfamethoxazole concentration, isolates persisted regardless of integron and mobilization gene designation, whereas in the presence of trimethoprim, the presence of both integron and mobilization genes made isolates less persistent than in the absence of both or the presence of a gene from either group individually. Regardless, isolates persisted in large concentrations throughout the experiment. Treated effluent containing antibiotic resistant bacteria may be an important source of integrase and mobilization genes into the stream environment. Sulfamethoxazole-trimethoprim resistant bacteria may have a high degree of genetic redundancy and diversity carrying resistance to each antibiotic, although the role of integrase and mobilization genes towards persistence is unclear. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparative genomics of pathogenic lineages of Vibrio nigripulchritudo identifies virulence-associated traits

PubMed Central

Goudenège, David; Labreuche, Yannick; Krin, Evelyne; Ansquer, Dominique; Mangenot, Sophie; Calteau, Alexandra; Médigue, Claudine; Mazel, Didier; Polz, Martin F; Le Roux, Frédérique

2013-01-01

Vibrio nigripulchritudo is an emerging pathogen of farmed shrimp in New Caledonia and other regions in the Indo-Pacific. The molecular determinants of V. nigripulchritudo pathogenicity are unknown; however, molecular epidemiological studies have suggested that pathogenicity is linked to particular lineages. Here, we performed high-throughput sequencing-based comparative genome analysis of 16 V. nigripulchritudo strains to explore the genomic diversity and evolutionary history of pathogen-containing lineages and to identify pathogen-specific genetic elements. Our phylogenetic analysis revealed three pathogen-containing V. nigripulchritudo clades, including two clades previously identified from New Caledonia and one novel clade comprising putatively pathogenic isolates from septicemic shrimp in Madagascar. The similar genetic distance between the three clades indicates that they have diverged from an ancestral population roughly at the same time and recombination analysis indicates that these genomes have, in the past, shared a common gene pool and exchanged genes. As each contemporary lineage is comprised of nearly identical strains, comparative genomics allowed differentiation of genetic elements specific to shrimp pathogenesis of varying severity. Notably, only a large plasmid present in all highly pathogenic (HP) strains encodes a toxin. Although less/non-pathogenic strains contain related plasmids, these are differentiated by a putative toxin locus. Expression of this gene by a non-pathogenic V. nigripulchritudo strain resulted in production of toxic culture supernatant, normally an exclusive feature of HP strains. Thus, this protein, here termed ‘nigritoxin', is implicated to an extent that remains to be precisely determined in the toxicity of V. nigripulchritudo. PMID:23739050

The rapid spread of carbapenem-resistant Enterobacteriaceae

PubMed Central

Potter, Robert F.; D’Souza, Alaric W.; Dantas, Gautam

2016-01-01

Carbapenems, our one-time silver bullet for multidrug resistant bacterial infections, are now threatened by widespread dissemination of carbapenem-resistant Enterobacteriaceae (CRE). Successful expansion of Enterobacteriaceae clonal groups and frequent horizontal gene transfer of carbapenemase expressing plasmids are causing increasing carbapenem resistance. Recent advances in genetic and phenotypic detection facilitate global surveillance of CRE diversity and prevalence. In particular, whole genome sequencing enabled efficient tracking, annotation, and study of genetic elements colocalized with carbapenemase genes on chromosomes and on plasmids. Improved characterization helps detail the co-occurrence of other antibiotic resistance genes in CRE isolates and helps identify pan-drug resistance mechanisms. The novel β-lactamase inhibitor, avibactam, combined with ceftazidime or aztreonam, is a promising CRE treatment compared to current colistin or tigecycline regimens. To halt increasing CRE-associated morbidity and mortality, we must continue quality, cooperative monitoring and urgently investigate novel treatments. PMID:27912842