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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.

References

  1. Sawitzke, J.A. et al. Recombineering: in vivo genetic engineering in E. coli, S. enterica, and beyond. Meth. Enzymol. 421, 171–199 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Zhang, Y., Muyrers, J.P.P., Testa, G. & Stewart, A.F. DNA cloning by homologous recombination in Escherichia coli. Nat. Biotechnol. 18, 1314–1317 (2000).

    Article  CAS  PubMed  Google Scholar 

  3. Datsenko, K.A. & Wanner, B.L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97, 6640–6645 (2000).

    Article  CAS  PubMed  Google Scholar 

  4. Sun, W., Wang, S. & Curtiss, R. III. Highly efficient methods for introducing successive multiple scarless gene deletions and markerless gene insertions into the Yersinia pestis chromosome. Appl. Environ. Microbiol. 74, 4241–4245 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Lesic, B. & Rahme, L.G. Use of the lambda Red recombinase system to rapidly generate mutants in Pseudomonas aeruginosa. BMC Mol. Biol. 9, 20 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Wiersinga, W.J., van der Poll, T., White, N.J., Day, N.P. & Peacock, S.J. Melioidosis: insight into the pathogenicity of Burkholderia pseudomallei. Nat. Rev. Microbiol. 4, 272–282 (2006).

    Article  CAS  PubMed  Google Scholar 

  7. Thongdee, M. et al. Targeted mutagenesis of Burkholderia pseudomallei and Burkholderia thailandensis through natural transformation of PCR fragments. Appl. Environ. Microbiol. 74, 2985–2989 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Barrett, A.R. et al. Genetic tools for allelic replacement in Burkholderia species. Appl. Environ. Microbiol. 74, 4498–4508 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Norris, M.H., Kang, Y., Lu, D., Wilcox, B.A. & Hoang, T.T. Glyphosate resistance as a novel select-agent-compliant, non-antibiotic selectable marker in chromosomal mutagenesis of the essential genes asd and dapB of Burkholderia pseudomallei. Appl. Environ. Microbiol. 75, 6062–6075 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Moore, R.A. et al. Contribution of gene loss to the pathogenic evolution of Burkholderia pseudomallei and Burkholderia mallei. Infect. Immun. 72, 4172–4187 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Chandler, J.R. et al. Mutational analysis of Burkholderia thailandensis quorum sensing and self-aggregation. J. Bacteriol. 191, 5901–5909 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Choi, K.-H., Kumar, A. & Schweizer, H.P. A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for the DNA fragment transfer between chromosomes and plasmid transformation. J. Microbiol. Meth. 64, 391–397 (2006).

    Article  CAS  Google Scholar 

  13. Mack, K. & Titball, R.W. Transformation of Burkholderia pseudomallei by electroporation. Anal. Biochem. 242, 73–76 (1996).

    Article  CAS  PubMed  Google Scholar 

  14. Lopez, C.M., Rholl, D.A., Trunck, L.A. & Schweizer, H.P. Versatile dual-technology system for markerless allele replacement in Burkholderia pseudomallei. Appl. Environ. Microbiol. 75, 6496–6503 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Choi, K.H. et al. Genetic tools for select-agent-compliant manipulation of Burkholderia pseudomallei. Appl. Environ. Microbiol. 74, 1064–1075 (2008).

    Article  CAS  PubMed  Google Scholar 

  16. Antoine, R. & Locht, C. Isolation and molecular characterization of a novel broad-host-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from Gram-positive organisms. Mol. Microbiol. 6, 1785–1799 (1991).

    Article  Google Scholar 

  17. Nakayama, M. & Ohara, O. Improvement of recombination efficiency by mutation of Red proteins. BioTechniques 38, 917–924 (2005).

    Article  CAS  PubMed  Google Scholar 

  18. Alice, A.F., Lopez, C.S., Lowe, C.A., Ledesma, M.A. & Crosa, J.H. Genetic and transcriptional analysis of the siderophore malleobactin biosynthesis and transport genes in the human pathogen Burkholderia pseudomallei K96243. J. Bacteriol. 188, 1551–1566 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Sambrook, J. & Russell, D.W. Molecular Cloning: A Laboratory Manual 2nd edn, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2001).

  20. Wilson, D.E. & Chosewood, L.C. Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th edn. (Centers for Disease Control and Prevention, Atlanta, Georgia, USA, 2007).

  21. Rholl, D.A., Trunck, L.A. & Schweizer, H.P. Himar1 in vivo transposon mutagenesis of Burkholderia pseudomallei. Appl. Environ. Microbiol. 74, 7529–7535 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Yanisch-Perron, C., Vieira, J. & Messing, J. Improved M13 cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103–119 (1985).

    Article  CAS  PubMed  Google Scholar 

  23. Kovach, M.E. et al. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166, 175–176 (1995).

    Article  CAS  PubMed  Google Scholar 

  24. Schweizer, H.P., Klassen, T.R. & Hoang, T. Improved methods for gene analysis and expression in Pseudomonas. In Molecular Biology of Pseudomonads. (eds. Nakazawa, T., Furukawa, K., Haas, D. & Silver, S.) 229–237 (American Society for Microbiology, Washington, D.C., USA, 1996).

  25. Cardona, S.T. & Valvano, M.A. An expression vector containing a rhamnose-inducible promoter provides tightly regulated gene expression in Burkholderia cenocepacia. Plasmid 54, 219–228 (2005).

    Article  CAS  PubMed  Google Scholar 

  26. Yu, M. & Tsang, J.S.H. Use of ribosomal promoters from Burkholderia cenocepacia and Burkholderia cepacia for improved expression of transporter protein in Escherichia coli. Protein Expr. Purif. 49, 219–227 (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Schwyn, B. & Neilands, J.B. Universal chemical assay for the detection and determination of siderophores. Anal. Biochem. 160, 47–56 (1987).

    Article  CAS  PubMed  Google Scholar 

  28. Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006).

  29. DeShazer, D., Brett, P.J., Carlyon, R. & Woods, D.E. Mutagenesis of Burkholderia pseudomallei with Tn5-OT182: isolation of motility mutant and molecular characterization of the flagellin structural gene. J. Bacteriol. 179, 2116–2125 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Brett, P.J., DeShazer, D. & Woods, D.E. Burkholderia thailandensis sp. nov., description of Burkholderia pseudomallei-like species. Int. J. Syst. Bacteriol. 48, 317–320 (1998).

    Article  CAS  PubMed  Google Scholar 

  31. Kang, Y., Norris, M.H., Barrett, A.R., Wilcox, B.A. & Hoang, T.T. Engineering of tellurite-resistant genetic tools for single-copy chromosomal analysis of Burkholderia spp. and characterization of the B. thailandensis betBA-operon. Appl. Environ. Microbiol. 75, 4015–4027 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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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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