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Transposon mutagenesis

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https://www.readbyqxmd.com/read/29032547/genome-wide-mutagenesis-in-borrelia-burgdorferi
#1
Tao Lin, Lihui Gao
Signature-tagged mutagenesis (STM) is a functional genomics approach to identify bacterial virulence determinants and virulence factors by simultaneously screening multiple mutants in a single host animal, and has been utilized extensively for the study of bacterial pathogenesis, host-pathogen interactions, and spirochete and tick biology. The signature-tagged transposon mutagenesis has been developed to investigate virulence determinants and pathogenesis of Borrelia burgdorferi. Mutants in genes important in virulence are identified by negative selection in which the mutants fail to colonize or disseminate in the animal host and tick vector...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29026163/revealing-the-saline-adaptation-strategies-of-the-halophilic-bacterium-halomonas-beimenensis-through-high-throughput-omics-and-transposon-mutagenesis-approaches
#2
Yan-Huey Chen, Chia-Wei Lu, Yuan-Tay Shyu, Shih-Shun Lin
Studies on the halotolerance of bacteria are attractive to the fermentation industry. However, a lack of sufficient genomic information has precluded an investigation of the halotolerance of Halomonas beimenensis. Here, we describe the molecular mechanisms of saline adaptation in H. beimenensis based on high-throughput omics and Tn5 transposon mutagenesis. The H. beimenensis genome is 4.05 Mbp and contains 3,807 genes, which were sequenced using short and long reads obtained via deep sequencing. Sixteen Tn5 mutants with a loss of halotolerance were identified...
October 12, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29018421/himar1-transposon-for-efficient-random-mutagenesis-in-aggregatibacter-actinomycetemcomitans
#3
Qinfeng Ding, Kai Soo Tan
Aggregatibacter actinomycetemcomitans is the primary etiological agent of aggressive periodontal disease. Identification of novel virulence factors at the genome-wide level is hindered by lack of efficient genetic tools to perform mutagenesis in this organism. The Himar1 mariner transposon is known to yield a random distribution of insertions in an organism's genome with requirement for only a TA dinucleotide target and is independent of host-specific factors. However, the utility of this system in A. actinomycetemcomitans is unknown...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28994778/creation-of-a-dense-transposon-insertion-library-using-bacterial-conjugation-in-enterobacterial-strains-such-as-escherichia-coli-or-shigella-flexneri
#4
Nikki E Freed
Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria...
September 23, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28993850/expression-of-collagenase-is-regulated-by-the-vars-vara-two-component-regulatory-system-in-vibrio-alginolyticus
#5
Takehiko Mima, Kazuyoshi Gotoh, Yumiko Yamamoto, Keiko Maeda, Taku Shirakawa, Shunsuke Matsui, Yumi Murata, Takaki Koide, Hiroshi Tokumitsu, Osamu Matsushita
Vibrio alginolyticus is an opportunistic pathogen in both humans and marine animals. Collagenase encoded by colA is considered to be one of the virulence factors. Expression of colA is regulated by multiple environmental factors, e.g., temperature, growth phase, and substrate. To elucidate the mechanism of regulation of colA expression, transposon mutagenesis was performed. VarS, a sensor histidine kinase of the two-component regulatory system, was demonstrated to regulate the expression of colA. VarA, a cognate response regulator of VarS, was also identified and shown to be involved in the regulation of colA expression...
October 9, 2017: Journal of Membrane Biology
https://www.readbyqxmd.com/read/28993411/sleeping-beauty-insertional-mutagenesis-in-mice-identifies-drivers-of-steatosis-associated-hepatic-tumor
#6
Barbara R Tschida, Nuri A Temiz, Timothy P Kuka, Lindsey A Lee, Jesse D Riordan, Carlos A Tierrablanca, Robert Hullsiek, Sandra Wagner, Wendy A Hudson, Michael A Linden, Khalid Amin, Pauline J Beckmann, Rachel A Heuer, Aaron L Sarver, Ju Dong Yang, Lewis R Roberts, Joseph H Nadeau, Adam J Dupuy, Vincent W Keng, David Largaespada
Hepatic steatosis is a strong risk factor for the development of hepatocellular carcinoma (HCC), yet little is known about the molecular pathology associated with this factor. In this study, we performed a forward genetic screen using Sleeping Beauty (SB) transposon insertional mutagenesis in mice treated to induce hepatic steatosis, and compared the results to human HCC data. In humans, we determined that steatosis increased the proportion of female HCC patients, a pattern also reflected in mice. Our genetic screen identified 203 candidate steatosis-associated HCC genes, many of which are altered in human HCC and are members of established HCC-driving signaling pathways...
October 9, 2017: Cancer Research
https://www.readbyqxmd.com/read/28978631/genetic-screening-for-ems-induced-maize-embryo-specific-mutants-altered-in-embryo-morphogenesis
#7
Dale C Brunelle, Janice K Clark, William F Sheridan
We have previously identified embryo-specific (emb) mutations that resulted in maize kernels containing abnormal embryos with normal appearing endosperm among the progeny of active Robertson's Mutator stocks.  Our rationale for the mutant screen described here is that it should be possible to produce ethyl methane sulfonate (EMS)-induced embryo-specific mutations at a frequency higher than that obtained by transposon mutagenesis and with greater ease. This proved to be the case when we screened for mutations that are embryo-specific among progeny of materials generated with EMS-treated pollen...
October 4, 2017: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/28967994/transposon-insertion-site-sequencing-tis-seq-an-efficient-and-high-throughput-method-for-determining-transposon-insertion-site-s-and-their-relative-abundances-in-a-piggybac-transposon-mutant-pool-by-next-generation-sequencing
#8
Yaligara Veeranagouda, Michel Didier
The PiggyBac (PB) transposon has emerged as a novel mutagenesis tool for understanding gene function and for phenotypic screening in eukaryotes. Successful screening of PB transposon mutants relies on efficient identification of transposon insertion site(s) (TIS) in mutant cells. However, currently available methods suffer from time-consuming steps. Here, we present the method for transposon insertion site sequencing (TIS-Seq) for high-throughput identification of TIS in transposon mutants. TIS-Seq provides qualitative and quantitative information on mutants present in a given PB transposon mutant library...
October 2, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28961327/chronic-liver-injury-alters-driver-mutation-profiles-in-hepatocellular-carcinoma
#9
Jesse D Riordan, Charlotte R Feddersen, Barbara R Tschida, Pauline Jackson, Vincent W Keng, Michael A Linden, Khalid Amin, Christopher S Stipp, David A Largaespada, Adam J Dupuy
Most hepatocellular carcinomas (HCCs) develop in a chronically injured liver, yet the extent to which this microenvironment promotes neoplastic transformation or influences selective pressures for genetic drivers of HCC remains unclear. We sought to determine the impact of hepatic injury in an established mouse model of HCC induced by Sleeping Beauty transposon mutagenesis. Chemically-induced chronic liver injury dramatically increased tumor penetrance and significantly altered driver mutation profiles, likely reflecting distinct selective pressures...
September 29, 2017: Hepatology: Official Journal of the American Association for the Study of Liver Diseases
https://www.readbyqxmd.com/read/28955306/the-laci-family-transcription-factor-rbsr-is-a-pleiotropic-regulator-of-motility-virulence-siderophore-and-antibiotic-production-gas-vesicle-morphogenesis-and-flotation-in-serratia
#10
Chin M Lee, Rita E Monson, Rachel M Adams, George P C Salmond
Gas vesicles (GVs) are proteinaceous, gas-filled organelles used by some bacteria to enable upward movement into favorable air/liquid interfaces in aquatic environments. Serratia sp. ATCC39006 (S39006) was the first enterobacterium discovered to produce GVs naturally. The regulation of GV assembly in this host is complex and part of a wider regulatory network affecting various phenotypes, including antibiotic biosynthesis. To identify new regulators of GVs, a comprehensive mutant library containing 71,000 insertion mutants was generated by random transposon mutagenesis and 311 putative GV-defective mutants identified...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28942835/a-novel-soxo-gene-encoding-a-glutathione-disulfide-reductase-is-essential-for-tetrathionate-oxidation-in-advenella-kashmirensis
#11
Prosenjit Pyne, Masrure Alam, Wriddhiman Ghosh
Molecular mechanisms of chemolithotrophic tetrathionate oxidation are not clearly understood. Here we used transposon(Tn5-mob)-insertion mutagenesis to search for novel tetrathionate oxidation genes in the facultatively chemolithoautotrophic betaproteobacterium Advenella kashmirensis that not only oxidizes tetrathionate, but also produces the same as an intermediate during thiosulfate oxidation. Genome-wide random insertion of Tn5-mob occurred at a frequency of one per 10(4) donor E. coli cells. A library of 8000 transconjugants yielded five tetrathionate-oxidation-impaired mutants, of which, the one named Ak_Tn_16 was studied here in detail...
