Burkholderia thailandensis: Genetic Manipulation

Erin C. Garcia1

1 Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, Kentucky
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 4C.2
DOI:  10.1002/cpmc.27
Online Posting Date:  May, 2017
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Abstract

Burkholderia thailandensis is a Gram‐negative bacterium endemic to Southeast Asian and northern Australian soils. It is non‐pathogenic; therefore, it is commonly used as a model organism for the related human pathogens Burkholderia mallei and Burkholderia pseudomallei. B. thailandensis is relatively easily genetically manipulated and a variety of robust genetic tools can be used in this organism. This unit describes protocols for conjugation, natural transformation, mini‐Tn7 insertion, and allelic exchange in B. thailandensis. © 2017 by John Wiley & Sons, Inc.

Keywords: allelic exchange; conjugation; mini‐Tn7; mutation; transformation Burkholderia thailandensis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Introduction of Plasmids by Conjugation
  • Basic Protocol 2: Site‐Specific Chromosome Insertion by Mini‐Tn7 Transposition
  • Basic Protocol 3: Homologous Recombination by Natural Transformation with Linear DNA
  • Basic Protocol 4: Flp Recombinase–Mediated Marker Excision
  • Basic Protocol 5: Allelic Exchange Using SACB Counterselection
  • Support Protocol 1: Colony PCR
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Introduction of Plasmids by Conjugation

  Materials
  • B. thailandensis frozen stock
  • Donor E. coli RHO3 strain(s) carrying plasmid(s) to be transferred
  • Low‐salt LB (LSLB) plates with or without 200 μg/ml 2,6‐diaminopimelic acid (DAP) and/or with or without appropriate antibiotics (see recipe; Table 4.2.1)
  • Sterile swabs
  • 37ºC incubator
Table 4.0.1   MaterialsAntibiotic and Media Supplement Concentrations for Use with B. thailandensis Genetic Tools

Stock Final
Supplement Solvent concentration a concentration b
2,6‐Diaminopimelic acid (DAP) 1 M NaOH 100 mg/ml 200 μg/ml
Chloramphenicol Ethanol 20 mg/ml 20 μg/ml
Kanamycin Water 125 mg/ml 250 μg/ml
Tetracycline c Ethanol 10 mg/ml 50 μg/ml
Ampicillin d Water 100 mg/ml 100 μg/ml
X‐gluc DMF or DMSO 50 mg/ml 50 μg/ml
Sucrose Water 50% (w/v) 15% (w/v)
Rhamnose c Water 20% (w/v) 0.2% (w/v)

 aMost stock solutions should be filter‐sterilized (0.22‐μm filter) and stored at −20ºC. Sucrose and rhamnose solutions may be stored at room temperature.
 bFinal working concentration in liquid medium or agar plates. Media should be cooled to 55ºC before the addition of antibiotics or supplements.
 cLight‐sensitive. Stock solution and prepared media should be protected from light.
 dFor use with E. coli only. B. thailandensis is resistant to ampicillin.
 eX‐gluc, 5‐bromo‐4‐chloro‐3‐indoxyl‐beta‐D‐glucuronide.

Basic Protocol 2: Site‐Specific Chromosome Insertion by Mini‐Tn7 Transposition

  Materials
  • B. thailandensis frozen stock
  • Donor E. coli RHO3 carrying mini‐Tn7 plasmid (see Table 4.2.2)
  • Donor E. coli RHO3 carrying helper plasmid pTNS3 (see Table 4.2.2)
  • Low‐salt LB (LSLB) plates supplemented with DAP and/or appropriate antibiotics (see recipe; Table 4.2.1)
  • Sterile toothpicks or inoculating loops
  • 37ºC incubator
Table 4.0.2   MaterialsPlasmids Used Routinely for Genetic Manipulation of B. thailandensis a

