In Vivo Himar1 Transposon Mutagenesis of Burkholderia pseudomallei
| العنوان: | In Vivo Himar1 Transposon Mutagenesis of Burkholderia pseudomallei |
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| المؤلفون: | Lily A. Trunck, Drew A. Rholl, Herbert P. Schweizer |
| المصدر: | Applied and Environmental Microbiology. 74:7529-7535 |
| بيانات النشر: | American Society for Microbiology, 2008. |
| سنة النشر: | 2008 |
| مصطلحات موضوعية: | DNA, Bacterial, Transposable element, Burkholderia pseudomallei, Kanamycin Resistance, Molecular Sequence Data, Mutagenesis (molecular biology technique), Origin of replication, Applied Microbiology and Biotechnology, Plasmid maintenance, Microbiology, Plasmid, Methods, Recombination, Genetic, Genetics, Ecology, biology, Sequence Analysis, DNA, Chromosomes, Bacterial, biology.organism_classification, Blotting, Southern, Mutagenesis, Insertional, Burkholderia, DNA Transposable Elements, bacteria, Transposon mutagenesis, Plasmids, Food Science, Biotechnology |
| الوصف: | Burkholderia psedudomallei is the etiologic agent of melioidosis, and the bacterium is listed as a potential agent of bioterrorism because of its low infectious dose, multiple infectious routes, and intrinsic antibiotic resistance. To further accelerate research with this understudied bacterium, we developed a Himar1 -based random mutagenesis system for B. pseudomallei ( HimarBP ). The transposons contain a Flp recombinase-excisable, approved kanamycin resistance selection marker and an R6K origin of replication for transposon rescue. In vivo mutagenesis of virulent B. pseudomallei strain 1026b was highly efficient, with up to 44% of cells transformed with the delivery plasmid harboring chromosomal HimarBP insertions. Southern analyses revealed single insertions with no evidence of delivery plasmid maintenance. Sequence analysis of rescued HimarBP insertions revealed random insertions on both chromosomes within open reading frames and intergenic regions and that the orientation of insertions was largely unbiased. Auxotrophic mutants were obtained at a frequency of 0.72%, and nutritional supplementation experiments supported the functional assignment of genes within the respective biosynthetic pathways. HimarBP insertions were stable in the absence of selection and could be readily transferred between naturally transformable strains. Experiments with B. thailandensis suggest that the newly developed HimarBP transposons can also be used for random mutagenesis of other Burkholderia spp., especially the closely related species B. mallei . Our results demonstrate that comprehensive transposon libraries of B. pseudomallei can be generated, providing additional tools for the study of the biology, pathogenesis, and antibiotic resistance of this pathogen. |
| تدمد: | 1098-5336 0099-2240 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::b6039e035d41c4b96c4c3676bedb57e9 https://doi.org/10.1128/aem.01973-08 |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....b6039e035d41c4b96c4c3676bedb57e9 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 10985336 00992240 |
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