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Applied and Environmental Microbiology, December 2008, p. 7529-7535, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.01973-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

In Vivo Himar1 Transposon Mutagenesis of Burkholderia pseudomallei{triangledown}

Drew A. Rholl, Lily A. Trunck, and Herbert P. Schweizer*

Department of Microbiology, Immunology and Pathology, Rocky Mountain Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research, Colorado State University, Fort Collins, Colorado 80523-1690

Received 25 August 2008/ Accepted 13 October 2008

Burkholderia psedudomallei is the etiologic agent of melioidosis, and the bacterium is listed as a potential agent of bioterrorism because of its low infectious dose, multiple infectious routes, and intrinsic antibiotic resistance. To further accelerate research with this understudied bacterium, we developed a Himar1-based random mutagenesis system for B. pseudomallei (HimarBP). The transposons contain a Flp recombinase-excisable, approved kanamycin resistance selection marker and an R6K origin of replication for transposon rescue. In vivo mutagenesis of virulent B. pseudomallei strain 1026b was highly efficient, with up to 44% of cells transformed with the delivery plasmid harboring chromosomal HimarBP insertions. Southern analyses revealed single insertions with no evidence of delivery plasmid maintenance. Sequence analysis of rescued HimarBP insertions revealed random insertions on both chromosomes within open reading frames and intergenic regions and that the orientation of insertions was largely unbiased. Auxotrophic mutants were obtained at a frequency of 0.72%, and nutritional supplementation experiments supported the functional assignment of genes within the respective biosynthetic pathways. HimarBP insertions were stable in the absence of selection and could be readily transferred between naturally transformable strains. Experiments with B. thailandensis suggest that the newly developed HimarBP transposons can also be used for random mutagenesis of other Burkholderia spp., especially the closely related species B. mallei. Our results demonstrate that comprehensive transposon libraries of B. pseudomallei can be generated, providing additional tools for the study of the biology, pathogenesis, and antibiotic resistance of this pathogen.

* Corresponding author. Mailing address: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1690. Phone: (970) 491-3536. Fax: (970) 491-8708. E-mail: Herbert.Schweizer{at}colostate.edu

{triangledown} Published ahead of print on 24 October 2008.

Applied and Environmental Microbiology, December 2008, p. 7529-7535, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.01973-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Lopez, C. M., Rholl, D. A., Trunck, L. A., Schweizer, H. P. (2009). Versatile Dual-Technology System for Markerless Allele Replacement in Burkholderia pseudomallei. Appl. Environ. Microbiol. 75: 6496-6503 [Abstract] [Full Text]  
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