Skip to main content
SpringerLink
Log in

Generation of Conditional Mutants in Borrelia burgdorferi

  • Protocol
  • First Online:
Borrelia burgdorferi

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1690))

Abstract

Mutational studies aimed at characterizing the function(s) of bacterial genes required for growth or viability are constrained by the inability to generate deletion strains lacking the gene of interest. To circumvent this limitation, it is possible to generate conditional mutants in which a copy of the gene of interest is introduced into the bacteria to compensate for the loss of the native allele. Expression of the non-native copy of the target gene is typically under control of an inducible promoter, which allows for controllable and regulated production of the gene of interest. Conditional mutants are also broadly useful for phenotypic analyses of genes that require a tightly regulated and artificially inducible copy of the target gene. Herein, we describe the methods used to generate and confirm conditional mutant clones in Borrelia burgdorferi utilizing the Borrelia-adapted lac operator/repressor system.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Falkow S (1988) Molecular Koch’s postulates applied to microbial pathogenicity. Rev Infect Dis 10(Suppl 2):S274–S276

    Article  PubMed  Google Scholar 

  2. Walker JR, Kovarik A, Allen JS, Gustafson RA (1975) Regulation of bacterial cell division: temperature-sensitive mutants of Escherichia coli that are defective in septum formation. J Bacteriol 123(2):693–703

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Eidlic L, Neidhardt FC (1965) Protein and nucleic acid synthesis in two mutants of Escherichia coli with temperature-sensitive aminoacyl ribonucleic acid synthetases. J Bacteriol 89:706–711

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Pringle JR (1975) Induction, selection, and experimental uses of temperature-sensitive and other conditional mutants of yeast. Methods Cell Biol 12:233–272

    Article  CAS  PubMed  Google Scholar 

  5. Kamionka A, Bertram R, Hillen W (2005) Tetracycline-dependent conditional gene knockout in Bacillus subtilis. Appl Environ Microbiol 71(2):728–733. doi:10.1128/AEM.71.2.728-733.2005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Groshong AM, Gibbons NE, Yang XF, Blevins JS (2012) Rrp2, a prokaryotic enhancer-like binding protein, is essential for viability of Borrelia burgdorferi. J Bacteriol 194(13):3336–3342. doi:10.1128/JB.00253-12

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Bandy NJ, Salman-Dilgimen A, Chaconas G (2014) Construction and characterization of a Borrelia burgdorferi strain with conditional expression of the essential telomere resolvase, ResT. J Bacteriol 196(13):2396–2404. doi:10.1128/JB.01435-13

    Article  PubMed  PubMed Central  Google Scholar 

  8. Dubytska L, Godfrey HP, Cabello FC (2006) Borrelia burgdorferi ftsZ plays a role in cell division. J Bacteriol 188(5):1969–1978. doi:10.1128/JB.188.5.1969-1978.2006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Hyde JA, Shaw DK, Smith R 3rd, Trzeciakowski JP, Skare JT (2010) Characterization of a conditional bosR mutant in Borrelia burgdorferi. Infect Immun 78(1):265–274. doi:10.1128/IAI.01018-09

    Article  CAS  PubMed  Google Scholar 

  10. Ouyang Z, Zhou J, Norgard MV (2014) Synthesis of RpoS is dependent on a putative enhancer binding protein Rrp2 in Borrelia burgdorferi. PLoS One 9(5):e96917. doi:10.1371/journal.pone.0096917

    Article  PubMed  PubMed Central  Google Scholar 

  11. Ye M, Zhang JJ, Fang X, Lawlis GB, Troxell B, Zhou Y, Gomelsky M, Lou Y, Yang XF (2014) DhhP, a cyclic di-AMP phosphodiesterase of Borrelia burgdorferi, is essential for cell growth and virulence. Infect Immun 82(5):1840–1849. doi:10.1128/IAI.00030-14

    Article  PubMed  PubMed Central  Google Scholar 

  12. Sze CW, Morado DR, Liu J, Charon NW, Xu H, Li C (2011) Carbon storage regulator A (CsrA(Bb)) is a repressor of Borrelia burgdorferi flagellin protein FlaB. Mol Microbiol 82(4):851–864. doi:10.1111/j.1365-2958.2011.07853.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Gilbert MA, Morton EA, Bundle SF, Samuels DS (2007) Artificial regulation of ospC expression in Borrelia burgdorferi. Mol Microbiol 63(4):1259–1273. doi:10.1111/j.1365-2958.2007.05593.x

    Article  CAS  PubMed  Google Scholar 

  14. Xu Q, Shi Y, Dadhwal P, Liang FT (2012) RpoS regulates essential virulence factors remaining to be identified in Borrelia burgdorferi. PLoS One 7(12):e53212. doi:10.1371/journal.pone.0053212

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Tilly K, Elias AF, Bono JL, Stewart P, Rosa P (2000) DNA exchange and insertional inactivation in spirochetes. J Mol Microbiol Biotechnol 2(4):433–442

    CAS  PubMed  Google Scholar 

  16. Rosa PA, Tilly K, Stewart PE (2005) The burgeoning molecular genetics of the Lyme disease spirochaete. Nat Rev Microbiol 3(2):129–143. doi:10.1038/nrmicro1086

