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Genetic Tools for Select-Agent-Compliant Manipulation of Burkholderia pseudomallei

Kyoung-Hee Choi,¹ Takehiko Mima,¹ Yveth Casart,^2, Drew Rholl,¹ Ayush Kumar,^1, Ifor R. Beacham,² and Herbert P. Schweizer^1*

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 80523-1682,¹ School of Medical Science and Institute for Glycomics, Griffith University—Gold Coast Campus, Gold Coast, Queensland 9726, Australia²

Received 28 October 2007/ Accepted 13 December 2007

Because of Burkholderia pseudomallei's classification as a select agent in the United States, genetic manipulation of this bacterium is strictly regulated. Only a few antibiotic selection markers, including gentamicin, kanamycin, and zeocin, are currently approved for use with this bacterium, but wild-type strains are highly resistant to these antibiotics. To facilitate routine genetic manipulations of wild-type strains, several new tools were developed. A temperature-sensitive pRO1600 broad-host-range replicon was isolated and used to construct curable plasmids where the Flp and Cre recombinase genes are expressed from the rhamnose-regulated Escherichia coli P_BAD promoter and kanamycin (nptI) and zeocin (ble) selection markers from the constitutive Burkholderia thailandensis ribosomal P_S12 or synthetic bacterial P_EM7 promoter. Flp and Cre site-specific recombination systems allow in vivo excision and recycling of nptII and ble selection markers contained on FRT or loxP cassettes. Finally, expression of Tn7 site-specific transposase from the constitutive P1 integron promoter allowed development of an efficient site-specific chromosomal integration system for B. pseudomallei. In conjunction with a natural transformation method, the utility of these new tools was demonstrated by isolating an unmarked (amrRAB-oprA) efflux pump mutant. Exploiting natural transformation, chromosomal DNA fragments carrying this mutation marked with zeocin resistance were transferred between the genomes of two different B. pseudomallei strains. Lastly, the deletion mutation was complemented by a chromosomally integrated mini-Tn7 element carrying the amrAB-oprA operon. The new tools allow routine select-agent-compliant genetic manipulations of B. pseudomallei and other Burkholderia species.

Published ahead of print on 21 December 2007.

Present address: Departamento de Biología Estructural, Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela.

Present address: Faculty of Health Sciences, 2000 Simcoe St. N, University of Ontario Institute of Technology, Oshawa, Ontario L1H 7K4, Canada.