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Applied and Environmental Microbiology, May 2008, p. 2985-2989, Vol. 74, No. 10
0099-2240/08/$08.00+0     doi:10.1128/AEM.00030-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Targeted Mutagenesis of Burkholderia thailandensis and Burkholderia pseudomallei through Natural Transformation of PCR Fragments{triangledown} ,{dagger}

Metawee Thongdee,1,2 Larry A. Gallagher,2 Mark Schell,3 Tararaj Dharakul,1 Sirirurg Songsivilai,1 and Colin Manoil2*

Department of Immunology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand,1 Department of Genome Sciences, University of Washington, Seattle, Washington 98195,2 Department of Microbiology, University of Georgia, Athens, Georgia 306023

Received 4 January 2008/ Accepted 20 February 2008

Burkholderia pseudomallei is the causative agent of melioidosis, an overwhelming, rapidly fatal septic infection, and B. thailandensis is a closely related, less virulent species. Both organisms are naturally competent for DNA transformation, and this report describes a procedure exploiting this property for the rapid generation of marked deletion mutations by using PCR products. The method was employed to create 61 mutant strains. Several selectable elements were employed, including elements carrying loxP and FRT recombinase recognition sites to facilitate resistance marker excision. Chromosomal mutations could also be transferred readily between strains by transformation. The availability of simple procedures for creating defined chromosomal mutations and moving them between strains should facilitate genetic analysis of virulence and other traits of these two Burkholderia species.

* Corresponding author. Mailing address: Department of Genome Sciences, Box 355065, University of Washington, Seattle, WA 98195-5065. Phone: (206) 543-7800. Fax: (206) 685-7301. E-mail: manoil{at}u.washington.edu

{triangledown} Published ahead of print on 29 February 2008.

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

Applied and Environmental Microbiology, May 2008, p. 2985-2989, Vol. 74, No. 10
0099-2240/08/$08.00+0     doi:10.1128/AEM.00030-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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