    Article  CAS  PubMed  Google Scholar 

  17. Liu P, Jenkins NA, Copeland NG (2003) A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Res 13(3):476–484. doi:10.1101/gr.749203

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Dancz CE, Haraga A, Portnoy DA, Higgins DE (2002) Inducible control of virulence gene expression in Listeria monocytogenes: temporal requirement of listeriolysin O during intracellular infection. J Bacteriol 184(21):5935–5945. doi:10.1128/JB.184.21.5935-5945.2002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Yansura DG, Henner DJ (1984) Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis. Proc Natl Acad Sci U S A 81(2):439–443

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Blevins JS, Revel AT, Smith AH, Bachlani GN, Norgard MV (2007) Adaptation of a luciferase gene reporter and lac expression system to Borrelia burgdorferi. Appl Environ Microbiol 73(5):1501–1513. doi:10.1128/AEM.02454-06

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Fraser CM, Casjens S, Huang WM, Sutton GG, Clayton R, Lathigra R, White O, Ketchum KA, Dodson R, Hickey EK, Gwinn M, Dougherty B, Tomb JF, Fleischmann RD, Richardson D, Peterson J, Kerlavage AR, Quackenbush J, Salzberg S, Hanson M, van Vugt R, Palmer N, Adams MD, Gocayne J, Weidman J, Utterback T, Watthey L, McDonald L, Artiach P, Bowman C, Garland S, Fuji C, Cotton MD, Horst K, Roberts K, Hatch B, Smith HO, Venter JC (1997) Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi. Nature 390(6660):580–586. doi:10.1038/37551

    Article  CAS  PubMed  Google Scholar 

  22. Yin Y, Yang Y, Xiang X, Wang Q, Yang ZN, Blevins J, Lou Y, Yang XF (2016) Insight into the dual functions of bacterial enhancer-binding protein Rrp2 of Borrelia burgdorferi. J Bacteriol 198(10):1543–1552. doi:10.1128/JB.01010-15

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Barbour AG (1984) Isolation and cultivation of Lyme disease spirochetes. Yale J Biol Med 57(4):521–525

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Hyde JA, Weening EH, Skare JT (2011) Genetic transformation of Borrelia burgdorferi. Curr Protoc Microbiol Chapter 12:Unit 12C 14. doi:10.1002/9780471729259.mc12c04s20

  25. Samuels DS (1995) Electrotransformation of the spirochete Borrelia burgdorferi. Methods Mol Biol 47:253–259. doi:10.1385/0-89603-310-4:253

    CAS  PubMed  Google Scholar 

  26. Yang XF, Pal U, Alani SM, Fikrig E, Norgard MV (2004) Essential role for OspA/B in the life cycle of the Lyme disease spirochete. J Exp Med 199(5):641–648. doi:10.1084/jem.20031960

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Coller HA, Coller BS (1986) Poisson statistical analysis of repetitive subcloning by the limiting dilution technique as a way of assessing hybridoma monoclonality. Methods Enzymol 121:412–417. doi:10.1016/0076-6879(86)21039-3

    Article  CAS  PubMed  Google Scholar 

  28. Blevins JS, Hagman KE, Norgard MV (2008) Assessment of decorin-binding protein a to the infectivity of Borrelia burgdorferi in the murine models of needle and tick infection. BMC Microbiol 8:82. doi:10.1186/1471-2180-8-82

    Article  PubMed  PubMed Central  Google Scholar 

  29. Eggers CH, Caimano MJ, Clawson ML, Miller WG, Samuels DS, Radolf JD (2002) Identification of loci critical for replication and compatibility of a Borrelia burgdorferi cp32 plasmid and use of a cp32-based shuttle vector for the expression of fluorescent reporters in the Lyme disease spirochaete. Mol Microbiol 43(2):281–295. doi:10.1046/j.1365-2958.2002.02758.x

    Article  CAS  PubMed  Google Scholar 

  30. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77(1):51–59. doi:10.1016/0378-1119(89)90358-2

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by funding to J.S.B. through the Arkansas Biosciences Institute (major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000), NIH/NIAID R01-AI087678, NIH/NIAID R21-AI 119532, as well as support through the Translational Research Institute (UL1-TR000039; NIH National Center for Research Resources and National Center for Advancing Translational Sciences) and the UAMS Center for Microbial Pathogenesis and Host Inflammatory Responses (P20-GM103625).

Author information

Authors and Affiliations

  1. Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR, 72205, USA

    Jacob I. Latham & Jon S. Blevins

Authors
  1. Jacob I. Latham
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jon S. Blevins
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jon S. Blevins .

Editor information

Editors and Affiliations

  1. Department of Veterinary Medicine, University of Maryland, College Park, Maryland, USA

    Utpal Pal

  2. Department of Microbiology, Ondokuz Mayis University, Samsun, Turkey

    Ozlem Buyuktanir

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Latham, J.I., Blevins, J.S. (2018). Generation of Conditional Mutants in Borrelia burgdorferi . In: Pal, U., Buyuktanir, O. (eds) Borrelia burgdorferi. Methods in Molecular Biology, vol 1690. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7383-5_17

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7383-5_17

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7382-8

  • Online ISBN: 978-1-4939-7383-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation