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Transposon mutagenesis

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https://www.readbyqxmd.com/read/28228480/a-stable-but-reversible-integrated-surrogate-reporter-for-assaying-crispr-cas9-stimulated-homology-directed-repair
#1
Yahong Wen, Grace Liao, Thomas Pritchard, Ting-Ting Zhao, Jon P Connelly, Shondra M Pruett-Miller, Valerie Blanc, Nicholas O Davidson, Blair B Madison
The discovery and application of CRISPR/Cas9 technology for genome editing has greatly accelerated targeted mutagenesis in a variety of organisms. CRISPR/Cas9-mediated site-specific cleavage is typically exploited for the generation of insertions or deletions (indels) following aberrant dsDNA repair via the endogenous non-homology end-joining (NHEJ) pathway, or alternatively, for enhancing homology directed repair (HDR) to facilitate the generation of a specific mutation (or knock-in). However, there is a need for efficient cellular assays that can measure Cas9/guide RNA (gRNA) activity...
February 22, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28219662/fumarase-deficiency-causes-protein-and-metabolite-succination-and-intoxicates-mycobacterium-tuberculosis
#2
Nadine Ruecker, Robert Jansen, Carolina Trujillo, Susan Puckett, Pradeepa Jayachandran, Gerardo G Piroli, Norma Frizzell, Henrik Molina, Kyu Y Rhee, Sabine Ehrt
Enzymes of central carbon metabolism are essential mediators of Mycobacterium tuberculosis (Mtb) physiology and pathogenicity, but are often perceived to lack sufficient species selectivity to be pursued as potential drug targets. Fumarase (Fum) is an enzyme of the canonical tricarboxylic acid cycle and is dispensable in many organisms. Transposon mutagenesis studies in Mtb, however, indicate that Fum is required for optimal growth. Here, we report the generation and characterization of a genetically engineered Mtb strain in which Fum expression is conditionally regulated...
February 9, 2017: Cell Chemical Biology
https://www.readbyqxmd.com/read/28188204/siderophore-product-ornibactin-is-required-for-the-bactericidal-activity-of-burkholderia-contaminans-ms14
#3
Peng Deng, Adam Foxfire, Jianhong Xu, Sonya M Baird, Jiayuan Jia, Keren H Delgado, Ronald Shin, Leif Smith, Shi-En Lu
Burkholderia contaminans MS14 was isolated from soil in Mississippi. When cultivated on nutrient broth-yeast extract (NBY) agar medium, the colonies exhibit bactericidal activity against a wide range of plant pathogenic bacteria. Only a bacteriostatic compound with siderophore activity was successfully purified, which was determined by nuclear magnetic resonance (NMR) to be ornibactin. The isolation of the bactericidal compound has not yet been achieved, thus, the exact nature of the bactericidal compound is still unknown...
February 10, 2017: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/28170428/comparative-study-on-the-in-vitro-effects-of-pseudomonas-aeruginosa-and-seaweed-alginates-on-human-gut-microbiota
#4
Shaofeng Bai, Huahai Chen, Liying Zhu, Wei Liu, Hongwei D Yu, Xin Wang, Yeshi Yin
Alginates pertain to organic polysaccharides that have been extensively used in food- and medicine-related industries. The present study obtained alginates from an alginate overproducing Pseudomonas aeruginosa PAO1 mutant by screening transposon mutagenesis libraries. The interaction between bacterial and seaweed alginates and gut microbiota were further studied by using an in vitro batch fermentation system. Thin-layer chromatography (TLC) analysis indicated that both bacterial and seaweed alginates can be completely degraded by fecal bacteria isolated from study volunteers, indicating that a minor structural difference between bacterial and seaweed alginates (O-acetylation and lack of G-G blocks) didn't affect the digestion of alginates by human microbiota...
2017: PloS One
https://www.readbyqxmd.com/read/28165493/a-noise-trimming-and-positional-significance-of-transposon-insertion-system-to-identify-essential-genes-in-yersinia-pestis
#5
Zheng Rong Yang, Helen L Bullifent, Karen Moore, Konrad Paszkiewicz, Richard J Saint, Stephanie J Southern, Olivia L Champion, Nicola J Senior, Mitali Sarkar-Tyson, Petra C F Oyston, Timothy P Atkins, Richard W Titball
Massively parallel sequencing technology coupled with saturation mutagenesis has provided new and global insights into gene functions and roles. At a simplistic level, the frequency of mutations within genes can indicate the degree of essentiality. However, this approach neglects to take account of the positional significance of mutations - the function of a gene is less likely to be disrupted by a mutation close to the distal ends. Therefore, a systematic bioinformatics approach to improve the reliability of essential gene identification is desirable...
