Genetic tools for select-agent-compliant manipulation of Burkholderia pseudomallei

Kyoung Hee Choi, Takehiko Mima, Yveth Casart, Drew Rholl, Ayush Kumar, Ifor R. Beacham, Herbert P. Schweizer

Research output: Contribution to journalArticle

126 Citations (Scopus)

Abstract

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 PBAD promoter and kanamycin (nptI) and zeocin (ble) selection markers from the constitutive Burkholderia thailandensis ribosomal PS12 or synthetic bacterial PEM7 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 Δ(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.

Original languageEnglish
Pages (from-to)1064-1075
Number of pages12
JournalApplied and environmental microbiology
Volume74
Issue number4
DOIs
Publication statusPublished - Feb 2008
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Food Science
  • Applied Microbiology and Biotechnology
  • Ecology

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    Choi, K. H., Mima, T., Casart, Y., Rholl, D., Kumar, A., Beacham, I. R., & Schweizer, H. P. (2008). Genetic tools for select-agent-compliant manipulation of Burkholderia pseudomallei. Applied and environmental microbiology, 74(4), 1064-1075. https://doi.org/10.1128/AEM.02430-07