TY - JOUR
T1 - Methods for genetic manipulation of Burkholderia gladioli pathovar cocovenenans
AU - Somprasong, Nawarat
AU - McMillan, Ian
AU - Karkhoff-Schweizer, Roxann R.
AU - Mongkolsuk, Skorn
AU - Schweizer, Herbert P.
N1 - Funding Information:
NS was supported by a Royal Golden Jubilee predoctoral fellowship from the Thailand Research Foundation. We thank Marissa Castillo for assisting with antimicrobial susceptibility assays. This research was supported by indirect cost recovery funds made possible by several NIH research grants awarded to HPS.
PY - 2010
Y1 - 2010
N2 - 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-Tn7 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.
AB - 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-Tn7 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.
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U2 - 10.1186/1756-0500-3-308
DO - 10.1186/1756-0500-3-308
M3 - Article
AN - SCOPUS:78149494202
SN - 1756-0500
VL - 3
JO - BMC Research Notes
JF - BMC Research Notes
M1 - 308
ER -