Abstract
The disciplines of Borrelia (Borreliella) burgdorferi microbiology and Lyme disease pathogenesis have come to depend on the genetic manipulation of the spirochete. Generating mutants in these recalcitrant bacteria, while not straightforward, is routinely accomplished in numerous laboratories, although there are several crucial caveats to consider. This chapter describes the design of basic molecular genetic experiments as well as the detailed methodologies to prepare and transform competent cells, select for and isolate transformants, and complement or genetically restore mutants.
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References
Samuels DS, Mach KE, Garon CF (1994) Genetic transformation of the Lyme disease agent Borrelia burgdorferi with coumarin-resistant gyrB. J Bacteriol 176:6045–6049
Samuels DS (1995) Electrotransformation of the spirochete Borrelia burgdorferi. In: Nickoloff JA (ed) Electroporation protocols for microorganisms, Methods in molecular biology, vol 47. Humana Press, Totowa, NJ, pp 253–259.
Brisson D, Drecktrah D, Eggers CH, Samuels DS (2012) Genetics of Borrelia burgdorferi. Annu Rev Genet 46:515–536
Groshong AM, Blevins JS (2014) Insights into the biology of Borrelia burgdorferi gained through the application of molecular genetics. Adv Appl Microbiol 86:41–143
Lin T, Troy EB, Hu LT, Gao L, Norris SJ (2014) Transposon mutagenesis as an approach to improved understanding of Borrelia pathogenesis and biology. Front Cell Infect Microbiol 4:63
Rosa PA, Cabello F, Samuels DS (2010) Genetic manipulation of Borrelia burgdorferi. In: Samuels DS, Radolf JD (eds) Borrelia: molecular biology, host interaction and pathogenesis. Caister Academic Press, Norfolk, UK, pp 189–219
Rosa PA, Tilly K, Stewart PE (2005) The burgeoning molecular genetics of the Lyme disease spirochaete. Nat Rev Microbiol 3:129–143
Samuels DS (2006) Antibiotic resistance in Borrelia burgdorferi: applications for genetic manipulation and implications for evolution. In: Cabello FC, Hulinska D, Godfrey HP (eds) Molecular biology of spirochetes, NATO science series: life and behavioural sciences, vol 373. IOS Press, Amsterdam, Netherlands, pp 56–70
Casjens S, Palmer N, van Vugt R, Huang WM, Stevenson B, Rosa P, Lathigra R, Sutton G, Peterson J, Dodson RJ, Haft D, Hickey E, Gwinn M, White O, Fraser CM (2000) A bacterial genome in flux: the twelve linear and nine circular extrachromosomal DNAs in an infectious isolate of the Lyme disease spirochete Borrelia burgdorferi. Mol Microbiol 35:490–516
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 J-F, Fleischmann RD, Richardson D, Peterson J, Kerlavage AR, Quakenbush J, Salzberg S, Hanson M, van Vugt R, Palmer N, Adams MK, Gocayne J, Weidman J, Utterback T, Watthey L, McDonald L, Artiach P, Bowman C, Garland S, Fujii C, Cotton MD, Horst K, Roberts K, Hatch B, Smith HO, Venter JC (1997) Genomic sequence of a Lyme disease spirochete, Borrelia burgdorferi. Nature 390:580–586
Bono JL, Elias AF, Kupko JJ III, Stevenson B, Tilly K, Rosa P (2000) Efficient targeted mutagenesis in Borrelia burgdorferi. J Bacteriol 182:2445–2452
Elias AF, Bono JL, Kupko JJ 3rd, Stewart PE, Krum JG, Rosa PA (2003) New antibiotic resistance cassettes suitable for genetic studies in Borrelia burgdorferi. J Mol Microbiol Biotechnol 6:29–40
Frank KL, Bundle SF, Kresge ME, Eggers CH, Samuels DS (2003) aadA confers streptomycin-resistance in Borrelia burgdorferi. J Bacteriol 185:6723–6727
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 expression of fluorescent reporters in the Lyme disease spirochaete. Mol Microbiol 43:281–296
Sartakova M, Dobrikova E, Cabello FC (2000) Development of an extrachromosomal cloning vector system for use in Borrelia burgdorferi. Proc Natl Acad Sci U S A 97:4850–4855
Stewart P, Thalken R, Bono J, Rosa P (2001) Isolation of a circular plasmid region sufficient for autonomous replication and transformation of infectious Borrelia burgdorferi. Mol Microbiol 39:714–721
Lin T, Gao L, Zhang C, Odeh E, Jacobs MB, Coutte L, Chaconas G, Philipp MT, Norris SJ (2012) Analysis of an ordered, comprehensive STM mutant library in infectious Borrelia burgdorferi: insights into the genes required for mouse infectivity. PLoS One 7:e47532
Morozova OV, Dubytska LP, Ivanova LB, Moreno CX, Bryksin AV, Sartakova ML, Dobrikova EY, Godfrey HP, Cabello FC (2005) Genetic and physiological characterization of 23S rRNA and ftsJ mutants of Borrelia burgdorferi isolated by mariner transposition. Gene 357:63–72
Stewart PE, Hoff J, Fischer E, Krum JG, Rosa PA (2004) Genome-wide transposon mutagenesis of Borrelia burgdorferi for identification of phenotypic mutants. Appl Environ Microbiol 70:5973–5979
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 73:1501–1513
Gilbert MA, Morton EA, Bundle SF, Samuels DS (2007) Artificial regulation of ospC expression in Borrelia burgdorferi. Mol Microbiol 63:1259–1273
Whetstine CR, Slusser JG, Zückert WR (2009) Development of a single-plasmid-based regulatable gene expression system for Borrelia burgdorferi. Appl Environ Microbiol 75:6553–6558
Carroll JA, Stewart PE, Rosa P, Elias AF, Garon CF (2003) An enhanced GFP reporter system to monitor gene expression in Borrelia burgdorferi. Microbiology 149:1819–1828
Hayes BM, Jewett MW, Rosa PA (2010) lacZ reporter system for use in Borrelia burgdorferi. Appl Environ Microbiol 76:7407–7412
Hyde JA, Weening EH, Chang M, Trzeciakowski JP, Höök M, Cirillo JD, Skare JT (2011) Bioluminescent imaging of Borrelia burgdorferi in vivo demonstrates that thefibronectin-bindingprotein BBK32 is required for optimal infectivity. Mol Microbiol 82:99–113
Drecktrah D, Douglas JM, Samuels DS (2010) Use of rpsL as a counterselectable marker in Borrelia burgdorferi. Appl Environ Microbiol 76:985–987
Barbour AG (1984) Isolation and cultivation of Lyme disease spirochetes. Yale J Biol Med 57:521–525
Burgdorfer W, Barbour AG, Hayes SF, Benach JL, Grunwaldt E, Davis JP (1982) Lyme disease—a tick-borne spirochetosis? Science 216:1317–1319
Nickoloff JA (ed) (1995) Electroporation protocols for microorganisms, vol 47. Methods in molecular biology. Humana Press, Totowa, NJ
Samuels DS, Garon CF (1997) Oligonucleotide-mediated genetic transformation of Borrelia burgdorferi. Microbiology 143:519–522
Shigekawa K, Dower WJ (1988) Electroporation of eukaryotes and prokaryotes: a general approach to the introduction of macromolecules into cells. Biotechniques 6:742–751
Trevors JT, Chassy BM, Dower WJ, Blaschek HP (1992) Electrotransformation of bacteria by plasmid DNA. In: Chang DC, Chassy BM, Saunders JA, Sowers AE (eds) Guide to electroporation and electrofusion. Academic Press, San Diego, pp 265–290