Plasmid Description/features Antibiotic resistance Reference
pTNS3 Helper plasmid for mini‐Tn7, oriT, R6K ori Amp (Choi et al., )
pUC18T‐miniTn7T‐Km attTn7 site delivery plasmid with MCS Kan (mini‐Tn7), Amp (backbone) (Choi et al., )
miniTn7‐kan‐gfp Delivers gfp to attTn7 site Kan (Norris et al., )
mini‐Tn7‐kan‐rfp Delivers rfp to attTn7 site Kan (Norris et al., )
pFlpe4 Rham‐inducible flp, TS ori Kan (Choi et al., )
pFlpTet Rham‐inducible flp, TS ori Tet (Garcia et al., )
pEXKm5 Allelic exchange vector Kan (López et al., )

 aMCS, multiple cloning site; TS, temperature‐sensitive; Rham, rhamnose; Amp, ampicillin; Kan, kanamycin; Tet, tetracycline.

Basic Protocol 3: Homologous Recombination by Natural Transformation with Linear DNA

  Materials
  • B. thailandensis frozen stock
  • Low‐salt LB (LSLB) medium (see recipe)
  • M63 minimal medium (see recipe)
  • DNA containing antibiotic‐resistance cassette flanked by >500‐bp regions of homology to B. thailandensis genome (PCR product or B. thailandensis chromosomal DNA); if subsequent antibiotic‐resistance marker removal is desired, first flank cassette with FRT sequences (see protocol 4)
  • Low‐salt LB (LSLB) plates supplemented with appropriate antibiotics (see recipe; Table 4.2.1)
  • Sterile sticks or inoculating loops
  • Sterile, capped glass or plastic culture tubes (15 mm × 125 mm)
  • 37ºC shaking incubator
  • Spectrophotometer and cuvettes
  • Microcentrifuge and plastic tubes
  • Sterile cell spreader or glass beads

Basic Protocol 4: Flp Recombinase–Mediated Marker Excision

  Materials
  • B. thailandensis with a chromosomal FRT‐flanked antibiotic‐resistance cassette
  • Donor E. coli RHO3 containing pFlpe4 or pFlpTet (see Table 4.2.2)
  • Low‐salt LB (LSLB) plates (see recipe)
  • LSLB plates supplemented with 200 μg/ml 2,6‐diaminopimelic acid (DAP), 250 μg/ml kanamycin (Kan) or 50 μg/ml tetracycline (Tet), and/or 0.2% rhamnose (see recipe; Table 4.2.1)
  • 30ºC incubator
  • Sterile applicator sticks or inoculating loops
  • Additional reagents and equipment for mating (see protocol 1)

Basic Protocol 5: Allelic Exchange Using SACB Counterselection

  Materials
  • B. thailandensis frozen stock
  • Donor E. coli RHO3 containing pEXKm5 (see Table 4.2.2) with recombinant allele and homology regions
  • Low‐salt LB (LSLB) plates (see recipe)
  • LSLB plates supplemented with 200 μg/ml 2,6‐diaminopimelic acid (DAP), 250 μg/ml kanamycin (Kan), and/or 50 μg/ml X‐gluc (see recipe; Table 4.2.1)
  • YT plates supplemented with 15% sucrose and either 250 μg/ml Kan or 50 μg/ml X‐gluc (see recipe; Table 4.2.1)
  • YT broth (see recipe)
  • Sterile swabs
  • 30º and 37ºC incubators with and without shaking
  • Sterile applicator sticks or inoculating loops
  • Sterile capped glass or plastic culture tubes
  • Sterile cell spreader or glass beads

Support Protocol 1: Colony PCR

  Materials
  • Sterile, deionized water
  • 5× GoTaq buffer (Promega) or alternative Taq buffer
  • 10 mM dNTP mix
  • Primers (10 μM each forward and reverse)
  • Dimethyl sulfoxide (DMSO)
  • GoTaq polymerase (Promega) or alternative Taq polymerase
  • B. thailandensis colonies
  • Restriction enzyme(s) and appropriate buffer (optional)
  • 6× DNA gel loading dye
  • Agarose and 0.5× TAE buffer
  • Ethidium bromide or other DNA gel stain
  • 0.2‐ml tubes
  • Sterile toothpick
  • Thermal cycler
  • Agarose gel electrophoresis apparatus
  • UV transilluminator
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Figures

