AEM Accepts, published online ahead of print on 23 May 2008

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Appl. Environ. Microbiol. doi:10.1128/AEM.00531-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Genetic Tools for Allelic-Replacement in Burkholderia Species

Ashley R. Barrett, Yun Kang, 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

^* To whom correspondence should be addressed. Email: tongh{at}hawaii.edu.

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-negatives is non-selective on sucrose in many Burkholderia species. In addition, the use of antibiotic resistant 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 (-subunit of phenylalanyl tRNA synthase) 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 (Tel^r) and trimethoprim (Tp^r) resistance that are excisable by flanking unique-FRT sequences. As proof-of-concept, we utilized one of these gene-replacement vectors (pBAKA) and the Tel^r-FRT cassette to produce a chromosomal mutation in the Burkholderia thailandensis betBA-operon, which encodes for betaine-aldehyde dehydrogenase and choline dehydrogenase. Chromosomal resistant markers could be excised by the introduction of pFLP-AB5 (Tp^r), which is one of two constructed flp-containing plasmids pFLP-AB4 (Tel^r) and pFLP-AB5 (Tp^r). These flp-containing plasmids harbor the mutant pheS gene, allowing self-curing on media that contain p-chlorophenylalanine after Flp-FRT excision. Characterization of the betBA::Tel^r-FRT and betBA::FRT mutants indicated a defect in growth with choline as a sole carbon source, while these mutants grew as well as wild-type with succinate and glucose as alternative carbon sources.

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