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
Google Scholar
Right arrow Articles by López, C. M.
Right arrow Articles by Schweizer, H. P.
PubMed
Right arrow PubMed Citation
Right arrow Articles by López, C. M.
Right arrow Articles by Schweizer, H. P.
Agricola
Right arrow Articles by López, C. M.
Right arrow Articles by Schweizer, H. P.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, October 2009, p. 6496-6503, Vol. 75, No. 20
0099-2240/09/$08.00+0     doi:10.1128/AEM.01669-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Versatile Dual-Technology System for Markerless Allele Replacement in Burkholderia pseudomallei{triangledown} ,{dagger}

Carolina M. López,{ddagger} Drew A. Rholl,{ddagger} 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 80523-2025

Received 14 July 2009/ Accepted 14 August 2009

Burkholderia pseudomallei is the etiologic agent of melioidosis, a rare but serious tropical disease. In the United States, genetic research with this select agent bacterium is strictly regulated. Although several select agent compliant methods have been developed for allelic replacement, all of them suffer from some drawbacks, such as a need for specific host backgrounds or use of minimal media. Here we describe a versatile select agent compliant allele replacement system for B. pseudomallei based on a mobilizable vector, pEXKm5, which contains (i) a multiple cloning site within a lacZ{alpha} gene for facile cloning of recombinant DNA fragments, (ii) a constitutively expressed gusA indicator gene for visual detection of merodiploid formation and resolution, and (iii) elements required for resolution of merodiploids using either I-SceI homing endonuclease-stimulated recombination or sacB-based counterselection. The homing endonuclease-based allele replacement system is completed by pBADSce, which contains an araC-PBAD-I-sceI expression cassette for arabinose-inducible I-SceI expression and a temperature-sensitive pRO1600 replicon for facile plasmid curing. Complementing these systems is the improved {Delta}asd Escherichia coli mobilizer strain RHO3. This strain is susceptible to commonly used antibiotics and allows nutritional counterselection on rich media because of its diaminopimelic acid auxotrophy. The versatility of the I-SceI- and sacB-based methods afforded by pEXKm5 in conjunction with E. coli RHO3 was demonstrated by isolation of diverse deletion mutants in several clinical, environmental, and laboratory B. pseudomallei strains. Finally, sacB-based counterselection was employed to isolate a defined chromosomal fabD(Ts) allele that causes synthesis of a temperature-sensitive FabD, an essential fatty acid biosynthesis enzyme.

* Corresponding author. Mailing address: Department of Microbiology, Immunology and Pathology, Colorado State University, IDRC at Foothills Campus, Campus Delivery 2025, Fort Collins, CO 80523-2025. Phone: (970) 491-3536. Fax: (970) 491-8708. E-mail: Herbert.Schweizer{at}colostate.edu

{triangledown} Published ahead of print on 21 August 2009.

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

{ddagger} C.M.L. and D.A.R. contributed equally to this work.

Applied and Environmental Microbiology, October 2009, p. 6496-6503, Vol. 75, No. 20
0099-2240/09/$08.00+0     doi:10.1128/AEM.01669-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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