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Applied and Environmental Microbiology, July 2008, p. 4498-4508, Vol. 74, No. 14
0099-2240/08/$08.00+0     doi:10.1128/AEM.00531-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Genetic Tools for Allelic Replacement in Burkholderia Species{triangledown} ,{dagger}

Ashley R. Barrett,{ddagger} Yun Kang,{ddagger} Ken S. Inamasu, Mike S. Son, Joseph M. Vukovich, and Tung T. Hoang*

Department of Microbiology, College of Natural Sciences, University of Hawaii at Manoa, Honolulu, Hawaii

Received 4 March 2008/ Accepted 13 May 2008

Allelic replacement in the Burkholderia genus has been problematic due to the lack of appropriate counter-selectable and selectable markers. The counter-selectable marker sacB, commonly used in gram-negative bacteria, is nonselective on sucrose in many Burkholderia species. In addition, the use of antibiotic resistance markers of clinical importance for the selection of desirable genetic traits is prohibited in the United States for two potential bioterrorism agents, Burkholderia mallei and Burkholderia pseudomallei. Here, we engineered a mutated counter-selectable marker based on the B. pseudomallei PheS (the {alpha}-subunit of phenylalanyl tRNA synthase) protein and tested its effectiveness in three different Burkholderia species. The mutant PheS protein effectively killed 100% of the bacteria in the presence of 0.1% p-chlorophenylalanine. We assembled the mutant pheS on several allelic replacement vectors, in addition to constructing selectable markers based on tellurite (Telr) and trimethoprim (Tpr) resistance that are excisable by flanking unique FLP recombination target (FRT) sequences. As a proof of concept, we utilized one of these gene replacement vectors (pBAKA) and the Telr-FRT cassette to produce a chromosomal mutation in the Burkholderia thailandensis betBA operon, which codes for betaine aldehyde dehydrogenase and choline dehydrogenase. Chromosomal resistance markers could be excised by the introduction of pFLP-AB5 (Tpr), which is one of two constructed flp-containing plasmids, pFLP-AB4 (Telr) and pFLP-AB5 (Tpr). These flp-containing plasmids harbor the mutant pheS gene and allow self curing on media that contain p-chlorophenylalanine after Flp-FRT excision. The characterization of the {Delta}betBA::Telr-FRT and {Delta}betBA::FRT mutants indicated a defect in growth with choline as a sole carbon source, while these mutants grew as well as the wild type with succinate and glucose as alternative carbon sources.

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

{triangledown} Published ahead of print on 23 May 2008.

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

{ddagger} These authors contributed equally to this research.

Applied and Environmental Microbiology, July 2008, p. 4498-4508, Vol. 74, No. 14
0099-2240/08/$08.00+0     doi:10.1128/AEM.00531-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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