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Transposon mutagenesis

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https://www.readbyqxmd.com/read/28097405/role-of-exopolysaccharide-in-salt-stress-resistance-and-cell-motility-of-mesorhizobium-alhagi-ccnwxj12-2-t
#1
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
#2
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
#3
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
#4
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
#5
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-through-quantitative-variation-in-nodulation
#6
Shaun J Curtin, Peter Tiffin, Joseph Guhlin, Diana I Trujillo, Liana T Burghardt, Paul Atkins, Nicholas J Baltes, Roxanne Denny, Daniel F Voytas, Robert M Stupar, Nevin Dale 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 retro-transposons, hairpin RNA-interference constructs, and CRISPR/Cas9 nucleases) together with randomized, well-replicated experiments to evaluate the function of genes that an earlier GWAS in Medicago truncatula had identified as candidates contributing to variation in the symbiosis between legumes and rhizobia...
January 5, 2017: Plant Physiology
https://www.readbyqxmd.com/read/28049432/identification-of-genes-affecting-alginate-biosynthesis-in-pseudomonas-fluorescens-by-screening-a-transposon-insertion-library
#7
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
#8
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
#9
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...
December 13, 2016: Cell Chemical Biology
https://www.readbyqxmd.com/read/28011697/incidental-mutations-occur-frequently-during-transposon-mutagenesis
#10
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
https://www.readbyqxmd.com/read/28002452/the-effector-cig57-hijacks-fcho-mediated-vesicular-trafficking-to-facilitate-intracellular-replication-of-coxiella-burnetii
#11
Eleanor A Latomanski, Patrice Newton, Chen Ai Khoo, Hayley J Newton
Coxiella burnetii is an intracellular bacterial pathogen that infects alveolar macrophages and replicates within a unique lysosome-derived vacuole. When Coxiella is trafficked to a host cell lysosome the essential Dot/Icm type IV secretion system is activated allowing over 130 bacterial effector proteins to be translocated into the host cytosol. This cohort of effectors is believed to manipulate host cell functions to facilitate Coxiella-containing vacuole (CCV) biogenesis and bacterial replication. Transposon mutagenesis has demonstrated that the Dot/Icm effector Cig57 is required for CCV development and intracellular replication of Coxiella...
December 2016: PLoS Pathogens
https://www.readbyqxmd.com/read/27995867/poly-a-polymerase-i-participates-in-the-indole-regulatory-pathway-of-pantoea-agglomerans-ys19
#12
Zihua Li, Jing Jiang, Xuemei Yu, Cunxiang Wu, Delong Shen, Yongjun Feng
Pantoea agglomerans YS19 is a preponderant endophytic bacterium isolated from rice. It is characterized by the formation of symplasmata, a type of multicellular aggregate structure, contributing to a strong stress-resistance and specific adaptation of YS19 in endophyte-host associations. Indole is an important signal molecule in intraspecies or interspecies relationships, regulating a variety of bacterial behaviors such as cell aggregation and stress-resistance; however, the regulatory mechanism remains an on-going area of investigation...
December 20, 2016: Microbiology
https://www.readbyqxmd.com/read/27992504/pptab-exports-rgg-quorum-sensing-peptides-in-streptococcus
#13
Jennifer C Chang, Michael J Federle
A transposon mutagenesis screen designed to identify mutants that were defective in peptide-pheromone signaling of the Rgg2/Rgg3 pathway in Streptococcus pyogenes generated insertions in sixteen loci displaying diminished reporter activity. Fourteen unique transposon insertions were mapped to pptAB, an ABC-type transporter recently described to export sex pheromones of Enterococcus faecalis. Consistent with an idea that PptAB exports signaling peptides, the pheromones known as SHPs (short hydrophobic peptides) were no longer detected in cell-free culture supernatants in a generated deletion mutant of pptAB...
2016: PloS One
https://www.readbyqxmd.com/read/27986717/flra-represses-transcription-of-biofilm-associated-bpfa-operon-in-shewanella-putrefaciens
#14
Yuan-Yuan Cheng, Chao Wu, Jia-Yi Wu, Hui-Ling Jia, Ming-Yu Wang, Huan-Yu Wang, Si-Min Zou, Rui-Rui Sun, Rong Jia, Ya-Zhong Xiao
: Manipulation of biofilm formation in Shewanella is beneficial for applications in industrial and environmental biotechnology. BpfA is an adhesin largely responsible for biofilm formation in many Shewanella species. However, the mechanism underlying the BpfA production and resulting biofilm remains vaguely understood. We previously described that BpfA expression is enhanced by DosD, an oxygen-stimulated diguanylate cyclase, under aerobic growth. In the present work, we identified FlrA as a critical transcription regulator of the bpfA operon in Shewanella putrefaciens CN32 by transposon mutagenesis...
December 16, 2016: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/27965645/glucose-induces-ecf-sigma-factor-genes-sigx-and-sigm-independent-of-cognate-anti-sigma-factors-through-acetylation-of-csha-in-bacillus-subtilis
#15
Mitsuo Ogura, Kei Asai
Extracytoplasmic function (ECF) σ factors have roles related to cell envelope and/or cell membrane functions, in addition to other cellular functions. Without cell-surface stresses, ECF σ factors are sequestered by the cognate anti-σ factor, leading to inactivation and the resultant repression of regulons due to the inhibition of transcription of their own genes. Bacillus subtilis has seven ECF σ factors including σ(X) and σ(M) that transcribe their own structural genes. Here, we report that glucose addition to the medium induced sigX and sigM transcription independent of their anti-σ factors...
