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Plasmid curing

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https://www.readbyqxmd.com/read/28922955/comparison-of-zfns-versus-crispr-specific-nucleases-for-genome-edition-of-the-wiskott-aldrich-syndrome-locus
#1
Alejandra Gutierrez-Guerrero, Sabina Sanchez-Hernandez, Giuseppe Galvani, Javier Pinedo-Gomez, Almudena Sanchez-Gilabert, Rocio Martin-Guerra, Marien Cobo, Philip Gregory, Michael Holmes, Karim Benabdellah, Francisco Martin
Primary immunodeficiencies (PID), including Wiskott-Aldrich syndrome (WAS), are a main target for genome editing (GE) strategies using specific nucleases (SNs) since a small number of corrected hematopoietic stem cells (HSCs) could cure patients. In this work, we have designed different WAS gene-specific CRISPR/Cas9 systems and compared their efficiency and specificity with homodimeric and heterodimeric WAS-specific Zinc Finger Nucleases (ZFNs) using K562 cells as a cellular model and plasmid nucleofection or integrative-deficient Lentiviral Vectors (IDLVs) for delivery...
September 19, 2017: Human Gene Therapy
https://www.readbyqxmd.com/read/28918634/engineered-probiotic-for-the-inhibition-of-salmonella-via-tetrathionate-induced-production-of-microcin-h47
#2
Jacob D Palmer, Emma Piattelli, Beth A McCormick, Mark W Silby, Christopher J Brigham, Vanni Bucci
Complications arising from antibiotic-resistant bacteria are becoming one of the key issues in modern medicine. Members of drug-resistant Enterobacteriaceae spp. include opportunistic pathogens (e.g., Salmonella spp.) that are among the leading causes of morbidity and mortality worldwide. Overgrowth of these bacteria is considered a hallmark of intestinal dysbiosis. Microcins (small antimicrobial peptides) produced by some gut commensals can potentially cure these conditions by inhibiting these pathogens and have been proposed as a viable alternative to antibiotic treatment...
September 20, 2017: ACS Infectious Diseases
https://www.readbyqxmd.com/read/28888103/using-crispr-cas-systems-as-antimicrobials
#3
REVIEW
David Bikard, Rodolphe Barrangou
Although CRISPR-Cas systems naturally evolved to provide adaptive immunity in bacteria and archaea, Cas nucleases can be co-opted to target chromosomal sequences rather than invasive genetic elements. Although genome editing is the primary outcome of self-targeting using CRISPR-based technologies in eukaryotes, self-targeting by CRISPR is typically lethal in bacteria. Here, we discuss how DNA damage introduced by Cas nucleases in bacteria can efficiently and specifically lead to plasmid curing or drive cell death...
September 5, 2017: Current Opinion in Microbiology
https://www.readbyqxmd.com/read/28856222/comparison-of-clinical-methods-for-detecting-carbapenem-resistant-enterobacteriaceae
#4
Kirsten Kost, Jumi Yi, Beverly Rogers, Robert Jerris
We evaluated detection of carbapenem-resistant Enterobacteriaceae (CRE) by routine minimal inhibitory concentration (MIC) testing, polymerase chain reaction (PCR) using Xpert® Carba-R assay, hydrolysis of ertapenem and imipenem detected by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), and hydrolysis by colorimetry using the EPI-CRE assay. Ninety-six Enterobacteriaceae isolates possessing carbapenemase genes and 29 carbapenem-susceptible Enterobacteriaceae were available for testing...
August 2017: Pract Lab Med
https://www.readbyqxmd.com/read/28854680/the-ctx-m-14-plasmid-phk01-encodes-novel-small-rnas-and-influences-host-growth-and-motility
#5
Xinlei Jiang, Xuan Liu, Carmen O K Law, Ya Wang, Wai U Lo, Xing Weng, Ting Fung Chan, P L Ho, Terrence C K Lau
The dissemination of extended-spectrum β-lactamases (ESBLs) genes among bacteria is commonly achieved by plasmid conjugation. In the last decade, the CTX-M type enzyme was the most widespread and prevalent ESBLs in the world. In Hong Kong and mainland China, among the commonly found CTX-M-carrying plasmids were pHK01 and pHK01-like plasmids, which belong to incompatibility group FII (IncFII). In this work, we studied the physiological effect caused by the pHK01 plasmid in bacterial host Escherichia coli J53...