February 6, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28127946/ribose-operon-repressor-rbsr-contributes-to-the-adhesion-of-aeromonas-hydrophila-to-anguilla-japonica-mucus
#6
Guifang Lin, Wenbo Chen, Yongquan Su, Yingxue Qin, Lixing Huang, Qingpi Yan
The characterization of adhesion between pathogenic bacteria and the host is critical. Pathogenic Aeromonas hydrophila was shown to adhere in vitro to the mucus of Anguilla japonica. To further investigate the adhesion mechanisms of A. hydrophila, a mini-Tn10 transposon mutagenesis system was used to generate an insertion mutant library by cell conjugation. Seven mutants that were impaired in adhesion to mucus were selected out of 332 individual colonies, and mutant M196 was the most severely impaired strain...
January 27, 2017: MicrobiologyOpen
https://www.readbyqxmd.com/read/28122981/transgenic-crispr-cas9-mediated-viral-gene-targeting-for-antiviral-therapy-of-bombyx-mori-nucleopolyhedrovirus-bmnpv
#7
Shuqing Chen, Chengxiang Hou, Honglun Bi, Yueqiang Wang, Jun Xu, Muwang Li, Anthony A James, Yongping Huang, Anjiang Tan
: We developed a novel anti-viral strategy by combining transposon-based transgenesis and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system for direct cleavage of the Bombyx mori nucleopolyhedrovirus (BmNPV) genome DNA to promote virus clearance in silkworms. We demonstrate that transgenic silkworms constitutively-expressing Cas9 and guide RNAs (gRNAs) targeting the BmNPV immediate early-1 (ie-1) gene and me53 gene effectively induce target-specific cleavage and subsequent mutagenesis, especially large segment deletions (∼ 7 kilobase-pairs [kb]) in BmNPV genomes and thus exhibit a robust suppression of BmNPV proliferation...
January 25, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28110783/identification-of-novel-virulence-related-genes-in-aeromonas-hydrophila-by-screening-transposon-mutants-in-a-tetrahymena-infection-model
#8
Maoda Pang, Xing Xie, Yuhao Dong, Hechao Du, Nannan Wang, Chengping Lu, Yongjie Liu
Outbreaks of motile Aeromonad septicemia (MAS) in fish caused by sequence type (ST) 251 Aeromonas hydrophila have become a prominent problem for the aquaculture industry. The pathogenesis of A. hydrophila is very complicated, and some virulence factors remain to be identified. In this study, to identify novel virulence-related factors, ST251 A. hydrophila strain NJ-35 was used as the parental strain to construct a mutant library comprising 1030 mutant strains by transposon insertion mutagenesis. Subsequently, 33 virulence-attenuated transposon insertion mutants were identified using Tetrahymena and zebrafish as model hosts in sequence...
February 2017: Veterinary Microbiology
https://www.readbyqxmd.com/read/28110506/antibiotic-discovery-throughout-the-small-world-initiative-a-molecular-strategy-to-identify-biosynthetic-gene-clusters-involved-in-antagonistic-activity
#9
Elizabeth Davis, Tyler Sloan, Krista Aurelius, Angela Barbour, Elijah Bodey, Brigette Clark, Celeste Dennis, Rachel Drown, Megan Fleming, Allison Humbert, Elizabeth Glasgo, Trent Kerns, Kelly Lingro, MacKenzie McMillin, Aaron Meyer, Breanna Pope, April Stalevicz, Brittney Steffen, Austin Steindl, Carolyn Williams, Carmen Wimberley, Robert Zenas, Kristen Butela, Hans Wildschutte
The emergence of bacterial pathogens resistant to all known antibiotics is a global health crisis. Adding to this problem is that major pharmaceutical companies have shifted away from antibiotic discovery due to low profitability. As a result, the pipeline of new antibiotics is essentially dry and many bacteria now resist the effects of most commonly used drugs. To address this global health concern, citizen science through the Small World Initiative (SWI) was formed in 2012. As part of SWI, students isolate bacteria from their local environments, characterize the strains, and assay for antibiotic production...
January 22, 2017: MicrobiologyOpen
https://www.readbyqxmd.com/read/28100651/stabilization-of-bacillus-circulans-xylanase-by-combinatorial-insertional-fusion-to-a-thermophilic-host-protein
#10
Vandan Shah, Brennal Pierre, Tamari Kirtadze, Seung Shin, Jin Ryoun Kim
High thermostability of an enzyme is critical for its industrial application. While many engineering approaches such as mutagenesis have enhanced enzyme thermostability, they often suffer from reduced enzymatic activity. A thermally stabilized enzyme with unchanged amino acids is preferable for subsequent functional evolution necessary to address other important industrial needs. In the research presented here, we applied insertional fusion to a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) in order to improve the thermal stability of Bacillus circulans xylanase (BCX)...
