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Transposon mutagenesis

Rebecca L Lamason, Effie Bastounis, Natasha M Kafai, Ricardo Serrano, Juan C Del Álamo, Julie A Theriot, Matthew D Welch
Spotted fever group (SFG) rickettsiae are human pathogens that infect cells in the vasculature. They disseminate through host tissues by a process of cell-to-cell spread that involves protrusion formation, engulfment, and vacuolar escape. Other bacterial pathogens rely on actin-based motility to provide a physical force for spread. Here, we show that SFG species Rickettsia parkeri typically lack actin tails during spread and instead manipulate host intercellular tension and mechanotransduction to promote spread...
October 20, 2016: Cell
Kyle A Tipton, Marjan Farokhyfar, Philip N Rather
Colony opacity phase variation in Acinetobacter baumannii strain AB5075 is regulated by a reversible high-frequency switch. Transposon mutagenesis was used to generate mutations that decreased the opaque to translucent switch and a gene encoding a predicted periplasmic membrane fusion component of a resistance-nodulation-cell division (RND)-type efflux system was isolated. This gene was designated arpA and immediately downstream was a gene designated arpB that encodes a predicted membrane transporter of RND-type systems...
October 19, 2016: MicrobiologyOpen
Brian J Akerley
The property of transposons to randomly insert into target DNA has long been exploited for generalized mutagenesis and forward genetic screens. Newer applications that monitor the relative abundance of each transposon insertion in large libraries of mutants can be used to evaluate the roles in cellular fitness of all genes of an organism, provided that transposition is in fact random across all genes. In a recent article, Kimura and colleagues identified an important exception to the latter assumption [S. Kimura, T...
October 11, 2016: MBio
José Eduardo Vargas, Leonardo Chicaybam, Renato Tetelbom Stein, Amilcar Tanuri, Andrés Delgado-Cañedo, Martin H Bonamino
Gene therapy protocols require robust and long-term gene expression. For two decades, retrovirus family vectors have offered several attractive properties as stable gene-delivery vehicles. These vectors represent a technology with widespread use in basic biology and translational studies that require persistent gene expression for treatment of several monogenic diseases. Immunogenicity and insertional mutagenesis represent the main obstacles to a wider clinical use of these vectors. Efficient and safe non-viral vectors are emerging as a promising alternative and facilitate clinical gene therapy studies...
October 12, 2016: Journal of Translational Medicine
Gaurav Majumdar, Rendani Mbau, Vinayak Singh, Digby F Warner, Marte Singsås Dragset, Raju Mukherjee
TnSeq, or transposon (Tn) insertion sequencing, is a powerful method for identifying the essential-as well as conditionally essential-regions in a genome, both coding and noncoding. The advent of accessible massively parallel DNA sequencing technologies in particular has resulted in the increased use of TnSeq-based approaches to elucidate various aspects of bacterial physiology and metabolism. Moreover, the availability of detailed protocols has enabled even nonspecialist laboratories to adapt and develop TnSeq approaches to address specific research questions...
2017: Methods in Molecular Biology
Saija Kiljunen, Maria I Pajunen, Harri Savilahti
Archaea constitute the third domain of life, but studies on their physiology and other features have lagged behind bacteria and eukarya, largely due to the challenging biology of archaea and concomitant difficulties in methods development. The use of genome-wide en masse insertion mutagenesis is one of the most efficient means to discover the genes behind various biological functions, and such a methodology is described in this chapter for a model archaeon Haloferax volcanii. The strategy successfully employs efficient in vitro transposition in combination with gene targeting in vivo via homologous recombination...
2017: Methods in Molecular Biology
Alicia M Jones, Joshua T Atkinson, Jonathan J Silberg
Rearrangements that alter the order of a protein's sequence are used in the lab to study protein folding, improve activity, and build molecular switches. One of the simplest ways to rearrange a protein sequence is through random circular permutation, where native protein termini are linked together and new termini are created elsewhere through random backbone fission. Transposase mutagenesis has emerged as a simple way to generate libraries encoding different circularly permuted variants of proteins. With this approach, a synthetic transposon (called a permuteposon) is randomly inserted throughout a circularized gene to generate vectors that express different permuted variants of a protein...
2017: Methods in Molecular Biology
Esteban Martínez-García, Tomás Aparicio, Víctor de Lorenzo, Pablo I Nikel
The construction of microbial cell factories à la carte largely depends on specialized molecular biology and synthetic biology tools needed to reprogram bacteria for modifying their existing functions or for bestowing them with new-to-Nature tasks. In this chapter, we document the use of a series of broad-host-range mini-Tn5 vectors for the delivery of gene(s) into the chromosome of Gram-negative bacteria and for the generation of saturated, random mutagenesis libraries for studies of gene function. The application of these tailored mini-transposon vectors, which could also be used for chromosomal engineering of a wide variety of Gram-negative microorganisms, is demonstrated in the platform environmental bacterium Pseudomonas putida KT2440...
2017: Methods in Molecular Biology
J Mark Weber, Andrew Reeves, William H Cernota, Roy K Wesley
Transposon mutagenesis is an invaluable technique in molecular biology for the creation of random mutations that can be easily identified and mapped. However, in the field of microbial strain improvement, transposon mutagenesis has scarcely been used; instead, chemical and physical mutagenic methods have been traditionally favored. Transposons have the advantage of creating single mutations in the genome, making phenotype to genotype assignments less challenging than with traditional mutagens which commonly create multiple mutations in the genome...
2017: Methods in Molecular Biology
Robert Gerlai, Tanya L Poshusta, Mindy Rampersad, Yohaan Fernandes, Tammy M Greenwood, Margot A Cousin, Eric W Klee, Karl J Clark
The zebrafish enjoys several advantages over other model organisms. It is small, easy to maintain, prolific, and numerous genetic tools are available for it. For example, forward genetic screens have allowed investigators to identify important genes potentially involved in a variety of functions from embryogenesis to cancer. However, despite its sophisticated behavioral repertoire, behavioral methods have rarely been utilized in forward genetic screens. Here, we employ a two-tiered strategy, a proof of concept study, to explore the feasibility of behavioral screens...
October 5, 2016: Behavior Genetics
Mariane S Thøgersen, Marina W Delpin, Jette Melchiorsen, Mogens Kilstrup, Maria Månsson, Boyke Bunk, Cathrin Spröer, Jörg Overmann, Kristian F Nielsen, Lone Gram
It has previously been reported that some strains of the marine bacterium Pseudoalteromonas luteoviolacea produce the purple bioactive pigment violacein as well as the antibiotic compound indolmycin, hitherto only found in Streptomyces. The purpose of the present study was to determine the relative role of each of these two compounds as antibacterial compounds in P. luteoviolacea S4054. Using Tn10 transposon mutagenesis, a mutant strain that was significantly reduced in violacein production in mannose-containing substrates was created...
2016: Frontiers in Microbiology
M Nancy Salazar-Vidal, Edith Acosta-Segovia, Nidia Sánchez-León, Kevin R Ahern, Thomas P Brutnell, Ruairidh J H Sawers
Phosphorus is an essential nutrient for all plants, but also one of the least mobile, and consequently least available, in the soil. Plants have evolved a series of molecular, metabolic and developmental adaptations to increase the acquisition of phosphorus and to maximize the efficiency of use within the plant. In Arabidopsis (Arabidopsis thaliana), the AtPHO1 protein regulates and facilitates the distribution of phosphorus. To investigate the role of PHO1 proteins in maize (Zea mays), the B73 reference genome was searched for homologous sequences, and four genes identified that were designated ZmPho1;1, ZmPho1;2a, ZmPho1;2b and ZmPho1;3...
2016: PloS One
Federico Rosconi, Stefan P W de Vries, Abiyad Baig, Elena Fabiano, Andrew J Grant
: The interior of plants contains microorganisms (referred to as endophytes) distinct to those present in the root surface or in the surrounding soil. Herbaspirillum seropedicae strain SmR1, belonging to the β-Proteobacterium, is an endophyte that colonizes crops, including rice, maize, sugarcane and sorghum. Different approaches have revealed genes and pathways regulated during the interaction of H. seropedicae with its plant hosts. However, functional genomic analysis of transposon (Tn) mutants has been hampered by the lack of genetic tools...
September 2, 2016: Applied and Environmental Microbiology
Tuan Ngoc Nguyen, Chen-Wei Yeh, Po-Chun Tsai, Kyoung Lee, Shir-Ly Huang
Pseudomonas nitroreducens TX1 is of special interest because of its ability to utilize 0.05% to 20% octylphenol polyethoxylates (OPEOn) as sole carbon source. In this study, a library containing 30,000 Tn5-insertion mutants of the wild-type strain TX1 was constructed and screened for the OPEOn utilization and 93 mutants were found to be unable to grow on OPEOn In total 42 separate disrupted genes were identified and the proteins encoded by the genes were then classified into various categories, namely information storage and processing (14...