July 1, 2017: FEMS Microbiology Ecology
https://www.readbyqxmd.com/read/28842543/lactolisterin-bu-a-novel-class-ii-broad-spectrum-bacteriocin-from-lactococcus-lactis-subsp-lactis-bv-diacetylactis-bgbu1-4
#6
Jelena Lozo, Nemanja Mirkovic, Paula M O'Connor, Milka Malesevic, Marija Miljkovic, Natalija Polovic, Branko Jovcic, Paul D Cotter, Milan Kojic
Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 produces a novel bacteriocin, lactolisterin BU, with strong antimicrobial activity against many species of Gram-positive bacteria, including important food spoilage and food-borne pathogens such as Listeria monocytogenes, Staphylococcus aureus, Bacillus sp. and streptococci. Lactolisterin BU was extracted from the cell surface of BGBU1-4 by propan-2-ol and purified to homogeneity by C18 solid phase extraction and reversed phase HPLC. The molecular mass of the purified lactolisterin BU was 5160...
August 25, 2017: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/28771369/isolation-and-characterization-of-2-4-d-butyl-ester-degrading-acinetobacter-sp-zx02-from-a-chinese-ginger-cultivated-soil
#7
Lin Xiao, Hai-Fei Jia, In-Hong Jeong, Young-Joon Ahn, Yong-Zhe Zhu
Strain ZX02 was isolated from Chinese ginger cultivated soil contaminated with various pesticides, which could utilize 2,4-dichlorophenoxyacetic acid butyl ester (2,4-D butyl ester) as the sole carbon source. On the basis of the sequence analysis of 16S rRNA gene as well as the morphological, biochemical, and physiological characteristics of strain ZX02, the organism belonged to Gram-negative bacterium and was identified as Acinetobacter sp. ZX02. The strain ZX02 showed a remarkable performance in 2,4-D butyl ester degradation (100% removal in <96 h) in pure culture...
August 30, 2017: Journal of Agricultural and Food Chemistry
https://www.readbyqxmd.com/read/28771154/multiresistant-opportunistic-pathogenic-bacteria-isolated-from-polluted-rivers-and-first-detection-of-nontuberculous-mycobacteria-in-the-algerian-aquatic-environment
#8
Lydia Neïla Djouadi, Okba Selama, Ahmed Abderrahmani, Amel Bouanane-Darenfed, Lamia Abdellaziz, Meriam Amziane, Marie-Laure Fardeau, Farida Nateche
Opportunistic infections constitute a major challenge for modern medicine mainly because the involved bacteria are usually multiresistant to antibiotics. Most of these bacteria possess remarkable ability to adapt to various ecosystems, including those exposed to anthropogenic activities. This study isolated and identified 21 multiresistant opportunistic bacteria from two polluted rivers, located in Algiers. Cadmium, lead, and copper concentrations were determined for both water samples to evaluate heavy metal pollution...
August 2017: Journal of Water and Health
https://www.readbyqxmd.com/read/28764701/a-versatile-one-step-crispr-cas9-based-approach-to-plasmid-curing
#9
Ida Lauritsen, Andreas Porse, Morten O A Sommer, Morten H H Nørholm
BACKGROUND: Plasmids are widely used and essential tools in molecular biology. However, plasmids often impose a metabolic burden and are only temporarily useful for genetic engineering, bio-sensing and characterization purposes. While numerous techniques for genetic manipulation exist, a universal tool enabling rapid removal of plasmids from bacterial cells is lacking. RESULTS: Based on replicon abundance and sequence conservation analysis, we show that the vast majority of bacterial cloning and expression vectors share sequence similarities that allow for broad CRISPR-Cas9 targeting...
August 2, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/28690290/rescue-of-an-intracellular-avirulent-rhodococcus-equi-replication-defect-by-the-extracellular-addition-of-virulence-associated-protein-a
#10
Nuttapone Sangkanjanavanich, Masanori Kawai, Tsutomu Kakuda, Shinji Takai
Rhodococcus equi is a facultative intracellular bacterium that can escape from bactericidal mechanisms associated with phagocytosis. Virulence-associated protein A (VapA), encoded on a virulence-associated plasmid, is essential for intracellular survival in macrophages, but its function is not known. Here, we show that the extracellular addition of recombinant glutathione S-transferase (GST)-VapA fusion protein rescued the intracellular replication defect of a mutant lacking the vapA gene. Furthermore, the virulence-plasmid-cured strain could also multiply to nearly wild-type levels by the addition of GST-VapA...