Tilly K, Elias AF, Bono JL, Stewart P, Rosa P (2000) DNA exchange and insertional inactivation in spirochetes. J Mol Microbiol Biotechnol 2:433–442
Kawabata H, Norris SJ, Watanabe H (2004) BBE02 disruption mutants of Borrelia burgdorferi B31 have a highly transformable, infectious phenotype. Infect Immun 72:7147–7754
Labandeira-Rey M, Skare JT (2001) Decreased infectivity in Borrelia burgdorferi strain B31 is associated with loss of linear plasmid 25 or 28-1. Infect Immun 69:446–455
Lawrenz MB, Kawabata H, Purser JE, Norris SJ (2002) Decreased electroporation efficiency in Borrelia burgdorferi containing linear plasmids lp25 and lp56: impact on transformation of infectious B. burgdorferi. Infect Immun 70:4798–4804
Purser JE, Norris SJ (2000) Correlation between plasmid content and infectivity in Borrelia burgdorferi. Proc Natl Acad Sci U S A 97:13865–13870
Elias AF, Stewart PE, Grimm D, Caimano MJ, Eggers CH, Tilly K, Bono JL, Akins DR, Radolf JD, Schwan TG, Rosa P (2002) Clonal polymorphism of Borrelia burgdorferi strain B31 MI: implications for mutagenesis in an infectious strain background. Infect Immun 70:2139–2150
Caimano MJ, Eggers CH, Hazlett KRO, Radolf JD (2004) RpoS is not central to the general stress response in Borrelia burgdorferi but does control expression of one or more essential virulence determinants. Infect Immun 72:6433–6445
Hübner A, Yang X, Nolen DM, Popova TG, Cabello FC, Norgard MV (2001) Expression of Borrelia burgdorferi OspC and DbpA is controlled by a RpoN-RpoS regulatory pathway. Proc Natl Acad Sci U S A 98:12724–12729
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 199:641–648
Chan K, Alter L, Barthold SW, Parveen N (2015) Disruption of bbe02 by insertion of a luciferase gene increases transformation efficiency of Borrelia burgdorferi and allows live imaging in Lyme disease susceptible C3H mice. PLoS One 10:e0129532
Parveen N, Cornell KA, Bono JL, Chamberland C, Rosa P, Leong JM (2006) Bgp, a secreted glycosaminoglycan-binding protein of Borrelia burgdorferi strain N40, displays nucleosidase activity and is not essential for infection of immunodeficient mice. Infect Immun 74:3016–3020
Fingerle V, Goettner G, Gern L, Wilske B, Schulte-Spechtel U (2007) Complementation of a Borrelia afzelii OspC mutant highlights the crucial role of OspC for dissemination of Borrelia afzelii in Ixodes ricinus. Int J Med Microbiol 297:97–107
Eggers CH, Caimano MJ, Radolf JD (2006) Sigma factor selectivity in Borrelia burgdorferi: RpoS recognition of the ospE/ospF/elp promoters is dependent on the sequence of the −10 region. Mol Microbiol 59:1859–1875
Johnson RC, Schmid GP, Hyde FW, Steigerwalt AG, Brenner DJ (1984) Borrelia burgdorferi sp. nov.: etiologic agent of Lyme disease. Int J Syst Bacteriol 34:496–497
Di L, Pagan PE, Packer D, Martin CL, Akther S, Ramrattan G, Mongodin EF, Fraser CM, Schutzer SE, Luft BJ, Casjens SR, Qiu W-G (2014) BorreliaBase: a phylogeny-centered browser of Borrelia genomes. BMC Bioinformatics 15:233
Casjens SR, Mongodin EF, Qiu W-G, Luft BJ, Schutzer SE, Gilcrease EB, Huang WM, Vujadinovic M, Aron JK, Vargas LC, Freeman S, Radune D, Weidman JF, Dimitrov GI, Khouri HM, Sosa JE, Halpin RA, Dunn JJ, Fraser CM (2012) Genome stability of Lyme disease spirochetes: comparative genomics of Borrelia burgdorferi plasmids. PLoS One 7:e33280
