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Plasmid curing

Mariya Tarazanova, Marke Beerthuyzen, Roland Siezen, Marcela M Fernandez-Gutierrez, Anne de Jong, Sjoerd van der Meulen, Jan Kok, Herwig Bachmann
Lactococcus lactis MG1363 is an important gram-positive model organism. It is a plasmid-free and phage-cured derivative of strain NCDO712. Plasmid-cured strains facilitate studies on molecular biological aspects, but many properties which make L. lactis an important organism in the dairy industry are plasmid encoded. We sequenced the total DNA of strain NCDO712 and, contrary to earlier reports, revealed that the strain carries 6 rather than 5 plasmids. A new 50-kb plasmid, designated pNZ712, encodes functional nisin immunity (nisCIP) and copper resistance (lcoRSABC)...
2016: PloS One
You Chen, Arnaud Taton, Michaela Go, Ross E London, Lindsey M Pieper, Susan S Golden, James W Golden
To facilitate development of synthetic biology tools for genetic engineering of cyanobacterial strains, we constructed pANS-derived self-replicating shuttle vectors that are based on the minimal replication element of the Synechococcus elongatus strain PCC 7942 plasmid pANS. To remove the possibility of homologous recombination events between the shuttle plasmids and the native pANS plasmid, the endogenous pANS was cured through plasmid incompatibility-mediated spontaneous loss. A heterologous toxin-antitoxin cassette was incorporated into the shuttle vectors for stable plasmid maintenance in the absence of antibiotic selection...
December 2016: Microbiology
Qing-Hua Cao, Huan-Huan Shao, Hui Qiu, Tao Li, Yi-Zheng Zhang, Xue-Mei Tan
The CRISPR/Cas system can be used to simply and efficiently edit the genomes of various species, including animals, plants, and microbes. Zymomonas mobilis ZM4 is a highly efficient, ethanol-producing bacterium that contains five native plasmids. Here, we constructed the pSUZM2a-Cas9 plasmid and a single-guide RNA expression plasmid. The pSUZM2a-Cas9 plasmid was used to express the Cas9 gene cloned from Streptococcus pyogenes CICC 10464. The single-guide RNA expression plasmid pUC-T7sgRNA, with a T7 promoter, can be used for the in vitro synthesis of single-guide RNAs...
November 30, 2016: Bioscience, Biotechnology, and Biochemistry
Claudia Minosse, Sabrina Coen, Ubaldo Visco Comandini, Raffaella Lionetti, Marzia Montalbano, Stefano Cerilli, Donatella Vincenti, Andrea Baiocchini, Maria R Capobianchi, Stefano Menzo
BACKGROUND: A functional cure of chronic hepatitis B (CHB) is feasible, but a clear view of the intrahepatic viral dynamics in each patient is needed. Intrahepatic covalently closed circular DNA (cccDNA) is the stable form of the viral genome in infected cells, and represents the ideal marker of parenchymal colonization. Its relationships with easily accessible peripheral parameters need to be elucidated in order to avoid invasive procedures in patients. OBJECTIVES: The goal of this study was to design, set up, and validate a reliable and straightforward method for the quantification of the cccDNA and total DNA of the hepatitis B virus (HBV) in a variety of clinical samples...
October 2016: Hepatitis Monthly
Yi Pan, Nan Shen, Sabine Jung-Klawitter, Christian Betzen, Georg F Hoffmann, Jörg D Hoheisel, Nenad Blau
The CRISPR/Cas9 system is a recently developed genome editing technique. In this study, we used a modified CRISPR system, which employs the fusion of inactive Cas9 (dCas9) and the FokI endonuclease (FokI-dCas9) to correct the most common variant (allele frequency 21.4%) in the phenylalanine hydroxylase (PAH) gene - c.1222C>T (p.Arg408Trp) - as an approach toward curing phenylketonuria (PKU). PKU is the most common inherited diseases in amino acid metabolism. It leads to severe neurological and neuropsychological symptoms if untreated or late diagnosed...
October 27, 2016: Scientific Reports
Wenyao Chen, Tingzi Fang, Xiujuan Zhou, Daofeng Zhang, Xianming Shi, Chunlei Shi
The wide usage of antibiotics contributes to the increase in the prevalence of antibiotic-resistant Salmonella. Plasmids play a critical role in horizontal transfer of antibiotic resistance markers in Salmonella. This study aimed to screen and characterize plasmid profiles responsible for antibiotic resistance in Salmonella and ultimately to clarify the molecular mechanism of transferable plasmid-mediated antibiotic resistance. A total of 226 Salmonella isolates were examined for antimicrobial susceptibility by a disk diffusion method...
2016: Frontiers in Microbiology
Issei Saitoh, Masahiro Sato, Miki Soda, Emi Inada, Yoko Iwase, Tomoya Murakami, Hayato Ohshima, Haruaki Hayasaki, Hirofumi Noguchi
Type 1 diabetes occurs due to the autoimmune destruction of pancreatic β-cells in islets. Transplantation of islets is a promising option for the treatment of patients with type 1 diabetes that experience hypoglycemic unawareness despite maximal care, but the present shortage of donor islets hampers such transplantation. Transplantation of insulin-producing cells derived from the patients themselves would be one of the most promising approaches to cure type 1 diabetes. Previously, we demonstrated that insulin-producing cells could be produced by transfecting murine pancreatic cells with Yamanaka's reprogramming factors...