Videos

Literature Cited

  Anderson, M. S., Garcia, E. C., & Cotter, P. A. (2012). The Burkholderia bcpAIOB genes define unique classes of Two‐Partner secretion and contact dependent growth inhibition systems. PLoS Genetics, 8, e1002877. doi: 10.1371/journal.pgen.1002877
  Barrett, A. R., Kang, Y., Inamasu, K. S., Son, M. S., Vukovich, J. M., & Hoang, T. T. (2008). Genetic tools for allelic replacement in Burkholderia species. Applied and Environmental Microbiology, 74, 4498–4508. doi: 10.1128/AEM.00531‐08
  Chandler, J. R., Duerkop, B. A., Hinz, A., West, T. E., Herman, J. P., Churchill, M. E. A., … Greenberg, E. P. (2009). Mutational analysis of Burkholderia thailandensis quorum sensing and self‐aggregation. Journal of Bacteriology, 191, 5901–5909. doi: 10.1128/JB.00591‐09
  Choi, K.‐H., Gaynor, J. B., White, K. G., Lopez, C., Bosio, C. M., Karkhoff‐Schweizer, R. R., & Schweizer, H. P. (2005). A Tn7‐based broad‐range bacterial cloning and expression system. Nature Methods, 2, 443–448. doi: 10.1038/nmeth765
  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, 1064–1075. doi: 10.1128/AEM.01362‐08
  French, C. T., Toesca, I., Wu, T.‐H., Teslaa, T., Beaty, S. M., Wong, W., … Miller, J. F. (2011). Dissection of the Burkholderia intracellular life cycle using a photothermal nanoblade. Proceedings of the National Academy of Sciences of the United States of America, 108, 10295–12100. doi: 10.1073/pnas.1107183108
  Gallagher, L. A., Ramage, E., Patrapuvich, R., Weiss, E., Brittnacher, M., & Manoil, C. (2013). Sequence‐defined transposon mutant library of Burkholderia thailandensis. mBio, 4, e00604–13. doi: 10.1128/mBio.00604‐13
  Garcia, E. C., Anderson, M. S., Hagar, J. A., & Cotter, P. A. (2013). Burkholderia BcpA mediates biofilm formation independently of interbacterial contact‐dependent growth inhibition. Molecular Microbiology, 89, 1213–1225. doi: 10.1111/mmi.12339
  Hamad, M. A., Zajdowicz, S. L., Holmes, R. K., & Voskuil, M. I. (2009). An allelic exchange system for compliant genetic manipulation of the select agents Burkholderia pseudomallei and Burkholderia mallei. Gene, 430, 123–131. doi: 10.1016/j.gene.2008.10.011
  Haraga, A., West, T. E., Brittnacher, M. J., Skerrett, S. J., & Miller, S. I. (2008). Burkholderia thailandensis as a model system for the study of the virulence‐associated type III secretion system of Burkholderia pseudomallei. Infection and Immunity, 76, 5402–5411. doi: 10.1128/IAI.00626‐08
  Holden, M. T. G., Titball, R. W., Peacock, S. J., Cerdeno‐Tarraga, A. M., Atkins, T., Crossman, L. C., … Parkhill, J. (2004). Genomic plasticity of the causative agent of melioidosis, Burkholderia pseudomallei. Proceedings of the National Academy of Sciences of the United States of America, 101, 14240–14245. doi: 10.1073/pnas.0403302101
  Kespichayawattana, W., Rattanachetkul, S., Wanun, T., Utaisincharoen, P., & Sirisinha, S. (2000). Burkholderia pseudomallei induces cell fusion and actin‐associated membrane protrusion: A possible mechanism for cell‐to‐cell spreading. Infection and Immunity, 68, 5377–5384. doi: 10.1128/IAI.68.9.5377‐5384.2000
