ArticleApplied and environmental microbiology2009
Engineering of tellurite-resistant genetic tools for single-copy chromosomal analysis of Burkholderia spp. and characterization of the Burkholderia thailandensis betBA operon.
Article in Applied and environmental microbiology, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.
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16 citing papers in PubMed, 16 citations in OpenAlex.
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- The Burkholderia pseudomallei intracellular 'TRANSITome'.Nature communications · 2021Article
- The heritable natural competency trait of Burkholderia pseudomallei in other Burkholderia species through comE and crp.Scientific reports · 2018Article
- Spatial transcriptomes within the Pseudomonas aeruginosa biofilm architecture.Molecular microbiology · 2017Article
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- Choline Catabolism in Burkholderia thailandensis Is Regulated by Multiple Glutamine Amidotransferase 1-Containing AraC Family Transcriptional Regulators.Journal of bacteriology · 2016Article
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- The Burkholderia pseudomallei Δasd mutant exhibits attenuated intracellular infectivity and imparts protection against acute inhalation melioidosis in mice.Infection and immunity · 2011Article
- Knockout and pullout recombineering for naturally transformable Burkholderia thailandensis and Burkholderia pseudomallei.Nature protocols · 2011Article
- Stable, site-specific fluorescent tagging constructs optimized for burkholderia species.Applied and environmental microbiology · 2010Article
- Glyphosate resistance as a novel select-agent-compliant, non-antibiotic-selectable marker in chromosomal mutagenesis of the essential genes asd and dapB of Burkholderia pseudomallei.Applied and environmental microbiology · 2009Article
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Authors and funding
5 authors at 1 institution in 1 country.
Funding
Abstract
There are few appropriate single-copy genetic tools for most Burkholderia species, and the high level of antibiotic resistance in this genus further complicates the development of genetic tools. In addition, the utilization of resistance genes for clinically important antibiotics is prohibited for the bioterrorism agents Burkholderia pseudomallei and Burkholderia mallei, necessitating the development of additional nonantibiotic-based genetic tools. Three single-copy systems devoid of antibiotic selection based on two nonantibiotic selectable markers, tellurite resistance (Tel(r)) and Escherichia coli aspartate-semialdehyde dehydrogenase (asd(Ec)), were developed to facilitate genetic manipulation in Burkholderia species. These systems include one mariner transposon, a mini-Tn7-derived site-specific transposon, and six FRT reporter fusion vectors based on the lacZ, gfp, and luxCDABE reporter genes. Initially, we showed that the random mariner transposon pBT20-Deltabla-Tel(r)-FRT efficiently transposed within Burkholderia cenocepacia, Burkholderia thailandensis, B. pseudomallei, and B. mallei. We then utilized the mini-Tn7-Tel(r)-based transposon vector (mini-Tn7-Tel(r)-betBA) and a transposase-containing helper plasmid (pTNS3-asd(Ec)) to complement the B. thailandensis DeltabetBA mutation. Next, one of the FRT-lacZ fusion vectors (pFRT1-lacZ-Tel(r)) was integrated by Flp (encoded on a helper plasmid, pCD13SK-Flp-oriT-asd(Ec)) to construct the B. thailandensis DeltabetBA::FRT-lacZ-Tel(r) reporter fusion strain. The betBA operon was shown to be induced in the presence of choline and under osmotic stress conditions by performing beta-galactosidase assays on the B. thailandensis DeltabetBA::FRT-lacZ-Tel(r) fusion strain. Finally, we engineered B. thailandensis DeltabetBA::FRT-gfp-Tel(r) and DeltabetBA::FRT-lux-Tel(r) fusion strains by utilizing fusion vectors pFRT1-gfp-Tel(r) and pFRT1-lux-Tel(r), respectively. The induction of the betBA operon by choline and osmotic stress was confirmed by performing fluorescent microscopy and bioluminescent imaging analyses.
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