%0 Journal Article
%T Methods for genetic manipulation of Burkholderia gladioli pathovar cocovenenans
%A Nawarat Somprasong
%A Ian McMillan
%A RoxAnn R Karkhoff-Schweizer
%A Skorn Mongkolsuk
%A Herbert P Schweizer
%J BMC Research Notes
%D 2010
%I BioMed Central
%R 10.1186/1756-0500-3-308
%X 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.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.Burkholderia gladioli pathovar cocovenenans was first described in the 1960 s as a bacterium that caused severe cases of food-poisoning and was then named Pseudomonas cocovenenans [1]. It has been re-named several times since. In 1995 the bacterium was named B. cocovenenans [2,3] and in 1999 it was combined with B. gladioli, a phytopathogenic bacterium [4]. Though later analyses confirmed that B. cocovenenans and B. gladioli should be categorized as a single species, it was deemed necessary to distinguish those strains that are phytogenic from those that also produce toxins lethal to mammals. This resulted in designation of a new pathovar, B. gladioli pathovar cocovenenans (BGC) in 2003 [5].Outbreaks of severe BGC-caused food-poisoning are sporadic and seem to be confined to Asia. In endemic regions, fermented coconut can be contaminated with BGC
%U http://www.biomedcentral.com/1756-0500/3/308