Methods for genetic manipulation of Burkholderia gladioli pathovar cocovenenans
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- 作者：Nawarat Somprasong (1) (2)
Ian McMillan (2)
RoxAnn R Karkhoff-Schweizer (2)
Skorn Mongkolsuk (1)
Herbert P Schweizer (1) (2) - 刊名：BMC Research Notes
- 出版年：2010
- 出版时间：December 2010
- 年：2010
- 卷：3
- 期：1
- 全文大小：724KB
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Ian McMillan (2)
RoxAnn R Karkhoff-Schweizer (2)
Skorn Mongkolsuk (1)
Herbert P Schweizer (1) (2)
1. Department of Biotechnology, Faculty of Science, Mahidol University, 10400, Ratchathewi, Bangkok, Thailand
2. Department of Microbiology, Immunology and Pathology, Colorado State University, IDRC at Foothills Campus, 0922 Campus Delivery, 80523, Fort Collins, CO, USA
文摘
Background Burkholderia gladioli pathovar cocovenenans (BGC) is responsible for sporadic food-poisoning outbreaks with high morbidity and mortality in Asian countries. Little is known about the regulation of virulence factor and toxin production in BGC, and studies in this bacterium have been hampered by lack of genetic tools. Findings Establishment of a comprehensive antibiotic susceptibility profile showed that BGC strain ATCC33664 is susceptible to a number of antibiotics including aminoglycosides, carbapenems, fluoroquinolones, tetracyclines and trimethoprim. In this study, we established that gentamicin, kanamycin and trimethoprim are good selection markers for use in BGC. Using a 10 min method for preparation of electrocompetent cells, the bacterium could be transformed by electroporation at high frequencies with replicative plasmids containing the pRO1600-derived origin of replication. These plasmids exhibited a copy number of > 100 in BGC. When co-conjugated with a transposase expressing helper plasmid, mini-Tn 7 vectors inserted site- and orientation-specifically at a single glmS -associated insertion site in the BGC genome. Lastly, a Himar1 transposon was used for random transposon mutagenesis of BGC. Conclusions A series of genetic tools previously developed for other Gram-negative bacteria was adapted for use in BGC. These tools now facilitate genetic studies of this pathogen and allow establishment of toxin biosynthetic pathways and their genetic regulation.