September 2, 2016: Applied and Environmental Microbiology
Yang Luo, Erica Y Jacobs, Todd M Greco, Kevin D Mohammed, Tommy Tong, Sarah Keegan, James M Binley, Ileana M Cristea, David Fenyö, Michael P Rout, Brian T Chait, Mark A Muesing
Although genetically compact, HIV-1 commandeers vast arrays of cellular machinery to sustain and protect it during cycles of viral outgrowth. Transposon-mediated saturation linker scanning mutagenesis was used to isolate fully replication-competent viruses harbouring a potent foreign epitope tag. Using these viral isolates, we performed differential isotopic labelling and affinity-capture mass spectrometric analyses on samples obtained from cultures of human lymphocytes to classify the vicinal interactomes of the viral Env and Vif proteins as they occur during natural infection...
May 23, 2016: Nature Microbiology
Dong Yang, Ruiqi Liao, Yun Zheng, Ling Sun, Tian Xu
Transposons and retroviruses are important pathogenic agents and tools for mutagenesis and transgenesis. Insertion target selection is a key feature for a given transposon or retrovirus. The piggyBac (PB) transposon is highly active in mice and human cells, which has a much better genome-wide distribution compared to the retrovirus and P-element. However, the underlying reason is not clear. Utilizing a tagged functional PB transposase (PBase), we were able to conduct genome-wide profiling for PBase binding sites in the mouse genome...
2016: International Journal of Biological Sciences
Satoru Fujimoto, Sachihiro Matsunaga, Minoru Murata
Insertion mutagenesis using known DNA sequences such as T-DNA and transposons is an important tool for studies on gene function in plant sciences. The transposons Activator (Ac)/Dissociation (Ds) have been systematically used to manipulate plant chromosomes. For both of these applications, the recovery of genomic DNA sequences flanking the insertions is required to estimate the sizes and/or scales of the reconstituted chromosomes. In this chapter, we describe the protocols for thermal asymmetric interlaced PCR (TAIL-PCR) for isolation of genomic sequences flanking DNA inserts in plant genomes...
2016: Methods in Molecular Biology
Minoru Murata, Asaka Kanatani, Kazunari Kashihara
The combination of the DNA sequence-specific recombination system Cre/LoxP and the DNA transposon system Activator (Ac)/Dissociation (Ds) has been used for insertional and deletional mutagenesis, as well as for the generation of artificial ring chromosomes in model plants such as Arabidopsis and tobacco. However, it takes a long time to complete this process, even in Arabidopsis. To overcome this issue, a new binary vector, pDLHC, has been developed to induce chromosomal rearrangements for a short time in rice...
2016: Methods in Molecular Biology
Robin Tecon, Johan H J Leveau
Microorganisms are capable of remarkable social behaviours, such as forming transient multicellular assemblages with properties and adaptive abilities exceeding those of individual cells. Here, we report on the formation and structure of genets known as symplasmata produced by Pantoea eucalypti bacteria. Each symplasmatum develops clonally and stochastically from a single bacterium into a membrane-delimited, capsule-embedded cluster of progeny cells and with a frequency that depends on temperature, pH, and nutrient availability...
2016: Scientific Reports
Tiffany Bonnefois, Marie-Stéphanie Vernerey, Valérie Rodrigues, Philippe Totté, Carinne Puech, Chantal Ripoll, François Thiaucourt, Lucía Manso-Silván
Fluorescence expression tools for stable and innocuous whole mycoplasma cell labelling have been developed. A Tn4001-derivative mini-transposon affording unmarked, stable mutagenesis in mycoplasmas was modified to allow the constitutive, high-level expression of mCherry, mKO2 and mNeonGreen. These tools were used to introduce the respective fluorescent proteins as chromosomal tags in the phylogenetically distant species Mycoplasma mycoides subsp. mycoides and Mycoplasma bovis. The production, selection and characterisation of fluorescent clones were straightforward and resulted in the unprecedented observation of red and green fluorescent mycoplasma colonies in the two species, with no apparent cytotoxicity...
October 20, 2016: Journal of Biotechnology
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