2016: PloS One
Tetsushi Sakuma, Keiichi Masaki, Hiromi Abe-Chayama, Keiji Mochida, Takashi Yamamoto, Kazuaki Chayama
CRISPR-Cas9-mediated genome-editing technology contributes not only to basic genomic studies but also to clinical studies such as genetic correction and virus inactivation. Hepatitis B virus (HBV) is a major target for potential application of CRISPR-Cas9 in eliminating viral DNA from human cells. However, the high stability of covalently closed circular DNA (cccDNA) makes it difficult to completely clear HBV infection. Here, we report highly multiplexed CRISPR-Cas9-nuclease and Cas9-nickase vector systems that simultaneously target three critical domains of the HBV genome...
November 2016: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Nayyer Taheri, Anna Fahlgren, Maria Fällman
Neutrophils are essential components of immunity and are rapidly recruited to infected or injured tissue. Upon their activation, neutrophils release granules to the cell's exterior, through a process called degranulation. These granules contain proteins with antimicrobial properties that help combat infection. The enteropathogenic bacterium Yersinia pseudotuberculosis successfully persists as an extracellular bacterium during infection by virtue of its translocation of virulence effectors (Yersinia outer proteins [Yops]) that act in the cytosol of host immune cells to subvert phagocytosis and proinflammatory responses...
December 2016: Infection and Immunity
Lili Li, Rikke Heidemann Olsen, Lei Shi, Lei Ye, Jianhua He, Hecheng Meng
A multi-drug resistant (MDR) Listeria monocytogenes isolate (serotype 1/2c) was recovered from a quick-frozen rice flour product collected from Langfang city in northern China. PCR screening identified the presence of cat, ermB and tetS genes. The plasmid profile of the strain showed the presence of an approximately 22.4-kb plasmid. Curing of this plasmid resulted in the loss of cat, ermB and tetS genes and increased susceptibility to several antibiotics, suggesting the involvement of the plasmid in multiple antibiotic resistances...
December 5, 2016: International Journal of Food Microbiology
V Pascual, N Alonso, M Simó, G Ortiz, M C Garcia, M Xercavins, A Rivera, M A Morera, E Miró, E Espejo, F Navarro, M Gurguí, J Pérez, M Rodríguez-Carballeira, J Garau, E Calbo
The aim of the study was to investigate the epidemiology and clinical features of bloodstream infections due to Escherichia coli producing AmpC β-lactamases (AmpC-Ec-BSI). In a multi-centre case-control study, all third-generation-cephalosporin-resistant Escherichia coli BSI (3GC-Ec-BSI) isolates were analysed. Acquired bla AmpC (bla ac-AmpC) detection was done by polymerase chain reaction (PCR) and sequencing. Chromosomal bla AmpC (bla c-AmpC) expression was quantified by real-time PCR. Cases were patients with AmpC-Ec-BSI...
December 2016: European Journal of Clinical Microbiology & Infectious Diseases
Jean-Paul Madeira, Béatrice Alpha-Bazin, Jean Armengaud, Hélène Omer, Catherine Duport
This data article reports changes in the cellular and exoproteome of B. cereus cured from pBClin15.Time-course changes of proteins were assessed by high-throughput nanoLC-MS/MS. We report all the peptides and proteins identified and quantified in B. cereus with and without pBClin15. Proteins were classified into functional groups using the information available in the KEGG classification and we reported their abundance in term of normalized spectral abundance factor. The repertoire of experimentally confirmed proteins of B...
September 2016: Data in Brief
Meenu Maheshwari, Nawar Hadi Yaser, Suraiya Naz, Mansha Fatima, Iqbal Ahmad
This study aimed to evaluate the incidence of ciprofloxacin-resistant extended-spectrum β-lactamase (ESBL)-producing enteric bacteria in hospital wastewater and clinical sources. Enteric bacteria, mainly Escherichia coli, were isolated from clinical sources (urinary tract and gastrointestinal tract infections; 80 isolates) and hospital wastewater (103 isolates). The antibiotic resistance profile and ESBL production of the isolates were investigated by disc diffusion assay and combined disc diffusion test, respectively...
June 2016: Journal of Global Antimicrobial Resistance
Carla Barría-Loaiza, Andrea Pincheira, Mario Quezada, Alejandra Vera, Pedro Valenzuela, Mariana Domínguez, Celia A Lima, Ingrid Araya, Pamela Araya, Soledad Prat, Carolina Aguayo, Jorge Fernández, Juan Carlos Hormazábal, Helia Bello-Toledo, Gerardo González-Rocha
The aim of this work was to determine the genetic environment and transferability of blaKPC as well as the pulsotypes of KPC-producing Klebsiella pneumoniae strains isolated from clinical samples in Chilean hospitals. Seventeen strains, principally isolated in Santiago (the capital of Chile) during the years 2012 and 2013, were included. The genetic environment of blaKPC was elucidated by PCR mapping and sequencing. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST)...