December 2017: Microbiological Research
https://www.readbyqxmd.com/read/28938018/genome-wide-identification-of-bacterial-plant-colonization-genes
#12
Benjamin J Cole, Meghan E Feltcher, Robert J Waters, Kelly M Wetmore, Tatiana S Mucyn, Elizabeth M Ryan, Gaoyan Wang, Sabah Ul-Hasan, Meredith McDonald, Yasuo Yoshikuni, Rex R Malmstrom, Adam M Deutschbauer, Jeffery L Dangl, Axel Visel
Diverse soil-resident bacteria can contribute to plant growth and health, but the molecular mechanisms enabling them to effectively colonize their plant hosts remain poorly understood. We used randomly barcoded transposon mutagenesis sequencing (RB-TnSeq) in Pseudomonas simiae, a model root-colonizing bacterium, to establish a genome-wide map of bacterial genes required for colonization of the Arabidopsis thaliana root system. We identified 115 genes (2% of all P. simiae genes) with functions that are required for maximal competitive colonization of the root system...
September 2017: PLoS Biology
https://www.readbyqxmd.com/read/28923910/a-transposon-screen-identifies-loss-of-primary-cilia-as-a-mechanism-of-resistance-to-smo-inhibitors
#13
Xuesong Zhao, Ekaterina Pak, Kimberly J Ornell, Maria F Pazyra-Murphy, Ethan L MacKenzie, Emily J Chadwick, Tatyana Ponomaryov, Joseph F Kelleher, Rosalind A Segal
Drug resistance poses a great challenge to targeted cancer therapies. In Hedgehog pathway-dependent cancers, the scope of mechanisms enabling resistance to Smo-inhibitors is not known. Here, we performed a transposon mutagenesis screen in medulloblastoma and identified multiple modes of resistance. Surprisingly, mutations in ciliogenesis genes represent a frequent cause of resistance, and patient datasets indicate that cilia loss constitutes a clinically relevant category of resistance. Conventionally, primary cilia are thought to enable oncogenic Hedgehog signaling...
September 18, 2017: Cancer Discovery
https://www.readbyqxmd.com/read/28904414/identification-of-arsenic-resistance-genes-from-marine-sediment-metagenome
#14
Nar Singh Chauhan, Sonam Nain, Rakesh Sharma
A metagenomic library of sea sediment metagenome containing 245,000 recombinant clones representing ~ 2.45 Gb of sea sediment microbial DNA was constructed. Two unique arsenic resistance clones, A7 and A12, were identified by selection on sodium arsenite containing medium. Clone A7 showed a six-fold higher resistance to arsenate [As(V)], a three-fold higher resistance to arsenite [As(III)] and significantly increased resistance to antimony [Sb(III)], while clone A12 showed increased resistance only to sodium arsenite and not to the other two metalloids...
September 2017: Indian Journal of Microbiology
https://www.readbyqxmd.com/read/28892254/transcriptional-regulation-of-ectoine-catabolism-in-response-to-multiple-metabolic-and-environmental-cues
#15
Annina Schulz, Lucas Hermann, Sven-Andreas Freibert, Tobias Bönig, Tamara Hoffmann, Ramona Riclea, Jeroen S Dickschat, Johann Heider, Erhard Bremer
Ectoine and hydroxyectoine are effective microbial osmostress protectants, but can also serve as versatile nutrients for bacteria. We have studied the genetic regulation of ectoine and hydroxyectoine import and catabolism in the marine Roseobacter species Ruegeria pomeroyi and identified three transcriptional regulators involved in these processes: the GabR/MocR-type repressor EnuR, the feast and famine-type regulator AsnC and the two-component system NtrYX. The corresponding genes are widely associated with ectoine and hydroxyectoine uptake and catabolic gene clusters (enuR, asnC), and with microorganisms predicted to consume ectoines (ntrYX)...