2016: Frontiers in Microbiology
https://www.readbyqxmd.com/read/27929521/efficient-footprint-free-human-ipsc-genome-editing-by-consolidation-of-cas9-crispr-and-piggybac-technologies
#16
Gang Wang, Luhan Yang, Dennis Grishin, Xavier Rios, Lillian Y Ye, Yong Hu, Kai Li, Donghui Zhang, George M Church, William T Pu
Genome editing of human induced pluripotent stem cells (hiPSCs) offers unprecedented opportunities for in vitro disease modeling and personalized cell replacement therapy. The introduction of Cas9-directed genome editing has expanded adoption of this approach. However, marker-free genome editing using standard protocols remains inefficient, yielding desired targeted alleles at a rate of ∼1-5%. We developed a protocol based on a doxycycline-inducible Cas9 transgene carried on a piggyBac transposon to enable robust and highly efficient Cas9-directed genome editing, so that a parental line can be expeditiously engineered to harbor many separate mutations...
January 2017: Nature Protocols
https://www.readbyqxmd.com/read/27922098/comparison-of-432-pseudomonas-strains-through-integration-of-genomic-functional-metabolic-and-expression-data
#17
Jasper J Koehorst, Jesse C J van Dam, Ruben G A van Heck, Edoardo Saccenti, Vitor A P Martins Dos Santos, Maria Suarez-Diez, Peter J Schaap
Pseudomonas is a highly versatile genus containing species that can be harmful to humans and plants while others are widely used for bioengineering and bioremediation. We analysed 432 sequenced Pseudomonas strains by integrating results from a large scale functional comparison using protein domains with data from six metabolic models, nearly a thousand transcriptome measurements and four large scale transposon mutagenesis experiments. Through heterogeneous data integration we linked gene essentiality, persistence and expression variability...
December 6, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27911809/unique-attributes-of-cyanobacterial-metabolism-revealed-by-improved-genome-scale-metabolic-modeling-and-essential-gene-analysis
#18
Jared T Broddrick, Benjamin E Rubin, David G Welkie, Niu Du, Nathan Mih, Spencer Diamond, Jenny J Lee, Susan S Golden, Bernhard O Palsson
The model cyanobacterium, Synechococcus elongatus PCC 7942, is a genetically tractable obligate phototroph that is being developed for the bioproduction of high-value chemicals. Genome-scale models (GEMs) have been successfully used to assess and engineer cellular metabolism; however, GEMs of phototrophic metabolism have been limited by the lack of experimental datasets for model validation and the challenges of incorporating photon uptake. Here, we develop a GEM of metabolism in S. elongatus using random barcode transposon site sequencing (RB-TnSeq) essential gene and physiological data specific to photoautotrophic metabolism...
December 20, 2016: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/27909217/the-small-foxp1-isoform-predominantly-expressed-in-activated-b-cell-like-diffuse-large-b-cell-lymphoma-and-full-length-foxp1-exert-similar-oncogenic-and-transcriptional-activity-in-human-b-cells
#19
Martine van Keimpema, Leonie J Grüneberg, Esther J M Schilder-Tol, Monique E C M Oud, Esther Beuling, Paul J Hensbergen, Johann de Jong, Steven T Pals, Marcel Spaargaren
The forkhead transcription factor FOXP1 is generally regarded as an oncogene in activated B cell-like diffuse large B-cell lymphoma. Previous studies have suggested that a small isoform of FOXP1 (FOXP1-iso), rather than full-length FOXP1 (FOXP1-FL), may possess this oncogenic activity. Corroborating those studies, we here show that activated B cell-like diffuse large B-cell lymphoma cell-lines and primary activated B cell-like diffuse large B-cell lymphoma cells predominantly express FOXP1-iso and that the 5-end of the Foxp1 gene is a common insertion site in murine lymphomas in leukaemia virus- and transposon-mediated insertional mutagenesis screens...
December 1, 2016: Haematologica
https://www.readbyqxmd.com/read/27895128/characterization-of-the-pathogenicity-of-streptococcus-intermedius-tyg1620-isolated-from-a-human-brain-abscess-based-on-the-complete-genome-sequence-with-transcriptome-analysis-and-transposon-mutagenesis-in-a-murine-subcutaneous-abscess-model
#20
Noriko Hasegawa, Tsuyoshi Sekizuka, Yutaka Sugi, Nobuhiro Kawakami, Yumiko Ogasawara, Kengo Kato, Akifumi Yamashita, Fumihiko Takeuchi, Makoto Kuroda
Streptococcus intermedius is known to cause periodontitis and pyogenic infections in the brain and liver. Here we report the complete genome sequence of strain TYG1620 (genome size: 2,006,877 bp; GC content: 37.6%; 2,020 predicted ORFs) isolated from a brain abscess in an infant. Comparative genome analysis of S. intermedius genome sequences suggested that TYG1620 carries a notable type VII secretion system (T7SS), two long-repeat regions and 19 ORFs for cell-wall-anchored proteins (CWAPs). To elucidate genes responsible for the pathogenicity of TYG1620, transcriptome analysis was performed in a murine subcutaneous abscess model...
November 28, 2016: Infection and Immunity
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