January 17, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28097405/role-of-exopolysaccharide-in-salt-stress-resistance-and-cell-motility-of-mesorhizobium-alhagi-ccnwxj12-2-t
#11
Xiaodong Liu, Yantao Luo, Zhefei Li, Jiamei Wang, Gehong Wei
Mesorhizobium alhagi, a legume-symbiont soil bacterium that forms nodules with the desert plant Alhagi sparsifolia, can produce large amounts of exopolysaccharide (EPS) using mannitol as carbon source. However, the role of EPS in M. alhagi CCNWXJ12-2(T), an EPS-producing rhizobium with high salt resistance, remains uncharacterized. Here, we studied the role of EPS in M. alhagi CCNWXJ12-2(T) using EPS-deficient mutants constructed by transposon mutagenesis. The insertion sites of six EPS-deficient mutants were analyzed using single primer PCR, and two putative gene clusters were found to be involved in EPS synthesis...
January 17, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28096490/comprehensive-essentiality-analysis-of-the-mycobacterium-tuberculosis-genome-via-saturating-transposon-mutagenesis
#12
Michael A DeJesus, Elias R Gerrick, Weizhen Xu, Sae Woong Park, Jarukit E Long, Cara C Boutte, Eric J Rubin, Dirk Schnappinger, Sabine Ehrt, Sarah M Fortune, Christopher M Sassetti, Thomas R Ioerger
: For decades, identifying the regions of a bacterial chromosome that are necessary for viability has relied on mapping integration sites in libraries of random transposon mutants to find loci that are unable to sustain insertion. To date, these studies have analyzed subsaturated libraries, necessitating the application of statistical methods to estimate the likelihood that a gap in transposon coverage is the result of biological selection and not the stochasticity of insertion. As a result, the essentiality of many genomic features, particularly small ones, could not be reliably assessed...
January 17, 2017: MBio
https://www.readbyqxmd.com/read/28079877/genome-wide-transposon-screening-and-quantitative-insertion-site-sequencing-for-cancer-gene-discovery-in-mice
#13
Mathias J Friedrich, Lena Rad, Iraad F Bronner, Alexander Strong, Wei Wang, Julia Weber, Matthew Mayho, Hannes Ponstingl, Thomas Engleitner, Carolyn Grove, Anja Pfaus, Dieter Saur, Juan Cadiñanos, Michael A Quail, George S Vassiliou, Pentao Liu, Allan Bradley, Roland Rad
Transposon-mediated forward genetics screening in mice has emerged as a powerful tool for cancer gene discovery. It pinpoints cancer drivers that are difficult to find with other approaches, thus complementing the sequencing-based census of human cancer genes. We describe here a large series of mouse lines for insertional mutagenesis that are compatible with two transposon systems, PiggyBac and Sleeping Beauty, and give guidance on the use of different engineered transposon variants for constitutive or tissue-specific cancer gene discovery screening...
February 2017: Nature Protocols
https://www.readbyqxmd.com/read/28062460/large-scale-transposition-mutagenesis-of-streptomyces-coelicolor-identifies-hundreds-of-genes-influencing-antibiotic-biosynthesis
#14
Zhong Xu, Yemin Wang, Keith F Chater, Hong-Yu Ou, H Howard Xu, Zixin Deng, Meifeng Tao
: Gram-positive Streptomyces bacteria produce thousands of bioactive secondary metabolites including antibiotics. To systematically investigate genes affecting secondary metabolism, we developed a hyperactive transposase-based Tn5 transposition system and employed it to mutagenize the model species Streptomyces coelicolor, leading to the identification of 51,443 transposition insertions. These insertions were distributed randomly along the chromosome except for some preferred regions associated with relatively low GC content in the chromosomal core...
January 6, 2017: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/28062003/identification-of-genes-involved-in-mycoplasma-gallisepticum-biofilm-formation-using-mini-tn4001-sgm-transposon-mutagenesis
#15
Yang Wang, Li Yi, Fanqing Zhang, Xusheng Qiu, Lei Tan, Shengqing Yu, Xiangchao Cheng, Chan Ding
Mycoplasma gallisepticum (MG) is an important pathogen that can cause chronic respiratory disease in chickens and infectious sinusitis in turkeys. MG has the ability to form biofilms. The molecular mechanisms underlying MG biofilm formation are complex and poorly understood. To better understand the mechanisms involved in biofilm formation, mini-Tn4001-SGM, a novel transposon vector containing the gentamicin gene was constructed and electroporated into MG strain Rlow. Of the 738 mutants obtained, 12 had significantly reduced capacity to form biofilms in a polystyrene microtiter-plate biofilm assay...