  Logue, C. A., Peak, I. R. A., & Beacham, I. R. (2009). Facile construction of unmarked deletion mutants in Burkholderia pseudomallei using sacB counter‐selection in sucrose‐resistant and sucrose‐sensitive isolates. Journal of Microbiological Methods, 76, 320–323. doi: 10.1016/j.mimet.2008.12.007
  López, C. M., Rholl, D. A., Trunck, L. S., & Schweizer, H. P. (2009). Versatile dual‐technology system for markerless allele replacement in Burkholderia pseudomallei. Applied and Environmental Microbiology, 75, 6496. doi: 10.1128/AEM.01669‐09
  Norris, M. H., Kang, Y., Wilcox, B., & Hoang, T. T. (2010). Stable, site‐specific fluorescent tagging constructs optimized for Burkholderia species. Applied and Environmental Microbiology, 76, 7635–7640. doi: 10.1128/AEM.01188‐10
  Peters, J. E. (2014). Tn7. Microbiology Spectrum 2:MDNA3–0010–2014. doi: 10.1128/microbiolspec.MDNA3‐0010‐2014
  Pósfai, G., Kolisnychenko, V., Bereczki, Z., & Blattner, F. R. (1999). Markerless gene replacement in Escherichia coli stimulated by a double‐strand break in the chromosome. Nucleic Acids Research, 27, 4409–4415. doi: 10.1093/nar/27.22.4409
  Schwarz, S., West, T. E., Boyer, F., Chiang, W.‐C., Carl, M. A., Hood, R. D., … Mougous, J. D. (2010). Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions. PLoS Pathogens, 6, e1001068. doi: 10.1371/journal.ppat.1001068
  Stevens, J. M., Ulrich, R. L., Taylor, L. A., Wood, M. W., Deshazer, D., Stevens, M. P., & Galyov, E. E. (2005). Actin‐binding proteins from Burkholderia mallei and Burkholderia thailandensis can functionally compensate for the actin‐based motility defect of a Burkholderia pseudomallei bimA mutant. Journal of Bacteriology, 187, 7857–7862. doi: 10.1128/JB.187.22.7857‐7862.2005
  Thongdee, M., Gallagher, L. A., Schell, M., Dharakul, T., Songsivilai, S., & Manoil, C. (2008). Targeted mutagenesis of Burkholderia thailandensis and Burkholderia pseudomallei through natural transformation of PCR fragments. Applied and Environmental Microbiology, 74, 2985–2989. doi: 10.1128/AEM.00030‐08
  Tuanyok, A., Leadem, B. R., Auerbach, R. K., Beckstrom‐Sternberg, S. M., Beckstrom‐Sternberg, J. S., Mayo, M., … Keim, P. (2008). Genomic islands from five strains of Burkholderia pseudomallei. BMC Genomics, 9, 566. doi: 10.1186/1471‐2164‐9‐566
  Yu, Y., Kim, H. S., Chua, H. H., Lin, C. H., Sim, S. H., Lin, D., … Tan, P. (2006). Genomic patterns of pathogen evolution revealed by comparison of Burkholderia pseudomallei, the causative agent of melioidosis, to avirulent Burkholderia thailandensis. BMC Microbiology, 6, 46. doi: 10.1186/1471‐2180‐6‐46
Internet Resources
  http://www.burkholderia.com
  The Burkholderia Genome Database contains a comprehensive collection of sequences, annotations, and comparative tools for completed and draft genomes within the Burkholderia genus, including B. thailandensis.
  http://tools.nwrce.org/tn_mutants/
  A useful tool for B. thailandensis researchers, the laboratory of Colin Manoil has generated a near‐saturation transposon mutant library in B. thailandensis E264, consisting of 87,000 unique mutants (7.5 mutants per gene) (Gallagher et al., ). The Transposon Mutant Library Browser displays available mutants and provides links for information on how to request mutants.
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