Chen Q, Fischer JR, Benoit VM, Dufour NP, Youderian P, Leong JM (2008) In vitro CpG methylation increases the transformation efficiency of Borrelia burgdorferi strains harboring the endogenous linear plasmid lp56. J Bacteriol 190:7885–7891
Rego ROM, Bestor A, Rosa PA (2011) Defining the plasmid-borne restriction-modification systems of the Lyme disease spirochete Borrelia burgdorferi. J Bacteriol 193:1161–1171
Purser JE, Lawrenz MB, Caimano MJ, Howell JK, Radolf JD, Norris SJ (2003) A plasmid-encoded nicotinamidase (PncA) is essential for infectivity of Borrelia burgdorferi in a mammalian host. Mol Microbiol 48:753–764
Jewett MW, Lawrence K, Bestor AC, Tilly K, Grimm D, Shaw P, VanRaden M, Gherardini F, Rosa PA (2007) The critical role of the linear plasmid lp36 in the infectious cycle of Borrelia burgdorferi. Mol Microbiol 64:1358–1374
Samuels DS, Garon CF (1993) Coumermycin A1 inhibits growth and induces relaxation of supercoiled plasmids in Borrelia burgdorferi, the Lyme disease agent. Antimicrob Agents Chemother 37:46–50
Samuels DS, Marconi RT, Huang WM, Garon CF (1994) gyrB mutations in coumermycin A1-resistant Borrelia burgdorferi. J Bacteriol 176:3072–3075
Knight SW, Kimmel BJ, Eggers CH, Samuels DS (2000) Disruption of the Borrelia burgdorferi gac gene, encoding the naturally synthesized GyrA C-terminaldomain. J Bacteriol 182:2048–2051
Rosa P, Samuels DS, Hogan D, Stevenson B, Casjens S, Tilly K (1996) Directed insertion of a selectable marker into a circular plasmid of Borrelia burgdorferi. J Bacteriol 178:5946–5953
Alverson J, Samuels DS (2002) groEL expression in gyrB mutants of Borrelia burgdorferi. J Bacteriol 184:6069–6072
Alverson J, Bundle SF, Sohaskey CD, Lybecker MC, Samuels DS (2003) Transcriptional regulation of the ospAB and ospC promoters from Borrelia burgdorferi. Mol Microbiol 48:1665–1677
Sohaskey CD, Barbour AG (1999) Esterases in serum-containing growth media counteract chloramphenicol acetyltransferase activity in vitro. Antimicrob Agents Chemother 43:655–660
Criswell D, Tobiason VL, Lodmell JS, Samuels DS (2006) Mutations conferring aminoglycoside and spectinomycin resistance in Borrelia burgdorferi. Antimicrob Agents Chemother 50:445–452
Drecktrah D, Lybecker M, Popitsch N, Rescheneder P, Hall LS, Samuels DS (2015) The Borrelia burgdorferi RelA/SpoT homolog and stringent response regulate survival in the tick vector and global gene expression during starvation. PLoS Pathog 11:e1005160
Hoon-Hanks LL, Morton EA, Lybecker MC, Battisti JM, Samuels DS, Drecktrah D (2012) Borrelia burgdorferi malQ mutants utilize disaccharides and traverse the enzootic cycle. FEMS Immunol Med Microbiol 66:157–165
Lybecker MC, Abel CA, Feig AL, Samuels DS (2010) Identification and function of the RNA chaperone Hfq in the Lyme disease spirochete Borrelia burgdorferi. Mol Microbiol 78:622–635
Lybecker MC, Samuels DS (2007) Temperature-induced regulation of RpoS by a small RNA in Borrelia burgdorferi. Mol Microbiol 64:1075–1089
Sultan SZ, Pitzer JE, Miller MR, Motaleb MA (2010) Analysis of a Borrelia burgdorferi phosphodiesterase demonstrates a role for cyclic-di-guanosine monophosphate in motility and virulence. Mol Microbiol 77:128–142
Ge Y, Old IG, Saint Girons I, Charon NW (1997) Molecular characterization of a large Borrelia burgdorferi motility operon which is initiated by a consensus σ70 promoter. J Bacteriol 179:2289–2299
Babitzke P, Yealy J, Campanelli D (1996) Interaction of the trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis with RNA: effects of the number of GAG repeats, the nucleotides separating adjacent repeats, and RNA secondary structure. J Bacteriol 178:5159–5163
Drecktrah D, Hall LS, Hoon-Hanks LL, Samuels DS (2013) An inverted repeat in the ospC operator is required for induction in Borrelia burgdorferi. PLoS One 8:e68799
Earnhart CG, LeBlanc DV, Alix KE, Desrosiers DC, Radolf JD, Marconi RT (2010) Identification of residues within ligand-binding domain 1 (LBD1) of the Borrelia burgdorferi OspC protein required for function in the mammalian environment. Mol Microbiol 76:393–408
Yang XF, Alani SM, Norgard MV (2003) The response regulator Rrp2 is essential for the expression of major membrane lipoproteins in Borrelia burgdorferi. Proc Natl Acad Sci U S A 100:11001–11006
Beaurepaire C, Chaconas G (2005) Mapping of essential replication functions of the linear plasmid lp17 of B. burgdorferi by targeted deletion walking. Mol Microbiol 57:132–142
Grimm D, Eggers CH, Caimano MJ, Tilly K, Stewart PE, Elias AF, Radolf JD, Rosa PA (2004) Experimental assessment of the roles of linear plasmids lp25 and lp28-1 of Borrelia burgdorferi throughout the infectious cycle. Infect Immun 72:5938–5946
Bundoc VG, Barbour AG (1989) Clonal polymorphisms of outer membrane protein OspB of Borrelia burgdorferi. Infect Immun 57:2733–2741
Kurtti TJ, Munderloh UG, Johnson RC, Ahlstrand GG (1987) Colony formation and morphology in Borrelia burgdorferi. J Clin Microbiol 25:2054–2058
Rosa PA, Hogan DM (1992) Colony formation by Borrelia burgdorferi in solid medium: clonal analysis of osp locus variants. In: Munderloh UG, Kurtti TJ (eds) First international conference on tick-borne pathogens at the host-vector Interface: an agenda for research. University of Minnesota, St. Paul, pp 95–103
Bunikis I, Kutschan-Bunikis S, Bonde M, Bergström S (2011) Multiplex PCR as a tool for validating plasmid content of Borrelia burgdorferi. J Microbiol Methods 86:243–247
Tilly K, Krum JG, Bestor A, Jewett MW, Grimm D, Bueschel D, Byram R, Dorward D, Vanraden MJ, Stewart P, Rosa P (2006) Borrelia burgdorferi OspC protein required exclusively in a crucial early stage of mammalian infection. Infect Immun 74:3554–3564
Marconi RT, Samuels DS, Garon CF (1993) Transcriptional analyses and mapping of the ospC gene in Lyme disease spirochetes. J Bacteriol 175:926–932
Acknowledgments
We thank Phil Stewart for thoughtful review of the manuscript, Rich Marconi, Patti Rosa, Tom Schwan, and Kit Tilly for advice during development of the original protocol in the early 1990s, Frank Yang for suggesting the cloning-by-limiting-dilution protocol, and Christian Eggers, Mike Gilbert, Meghan Lybecker and the other members, past and present, of our laboratory for useful discussions. Genetic transformation and complementation experiments in our laboratory are supported by National Institutes of Health grant AI051486 (to D.S.S.).
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Samuels, D.S., Drecktrah, D., Hall, L.S. (2018). Genetic Transformation and Complementation. 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_15
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