August 4, 2017: Journal of Veterinary Medical Science
https://www.readbyqxmd.com/read/28690133/the-complete-genome-sequence-of-bacillus-velezensis-strain-gh1-13-reveals-agriculturally-beneficial-properties-and-a-unique-plasmid
#11
Sang Yoon Kim, Hajin Song, Mee Kyung Sang, Hang-Yeon Weon, Jaekyeong Song
The bacterial strain Bacillus velezensis GH1-13, isolated from rice paddy soil in Korea, has been shown to promote plant growth and have strong antagonistic activities against pathogens. Here, we report the complete genome sequence of GH1-13, revealing that it possesses a single 4,071,980-bp circular chromosome with 46.2% GC-content. The chromosome encodes 3,930 genes, and we have also identified a unique plasmid in the strain that encodes a further 104 genes (71,628bp and 31.7% GC-content). The genome was found to contain various enzyme-encoding operons, including indole-3-acetic acid (IAA) biosynthesis proteins, 2,3-butanediol dehydrogenase, various non-ribosomal peptide synthetases, and several polyketide synthases...
July 6, 2017: Journal of Biotechnology
https://www.readbyqxmd.com/read/28649005/crispr-cas9-coupled-recombineering-for-metabolic-engineering-of-corynebacterium-glutamicum
#12
Jae Sung Cho, Kyeong Rok Choi, Cindy Pricilia Surya Prabowo, Jae Ho Shin, Dongsoo Yang, Jaedong Jang, Sang Yup Lee
Genome engineering of Corynebacterium glutamicum, an important industrial microorganism for amino acids production, currently relies on random mutagenesis and inefficient double crossover events. Here we report a rapid genome engineering strategy to scarlessly knock out one or more genes in C. glutamicum in sequential and iterative manner. Recombinase RecT is used to incorporate synthetic single-stranded oligodeoxyribonucleotides into the genome and CRISPR/Cas9 to counter-select negative mutants. We completed the system by engineering the respective plasmids harboring CRISPR/Cas9 and RecT for efficient curing such that multiple gene targets can be done iteratively and final strains will be free of plasmids...
July 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/28646112/crispr-cas12a-assisted-recombineering-in-bacteria
#13
Mei-Yi Yan, Hai-Qin Yan, Gai-Xian Ren, Ju-Ping Zhao, Xiao-Peng Guo, Yi-Cheng Sun
Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a (Cpf1) has emerged as an effective genome editing tool in many organisms. Here, we developed and optimized a CRISPR-Cas12a-assisted recombineering system to facilitate genetic manipulation in bacteria. Using this system, point mutations, deletions, insertions, and gene replacements can be easily generated on the chromosome or native plasmids in Escherichia coli, Yersinia pestis, and Mycobacterium smegmatis Because CRISPR-Cas12a-assisted recombineering does not require introduction of an antibiotic resistance gene into the chromosome to select for recombinants, it is an efficient approach for generating markerless and scarless mutations in bacteria...
September 1, 2017: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/28540425/contribution-of-plasmid-encoded-peptidase-s8-prtp-to-adhesion-and-transit-in-the-gut-of-lactococcus-lactis-ibb477-strain
#14
Joanna Maria Radziwill-Bienkowska, Véronique Robert, Karolina Drabot, Florian Chain, Claire Cherbuy, Philippe Langella, Muriel Thomas, Jacek Karol Bardowski, Muriel Mercier-Bonin, Magdalena Kowalczyk
The ability of Lactococcus lactis to adhere to the intestinal mucosa can potentially prolong the contact with the host, and therefore favour its persistence in the gut. In the present study, the contribution of plasmid-encoded factors to the adhesive and transit properties of the L. lactis subsp. cremoris IBB477 strain was investigated. Plasmid-cured derivatives as well as deletion mutants were obtained and analysed. Adhesion tests were performed using non-coated polystyrene plates, plates coated with mucin or fibronectin and mucus-secreting HT29-MTX intestinal epithelial cells...
July 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28526263/n-stearoyl-l-tyrosine-inhibits-the-cell-senescence-and-apoptosis-induced-by-h2o2-in-hek293-tau-cells-via-the-cb2-receptor
#15
Yue Hu, Kai-Yi Zhou, Ze-Jian Wang, Yang Lu, Ming Yin
Although considerable energy and money have been spent trying to inhibit Aβ production and its related metabolic enzyme activities, there are still no drug treatments available to cure even slow for Alzheimer's disease. Therefore, tau protein has been focused recently as the new target for the treatment of Alzheimer's disease. The transfected human embryonic kidney 293 (HEK 293) cells with or without Tau 411 plasmid were used to evaluate the effect of tau protein on cell viability. H2O2 was added to simulate microenvironment of oxidative stress (OS) during aging...