September 11, 2017: Environmental Microbiology
https://www.readbyqxmd.com/read/28892231/genome-wide-analysis-of-bacterial-determinants-of-plant-growth-promotion-and-induced-systemic-resistance-by-pseudomonas-fluorescens
#16
Xu Cheng, Desalegn W Etalo, Judith E van de Mortel, Ester Dekkers, Linh Nguyen, Marnix H Medema, Jos M Raaijmakers
Pseudomonas fluorescens strain SS101 (Pf.SS101) promotes growth of Arabidopsis thaliana, enhances greening and lateral root formation, and induces systemic resistance (ISR) against the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). Here, targeted and untargeted approaches were adopted to identify bacterial determinants and underlying mechanisms involved in plant growth promotion and ISR by Pf.SS101. Based on targeted analyses, no evidence was found for volatiles, lipopeptides and siderophores in plant growth promotion by Pf...
September 11, 2017: Environmental Microbiology
https://www.readbyqxmd.com/read/28888423/transposons-as-tools-for-functional-genomics-in-vertebrate-models
#17
REVIEW
Koichi Kawakami, David A Largaespada, Zoltán Ivics
Genetic tools and mutagenesis strategies based on transposable elements are currently under development with a vision to link primary DNA sequence information to gene functions in vertebrate models. By virtue of their inherent capacity to insert into DNA, transposons can be developed into powerful tools for chromosomal manipulations. Transposon-based forward mutagenesis screens have numerous advantages including high throughput, easy identification of mutated alleles, and providing insight into genetic networks and pathways based on phenotypes...
September 6, 2017: Trends in Genetics: TIG
https://www.readbyqxmd.com/read/28855965/thermotolerant-genes-essential-for-survival-at-a-critical-high-temperature-in-thermotolerant-ethanologenic-zymomonas-mobilis-tistr-548
#18
Kannikar Charoensuk, Tomoko Sakurada, Amina Tokiyama, Masayuki Murata, Tomoyuki Kosaka, Pornthap Thanonkeo, Mamoru Yamada
BACKGROUND: High-temperature fermentation (HTF) technology is expected to reduce the cost of bioconversion of biomass to fuels or chemicals. For stable HTF, the development of a thermotolerant microbe is indispensable. Elucidation of the molecular mechanism of thermotolerance would enable the thermal stability of microbes to be improved. RESULTS: Thermotolerant genes that are essential for survival at a critical high temperature (CHT) were identified via transposon mutagenesis in ethanologenic, thermotolerant Zymomonas mobilis TISTR 548...
2017: Biotechnology for Biofuels
https://www.readbyqxmd.com/read/28855524/rnai-combining-sleeping-beauty-transposon-system-inhibits-ex-vivo-expression-of-foot-and-mouth-disease-virus-vp1-in-transgenic-sheep-cells
#19
Shoulong Deng, Guangdong Li, Kun Yu, Xiuzhi Tian, Feng Wang, Wenting Li, Wuqi Jiang, Pengyun Ji, Hongbing Han, Juncai Fu, Xiaosheng Zhang, Jinlong Zhang, Yixun Liu, Zhengxing Lian, Guoshi Liu
Foot and mouth disease, which is induced by the foot and mouth disease virus (FMDV), takes its toll on the cloven-hoofed domestic animals. The VP1 gene in FMDV genome encodes the viral capsid, a vital element for FMDV replication. Sleeping Beauty (SB) is an active DNA-transposon system responsible for genetic transformation and insertional mutagenesis in vertebrates. In this study, a conserved VP1-shRNA which specifically targets the ovine FMDV-VP1 gene was constructed and combined with SB transposase and transposon...
August 30, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28854370/generation-of-mouse-haploid-somatic-cells-by-small-molecules-for-genome-wide-genetic-screening
#20
Zheng-Quan He, Bao-Long Xia, Yu-Kai Wang, Jing Li, Gui-Hai Feng, Lin-Lin Zhang, Yu-Huan Li, Hai-Feng Wan, Tian-Da Li, Kai Xu, Xue-Wei Yuan, Yu-Fei Li, Xin-Xin Zhang, Ying Zhang, Liu Wang, Wei Li, Qi Zhou
The recent success of derivation of mammalian haploid embryonic stem cells (haESCs) has provided a powerful tool for large-scale functional analysis of the mammalian genome. However, haESCs rapidly become diploidized after differentiation, posing challenges for genetic analysis. Here, we show that the spontaneous diploidization of haESCs happens in metaphase due to mitotic slippage. Diploidization can be suppressed by small-molecule-mediated inhibition of CDK1 and ROCK. Through ROCK inhibition, we can generate haploid somatic cells of all three germ layers from haESCs, including terminally differentiated neurons...
August 29, 2017: Cell Reports
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