March 2016: Journal of Global Antimicrobial Resistance
Shanze Yi, Dewei Niu, Fang Bai, Shuaiguang Li, Luyuan Huang, Wenyan He, Anand Prasad, Alexander Czachor, Lee Charles Tan, Narasaiah Kolliputi, Feng Wang
Manganese superoxide dismutase (MnSOD) is a vital enzyme that protects cells from free radicals through eliminating superoxide radicals (O²⁻). Hirudin, a kind of small active peptide molecule, is one of the strongest anticoagulants that can effectively cure thrombus diseases. In this study, we fused Hirudin to the C terminus of human MnSOD with the GGGGS linker to generate a novel dual-feature fusion protein, denoted as hMnSOD-Hirudin. The hMnSOD-Hirudin gene fragment was cloned into the pET15b (SmaI, CIAP) vector, forming a recombinant pET15b-hMnSOD-Hirudin plasmid, and then was transferred into Escherichia coli strain Rosetta-gami for expression...
November 28, 2016: Journal of Microbiology and Biotechnology
B Thilakan, K Chakraborty, R D Chakraborty
In this study, 234 bacterial strains were isolated from 7 seaweed species in the Gulf of Mannar on the southeast coast of India. The strains having consistent antimicrobial activity were chosen for further studies, and this constituted about 9.8% of the active strains isolated. Phylogenetic analysis using 16S rDNA sequencing with the help of classical biochemical identification indicated the existence of 2 major phyla, Firmicutes and Proteobacteria. Antimicrobial activity analysis combined with the results of amplifying genes encoding for polyketide synthetase and nonribosomal peptide synthetase showed that seaweed-associated bacteria had broad-spectrum antimicrobial activity...
August 2016: Canadian Journal of Microbiology
Martin Ungerer, Julia Faßbender, Zhongmin Li, Götz Münch, Hans-Peter Holthoff
Various approaches have been used to model human Graves' disease in mice, including transfected fibroblasts, and plasmid or adenoviral immunisations with the extracellular A subunit of the human thyrotropin receptor (TSHR). Some of these models were only observed for a short time period or were self-limiting. A long-term model for human Graves' disease was established in mice using continuing immunisations (4-weekly injections) with recombinant adenovirus expressing TSHR. Generation of TSHR binding cAMP-stimulatory antibodies, thyroid enlargement and alterations, elevated serum thyroxin levels, tachycardia and cardiac hypertrophy were maintained for at least 9 months in all Ad-TSHR-immunised mice...
July 2, 2016: Clinical Reviews in Allergy & Immunology
Kristen E Wendt, Justin Ungerer, Ryan E Cobb, Huimin Zhao, Himadri B Pakrasi
BACKGROUND: As autotrophic prokaryotes, cyanobacteria are ideal chassis organisms for sustainable production of various useful compounds. The newly characterized cyanobacterium Synechococcus elongatus UTEX 2973 is a promising candidate for serving as a microbial cell factory because of its unusually rapid growth rate. Here, we seek to develop a genetic toolkit that enables extensive genomic engineering of Synechococcus 2973 by implementing a CRISPR/Cas9 editing system. We targeted the nblA gene because of its important role in biological response to nitrogen deprivation conditions...
June 23, 2016: Microbial Cell Factories
Sidney Hayes, Wen Wang, Karthic Rajamanickam, Audrey Chu, Anirban Banerjee, Connie Hayes
The bacteriophage lambda replication initiation protein P exhibits a toxic effect on its Escherichia coli (E. coli) host, likely due to the formation of a dead-end P-DnaB complex, sequestering the replicative DnaB helicase from further activity. Intracellular expression of P triggers SOS-independent cellular filamentation and rapidly cures resident ColE1 plasmids. The toxicity of P is suppressed by alleles of P or dnaB. We asked whether P buildup within a cell can influence E. coli replication fidelity. The influence of P expression from a defective prophage, or when cloned and expressed from a plasmid was examined by screening for auxotrophic mutants, or by selection for rifampicin resistant (Rif(R)) cells acquiring mutations within the rpoB gene encoding the β-subunit of RNA polymerase (RNAP), nine of which proved unique...
2016: Viruses
Jean-Paul Madeira, Hélène Omer, Béatrice Alpha-Bazin, Jean Armengaud, Catherine Duport
UNLABELLED: The pathogen, Bacillus cereus, is able to adapt its metabolism to various environmental conditions. The reference strain, Bacillus cereus ATCC 14579, harbors a linear plasmid, pBClin15, which displays a cryptic prophage behavior. Here, we studied the impact of pBClin15 on the aerobic respiratory metabolism of B. cereus by curing its host strain. We compared, by means of a high-throughput shotgun proteomic approach, both the cellular proteome and the exoproteome of B. cereus ATCC 14579 in the presence and absence of pBClin15 at the early, late and stationary growth phases...
September 2, 2016: Journal of Proteomics
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