This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Google Scholar
Right arrow Articles by Kang, Y.
Right arrow Articles by Hoang, T. T.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kang, Y.
Right arrow Articles by Hoang, T. T.
Agricola
Right arrow Articles by Kang, Y.
Right arrow Articles by Hoang, T. T.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, June 2009, p. 4015-4027, Vol. 75, No. 12
0099-2240/09/$08.00+0     doi:10.1128/AEM.02733-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Engineering of Tellurite-Resistant Genetic Tools for Single-Copy Chromosomal Analysis of Burkholderia spp. and Characterization of the Burkholderia thailandensis betBA Operon{triangledown} ,{dagger}

Yun Kang,2 Michael H. Norris,2 Ashley R. Barrett,1 Bruce A. Wilcox,3 and Tung T. Hoang1,2*

Department of Microbiology,1 Department of Molecular Biosciences and Bioengineering,2 Department of Ecology and Health, University of Hawaii at Manoa, Honolulu, Hawaii 968223

Received 30 November 2008/ Accepted 7 April 2009

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 (Telr) and Escherichia coli aspartate-semialdehyde dehydrogenase (asdEc), 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-{Delta}bla-Telr-FRT efficiently transposed within Burkholderia cenocepacia, Burkholderia thailandensis, B. pseudomallei, and B. mallei. We then utilized the mini-Tn7-Telr-based transposon vector (mini-Tn7-Telr-betBA) and a transposase-containing helper plasmid (pTNS3-asdEc) to complement the B. thailandensis {Delta}betBA mutation. Next, one of the FRT-lacZ fusion vectors (pFRT1-lacZ-Telr) was integrated by Flp (encoded on a helper plasmid, pCD13SK-Flp-oriT-asdEc) to construct the B. thailandensis {Delta}betBA::FRT-lacZ-Telr reporter fusion strain. The betBA operon was shown to be induced in the presence of choline and under osmotic stress conditions by performing β-galactosidase assays on the B. thailandensis {Delta}betBA::FRT-lacZ-Telr fusion strain. Finally, we engineered B. thailandensis {Delta}betBA::FRT-gfp-Telr and {Delta}betBA::FRT-lux-Telr fusion strains by utilizing fusion vectors pFRT1-gfp-Telr and pFRT1-lux-Telr, respectively. The induction of the betBA operon by choline and osmotic stress was confirmed by performing fluorescent microscopy and bioluminescent imaging analyses.

* Corresponding author. Mailing address: Department of Microbiology, University of Hawaii at Manoa, 2538 The Mall-Snyder 310, Honolulu, HI 96822. Phone: (808) 956-3522. Fax: (808) 956-5339. E-mail: tongh{at}hawaii.edu

{triangledown} Published ahead of print on 17 April 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, June 2009, p. 4015-4027, Vol. 75, No. 12
0099-2240/09/$08.00+0     doi:10.1128/AEM.02733-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Norris, M. H., Kang, Y., Lu, D., Wilcox, B. A., Hoang, T. T. (2009). 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. Appl. Environ. Microbiol. 75: 6062-6075 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»