June 25, 2017: Chemico-biological Interactions
https://www.readbyqxmd.com/read/28481933/the-limits-to-growth-energetic-burden-of-the-endogenous-antibiotic-tropodithietic-acid-in-phaeobacter-inhibens-dsm-17395
#16
Sabine Eva Will, Meina Neumann-Schaal, Raymond Leopold Heydorn, Pascal Bartling, Jörn Petersen, Dietmar Schomburg
Phaeobacter inhibens DSM 17395, a model organism for marine Roseobacter group, was studied for its response to its own antimicrobial compound tropodithietic acid (TDA). TDA biosynthesis is encoded on the largest extrachromosomal element of P. inhibens, the 262 kb plasmid, whose curation leads to an increased growth and biomass yield. In this study, the plasmid-cured strain was compared to the wild-type strain and to transposon mutants lacking single genes of the TDA biosynthesis. The data show that the growth inhibition of the wild-type strain can be mainly attributed to the TDA produced by P...
2017: PloS One
https://www.readbyqxmd.com/read/28474344/differential-expression-of-duplicated-binary-toxin-genes-bina-binb-in-lysinibacillus-sphaericus-c3-41
#17
P Fu, X Xiang, Y Ge, Z Yuan, X Hu
Due to the self-produced binary toxins BinA/BinB, some Lysinibacillus sphaericus isolates exhibit high toxicity against mosquito larvae and are utilized in mosquito control programmes. Previous whole-genome sequencing revealed the presence of a large plasmid pBsph in L. sphaericus C3-41, which contains a 30·5-kb duplication of the genome including the binary toxin genes binA/binB. This was confirmed by Southern blot and qPCR experiments in this study. Mutants of L. sphaericus C3-41, bearing disruptions of the chromosomal-bin (C3-41ΔCab ) or the plasmid-bin (C3-41ΔPab ) and a plasmid-cured strain G725, were generated...
May 5, 2017: Letters in Applied Microbiology
https://www.readbyqxmd.com/read/28433723/elimination-of-the-cryptic-plasmid-in-leuconostoc-citreum-by-crispr-cas9-system
#18
Ye-Ji Jang, Seung-Oh Seo, Seul-Ah Kim, Ling Li, Tae-Jip Kim, Sun Chang Kim, Yong-Su Jin, Nam Soo Han
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...
April 19, 2017: Journal of Biotechnology
https://www.readbyqxmd.com/read/28432094/use-of-synthetic-hybrid-strains-to-determine-the-role-of-replicon-3-in-virulence-of-the-burkholderia-cepacia-complex
#19
Kirsty Agnoli, Roman Freitag, Margarida C Gomes, Christian Jenul, Angela Suppiger, Olga Mannweiler, Carmen Frauenknecht, Daniel Janser, Annette C Vergunst, Leo Eberl
The Burkholderia cepacia complex (Bcc) displays a wealth of metabolic diversity with great biotechnological potential, but the utilization of these bacteria is limited by their opportunistic pathogenicity to humans. The third replicon of the Bcc, megaplasmid pC3 (0.5 to 1.4 Mb, previously chromosome 3), is important for various phenotypes, including virulence, antifungal, and proteolytic activities and the utilization of certain substrates. Approximately half of plasmid pC3 is well conserved throughout sequenced Bcc members, while the other half is not...
July 1, 2017: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/28330200/use-of-ca-alginate-immobilized-pseudomonas-aeruginosa-for-repeated-batch-and-continuous-degradation-of-endosulfan
#20
Vijayalakshmi Pradeep, Usha Malavalli Subbaiah
The current investigation is taken up with the aim of studying repeated batch and continuous degradation of Endosulfan, using Ca-alginate immobilized cells of Pseudomonas aeruginosa isolated from an agricultural soil. The work involves the study of genes and enzymes involved in the degradation of the pesticide and was carried out with an objective of reducing the toxicity of Endosulfan by degrading it to less toxic metabolites. The long-term stability of Endosulfan degradation was studied during its repeated batch degradation, carried out over a period of 35 days...
December 2016: 3 Biotech
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