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Knockout and pullout recombineering for naturally transformable Burkholderia thailandensis and Burkholderia pseudomallei

Nature Protocols volume 6, pages 1085–1104 (2011)Cite this article

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Abstract

Phage λ-Red proteins are powerful tools for pulling and knocking out chromosomal fragments but have been limited to the γ-proteobacteria. Procedures are described here to easily knock out (KO) and pull out (PO) chromosomal DNA fragments from naturally transformable Burkholderia thailandensis and Burkholderia pseudomallei. This system takes advantage of published compliant counterselectable and selectable markers (sacB, pheS, gat and the arabinose-utilization operon) and λ-Red mutant proteins. pheS-gat (KO) or oriT-ColE1ori-gat-ori1600-rep (PO) PCR fragments are generated with flanking 40- to 45-bp homologies to targeted regions incorporated on PCR primers. One-step recombination is achieved by incubation of the PCR product with cells expressing λ-Red proteins and subsequent selection on glyphosate-containing medium. This procedure takes ∼10 d and is advantageous over previously published protocols: (i) smaller PCR products reduce primer numbers and amplification steps, (ii) PO fragments suitable for downstream manipulation in Escherichia coli are obtained and (iii) chromosomal KO increases flexibility for downstream processing.

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Figure 1: Genetic constructs for KO and PO recombineering in B. thailandensis and B. pseudomallei.
Figure 2: Overview of an example of PO recombineering in B. pseudomallei.
Figure 3: KO recombineering strategy.
Figure 4: λ-Red recombineering scheme for PO/KO in B. thailandensis and B. pseudomallei.
Figure 5: PCR confirmation for pullout and knockout in B. thailandensis and B. pseudomallei.
Figure 6: Phenotypic and PCR confirmation for the mba-cluster knockouts in the B. pseudomallei.
Figure 7: KO recombineering in different B. pseudomallei strains.

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Acknowledgements

The project described was supported by Award Number AI065359 from the US National Institute of Allergy and Infectious Diseases and in part by the Center of Biomedical Research Excellence grant P20RR018727 from the National Center for Research Resources (both components of the NIH). We are grateful to H.P. Schweizer for the generous gift of constructs containing a modified sacB gene and the rhamnose-inducible promoter.

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Authors and Affiliations

  1. Department of Molecular Biosciences and Bioengineering University of Hawaii at Manoa, University of Hawaii at Manoa, Honolulu, Hawaii, USA

    Yun Kang, Michael H Norris & Tung T Hoang

  2. Department of Ecology and Health, University of Hawaii at Manoa, Honolulu, Hawaii, USA

    Bruce A Wilcox

  3. Translational Genomics Research Institute, Flagstaff, Arizona, USA

    Apichai Tuanyok & Paul S Keim

  4. Northern Arizona University, Flagstaff, Arizona, USA

    Paul S Keim

  5. Department of Microbiology, University of Hawaii at Manoa, Honolulu, Hawaii, USA

    Tung T Hoang

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Contributions

Y.K. created the constructs and performed the experiments in B. pseudomallei. M.H.N. performed the experiments in B. thailandensis. B.A.W. provided guidance for M.H.N. in this project. A.T. and P.S.K. isolated and sequenced the B. pseudomallei clinical and environmental isolates on Table 1. T.T.H. designed and supervised the experiments. Y.K., M.H.N. and T.T.H. wrote this manuscript.

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Correspondence to Tung T Hoang.

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Kang, Y., Norris, M., Wilcox, B. et al. Knockout and pullout recombineering for naturally transformable Burkholderia thailandensis and Burkholderia pseudomallei. Nat Protoc 6, 1085–1104 (2011). https://doi.org/10.1038/nprot.2011.346

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