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Transposon mutagenesis

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https://www.readbyqxmd.com/read/29691334/forward-genetic-dissection-of-biofilm-development-by-fusobacterium-nucleatum-novel-functions-of-cell-division-proteins-ftsx-and-envc
#1
Chenggang Wu, Abu Amar Mohamed Al Mamun, Truc Thanh Luong, Bo Hu, Jianhua Gu, Ju Huck Lee, Melissa D'Amore, Asis Das, Hung Ton-That
Fusobacterium nucleatum is a key member of the human oral biofilm. It is also implicated in preterm birth and colorectal cancer. To facilitate basic studies of fusobacterial virulence, we describe here a versatile transposon mutagenesis procedure and a pilot screen for mutants defective in biofilm formation. Out of 10 independent biofilm-defective mutants isolated, the affected genes included the homologs of the Escherichia coli cell division proteins FtsX and EnvC, the electron transport protein RnfA, and four proteins with unknown functions...
April 24, 2018: MBio
https://www.readbyqxmd.com/read/29683762/regulation-of-hemolysin-in-uropathogenic-escherichia-coli-fine-tunes-killing-of-human-macrophages
#2
Ambika M V Murthy, Minh-Duy Phan, Kate M Peters, Nguyen Thi Khanh Nhu, Rodney A Welch, Glen C Ulett, Mark A Schembri, Matthew J Sweet
Uropathogenic E. coli (UPEC) causes the majority of urinary tract infections (UTIs), which are a major global public health concern. UPEC uses numerous mechanisms to subvert the innate immune system, including targeting macrophage functions. We recently showed that some UPEC strains rapidly kill human macrophages via an NLRP3-independent pathway, and also trigger NLRP3-dependent IL-1β processing. In this study, we used random transposon mutagenesis in the reference strain CFT073 to identify UPEC genes that mediate human macrophage cell death...
April 23, 2018: Virulence
https://www.readbyqxmd.com/read/29678883/ribosome-maturation-by-the-endoribonuclease-ybey-stabilises-a-type-iii-secretion-system-transcript-required-for-virulence-of-enterohemorrhagic-escherichia-coli
#3
Sean P McAteer, Brandon M Sy, Julia L Wong, David Tollervey, David L Gally, Jai J Tree
Enterohemorrhagic Escherichia coli (EHEC) is a significant human pathogen that colonizes humans and its reservoir host, cattle. Colonization requires the expression of a type III secretion (T3S) system that injects a cocktail of effector proteins into host cells, to promote bacterial attachment and disease progression. The T3S system is tightly regulated by a complex network of transcriptional and post-transcriptional regulators. Using transposon mutagenesis, here we identified the ybeZYX-int operon as being required for normal T3S levels...
April 20, 2018: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29662477/new-genes-involved-in-mild-stress-response-identified-by-transposon-mutagenesis-in-lactobacillus-paracasei
#4
Aurore Palud, Hélène Scornec, Jean-François Cavin, Hélène Licandro
Lactic acid bacteria (LAB) are associated with various plant, animal, and human niches and are also present in many fermented foods and beverages. Thus, they are subjected to several stress conditions and have developed advanced response mechanisms to resist, adapt, and grow. This work aimed to identify the genes involved in some stress adaptation mechanisms in LAB. For this purpose, global reverse genetics was applied by screening a library of 1287 Lactobacillus paracasei transposon mutants for mild monofactorial stresses...
2018: Frontiers in Microbiology
https://www.readbyqxmd.com/read/29660854/the-butanol-producing-microbe-clostridium-beijerinckii-ncimb-14988-manipulated-using-forward-and-reverse-genetic-tools
#5
Gareth T Little, Benjamin J Willson, John T Heap, Klaus Winzer, Nigel P Minton
The solventogenic anaerobe Clostridium beijerinckii has potential for use in the sustainable bioconversion of plant-derived carbohydrates into solvents, such as butanol or acetone. However, relatively few strains have been extensively characterised either at the genomic level or through exemplification of a complete genetic toolkit. To remedy this situation, a new strain of C. beijerinckii, NCIMB 14988, was selected from amongst a total of 55 new clostridial isolates capable of growth on hexose and pentose sugars...
April 16, 2018: Biotechnology Journal
https://www.readbyqxmd.com/read/29657968/inferring-the-minimal-genome-of-mesoplasma-florum-by-comparative-genomics-and-transposon-mutagenesis
#6
Vincent Baby, Jean-Christophe Lachance, Jules Gagnon, Jean-François Lucier, Dominick Matteau, Tom Knight, Sébastien Rodrigue
The creation and comparison of minimal genomes will help better define the most fundamental mechanisms supporting life. Mesoplasma florum is a near-minimal, fast-growing, nonpathogenic bacterium potentially amenable to genome reduction efforts. In a comparative genomic study of 13 M. florum strains, including 11 newly sequenced genomes, we have identified the core genome and open pangenome of this species. Our results show that all of the strains have approximately 80% of their gene content in common. Of the remaining 20%, 17% of the genes were found in multiple strains and 3% were unique to any given strain...
May 2018: MSystems
https://www.readbyqxmd.com/read/29587177/advances-in-functional-genetic-screening-with-transposons-and-crispr-cas9-to-illuminate-cancer-biology
#7
REVIEW
Kathryn A O'Donnell
Large-scale genome sequencing studies have identified a wealth of mutations in human tumors and have dramatically advanced the field of cancer genetics. However, the functional consequences of an altered gene in tumor progression cannot always be inferred from mutation status alone. This underscores the critical need for complementary methods to assign functional significance to mutated genes in cancer. Transposons are mobile genetic elements that serve as powerful tools for insertional mutagenesis. Over the last decade, investigators have employed mouse models with on-demand transposon-mediated mutagenesis to perform unbiased genetic screens to identify clinically relevant genes that participate in the pathogenesis of human cancer...
March 24, 2018: Current Opinion in Genetics & Development
https://www.readbyqxmd.com/read/29580289/a-multi-host-approach-to-identify-a-transposon-mutant-of-pseudomonas-aeruginosa-lesb58-lacking-full-virulence
#8
Cynthia Gagné-Thivierge, Irena Kukavica-Ibrulj, Geneviève Filion, Valérie Dekimpe, Sok Gheck E Tan, Antony T Vincent, Éric Déziel, Roger C Levesque, Steve J Charette
OBJECTIVE: Pseudomonas aeruginosa is an opportunistic bacterial pathogen well known to cause chronic lung infections in individuals with cystic fibrosis (CF). Some strains adapted to this particular niche show distinct phenotypes, such as biofilm hyperproduction. It is necessary to study CF clinical P. aeruginosa isolates, such as Liverpool Epidemic Strains (LES), to acquire a better understanding of the key genes essential for in vivo maintenance and the major virulence mechanisms involved in CF lung infections...
March 27, 2018: BMC Research Notes
https://www.readbyqxmd.com/read/29554276/collection-of-homozygous-mutant-mouse-embryonic-stem-cells-arising-from-autodiploidization-during-haploid-gene-trap-mutagenesis
#9
Ayako Yamanishi, Atsushi Matsuba, Ryohei Kondo, Rie Akamatsu, Sachiyo Tanaka, Masahiro Tokunaga, Kyoji Horie, Chikara Kokubu, Yasumasa Ishida, Junji Takeda
Haploid mouse embryonic stem cells (ESCs), in which a single hit mutation is sufficient to produce loss-of-function phenotypes, have provided a powerful tool for forward genetic screening. This strategy, however, can be hampered by undesired autodiploidization of haploid ESCs. To overcome this obstacle, we designed a new methodology that facilitates enrichment of homozygous mutant ESC clones arising from autodiploidization during haploid gene trap mutagenesis. Haploid mouse ESCs were purified by fluorescence-activated cell sorting to maintain their haploid property and then transfected with the Tol2 transposon-based biallelically polyA-trapping (BPATrap) vector that carries an invertible G418 plus puromycin double selection cassette...
March 15, 2018: Nucleic Acids Research
https://www.readbyqxmd.com/read/29549432/mexef-oprn-multidrug-efflux-pump-transporter-negatively-controls-n-acyl-homoserine-lactone-accumulation-in-pseudomonas-syringae-pv-tabaci-6605
#10
Takahiro Sawada, Miho Eguchi, Seiya Asaki, Ryota Kashiwagi, Kousuke Shimomura, Fumiko Taguchi, Hidenori Matsui, Mikihiro Yamamoto, Yoshiteru Noutoshi, Kazuhiro Toyoda, Yuki Ichinose
Our previous studies revealed that flagellar-motility-defective mutants such as ∆fliC of Pseudomonas syringae pv. tabaci 6605 (Pta6605) have remarkably reduced production of N-acyl-homoserine lactones (AHL), quorum-sensing molecules. To investigate the reason of loss of AHL production in ∆fliC mutant, we carried out transposon mutagenesis. Among approximately 14,000 transconjugants, we found 11 AHL production-recovered (APR) strains. In these APR strains, a transposon was inserted into either mexE or mexF, genes encoding for the multidrug efflux pump transporter MexEF-OprN, and mexT, a gene encoding a putative transcriptional activator for mexEF-oprN...
March 16, 2018: Molecular Genetics and Genomics: MGG
https://www.readbyqxmd.com/read/29542085/the-enhancer-trap-in-ciona
#11
Yasunori Sasakura
Enhancer trap is a famous application of transposons. This method is useful for the creation of marker transgenic lines that express a reporter gene in tissue- or organ-specific manner, characterization of enhancers in the genome, finding novel patterns of gene expression, and mutagenesis. In Ciona intestinalis, efficient enhancer traps with Minos and Sleeping beauty transposons have been reported. With the enhancer trap lines, the intronic enhancers regulating the expression of the Musashi gene, the compartment in the digestive tube, the presence of enhancers sensitive to the orientation of the gene that they regulate, and the functions of the Hox1 gene have been revealed...
2018: Advances in Experimental Medicine and Biology
https://www.readbyqxmd.com/read/29539877/effects-of-flac-mutation-on-stringent-response-mediated-bacterial-growth-toxin-production-and-motility-in-vibrio-cholerae
#12
Hwa Young Kim, Sang-Mi Yu, Sang Chul Jeong, Sang Sun Yoon, Young Taek Oh
The stringent response (SR), which is activated by accumulation of (p)ppGpp under conditions of growth-inhibiting stresses, plays an important role ongrowth and virulence in Vibrio cholerae . Herein, we carried out a genome-wide screen using transposon random mutagenesis to identify genes controlled by SR in (p)ppGpp-overproducing mutant strain. One of the identified SR target genes was the flaC encoding flagellin. Genetic studies using flaC and SR mutants demonstrated that FlaC involved in bacterial growth, toxin production, and normal flagellum function under conditions of high (p)ppGpp levels, suggesting FlaC plays an important role in SR-induced pathogenicityin V...
March 15, 2018: Journal of Microbiology and Biotechnology
https://www.readbyqxmd.com/read/29505613/tnseq-of-mycobacterium-tuberculosis-clinical-isolates-reveals-strain-specific-antibiotic-liabilities
#13
Allison F Carey, Jeremy M Rock, Inna V Krieger, Michael R Chase, Marta Fernandez-Suarez, Sebastien Gagneux, James C Sacchettini, Thomas R Ioerger, Sarah M Fortune
Once considered a phenotypically monomorphic bacterium, there is a growing body of work demonstrating heterogeneity among Mycobacterium tuberculosis (Mtb) strains in clinically relevant characteristics, including virulence and response to antibiotics. However, the genetic and molecular basis for most phenotypic differences among Mtb strains remains unknown. To investigate the basis of strain variation in Mtb, we performed genome-wide transposon mutagenesis coupled with next-generation sequencing (TnSeq) for a panel of Mtb clinical isolates and the reference strain H37Rv to compare genetic requirements for in vitro growth across these strains...
March 2018: PLoS Pathogens
https://www.readbyqxmd.com/read/29504631/wxcx-is-involved-in-bacterial-attachment-and-virulence-in-xanthomonas-campestris-pv-campestris
#14
Yu-Fan Liu, Chao-Tsai Liao, Ying-Chuan Chiang, Chih-En Li, Yi-Min Hsiao
Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot in crucifers. Here, one EZ-Tn5 transposon mutant of Xcc, altered in bacterial attachment, was isolated. Further analysis revealed that the transposon was inserted in the wxcX gene (encodes a hypothetical protein) of the transposon mutant. Sequence analysis revealed that WxcX is highly conserved in Xanthomonas, but none has been characterized. In this study, it was indicated that mutation of wxcX resulted in enhanced bacterial attachment, reduced virulence on the host cabbage, and increased sensitivity to sodium dodecyl sulfate...
March 5, 2018: Journal of Basic Microbiology
https://www.readbyqxmd.com/read/29493878/developing-a-piggybac-transposon-system-and-compatible-selection-markers-for-insertional-mutagenesis-and-genome-engineering-in-yarrowia-lipolytica
#15
James M Wagner, Eden V Williams, Hal S Alper
Yarrowia lipolytica is a non-conventional yeast of interest to the biotechnology industry. However, the physiology, metabolism, and genetic regulation of Y. lipolytica diverge significantly from more well-studied and characterized yeasts such as Saccharomyces cerevisiae. To develop additional genetic tools for this industrially relevant host, we adapt the piggyBac transposon system to enable efficient generation of genome-wide insertional mutagenesis libraries and introduction of scarless, footprint-free genomic modifications in Y...
March 1, 2018: Biotechnology Journal
https://www.readbyqxmd.com/read/29472909/metagenomic-profiling-of-soil-microbes-to-mine-salt-stress-tolerance-genes
#16
Vasim Ahmed, Manoj K Verma, Shashank Gupta, Vibha Mandhan, Nar S Chauhan
Osmotolerance is one of the critical factors for successful survival and colonization of microbes in saline environments. Nonetheless, information about these osmotolerance mechanisms is still inadequate. Exploration of the saline soil microbiome for its community structure and novel genetic elements is likely to provide information on the mechanisms involved in osmoadaptation. The present study explores the saline soil microbiome for its native structure and novel genetic elements involved in osmoadaptation...
2018: Frontiers in Microbiology
https://www.readbyqxmd.com/read/29470858/genes-affecting-progression-of-bacteriophage-p22-infection-in-salmonella-identified-by-transposon-and-single-gene-deletion-screens
#17
Kaitlynne Bohm, Steffen Porwollik, Weiping Chu, John A Dover, Eddie B Gilcrease, Sherwood R Casjens, Michael McClelland, Kristin N Parent
Bacteriophages rely on their hosts for replication, and many host genes critically determine either viral progeny production or host success via phage resistance. A random insertion transposon library of 240,000 mutants in Salmonella enterica serovar Typhimurium was used to monitor effects of individual bacterial gene disruptions on bacteriophage P22 lytic infection. These experiments revealed candidate host genes that alter the timing of phage P22 propagation. Using a False Discovery Rate of <0.1, mutations in 235 host genes either blocked or delayed progression of P22 lytic infection, including many genes for which this role was previously unknown...
February 22, 2018: Molecular Microbiology
https://www.readbyqxmd.com/read/29463657/the-essential-genome-of-escherichia-coli-k-12
#18
Emily C A Goodall, Ashley Robinson, Iain G Johnston, Sara Jabbari, Keith A Turner, Adam F Cunningham, Peter A Lund, Jeffrey A Cole, Ian R Henderson
Transposon-directed insertion site sequencing (TraDIS) is a high-throughput method coupling transposon mutagenesis with short-fragment DNA sequencing. It is commonly used to identify essential genes. Single gene deletion libraries are considered the gold standard for identifying essential genes. Currently, the TraDIS method has not been benchmarked against such libraries, and therefore, it remains unclear whether the two methodologies are comparable. To address this, a high-density transposon library was constructed in Escherichia coli K-12...
February 20, 2018: MBio
https://www.readbyqxmd.com/read/29454685/genes-involved-in-tolerance-to-osmotic-stress-by-random-mutagenesis-in-cronobacter-malonaticus
#19
Maofeng Zhang, Xiyan Zhang, Liaowang Tong, Yaping Wang, Dexin Ou, Jumei Zhang, Qingping Wu, Yingwang Ye
Cronobacter malonaticus is one of the opportunistic food-borne pathogens in powdered infant formula and has unusual abilities to survive under environmental stresses such as osmotic conditions. However, the genes involved in osmotic stress have received little attention in C. malonaticus. Here, genes involved in osmotic stress were determined in C. malonaticus using a transposon mutagenesis approach. According to the growth of mutants (n = 215) under 5.0% NaCl concentration, the survival of 5 mutants under osmotic stress was significantly decreased compared with that of the wild type strain...
February 14, 2018: Journal of Dairy Science
https://www.readbyqxmd.com/read/29445422/sleeping-beauty-transposon-integrates-into-non-ta-dinucleotides
#20
Yabin Guo, Yin Zhang, Kaishun Hu
Background: Sleeping Beauty transposon (SB) has become an increasingly important genetic tool for generating mutations in vertebrate cells. It is widely thought that SB exclusively integrates into TA dinucleotides. However, this strict TA-preference has not been rigorously tested in large numbers of insertion sites that now can be detected with next generation sequencing. Li et al. found 71 SB insertions in non-TA dinucleotides in 2013, suggesting that TA dinucleotides are not the only sites of SB integration, yet further studies on this topic have not been carried out...
2018: Mobile DNA
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