January 2017: Veterinary Microbiology
https://www.readbyqxmd.com/read/28057894/validating-genome-wide-association-candidates-controlling-quantitative-variation-in-nodulation
#16
Shaun J Curtin, Peter Tiffin, Joseph Guhlin, Diana Trujillo, Liana Burghart, Paul Atkins, Nicholas J Baltes, Roxanne Denny, Daniel F Voytas, Robert M Stupar, Nevin D Young
Genome-wide association (GWA) studies offer the opportunity to identify genes that contribute to naturally occurring variation in quantitative traits. However, GWA relies exclusively on statistical association, so functional validation is necessary to make strong claims about gene function. We used a combination of gene-disruption platforms (Tnt1 retrotransposons, hairpin RNA-interference constructs, and CRISPR/Cas9 nucleases) together with randomized, well-replicated experiments to evaluate the function of genes that an earlier GWA study in Medicago truncatula had identified as candidates contributing to variation in the symbiosis between legumes and rhizobia...
February 2017: Plant Physiology
https://www.readbyqxmd.com/read/28049432/identification-of-genes-affecting-alginate-biosynthesis-in-pseudomonas-fluorescens-by-screening-a-transposon-insertion-library
#17
Helga Ertesvåg, Håvard Sletta, Mona Senneset, Yi-Qian Sun, Geir Klinkenberg, Therese Aursand Konradsen, Trond E Ellingsen, Svein Valla
BACKGROUND: Polysaccharides often are necessary components of bacterial biofilms and capsules. Production of these biopolymers constitutes a drain on key components in the central carbon metabolism, but so far little is known concerning if and how the cells divide their resources between cell growth and production of exopolysaccharides. Alginate is an industrially important linear polysaccharide synthesized from fructose 6-phosphate by several bacterial species. The aim of this study was to identify genes that are necessary for obtaining a normal level of alginate production in alginate-producing Pseudomonas fluorescens...
January 3, 2017: BMC Genomics
https://www.readbyqxmd.com/read/28036242/identification-of-new-regulatory-genes-involved-in-the-pathogenic-functions-of-the-rice-pathogenic-bacterium-burkholderia-glumae
#18
Rebecca A Melanson, Inderjit Barphagha, Surendra Osti, Tiago P Lelis, Hari S Karki, Ruoxi Chen, Bishnu K Shrestha, Jong Hyun Ham
Burkholderia glumae is an emerging plant-pathogenic bacterium that causes disease in rice in several of the major of rice-producing areas throughout the world. In the southern United States, B. glumae is the major causal agent of bacterial panicle blight of rice and has caused severe yield losses in recent decades. Despite its importance, few management options are available for diseases caused by B. glumae, and knowledge of how this pathogen causes disease is limited. In an effort to identify novel factors that contribute to the pathogenicity of B...
December 30, 2016: Microbiology
https://www.readbyqxmd.com/read/28017601/identification-and-characterization-of-the-sulfazecin-monobactam-biosynthetic-gene-cluster
#19
Rongfeng Li, Ryan A Oliver, Craig A Townsend
The monobactams, exemplified by the natural product sulfazecin, are the only class of β-lactam antibiotics not inactivated by metallo-β-lactamases, which confer bacteria with extended-spectrum β-lactam resistance. We screened a transposon mutagenesis library from Pseudomonas acidophila ATCC 31363 and isolated a sulfazecin-deficient mutant that revealed a gene cluster encoding two non-ribosomal peptide synthetases (NRPSs), a methyltransferase, a sulfotransferase, and a dioxygenase. Three modules and an aberrant C-terminal thioesterase (TE) domain are distributed across the two NRPSs...
January 19, 2017: Cell Chemical Biology
https://www.readbyqxmd.com/read/28011697/incidental-mutations-occur-frequently-during-transposon-mutagenesis
#20
Guoqiang Liu, Yingying Kong, Hualin Liu, Donghai Peng, Lifang Ruan, Yiming Wu, Ming Sun
Transposon mutagenesis and subsequent phenotype-driven screening have been extensively used to annotate gene function and uncover the mechanisms of biological phenomena. During this process, the resulting phenotypic differences are specifically attributable to the disrupted genes. In this study, the complementation of the mini-Tn10 transposon-disrupted gene gntP did not restore the germination ability of the Bacillus thuringiensis MT1518-1 mutant to the wild-type level. Genome sequencing identified 186 unlinked mutations on the chromosome of the mutant MT1518-1, including one frameshift mutation in the germination-related gene exsA We randomly selected 19 mini-Tn10 mutants for high through-put genome sequencing...
December 22, 2016: FEMS Microbiology Letters
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